Thrombin-thrombomodulin activation of protein C facilitates the activation of progelatinase A.

The activation of the matrix metalloproteinase progelatinase A (MMP-2) has been of keen interest because an increase in MMP-2 activity has been implicated in disease states such as cancer and atherosclerosis. Activation of MMP-2 occurs on the surface of specific cell types in two steps. In the first step, primary cleavage of MMP-2 by a membrane-type matrix metalloproteinase generates an intermediate. A secondary cleavage and activation of the intermediate is thought to occur autocatalytically. Previous studies have shown that thrombin can also activate progelatinase A in the presence of endothelial cells. We show that this cell-dependent mechanism of MMP-2 activation also occurs with THP-1 cells and involves binding of thrombin to thrombomodulin present on the cell surface and generation of the anti-coagulant protein, activated protein C. We demonstrate that activated protein C is directly responsible for activation and cleavage of the gelatinase A intermediate. This work contributes new mechanistic insights into the activation of MMP-2 and provides a novel link between matrix metalloproteinase activation and anti-coagulation.

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture.

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.

Transforming growth factor-beta is involved in the pathogenesis of dialysis-related amyloidosis.

BACKGROUND: Advanced glycation end product-modified beta2-microglobulin (AGE-beta2m) is an important component of dialysis-related amyloidosis (DRA). Its presence induces monocyte chemotaxis and the release of the proinflammatory cytokines through macrophage activation. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that also has chemotactic activity for monocytes at very low (0.1 to 10 pg/mL) concentrations and inhibits proinflammatory cytokine production of macrophages. In this study, we investigated the role of TGF-beta in the pathogenesis of DRA. METHODS: We performed an immunohistochemical study of DRA tissues (8 cases) to confirm the existence of TGF-betas and their receptors; we also performed a chemotaxis assay of human monocytes as well as enzyme-linked immunosorbent assay (ELISA) of TGF-beta1, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-1 receptor antagonist (IL-1Ra) in the supernatant of human monocyte-derived macrophage cell culture under varying conditions of incubation with TGF-beta1, AGE-beta2m, and TGF-beta1 antibody additions. RESULTS: There was positive staining for TGF-betas (types 1, 2, and 3) and their receptors (types I, II, and III) in infiltrated macrophages (CD68+), synovial lining cell, as well as vascular walls around amyloid deposition. AGE-beta2m also induced TGF-beta1 production by macrophages in a dose-dependent manner (410 +/- 80 pg/mL at 12.5 microg/mL, 621 +/- 62 pg/mL at 25 microg/mL, and 776 +/- 62 pg/mL at 50 microg/mL of AGE-beta2m). AGE-beta2m induced significant TNF-alpha and IL-1Ra production by macrophage. The addition of exogenous TGF-beta1 (0.1 to 10 ng/mL) decreased AGE-beta2m-induced TNF-alpha production and increased IL-1Ra production in a dose-dependent fashion. IL-1beta production was not effected by any experimental conditions. In chemotaxis assay, anti-TGF-beta1 antibody (0.1 to 10 microg/mL) attenuated AGE-beta2m-induced monocyte chemotaxis. CONCLUSIONS: These results provide the first evidence to our knowledge for the presence of TGF-beta in DRA tissue, as well as the stimulatory action of AGE-beta2m on tissue macrophages. In turn, TGF-beta suppresses the proinflammatory activation of macrophages, suggesting a dual role for TGF-beta in the inflammatory process of DRA. These observations may provide a pathophysiologic link between TGF-beta and DRA.

Interactions of monocytic cells with human endothelial cells stimulate monocytic metalloproteinase production.

Monocyte-endothelial cell interactions play an important role in the early stages of atherosclerosis, and it is hypothesized that regulation of metalloproteinase production by these interactions contributes to this pathological process. The effects of monocytic cell-endothelial cell interactions on monocytic metalloproteinase production were investigated using an in vitro system, focusing on the role of endothelial cell secretions and physical contact as effectors in the regulation of monocytic metalloproteinase expression. Human umbilical vein endothelial cells (HUVECs) and the human monocytic cell line THP-1 were used, and changes in the levels of THP-1 metalloproteinase secretion and mRNA were measured. When THP-1 cells were incubated for 18 hours with HUVEC conditioned medium (CM), a four- to eightfold induction of the metalloproteinase MMP-9 was observed at both the mRNA and protein levels; however, levels of another metalloproteinase, MMP-2, were unaffected. The induction of MMP-9 by HUVEC CM was confirmed using freshly isolated human monocytes. A sevenfold increase in MMP-9 levels was observed with apically collected HUVEC CM but not with basally collected CM. THP-1 cells incubated with paraformaldehyde-fixed HUVECs and isolated HUVEC plasma membranes showed an eightfold increase in MMP-9 levels, and measurements of MMP-9 activity found in THP-1 conditioned medium due to either HUVEC contact or HUVEC CM showed a threefold increase. The molecular weight of the endothelial secreted effector molecule(s) was determined to be 30 +/- 6 kd. The data show that endothelial cells through the release of soluble factors and through direct contact with monocytic cells regulate monocytic metalloproteinase production, which has implications for the atherogenic process.

