Connective tissue growth factor induces extracellular matrix in asthmatic airway smooth muscle

Transforming growth factor (TGF)-beta and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-beta increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF-beta and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF-beta and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase-dependent in both cell types but phosphoinositide-3 kinase-dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF-beta induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.     

Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: role of TGF-beta(1)

Elevated blood pressure imposes increased mechanical stress on the vascular wall, and mechanical strain is a mitogenic stimulus for vascular smooth muscle (VSM) cells. The role of mechanical forces in regulating the production of noncellular material by VSM cells for VSM cells of human origin remains undefined. We thus investigated the effects of chronic cyclical mechanical strain on extracellular matrix (ECM) protein production by cultured human VSM cells. To simulate a blood pressure of 120/80 mm Hg, human VSM cells were repetitively stretched and relaxed by 10% to 16% of their original length with the Flexercell apparatus. Fibronectin and collagen protein concentrations, matrix metalloproteinase (MMP) activity, and transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression by human VSM cells were measured in response to mechanical strain. Exposing human VSM cells to 5 days of chronic cyclical mechanical strain increased fibronectin (+48%, P:<0.01) and collagen (+50%, P:<0.001) concentrations when compared with cells grown in static conditions. Mechanical strain also increased MMP-2 activity, the predominant matrix-degrading isoform (+11%, P:<0.05) in human VSM cells, thus strain-induced ECM accumulation was not due to inhibition of ECM protein degradation. Strain also increased TGF-beta(1) mRNA expression and the production of a soluble factor that increased ECM protein production. Moreover, a TGF-beta-blocking antibody inhibited the effect of strain-conditioned media on matrix production by human VSM cells. These results suggest that chronic cyclical mechanical strain can directly modulate the fibrogenic activity of human VSM cells by inducing ECM protein synthesis and MMP activity. This occurs, at least in part, through mechanical strain-induced TGF-beta(1) production, a mechanism that could explain the increased vascular ECM deposition in hypertension.     

Incorporation of cellular and plasma fibronectins into smooth muscle cell extracellular matrix in vitro.

Fibronectins isolated from the conditioned medium produced by cultures of undifferentiated (monolayer) and differentiated (nodular) swine vascular smooth muscle cells are similar but not identical. In general, the nodular-cell fibronectin has a smaller molecular mass than monolayer-cell fibronectin and appears to lack the COOH-terminal interchain disulfide linkage. We studied the incorporation of cellular and plasma fibronectins into the cell layer. Smooth muscle cells bound 2.5 times more monolayer-cell fibronectin than nodular-cell fibronectin. Polypeptide fragments of human plasma fibronectin were used as a model system to investigate fibronectin incorporation into the cell layer. Only intact molecules were incorporated into the cell layer and subsequently organized into fibers. Polypeptide fragments of molecular mass 205 kDa and 185 kDa were not incorporated even though they retained the collagen-, cell-, and heparin-binding regions. Incorporation appears to require an activity associated with either the NH2-terminal or COOH-terminal domains. We propose that fibronectin activity is lost during differentiation of smooth muscle cells.     

Quantitative assay for morphogenesis indicates the role of extracellular matrix components and G proteins.

A quantitative assay for morphogenesis is described that involves counting the organizing centers (swirling patterns) formed by many cultured fibroblasts. Organizing centers, which are found in vivo, represent one of the smallest units of morphogenesis. We show that macroscopically visible organizing centers form by the merger of smaller organizing centers. Parallel orientation of cells on plastic substrata requires cell-cell contact, but organizing centers can develop without cell-cell contact on collagen gels. On collagen gels, the orientation of collagen fibers determines the orientation of cells with respect to one another. Although organizing centers resemble fingerprints, we have shown that a stochastic process determines the spatial orientation of organizing centers. Treatment of transformed cell lines with agents that increase cAMP levels or alter the activity of guanine nucleotide binding proteins resulted in the generation of organizing centers. Cholesterol precursors involved in protein isoprenylation were found to be potent reverse-transformation agents that could alter the two-dimensional morphogenesis of cells. The simple assay described should permit the analysis of morphogenesis at the molecular and cellular levels.     

External beam radiation after stent implantation increases neointimal hyperplasia by augmenting smooth muscle cell proliferation and extracellular matrix accumulation.

