elastin and Hyperhomocysteinemia

Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation.. ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-β2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into μMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-β2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining.. Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-β2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

Topics: Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Autoantibodies; B-Lymphocytes; beta 2-Glycoprotein I; Calcium Phosphates; Cells, Cultured; Disease Models, Animal; Elastin; Hyperhomocysteinemia; Macrophages; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Elastase; Toll-Like Receptor 4

This study investigated the role and mechanism of Hyperhomocysteinemia (HHcy) on vascular remodeling in mice. We assessed the effect of HHcy on vascular remodeling using a carotid arterial vein patch model in mice with the gene deletion of cystathionine-beta-synthase (Cbs). Vein grafts were harvested 4 weeks after surgery. Cross sections were analyzed using Verhoeff-van Gieson staining, Masson`s Trichrome staining, and immunostaining for morphological analysis and protein level assessment. The effect of Hcy on collagen secretion was examined in cultured rat aortic smooth muscle cells (RASMC). We found that Cbs-/- mice with severe HHcy exhibited thicker neointima and a higher percentage of luminal narrowing in vein grafts. In addition, severe HHcy increased elastin and collagen deposition in the neointima. Further, severe HHcy increases CD45 positive cells and proliferative cells in vein grafts. Finally, Hcy increases collagen secretion in RASMC. These results demonstrate that HHcy increases neointima formation, elastin and collagen deposition following a carotid arterial vein patch. The capacity of Hcy to promote vascular fibrosis and inflammation may contribute to the development of vascular remodeling.

Topics: Animals; Blotting, Western; Carotid Arteries; Cell Proliferation; Cells, Cultured; Collagen; Cystathionine beta-Synthase; Elastin; Homocysteine; Hyperhomocysteinemia; Jugular Veins; Leukocyte Common Antigens; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Rats; Vascular Remodeling

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.

Topics: Abdominal Pain; Amidohydrolases; Animals; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blotting, Western; Connexins; Cystathionine beta-Synthase; Disease Models, Animal; Elasticity; Elastin; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Gap Junction alpha-5 Protein; Homocysteine; Hyperhomocysteinemia; Hypertension; Male; Matrix Metalloproteinase 9; Mesenteric Artery, Superior; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Microscopy, Video; Nitric Oxide Synthase Type III; Nitrites; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Splanchnic Circulation; Telemetry; Vascular Resistance

In the present study we tested the hypothesis whether hyperhomocysteinemia, an elevated homocysteine level, induces venous phenotype in artery. To test our hypothesis, we employed wild type (WT) and cystathionine β-synthase heterozygous (+/-) (CBS+/-) mice treatment with or without folic acid (FA). Aortic blood flow and velocity were significantly lower in CBS+/-mice compared to WT. Aortic lumen diameter was significantly decreased in CBS+/-mice, whereas FA treatment normalized it. Medial thickness and collagen were significantly increased in CBS+/-aorta, whereas elastin/collagen ratio was significantly decreased. Superoxide and gelatinase activity was significantly high in CBS+/-aorta vs WT. Western blot showed significant increase in MMP-2, -9,-12, TIMP-2 and decrease in TIMP-4 in aorta. RT-PCR revealed significant increase of vena cava marker EphB4, MMP-13 and TIMP-3 in aorta. We summarize that chronic HHcy causes vascular remodelling that transduces changes in vascular wall in a way that artery expresses vein phenotype.

Topics: Animals; Aorta; Blotting, Western; Chronic Disease; Collagen; Cystathionine beta-Synthase; Elastin; Ephrin-B2; Folic Acid; Gene Expression; Genotype; Hemorheology; Hyperhomocysteinemia; Male; Matrix Metalloproteinases; Mice; Mice, Transgenic; Phenotype; Receptor, EphB4; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases; Tunica Intima; Veins

Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft(-/-) mice deficient in intestinal folate transport compared with wild-type Pcft(+/+) animals.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Desmosine; Elastin; Hyperhomocysteinemia; Isodesmosine; Limit of Detection; Lung; Male; Mice; Mice, Inbred C57BL; Mice, SCID; Severe Combined Immunodeficiency; Swine; Time Factors