Vascular endothelial growth factor is expressed in ovine pulmonary vascular smooth muscle cells in vitro and regulated by hypoxia and dexamethasone.

Chronic lung disease in neonates results from both lung injury and inadequate repair processes. Little is known about the growth factors involved in lung injury and repair, but vascular endothelial growth factor (VEGF) has recently been reported in several animal models of lung injury. VEGF is an endothelial cell-specific mitogen, which is also known as vascular permeability factor because of its ability to induce vascular leak in some tissues. Chronic lung disease is complicated by increased vascular permeability, which can be improved by avoidance of hypoxia and in some cases by dexamethasone therapy. In many cells, hypoxia stimulates VEGF expression. Also, in some cases, dexamethasone blocks VEGF expression. This study examined the role of hypoxia and dexamethasone in regulating the expression of VEGF in pulmonary artery smooth muscle cells. An ovine VEGF cDNA fragment (453 bp) was cloned and found to be highly homologous to known human sequences for VEGF165. Sheep pulmonary artery smooth muscle cells were cultured and exposed to room air, hypoxia, and dexamethasone, alone or in combination for 6 h. At baseline these cells expressed VEGF mRNA at approximately 3.9 kb. The half-life of VEGF mRNA in the smooth muscle cells was 171 min, more than 3-fold longer than previous reports for epithelial cells. Exposure to hypoxia caused a 3-fold increase in mRNA abundance, primarily through transcriptional up-regulation. Dexamethasone blocked the hypoxia-induced increase in VEGF mRNA. The results demonstrate that hypoxia and dexamethasone are regulators of VEGF expression in ovine pulmonary artery smooth muscle cells. It is not known whether VEGF derived from these cells is involved in lung injury and/or normal homeostatsis.

TGF-beta and PDGF act synergistically in affecting the growth of human osteoblast-enriched cultures.

Transforming growth factor beta (TGF-beta) is a polypeptide found in high concentrations in bone and is produced by and acts on primary adult human derived osteoblast-enriched cultures (PHO cells). Receptors for TGF-beta are present on PHO cells and TGF-beta is mitogenic for these cells. Results of these studies in conjunction with those of others suggest that TGF-beta may have an important therapeutic role in orthopaedic surgery; however, with respect to its mitogenic actions, further studies were needed to establish whether TGF-beta was acting directly to stimulate the growth of PHO cells. TGF-beta has been found in other systems to act as an indirect mitogen, stimulating growth via secretion of another growth factor, platelet-derived growth factor (PDGF). In an effort to determine whether the TGF-beta growth stimulation was mediated directly or indirectly, we have examined the growth stimulation of PHO cells by PDGF alone and in combination with TGF-beta. These studies revealed that TGF-beta in combination with either PDGF-AA or BB led to stimulation greater than that observed with either growth factor alone. TGF-beta in combination with PDGF-BB led to a synergistic stimulatory response while that observed with the AA isoform was more nearly additive. Further studies demonstrated that TGF-beta was capable of up-regulating the protein levels of the PDGF alpha (alpha) receptor within thirty minutes of TGF-beta pretreatment. Thus, TGF-beta appears to have both direct and indirect mechanisms of action as a mitogen in the PHO system. Finally, we showed that both the positive and negative alkaline phosphatase staining PHO cells were responsive to the mitogenic effects of both growth factor singly and in combination.

Induction of manganese superoxide dismutase by glucocorticoids in glomerular cells.

