transforming-growth-factor-beta and Calcinosis

Calcinosis cutis, defined as sub-epidermal deposition of calcium salts, is a major clinical problem in patients with SSc, affecting 20-40% of patients. A number of recognized factors associated with calcinosis have been identified, including disease duration, digital ischaemia and acro-osteolysis. Yet, to date, the pathogenesis of SSc-related calcinosis remains unknown, and currently there is no effective disease-modifying pharmacotherapy. Following onset of SSc, there are marked changes in the extracellular matrix (ECM) of the skin, notably a breakdown in the microfibrillar network and accumulation of type I collagen. Our hypothesis is that these pathological changes reflect a changing cellular phenotype and result in a primed microenvironment for soft tissue calcification, with SSc fibroblasts adopting a pro-osteogenic profile, and specific driving forces promoting tissue mineralization. Considering the role of the ECM in disease progression may help elucidate the mechanism(s) behind SSc-related calcinosis and inform the development of future therapeutic interventions.

Topics: Calcinosis; Cell Differentiation; Cell Hypoxia; Cellular Microenvironment; Collagen Type I; Disease Progression; Elastin; Extracellular Matrix; Fibrillin-1; Fibroblasts; Glucose Transporter Type 1; Humans; Mesenchymal Stem Cells; Myofibroblasts; Osteoblasts; Osteogenesis; Osteolysis; Phenotype; Phosphates; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor β (TGFβ) family members are involved in a wide range of diverse functions and play key roles in embryogenesis, development and tissue homeostasis. Perturbation of TGFβ signaling may lead to vascular and other diseases. In vitro studies have provided evidence that TGFβ family members have a wide range of diverse effects on vascular cells, which are highly dependent on cellular context. Consistent with these observations genetic studies in mice and humans showed that TGFβ family members have ambiguous effects on the function of the cardiovascular system. In this review we discuss the recent advances on TGFβ signaling in (cardio)vascular diseases, and describe the value of TGFβ signaling as both a disease marker and therapeutic target for (cardio)vascular diseases.

Topics: Animals; Atherosclerosis; Calcinosis; Cardiovascular Diseases; Humans; Ligands; Mice; Models, Biological; Signal Transduction; Transforming Growth Factor beta

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. A full understanding of the signalling pathways mediating vascular calcification is crucial not just because of the importance of this pathology in disease, but also for exploring potential therapeutic targets. Clinically there is a need to develop therapies to prevent or even reverse calcification in situations of atherosclerosis, chronic kidney disease, diabetes, and aging. In this brief review, we intend to explore the initial triggers, which are commonly related to calcification in different disease scenarios and examine the downstream signalling pathways that instigate the process of vascular calcification. In particular, we try to dissect these pathways and also examine cross-talk between different signalling pathways. Our focus is the vascular smooth muscle cell (VSMC) as it is ultimately the phenotypic modulation of these cells that may drive the calcification process.

Topics: Aging; Bone Morphogenetic Proteins; Calcinosis; Humans; Hypercalcemia; Hyperphosphatemia; Inflammation; Interleukin-6; Muscle, Smooth, Vascular; Oxidative Stress; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factors; Vascular Diseases; Wnt Proteins

Recently, many investigators have reported that treatment with vitamin D improves outcomes of patients with chronic kidney disease. Though the detailed mechanisms have remained unclear, it has been speculated that such a treatment may prevent progression of chronic kidney disease and cardiovascular disease. It has been reported that Vitamin D may attenuate renal injury and ameliorate renal function and proteinuria. In addition, several studies have shown that vitamin D may prevent progression of atherosclerosis, vascular calcification and left ventricular hypertrophy. The emerging experimental and clinical evidence has suggested that vitamin D may protect kidney and cardiovascular system.

Topics: Atherosclerosis; Calcinosis; Cardiovascular Diseases; Chronic Disease; Humans; Hyperparathyroidism, Secondary; Hypertrophy, Left Ventricular; Kidney; Kidney Diseases; Receptors, Calcitriol; Renin-Angiotensin System; Th1 Cells; Transforming Growth Factor beta; Vitamin D; Vitamin D-Binding Protein

In the last decade, the nephrology community has focused its attention on the main cause of morbidity and mortality in chronic renal failure patients: cardiovascular disease. In addition, recent studies pointed out that vascular calcification (VC) is a major cause of cardiovascular disease in the dialysis population. Interestingly, the pathogenesis of VC and soft tissue calcification in chronic kidney disease (CKD) has been extensively investigated. Nowadays we know that VC is associated not only with passive calcium phosphate deposition, but also with an active, cell-mediated process. To better understand the pathogenesis of VC in CKD, numerous regulatory proteins have been studied, because of their ability to inhibit mineral deposition in the vessels. We here examine the state of the art of those substances recognized as regulatory key factors in preventing VC in uremic conditions, such as fetuin A (alpha2-Heremans-Schmid glycoprotein), matrix gamma-carboxyglutamic acid protein, pyrophosphate, osteoprotegerin and bone morphogenetic protein. We conclude that at present it is too early to introduce these novel markers into clinical practice.

Topics: 1-Carboxyglutamic Acid; alpha-Fetoproteins; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcinosis; Diphosphates; Humans; Kidney Failure, Chronic; Models, Biological; Osteoprotegerin; Risk Factors; Transforming Growth Factor beta; Uremia; Vascular Diseases

Arterial calcification is common in patients with type 2 diabetes mellitus (DM), chronic kidney disease (CKD), and other chronic inflammatory disorders. Arterial calcification is associated with significant morbidity and increased early mortality. The molecular signature of vascular calcification in diabetes is strikingly similar to that of CKD. Low-grade arterial inflammation is common to both conditions, and increased levels of tumor necrosis factor-alpha (TNF-alpha) have been reported in both DM and CKD. Recently, we described a novel TNF-alpha regulated Msx2-Wnt osteogenic program that regulates arterial calcification in an animal model of type 2 DM. TNF-alpha induces the osteogenic bone morphogenetic protein-2 (BMP-2), Msx2, Wnt3a, and Wnt7a mRNAs and leads to increased aortic calcium accumulation. Treatment with the TNF-alpha neutralizing antibody infliximab abrogates aortic BMP-2-Msx2-Wnt3a and Wnt7a signaling and attenuates aortic calcium accumulation significantly. Mice with vascular TNF-alpha augmented by the SM22-TNF-alpha transgene upregulate the aortic Msx2-Wnt3a/Wnt7a axis. Furthermore, SM22-TNF-alphaTg;TOPGAL mice exhibit greater beta-galactosidase reporter staining versus TOPGAL siblings in the aorta and coronaries, which indicates enhanced mural Wnt signaling in response to TNF-alpha. Thus, inflammatory TNF-alpha signals promote aortic osteogenic Msx2-Wnt programs in type 2 DM, and arterial calcification in this model is a TNF-alpha-driven Wnt-opathy. Having established the role of TNF-alpha in diabetic vascular calcification, an unmet need exists to evaluate the role of TNF-alpha and Msx2-Wnt signals in CKD-related calcification models. If validated in these models, then these findings will have significant therapeutic applications.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Gene Expression; Humans; Microfilament Proteins; Models, Biological; Muscle Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Diseases; Wnt Proteins

Interactions between endothelial cells and mural cells (pericytes and vascular smooth muscle cells) in the blood vessel wall have recently come into focus as central processes in the regulation of vascular formation, stabilization, remodeling, and function. Failure of the interactions between the 2 cell types, as seen in numerous genetic mouse models, results in severe and often lethal cardiovascular defects. Abnormal interactions between the 2 cell types are also implicated in a number of human pathological conditions, including tumor angiogenesis, diabetic microangiopathy, ectopic tissue calcification, and stroke and dementia syndrome CADASIL. In the present review, we summarize current knowledge concerning the identity, characteristics, diversity, ontogeny, and plasticity of pericytes. We focus on the advancement in recent years of the understanding of intercellular communication between endothelial and mural cells with a focus on transforming growth factor beta, angiopoietins, platelet-derived growth factor, spingosine-1-phosphate, and Notch ligands and their respective receptors. We finally highlight recent important data contributing to the understanding of the role of pericytes in tumor angiogenesis, diabetic retinopathy, and hereditary lymphedema.

Topics: Angiopoietin-1; Animals; Becaplermin; Calcinosis; Cell Communication; Cell Differentiation; Endothelial Cells; Humans; Lysophospholipids; Membrane Proteins; Muscle, Smooth, Vascular; Pericytes; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptor, Platelet-Derived Growth Factor beta; Receptor, TIE-2; Receptors, Lysosphingolipid; Receptors, Notch; Signal Transduction; Sphingosine; Transforming Growth Factor beta

Vascular calcification is a prominent feature of atherosclerosis but the mechanisms underlying vascular calcification are still obscure. Since bone-associated proteins such as osteonectin, osteocalcin, and matrix Gla protein have been detected in calcified vascular tissues, calcification has been considered to be an organized, regulated process similar to mineralization in bone tissue. Vascular smooth muscle cells (VSMCs) are currently considered to be responsible for the formation of vascular calcifications. Apoptosis of VSMCs appears to be a key factor in this process, while other factors including cell-cell interactions (macrophages and VSMCs), lipids, and plasma inorganic phosphate levels modulate the calcification process. The focus of this review is on the role of VSMCs in the development of calcifications in atherosclerotic plaques.

Topics: Apoptosis; Arteriosclerosis; Bone Development; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium Phosphates; Cell Differentiation; Cytoplasmic Vesicles; Lipid Metabolism; Macrophages; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteopontin; Sialoglycoproteins; Transforming Growth Factor beta

When last reviewed, bone morphogenetic protein 7 was presented as a potential new renal therapeutic agent, with multiple efficacies in chronic kidney disease. The object of this review is to describe progress from many sources since then in support or denial of the hypothesis.. Bone morphogenetic protein 7 has been shown to be an effective defence in several forms of chronic kidney disease in animal models, and its mechanisms of action have begun to be elucidated. Bone morphogenetic protein 7 inhibits tubular epithelial cell de-differentiation, mesenchymal transformation and apoptosis stimulated by various renal injuries. Bone morphogenetic protein 7 preserves glomerular integrity and inhibits injury-mediated mesangial matrix accumulation. In renal osteodystrophy, bone morphogenetic protein 7 affects osteoblast morphology and number, eliminates peritrabecular fibrosis, decreases bone resorption, and increases bone formation in secondary hyperparathyroidism. Bone morphogenetic protein 7 restores normal rates of bone formation in the adynamic bone disorder. Bone morphogenetic protein 7 is broadly efficacious in renal osteodystrophy, and importantly increases the skeletal deposition of ingested phosphorus and calcium, improving ion homeostasis in chronic kidney disease. Bone morphogenetic protein 7 was shown to prevent vascular calcification in a model of chronic kidney disease associated with the restoration of osteocalcin expression to normal tissue-restricted sites.. Bone morphogenetic protein 7 may be a powerful new therapeutic agent for chronic kidney disease, with the novel attribute of not only treating the kidney disease itself, but also directly inhibiting some of the most important complications of the disease state.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcinosis; Chronic Kidney Disease-Mineral and Bone Disorder; Diabetic Nephropathies; Fibrosis; Humans; Kidney Diseases; Kidney Failure, Chronic; Lupus Nephritis; Nephritis, Hereditary; Transforming Growth Factor beta; Vascular Diseases

Vascular calcification is a pathological calcification process. Its pathogenesis involves active mineralization by chondrogenic and osteogenic cells. Since cartilaginous metaplasia has been found in several vascular diseases, this process may represent one of vascular remodeling in response to vascular injury. Endochondral ossification following cartilaginous metaplasia play an important role in the progression of vascular calcification.

Topics: Animals; Arteriosclerosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cell Differentiation; Chondrocytes; Chondrogenesis; Collagen Type II; Cytokines; Diabetes Complications; High Mobility Group Proteins; Humans; Metaplasia; Muscle, Smooth, Vascular; Neoplasm Proteins; SOX9 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Diseases

Vascular calcification in dialysis patients is associated with morbidity and mortality risks. Recent evidence suggests that vascular calcification is an active process resembling osteogenesis and chondrogenesis process. In this process, hyperphosphatemia is one of the important regulators. Inorganic phosphates directly regulate vascular calcification in vitro through a sodium-dependent phosphate cotransporter and promote expression of the osteoblastic differentiation markers.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium-Binding Proteins; Dialysis; Extracellular Matrix Proteins; Humans; Matrix Gla Protein; Neoplasm Proteins; Osteopontin; Phosphorus; Phosphorus Metabolism Disorders; Risk; Sialoglycoproteins; Sodium-Phosphate Cotransporter Proteins; Symporters; Transcription Factors; Transforming Growth Factor beta; Vascular Diseases

Recent progress in genetics and mouse genomics enables researchers to unveil the molecular basis for mouse phenotypes that express pathologic calcification in soft tissue and/or articular tissues. A newly identified multipass transmembrane protein, ANK, appears to function as an inorganic pyrophosphate (PPi) transporter or regulator of PPi transport. Abnormal extracellular PPi (ePPi) metabolism has been implicated in abnormal calcification, decreased concentrations predisposing to basic calcium phosphate (BCP) deposition, and increased concentrations promoting calcium pyrophosphate dihydrate (CPPD) crystal deposition in articular tissues. The chromosomal location of human ANK overlaps the locus identified in several kindreds affected with familial chondrocalcinosis. Deficient generation of ePPi by the ectoenzyme nucleoside triphosphate pyrophosphohydrolase also results in excessive ossification and ectopic deposition of BCP crystals in tiptoe-walking mice and PC-1 null mice. Recent studies reinforce the important regulatory role of ePPi in pathologic and physiologic calcification.

