Search

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. [ABSTRACT FROM AUTHOR]

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 increasedHAlevels 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 implicateHAandHC-HAin the pathogenesis of asthma severity that may occur in part due to repetitive asthma exacerbations over the course of the disease. [ABSTRACT FROM AUTHOR]

Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat. Am J Physiol Regulatory Integrative Comp Physiol 281: R1710-R1717, 2001.--To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 [+ or -] 5%; 14 days HU 53 [+ or -] 4%; 28 days HU 53 [+ or -] 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 [+ or -] 5%; 14 days HU 47 [+ or -] 4%; 28 days HU 48 [+ or -] 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 [+ or -] 2%; 14 days HU 86 [+ or -] 1%; 28 days HU 83 [+ or -] 1 %), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 [+ or -] 2%; 28 days HU 14 [+ or -] 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation. hindlimb unweighting; extracellular matrix; fiber type; microgravity; tail suspension

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.--Zhou, J., Werstuck, G. H., Lhoták, Š., Shi, Y. Y., Tedesco, V., Trigatti, B., Dickhout, J., Majors, A. K., DiBello, P. M., Jacobsen, D. W., Austin, R. C. Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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 resuits indicate that ER stress may contribute to chronic inflammation by forming a hyaluronan-rich extracellular matrix that is conducive to leukocyte binding. [ABSTRACT FROM AUTHOR]

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 μM 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 H2O2 (up to 2 μM) 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 H2O2, did not increase collagen accumulation; and (3) the direct addition of H2O2 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 H2O2. [Copyright &y& Elsevier]

Autosomal recessive and autosomal dominant forms of Weill- Marchesani syndrome, an inherited connective tissue disorder, are caused by mutations in ADAMTS10 (encoding a secreted met- alloprotease) 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. [ABSTRACT FROM AUTHOR]

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 acidpolycytidylic 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 ∼10 h, reaching a plateau level ∼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 ∼10 h, but only reach a plateau level ∼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 ∼4000 kDa and not different in size than hyaluronan made by untreated cells. [ABSTRACT FROM AUTHOR]

Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Büdy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.

An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds. It is known that albumin combines with cysteine in circulation to form albumin-Cys(34)-S-S-Cys. Studies are now presented to show that the formation of albumin-bound homocysteine proceeds through the generation of an albumin thiolate anion. Incubation of human plasma with l-(35)S-homocysteine results in the association of >90% of the protein-bound (35)S-homocysteine with albumin as shown by nonreduced SDS-polyacrylamide gel electrophoresis. Treatment of the complex with beta-mercaptoethanol results in near quantitative release of the bound l-(35)S-homocysteine, demonstrating that the binding of homocysteine to albumin is through a disulfide bond. Furthermore, using an in vitro model system to study the mechanisms of this disulfide bond formation, we show that homocysteine binds to albumin in two steps. In the first step homocysteine rapidly displaces cysteine from albumin-Cys(34)-S-S-Cys, forming albumin-Cys(34) thiolate anion and homocysteine-cysteine mixed disulfide. In the second step, albumin thiolate anion attacks homocysteine-cysteine mixed disulfide to yield primarily albumin-Cys(34)-S-S-Hcy and to a much lesser extent albumin-Cys(34)-S-S-Cys. The results clearly suggest that when reduced homocysteine enters circulation, it attacks albumin-Cys(34)-S-S-Cys to form albumin-Cys(34) thiolate anion, which in turn, reacts with homocysteine-cysteine mixed disulfide or homocystine to form albumin-bound homocysteine.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Greater than 70% of homocysteine in circulation is protein-bound. An in vitro model system using human plasma has been developed to study mechanisms of protein-bound homocysteine formation and establish the equilibrium binding capacities of plasma for homocysteine. Addition of homocysteine to plasma caused an initial rapid displacement of cysteine and a subsequent increase in protein-bound homocysteine. This rapid reaction was followed by a slower oxygen-dependent reaction forming additional protein-bound homocysteine. To determine the equilibrium binding capacity of plasma proteins for homocysteine, plasma was treated with 0.5-10 mM dl-homocysteine for 4 h at 37 degrees C under aerobic conditions. Under these conditions the equilibrium binding capacity was 4.88 +/- 0.51 and 4.74 +/- 0.68 micromol/g protein for male (n = 10) and female (n = 10) donors, respectively. The mechanism of protein-bound homocysteine formation involves both thiol-disulfide exchange and thiol oxidation reactions. We conclude that plasma proteins have a high capacity for binding homocysteine in vitro., (Copyright 2000 Academic Press.)

Cystathionine beta-synthase (CBS) deficiency is an inborn error of amino acid metabolism that has pleiotropic manifestations and is commonly called "homocystinuria." The features include skeletal, ocular, and vascular defects, some of which are reminiscent of those found in Marfan syndrome (MFS). Because of the spectrum of clinical effects, the pathogenesis of homocystinuria has long been thought to involve the extracellular matrix (ECM), and the condition has been classified as a heritable disorder of connective tissue. Because of the superficial similarities with MFS, we and others (Pyeritz, in McKusicks Heritable Disorders of Connective Tissue, St. Louis, Mosby-Year Book Inc., 5th ed., pp 137-178, 1993; Pyeritz, in Principles and Practice of Medical Genetics, New York, Churchill Livingstone, 3rd ed., pp 1027-1066, 1997; Mudd, Levy, and Skovby, in The Metabolic and Molecular Bases of Inherited Disease, New York, McGraw-Hill Publishing Co., 7th ed., pp 1279-1327, 1995) have speculated how CBS deficiency might affect fibrillin-1, the protein altered in MFS. For example, the altered plasma concentrations of homocysteine and/or cysteine in patients with CBS deficiency may hinder fibrillin-1 synthesis, deposition, or both. When arterial smooth muscle cells were cultured under conditions of cysteine deficiency, fibrillin-1 deposition into the ECM was greatly diminished as revealed by immunocytochemistry. Excessive homocysteine, in contrast, had little, if any, effect on fibrillin-1 deposition. When cysteine concentrations were returned to normal, the smooth muscle cells began to accumulate a matrix rich in fibrillin-1. Type I collagen, the major matrix component synthesized by these smooth muscle cells, was not reduced by low cysteine concentrations nor high homocysteine concentrations. These results demonstrate that a deficiency of cysteine and subsequent inhibition of fibrillin-1 accumulation in CBS deficient patients may be at least partly responsible for their phenotype, and suggest that maintenance of normal plasma cyst(e)ine levels may be an important therapeutic goal., (Copyright 2000 Academic Press.)

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.