Our previous in vivo study demonstrated that methylprednisolone (MP) activates glomerular antioxidant enzymes and attenuates glomerular oxidant injuries, including those in experimental nephrosis. The present study investigates the cellular mechanism of the MP-induced activation of antioxidant enzymes and their contribution to the attenuation of cellular oxidant toxicity. When bovine glomerular endothelial cells (GECs) were treated with 10 microM MP, cellular manganese superoxide dismutase (Mn-SOD, 3.95 +/- 0.33 mu/mg protein, M +/- SE) and catalase (1.64 +/- 0.06 k/mg protein) activities were significantly (P < 0.05) elevated above control GECs (2.23 +/- 0.43 mu/mg protein and 1.06 +/- 0.09 k/mg protein, respectively). When GECs pretreated with MP (10 microM 24 hrs) were exposed to xanthine (0.1 mM)+xanthine oxidase (5 mU/ml) for four hours, levels of specific membrane lipid peroxidation products, that is, phosphatidylcholine- and phosphatidylethanolamine-hydroperoxides, remained at levels 10 to 25% of those measured in non-MP-treated (xanthine/xanthine oxidase-exposed) control cells. Moreover, the degree of cell damage following exposure to the superoxide generating system, assessed by 51Cr release, was significantly attenuated in MP-treated cells (approximately 50% of MP-non-treated controls, N = 6). Thus, MP-treated GECs with elevated antioxidant enzyme activities by MP were more resistant to the toxic effect of reactive oxygen metabolites. The mechanism of antioxidant enzyme induction by MP was studied for Mn-SOD. MP was shown to enhance Mn-SOD mRNA in bovine GECs and rat glomerular mesangial cells (GMCs) in dose-dependent manners.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells.

We investigated whether cell volume decrease per se can activate intracellular mechanisms leading to mesangial cell contraction. For this purpose, we applied hyperosmotic stress to cultured glomerular mesangial cells and examined the effects on phosphorylation of myosin light chain (MLCP). Compared with control cells, hyperosmotic stress (390 mosmol/kg) attained by either NaCl or raffinose significantly increased MLCP to 140.7 +/- 7.0% (n = 5) and 134.8 +/- 7.7% (n = 4), respectively, in parallel with a decrease in the cell volume. This increase was comparable to that achieved by the following agonists: arginine vasopressin (AVP, 100 nM; n = 5) and endothelin-1 (ET, 10 nM; n = 5). By using two-dimensional tryptic phosphopeptide mapping, contribution of myosin light-chain kinase (MLCK) and protein kinase C (PKC) to the observed phosphorylation was examined by identifying phosphorylation at serine-19 (by MLCK) and at serine-1 or serine-2 (by PKC). Under resting conditions, relative distribution of phosphorylation between MLCK and PKC sites was 60.1 +/- 8.4 and 39.9 +/- 8.4%. The relative contribution by these enzymes remained similar during hyperosmotic stress or agonist stimulation. Since cytosolic Ca2+ concentration ([Ca2+]i) is an important determinant of MLCP, we also examined [Ca2+]i in these settings. While AVP and ET-induced a characteristic transient spike in [Ca2+]i, hyperosmotic stress caused a gradual and modest increase in [Ca2+]i. These studies show that, in mesangial cells, reduction in cell volume induces MLCP through mechanisms distinct from those involved in agonist-induced events.

Dietary fatty acid modulates glomerular atrial natriuretic peptide receptor.

Modification of dietary fatty acid (FA) has been shown to affect the incidence of hypertension and coronary artery disease. We studied whether these effects involve changes in the receptor characteristics of vasoactive substance. Characteristics of atrial natriuretic peptide (ANP) receptors were examined in glomeruli isolated from rats fed a diet containing 5% in weight omega 6, 5% omega 3, 20% omega 6, 20% omega 3 polyunsaturated FA or 20% saturated FA (SFA) for greater than 4 weeks. The FA composition of phospholipids in isolated glomeruli showed an elevation in 20:4 omega 6 (arachidonic acid, AA) in 5% omega 6, 20% omega 6 and 20% SFA, and elevations in 20:5 omega 3 (eicosapentaenoic acid, EPA) in 5% omega 3 and 20% omega 3 groups. The radioligand binding study revealed: (1) in 20% FA group, receptor density (Ro, fmol/mg prot) of ANP was significantly decreased compared to 5% group (262 +/- 13, n = 8 to 120 +/- 13, n = 12) without changes in equilibrium dissociation constant (KD), (2) among high FA (20%) groups, type of FA was essential for determining Ro; higher omega 6 was associated with a lower ANP Ro (177 +/- 11 vs. 103 +/- 3 fmol/mg prot, P less than 0.05) and KD (0.43 +/- .04 vs. 0.27 +/- .02 nM, P less than 0.05). To examine whether the alteration in receptor characteristics is mediated by FA, effects of FA were examined in vitro. In cultured mesangial cells, AA, but not EPA, decreased Ro of ANP receptors (48.7 +/- 4.8% of control, P less than 0.05) without affecting KD.(ABSTRACT TRUNCATED AT 250 WORDS)

Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thromboxane A2 receptors.