Topics: Animals; Calcinosis; Diphosphates; Disease Models, Animal; Homeostasis; Humans; Membrane Proteins; Mice; Mice, Knockout; Mice, Transgenic; Phosphate Transport Proteins; Pyrophosphatases; Transforming Growth Factor beta

Topics: Adult; Aged; Arteriosclerosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium-Binding Proteins; Coronary Stenosis; Extracellular Matrix Proteins; Humans; Macrophages; Matrix Gla Protein; Middle Aged; Muscle, Smooth, Vascular; Neoplasm Proteins; Osteogenesis; Transcription Factors; Transforming Growth Factor beta; Tunica Intima

A case of gastric carcinoma with psammomatous calcification arising in the remnant stomach after Billroth II reconstruction is reported. Borrmann type 1 gastric carcinoma was detected in the remnant stomach of an 82-year-old woman, who had a past history of distal partial gastrectomy for a perforated gastric ulcer, with Billroth II reconstruction at 40 years of age. Histologically, the tumor was a tubular adenocarcinoma that invaded the muscularis propria. Numerous psammoma bodies were found in the lumens of the tumor glands. Dystrophic calcification of gastric cancer is rare and psammomatous calcification of gastric cancer has only been reported in five cases previously. To our knowledge, this is the first case of gastric carcinoma with psammomatous calcification arising in the remnant stomach. We also review previously published reports regarding gastric carcinoma with psammomatous calcification.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Proteins; Calcinosis; Female; Gastrectomy; Humans; Immunohistochemistry; Osteopontin; Sialoglycoproteins; Stomach Neoplasms; Transforming Growth Factor beta

Nowadays there is a strong necessity in identifying patients who may be exposed to the risk for future cardiovascular events like progressive atherosclerotic disease. Biomarkers are valuable tools for this purpose. Coronary artery calcification (CAC) is utilized as an important tool for the global risk assessment of cardiovascular events in individuals with intermediate risk. Decorin (DCN) is a small leucine-rich proteoglycan that induces calcification of arterial smooth muscle cell and localizes to mineral deposition in human atherosclerotic plaque. The main purpose of this clinical study was to find out the correlation between Decorin serum concentration and CAC in human for the first time.. In this study 84 patients with coronary artery disease who fulfilled inclusion and exclusion criteria, entered the study. For all patients a questionnaire consisting demographic data and traditional cardiovascular risk factors were completed. CT-Angiography was carried out to determine coronary artery calcium score and ELISA method was used for measuring DCN serum concentrations.. No significant correlation between DCN serum concentration and total CAC score and also CAC of left anterior descending, right coronary artery, left main coronary artery and circumflex was found in the study population (P>0.05).. On the basis of our results DCN serum concentration is not a suitable biomarker of coronary artery disease. However, more studies with higher sample size are necessary for its confirmation.

Topics: Aged; Biomarkers; Calcinosis; Comorbidity; Computed Tomography Angiography; Coronary Artery Disease; Decorin; Female; Humans; Lipids; Male; Middle Aged; Myocardial Ischemia; Pilot Projects; Risk Factors; Surveys and Questionnaires; Transforming Growth Factor beta

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-β1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-β1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-β-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-β1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-β are found in aVICs. TGF-β transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-β-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

Topics: Animals; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Cell Differentiation; Cells, Cultured; Cellular Senescence; Dogs; Mitral Valve; Myofibroblasts; Phenotype; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

To explore the mechanism of CircANKRD36 regulating cell heterogeneity and endothelial mesenchymal transition in aortic valve stromal cells by regulating miR-599 and TGF-β signaling pathway.. Human tissue specimens were divided into Control group (n = 25) and CAVD group (n = 25). The mRNA expressions of CircANKRD36 and miR-599 in tissue samples were analyzed by qRT-PCR. Western blot was used to analyze the protein expression of osteogenic differentiation related factors induced by OM.The expressions of ALP, osteocalcin, osteopontin, Runx2 and Cadherin11 were detected by Western blot.. The expression of CircANKRD36mRNA in CAVD tissue was lower than that in Control tissue (P < 0.05), and the expression of miR-599mRNA in CAVD tissue was higher than that in Control tissue (P < 0.05). CircANKRD36 was negatively correlated with ALP, osteocalcin, osteopontin, Runx2, Cadherin11 expression level after OM induced osteogenic differentiation. The expression level of miR-599 was positively correlated with ALP, osteocalcin, osteopontin, Runx2 and Cadherin11 after OM induced osteogenic differentiation.The expression of ALP, osteocalcin, osteopontin, Runx2 and Cadherin11 protein in circ+miR-599 group was lower than that in circ+miR-NC group (P < 0.05). Compared with Vector+miR-NC group, the protein expressions of TGF-β1, TGF-β2 and SMAD4 in circ+miR-NC group decreased (P < 0.05), while the protein expressions of TGF-β1, TGF-β2 and SMAD4 in circ+miR-599 group increased (P < 0.05).. CircANKRD36 can inhibit the expression of miR-599 and the activation of TGF-β signaling pathway, thus inhibiting the expression of differentiation-related factors of VIC osteogenesis and the formation of calcified nodules. Therefore, circANKRD36-miR-599-TGF-β axis can be a new theoretical basis for treating CAVD.

Topics: Aortic Diseases; Aortic Valve; Calcinosis; Cell Differentiation; Cells, Cultured; Humans; MicroRNAs; Nuclear Proteins; Osteogenesis; RNA, Circular; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Heart valve disease is a growing problem worldwide. Though very common in older adults, the mechanisms behind the development of the disease aren't well understood, and at present the only therapeutic option is valve replacement. Valvular interstitial cells (VICs) may hold the answer. These cells can undergo pathological differentiation into contractile myofibroblasts or osteoblasts, leading to thickening and calcification of the valve tissue. Our study aimed to characterise the effect of fibroblast growth factor 2 (FGF-2) on the differentiation potential of VICs. We isolated VICs from diseased human valves and treated these cells with FGF-2 and TGF-β to elucidate effect of these growth factors on several myofibroblastic outcomes, in particular immunocytochemistry and gene expression. We used TGF-β as a positive control for myofibroblastic differentiation. We found that FGF-2 promotes a 'quiescent-type' morphology and inhibits the formation of α-smooth muscle actin positive myofibroblasts. FGF-2 reduced the calcification potential of VICs, with a marked reduction in the number of calcific nodules. FGF-2 interrupted the 'canonical' TGF-β signalling pathway, reducing the nuclear translocation of the SMAD2/3 complex. The panel of genes assayed revealed that FGF-2 promoted a quiescent-type pattern of gene expression, with significant downregulations in typical myofibroblast markers α smooth muscle actin, extracellular matrix proteins, and scleraxis. We did not see evidence of osteoblast differentiation: neither matrix-type calcification nor changes in osteoblast associated gene expression were observed. Our findings show that FGF-2 can reverse the myofibroblastic phenotype of VICs isolated from diseased valves and inhibit the calcification potential of these cells.

Topics: Actins; Aged; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Cell Differentiation; Cells, Cultured; Fibroblast Growth Factor 2; Humans; Myofibroblasts; Transforming Growth Factor beta

The BRAF V600E mutation is the most common genetic variant in papillary thyroid cancer (PTC), but the relationship between the BRAF V600E mutation in PTC and cervical lymph node metastasis (LNM) remains controversial.. To estimate risk factors for neck nodal metastasis in PTC with BRAF V600E mutation.. A total of 292 patients diagnosed with BRAF V600E mutation related PTC were admitted.. In this retrospective study, data from 292 patients, including clinical, molecular, and ultrasonic characteristics, were analyzed. Univariate and multivariate logistic regression analyses were applied to identify risk factors for LNM in PTC with the. In the univariate analysis of all PTC patients with the BRAF V600E mutation, the LNM was found to be significantly associated with age (. This study revealed that several significant risk factors for LNM in PTCs with the BRAF V600E mutation included: age ≤ 45 years, size ≥ 1cm, LNM in US, bilaterality, ill-defined margins in US, and microcalcification in US.

Topics: Calcinosis; Factor IX; Humans; Lymphatic Metastasis; Middle Aged; Mutation; Proto-Oncogene Proteins B-raf; Retrospective Studies; Risk Factors; Thyroid Cancer, Papillary; Thyroid Neoplasms; Transforming Growth Factor beta

TGFβ1 plays an important role in the metabolism of articular cartilage and bone; however, the pathological mechanism and targets of TGFβ1 in cartilage degradation and uncoupling of subchondral bone remodeling remain unclear. Therefore, in this study, we investigated the relationship between TGFβ1 and major protein-degrading enzymes, and evaluated the role of high levels of active TGFβ1 in the thickening of subchondral bone and calcification of articular cartilage.. The expression of TGFβ1 and protein-degrading enzymes in clinical samples of articular cartilage and subchondral bone obtained from the knee joint of patients with osteoarthritis was detected by immunohistochemistry. The expression levels of TGFβ1, MMP-3, MMP-13 and IL-1β in cartilage and subchondral bone tissues were detected by absolute real-time quantitative RT-PCR. The expression of TGFβ1, nestin and osterix in subchondral bone was detected by Western blot analysis and immunohistochemistry. The degree of subchondral bone thickening was determined by micro-computed tomography (CT) imaging.. Expression of TGFβ1 and cartilage-degrading enzymes was higher in the cartilage-disrupted group than that in the intact group. Furthermore, expression of TGFβ1, nestin and osterix was significantly higher in the OA group than that in the control group. Micro-CT imaging showed that in the OA group, the subchondral bone plate is thickened and the density is increased. The trabecular bone structure is thick plate-like structure, the thickness of the trabecular bone is increased and the gap is small.. The data suggest that highly active TGFβ1 activates the expression of cartilage-degrading enzymes. Abnormally activated TGFβ1 may induce formation of the subchondral bone and expansion of the calcified cartilage area, eventually leading to degradation of the cartilage tissue.. ZIEL: TGFβ1 spielt eine wichtige Rolle im Stoffwechsel von Gelenkknorpel und Knochen; jedoch bleiben der pathologische Mechanismus und die Ziele von TGFβ1 beim Knorpelabbau und Entkopplung des subchondralen Knochenumbaus unklar. Daher untersuchten wir in dieser Studie die Beziehung zwischen TGFβ1 und wichtigen proteinabbauenden Enzymen und der Rolle hoher Konzentrationen an aktivem TGFβ1 bei der Verdickung des subchondralen Knochens und Verkalkung des Gelenkknorpels.. Die Expression von TGFβ1 und der Abbau von Enzymen in klinischen Proben von Gelenkknorpel und subchondralen Knochen aus dem Kniegelenk von Patienten mit Arthrose wurden immunhistochemisch nachgewiesen. Die Expressionsniveaus von TGFβ1, MMP-3, MMP-13 und IL-1β in Knorpel- und subchondralen Knochengeweben wurden durch absolute quantitative RT-PCR in Echtzeit nachgewiesen. Die Expression von TGFβ1, Nestin und Osterix im subchondralen Knochen wurde durch Western-Blot-Analyse und Immunhistochemie nachgewiesen. Der Grad der subchondralen Knochenverdickung wurde durch Mikrocomputertomografie (CT) bestimmt.. Die Expression von TGFβ1 und knorpelabbauenden Enzymen war in der Gruppe mit Knorpelstörungen höher als in der intakten Gruppe. Weiterhin war die Expression von TGFβ1, Nestin und Osterix in der OA-Gruppe signifikant höher als in der Kontrollgruppe. Die Mikro-CT-Bildgebung zeigte, dass in der OA-Gruppe die subchondrale Knochenplatte verdickt und die Dichte erhöht ist. Die trabekuläre Knochenstruktur ist dick plättchenförmig, die Dicke des Trabekelknochens erhöht und die Lücke ist klein.. Die Daten legen nahe, dass hochaktives TGFβ1 die Expression von knorpelabbauenden Enzymen aktiviert. Ungewöhnlich aktiviertem TGFβ1 kann die Bildung des subchondralen Knochens und Ausdehnung des verkalkten Knorpelbereichs induzieren, was zu einem Abbau des Knorpelgewebes führt.

Topics: Bones of Lower Extremity; Calcinosis; Cartilage, Articular; Enzymes; Extracellular Matrix Proteins; Humans; Immunohistochemistry; Knee Joint; Osteoarthritis, Knee; Proteins; Transforming Growth Factor beta; X-Ray Microtomography

Dystrophic calcification (DC) is the deposition of calcium in degenerated tissue which occurs as a reaction to tissue damage. Sometimes if tissue repair fails, it can progress into heterotopic ossification (HO), a pathological condition of abnormal bone formation. HO happens frequently in severe trauma patients such as in blast injury, central nervous system injury and burn injury, in which excessive endogenous glucocorticoid production has always been found. Glucocorticoids have a big impact on bone and muscle. However, few studies have investigated the impact of glucocorticoids on DC/HO formation in muscle. This study aimed to determine the role of glucocorticoids in DC/HO pathogenesis following muscular injury and the possible underlying mechanism. In this study, we administered a high dose of a synthetic glucocorticoid, dexamethasone (DEX), to animals with muscle injury induced by cardiotoxin (CTX) injection to mimic a glucocorticoid excess state following severe muscle trauma. The findings reported here showed that DEX treatment together with CTX-induced muscle injury led to a significant amount of DC in muscle. This effect was likely related to protein level alterations in the fibrinolytic system and resultant decreased circulating transforming growth factor-beta 1 (TGF-β1), given that supplementation of recombinant TGF-β1 markedly rescued this phenomenon. In summary, our results suggest that glucocorticoid excess impairs muscle regeneration and promotes DC/HO, and that TGF-β1 could be a key factor in modulating this process.

Topics: Animals; Bone and Bones; Calcinosis; Glucocorticoids; Humans; Ossification, Heterotopic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Calcific aortic valve disease (CAVD)-the most common valvular heart disease-is accelerated in diabetes and has no pharmacotherapy. Although it is known that early CAVD is associated with inflammation and osteogenesis, the molecular mechanisms involved in diabetes-associated CAVD still need to be uncovered. In this context, we have developed a 3D construct based on gelatin populated with human valvular endothelial cells (VEC) and valvular interstitial cells (VIC) and evaluated the effect of high glucose (HG) concentration on osteogenic molecules expression and on calcification mechanisms. First, we characterized the 3D model and assessed VIC remodelling properties at different time-points. Then, we exposed it to normal glucose (NG) or high glucose (HG) for 7, 14 and 21 days after which the cells were isolated, separated and investigated individually. Our results showed that encapsulated VIC actively remodel the hydrogel, as demonstrated by an increased expression of extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs). Moreover, exposure of the construct to HG triggered bone morphogenetic protein (BMP) and TGF-β signalling pathways, up-regulating expression of osteogenic molecules-BMP-2/-4, osteocalcin, osteopontin, SMADs and Runt-related transcription factor (Runx-2)-and increased calcium deposits in an osteogenic environment. These findings underline the potential of the developed 3D model as a suitable system to investigate the mechanisms of human CAVD and may help to better understand the calcification mechanisms in CAVD associated to diabetes.

