IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility.

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

Nitric oxide alters hyaluronan deposition by airway smooth muscle cells.

Asthma is a chronic inflammatory disease that is known to cause changes in the extracellular matrix, including changes in hyaluronan (HA) deposition. However, little is known about the factors that modulate its deposition or the potential consequences. Asthmatics with high levels of exhaled nitric oxide (NO) are characterized by greater airway reactivity and greater evidence of airway inflammation. Based on these data and our previous work we hypothesized that excessive NO promotes the pathologic production of HA by airway smooth muscle cells (SMCs). Exposure of cultured SMCs to various NO donors results in the accumulation of HA in the form of unique, cable-like structures. HA accumulates rapidly after exposure to NO and can be seen as early as one hour after NO treatment. The cable-like HA in NO-treated SMC cultures supports the binding of leukocytes. In addition, NO produced by murine macrophages (RAW cells) and airway epithelial cells also induces SMCs to produce HA cables when grown in co-culture. The modulation of HA by NO appears to be independent of soluble guanylate cyclase. Taken together, NO-induced production of leukocyte-binding HA by SMCs provides a new potential mechanism for the non-resolving airway inflammation in asthma and suggests a key role of non-immune cells in driving the chronic inflammation of the submucosa. Modulation of NO, HA and the consequent immune cell interactions may serve as potential therapeutic targets in asthma.

TNF-stimulated gene 6 promotes formation of hyaluronan-inter-α-inhibitor heavy chain complexes necessary for ozone-induced airway hyperresponsiveness.

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA-HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6-/- and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6-/- or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil-containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6-/- tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6-mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA-HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.

Hyaluronan and Its Heavy Chain Modification in Asthma Severity and Experimental Asthma Exacerbation.

Hyaluronan (HA) is a large (>1500 kDa) polysaccharide of the extracellular matrix that has been linked to severity and inflammation in asthma. During inflammation, HA becomes covalently modified with heavy chains (HC-HA) from inter-α-inhibitor (IαI), which functions to increase its avidity for leukocytes. Our murine model of allergic pulmonary inflammation suggested that HC-HA may contribute to inflammation, adversely effecting lower airway remodeling and asthma severity. Our objective was to characterize the levels of HA and HC-HA in asthmatic subjects and to correlate these levels with asthma severity. We determined the levels and distribution of HA and HC-HA (i) from asthmatic and control lung tissue, (ii) in bronchoalveolar lavage fluid obtained from non-severe and severe asthmatics and controls, and (iii) in serum and urine from atopic asthmatics after an experimental asthma exacerbation. HC-HA distribution was observed (i) in the thickened basement membrane of asthmatic lower airways, (ii) around smooth muscle cells of the asthmatic submucosa, and (iii) around reserve cells of the asthmatic epithelium. Patients with severe asthma had increased HA levels in bronchoalveolar lavage fluid that correlated with pulmonary function and nitric oxide levels, whereas HC-HA was only observed in a patient with non-severe asthma. After an experimental asthma exacerbation, serum HA was increased within 4 h after challenge and remained elevated through 5 days after challenge. Urine HA and HC-HA were not significantly different. These data implicate HA and HC-HA in the pathogenesis of asthma severity that may occur in part due to repetitive asthma exacerbations over the course of the disease.

Novel insights in mammalian catalase heme maturation: effect of NO and thioredoxin-1.

Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorporation was associated with defective oligomerization of catalase, such that inactive catalase monomers and dimers accumulated in place of the mature tetrameric enzyme. We also found that GAPDH plays a key role in mediating these NO effects on the structure and activity of catalase. Moreover, the NO sensitivity of catalase maturation could be altered up or down by manipulating the cellular expression level or activity of thioredoxin-1, a known protein-SNO denitrosylase enzyme. In a mouse model of allergic inflammatory asthma, we found that lungs from allergen-challenged mice contained a greater percentage of dimeric catalase relative to tetrameric catalase in the unchallenged control, suggesting that the mechanisms described here are in play in the allergic asthma model. Together, our study shows how maturation of active catalase can be influenced by NO, S-nitrosylated GAPDH, and thioredoxin-1, and how maturation may become compromised in inflammatory conditions such as asthma.

ADAMTSL4, a secreted glycoprotein widely distributed in the eye, binds fibrillin-1 microfibrils and accelerates microfibril biogenesis.