A decrease in vascular elasticity and an increase in pulse wave velocity in hyperhomocysteinemic (HHcy) cystathionine-beta-synthase heterozygote knockout (CBS(-/+)) mice has been observed. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-tissue inhibitor of metalloproteinase (TIMP) inhibitory tertiary complex. However, the contribution of the nitric oxide synthase (NOS) isoforms eNOS and iNOS in the activation of latent MMP is unclear. We hypothesize that the differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP, resulting in vascular remodeling in response to HHcy. The overall goal is to elucidate the contribution of the NOS isoforms, endothelial and inducible, in the collagen/elastin switch. Experiments were performed on six groups of animals [wild-type (WT), eNOS(-/-), and iNOS(-/-) with and without homocysteine (Hcy) treatment (0.67 g/l) for 8-12 wk]. In vivo echograph was performed to assess aortic timed flow velocity for indirect compliance measurement. Histological determination of collagen and elastin with trichrome and van Gieson stains, respectively, was performed. In situ measurement of superoxide generation using dihydroethidium was used. Differential expression of eNOS, iNOS, nitrotyrosine, MMP-2 and -9, and elastin were measured by quantitative PCR and Western blot analyses. The 2% gelatin zymography was used to assess MMP activity. The increase in O(2)(-) and robust activity of MMP-9 in eNOS(-/-), WT+Hcy, and eNOS(-/-)+Hcy was accompanied by the gross disorganization and thickening of the ECM along with extensive collagen deposition and elastin degradation (collagen/elastin switch) resulting in a decrease in aortic timed flow velocity. Results show that an increase in iNOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance.

Topics: Animals; Aorta; Blood Flow Velocity; Collagen; Compliance; Elastin; Extracellular Matrix; Hyperhomocysteinemia; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Tissue Inhibitor of Metalloproteinases; Tyrosine; Ultrasonography

Coronary artery remodeling implies structural changes in the vessel wall in response to various pathophysiologic conditions. However, the classification of remodeling is unclear. We hypothesized that the adaptive, positive-outward remodeling is a reactive and compensatory response to the stress. The maladaptive negative-inward constrictive remodeling is a passive atherosclerotic condition in which the vessel becomes stiffer.. Patients with atherosclerotic lesions underwent intravascular ultrasound (IVUS) scans. The size of the vessels distal to and proximal to plaques were analyzed by IVUS. Diabetes was created in mice by an intraperitoneal injection of alloxan (65 mg/kg). To reduce remodeling, mice received ciglitazone, an agonist of peroxisome proliferators activated receptor-gamma (PPARgamma) in drinking water. After 8 weeks, atherosclerotic vessels were analyzed for collagen and elastin.. IVUS data suggest an adaptive coronary arterial remodeling was a positive compensatory response to various pathologic stimuli; for example, with the deposition of atherosclerotic plaque, coronary arterial segments enlarged to maintain luminal area. This phenomenon was commonly observed during the initial phases of the development of atherosclerosis. However, negative coronary artery remodeling, or a decrease in vessel area with the formation of atherosclerotic plaque, was maladaptive and was associated with smoking, hypertension, hyperhomocysteinemia, diabetes mellitus, and also after percutaneous coronary interventions (restenosis). In diabetic mice, there was increased collagen and decreased elastin contents; however, treatment with ciglitazone ameliorated the decrease in elastin contents.. Global enlargement of the coronary vascular tree occurs during pressure and volume overload associated with ventricular hypertrophic states such as athletic conditioning, hypertensive heart disease, and dilated cardiomyopathy. On the other hand, maladaptive coronary arterial remodeling occurs in patients with severe deconditioning, diabetes mellitus, after coronary artery bypass surgery, and in some instances, postintervention.