Topics: Aortic Valve; Aortic Valve Stenosis; Bone Morphogenetic Protein 2; Calcinosis; Calcium; Cells, Cultured; Endothelial Cells; Gene Expression Regulation; Glucose; Humans; Models, Biological; Osteogenesis; Phenotype; RNA, Messenger; Tissue Scaffolds; Transforming Growth Factor beta; Vascular Remodeling

Topics: Actins; Animals; Aortic Valve; Aortic Valve Stenosis; Apoptosis; beta Catenin; Calcinosis; Calcium; Cells, Cultured; Cyclin D1; Fibrosis; Sheep; Smad7 Protein; Transforming Growth Factor beta; Wnt Signaling Pathway

Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004;

Topics: Aged; Aged, 80 and over; Animals; Aortic Valve; Aortic Valve Stenosis; Apoptosis; Calcinosis; Celecoxib; Cyclooxygenase 2; Female; Gene Expression Regulation; Humans; Inflammation; Male; Mice; Middle Aged; RNA, Messenger; Transforming Growth Factor beta

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Topics: Calcinosis; Child; Child, Preschool; Clubfoot; Extracellular Matrix Proteins; Female; Humans; Male; Mass Spectrometry; Prospective Studies; Proteome; Transforming Growth Factor beta

The calcification of adamantinomatous craniopharyngioma (ACP) often creates difficulties for surgical therapy. Nevertheless, the mechanism of ACP calcification is unclear. Our previous studies demonstrated that osteoblastic factors might play important roles in ACP calcification.. We examined the effects of recombinant human Bmp2 on ACP cell differentiation by testing osteoblastic proteins and calcium deposition.. The expression of osteoblastic factors including osteopontin (OPN), Runx2, and osterix in Bmp2-treated ACP cells was examined by western blot and/or real time PCR. ALP activity and calcium deposition after Bmp2 induction were also tested.. Bmp2 significantly amplified the expression of Runx2, Osterix and OPN, as well as ALP activity. Both of these effects could be repressed by noggin treatment. Bmp2 also significantly induced the calcification of ACP, and noggin inhibited this calcium deposition.. Our study demonstrated for the first time that ACP cells could differentiate into an osteoblastic lineage via induction by Bmp2. The mechanism of ACP calcification likely involves osteoblastic differentiation modulated by Bmp2. Further studies targeting Bmp2 cascades could result in novel therapeutic interventions for recurrent ACP.

Topics: Adolescent; Adult; Alkaline Phosphatase; Bone Morphogenetic Protein 2; Calcinosis; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Craniopharyngioma; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Osteoblasts; Osteopontin; Pituitary Neoplasms; Recombinant Proteins; Sp7 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The study was designed to investigate whether endogenous sulfur dioxide (SO₂) plays a role in vascular calcification (VC) in rats and its possible mechanisms. In vivo medial vascular calcification was induced in rats by vitamin D3 and nicotine for four weeks. In vitro calcification of cultured A7r5 vascular smooth muscle cells (VSMCs) was induced by calcifying media containing 5 mmol/L CaCl₂. Aortic smooth muscle (SM) α-actin, runt-related transcription factor 2 (Runx2), transforming growth factor-β (TGF-β) and Smad expression was measured. VC rats showed dispersed calcified nodules among the elastic fibers in calcified aorta with increased aortic calcium content and alkaline phosphatase (ALP) activity. SM α-actin was markedly decreased, but the osteochondrogenic marker Runx2 concomitantly increased and TGF-β/Smad signaling was activated, in association with the downregulated SO₂/aspartate aminotransferase (AAT) pathway. However, SO₂ supplementation successfully ameliorated vascular calcification, and increased SM α-actin expression, but inhibited Runx2 and TGF-β/Smad expression. In calcified A7r5 VSMCs, the endogenous SO₂/AAT pathway was significantly downregulated. SO₂ treatment reduced the calcium deposits, calcium content, ALP activity and Runx2 expression and downregulated the TGF-β/Smad pathway in A7r5 cells but increased SM α-actin expression. In brief, SO₂ significantly ameliorated vascular calcification in association with downregulation of the TGF-β/Smad pathway.

Topics: Actins; Animals; Aspartate Aminotransferases; Calcinosis; Calcium; Cell Line; Core Binding Factor Alpha 1 Subunit; Male; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Sulfur Dioxide; Transforming Growth Factor beta

Similar risk factors and mediators are involved in calcific aortic stenosis (CAS) and atherosclerosis. Since normal valves harbour a low percentage of smooth muscle cells (SMCs), we hypothesize that the SMC phenotype participates in the pathogenesis of CAS.. We analysed 12 normal and 22 calcified aortic valves for SMC markers and the expression of co-activators of SMC gene expression, myocardin and myocardin-related transcription factors (MRTF-A/B). Transforming growth factor β (TGFβ1) was used to upregulate SMC markers and co-activators in valve interstitial cells (VICs) and transmission electron microscopy (TEM) was used to detect the presence of SMC in atypical regions of the valve leaflets. Smooth muscle cell markers and co-activators, myocardin, MRTF-A, and MRTF-B, demonstrated an increased incidence and aberrant expression around calcified nodules in all 22 calcified valves as well as in surface and microvessel endothelial cells. Smooth muscle cell markers and MRTF-A were significantly increased in calcified valves. Transforming growth factor β1 (TGFβ1) (10 ng/mL) was able to significantly upregulate the expression of some SMC markers and MRTF-A in VICs. Transmission electron microscopy of the fibrosa layer of calcified valves demonstrated the presence of bundles of SMCs and smooth muscle-derived foam cells.. Smooth muscle cell markers and co-activators, myocardin and MRTFs, were aberrantly expressed in calcified valves. Transforming growth factor β1 was able to significantly upregulate SMC markers and MRTF-A in VICs. Transmission electron microscopy unequivocally identified the presence of SMCs in calcified regions of valve leaflets. These findings provide evidence that the SMC phenotype plays a role in the development of CAS.

Topics: Adolescent; Adult; Aortic Valve; Aortic Valve Stenosis; Biomarkers; Calcinosis; Calcium-Binding Proteins; Calponins; Female; Foam Cells; Humans; Male; Microfilament Proteins; Microscopy, Electron, Transmission; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nuclear Proteins; Phenotype; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Young Adult

We studied the mechanistic links between fibrocalcific changes in the aortic valve and aortic valve function in mice homozygous for a hypomorphic epidermal growth factor receptor mutation (Wave mice). We also studied myocardial responses to aortic valve dysfunction in Wave mice.. At 1.5 months of age, before development of valve fibrosis and calcification, aortic regurgitation, but not aortic stenosis, was common in Wave mice. Aortic valve fibrosis, profibrotic signaling, calcification, osteogenic markers, lipid deposition, and apoptosis increased dramatically by 6 and 12 months of age in Wave mice. Aortic regurgitation remained prevalent, however, and aortic stenosis was rare, at all ages. Proteoglycan content was abnormally increased in aortic valves of Wave mice at all ages. Treatment with pioglitazone prevented abnormal valve calcification, but did not protect valve function. There was significant left ventricular volume overload, hypertrophy, and fetal gene expression, at all ages in Wave mice with aortic regurgitation. Left ventricular systolic function was normal until 6 months of age in Wave mice, but became impaired by 12 months of age. Myocardial transverse tubules were normal in the presence of left ventricular hypertrophy at 1.5 and 3 months of age, but became disrupted by 12 months of age.. We present the first comprehensive phenotypic and molecular characterization of spontaneous aortic regurgitation and volume-overload cardiomyopathy in an experimental model. In Wave mice, fibrocalcific changes are not linked to valve dysfunction and are epiphenomena arising from structurally incompetent myxomatous valves.

Topics: Actins; Animals; Aortic Valve; Aortic Valve Insufficiency; Calcinosis; Cell Death; Disease Progression; Fibrosis; Gene Expression; Heart Valve Diseases; Lipid Metabolism; Mice; Mice, Mutant Strains; Osteocalcin; Pioglitazone; Proteoglycans; Sp7 Transcription Factor; Systole; Thiazolidinediones; Transcription Factors; Transforming Growth Factor beta

To establish human heart valve interstitial cells calcification culture model in vitro, and observe the effect of bone morphogenetic protein-2 (BMP-2) on calcification of human heart valve interstitial cells.. Human heart valve interstitial cells were cultured in vitro, and divided into control group: cells were cultured in conventional media plus recombinant human BMP-2 treatment and experimental group: besides above treaments, calcification inducers ( recombinant human BMP-2, β-glycerophosphate, L-ascorbic acid, dexamethasone) were added to the culture media. The two group of cells were cultured for 14 days and were stained by Von Kossa, then the cell calcification was observed in this valvular interstitial cells calcification culture model in vitro. Protein expression of intercellular adhesion molecule 1 (ICAM-1), interleukin 8, BMP-2 and BMP-4 was determined by Western blot and BMP-2 secretion was measured by ELISA.. In the control group, the structure of human heart valve interstitial cells was clear, and the spindle and radial growth shaped cellular morphology was visible, and Von Kossa staining was negative. In the experimental group, the nuclei become darker in color, and granular sediment distribution was seen surrounding cells, and Von Kossa staining was positive, the cells were forming nodules of calcification. The protein expression of ICAM-1, interleukin 8, BMP-2 and BMP-4 in the experimental was significantly higher than that of the control group (all P < 0.05). The expression of BMP-2 in the experimental group was also significantly higher than that in control group ((92.5 ± 4.9) pg/ml vs. (22.2 ± 1.9) pg/ml, P < 0.05).. Human BMP-2, β-glycerophosphate, L-ascorbic acid, and dexamethasone can induce human heart valve interstitial cells calcification and enhance inflammation in vitro by stimulating the secretion of BMP-2.

Topics: Ascorbic Acid; Bone Morphogenetic Protein 2; Calcinosis; Cells, Cultured; Glycerophosphates; Heart Valve Diseases; Humans; Recombinant Proteins; Transforming Growth Factor beta

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1-/-) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1-/- aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1-/- aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-β signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD.

Topics: Animals; Aortic Valve; Bicuspid Aortic Valve Disease; Calcinosis; Cell Proliferation; Cutis Laxa; Disease Models, Animal; Disease Progression; Elastic Tissue; Fibrosis; Heart Defects, Congenital; Heart Valve Diseases; Inflammation; Membrane Glycoproteins; Mice; Models, Biological; Myofibroblasts; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta; Ultrasonography

Bone morphogenetic protein-2 (BMP-2) is a major regulator of aortic valve calcification. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression and miRNA-141 is a known repressor of BMP-2-mediated osteogenesis. We hypothesized that miRNA-141 is a key regulator of aortic valve calcification.. Porcine valvular interstitial cells were isolated, transfected with miRNA-141 or control, and stimulated with transforming growth factor-β. The BMP-2, extracellular signal-regulated kinase 1/2, and runt-related transcription factor 2 levels were determined by immunoblotting and reverse transcriptase polymerase chain reaction. To determine the role of miRNA-141 in bicuspid aortic valve disease, human bicuspid (n = 19) and tricuspid (n = 17) aortic valve leaflets obtained intraoperatively were submitted for GenoExplorer human microRNA array, immunoblotting, and histologic and immunohistochemical analyses.. Stimulation of porcine aortic valvular interstitial cells with transforming growth factor-β induced morphologic alterations consistent with myofibroblastic transformation, BMP-2 signaling, and calcification. Transfection with miRNA-141 restored transforming growth factor-β-induced valvular interstitial cell activation, BMP-2 signaling, and alkaline phosphatase activity (3.55 ± 0.18 vs 4.01 ± 0.21, P < .05), suggesting upstream regulation by miRNA-141. miRNA microarray demonstrated differential expression of 35 of 1583 miRNA sequences in the bicuspid versus tricuspid aortic valve leaflets, with a 14.5-fold decrease in miRNA-141 in the bicuspid versus tricuspid leaflets (P < .05). This was associated with significantly increased BMP-2 protein expression in bicuspid aortic valve compared with the tricuspid aortic valve leaflets (P < .001).. We report a completely novel role of miRNA-141 as a regulator of BMP-2-dependent aortic valvular calcification and demonstrate marked attenuation of miRNA-141 expression in patients with bicuspid aortic valve-associated aortic stenosis. Therapeutic targeting of miRNA-141 could serve as a novel strategy to limit progressive calcification in aortic stenosis.

Topics: Alkaline Phosphatase; Animals; Aortic Valve; Aortic Valve Stenosis; Blotting, Western; Bone Morphogenetic Protein 2; Calcinosis; Chi-Square Distribution; Core Binding Factor Alpha 1 Subunit; Humans; Immunohistochemistry; In Situ Nick-End Labeling; MicroRNAs; Real-Time Polymerase Chain Reaction; Signal Transduction; Swine; Transfection; Transforming Growth Factor beta

To investigate the correlation of the functional disequilibrium of regulatory T cells (Treg)/T-helper (Th17) cells with calcification and to explore the significance of their influence on the outcome of cardiovascular disease (CVD) in uremic patients after hemodialysis (HD).. Out of 66 uremia patients, 36 patients had CVD after HD (maintenance hemodialysis (MHD) group1) and 30 patients did not have CVD (MHD group2). Twenty healthy volunteers were selected as normal control group. Peripheral blood mononuclear cells were isolated and treated with recombinant human bone morphogenetic protein-2 (rhBMP-2). Treg and Th17 frequencies were measured by flow cytometry. Forkhead/winged helix transcription factor (Foxp3) and retinoic acid receptor-related orphan receptor-γt (ROR-γt) mRNA expressions were measured by real-time quantitative polymerase chain reaction. Levels of interleukin (IL)-10 and IL-17 were detected by enzyme-linked immunosorbent assay.. When compared with controls, rhBMP-2 upregulates Treg/Th17 functional disequilibrium in uremia patients, displaying higher Treg and Th17 frequencies, Foxp3 and ROR-γt expressions, and levels of cytokines (p < 0.05). These differences were also significant between MHD group1 and group2 (p < 0.05). It was also observed that Treg/Th17 functional disequilibrium was not only correlated with a calcification state but also consistent with the CVD.. The Treg/Th17 cell function disequilibrium might act synergistically with calcification in the high incidence of CVD after HD.

Topics: Analysis of Variance; Bone Morphogenetic Protein 2; Calcinosis; Cardiovascular Diseases; Case-Control Studies; China; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Interleukin-10; Interleukin-17; Male; Middle Aged; Real-Time Polymerase Chain Reaction; Receptors, Retinoic Acid; Recombinant Proteins; Renal Dialysis; Risk Factors; RNA, Messenger; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Uremia

Calcific aortic valve stenosis is the most common indication for surgical valve replacement. Inflammation appears to be one of the mechanisms involved in aortic valve calcification, and valve interstitial cells seem to contribute to that process. Although Toll-like receptors (TLRs) play an important role in the cellular inflammatory response, it is unknown whether human aortic valve interstitial cells (HAVICs) express functional TLRs. We examined the expression of TLR2 and TLR4 in human aortic valve leaflets and in isolated HAVICs and analyzed the response of cultured HAVICs to the TLR2 and TLR4 agonists peptidoglycan (PGN) and LPS. Abundant TLR2 and TLR4 proteins were found in human aortic valve leaflets and in isolated HAVICs, and both receptors were detected in the membrane and cytoplasm of cultured HAVICs. Stimulation by either PGN or LPS resulted in the activation of the NF-kappaB signaling pathway and the production of multiple proinflammatory mediators, including IL-6, IL-8, and ICAM-1. In addition, stimulation by either PGN or LPS upregulated the expression of bone morphogenetic protein-2 (BMP-2) and Runx2, factors associated with osteogenesis. This study demonstrates for the first time that HAVICs express TLR2 and TLR4 and that stimulation of HAVICs by PGN or LPS induces the expression of proinflammatory mediators and the upregulation of osteogenesis-associated factors. These results suggest that TLR2 and TLR4 may play a role in aortic valve inflammation and stenosis.