OBJECTIVES: We sought to examine the effects of high volume external beam radiation (EBR) after stent implantation on neointimal hyperplasia, smooth muscle cell (SMC) proliferation, presence of inflammatory cells and expression of extracellular matrix (ECM). BACKGROUND: Endovascular irradiation has been shown to reduce restenosis rates after angioplasty in preliminary trials, but conflicting results have been reported for the effects of external beam irradiation. METHODS: Forty-three Palmaz-Schatz stents were implanted into iliac arteries of New Zealand White rabbits. The arteries were externally irradiated after stent implantation with a single dose of 8 Gy (at day 3) or 16 Gy in two fractions (8 Gy at days 3 and 4) by means of a linear accelerator. In the control rabbits, no radiation was applied after stent implantation. Smooth muscle cells, macrophages and ECM were studied by immunohistochemistry at one and 12 weeks after stent implantation. Collagen type I and biglycan messenger ribonucleic acid (mRNA) levels were assessed by Northern blot analysis at one week. Neointimal cell densities and arterial lumen stenosis were measured by histomorphometry at 12 weeks. RESULTS: At 1 week, SMC proliferation at the site of stent implantation was increased after EBR with 8 and 16 Gy (26 +/- 5%, 32 +/- 3% vs. 17 +/- 8%; p < 0.01, 16 Gy vs. control). External beam radiation with 8 and 16 Gy augmented SMC proliferation proximal and distal to the angioplasty site (11 +/- 3%, 14 +/- 3 vs. 6 +/- 1%; p < 0.01, 16 Gy vs. control). Collagen type I and biglycan mRNA levels were elevated in stented arteries after EBR with 16 Gy. At 12 weeks, a marked decrease in neointimal cell density (248 +/- 97 vs. 498 +/- 117 SMCs/0.1 mm2 neointima; p < 0.005 vs. control) was noted after EBR with 16 Gy. Irradiation with 8 and 16 Gy increased arterial lumen stenosis compared with nonirradiated control rabbits (45 +/- 7%, 55 +/- 9% vs. 33 +/- 7%; p < 0.05, 8 Gy and p < 0.001, 16 Gy vs. control). CONCLUSIONS: High volume external beam radiation at doses of 8 or 16 Gy causes restenosis by augmenting proliferative activity at and adjacent to the site of stent implantation, and by dose-dependent up-regulation of extracellular matrix expression. The study suggests that excessive matrix accumulation is an important determinant of failure of radiation therapy to prevent restenosis.     

Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth

Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO)via the catabolism of heme by the enzyme heme oxygenase (HO). In thepresent study, we found that serum stimulated a time- andconcentration-dependent increase in the levels of HO-1 messenger RNA(mRNA) and protein in vascular SMCs. The induction of HO-1 expressionby serum was inhibited by actinomycin D or cycloheximide. In addition,serum stimulated HO activity, as reflected by an increase in theconcentration of bilirubin in the culture media. Treatment of vascularSMCs with serum stimulated DNA synthesis and this was potentiated bythe HO inhibitors, zinc and tin protoporphyrin-IX as well as by the COscavenger, hemoglobin. The iron chelator desferrioxamine had no effecton DNA synthesis. However, exposure of vascular SMCs to exogenous COinhibited serum-stimulated SMC proliferation and the phosphorylation ofretinoblastoma protein. In addition, CO arrested SMCs at theG₁/S transition phase of the cell cycle and selectivelyblocked the serum-stimulated expression of cyclin A mRNA and proteinwithout affecting the expression of cyclin D1 and E. CO also inhibitedthe serum-stimulated activation of cyclin A-associated kinase activityand cyclin-dependent kinase 2 activity. These results demonstrate thatserum stimulates HO-1 gene expression and CO synthesis. Furthermore,they show that CO acts in a negative feedback fashion to inhibitvascular SMC growth by regulating specific components of the cell cyclemachinery. The capacity of vascular mitogens to induce COsynthesis may provide a novel mechanism by which these agents modulatecell growth.     

Modulation of aortic smooth muscle cell membrane potential by extracellular calcium

Removal of extracellular calcium may result in depolarization of the resting cell membrane potential. This has been attributed to the stabilizing action of calcium on the ionic permeability of the cell membrane. It is unknown whether this phenomenon is exclusively mediated by extracellular calcium or through associated changes in intracellular calcium. To examine this, we exposed rat aortic smooth muscle cells in culture to different calcium concentrations and studied their effects on the resting membrane potential and intracellular calcium activity. The resting membrane potential was dependent on the extracellular potassium concentration. Exposure to reduced extracellular calcium concentrations (0.25 and 0.5 mM) caused a steep and reversible depolarization of the membrane potential, but intracellular calcium, measured with fura 2-AM, was not reduced below that measured in control conditions (1.8 mM). Atomic absorption spectrophotometric measurements did not indicate a measurable gain in cell sodium after reduction of extracellular calcium levels. We conclude that extracellular calcium controls the resting cell membrane potential of vascular smooth muscle through a mechanism that is independent of cytosolic Ca2+ activity.     