8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha) and related compounds are novel prostanoid produced by a noncyclooxygenase mechanism involving lipid peroxidation. Renal ischemia-reperfusion injury increased urinary excretion of these compounds by 300% over baseline level. Intrarenal arterial infusion at 0.5, 1, and 2 micrograms/kg per min induced dose-dependent reductions in glomerular filtration rate (GFR) and renal plasma flow, with renal function ceasing at the highest dose. Micropuncture measurements (0.5 microgram/kg per min) revealed a predominant increase in afferent resistance, resulting in a decrease in transcapillary hydraulic pressure difference, and leading to reductions in single nephron GFR and plasma flow. These changes were completely abolished or reversed by a TxA2 receptor antagonist, SQ 29,548. Competitive radioligand binding studies demonstrated that 8-epi-PGF2 alpha is a potent competitor for [3H]SQ 29,548 binding to rat renal arterial smooth muscle cells (RASM) in culture. Furthermore, addition of 8-epi-PGF2 alpha to RASM or isolated glomeruli was not associated with stimulation of arachidonate cyclooxygenase products. Therefore, 8-epi-PGF2 alpha is a potent preglomerular vasoconstrictor acting principally through TxA2 receptor activation. These findings may explain, in part, the beneficial effects of antioxidant therapy and TxA2 antagonism observed in numerous models of renal injury induced by lipid peroxidation.

Interactions between live Bartonella bacilliformis and endothelial cells.

Bartonella bacilliformis, a gram-negative, flagellated, motile bacterium, is the etiologic agent of verruca peruana. It is found within the verruca, where it can form large cytoplasmic (Rocha-Lima) inclusions in endothelial cells. Previously, an activity has been described in homogenates of B. bacilliformis that in vitro increases the proliferation of endothelial cells and their production of tissue-type plasminogen activator (t-PA) and in vivo is angiogenic. The aim of the present study was to determine if live B. bacilliformis similarly stimulated endothelial cells and produced the Rocha-Lima inclusion. By measuring proliferation of cells and the production of t-PA in vitro, it was found that the live bacteria increased both parameters in a fashion similar to the homogenates of B. bacilliformis. Interaction between the bacteria and endothelial cells appeared to be necessary for proliferation. On electron microscopy, bacteria penetrated the endothelial cell within 1 h, forming a small membrane-bound inclusion. By 12 h, a large membrane-bound inclusion, similar to the Rocha-Lima inclusion, containing numerous bacteria was present. These data provide further evidence that B. bacilliformis has an angiogenic activity and that the bacteria are at least in part responsible for the vascular proliferation of the verruca.

Endothelin-1 receptor antagonist: effects on endothelin- and cyclosporine-treated mesangial cells.

Endothelin-1 (Et) has profound effects on glomerular microcirculation and mesangial cell contraction. A parameter of mesangial cell contraction was examined by measuring myosin light chain phosphorylation (MLCP) in glomerular mesangial cells in the presence and absence of a newly developed endothelin-1 receptor antagonist (EtA). Addition of Et alone (10 nM) caused a marked increase in MLCP, which, on average, rose by 53 +/- 6% above the level in cells exposed to vehicle (P less than 0.0005). This effect was shown to continue for at least one hour; MLCP at 60 minutes was 64 +/- 12% higher than controls, (P less than 0.025), constituting a unique observation of an in vitro parameter which parallels the characteristic in vivo effect of Et. Treatment of cells with EtA virtually abolished this Et-induced increase in MLCP, which rose by only 2 +/- 3% and -1 +/- 4% for doses of EtA of 44 nM and 66 nM, respectively. Examination of the intracellular calcium concentration, [Ca2+]i, revealed that EtA almost completely abolished the transient increase in [Ca2+]i evoked by Et and also suppressed the early portions of the sustained increase in [Ca2+]i. EtA was ineffective in abolishing [Ca2+]i increase in response to arginine vasopressin. Finally, to evaluate EtA's efficacy in a pathophysiologic setting, we also studied mesangial cells exposed to cyclosporine (Cs). Exposure of mesangial cells to Cs (10(-5) M) for 60 minutes caused a significant increase in MLCP, on average, by 38 +/- 6% above control (P less than 0.0005), while cells exposed to Cs in the presence of EtA increased MLCP significantly less, by only 15 +/- 9%. These data provide further evidence for Et's long-lasting cellular actions, and demonstrate inhibitory effects of an Et receptor antagonist after direct cellular exposure to Et and also after Cs exposure, a pathophysiologic setting which likely involves Et.