Topics: Aortic Valve; Aortic Valve Stenosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Humans; Immunity, Innate; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Lipopolysaccharides; NF-kappa B; Peptidoglycan; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4; Transforming Growth Factor beta; Up-Regulation

Vascular calcification often occurs with advancing age, atherosclerosis, and metabolic disorders such as diabetes mellitus and end-stage renal disease. Vascular calcification is associated with cardiovascular events and increased mortality. Nitric oxide (NO) is crucial for maintaining vascular function, but little is known about how NO affects vascular calcification. The aim of this study was to examine the effect of NO on vascular calcification.. In this study, we examined the inhibitory effects of NO on calcification of murine vascular smooth muscle cells (VSMCs) in vitro. We measured calcium concentration, alizarin red staining, and alkaline phosphatase activity to examine the effect of NO on calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. We also determined gene expression and levels of phosphorylation of Smad2/3 by RT-PCR and western blotting. NO inhibited calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. An inhibitor of cyclic guanosine monophosphate (cGMP)-dependent protein kinase restored the inhibition by NO of osteoblastic differentiation and calcification of VSMCs. NO inhibited transforming growth factor-beta (TGF-beta)-induced phosphorylation of Smad2/3 and expression of TGF-beta-induced genes such as plasminogen activator inhibitor-1. In addition, NO inhibited expression of the TGF-beta receptor ALK5.. Our data show that NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-beta signalling through a cGMP-dependent pathway. Our findings suggest that NO may play a beneficial role in atherogenesis in part by limiting vascular calcification.

Topics: Animals; Calcinosis; Cell Differentiation; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Nitric Oxide; Osteoblasts; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases

The transformation of smooth muscle cells (VSMCs) in the vessel wall to osteoblast like cells is known to precede arterial calcification which may cause bleeding complications. The vitamin K-dependent protein MGP has been identified as an inhibitor of this process by binding BMP-2, a growth factor known to trigger the transformation. In this study, we determined if the vitamin K-dependent Gla region in MGP by itself can inhibit the growth factor activity of BMP-2 and if menaquinone-4 (MK4) regulates gene expression in VSMCs.. A synthetic gamma-carboxyglutamic acid (Gla) containing peptide covering the Gla region in human MGP was used to test its ability to inhibit BMP-2 induced transformation of mouse pro-myoblast C2C12 cells into osteoblasts. MK4 was tested by microarray analysis as a gene regulatory molecule in VSMCs.. The results show that the Gla - but not the Glu-peptide inhibited the transformation which provide evidence that the Gla region in MGP is directly involved in the BMP-2/MGP interaction and emphasizes the importance of the vitamin K-dependent modification of MGP. From the data obtained from the microarray analysis, we focused on two quantitatively altered cDNAs representing proteins known to be associated with vessel wall calcification. DT-diaphorase of the vitamin K-cycle, showed increased gene expression with a 4.8-fold higher specific activity in MK4 treated cells. Osteoprotegrin gene expression was down regulated and osteoprotegrin protein secretion from the MK4 treated cells was lowered to 1.8-fold. These findings suggest that MK4 acts as an anti-calcification component in the vessel wall.

Topics: Animals; Antifibrinolytic Agents; Aorta, Thoracic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium-Binding Proteins; Cell Differentiation; Cell Line; Extracellular Matrix Proteins; Humans; Matrix Gla Protein; Mice; Muscle, Smooth, Vascular; Myoblasts; Osteoblasts; Peptide Fragments; Rats; Transforming Growth Factor beta; Vitamin K; Vitamin K 2

Isolated systolic hypertension is associated with increased elastase activity, vascular calcification, and vascular stiffness. We sought to determine the importance of elastase activity and matrix degradation in the development of elastocalcinosis.. Elastocalcinosis was induced in vivo and ex vivo using warfarin. Hemodynamic parameters, calcium deposition, elastin degradation, transforming growth factor (TGF)-beta signaling, and elastase activity were evaluated at different time points in the in vivo model. Metalloproteinases, serine proteases, and cysteine proteases were blocked to measure their relative implication in elastin degradation. Gradual elastocalcinosis was obtained, and paralleled the elastin degradation pattern. Matrix metalloproteinase (MMP)-9 activity was increased at 5 days of warfarin treatment, whereas TGF-beta signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-beta signaling with SB-431542 were able to prevent calcification.. Early MMP-9 activation precedes the increase of TGF-beta signaling, and overt vascular elastocalcinosis and stiffness. Modulation of matrix degradation could represent a novel therapeutic avenue to prevent the gradual age-related stiffening of large arteries, leading to isolated systolic hypertension.

Topics: Animals; Aorta, Abdominal; Calcinosis; Collagen; Disease Models, Animal; Durapatite; Elastin; Femoral Artery; Male; Matrix Metalloproteinase 9; Osteopontin; Rats; Rats, Wistar; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases; Warfarin

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cell Differentiation; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Osteoblasts; Transforming Growth Factor beta; Vascular Diseases

The present study examined histological difference between ossifying fibromas (OF, n=5) and peripheral cemento-ossifying fibromas (PCOF, n=7). Bone morphogenetic proteins (BMP)-2 and -4, osteopontin (OPN), osteocalcin (OCN) and proliferating cell nuclear antigen (PCNA) were used for the immunohistochemical examinations. Oxytalan fibers present at the periodontal tissue were stained to determine the tumor cell origin. Many OFs showed high immunohistochemical reactions for BMP-2, -4 and OPN compared to those of PCOFs. PCNA index (IP) of OFs was significantly higher than that of PCOFs. All the PCOFs showed a high expression of oxytalan fibers. Only two OFs exhibited a small number of oxytalan fibers. These results suggest that PCOF has only little ability to form hard tissue and seems to be a reactive lesion. The expression of oxytalan fibers reveals that OF does not only originate from periodontal tissue.

Topics: Adolescent; Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Calcinosis; Child; Female; Fibroma, Ossifying; Gingival Hyperplasia; Gingival Neoplasms; Humans; Immunohistochemistry; Jaw Neoplasms; Male; Middle Aged; Osteocalcin; Osteopontin; Proliferating Cell Nuclear Antigen; Transforming Growth Factor beta

Age-related medial calcification (elastocalcinosis) of large arteries is accelerated in diabetes and appears mainly in distal arteries. The aim was to devise a rat model of elastocalcinosis in association with diabetes to examine the hypothesis that diabetes accelerates vascular calcification experimentally.. Male Wistar rats received a high fat diet during 2 months followed by a low dose of streptozotocin to induce diabetes (D). Elastocalcinosis was facilitated by 3 weeks of treatment with warfarin and vitamin K (WVK). We started WVK treatment 1 week (D4WVK) and 4 weeks (D7WVK) after the injection of streptozotocin and in age-matched healthy rats. Measurements of hemodynamic and metabolic parameters, aortic and femoral calcium content, and immunohistochemistry for alkaline phosphatase, osteopontin, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-TGF-beta were performed.. Three weeks of WVK treatment alone did not increase the calcium content in the aorta and femoral arteries. However, in the D7WVK group, femoral calcification, but not aortic calcium content, increased significantly as compared to the WVK group. This response was not observed in the D4WVK group. In femoral arteries, strong immunostaining for alkaline phosphatase and osteopontin was observed in the D7WVK group. TNF-alpha and TGF-beta expressions were mainly localized in the adventitia of arteries from diabetic rats.. We have established a model of accelerated elastocalcinosis in diabetes related to its duration and localized in distal arteries. The modification of local protein expression is also in accordance with clinical data, suggesting that this model could be useful to investigate mechanisms related to this important clinical macrovascular complication of diabetes.

Topics: Alkaline Phosphatase; Animals; Aorta; Arteriosclerosis; Calcinosis; Calcium; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Femoral Artery; Immunohistochemistry; Male; Models, Animal; Osteopontin; Rats; Rats, Wistar; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vitamin K; Warfarin

Clinical studies demonstrate that mineralocorticoid receptor (MR) antagonism improves outcomes in cardiovascular patients and that vascular calcification correlates with adverse cardiac events. We have recently demonstrated that human vascular smooth muscle cells (VSMCs) express functional MRs that, in response to aldosterone, modulate expression of osteogenic genes including alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP2). This study examines the effects of MR activation by aldosterone on the process of in vitro vascular calcification.. Using immunoblotting and adenoviral promoter-reporter assays, we demonstrated that calcifying vascular cells (CVCs), an in vitro model of vascular calcification, express MRs that mediate both aldosterone- and cortisol-stimulated gene transcription. In this model, aldosterone stimulated ALP activity, an early marker of osteoblastic differentiation, as well as mineralization. Aldosterone antagonism with spironolactone abolished both effects implicating CVC MRs in the mechanism of aldosterone-stimulated vascular calcification. Inhibition of BMP2 signaling by overexpression of dominant negative BMP2 receptor did not attenuate aldosterone-induced osteoblastic differentiation.. Aldosterone activation of MR promotes osteoblastic differentiation and mineralization of VSMCs independent of BMP2 signaling. These data provide a mechanistic link between hormone-mediated VSMC MR activation and vascular calcification, two processes associated with increased risk of cardiovascular ischemic events in humans.

Topics: 11-beta-Hydroxysteroid Dehydrogenase Type 2; Adrenal Cortex Hormones; Aldosterone; Alkaline Phosphatase; Animals; Aorta; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cattle; Cells, Cultured; Coronary Vessels; Humans; Minerals; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Receptors, Mineralocorticoid; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases

The genetic factors contributing to the complex disorder of myocardial calcification are largely unknown. Using a mouse model, we fine-mapped the major locus (Dyscalc1) contributing to the dystrophic cardiac calcification (DCC) to an 840-kb interval containing 38 genes. We then identified the causal gene by using an approach integrating genetic segregation and expression array analyses to identify, on a global scale, cis-acting DNA variations that perturb gene expression. By studying two intercrosses, in which the DCC trait segregates, a single candidate gene (encoding the ATP-binding cassette transporter ABCC6) was identified. Transgenic complementation confirmed Abcc6 as the underlying causal gene for Dyscalc1. We demonstrate that in the cross, the expression of Abcc6 is highly correlated with the local mineralization regulatory system and the BMP2-Wnt signaling pathway known to be involved in the systemic regulation of calcification, suggesting potential pathways for the action of Abcc6 in DCC. Our results demonstrate the power of the integrative genomics in discovering causal genes and pathways underlying complex traits.

Topics: Animals; ATP-Binding Cassette Transporters; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Gene Expression Regulation; Genomics; Heart Diseases; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Multidrug Resistance-Associated Proteins; Myocardium; Osteopontin; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Sclerotic calcification of the aortic valve is a common disease in advanced age. Its pathophysiology is unclear. However, pathobiological similarities to atherosclerosis have been shown in several studies. The current study assesses gene profiling of severe calcified stenotic human aortic valves identifying transforming growth factor (TGF)-beta, Eotaxin3, vascular adhesion protein-1 (VAP-1) and monokine induced by interferon-gamma (MIG) as potential atherosclerotic target genes in severe calcified and stenotic aortic valves, and analyzes the effects of statins on their expression as part of an anti-inflammatory treatment strategy. We collected human severe calcified and stenotic aortic valves with (CSAV+) or without (CSAV-) statin pre-treatment prior to valve replacement and investigated gene profiling by using micro-array technique and real-time PCR for the TGF-beta, Eotaxin3, VAP-1 and MIG expression. In comparison to atherosclerotic plaques of carotid arteries, immunohistochemical staining was investigated. Results were contrasted to human normal non-calcified aortic valves as controls (C). As compared to C, TGF-beta, Eotaxin3, MIG or VAP-1 was significantly upregulated in CSAV-. In CSAV+ no significant change in gene expression was found for Eotaxin3 and MIG. In contrast, VAP-1 and TGF-beta were still upregulated. Corresponding gene expression was confirmed on atherosclerotic plaque formations of carotid arteries. Monocyte/Macrophage infiltration (presence of CD68) on aortic valves (CSAV+, CSAV-, or C) confirmed inflammatory nature of the disease. Our data support further evidence for atherosclerotic inflammation as a trigger for sclerosis in end-stage calcified stenotic aortic valves by showing upregulation of gene expression for TGF-beta, VAP-1, MIG and Eotaxin3, which is only partially inhibited by previous statin therapy. Potent benefits of statin treatment on early stages of valve disease are still propagated.

Topics: Amine Oxidase (Copper-Containing); Aortic Valve; Aortic Valve Stenosis; Atherosclerosis; Calcinosis; Cell Adhesion Molecules; Chemokine CCL26; Chemokine CXCL9; Chemokines, CC; Gene Expression Profiling; Heart Valve Prosthesis Implantation; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Transforming Growth Factor beta

Coronary calcification is a potent predictor of cardiac events. In patients with chronic renal disease, both prevalence and intensity of coronary calcification are increased. It has remained uncertain whether it is the intima of the coronaries or the media that is calcified and whether the morphologic details of calcified plaques differ between renal and nonrenal patients. Autopsy samples of coronaries were obtained from standard sites in 23 renal and 23 age- and gender-matched nonuremic patients. Specimens were examined using light and electron microscopy, immunohistochemistry, backscatter imaging, and x-ray analysis. In coronaries, calcified plaques occupied a similar proportion of the intima area in renal versus nonrenal patients (17.3 +/- 11.9 versus 18.1 +/- 11.9%) but occupied a significantly higher proportion of the media (16.6 +/- 10.6 versus 3.8 +/- 2.31%). Expression of the proteins osteocalcin, C-reactive protein, TGF-beta, and collagen IV was significantly more intensive around coronary plaques of renal compared with nonrenal patients. The non-plaque-bearing intima of renal patients showed minimal staining for fetuin, but fetuin staining was seen surrounding calcified plaques. In addition, more pronounced deposition of C5b-9 was found around coronary plaques of renal patients, and glycophorin deposition pointed to more past intraplaque hemorrhage in renal patients. Calcification by electron backscatter analysis is more intense in the coronary media, but not if the intima is more intense in renal compared with nonrenal patients. A more marked inflammatory response in renal patients is suggested by more frequent presence and greater intensity of markers of inflammation.

Topics: Aged; Aged, 80 and over; Biomarkers; C-Reactive Protein; Calcinosis; Collagen Type IV; Complement Membrane Attack Complex; Coronary Angiography; Coronary Artery Disease; Coronary Vessels; Endothelium, Vascular; Female; Glycophorins; Humans; Hypoxia; Immunohistochemistry; Kidney Diseases; Macrophages; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Middle Aged; Transforming Growth Factor beta; Tunica Intima; Tunica Media

Aortic calcification is prevalent in type II diabetes (T2DM), enhancing morbidity and tracking metabolic syndrome parameters. Ldlr(-/-) mice fed high-fat "Westernized" diets (HFD) accumulate aortic calcium primarily in the tunica media, mediated via osteogenic morphogens and transcriptional programs that induce aortic alkaline phosphatase (ALP). Because elevated TNF-alpha is characteristic of obesity with T2DM, we examined contributions of this inflammatory cytokine.. HFD promoted obesity, hyperglycemia, and hyperlipidemia, and upregulated serum TNF-alpha in Ldlr(-/-) mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF-alpha neutralizing antibody infliximab did not reduce obesity, hypercholesterolemia, or hyperglycemia; however, haptoglobin, aortic BMP2, Msx2, Wnt3a, and Wnt7a and aortic calcium accumulation were downregulated by infliximab. Mice with vascular TNF-alpha augmented by a transgene (SM22-TNFalphaTg) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNFalphaTg;TOPGAL mice exhibited greater aortic beta-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF-alpha upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions.. TNF-alpha promote aortic Msx2-Wnt programs that contribute to aortic calcium accumulation in T2DM.

Topics: Alkaline Phosphatase; Animals; Anti-Inflammatory Agents; Antibodies, Monoclonal; Aorta; Aortic Diseases; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cells, Cultured; Diabetes Mellitus, Type 2; Dietary Fats; Disease Models, Animal; DNA-Binding Proteins; Fibroblasts; Haptoglobins; Homeodomain Proteins; Inflammation; Infliximab; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microfilament Proteins; Muscle Proteins; Promoter Regions, Genetic; Receptors, LDL; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were increased by both BMPs. Members of the TGF-superfamily, i.e. TGF-beta1, BMP-2 and BMP-7 exert effects on OPG and its ligands, indicating that these peptides may be involved in the development of vascular calcifications. The downregulation of OPG by these peptides does, however, not suggest that these factors are directly involved in OPG accumulation in diabetes.

Topics: Actins; Bone Morphogenetic Proteins; Calcinosis; Cells, Cultured; Diabetes Mellitus; Humans; Muscle, Smooth, Vascular; Osteoprotegerin; RANK Ligand; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta

Calcification of vascular elastin occurs in patients with arteriosclerosis, renal failure, diabetes, and vascular graft implants. We hypothesized that pathological elastin calcification is related to degenerative and osteogenic mechanisms. To test this hypothesis, the temporal expression of genes and proteins associated with elastin degradation and osteogenesis was examined in the rat subdermal calcification model by quantitative real-time reverse transcription-polymerase chain reaction and specific protein assays. Purified elastin implanted subdermally in juvenile rats exhibited progressive calcification in a time-dependent manner along with fibroblast and macrophage infiltration. Reverse transcription-polymerase chain reaction analysis showed that relative gene expression levels of matrix metalloproteinases (MMP-2 and MMP-9) and transforming growth factor-beta1 were increased in parallel with calcification. Gelatin zymography showed strong MMP activities at early time points, which were associated with high levels of soluble elastin peptides. Gene expression of core binding factor alpha-1, an osteoblast-specific transcription factor, increased in parallel with elastin calcification and attained approximately 9.5-fold higher expression at 21 days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontin and alkaline phosphatase also increased progressively, but osteocalcin levels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.

Topics: Alkaline Phosphatase; Animals; Calcinosis; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Elastin; Fibroblasts; Gene Expression Regulation; Heart; Macrophages; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Osteogenesis; Osteopontin; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Swine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

The matrix GLA (MGP) gene has been found to be among the 10 most highly expressed genes in the human trabecular meshwork (TM), and its expression is affected by conditions associated with glaucoma. Because MGP protein has been shown to play a key role in inhibiting calcification in cartilage and arterial vessels, MGP's function in human TM was investigated.. Perfused TM tissue and primary human TM (HTM) cells originated from donors of nonglaucomatous eyes. MGP mRNA was assayed by relative quantitative and real-time PCR. AdhMGP recombinant adenovirus was generated by bacterial transposition. Western blot analyses were cross-reacted with MGP N-terminal- and conformational-specific antibodies. MGP/BMP2 colocalization was analyzed by confocal microscopy. gamma-Carboxylation activity was measured by incorporation of 14CO2 into FLEEL synthetic peptide. Alkaline phosphatase (ALP) activity was used as a marker of osteogenic differentiation and a calcification precursor. Calcification was assessed by measuring direct calcium (o-cresolphthalein). Normalization was conducted with a telomerase probe (genomic DNA).. HTM cells contained high levels of gamma-carboxylase activity and were able to convert MGP to its active conformation. Overexpression of MGP in HTM cells reduced ALP activity in a model of BMP2-induced osteogenesis. MGP colocalized intracellularly with BMP2. HTM cells aged in culture exhibited increased calcium content, increased ALP, decreased normalized MGP expression and lower gamma-carboxylase activity.. MGP protein is active and functions as an inhibitor of BMP2-induced ALP activity in the HTM cells. The human TM may undergo a calcification process with age. Inhibition of the calcification mechanism mediated by MGP could be used to regulate resistance and elevated IOP.

Topics: Adenoviridae; Adolescent; Adult; Alkaline Phosphatase; Blotting, Western; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium; Calcium-Binding Proteins; Carbon-Carbon Ligases; Cell Differentiation; Cells, Cultured; Extracellular Matrix Proteins; Fluorescent Antibody Technique, Indirect; Genetic Vectors; Humans; Matrix Gla Protein; Microscopy, Confocal; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trabecular Meshwork; Transforming Growth Factor beta

How T-cells, attracted to local sites of inflammation in arthritides, affect heterotopic ossification is presently unknown. Here, we tested the hypothesis that T-cell cytokines play a role in the differentiation of human mesenchymal stromal cells (HMSC) into the osteoblast phenotype by inducing autologous BMP-2, providing a possible mechanism for heterotopic ossification. HMSC from multiple donor bones were treated with either activated T-cell conditioned medium (ACTTCM) or physiological concentrations of the major inflammatory cytokines, TNF-alpha, TGF-beta, IFN-gamma, and IL-17 (TTII), individually or in combinations. ACTTCM induced BMP-2 protein in a time-dependent manner over a 48 h period and alkaline phosphatase (AlkP) within 7 days. In combination, TTII, like ACTTCM, induced AlkP and synergistically induced BMP-2 protein. Either individually, or in combinations of up to three, the T-cell cytokines failed to induce BMP-2 above control levels while a combination of all four cytokines synergistically induced BMP-2 10-fold as assessed by ELISA. TTII induced mineralized matrix as effectively as dexamethasone. Inhibition of p38 MAPK completely inhibited TTII-induced BMP-2 production and matrix mineralization. Real time RT-PCR analysis demonstrated a striking early (within 4 h) increase in BMP-2 gene expression by TTII, which was suppressed by p38 MAP kinase inhibition. In localized chronic inflammatory diseases, T-cell cytokines released at localized sites of inflammation may be the driving force for differentiation of local mesenchymal stromal cells into the osteoblast phenotype thereby playing a significant role in the heterotopic ossification observed in these diseases.

Topics: Arthritis; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cell Differentiation; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Cytokines; Humans; Inflammation; Signal Transduction; Stromal Cells; T-Lymphocytes; Tissue Donors; Transforming Growth Factor beta

Calcific aortic valve stenosis is a common disease in the elderly and is characterized by progressive calcification and fibrous thickening of the valve, but the cellular and molecular mechanisms are not fully understood. We hypothesized that human valve interstitial cells (ICs) are able to differentiate into osteoblast-like cells through the influence of defined mediators and that this process can be modulated pharmacologically.. To test this hypothesis, we treated primary cultures of human aortic valve ICs with osteogenic media, bone morphogenic proteins ([BMPs] BMP-2, BMP-4, and BMP-7), and tissue growth factor-beta ([TGF-beta] TGF-beta1 and TGF-beta3) for 21 days. These mediators induced osteoblast differentiation of valve ICs by significantly increasing the activity and expression of alkaline phosphatase ([ALP] P<0.001). A cytokine protein array revealed that atorvastatin treatment (100 micromol/L) of human valve ICs caused a downregulation in levels of expression of BMP-2, BMP-6, TGF-beta1, and TGF-beta3 after 24 hours. In addition, human valve ICs treated with atorvastatin in the presence of osteogenic media showed a significant reduction in ALP activity in comparison to cells treated with osteogenic media only (P=<0.001). This was further confirmed with immunocytochemical staining of valve ICs, whereby atorvastatin markedly reduced the expression of ALP and osteocalcin induced by osteogenic media in comparison to untreated cells.. These findings suggest that human valve ICs are capable of osteoblastic differentiation, by potential mediators which can be pharmacologically targeted by atorvastatin.

Topics: Aged; Alkaline Phosphatase; Aortic Valve; Aortic Valve Stenosis; Atorvastatin; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcinosis; Cell Differentiation; Cells, Cultured; Connective Tissue Cells; Culture Media; Fibrosis; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Middle Aged; Osteoblasts; Osteocalcin; Pyrroles; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3

Topics: Biomedical Research; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcinosis; Fracture Healing; Fractures, Bone; Humans; Orthopedics; Transforming Growth Factor beta; United States

In two independent and separate studies, we have shown that renal injury and chronic kidney disease (CKD) directly inhibit skeletal anabolism, and that stimulation of bone formation decreased the serum phosphate. In the first study, the serum Ca PO(4), parathyroid hormone (PTH), and calcitriol were maintained normal after renal ablation in mice, and even mild renal injury equivalent to stage 3 CKD decreased bone formation rates. More recently, these observations were rediscovered in low-density lipoprotein receptor null (LDLR-/-) mice fed high-fat/cholesterol diets, a model of the metabolic syndrome (hypertension, obesity, dyslipidemia and insulin resistance). We demonstrated that these mice have vascular calcification (VC) of both the intimal atherosclerotic type and medial calcification. We have also shown that VC is made worse by CKD and ameliorated by bone morphogenetic protein-7 (BMP-7). The finding that high-fat fed LDLR-/- animals with CKD had hyperphosphatemia which was prevented in BMP-7-treated animals lead us to examine the skeletons of these mice. It was found that significant reductions in bone formation rates were associated with high-fat feeding, and superimposing CKD resulted in the adynamic bone disorder (ABD), while VC was made worse. The effect of CKD to decrease skeletal anabolism (decreased bone formation rates and reduced number of bone modelling units) occurred despite secondary hyperparathyroidism. The BMP-7 treatment corrected the ABD and hyperphosphatemia, owing to BMP-7-driven stimulation of skeletal phosphate deposition reducing plasma phosphate and thereby removing a major stimulus to VC. A pathological link between abnormal bone mineralization and VC through the serum phosphorus was demonstrated by the partial effectiveness of directly reducing the serum phosphate by a phosphate binder that had no skeletal action. Thus, in the metabolic syndrome with CKD, a reduction in bone forming potential of osteogenic cells leads to the ABD producing hyperphosphatemia and VC, processes ameliorated by BMP-7, in part through increased bone formation and skeletal deposition of phosphate and in part through direct actions on vascular smooth muscle cells. We have demonstrated that the processes leading to vascular calcification begin with even mild levels of renal injury affecting the skeleton before demonstrable hyperphosphatemia and that they are preventable and treatable. Therefore, early intervention in the skeletal disorder associated wi

Topics: Animals; Bone and Bones; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcinosis; Cartilage; Cell Differentiation; Chronic Kidney Disease-Mineral and Bone Disorder; Humans; Kidney; Kidney Failure, Chronic; Mice; Osteoblasts; Osteogenesis; Phosphates; Transforming Growth Factor beta; Vascular Diseases

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.

Topics: Animals; Animals, Newborn; Biomarkers; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Calcinosis; Calcium-Binding Proteins; Craniosynostoses; Glycoproteins; Humans; Male; Mice; Mice, Transgenic; Nerve Tissue Proteins; Osteoblasts; Osteocalcin; Osteogenesis; Rats; Receptor, Fibroblast Growth Factor, Type 2; Skull; Tissue Culture Techniques; Transforming Growth Factor beta; Up-Regulation

Vascular calcification is common in diabetes but the pathogenesis is poorly understood.. To investigate the pathogenesis, we first examined the histology of inferior epigastric arteries from diabetic and non-diabetic patients undergoing a renal transplant. To examine the role of hyperglycaemia, bovine vascular smooth muscle cells (BVSMCs) were incubated with normal (5 mM) or high glucose (25 mM) for 48 or 72 h.. The results demonstrated that diabetic patients, compared with non-diabetic patients, had significantly greater calcification and increased expression of the bone matrix proteins osteopontin, type I collagen, bone sialoprotein and alkaline phosphatase (ALP). The in vitro studies demonstrated that high glucose increased the expression of the osteoblast transcription factor core binding factor alpha subunit 1 (Cbfa1) and its downstream protein osteocalcin by 1.9-fold and 1.8-fold, respectively, and ALP activity by 1.5-fold. These findings were blunted in the presence of an inhibitor to protein kinase C. High glucose also significantly enhanced calcification in BVSMC in a time-dependent manner (2.20 +/- 0.50 vs 1.35 +/- 0.55 micromol/mg, day 7; 5.04 +/- 1.35 vs 3.12 +/- 0.92 micromol/mg, day 14; P < 0.05). High glucose also induced the secretion of bone morphogenetic protein-2, a known osteoinductive factor, and further increased the secretion normally seen during calcification by 43% at day 7 and 57% at day 14.. These results demonstrate that vascular calcification in patients with diabetes is a cell-mediated process characterized by a phenotypic change of VSMCs to osteoblast-like cells with increased bone matrix protein expression, and that hyperglycaemia may directly induce these changes.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cattle; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Diabetic Angiopathies; Glucose; Humans; Muscle, Smooth, Vascular; Transforming Growth Factor beta; Vascular Diseases

We report here a comparative study of the development and behavior of chondrocytes isolated from normal growth plate tissue, tibial dyschondroplasic lesions, and from articular cartilage. The objective of these studies was to determine whether the properties exhibited by chondrocytes in dysplasic lesions or in articular cartilage were due to their cellular phenotype, their environment, or both. We had previously analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes. Using these data, we constructed a culture medium (DATP5) in which growth plate cells essentially recapitulate their normal behavior in vivo. Here, we used DATP5 to examine the behavior of chondrocytes isolated from lesions of tibial dyschondroplasia (TD). We found that once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. These findings suggest that the cause of TD is local factors operating in vivo to prevent these cells from developing normally. With respect to articular chondrocytes, our data indicate that they more closely retain normal protein and proteoglycan synthesis when grown in serum-free media. These cells readily induced mineral formation in vitro, both in the presence and absence of serum. However, in serum-containing media, mineralization was significantly enhanced when the cells were exposed to retinoic acid (RA) or osteogenic protein-1 (OP-1). Our studies support previous work indicating the presence of autocrine factors produced by articular chondrocytes in vivo that prevent mineralization and preserve matrix integrity. The lack of inhibitory factors and the presence of supporting factors are likely reasons for the induction of mineralization by articular chondrocytes in vitro.