Human aortic vascular smooth muscle cells digest extracellular matrix by elaboration of plasminogen activators: Implications for atherogenesis

Migration and proliferation of vascular smooth muscle cells (SMCs) are hallmarks of atherogenesis and restenosis after angioplasty. Digestion of surrounding extracellular matrix (ECM) may be a critical link. To determine whether invasion of ECM by human aortic SMCs (HASMCs) depends on proteolytic digestion mediated by the cells themselves, we characterized ECM digestion in terms of solubilization of³H-proline-labeled ECM, produced by the use of rat aortic SMCs, by HASMCs under various conditions. Pasmin alone (10 g/ml) digested 80% of ECM in 2 hours. HASMCs in 10% fetal bovine serum cultured on ECM that was not exposed to plasmin digested 48% of the ECM in 7 days. When HASMCs were cultured on plasmin-pretreated ECM, only 14% of the residual ECM was digested. Conditioned media or cells cultured on porous membrane 1 mm removed from the ECM had no effect. Baseline secretion of tissue-type plasminogen activator (t-PA) into the media by HASMCs averaged 3.9 ng/10⁵ cells/24 hr and baseline secretion of type-1 plasminogen activator inhibitor (PAI-1) averaged 1300 ng/10⁵ cells/24 hr. Thrombin (5 U/ml) increased t-PA antigen production by 184% without altering PAI-1 activity and increased ECM degradation by 43% in 7 days. Transforming growth factor- (TGF-) decreased t-PA antigen production, increased PAI-1 activity, and decreased ECM degradation. These results suggest that (1) HASMCs can digest naturally produced ECM; (2) plasminogen-dependent mechanisms requiring cell contact are important in the initiation of this phenomenon; and (3) thrombin in the vicinity of clots may modulate the fibrinolytic and proteolytic properties of SMC through t-PA after vascular injury.     

Proteoglycans contribution to association of Lp(a) and LDL with smooth muscle cell extracellular matrix.

Lp(a) interference with fibrinolysis could contribute to atherothrombosis. Additionally, accumulation of Lp(a) and LDLs, could lead to cholesterol deposition and foam cell formation in atherogenesis. The interactions between Lp(a) and LDL could cause their entrapment in the extracellular matrix of lesions. We found that association of Lp(a) with matrix secreted by cultured human arterial smooth muscle cells increased 2 to 3 times the subsequent specific binding of radioactive LDL. Chondroitin sulfate proteoglycans seem responsible for formation of the specific matrix-Lp(a) and matrix-LDL aggregates. The proteoglycans appeared also to participate in a cooperative increase of radioactive LDL binding to matrix pretreated with Lp(a). In the matrix preincubated with LDL, approximately 50% of the additional lipoprotein was bound by ionic interactions. In the matrix preincubated with Lp(a), 20% of the additional LDL was held by ionic bonds, and the rest was held by strong nonionic associations. Binding analysis in physiological solutions confirmed that chondroitin sulfate-rich proteoglycans from the smooth muscle cell matrix have a high affinity for Lp(a) and LDL. The results provide an explanation to the observed localization of Lp(a) and LDL in the extracellular matrix of arterial lesions and suggest a mechanism for their cooperative accumulation there.     

Tyrosine-kinase dependent TGF-beta and extracellular matrix expression by mechanical stretch in vascular smooth muscle cells.

Vascular hypertrophy, which is characterized by proliferation of vascular smooth muscle cells (VSMC) and accumulation of extracellular matrix (ECM), is a major pathological change in blood vessels after chronic exposure to hypertension. Blood pressure is transmitted to the arterial walls and counterbalanced by mechanical stress, leading to stretching of circumferentially oriented VSMC, which may play some role in the pathogenesis of vascular hypertrophy. The present study was designed, therefore, to investigate the effect of mechanical stretch on the expression of ECM components and transforming growth factor-beta (TGF-beta), a potent stimulator for ECM production, and to examine the signal transduction mechanisms of the induction of TGF-beta in cultured rat VSMC. VSMC were subjected to cyclic stretch to provide a maximal elongation of 20% at a rate of 60 cycles per minute for up to 24 h. Mechanical stretch stimulated TGF-beta1 mRNA expression in a time- and elongation-dependent manner. Indeed, the secretion of TGF-beta proteins into the culture media was increased after stretch. Stretch also stimulated mRNA expression of the ECM components, type I and type IV collagen, and fibronectin, which was largely inhibited by addition of neutralizing antibody against TGF-beta. The tyrosine kinase inhibitors genistein and herbimycin A blocked the induction of TGF-beta1 and type I collagen by stretch, while protein kinase C inhibitors, the calcium channel blockers nitrendipine and gadolinium, or Ca removal from the media had no effect. These results suggest that stretch-induced, tyrosine kinase-mediated autocrine/paracrine production of TGF-8 may play a critical role in the progression of vascular remodeling associated with high blood pressure.     

Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin

Red blood cell (RBC) adhesion to the vascular endothelium is increased in several pathologic conditions, including sickle cell disease and malaria. However, RBC interactions with components of the subendothelial matrix are not well-characterized. Under in vitro flow conditions of 1 dyne/cm2, washed RBCs bound to the purified adhesive molecules thrombospondin (TSP) and laminin. Sickle RBCs had the greatest adhesion of all tested RBCs. The adhesion of sickle RBCs to immobilized TSP was inhibited by the anionic polysaccharides high molecular weight (MW) dextran sulfate and chondroitin sulfate A, but not other anionic polysaccharides of similar structure and/or charge density. These data were consistent with the RBC adhesive molecule being a sulfated glycolipid. Therefore, TSP-binding lipids from normal and sickle RBCs were isolated and characterized. The TSP-binding lipid was purified by alkaline methanolysis, anion exchange chromatography and preparative thin layer chromatography (TLC). A homogeneous band on TLC was identified using a specific overlay TSP-binding assay. TSP binding to the purified lipid was stable to bass and neuraminidase treatment, labile to acid treatment, and was inhibited by high MW dextran sulfate, similar to that seen with intact RBCs binding to immobilized TSP under conditions of flow. In addition, soluble laminin bound to the purified RBC lipid. This acidic TSP- and laminin-binding lipid(s) isolated from both sickle and normal RBC membranes may contribute to erythrocyte interactions with the subendothelial matrix, hereby participating in the pathogenesis of vaso-occlusive diseases.     

Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.     

Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis.

Plasma homocysteine levels are elevated in 20-30% of all patients with premature atherosclerosis. Although elevated homocysteine levels have been recognized as an independent risk factor for myocardial infarction and stroke, the mechanism by which these elevated levels cause atherosclerosis is unknown. To understand the role of homocysteine in the pathogenesis of atherosclerosis, we examined the effect of homocysteine on the growth of both vascular smooth muscle cells and endothelial cells at concentrations similar to those observed in clinical studies. As little as 0.1 mM homocysteine caused a 25% increase in DNA synthesis, and homocysteine at 1 mM increased DNA synthesis by 4.5-fold in rat aortic smooth muscle cells (RASMC). In contrast, homocysteine caused a dose-dependent decrease in DNA synthesis in human umbilical vein endothelial cells. Homocysteine increased mRNA levels of cyclin D1 and cyclin A in RASMC by 3- and 15-fold, respectively, indicating that homocysteine induced the mRNA of cyclins important for the reentry of quiescent RASMC into the cell cycle. Furthermore, homocysteine promoted proliferation of quiescent RASMC, an effect markedly amplified by 2% serum. The growth-promoting effect of homocysteine on vascular smooth muscle cells, together with its inhibitory effect on endothelial cell growth, represents an important mechanism to explain homocysteine-induced atherosclerosis.     

Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage, and smooth muscle cell content.

OBJECTIVE--To assess the size of the lipid pool and the number of smooth muscle cells and monocyte/macrophages in human aortic plaques that were intact and to compare the results with those in aortic plaques undergoing ulceration and thrombosis. DESIGN--The lipid pool was measured as a percentage of the total cross sectional area of the plaque. Immunohistochemistry was used to identify cell types (monocytes/macrophages (M phi) by EBM11 and HAM56, smooth muscle cells by alpha actin). The area of the tissue occupied by each cell type was measured by quantitative microscopy in the peripheral (shoulder) area of the plaque and the plaque cap. Absolute counts of each cell type were expressed as the ratio of SMC:M phi. MATERIAL--Aortas were obtained at necropsy from men aged less than 69 years who died suddenly (within 6 hours of the onset of symptoms) of ischaemic heart disease. 155 plaques from 13 aortas were studied. Four aortas showed intact plaques only (group A, n = 31). Nine aortas showed both intact plaques (group B, n = 79) and plaques that were undergoing thrombosis (group C, n = 45). RESULTS--In 41 (91.1%) of the 45 plaques undergoing thrombosis (group C) lipid pools occupied more than 40% of the cross sectional area of the plaque. Only 12 (10.9%) of the 110 intact plaques (groups A + B) had lipid pools of this size. The mean size of the lipid pool in plaques of groups A, B, and C was 12.7%, 27.3% and 56.7% respectively. Compared with intact plaques those undergoing thrombosis contained a smaller volume of smooth muscle cells (2.8% v 11.8%) and a larger volume of monocyte/macrophages (13.7% v 2.9%) in the plaque cap. The ratio of the number of smooth muscle cells to monocytes/macrophages was 7.8 in group A plaques, 4.1 in group B plaques, and 1.0 in group C plaques. This gradient was the result of an absolute increase in monocyte/macrophages and an absolute decrease in smooth muscle cells. CONCLUSIONS--In the aorta ulceration and thrombosis were characteristic of plaques with a high proportion of their volume occupied by extracellular lipid, and in which there was a shift toward a preponderance of monocyte/macrophages compared with smooth muscle cells in the cap.     