Secretion of a chemotactic substance(s) by AGE-stimulated human monocytes.

Receptors for products of non-enzymatic glycosylation have been identified previously on activated human monocytes. In this study we have found that medium conditioned by activated human monocytes following stimulation with AGE-BSA elicited an almost 3-fold greater chemotactic response from other activated monocytes than conditioned medium obtained following stimulation with control BSA (44 +/- 13 and 16 +/- 4.6, respectively; n = 9, P less than 0.05). The response elicited from AGE-BSA alone was not statistically significant. It appears that stimulation of the cells via the AGE-receptor results in the secretion of increased levels of a chemotactic substance(s) for monocytes/macrophages. This mechanism may help to explain the pathogenesis of atherosclerosis in diabetes, as monocyte accumulation within the vessel wall is an important step in fatty streak development.

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.

Metabolism of nitroglycerin by smooth muscle cells. Involvement of glutathione and glutathione S-transferase.

Metabolism of nitroglycerin (GTN) in the vascular smooth muscle is required for the drug to be effective in the treatment of angina pectoris and congestive heart failure. The usefulness of GTN is limited by the development of tolerance to the drug. The metabolism of GTN was studied in its target tissue, vascular smooth muscle. Inorganic nitrite was produced by cultured smooth muscle cells when GTN was added to the culture dish. Nitrite production increased with increasing GTN concentration and with incubation time. The enzymatic nature of GTN metabolism to nitrite was assessed by enzyme inhibition studies. Indocyanine green, a non-substrate inhibitor of glutathione S-transferase, inhibited GTN metabolism by smooth muscle cells. Cellular glutathione is also involved in GTN metabolism by the smooth muscle cell. Pretreatment with phorone, a glutathione S-transferase substrate, depleted cellular glutathione and decreased nitrite production from GTN. Pretreatment with buthionine sulfoximine, inhibitor of gamma-glutamylcysteine synthetase, decreased intracellular glutathione and caused decreased GTN metabolism in smooth muscle cells. Removal of cysteine from the smooth muscle cell incubation medium in combination with buthionine sulfoximine pretreatment decreased GTN metabolism to a lower level than buthionine sulfoximine pretreatment alone. This study shows that glutathione S-transferase and glutathione are involved in GTN metabolism by cultured smooth muscle cells.

Human monocyte interactions with non-enzymatically glycated collagen.

We have previously shown that receptors for advanced glycation end products are expressed on activated human monocytes. We now report that activated human monocytes exhibit increased adhesion to non-enzymatically glycated collagen substrates (+32% +/- 1, p less than 0.001), and the increased adhesion can be competitively inhibited with non-enzymatically glycated albumin. Non-activated monocytes, which do not express receptors for advanced glycation end products, exhibit decreased adhesion (-16% +/- 1, p less than 0.001). Similar results were observed with substrates of fibronectin and endothelial cell matrix proteins. As the presence of glycation adducts on collagen interferes with the normal binding of monocytes/macrophages, one possible role for advanced glycation adduct receptors on activated monocytes is to counterbalance such decreased adherence. Overcompensation for long periods of time may lead to pathological changes. Additionally, such receptors may play a role in monocyte-mediated remodelling of glycated matrix proteins, as we have observed increased degradation of non-enzymatically glycated collagen substrates by activated human monocytes at 2 h (+52% +/- 11, p = 0.01), 3 h (+49% +/- 10, 10, p = 0.01), and 4 h (+36% +/- 6, p less than 0.01) after adding activated monocytes to 125I-labelled substrates.

Identification and analysis of transforming growth factor beta receptors on primary osteoblast-enriched cultures derived from adult human bone.