PURPOSE: ADAMTSL4 mutations cause autosomal recessive isolated ectopia lentis (IEL) and ectopia lentis et pupillae. Dominant FBN1 mutations cause IEL or syndromic ectopia lentis (Marfan syndrome and Weill-Marchesani syndrome). The authors sought to characterize recombinant ADAMTSL4 and the ocular distribution of ADAMTSL4 and to investigate whether ADAMTSL4 influences the biogenesis of fibrillin-1 microfibrils, which compose the zonule. METHODS: ADAMTSL4 was expressed by the transfection of HEK293F cells. Protein extracts and paraffin sections from human eyes were analyzed by Western blot analysis and by immunoperoxidase staining, respectively. Immunofluorescence was used to evaluate fibrillin-1 deposition in the ECM of fetal bovine nuchal ligament cells after culture in ADAMTSL4-conditioned medium or control medium. Confocal microscopy was performed to investigate ADAMTSL4 and fibrillin-1 colocalization in these cultures. RESULTS: Western blot analysis identified ADAMTSL4 as a glycoprotein in HEK293F cells and as a major band of 150 kDa in ocular tissues including ciliary body, sclera, cornea, and retina. Immunoperoxidase staining showed a broad ocular distribution of ADAMTSL4, associated with both cells and fibrillar ECM. When cultured in ADAMTSL4-containing medium, fetal bovine nuchal ligament cells showed accelerated fibrillin-1 deposition in ECM. ADAMTSL4 colocalized with fibrillin-1 microfibrils in the ECM of these cells. CONCLUSIONS: ADAMTSL4 is a secreted glycoprotein that is widely distributed in the human eye. Enhanced fibrillin-1 deposition in the presence of ADAMTSL4 and colocalization of ADAMTSL4 with fibrillin-1 in the ECM of cultured fibroblasts suggest a potential role for ADAMTSL4 in the formation or maintenance of the zonule.

ADAMTS10 protein interacts with fibrillin-1 and promotes its deposition in extracellular matrix of cultured fibroblasts.

Autosomal recessive and autosomal dominant forms of Weill-Marchesani syndrome, an inherited connective tissue disorder, are caused by mutations in ADAMTS10 (encoding a secreted metalloprotease) and FBN1 (encoding fibrillin-1, which forms tissue microfibrils), respectively, yet they are clinically indistinguishable. This genetic connection prompted investigation of a potential functional relationship between ADAMTS10 and fibrillin-1. Specifically, fibrillin-1 was investigated as a potential ADAMTS10 binding partner and substrate, and the role of ADAMTS10 in influencing microfibril biogenesis was addressed. Using ligand affinity blotting and surface plasmon resonance, recombinant ADAMTS10 was found to bind to fibrillin-1 with a high degree of specificity and with high affinity. Two sites of ADAMTS10 binding to fibrillin-1 were identified, one toward the N terminus and another in the C-terminal half of fibrillin-1. Confocal microscopy and immunoelectron microscopy localized ADAMTS10 to fibrillin-1-containing microfibrils in human tissues. Furin-activated ADAMTS10 could cleave fibrillin-1, but innate resistance of ADAMTS10 zymogen to propeptide excision by furin was observed, suggesting that, unless activated, ADAMTS10 is an inefficient fibrillinase. To investigate the role of ADAMTS10 in microfibril biogenesis, fetal bovine nuchal ligament cells were cultured in the presence or absence of ADAMTS10. Exogenously added ADAMTS10 led to accelerated fibrillin-1 microfibril biogenesis. Conversely, fibroblasts obtained from a Weill-Marchesani syndrome patient with ADAMTS10 mutations deposited fibrillin-1 microfibrils sparsely compared with unaffected control cells. Taken together, these findings suggest that ADAMTS10 participates in microfibril biogenesis rather than in fibrillin-1 turnover.

Inhibition of the phosphatidylinositol-3-kinase pathway abrogates polyinosinic:polycytidylic acid-stimulated hyaluronan-mediated human mucosal smooth muscle cell binding of U937 monocytic cells.