Topics: Animals; Autocrine Communication; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcification, Physiologic; Calcinosis; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Chickens; Chondrocytes; Chondrogenesis; Culture Media; Culture Media, Serum-Free; Growth Plate; Osteochondrodysplasias; Proteoglycans; Tibia; Transforming Growth Factor beta; Tretinoin

Collagen triple helix repeat containing 1 (Cthrc1) was identified in a screen for differentially expressed sequences in balloon-injured versus normal arteries. Cthrc1 expression was not detectable in normal arteries. However, on injury it was transiently expressed by fibroblasts of the remodeling adventitia and by smooth muscle cells of the neointima. It was also found in the matrix of calcifying human atherosclerotic plaques. CTHRC1 is a secreted 28-kDa protein that is glycosylated and highly conserved from lower chordates to mammals. A short collagen motif with 12 Gly-X-Y repeats appears to be responsible for trimerization of the protein and this renders the molecule susceptible to cleavage by collagenase. Cthrc1 mRNA expression levels are increased in response to transforming growth factor-beta and bone morphogenetic protein-4. Cell migration assays performed with CTHRC1-overexpressing fibroblasts and smooth muscle cells demonstrate that increased CTHRC1 levels are associated with enhanced migratory ability. Furthermore, CTHRC1 overexpression caused a dramatic reduction in collagen type I mRNA and protein levels. Our data indicate that the novel molecule CTHRC1 is transiently expressed in the arterial wall in response to injury where it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Aorta; Biopolymers; Calcinosis; Carotid Artery Injuries; Carotid Stenosis; Catheterization; Cell Adhesion; Cell Differentiation; Cell Movement; CHO Cells; Collagenases; Cricetinae; Cricetulus; Evolution, Molecular; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Profiling; Glycoproteins; Humans; Molecular Sequence Data; Myoblasts; Myocytes, Smooth Muscle; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Species Specificity; Transforming Growth Factor beta; Vertebrates

LDL receptor (LDLR)-null mice fed high-fat/cholesterol diets, a model of the metabolic syndrome, have vascular calcification (VC) worsened by chronic kidney disease (CKD) and ameliorated by bone morphogenetic protein-7 (BMP-7), an efficacious agent in treating animal models of renal osteodystrophy. Here, LDLR-/- high-fat-fed mice without CKD were shown to have significant reductions in bone formation rates, associated with increased VC and hyperphosphatemia. Superimposing CKD resulted in a low turnover osteodystrophy, whereas VC worsened and hyperphosphatemia persisted. BMP-7 treatment corrected the hyperphosphatemia, corrected the osteodystrophy, and prevented VC, compatible with skeletal phosphate deposition leading to reduced plasma phosphate and removal of a major stimulus to VC. A pathologic link between abnormal bone mineralization and VC through the serum phosphorus was supported by the partial effectiveness of directly reducing the serum phosphate by a phosphate binder that had no skeletal action. Thus, in this model of the metabolic syndrome with CKD, a reduction in bone-forming potential of osteogenic cells leads to low bone turnover rates, producing hyperphosphatemia and VC, processes ameliorated by the skeletal anabolic agent BMP-7, in part through deposition of phosphate and increased bone formation.

Topics: Animals; Aorta; Aortic Diseases; Bone and Bones; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Remodeling; Calcinosis; Calcium; Chronic Disease; Chronic Kidney Disease-Mineral and Bone Disorder; Dietary Fats; Dose-Response Relationship, Drug; Female; Kidney Diseases; Male; Metabolic Syndrome; Mice; Mice, Inbred C57BL; Mice, Knockout; Parathyroid Glands; Phosphates; Receptors, LDL; Transforming Growth Factor beta

Dent's disease, an X-linked renal tubular disorder, is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and progressive renal failure. Dent's disease results from mutations of the voltage-gated chloride channel CLC-5.. We studied the effect of zero and high citrate diet on renal function of ClC-5 knockout mice and wild-type mice. The mice were placed in metabolic cages from which the urine was collected. Mice were sacrificed to obtain serum and tissues for analysis.. ClC-5 knockout mice fed zero or high citrate diet had significantly increased urinary calcium excretion compared with wild-type mice fed the same diets. Nine-month-old ClC-5 knockout mice on a zero citrate diet had significantly decreased glomerular filtration rate (GFR), whereas 9-month-old ClC-5 knockout mice on a high citrate diet had normal renal function. ClC-5 knockout mice fed a zero citrate diet had significantly increased tubular atrophy, interstitial fibrosis, cystic changes, and nephrocalcinosis compared to ClC-5 knockout mice fed a high citrate diet. Transforming growth factor-beta1 (TGF-beta1) was significantly increased in 9-month-old ClC-5 knockout mice on zero citrate diet compared to 9-month-old wild-type mice on the same diet.. High citrate diet preserved renal function and delayed progression of renal disease in ClC-5 knockout mice even in the apparent absence of stone formation. We conclude from this that long-term control of hypercalciuria is an important factor in preventing renal failure in these mice.

Topics: Animals; Blood Urea Nitrogen; Calcinosis; Calcium; Chloride Channels; Citrates; Creatinine; Diet; Disease Models, Animal; Glomerular Filtration Rate; Hydrogen-Ion Concentration; Kidney Calculi; Kidney Failure, Chronic; Mice; Mice, Knockout; Potassium; Sodium; Transforming Growth Factor beta; Transforming Growth Factor beta1

We have shown that renal injury and chronic kidney disease (CKD) directly inhibit skeletal anabolism, and that stimulation of bone formation decreases the serum phosphate. Most recently, these observations were rediscovered in low-density lipoprotein receptor null mice fed high-fat/cholesterol diets, a model of the metabolic syndrome (hypertension, obesity, dyslipidemia, and insulin resistance). We had demonstrated that these mice have vascular calcification (VC) of both the intimal atherosclerotic type and medial type. We have shown that VC is worsened by CKD and ameliorated by bone morphogenetic protein -7 (BMP-7). The finding that high-fat-fed low-density lipoprotein receptor null animals without CKD have hyperphosphatemia led us to examine the skeletons of these mice. We found significant reductions in bone formation rates, associated with increased VC and superimposing CKD results in the adynamic bone disorder (ABD), while VC was worsened and hyperphosphatemia persisted. A pathological link between abnormal bone mineralization and VC through the serum phosphorus was demonstrated by the partial effectiveness of directly reducing the serum phosphate by a phosphate binder that had no skeletal action. BMP-7 treatment corrected the ABD and corrected hyperphosphatemia, compatible with BMP-7-driven stimulation of skeletal phosphate deposition reducing plasma phosphate and thereby removing a major stimulus to VC. Thus, in the metabolic syndrome with CKD, a reduction in bone-forming potential of osteogenic cells leads to ABD producing hyperphosphatemia and VC, processes ameliorated by the skeletal anabolic agent BMP-7, in part through increased bone formation and skeletal deposition of phosphate, and in part through direct actions on vascular smooth muscle cells. We have demonstrated that the processes leading to vascular calcification begin with even mild levels of renal injury before demonstrable hyperphosphatemia, and they are preventable and treatable. Therefore, early intervention in CKD is warranted and may affect mortality of the disease.

Topics: Animals; Aorta, Thoracic; Bone Diseases; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcinosis; Calcium; Calcium Carbonate; Chronic Disease; Chronic Kidney Disease-Mineral and Bone Disorder; Dietary Fats; Disease Progression; Female; Hyperparathyroidism; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Osteoblasts; Osteogenesis; Phosphates; Renal Insufficiency, Chronic; Transforming Growth Factor beta

In idiopathic chondrocalcinosis and in osteoarthritis (OA), increased extracellular PP(i) (ecPP(i)) promotes calcification. In chromosome 5p-associated familial chondrocalcinotic degenerative arthropathy, certain mutations in the membrane protein ANK may chronically raise ecPP(i) via enhanced PP(i) channeling. Therefore, we assessed if dysregulated wild-type ANK expression could contribute to pathogenesis of idiopathic degenerative arthropathy through elevated ecPP(i).. Using cells with genetic alterations in expression of ANK and the PP(i)-generating nucleotide pyrophosphatase phosphodiestrase (NPP) PC-1, we examined how increased ANK expression elevates ecPPI, testing for codependent effects with PC-1. We also evaluated the effects of ANK expression on chondrocyte growth, matrix synthesis, and MMP-13 expression and we immunohistochemically examined ANK expression in situ in human knee OA cartilages.. Using cells expressing defective ANK, as well as PC-1 knockout cells, we demonstrated that ANK required PC-1 (and vice versa) to raise ecPP(i) and that the major ecPP(i) regulator TGFbeta required both ANK and PC-1 to elevate ecPP(i). Upregulation of wild-type ANK by transfection in normal chondrocytes not only raised ecPP(i) 5-fold to approximately 100nM but also directly stimulated matrix calcification and inhibited collagen and sulfated proteoglycans synthesis. In addition, upregulated ANK induced chondrocyte MMP-13, an effect that also was stimulated within 2h by treatment of chondrocytes with 100nM PP(i) alone. Finally, ANK expression was upregulated in situ in human knee OA cartilages.. Elevation of ecPP(i) by ANK critically requires the fraction of cellular PP(i) generated by PC-1. The upregulation of ANK expression in OA cartilage and the capacity of increased ANK expression to induce MMP-13 and to promote matrix loss suggest that increased ANK expression and ecPP(i) exert noxious effects in degenerative arthropathies beyond stimulation of calcification.

Topics: Animals; Calcinosis; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagen; Collagenases; Diphosphates; Extracellular Fluid; Humans; Immunohistochemistry; Knee Joint; Matrix Metalloproteinase 13; Mice; Mice, Inbred Strains; Osteoarthritis, Knee; Phosphoric Diester Hydrolases; Proteoglycans; Pyrophosphatases; Transfection; Transforming Growth Factor beta; Up-Regulation

It has been suggested that subchondral bone remodeling plays a role in the progression of osteoarthritis (OA). To test this hypothesis, we characterized the changes in the rat anterior cruciate ligament transection (ACLT) model of OA and evaluated the effects of alendronate (ALN), a potent inhibitor of bone resorption, on cartilage degradation and on osteophyte formation.. Male Sprague-Dawley rats underwent ACLT or sham operation of the right knee. Animals were then treated with ALN (0.03 and 0.24 microg/kg/week subcutaneously) and necropsied at 2 or 10 weeks postsurgery. OA changes were evaluated. Subchondral bone volume and osteophyte area were measured by histomorphometric analysis. Coimmunostaining for transforming growth factor beta (TGF beta), matrix metalloproteinase 9 (MMP-9), and MMP-13 was performed to investigate the effect of ALN on local activation of TGF beta.. ALN was chondroprotective at both dosages, as determined by histologic criteria and collagen degradation markers. ALN suppressed subchondral bone resorption, which was markedly increased 2 weeks postsurgery, and prevented the subsequent increase in bone formation 10 weeks postsurgery, in the untreated tibial plateau of ACLT joints. Furthermore, ALN reduced the incidence and area of osteophytes in a dose-dependent manner. ALN also inhibited vascular invasion into the calcified cartilage in rats with OA and blocked osteoclast recruitment to subchondral bone and osteophytes. ALN treatment reduced the local release of active TGF beta, possibly via inhibition of MMP-13 expression in articular cartilage and MMP-9 expression in subchondral bone.. Subchondral bone remodeling plays an important role in the pathogenesis of OA. ALN or other inhibitors of bone resorption could potentially be used as disease-modifying agents in the treatment of OA.

Topics: Acid Phosphatase; Alendronate; Animals; Anterior Cruciate Ligament; Bone Remodeling; Calcinosis; Cartilage, Articular; Collagen; Collagen Type I; Collagen Type II; Disease Models, Animal; Disease Progression; Extracellular Matrix Proteins; Glycoproteins; Isoenzymes; Male; Matrilin Proteins; Osteoarthritis, Knee; Osteoclasts; Peptides; Rats; Sclerosis; Severity of Illness Index; Tartrate-Resistant Acid Phosphatase; Transforming Growth Factor beta

Vascular calcification is a highly regulated process sharing features of bone mineralization. Since endothelium regulates many of the processes during atherogenesis, we monitored the expression of genes involved in calcification upon exposure of human coronary artery endothelial cells (HCAECs) to atherogenic stimuli. Genes studied were: core binding factor alpha-1 (Cbfa1/Runx2), a pivotal transcriptional regulator of osteogenesis; bone morphogenetic protein-2 (BMP2), an inducer of cartilage and bone; and matrix gla-protein (MGP), a potent inhibitor of calcification, which exerts its action by blocking BMP2. HCAECs were treated with oxidized-low density lipoprotein (ox-LDL, 80 microg/mL) or tumor necrosis factor-alpha (TNFalpha, 10 ng/mL), and the expression of Cbfa1, BMP2, and MGP was quantified by real-time PCR. Cbfa1 was expressed at low levels in untreated HCAECs, and its expression did not change with ox-LDL or TNFalpha treatment. The expression of BMP2 and MGP increased early after exposure to ox-LDL or TNFalpha (at 2-8 h), and the increase was not evident at 24 h. Ox-LDL exerted a stronger effect on MGP than on BMP2 expression. The effects of ox-LDL, but not TNFalpha, on MGP and BMP2 expression were inhibited by pretreatment of cells with an antibody directed at LOX-1, a lectin-like receptor for ox-LDL (10 microg/mL). Thus, the endothelium, when exposed to atherogenic stimuli, ox-LDL in particular, regulates the process of calcification by enhancing the expression of the bone inhibitory MGP, while the expression of Cbfa1 remains unchanged. Upregulation of BMP2 may represent a feedback upregulation in response to increase in MGP. The effect of ox-LDL appears to be mediated by LOX-1 activation.

Topics: Antibodies; Arteries; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium-Binding Proteins; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Core Binding Factors; Endothelium, Vascular; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Lipoproteins, LDL; Matrix Gla Protein; Neoplasm Proteins; Receptors, LDL; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Calcific aortic stenosis, the major heart valve disease encountered in the elderly, leads to massive calcium deposition in the valve leaflets that morphologically resembles bone formation. Recent studies have demonstrated the expression of various bone-associated proteins in stenotic valves, suggesting that valvular calcification may be an actively regulated process. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, and bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor cytokine superfamily, are known to participate in the regulation of bone development and maturation. Their pathogenetic role in calcific aortic stenosis is unknown.. Using an immunoperoxidase technique and antibodies against BSP and BMP-2, the expression of BSP and BMP-2 was examined in 16 human aortic valves with calcific aortic stenosis obtained at valve replacement, and in seven normal autopsy controls without signs of aortic stenosis.. By semiquantitative scoring, stenotic valves showed a significantly increased staining of BSP in cells and extracellular matrix as compared to control valves (2.7 +/- 0.1 versus 0.6 +/- 0.2 score units, p <0.001). Marked BMP-2 expression was detected in stenotic valves, mostly in cell-rich areas associated with focal calcium deposits, but no specific staining for BMP-2 was detected in control valves (1.5 +/- 0.2 versus 0.0 +/- 0.0 score units, p <0.001).. These results demonstrate for the first time that BSP and BMP-2 are differentially expressed in normal aortic valves and in aortic stenosis, thereby supporting the concept that valvular calcification might be based on an actively regulated process involving BSP and BMP-2.

Topics: Aged; Aortic Valve; Aortic Valve Stenosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium; Female; Humans; Immunohistochemistry; Integrin-Binding Sialoprotein; Male; Sialoglycoproteins; Staining and Labeling; Statistics as Topic; Transforming Growth Factor beta

Aortic valve stenosis characteristically progresses due to cuspal calcification, often necessitating valve replacement surgery. The present study investigated the hypothesis that TGF-beta1, a cytokine that causes calcification of vascular smooth muscle cells in culture, initiates apoptosis of valvular interstitial cells as a mechanistic event in cuspal calcification.. Noncalcified and calcified human aortic valve cusps were obtained at autopsy or at the time of cardiac surgery. The distributions within cusps of TGF-beta1, latent-TGF-beta1-associated peptide, and TGF-beta receptors were studied using immunohistochemistry. The effects of TGF-beta1 on mechanistic events contributing to aortic valve calcification were also investigated using sheep aortic valve interstitial cell (SAVIC) cultures.. Immunohistochemistry studies revealed that calcific aortic stenosis cusps characteristically contained within the extracellular matrix qualitatively higher levels of TGF-beta1 than noncalcified cusps. Noncalcified normal valves demonstrated only focal intracellular TGF-beta1. Addition of TGF-beta1 to SAVIC cultures led to a cascade of events, including: cellular migration, aggregation, formation of apoptotic-alkaline phosphatase enriched nodules, and calcification of these nodules. The time course of these events in the SAVIC culture system was rapid with nodule formation with apoptosis by 72 hours, and calcification after 7 days. Furthermore, ZVAD-FMK, an antiapoptosis agent (caspase inhibitor), significantly inhibited calcification and apoptosis induced by TGF-beta1, but had no effect on nodule formation. However, cytochalasin D, an actin-depolymerizing agent, inhibited nodule formation, but not calcification.. TGF-beta1 is characteristically present within calcific aortic stenosis cusps, and mediates the calcification of aortic valve interstitial cells in culture through mechanisms involving apoptosis.

Topics: Aged; Aged, 80 and over; Alkaline Phosphatase; Amino Acid Chloromethyl Ketones; Animals; Aortic Valve; Aortic Valve Stenosis; Apoptosis; Calcinosis; Caspase Inhibitors; Cells, Cultured; Disease Progression; Female; Humans; Immunohistochemistry; Male; Sheep; Transforming Growth Factor beta; Transforming Growth Factor beta1

Chronic renal failure is complicated by high cardiovascular mortality. One key contributor to this mortality is vascular calcification, for which no therapy currently exists. Bone morphogenetic protein 7 is an essential renal morphogen that maintains renal tubular differentiation in the adult and is downregulated in renal failure. Several studies have demonstrated its efficacy in treating various renal diseases in rodents, and it was hypothesized that it would also be an effective treatment of vascular calcification in this setting. Uremia was imposed on LDL receptor null mice (a model of atherosclerosis), which were then treated with bone morphogenetic protein 7 for 15 wk. Uremic animals had increased vascular calcification by histology and chemical analysis. Calcification in treated animals was similar to or less than non-uremic control animals. Cells exhibiting an osteoblast-like phenotype in the vessel wall may be important in the etiology of vascular calcification. Expression of osteocalcin was assessed as a marker of osteoblastic function, and it is shown that it is increased in untreated uremic animals but downregulated to levels similar to non-uremic control animals with treatment. The data are compatible with bone morphogenetic protein 7 deficiency as a pathophysiologic factor in chronic renal failure, and they demonstrate its efficacy as a potential treatment of vascular calcification.

Topics: Animals; Arteriosclerosis; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcinosis; Immunohistochemistry; Kidney Failure, Chronic; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteocalcin; Receptors, LDL; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Vascular Diseases

The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Here we present additional evidence that the Ca2+-induced conformer of the vitamin K-dependent Gla region in MGP is involved in BMP-2 binding. Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly gamma-carboxylated and did not bind BMP-2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP-2/MGP complex exists in vivo, consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-gamma-carboxylation of MGP, allowing unopposed BMP-2 activity.

Topics: Amino Acid Sequence; Animals; Aortic Diseases; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium; Calcium-Binding Proteins; Cattle; Extracellular Matrix Proteins; Female; Fluorescent Antibody Technique; Growth Hormone; Humans; Immunohistochemistry; Matrix Gla Protein; Microscopy, Confocal; Molecular Sequence Data; Nasal Mucosa; Nasal Septum; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta; Vitamin K

Calcification of homografts and vascular conduits is poorly understood. Mechanisms leading to calcification were studied in a rat model of aortic allografts.. Rat aortas from Lew1W (RT1(u)) were transplanted into Lew1A (RT1(a)). Animals were killed at 30 days and 180 days, and aortic grafts were removed and analyzed for histologic and immunohistologic studies.. Intimal surface increased progressively over 6 months and was the site of important modifications. Intimal cellular population changed from a leukocyte (CD45, OX1-OX30)- and macrophage (CD68, ED-1)-based population at 30 days to predominantly alpha-smooth muscle actin-expressing cells at 180 days. At 180 days, allografts were characterized by an abundant extracellular matrix composed of collagen and elastic fibers associated with extensive calcification (von Kossa staining) located in the intima and media. Osteoblastic activity was present in calcified lesion as shown by alkaline phosphatase activity. At 180 days, numerous chondrocytes (protein S100-positive and alpha-smooth muscle actin-negative) were present focally in the media. However, double immunostaining revealed that a cellular population within the media with a chondrocyte-like morphology was alpha-smooth muscle actin-positive and S100-negative. Active form of transforming growth factor beta1 was expressed from 30 to 80 days in the medial and intimal layers.. These observations suggest that alpha-smooth muscle actin-positive cells within aortic allografts are eventually transformed to a chondrocyte-like structure, leading to vascular cartilaginous metaplasia associated with the expression of transforming growth factor beta1 and could be a potential pathway leading to extensive vascular wall calcification in allografts through endochondral ossification.

Topics: Animals; Aorta, Thoracic; Biomarkers; Biopsy, Needle; Calcinosis; Chondrocytes; Disease Models, Animal; Immunohistochemistry; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred Lew; Sensitivity and Specificity; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Homologous

A biochemical and histochemical study investigating the role of CTGF/Hcs24 in the ossification of the posterior longitudinal ligament (OPLL) was conducted.. To clarify the involvement of CTGF/Hcs24 in ectopic bone formation in OPLL through endochondral ossification using human tissue.. Previous studies have shown that various cytokines are involved in the occurrence or development of ectopic bone formation in OPLL. Recently, the authors cloned an mRNA predominantly expressed in chondrocytes by differential display PCR and found that its gene, hcs24, is identical to that of connective tissue growth factor. It has been shown that CTGF/Hcs24 plays a major role in endochondral ossification.. Ossified ligament tissues were taken from seven male OPLL patients during surgery. Immunohistochemical staining was performed using an antibody specific for CTGF/Hcs24. Spinal ligament cells were isolated from five OPLL patients as well as five non-OPLL patients. The cells were incubated with recombinant human CTGF/Hcs24 or TGFbeta. The expression of ALP was analyzed by RT-PCR. For the effects of TGFbeta, the expression of CTGF/Hcs24 mRNA was analyzed.. Immunohistochemical staining showed that chondrocytes in the transitional region from nonossified to ossified ligament were stained with an antibody against CTGF/Hcs24. It was found that CTGF/Hcs24 enhanced the expression ALP mRNA in OPLL cells, whereas the expression remained unchanged in non-OPLL cells. The expression of CTGF/Hcs24 mRNA in OPLL and non-OPLL cell lines was increased by TGFbeta, and there was no significant difference between the two groups. However, TGFbeta and CTGF/Hcs24 enhanced the expression of ALP mRNA only in OPLL cells.. According to the study results, CTGF/Hcs24 may not only be an important factor in the development of endochondral ossification in OPLL, but may also be responsible for initiating osteogenesis in spinal ligament cells.

Topics: Adult; Aged; Alkaline Phosphatase; Antigens, Differentiation; Calcinosis; Cells, Cultured; Chondrocytes; Connective Tissue Growth Factor; Female; Growth Substances; Humans; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Longitudinal Ligaments; Male; Middle Aged; Ossification of Posterior Longitudinal Ligament; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Mice lacking the intermediate filament protein desmin demonstrate abnormal mitochondria behavior, disruption of muscle architecture, and myocardial degeneration with extensive calcium deposits and fibrosis. These abnormalities are associated with cardiomyocyte hypertrophy, cardiac chamber dilation and eventually with heart failure. In an effort to elucidate the molecular mechanisms leading to the observed pathogenesis, we have analyzed gene expression changes in cardiac tissue using differential display polymerase chain reaction and cDNA atlas array methods. The most substantial changes were found in genes coding the small extracellular matrix proteins osteopontin and decorin that are dramatically induced in the desmin-null myocardium. We further analyzed their expression pattern both at the RNA and protein levels and we compared their spatial expression with the onset of calcification. Extensive osteopontin localization is observed by immunohistochemistry in the desmin-null myocardium in areas with massive myocyte death, as well as in hypercellular regions with variable degrees of calcification and fibrosis. Osteopontin is consistently co-localized with calcified deposits, which progressively are transformed to psammoma bodies surrounded by decorin, especially in the right ventricle. These data together with the observed up-regulation of transforming growth factor-beta1 and angiotensin-converting enzyme, could explain the extensive fibrosis and dystrophic calcification observed in the heart of desmin-null mice, potentially crucial events leading to heart failure.

Topics: Animals; Calcinosis; Cardiomyopathies; Decorin; Desmin; Extracellular Matrix Proteins; Fibrosis; Gene Expression Regulation; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteopontin; Peptidyl-Dipeptidase A; Proteoglycans; Sialoglycoproteins; Transforming Growth Factor beta

Chronic cyclosporine (CsA) nephrotoxicity is characterized by tubulointerstitial fibrosis. Pirfenidone (PFD) is a novel antifibrotic compound that was shown to prevent and even reverse fibrosis. The mechanism of action of PFD is unclear but involves inhibition of transforming growth factor-beta (TGF-beta). Salt-depleted rats were administered CsA, CsA + PFD, vehicle (VH) or VH + PFD and sacrificed at 28days. Physiologic and histologic changes were studied in addition to TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and biglycan mRNA expressions by Northern blot. TGF-beta1 immunohistochemistry was also performed. Treatment with PFD ameliorated CsA-induced fibrosis by about 50% (p < 0.05). CsA-induced decrease in creatinine clearance improved with PFD but the difference was not significant. TGF-beta1, PAI-1 and biglycan mRNA expressions increased with CsA (p < 0.05 vs. VH) but strikingly improved with PFD treatment (p < 0.05 vs. CsA), which brought the levels down to VH levels. PFD treatment also decreased TGF-beta1 protein expression by 80%. These results demonstrate that PFD can attenuate renal fibrosis in this model. PFD was associated with a decrease in TGF-beta1 expression, which, in turn, was associated with a decrease in matrix deposition. These experiments suggest that PFD can be clinically useful for preventing chronic CsA nephrotoxicity and may prove to be helpful in other progressive renal diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biglycan; Calcinosis; Creatinine; Cyclosporine; Extracellular Matrix Proteins; Humans; Immunohistochemistry; Immunosuppressive Agents; Kidney; Kidney Diseases; Kidney Tubules; Male; Proteoglycans; Pyridones; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Prostate cancer frequently metastasizes to bone. However, unlike many other tumors that produce osteolytic lesions, prostate cancer produces osteoblastic lesions through unknown mechanisms. In the current study, we explored the ability and mechanism of an osteotropic prostate cancer cell line (C4-2B) to induce mineralization.. C4-2B cells were grown in promineralization media. Mineral deposition was characterized using von Kossa staining, calcium retention, alizarin red staining, Raman spectroscopy, and electron microscopy. Expression of osteoblast-related proteins was determined by RT-PCR. The nuclear level of the bone-specific transcription factor Cbfa1 was determined using western analysis and the effect of inhibiting Cbfa1 function, using a "decoy" Cbfa1 response element oligo, on mineralization was determined.. The studies demonstrated that C4-2B cells, but not its nonosteotropic parent cell line LNCaP, has an osteoblastlike phenotype including production of alkaline phosphatase, osteocalcin, osteonectin, bone sialoprotein, osteoprotegerin (OPG), and OPG ligand. Most importantly, the C4-2B cells produced hydroxyapatite mineral in vitro. Furthermore, C4-2B cells expressed high nuclear levels of the bone-specific transcription factor Cbfa1, compared to LNCaP cells, which accounts for their ability to produce bone-specific proteins. Inhibition of Cbfa1, using decoy DNA Cbfa1 response elements, abrogated the ability of C4-2B to produce mineral. Finally, we determined that C4-2B cells express bone morphogenic protein-7, a known inducer of Cbfa1 expression.. These data demonstrate a novel mechanism through which prostate cancer cells may directly contribute to the osteoblastic component that characterize their skeletal metastatic lesions. Prostate 47:212-221, 2001.