Primary human osteoblast-enriched (PHO) cultures derived from adult trabecular bone were analyzed to determine the presence or absence of transforming growth factor beta (TGF-beta) receptors. Saturation binding studies were performed with 125I-TGF-beta in the absence or presence of 200-fold excess cold TGF-beta. Cross-linking experiments utilizing 125-I-TGF-beta were performed to identify specific cell surface binding proteins for TGF-beta. The saturation binding studies demonstrated saturable binding for TGF-beta on PHO cells. TGF-beta was cross-linked to cell surface binding proteins of 50 to 110 KDa and a high molecular weight component. Thus, these receptors appear to be similar in affinity, number per cell, and molecular weight to those previously identified with other cell types. The potential biological effects of TGF-beta on the growth of PHO cultures were evaluated by both 3H-thymidine incorporation and cell number determination. Growth of PHO cells in the presence of TGF-beta resulted in an approximately two-fold stimulation in cell number as compared to control cells while the 3H-thymidine experiments demonstrated a two to four-fold increase in thymidine uptake in the presence of TGF-beta. Radiographic emulsion studies revealed that the alkaline phosphatase positive and negative cell populations were responsive to the TGF-beta mitogenic stimulation. The cumulative findings of saturable binding, specific cell surface binding proteins, and biological effects suggest that functional TGF-beta cell surface receptors are present on primary osteoblast-enriched cultures derived from adult human trabecular bone.

Characterization of myosin heavy and light chains in cultured mesangial cells.

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that postconfluent mesangial cells in primary culture expressed three myosin heavy chains (MHCs), 204 kD, 200 kD and 196 kD, in a manner similar to that of smooth muscle cells. The MHCs of 204 kD and 200 kD in mesangial cells reacted positively with antibodies raised against bovine aorta smooth muscle myosin while the 196 kD MHC reacted positively with antibodies against platelet myosin. Moreover, the combined content of the MHCs in cultured mesangial cells was remarkably similar in amount to that in cultured aortic smooth muscle cells. After three passages, cultured mesangial cells expressed only the 196 kD MHC as has been reported for cultured smooth muscle cells. Two phosphorylated proteins were found in the immunoprecipitate after incubation of the cell extract with antibodies against platelet myosin: a MHC of approximately 200 kD and myosin light chain (MLC) of 20 kD. The level of MLC phosphorylation was quantitated by scanning densitometry of autoradiograms. Arginine vasopressin (AVP) at 100 nM induced MLC phosphorylation with a maximum effect at 10 minutes. AVP enhanced MLC phosphorylation in a dose dependent manner: maximum response was observed with 100 nM and half maximum, at 3.5 nM. Similarly, angiotensin II (100 nM), endothelin-1 (10 nM) and the calcium ionophore, A23187 (1 microM), significantly enhanced MLC phosphorylation. Thus, although the expression of MHC was altered in quality after mesangial cells were placed in culture, the cells remained rich in myosin content and had an intact regulatory system for contraction which responded to a variety of vasoconstrictive agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of monocyte adherence to endothelium under hyperglycemic conditions.

Increased nonenzymatic glycation of proteins has been implicated in the pathogenesis of diabetic vascular disease. The authors have shown by 3H-NaBH4 reduction of nonenzymatic glycation adducts that endothelial cell membrane proteins undergo increased nonenzymatic glycation in vitro when exposed to elevated concentrations of glucose. Increased nonenzymatic glycation also was found in vivo for microvascular endothelial cells isolated from streptozotocin-induced diabetic rats compared with control rats. Cultured monocytes have previously been reported to express receptors for certain nonenzymatic glycation adducts. The authors have further investigated whether monocyte interactions with endothelium are altered by the presence of nonenzymatic glycation adducts on endothelium. Adherence assays were performed in the presence of elevated concentrations of glucose with decreased NaCl levels to maintain normal osmolarity (as occurs physiologically). Although monocyte adherence to endothelium and levels of early nonenzymatic glycation adducts increased under these conditions, the increased adherence appears to be due to the altered NaCl levels. In fact, freshly isolated monocytes (in contrast to what has been found for macrophages and activated monocytes) were shown not to express appreciable numbers of receptors for nonenzymatic glycation adducts.

Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 37 degrees C for 24 hours as evidenced by continuously increased amounts of PAI-1 released into the conditioned media. t-PA release, however, decreased during the 24-hour period at 37 degrees C after exposure to hyperthermia. No difference in PAI-1 antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 37 degrees C could be found. Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells.