calpain and Fibrosis

Eosinophilic esophagitis (EoE) is a chronic allergen-mediated inflammatory disease of the esophagus. This inflammation leads to feeding difficulties, failure to thrive and vomiting in young children, and causes food impaction and esophageal stricture in adolescents and adults. In the 20 years since EoE was first described, we have gained a great deal of knowledge regarding the genetic predisposition of disease, the inflammatory milieu associated with EoE and the long-term complications of chronic inflammation. Herein, we summarize the important breakthroughs in the field including both in vitro and in vivo analysis. We discuss insights that we have gained from large-scale unbiased genetic analysis, a multitude of genetically and chemically altered mouse models of EoE and most importantly, the results of clinical trials of various pharmacologic agents. Understanding these successes and failures may be the key to developing more effective therapeutic strategies.

Topics: Animals; Basophils; Calpain; Cellular Microenvironment; Cytokines; Dysbiosis; Eosinophilic Esophagitis; Esophagus; Fibrosis; Genetic Predisposition to Disease; Humans; Immunoglobulin E; Interleukin-13; Interleukin-5; Mast Cells; Mucous Membrane; Neoplasm Proteins; Nuclear Proteins; Receptors, Cytokine; Repressor Proteins; Th2 Cells; Thymic Stromal Lymphopoietin

Calpains are cytosolic calcium-activated cysteine proteases. Recently, they have been proposed to influence signal transduction processes leading to myocardial remodelling and heart failure. In this review, we will first describe some of these molecular mechanisms. Calpains may contribute to myocardial hypertrophy and inflammation, mainly through the activation of transcription factors such as NF-κB. They play an important role in the fibrosis process partly by activating transforming growth factor β. They are also implicated in cell death as they cause the breakdown of sarcolemma and sarcomeres. Nevertheless, a key to understanding the molecular basis of calpain-mediated myocardial remodelling likely lies in the identification of mechanisms involved in calpain secretion, since cytosolic and extracellular proteases would have different functions. Finally, we will provide an overview of the available evidence that calpains are indeed actively involved in the common causes of heart failure, including hypertension, diabetes, atherosclerosis, ischaemia-reperfusion, atrial fibrillation, congestive failure, and mechanical unloading.

Topics: Animals; Apoptosis; Calpain; Cardiomegaly; Fibrosis; Heart Failure; Humans; Inflammation; Necrosis; Transcription Factors; Ventricular Remodeling

Systemic sclerosis (SSc), with unclear pathophysiology, is a paradigmatic rheumatic disease of immunity dysfunction-driven multi-organ inflammation and ultimate fibrosis. Pathogenesis breakthroughs are urgently needed for available treatments halting its unremitting stiffness. This study aims to investigate whether ferroptosis can regulate the progressive SSc fibrosis.. In vivo, bleomycin (BLM)-induced mice model was subjected to ferroptosis detection using western blotting, malondialdehyde (MDA), and glutathione (GSH) assays. Pharmacological inhibitor of the acyl-CoA synthetase long-chain family member 4 (ACSL4) was utilized to explore its potential therapeutic effects for fibrosis, from histological, biochemical, and molecular analyses. In vitro, bone marrow-derived macrophages (BMDM) were activated into inflammatory phenotype and then the relationship was evaluated between activation level and ferroptosis sensitivity in lipopolysaccharide (LPS) incubation with gradient concentrations. The potential calpain/ACSL4 axis was analyzed after calpain knockdown or over-expression in Raw264.7.. Both skin and lung tissue ferroptosis were present in SSc mice with enhanced ACSL4 expression, while ACSL4 inhibition effectively halted fibrosis progressing and provides protection from inflammatory milieu. Meanwhile, a positive regulation relationship between LPS-induced macrophage activity and ferroptosis sensitivity can be observed. After calpain knockdown, both inflammatory macrophage ferroptosis sensitivity and ACSL4 expression decreased, while its over-expression renders ACSL4-envoking condition. Also, calpain pharmacological inhibition reduced both ferroptosis and fibrosis aptitude in mice.. ACSL4 induces inflammatory macrophage ferroptosis to aggravate fibrosis progressing. ACSL4 and its upregulators of calpains may be potential therapeutic targets for BLM model of SSc.