Topics: Anthraquinones; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Calcinosis; Calcium; Cell Division; Core Binding Factor Alpha 1 Subunit; Durapatite; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Male; Neoplasm Proteins; Osteoblasts; Osteoprotegerin; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Spectrophotometry, Infrared; Spectrum Analysis, Raman; Staining and Labeling; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The goal of this study was to determine the role of transforming growth factor-beta (TGF-beta) family members in arterial repair processes related to vascular injury.. TGF-beta plays important roles in many tissues including calcifying tissues and blood vessels. The family members of TGF-beta 1, -beta 2, and -beta 3 have overlapping functions and signal via the same receptor complex. To examine the role of TGF-beta in vascular remodeling and intimal hyperplasia we disrupted this signaling pathway using recombinant soluble TGF-beta receptor II (TGF-beta R:Fc).. The balloon catheter injury model of the rat carotid artery was used and TGF-beta R:Fc was injected every other day for a period of two weeks after which the vessels were harvested for analysis by histology, morphometry, and Northern blotting.. In situ hybridization showed TGF-beta receptor II expression in smooth muscle cells (SMC) of the injured vessel wall while the same cells also revealed abundant expression of all three TGF-beta ligands. Injection of TGF-beta R:Fc localized to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (65%) and an increase in lumen area (88%). The increase in lumen area was largely due to inhibition of negative remodeling which coincided with reduced adventitial fibrosis and collagen synthesis. Four days after injury, TGF-beta R:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells.. These results identify TGF-beta isoforms as the major mediators of adventitial fibrosis and negative remodeling after arterial injury, which is a major cause for restenosis following angioplasty.

Topics: Animals; Arteriosclerosis; Calcinosis; Catheterization; Cell Division; Fibromuscular Dysplasia; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

To investigate the expression and localisation of transforming growth factor betas (TGF beta s) and their receptor CD105 (endoglin) in relation to calcification and bone formation in atherosclerotic lesions of human carotid arteries.. The TGF beta family regulates cellular growth, differentiation and angiogenesis and plays a key role in enchondral bone formation. CD105 is part of the TGF beta receptor complex preferentially expressed on endothelial cells (EC).. Immunohistochemical methods were used to determine the localisation of TGF beta isoforms 1, 2 and 3 and their spatial expression patterns in relation to calcification and bone formation in atherosclerotic lesions. Cellular sources of TGF beta s and CD105 were assessed using cell-type specific antibodies.. There was marked variability in TGF beta expression in different cell types associated with calcification. Smooth muscle cells (SMC) in the atheroma cap showed higher levels of TGF beta 3 and 2 than 1, but in the deep musculoelastic intima there were higher levels of TGF beta 1 and alpha-actin. All three TGF beta isoforms were expressed in monocyte-macro-phages. Giant cells associated with calcifications showed intense staining for TGF beta 2. TGF beta 1 was most strongly expressed on matrix and cells associated with bone formation. CD105 expression on SMCs and monocyte-macrophages was lower on cells in close association with calcification. SMCs associated with bone formation expressed high levels of CD105.. The different TGF beta isoforms exhibit distinct but overlapping patterns of expression, and support the hypothesis that they are involved in the process of calcification and bone formation in human atherosclerotic lesions. Lower expression of CD105 on cells associated with calcification may represent their state of lower responsiveness to TGF beta s.

Topics: Antigens, CD; Arteriosclerosis; Calcinosis; Carotid Arteries; Carotid Stenosis; Endoglin; Endothelium, Vascular; Humans; Muscle, Smooth, Vascular; Ossification, Heterotopic; Receptors, Cell Surface; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

Matrix Gla Protein (MGP) is a small protein which is thought to be an inhibitor of tissue calcification and a regulator of cell differentiation. In this study we have examined the transcriptional regulation of MGP within rat vascular smooth muscle cells (VSMCs). We found that MGP transcription is downregulated by retinoic acid and transforming growth factor beta (TGF beta) whereas it is upregulated by vitamin D3 and cyclic AMP.

Topics: Animals; Arteriosclerosis; Calcinosis; Calcium-Binding Proteins; Cholecalciferol; Cyclic AMP; Down-Regulation; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Matrix Gla Protein; Muscle, Smooth, Vascular; Rats; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Up-Regulation

Topics: Calcinosis; CREST Syndrome; Female; Humans; Middle Aged; Scleroderma, Systemic; Skin Diseases; Transforming Growth Factor beta; Transforming Growth Factor beta3

We examined the relative contributions of foreign body granulation and de novo lesions to neointimal hyperplasia in atherectomized specimens of restenosis after coronary stenting.. Clinicopathological studies have suggested that smooth muscle cell (SMC) hyperplasia is the most likely cause of restenosis after coronary stenting. It is not yet fully understood how SMC hyperplasia occurs or how SMCs stimulation can lead to intimal hyperplasia. Although inflammation has been postulated to be a major contributor to restenosis after coronary stenting, there is a paucity of data on the relationsip between inflammation and subsequent neointimal formation in humans. Only in a porcine experimental model of stent restenosis, foreign body granulation tissue as a cause of inflammation in stent restenosis was reported.. Tissue specimens were retrieved by directional atherectomy from 11 patients in whom stent restenosis developed after percutaneous revascularization of coronary artery disease. For specimens preserved in 10% buffered formalin, analysis of cellular composition was performed quantitatively after cell-specific immunostaining, i.e. CD68, UCHL-1, HLA-DR, smooth muscle actin, vimentin, desmin, PCNA and TGF-beta.. Five of the 11 patients showed granulation tissues 3-6 months after stent implantation, of whom 3 patients revealed foreign body multinucleated giant cells around the stent struts where PCNA- and vimentin-positive SMCs were demonstrated. Calcification and de novo lesions in medial and adventitial tissues were observed in 3 other patients, and fresh and/or organized thrombi were documented in 3 of the 11 patients.. These findings support the notion that stent restenosis results from SMC hyperplasia and suggest that the foreign body granulation tissue against metals of the stents and de novo lesions could play an important role in chronic inflammation leading to intimal hyperplasia and subsequently to stent restenosis in some patients. Clinicians should thus consider whether a patient may be allergic to stent components with unknown reaction, e.g. haptens.

Topics: Aged; Atherectomy, Coronary; B-Lymphocytes; Calcinosis; Coronary Angiography; Coronary Disease; Female; Giant Cells, Foreign-Body; Graft Occlusion, Vascular; Granuloma, Foreign-Body; HLA-DR Antigens; Humans; Hyperplasia; Immunoenzyme Techniques; Male; Microfilament Proteins; Middle Aged; Muscle, Smooth, Vascular; Proliferating Cell Nuclear Antigen; Recurrence; Reoperation; Stents; T-Lymphocytes; Transforming Growth Factor beta; Tunica Intima

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Topics: Activin Receptors; Adult; Aged; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcinosis; Cartilage; Cell Size; Female; Humans; Immunohistochemistry; Ligamentum Flavum; Male; Middle Aged; Protein Serine-Threonine Kinases; Radioligand Assay; Receptors, Cell Surface; Receptors, Growth Factor; Transforming Growth Factor beta

To understand whether osteogenesis imperfecta (OI) could result from defective differentiation of osteoprogenitor cells, we investigated the osteogenic potential of bone marrow stromal cells from a mouse model of human OI (oim). Bone marrow was flushed from the femurs and tibias of oim and normal littermates using a syringe with Dulbecco's modified Eagle's medium, and cells were allowed to adhere to flasks. Adherent cells were trypsinized and passaged weekly at a 1:4 split. The established stromal cells were assessed for collagen synthesis, alkaline phosphatase, and osteocalcin production in the presence or absence of rhBMP-2. The stromal cells were also assessed for mineralization by Von-Kossa staining and for exogenous gene transfer using adeno-lacZ and a retroviral vector. The bone marrow stromal cells from oim mice synthesized alpha 1(I) homotrimers as expected, whereas the stromal cells from the normal littermates synthesized alpha 1(I)2 alpha 2(I) heterotrimers. The bone marrow stromal cells exhibited low levels of alkaline phosphatase activity under basal conditions: upon treatment with rhBMP-2, the level of the alkaline phosphatase activity increased approximately 40-fold. Cytochemical staining of the cells confirmed the expression of alkaline phosphatase by the oim stromal cells and its augmentation by rhBMP-2. Osteocalcin production in the stromal cells was also enhanced approximately threefold by rhBMP-2. oim stromal cells grown in the presence of beta-glycerophosphate and ascorbic acid demonstrated Von-Kossa-positive solid deposits after 3 weeks in culture. Ten days after infection with adeno-lacZ, approximately 70% of oim stromal cells expressed the transgene product, and after infection with a retrovirus, approximately 20% of the cells expressed the transgene. These data indicate that bone marrow stromal cells, have osteogenic potential, and also the potential to be transduced with exogenous genes. Under basal conditions, however, the stromal cells from oim mice exhibited significantly lower levels of alkaline phosphatase activity than their normal littermates.

Topics: Alkaline Phosphatase; Animals; Bone Marrow; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Cell Adhesion; Cells, Cultured; Collagen; Disease Models, Animal; Femur; Gene Expression Regulation, Viral; Humans; Lac Operon; Mice; Osteocalcin; Osteogenesis; Osteogenesis Imperfecta; Polymers; Recombinant Proteins; Retroviridae Infections; Signal Transduction; Stromal Cells; Tibia; Transforming Growth Factor beta

To elucidate the process of ossification in spinal ligaments, an aqueous solution containing recombinant human bone morphogenetic protein (BMP)-2 (40 micrograms/100 microL) was injected into murine ligamenta flava, and the ossification process was analyzed morphologically. In the control group, the solution administered lacked the protein; these flattened ligamentous fibroblasts possessing BMP receptors type IA and type II existed among type I collagen bundles. In the week immediately following the injection of BMP-2, ligamentous fibroblasts began to proliferate, differentiating into alkaline phosphatase-positive chondrocytes surrounded by an extracellular matrix composed of type I and II collagen. By the second week, differentiated chondrocytes of various stages were observed in type II collagen-rich matrix. These chondrocytes showed an abundance of BMP receptors type IA and II. The pathologically induced cartilage was resorbed by chondroclasts, permitting migration of blood vessels and osteogenic cells, as well as providing a site for endochondral ossification. By the third week, BMP-induced ossification had compressed the spinal cord, and by the sixth week, the ligamentous tissue had been almost completely replaced by bone. Ligamentous fibroblasts appeared to possess BMP receptors, as well as the potentiality to differentiate into chondrocytes. BMP receptors were upregulated during chondrification of ligamentous fibroblasts induced by exogenous BMP-2, suggesting that BMPs may play an important role in ossification of spinal ligaments.

Topics: Acid Phosphatase; Animals; Biomarkers; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Calcinosis; Cartilage; Cell Differentiation; Collagen; Extracellular Matrix; Fibroblasts; Humans; Immunohistochemistry; Isoenzymes; Ligamentum Flavum; Lumbar Vertebrae; Male; Mice; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Recombinant Proteins; Ribonucleases; Tartrate-Resistant Acid Phosphatase; Transforming Growth Factor beta

Abnormal metabolism of extracellular inorganic pyrophosphate (PPi) by articular cartilage contributes to calcium pyrophosphate dihydrate (CPPD) crystal formation and the resultant arthritis known as CPPD deposition disease. The factors causing excess PPi elaboration in affected cartilage remain poorly defined. Retinoic acid (RA), a naturally occurring vitamin A metabolite, promotes cartilage degeneration and mineralization, two correlates of CPPD crystal deposition. RA was examined as a potential modifier of cartilage PPi elaboration. All-trans RA (200-1000 nM) increased PPi levels in culture medium of normal porcine cartilage and chondrocytes 2-3-fold over control values at 96 hours of incubation (P < 0.01). IGF1 and anti-EGF antibody diminished the effects of RA on PPi elaboration. RA modestly increased activity of the PPi-generating ectoenzyme NTPPPH in culture medium (P < 0.01). As some RA effects are mediated through increased activity of TGFbeta, a known PPi stimulant, we examined the effect of anti-TGFbeta antibody on RA-induced PPi elaboration. PPi levels in medium were reduced from 30 +/- 7 microM in cartilage cultures with 500 nM RA to 14 +/- 4 microM PPi in cartilage cultures with RA and anti-TGFbeta. Anti-TGFbeta antibody, however, had no significant effect on RA-induced PPi elaboration in chondrocyte cultures. Thus, RA, along with TGFbeta and ascorbate, can now be included in the list of known PPi stimulants. All three of these factors promote mineralization in growth plate cartilage. These data support a central role for TGFbeta in CPPD disease, and provide further evidence linking processes of normal and pathologic calcification in cartilage.

Topics: Animals; Antibodies, Monoclonal; Arthritis; Calcinosis; Calcium Pyrophosphate; Cartilage, Articular; Cells, Cultured; Insulin-Like Growth Factor I; Keratolytic Agents; Knee Joint; Organ Culture Techniques; Pyrophosphatases; Swine; Transforming Growth Factor beta; Tretinoin

Previous studies in our laboratory demonstrated messenger RNA for bone morphogenetic protein-2a in human calcified plaque, suggesting that arterial calcification is a regulated process, similar to osteogenesis. To further test this hypothesis, we have isolated and cloned a subpopulation of cells from bovine aortic media that show osteoblastic potential. These novel cells are primarily distinguished from smooth muscle cells by expression of a surface marker preliminarily identified as a modified form of the ganglioside sialyl-lactosylceramide (GM3). Osteoblastic potential was indicated by high levels of alkaline phosphatase and collagen I, expression of osteopontin and osteonectin (SPARC), and production of bone-specific osteocalcin and hydroxyapatite. Cultures of these cells were stimulated to form increased numbers of calcium-mineral-producing nodules by the oxysterol 25-hydroxycholesterol as well as by transforming growth factor-beta 1, both known to be present in atherosclerotic lesions. The stimulation of calcifying vascular cells in the artery wall by these two factors suggests a possible mechanism for the colocalization of calcification with atherosclerosis in vivo.

Topics: Alkaline Phosphatase; Animals; Aorta; Arteriosclerosis; Biomarkers; Calcinosis; Cattle; Clone Cells; Cytokines; G(M3) Ganglioside; Histocytochemistry; Humans; Hydroxycholesterols; Osteoblasts; Osteocalcin; Transforming Growth Factor beta

Immunohistochemical detection of bone morphogenetic protein (BMP) in calcifying fibrous epulis was performed to elucidate the biological process of ossification and cemento-ossification. In a total 25 cases, 15 (60%) showed positive BMP staining in bone forming areas. Histopathological features of developing hard tissues were varied, consisting of structures such as woven bone and cemento-osseous formations. BMP immunostaining was limited to osteoblasts and fibrous connective tissue surrounding the bone matrix. BMP was concentrated in the periodontal fibres and in dense fibrous structures in the cemento-osseous masses. On the basis of histopathological and immunohistochemical features, the histogenesis of ossifying and cemento-ossifying processes appear to be of two possible origins; the excessive proliferation of periodontal ligament and a metaplastic process occurring in the connective tissue fibres (non-periodontal in origin), with the former being more common.

Topics: Animals; Bone Morphogenetic Proteins; Calcinosis; Gingival Hyperplasia; Humans; Immunohistochemistry; Mice; Ossification, Heterotopic; Periodontal Ligament; Proteins; Transforming Growth Factor beta