Topics: Animals; Collagen; Coronary Artery Disease; Coronary Vessels; Diabetes Complications; Diabetes Mellitus, Experimental; Elastin; Humans; Hyperhomocysteinemia; Male; Mice; PPAR gamma; Thiazolidinediones; Ultrasonography

Elevated plasma homocysteine has been associated with cardiovascular disease, although a causal relationship is unclear. The purpose of this study was to evaluate whether mild hyperhomocysteinemia (H-Hcy) may increase vascular stiffness of small arteries.. Wild-type (+/+) and heterozygous (+/-) methylenetetrahydrofolate reductase (Mthfr) knockout mice, a new model of mild H-Hcy, were treated with vehicle or angiotensin (Ang) II infusion (400 ng/kg per min s.c.). Second-order mesenteric arteries were studied on pressurized myograph. They were exposed to intraluminal pressures ranging from 3 to 140 mmHg. Media thickness and lumen diameter were measured at each pressure level to determine wall mechanical properties. Collagen type I/III and elastin deposition in the vascular wall were evaluated by confocal immunofluorescence microscopy.. Media-to-lumen ratio was similar in Mthfr and Mthfr mice, and significantly increased by Ang II. The stress-strain relationship was shifted to the left in small mesenteric arteries from Mthfr compared to Mthfr mice, indicating that mild H-Hcy is associated with stiffer vessels. Ang II treatment in Mthfr mice enhanced the leftward shift in the stress-strain relationship and significantly increased the elastic modulus, suggesting the presence of stiffer wall components in small arteries in these animals. Increased collagen type I/III accumulation and decreased elastin content in the media of mesenteric arteries was noted in Mthfr compared to Mthfr mice. Ang II infusion augmented vascular collagen deposition in both groups, more substantially in Mthfr mice.. Mild hyperhomocysteinemia is associated with stiffer small arteries with increased collagen deposition in the media. These changes are accentuated by Ang II-induced blood pressure elevation.

Topics: Angiotensin II; Animals; Blood Pressure; Collagen; Disease Models, Animal; Elastin; Female; Homocysteine; Hyperhomocysteinemia; Male; Mesenteric Arteries; Methylenetetrahydrofolate Reductase (NADPH2); Mice; Mice, Inbred BALB C; Mice, Knockout; Vasoconstrictor Agents

Homocysteinemia in humans is associated with vascular complications that increase the risk for atherosclerosis and stroke. Animal studies have shown that the disease is multifactorial and includes lesions associated with the elastin component of the extracellular matrix. In the following experiments we have used the aortas from rapidly growing chicks to assess the cause of the elastin defects resulting from homocysteinemia. Day-old chicks were fed diets containing varying amounts of DL-methionine, DL-homocysteine, homocysteine thiolactone or DL-cysteine for periods up to 9 wk. Three weeks after feeding 2% DL-methionine the plasma methionine was elevated > 20-fold, whereas plasma homocysteine was more than 3-fold normal plasma values. The aortas showed severe histopathology, evidenced by the pronounced separation of elastic lamellae with marked smooth muscle proliferation and, in some instances, aneurysms. There was no evidence of decreased desmosine content or a significant reduction in lysyl oxidase in the aortas from the treated groups compared to those from controls. Increasing other dietary factors such as the vitamins required for methionine metabolism had no effect on the development of the vascular lesions. Twenty to 30% of the chicks fed the high methionine diets exhibited severe neurological problems, expressed as tonic contractions or seizures. Electron microscopy revealed disordered aortic elastic fibrils, associated with either an absence of or disrupted assembly of microfibrils. Immunohistochemical studies demonstrated a loss of fibrillin-2 immunoreactivity in the aortas of chicks fed 2% methionine. The studies suggest that elevated plasma methionine or its metabolites disrupt normal microfibril configuration, leading to the assembly of aberrant elastic fibers.

Topics: Animal Feed; Animals; Calcium-Binding Proteins; Chickens; Diet; Dietary Supplements; Elasticity; Elastin; Fibrillin-2; Fibrillins; Hyperhomocysteinemia; Microfilament Proteins