Topics: Animals; Bleomycin; Calpain; Coenzyme A Ligases; Ferroptosis; Fibrosis; Macrophages; Mice; Scleroderma, Systemic

Topics: Animals; Calpain; Cytokines; Fibrosis; Humans; Ion Channels; Liver Cirrhosis; Macrophages; Mice

Calpain-1, a calcium-activated neutral cysteine proteases, has been reported to be involved in the formation of pulmonary hypertension. HIF-1α, an oxygen-sensitive transcription factor, has been reported to activate genes involved in cell proliferation and extracellular matrix recombination. This study was designed to investigate the effect of calpain-1 in hypoxic pulmonary hypertension (HPH) and to explore whether there is a relationship between calpain-1 and HIF-1α in this disease. In the hypoxia-induced model of HPH, we found that hypoxia resulted in increased right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodelling and collagen deposition in lung tissues of mice. The levels of calpain-1 and HIF-1α were up-regulated in the lung tissues of hypoxia-treated mice and pulmonary arterial smooth muscle cells (PASMCs). Knock-out of calpain-1 restrained haemodynamic and histological changes induced by chronic hypoxia in mice, and inhibition of calpain-1 also repressed the abnormal proliferation and migration of PASMCs. Besides, knock-out or inhibition of calpain-1 suppressed hypoxia-induced expression of HIF-1α, VEGF, PCNA, TGF-β1, MMP2 and collagen I in vivo and in vitro. While inhibition of HIF-1α abolished the above effects of calpain-1. Furthermore, we found that calpain-1 mediates the expression of HIF-1α through NF-κB (P65) under hypoxia conditions. In conclusion, our results suggest that calpain-1 plays a pivotal role in hypoxia-induced pulmonary vascular remodelling and fibrosis through HIF-1α, providing a better understanding of the pathogenesis of HPH.

Topics: Animals; Calpain; Cell Proliferation; Fibrosis; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Myocytes, Smooth Muscle; Pulmonary Artery; Vascular Remodeling

Calpains are a family of calcium-dependent thiol proteases that participate in a wide variety of biological activities. In our recent study, calpain is increased in the sera of scleroderma or systemic sclerosis (SSc). However, the role of calpain in interstitial lung disease (ILD) has not been reported. ILD is a severe complication of SSc, which is the leading cause of death in SSc. The pathogenesis of SSc-related ILD remains incompletely understood. This study investigated the role of myeloid cell calpain in SSc-related ILD.. A novel line of mice with myeloid cell-specific deletion of Capns1 (Capns1-ko) was created. SSc-related ILD was induced in Capns1-ko mice and their wild-type littermates by injection 0.l mL of bleomycin (0.4 mg/mL) for 4 weeks. In a separate experiment, a pharmacological inhibitor of calpain PD150606 (Biomol, USA, 3 mg/kg/day, i.p.) daily for 30 days was given to mice after bleomycin injection on daily basis. At the end of the experiment, the animals were killed, skin and lung tissues were collected for the following analysis. Inflammation, fibrosis and calpain activity and cytokines were assessed by histological examinations and ELISA, and immunohistochemical analyses, western blot analysis and Flow cytometry analysis.. Calpain activities increased in SSc-mouse lungs. Both deletion of Capns1 and administration of PD150606 attenuated dermal sclerosis as evidenced by a reduction of skin thickness and reduced interstitial fibrosis and inflammation in bleomycin model of SSc mice. These effects of reduced calpain expression or activity were associated with prevention of macrophage polarization toward M1 phenotype and consequent reduced production of pro-inflammatory cytokines including TNF-α, IL-12 and IL-23 in lung tissues of Capns1-ko mice with bleomycin model of SSc. Furthermore, inhibition of calpain correlated with an increase in the protein levels of PI3K and phosphorylated AKT1 in lung tissues of the bleomycin model of SSc mice.. This study for the first time demonstrates that the role of myeloid cell calpain may be promotion of macrophage M1 polarization and pro-inflammatory responses related PI3K/AKT1 signaling. Thus, myeloid cell calpain may be a potential therapeutic target for bleomycin model of SSc-related ILD.

Topics: Animals; Bleomycin; Calpain; Cytokines; Disease Models, Animal; Fibrosis; Inflammation; Lung; Lung Diseases, Interstitial; Macrophages; Mice; Myeloid Cells; Phenotype; Phosphatidylinositol 3-Kinases; Scleroderma, Systemic

Topics: Calpain; Fibrosis; Humans

Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium.. Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content.. In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters.. Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.

Topics: Animals; Calpain; Chemokine CCL2; Collagen; Coronary Artery Disease; Disease Models, Animal; Fibrosis; Glycoproteins; Hypercholesterolemia; Janus Kinase 2; Myocardial Ischemia; Myocardium; Signal Transduction; STAT Transcription Factors; Swine; Ventricular Remodeling

Topics: Animals; Calpain; Coronary Artery Disease; Drug Discovery; Fibrosis; Glycoproteins; Hypercholesterolemia; Myocardial Ischemia; Myocardium; Swine; Ventricular Remodeling

Topics: Actin Depolymerizing Factors; Animals; Bleomycin; Calpain; Cell Movement; Fibrosis; Humans; Idiopathic Pulmonary Fibrosis; Lung; Mice; Transforming Growth Factor beta1

The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cardiomegaly; Diabetes Mellitus, Experimental; Fibrosis; Male; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Proton-Translocating ATPases; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species

Topics: Actins; Animals; Calcinosis; Calcium; Calpain; Cataract; Collagen; Fibrosis; Heterozygote; Lens, Crystalline; Macrophages; Mice; Mice, Inbred C57BL; TRPM Cation Channels

To explore the role of calpain activation in the progression of peritoneal fibrosis.. Twenty-four male Sprague-Dawley rats were randomized equally into control group, MDL28170 (a calpain inhibitor)+normal saline group, peritoneal dialysis (PD) model group and PD + MDL28170 group. In the latter two groups, the rats received daily intraperitoneal injections of 100 mL/kg of 4.25% glucose PD solution, and those in PD+MDL28170 group and MDL28170 saline group received daily infusion of 4 mg/kg MDL28170 every other day. Eight weeks later, the rats were euthanized for pathological examination of the parietal peritoneum, and the visceral peritoneum was used for examining the activation status of calpain and the expressions of fibronectin (FN) and collagen I (COL-I). Calpain activation and expressions of FN, COL-I and α-SMA were also examined using Western blotting and immunofluorescence assay in primary cultures of rat peritoneal mesothelial cells treated with MDL28170, transforming growth factor-β (TGF-β), or both.. Compared with the control rats, the rats in PD model group showed significantly increased peritoneal peritoneum thickness, calpain activation in the peritoneal tissue, and expressions of FN and COL-I (. Peritoneal calpain activity and expressions FN and COL-I all increase significantly in rat models of PD-associated peritoneal fibrosis. Calpain activation can promote peritoneal fibrosis, and inhibition of calpain can alleviate peritoneal fibrosis.

Topics: Animals; Calpain; Fibrosis; Male; Peritoneal Fibrosis; Peritoneum; Rats; Rats, Sprague-Dawley; Renal Dialysis; Transforming Growth Factor beta1

Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth factor-β (TGFβ) to efficiently induce myofibroblast differentiation of cultured epithelial cells, endothelial cells, or quiescent fibroblasts is dependent on the induced expression and activity of dimeric calpains, a family of non-lysosomal cysteine proteases that regulate a variety of cellular events through posttranslational modification of diverse substrates. siRNA-based gene silencing demonstrated that TGFβ-induced mesenchymal transition of a murine breast epithelial cell line was dependent on induction of expression of calpain 9 (CAPN9), an isoform previously thought to be restricted to the gastrointestinal tract. Mice lacking functional CAPN9 owing to biallelic targeting of

Topics: Angiotensin II; Animals; Bleomycin; Calcium-Binding Proteins; Calpain; Carbon Tetrachloride; Cell Line; Dogs; Epithelial-Mesenchymal Transition; Fibrosis; Humans; Isoenzymes; Liver Cirrhosis; Male; Mice, Inbred C57BL; Molecular Targeted Therapy; Myocardium; Protein Biosynthesis; Protein Multimerization; RNA Stability; Signal Transduction; Transforming Growth Factor beta

Pleural fibrosis is associated with various inflammatory processes such as tuberculous pleurisy and bacterial empyema. There is currently no ideal therapeutic to attenuate pleural fibrosis. Some pro-fibrogenic mediators induce fibrosis through inflammatory processes, suggesting that blockage of these mediators might prevent pleural fibrosis. The MeT-5A human pleural mesothelial cell line (PMC) was used in this study as an in vitro model of fibrosis; and intra-pleural injection of bleomycin with carbon particles was used as an in vivo mouse model of pleural fibrosis. Calpain knockout mice, calpain inhibitor (calpeptin), and angiotensin (Ang) II type 1 receptor (AT

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Bleomycin; Calpain; Carbon; Cell Line; Collagen Type I; Dipeptides; Disease Models, Animal; Fibrosis; Humans; Losartan; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pleural Diseases

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Topics: 3T3 Cells; Adipocytes; Adipose Tissue; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Collagen; Diet, High-Fat; Disease Models, Animal; Fibrosis; Inflammation; Liver; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Obesity; Weight Gain

Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (ANG)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-ANG II in TPE resulted in activation of calpain and subsequent triggering of the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimens from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-κB signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.