The origin of inflammatory bowel disease (IBD) is unknown and likely to be multifactorial. Our laboratory has established that in human mucosal smooth muscle cells (M-SMCs), cellular stress induced by virus or the viral mimic double-stranded RNA (polyinosinic:polycytidylic acid [poly I:C]) increases cell surface hyaluronan (HA) deposition and the formation of long cable-like structures of HA that are important for leukocyte attachment. Since leukocyte accumulation and hyperplasia of the M-SMCs are characteristic pathological changes observed in IBD patients, and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways play established roles in cell survival, we investigated whether this pathway is involved in this unique HA-mediated leukocyte attachment. Poly I:C-stimulated M-SMCs bind significantly more monocytic cells than untreated cells and this response was inhibited in a dose-dependent manner by treatment with the PI3K inhibitor, LY294002. Since Akt is a critical downstream regulator of PI3K, we investigated the phosphorylation status of Akt in M-SMCs after treatment with poly I:C for 1 h and found that Akt was phosphorylated, but the phosphorylated Akt band was undetectable in LY294002 plus poly I:C-treated cultures. Confocal microscopy of M-SMCs stained for HA revealed that HA cable formation after poly I:C treatment was abrogated by LY294002. These results demonstrate that poly I:C-stimulated M-SMCs phosphorylate Akt, produce HA cables, and promote HA-mediated leukocyte adhesion through a PI3K/Akt-dependent manner.

Primary murine airway smooth muscle cells exposed to poly(I,C) or tunicamycin synthesize a leukocyte-adhesive hyaluronan matrix.

Asthmatic attacks often follow viral infections with subsequent airway smooth muscle cell proliferation and the formation of an abnormal hyaluronan extracellular matrix with infiltrated leukocytes. In this study, we show that murine airway smooth muscle cells (MASM) treated with polyinosinic acid-polycytidylic acid (poly(I,C)), a double-stranded RNA that simulates a viral infection, synthesize an abnormal hyaluronan matrix that binds leukocytes (U937 cells). Synthesis of this matrix is initiated rapidly and accumulates linearly for approximately 10 h, reaching a plateau level approximately 7-fold higher than control cultures. MASM cells treated with tunicamycin, to induce endoplasmic reticulum stress, also rapidly initiate synthesis of the abnormal hyaluronan matrix with linear accumulation for approximately 10 h, but only reach a plateau level approximately 2-fold higher than control cultures. In contrast to poly(I,C), the response to tunicamycin depends on cell density, with pre-confluent cells producing more abnormal matrix per cell. Furthermore, U937 cell adhesion per hyaluronan content is higher in the sparse matrix produced in response to tunicamycin, suggesting that the structure in the poly(I,C)-induced matrix masks potential binding sites. When MASM cells were exposed to tunicamycin and poly(I,C) at the same time, U937 cell adhesion was partially additive, implying that these two toxins stimulate hyaluronan synthesis through two different pathways. We also characterized the size of hyaluronan produced by MASM cells, in response to poly(I,C) and tunicamycin, and we found that it ranges from 1500 to 4000 kDa, the majority of which was approximately 4000 kDa and not different in size than hyaluronan made by untreated cells.

Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice.

A causal relationship between diet-induced hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE(-/-)) mice. However, it is not known whether the proatherogenic effect of HHcy in apoE(-/-) mice is independent of hyperlipidemia and/or deficiency of apoE. In this study, a comprehensive dietary approach using C57BL/6J mice was used to investigate whether HHcy is an independent risk factor for accelerated atherosclerosis or dependent on additional dietary factors that increase plasma lipids and/or inflammation. C57BL/6J mice at 4 wk of age were divided into 6 dietary groups: chow diet (C), chow diet + methionine (C+M), western-type diet (W), western-type diet + methionine (W+M), atherogenic diet (A), or atherogenic diet + methionine (A+M). After 2, 10, 20, or 40 wk on the diets, mice were sacrificed, and the levels of total plasma homocysteine, cysteine, and glutathione, as well as total plasma cholesterol and triglycerides were analyzed. Aortic root sections were examined for atherosclerotic lesions. HHcy was induced in all groups supplemented with methionine, compared to diet-matched control groups. Plasma total cholesterol was significantly increased in mice fed the W or A diet. However, the W diet increased LDL/IDL and HDL levels, while the A diet significantly elevated plasma VLDL and LDL/IDL levels without increasing HDL. No differences in plasma total cholesterol levels or lipid profiles were observed between methionine-supplemented groups and the diet-matched control groups. Early atherosclerotic lesions containing macrophage foam cells were only observed in mice fed the A or A + M diet. Furthermore, lesion size was significantly larger in the A + M group compared to the A group at 10 and 20 wk; however, mature lesions were never observed even after 40 wk on these diets. The presence of lymphocytes, increased hyaluronan staining, and the expression of endoplasmic reticulum (ER) stress markers were also increased in atherosclerotic lesions from the A + M group. Taken together, these results suggest that HHcy does not independently cause atherosclerosis in C57BL/6J mice even in the presence of increased total plasma lipids induced by the W diet. However, HHcy can accelerate atherosclerotic lesion development under dietary conditions that increase plasma VLDL levels and/or inflammation.