We examined effects of hyperhomocysteinemia on structure and mechanics of cerebral arterioles. We measured plasma total homocysteine (tHcy) and pressure, diameter, and cross-sectional area of the vessel wall in maximally dilated cerebral arterioles in heterozygous cystathionine beta-synthase-deficient (CBS(+/-)) mice and wild-type (CBS(+/+)) littermates that were provided with drinking water that was unsupplemented (control diet) or supplemented with 0.5% L-methionine (high-methionine diet). Plasma tHcy was 5.0+/-1.1 micro mol/L in CBS(+/+) mice and 8.3+/-0.9 micro mol/L in CBS(+/-) mice (P<0.05 versus CBS(+/+) mice) fed the control diet. Plasma tHcy was 17.2+/-4.6 micro mol/L in CBS(+/+) mice and 21.2+/-3.9 micro mol/L in CBS(+/-) mice (P<0.05) fed the high-methionine diet. Cross-sectional area of the vessel wall was significantly increased in CBS(+/-) (437+/-22 micro m(2)) mice fed control diet and CBS(+/+) (442+/-36 micro m(2)) and CBS(+/-) (471+/-46 micro m(2)) mice fed high-methionine diet relative to CBS(+/+) (324+/-18 micro m(2)) mice fed control diet (P<0.05). During maximal dilatation, the stress-strain curves in cerebral arterioles of CBS(+/-) mice on control diet and CBS(+/+) and CBS(+/-) mice on high-methionine diet were shifted to the right of the curve in cerebral arterioles of CBS(+/+) mice on control diet, an indication that distensibility of cerebral arterioles was increased in mice with elevated levels of plasma tHcy. Thus, hyperhomocysteinemia in mice was associated with hypertrophy and an increase in distensibility of cerebral arterioles. These findings suggest that hyperhomocysteinemia promotes cerebral vascular hypertrophy and altered cerebral vascular mechanics, both of which may contribute to the increased incidence of stroke associated with hyperhomocysteinemia.

Topics: Animals; Arterioles; Basement Membrane; Blood Pressure; Brain; Collagen; Cystathionine beta-Synthase; Diet; Disease Models, Animal; Elastin; Genotype; Heterozygote; Hyperhomocysteinemia; Hypertrophy; Methionine; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Vascular Patency

Hyperhomocysteinemia is a risk factor for arterial diseases, and the deterioration of the arterial elastic structures is one of the possible mechanisms underlying this epidemiological association. The aim of this paper is to quantitatively characterize such structural alterations and to explore their causes in a previous model of dietary induced mild hyperhomocysteinemia in minipigs. After four months, both a morphodensitometrical analysis of the elastic structure and a biochemical analysis of elastin and elastase activities were performed on the infrarenal abdominal aorta (IRAA) and the proximal left interventricular coronary artery (LIVCA) of control (C), hyperhomocysteinemic (H) and captopril-hydrochlorothiazide (Cp-Htz, 25 + 12.5 mg/d)-treated (H+/-Cp) minipigs (n = 8/group). Hyperhomocysteinemia was found to induce an increase in parietal elastolytic metalloproteinase activities. It resulted in opening and enlargement of fenestrae through the medial elastic laminae and in a decrease in medial elastin content (p < 10(-3)), expressed as well as volume density (%) as weight concentration (microg elastin/mg dry tissue). The thickness of the media and its basic lamellar organization was unchanged. The reduction in volume density was more dramatic in LIVCA (H: 4.7 +/- 0.9 vs C: 8.8 +/- 2.4), where it was evenly distributed within the media, than in IRAA (H: 6.7 +/- 1.1 vs C: 9.3 +/- 1.2), where the deep medial layers were less affected. Cp-Htz partly prevented the hyperhomocysteinemia-induced reduction of the medial elastin content in LIVCA (5.7 +/- 1.2) and IRAA (7.9 +/- 1.4). This effect, occurring in the subintimal layers of the media in both arteries but not in the deeper layers, resulted in a less beneficial effect in LIVCA than in IRAA. This result parallels the moderate beneficial therapeutic effect of ACE inhibitors against coronary atherosclerosis in humans. This paper reports for the first time a quantitative analysis of the arterial site-dependent deterioration of the elastic structure caused by mild hyperhomocysteinemia and the involvement of metalloproteinases in this process. These results confirm that the plaque-independent damage to elastic structure previously described in hyperhomocysteinemic-atherosclerotic minipigs was mainly due to homocysteine. This highlights that the metalloproteinase-related elastolysis and the subsequent structural deterioration is one of the major events underlying the epidemiological association between mild hyp

Topics: Animals; Antihypertensive Agents; Aorta, Abdominal; Captopril; Coronary Vessels; Disease Models, Animal; Elastic Tissue; Elasticity; Elastin; Hydrochlorothiazide; Hyperhomocysteinemia; Swine; Swine, Miniature