Inhibition of vascular smooth muscle cell migration by elevation of extracellular potassium concentration

The effect of potassium on the migration of vascular smooth muscle cells was analyzed in media made with extracellular potassium concentrations of 3, 4, 5, and 6 mmol/L. The migration of cultured porcine coronary artery cells was stimulated with platelet-derived growth factor (PDGF)-BB. In the first study, cells were exposed to PDGF-BB at concentrations of 0, 10, or 20 ng/mL for 5 hours with the use of a Boyden chamber. Cells were quiescent overnight in 0.5% fetal bovine serum in Dulbecco's modified Eagle's medium with an extracellular potassium concentration of 4 mmol/L. With increasing potassium concentration, migration was significantly inhibited (P<0. 02, 2-way ANOVA). In the cells exposed to 10 ng/mL PDGF-BB, migration ranged from 500+/-86% to 294+/-44% (value in wells with 0 ng/mL PDGF-BB and 4 mmol/L potassium concentration=100%) in medium containing 3 to 6 mmol/L extracellular potassium concentration (P<0. 03). Long-term potassium exposure was investigated in cells grown in 5% serum in Dulbecco's modified Eagle's medium with an extracellular potassium concentration of 3, 4, 5, or 6 mmol/L for 3 to 4 weeks. Migration was assessed with 0 or 20 ng/mL PDGF-BB. Migration was significantly inhibited by the elevation of extracellular potassium concentration (P<0.01, 2-way ANOVA). With 20 ng/mL PDGF-BB, the migration rates ranged from 152+/-11% in medium with 3 mmol/L potassium to 69+/-5% in 6 mmol/L potassium (P<0.01). Increases in extracellular potassium concentration within the physiological range significantly and directly inhibit vascular smooth muscle cell migration.     

Platelet-derived growth factor-BB-induced suppression of smooth muscle cell differentiation.

Previously, we demonstrated that treatment of postconfluent quiescent rat aortic smooth muscle cells (SMCs) with platelet-derived growth factor (PDGF)-BB dramatically reduced smooth muscle (SM) alpha-actin synthesis. In the present studies, we focused on the expression of two other SM-specific proteins, SM myosin heavy chain (SM-MHC) and SM alpha-tropomyosin (SM-alpha TM), to determine whether the actions of PDGF-BB were specific to SM alpha-actin or represented a global ability of PDGF-BB to inhibit expression of cell-specific proteins characteristic of differentiated SMCs. SM-MHC and SM-alpha TM expression were assessed by one- or two-dimensional gel electrophoretic analysis of proteins from cells labeled with [35S]methionine, as well as by Northern analysis of mRNA levels. Synthesis of both SM-specific proteins was decreased by 50-70% in PDGF-BB--treated cells as compared with cells treated with PDGF vehicle. Treatment of cells with 10% fetal bovine serum, which produced a mitogenic effect equivalent to that of PDGF-BB, decreased SM-MHC synthesis by 40% but increased SM-alpha TM synthesis. SM-MHC and SM-alpha TM mRNA expression was decreased by 80% at 24 hours in PDGF-BB--treated postconfluent SMCs, whereas treatment with 10% fetal bovine serum did not decrease the expression of SM-alpha TM mRNA but did inhibit SM-MHC mRNA expression by 36%. Consistent with the absence of detectable PDGF alpha-receptors on these cells, PDGF-AA had no effect on either mitogenesis or expression of SM-MHC or SM-alpha TM.(ABSTRACT TRUNCATED AT 250 WORDS)