Intracellular hyaluronan: a new frontier for inflammation?

A variety of obstacles have hindered the ultrastructural localization of hyaluronan (HA). These include a lack of adequate fixation techniques to prevent the loss of HA, the lack of highly sensitive and specific probes, and a lack of accessibility due to the masking of HA by HA-binding macromolecules such as proteoglycans and glycoproteins. Despite these problems, a number of studies, both biochemical and histochemical, have been published indicating that HA is not restricted to the extracellular milieu, but is also present intracellularly. This review focuses on the possible functions of intracellular HA, its potential relationships to extracellular HA structures, and implications for inflammatory processes.

Renal handling of homocysteine during normal pregnancy and preeclampsia.

OBJECTIVE: Maternal plasma homocysteine decreases in normal pregnancy and is significantly increased in preeclampsia. The goal of this study was to investigate the role of the maternal kidney in the changes of plasma homocysteine during normal pregnancy and preeclampsia. METHODS: Plasma and 24-hour urine samples were collected in the same women before, during (first, second, and third trimesters), and after normal pregnancy; and in a separate cross-sectional study of normal pregnant, preeclamptic and nonpregnant women and homocysteine concentrations were measured. RESULTS: Longitudinally, maternal plasma homocysteine decreased significantly by the first trimester compared with prepregnancy and postpartum levels (5.6 +/- 1.8 versus 6.8 +/- 0.5 and 7.4 +/- 0.4 microM, respectively, P<.05 by analysis of variance) and paralleled a significant increase in the renal clearance of homocysteine (2.9 +/- 0.4 versus 1.8 +/- 0.2 and 1.6 +/- 0.2 L/24 hours, respectively, P<.001). In addition, plasma homocysteine was significantly elevated in preeclampsia compared with normal pregnancy (4.4 +/- 0.6 versus 3.2 +/- 0.2 microM, P<.04); however, renal clearance was not different (1.2 +/- 0.1 versus 1.0 +/- 0.1 L/24 hours, P=.55). CONCLUSION: Increases in renal clearance contribute to the decrease in plasma homocysteine during normal pregnancy. However, changes in renal handling do not appear to contribute to the increase in plasma homocysteine in preeclampsia.

Endoplasmic reticulum stress induces hyaluronan deposition and leukocyte adhesion.

There is mounting evidence that perturbations in endoplasmic reticulum (ER) function play a key role in the pathogenesis of a broad range of diseases. We have examined the ability of ER stress to modulate leukocyte binding to colonic and aortic smooth muscle cells. In vitro, control smooth muscle cells bind few leukocytes, but treatment with compounds that induce ER stress, including tunicamycin, A23187, and thapsigargin, promotes leukocyte binding. Likewise, dextran sulfate, another agent capable of inducing ER stress and promoting inflammation in vivo, strongly induces leukocyte adhesion. The bound leukocytes are released by hyaluronidase treatment, indicating a critical role for hyaluronan-containing structures in mediating leukocyte binding. Affinity histochemistry demonstrated that hyaluronan accumulates and is present in cable-like structures in the treated, but not the untreated, cultures and that these structures serve as attachment sites for leukocytes. Hyaluronan-rich regions of both murine and human inflamed colon contain numerous cells that stain intensely for ER-resident chaperones containing the KDEL sequence, demonstrating a relationship between ER stress and hyaluronan deposition in vivo. These results indicate that ER stress may contribute to chronic inflammation by forming a hyaluronan-rich extracellular matrix that is conducive to leukocyte binding.

Changes in markers of vascular injury in response to transient hyperhomocysteinemia.

The purpose of this study was to test whether transient increases in homocysteine would promote changes in markers of endothelial injury, cellular fibronectin (cFN), and soluble vascular cell adhesion molecule 1 (sVCAM-1). Homocysteine, cFN, and sVCAM-1 concentrations increased significantly in response to a methionine load by 6 hours in human subjects. However, no correlation was observed between homocysteine and cFN or sVCAM-1. To directly test whether homocysteine can injure endothelial cells, human umbilical vein endothelial cells (HUVEC) were incubated with increasing concentrations of homocysteine, plasma, or serum from hyperhomocysteinemic mice or from the methionine-loaded test subjects. cFN release was increased from endothelial cells cultured with plasma (but not serum) of hyperhomocysteinemic transgenic mice or from methionine-loaded human subjects. These data suggest that very high homocysteine concentrations can promote endothelial injury; however, this effect is likely mediated by secondary effects that include a factor(s) present in plasma that affects endothelial cells.

Plasma homocysteine and malondialdehyde are correlated in an age- and gender-specific manner.

Homocysteine is an independent risk factor for peripheral vascular and coronary artery disease. The exact mechanism by which homocysteine promotes vascular dysfunction is unclear, but it is speculated to involve oxidative stress. Several studies have investigated the role of homocysteine in promoting oxidative stress and have obtained conflicting results. The age and gender of the subject populations in these studies may have influenced the outcome. Therefore, we investigated whether plasma homocysteine concentrations were correlated with plasma malondialdehyde (MDA, a marker of oxidative stress), and if the subject's age and gender affected this correlation. Plasma homocysteine and MDA were measured in 35 premenopausal women, 14 young men, 38 postmenopausal women, and 18 older men. Homocysteine was significantly higher in men than women (P <.01) and in older subjects versus younger. However, MDA was significantly greater only in the young men (P <.01). Furthermore, there was a significant correlation between homocysteine and MDA only in these young men (R(2) = 0.50, P <.01). Lastly, subjects undergoing a methionine load did not exhibit increased MDA despite significant increases in homocysteine. Since oxidative stress correlates with basal homocysteine only in young men and does not increase with acutely increased homocysteine, it is unlikely to be the result of a direct effect of homocysteine.

Homocysteine binds to human plasma fibronectin and inhibits its interaction with fibrin.

OBJECTIVE: More than 70% of circulating homocysteine is disulfide-bonded to protein, but little is known about the specific proteins that bind homocysteine and their function as a consequence of homocysteine binding. METHODS AND RESULTS: When human plasma was incubated with [(35)S]L-homocysteine, most of the homocysteine bound to albumin. However, additional homocysteine-binding proteins were detected, and 1 of them comigrated with fibronectin. Treatment with 2-mercaptoethanol removed the bound homocysteine, demonstrating the involvement of disulfide bonding. In contrast, [35S]L-cysteine did not bind to fibronectin. Purified fibronectin bound approximately 5 homocysteine molecules per fibronectin dimer. SDS-PAGE of a limited trypsin digestion of homocysteinylated fibronectin showed that several tryptic fragments contained [35S]homocysteine. Sequence analysis demonstrated that the fragments containing bound homocysteine had localized mainly to the C-terminal region, within and adjacent to the fibrin-binding domain. Homocysteinylation of fibronectin significantly inhibited its capacity to bind fibrin by 62% (P<0.005). In contrast, neither the binding of fibronectin to gelatin nor its capacity to serve as an attachment factor for aortic smooth muscle cells was affected. CONCLUSIONS: These results suggest that homocysteine may alter normal thrombosis and delay or interfere with wound healing by impairing the interaction of fibronectin with fibrin.

Upregulation of smooth muscle cell collagen production by homocysteine-insight into the pathogenesis of homocystinuria.

Patients with untreated homocystinuria have widespread premature atherosclerosis with intimal thickening and collagen-rich, fibrous plaques. We previously demonstrated that homocysteine (Hcy) upregulates collagen synthesis and accumulation by arterial smooth muscle cells (SMCs) [A. Majors, L.A. Ehrhart, E.H. Pezacka, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 2074-2081] but the underlying mechanisms are not known. Since many of the effects of Hcy on intact vessels and vascular cells are thought to involve reactive oxygen species generated from Hcy oxidation, we investigated the role of reactive oxygen species in the upregulation of collagen production by Hcy. Treatment of SMCs with 300 microM l-Hcy increased collagen accumulation 2-3-fold. When added to culture medium containing serum, the exogenous Hcy was rapidly oxidized with a half-life of approximately 1 h but only very low amounts of H(2)O(2) (up to 2 microM) were detected. Three lines of evidence demonstrate that the increased accumulation of collagen was not mediated by reactive oxygen species generated from Hcy oxidation: (1) catalase in the medium did not block the accumulation of collagen in Hcy-treated cultures; (2) the addition of xanthine/xanthine oxidase, a system that generates superoxide and H(2)O(2), did not increase collagen accumulation; and (3) the direct addition of H(2)O(2) did not substantially enhance collagen accumulation. In contrast, heparin, a potent modulator of SMC function, significantly blocked the accumulation of collagen in Hcy-treated cultures. Together, these results demonstrate that the increase in collagen accumulation in Hcy-treated cultures involves alternate mechanisms not involving H(2)O(2).