Topics: Adolescent; Adult; Aged; Angiotensin II; Calpain; Cell Proliferation; Cells, Cultured; Collagen Type I; Enzyme Activation; Epithelium; Female; Fibrosis; Humans; Male; Middle Aged; Pleura; Pleural Effusion, Malignant; Renin-Angiotensin System; Signal Transduction; Tuberculosis, Pleural; Young Adult

Viral myocarditis (VMC) treatment has long been lacking of effective methods. Our former studies indicated roles of calpain in VMC pathogenesis. This study aimed at verifying the potential of calpain in Coxsackievirus B3 (CVB3)-induced myocarditis treatment.. A transgenic mouse overexpressing the endogenous calpain inhibitor, calpastatin, was introduced in the study. VMC mouse model was established via intraperitoneal injection of CVB3 in transgenic and wild mouse respectively. Myocardial injury was assayed histologically (HE staining and pathology grading) and serologically (myocardial damage markers of CK-MB and cTnI). CVB3 replication was observed in vivo and in vitro via the capsid protein VP1 detection or virus titration. Inflammation/fibrotic factors of MPO, perforin, IFNγ, IL17, Smad3 and MMP2 were evaluated using western blot or immunohistology stain. Role of calpain in regulating fibroblast migration was studied in scratch assays.. Calpastatin overexpression ameliorated myocardial injury induced by CVB3 infection significantly in transgenic mouse indicated by reduced peripheral CK-MB and cTnI levels and improved histology injury. Comparing with CVB3-infected wild type mouse, the transgenic mouse heart tissue carried lower virus load. The inflammation factors of MPO, perforin, IFNγ and IL17 were down-regulated accompanied with fibrotic agents of Smad3 and MMP2 inhibition. And calpain participated in the migration of fibroblasts in vitro, which further proves its role in regulating fibrosis.. Calpain plays dual roles of facilitating CVB3 replication and inflammation promotion. Calpain inhibition in CVB3-induced myocarditis showed significant treatment effect. Calpain might be a novel target for VMC treatment in clinical practices.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Migration Inhibition; Coxsackievirus Infections; Disease Models, Animal; Enterovirus B, Human; Fibroblasts; Fibrosis; Inflammation; Mice; Myocarditis; Virus Replication

Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.. We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.. The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.. Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.

Topics: Animals; Calpain; Cardiomegaly; Collagen Type I; Collagen Type III; Cytoskeletal Proteins; Disease Models, Animal; Enzyme Activation; Female; Fibrosis; Gene Expression Regulation; Humans; Isoenzymes; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases, Secreted; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Infarction; Survival Analysis; Ventricular Function, Left; Ventricular Remodeling

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

Topics: Actins; Animals; Blood Vessels; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Differentiation; Cell Movement; Cells, Cultured; Cicatrix; Collagen; Female; Fibroblasts; Fibrosis; Granulation Tissue; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myofibroblasts; Platelet Endothelial Cell Adhesion Molecule-1; Skin; Wound Healing

Survival of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis, while the induction of HSC apoptosis may induce recovery. Activated HSC are resistant to many pro-apoptotic stimuli. To this issue, the role of Endoplasmic Reticulum (ER) stress in promoting apoptosis of HSCs and consequently fibrosis resolution is still debated.. To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution. HSCs were incubated with the ER stress agonists, tunicamycin or thapsigargin. In vivo, HSC were isolated from normal, bile duct-ligated (BDL) and bile duct-diverted (BDD) rats.. In activated HSC, the specific inhibitor of ER stress-induced apoptosis, calpastatin, is significantly increased vs. quiescent HSCs. Calpain is conversely reduced in activated HSCs. This pattern of protein expression provides HSCs resistance to the ER stress signals of apoptosis (apoptosis-resistant phenotype). However, both tunicamycin and thapsigargin are able to induce apoptosis in HSCs in vitro, completely reversing the calpain/calpastatin pattern expression. Furthermore, in vivo, the fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), leads to fibrosis resolution through a mechanism of HSCs apoptosis, potentially associated with ER stress: in fact, BDD rat liver shows an increased number of apoptotic HSCs associated with reduced calapstatin and increased calpain protein expression, leading to an apoptosis-sensible phenotype.. ER stress sensitizes HSC to apoptosis both in vitro and in vivo. Thus, ER stress represents a key target to trigger cell death in activated HSC and promotes fibrosis resolution.

Topics: Animals; Apoptosis; Bile Ducts; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 8; Endoplasmic Reticulum Stress; Fibrosis; Hepatic Stellate Cells; Immunohistochemistry; In Situ Nick-End Labeling; Ligation; Liver; Rats; RNA, Small Interfering; Thapsigargin; Tunicamycin

Age-associated central arterial wall stiffness is linked to extracellular matrix remodeling, including fibrosis and vascular calcification. Angiotensin II induces both matrix metalloproteinase 2 (MMP2) and calpain-1 expression and activity in the arterial wall. However, the role of calpain-1 in MMP2 activation and extracellular matrix remodeling remains unknown. Dual histo-immunolabeling demonstrates colocalization of calpain-1 and MMP2 within old rat vascular smooth muscle cells. Overexpression of calpain-1 induces MMP2 transcripts, protein levels, and activity, in part, by increasing the ratio of membrane type 1 MMPs to tissue inhibitor of metalloproteinases 2. These effects of calpain-1 overexpression-induced MMP2 activation are linked to increased collagen I and III production and vascular calcification. In addition, overexpression of calpain-1 also induces transforming growth factor-β1/Smad signaling, elastin degradation, alkaline phosphatase activation, and total calcium content but reduces the expression of calcification inhibitors, osteopontin, and osteonectin, in cultured vascular smooth muscle cells in vitro and in carotid artery rings ex vivo. Furthermore, both calpain-1 and collagen II increase with aging within human aortic intima. Interestingly, in aged human aortic wall, both calpain-1 and collagen II are highly expressed in artherosclerotic plaque areas compared with grossly normal areas. Cross-talk of 2 proteases, calpain-1 and MMP2, leads to secretion of active MMP2, which modulates extracellular matrix remodeling via enhancing collagen production and facilitating vascular calcification. These results establish calpain-1 as a novel molecular candidate to retard age-associated extracellular matrix remodeling and its attendant risk for hypertension and atherosclerosis.

Topics: Adolescent; Aged; Aging; Animals; Aorta; Blotting, Western; Calcinosis; Calpain; Cells, Cultured; Collagen; Elastin; Enzyme Activation; Fibrosis; Humans; Male; Matrix Metalloproteinase 2; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rats; Rats, Inbred BN; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-2; Young Adult

Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.. Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions.. Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose-induced proliferation and MMP activities were prevented by calpain inhibition.. Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Hypertrophic; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Cardiomyopathies; Disease Models, Animal; Fibrosis; Gene Expression Regulation; Heart; Hyperglycemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Molecular Targeted Therapy; Myocardium; Streptozocin

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Topics: Angiotensin II; Animals; Aorta; Blood Pressure; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fibrosis; Genetic Therapy; Hypertension; Hypertrophy; Hypertrophy, Left Ventricular; Inflammation; Infusion Pumps, Implantable; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocardium; NF-kappa B; NFATC Transcription Factors; Renal Artery; Time Factors; Ventricular Remodeling

Increased expression of critical components of the ubiquitin-dependent proteolytic pathway occurs in any muscle wasting condition so far studied in rodents where proteolysis rises. We have recently reported similar adaptations in head trauma patients [Mansoor et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2714-2718]. We demonstrate here that the increased muscle protein breakdown seen in mdx mice only correlated with enhanced expression of m-calpain, a Ca(2+)-activated proteinase. By contrast, no change in mRNA levels for components of the ubiquitin-proteasome proteolytic process was seen in muscles from both mdx mice and Duchenne muscular dystrophy patients. Thus, gene expression of components of this pathway is not regulated in the chronic wasting that characterizes muscular dystrophy.

Topics: Adolescent; Animals; Calpain; Cathepsin D; Cathepsin L; Cathepsins; Child; Cysteine Endopeptidases; Dystrophin; Endopeptidases; Female; Fibrosis; Gene Expression; Humans; Male; Mice; Mice, Inbred mdx; Multienzyme Complexes; Muscle, Skeletal; Muscular Dystrophies; Necrosis; Proteasome Endopeptidase Complex; Reference Values; RNA, Messenger; Transcription, Genetic; Ubiquitins