Porcine Vascular Smooth Muscle Cells Research Articles

Full-Length cDNA Sequence of a Progesterone Membrane-Binding Protein from Porcine Vascular Smooth Muscle Cells

A full-length cDNA clone for a progesterone membrane binding protein from porcine vascular smooth muscle cells was isolated and the complete nucleotide sequence determined. The cDNA encodes a protein of 194 amino acids with a transmembrane segment. This protein is likely to represent the first steroid membrane receptor or a part of it for which sequence information is available.

Expression of rho A and rho Kinase mRNAs in Porcine Vascular Smooth Muscle

It has recently been indicated that rho A and rho kinase may be involved in the mechanism for the increase in Ca2+sensitivity in smooth muscle myofilaments. In the present study, we investigated the mRNA expression of rho A and rho kinase in porcine vascular smooth muscle cells (aorta, coronary artery, pulmonary artery, and pulmonary vein). In reverse transcription–polymerase chain reaction (RT–PCR) experiments, using total RNA from these tissue specimens and the primers designed in the conserved regions of each mRNA in human and bovine, rho A and rho kinase mRNAs were detected in all of these smooth muscle cells. An analysis of these PCR products by direct sequencing indicated that they were derived from each mRNA. The finding that both rho A and rho kinase mRNAs were expressed in the various porcine vascular smooth muscle cells strongly supports the idea that these proteins are involved in the mechanism dealing with the increase in Ca2+sensitivity in the myofilaments of vascular smooth muscle cells.

Effect of hypercholesterolaemia on platelet growth factors.

Evidence from several sources suggests that important interactions occur between platelets and low-density lipoproteins. This study was undertaken to find out if diet-induced hypercholesterolaemia affects the growth factor content in circulating platelets. Minipigs were fed either normal diet supplemented with 2% cholesterol (n = 12) or normal diet alone (n = 12). After 4 months, mean platelet volume was significantly lower (P < 0.05) and monocyte count was significantly higher (P < 0.05) in the cholesterol group. Serum and intraplatelet levels of platelet-derived growth factor (BB homodimer) and transforming growth factor beta 1 were statistically unchanged after diet. Hypercholesterolaemia did not affect the proliferative effect of either serum or platelet lysates on porcine vascular smooth muscle cells and Swiss-3T3 cells in culture. A significant positive correlation between Swiss-3T3 and smooth muscle cell proliferation was present in both groups. These results suggest that the atherosclerosis-promoting effect of hypercholesterolaemia cannot be explained by its direct effect on smooth muscle cell proliferation or by changes in serum or intraplatelet concentrations of growth factors.

Formation of an F2-isoprostane in vascular smooth muscle cells by elevated glucose and growth factors.

Recently a series of non-cyclooxygenase-derived prostanoids were identified in vivo in humans and in animal models of free radical injury as products of free radical-catalyzed peroxidation of arachidonic acid. One of these, an F2-isoprostane, 8-epiprostaglandin F2 alpha (8-epi-PGF 2 alpha), is a potent renal vasoconstrictor and can increase vascular smooth muscle cell (VSMC) DNA synthesis. In the present study we have evaluated whether F2-isoprostanes play a role in diabetic vascular dysfunction by studying the formation of 8-epi-PGF2 alpha in porcine VSMC (PVSMC) cultured under hyperglycemic conditions. 8-Epi-PGF2 alpha levels were quantitated by a specific enzyme immunoassay. We also examined whether certain VSMC growth factors, such as angiotensin II, platelet-derived growth factor, and transforming growth factor-beta, could also regulate the formation of 8-epi-PGF2 alpha. We observed that PVSMC cultured under high glucose (HG) conditions produced significantly higher amounts of 8-epi-PGF2 alpha compared with normal glucose (NG) conditions (3.7 +/- 0.13 ng/10(6) cells in HG vs. 2.9 +/- 0.2 ng/10(6) cells in NG, P < 0.05). Furthermore, all three growth factors tested evoked significant dose-dependent formation of 8-epi-PGF2 alpha (ranging from 125 to 220% of control). These results suggest that 8-epi-PGF2 alpha formation, as a result of hyperglycemia or due to growth factor action, may lead to increased VSMC growth and contribute to the complications of diabetes and cardiovascular disease.

Insulin-like growth factor (IGF)-binding proteins inhibit the smooth muscle cell migration responses to IGF-I and IGF-II

Smooth muscle cells have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the role of IGF-binding proteins (IGFBPs) in this process has not been determined. As IGFBPs are synthesized and secreted by smooth muscle cells and bind to IGFs with high affinity, this study was undertaken to determine the ability of IGFBP-1 and -2 to modulate cellular migration in response to IGF-I and -II. Confluent monolayers of porcine vascular smooth muscle cells were wounded with a razor blade. After wounding, IGF-I and -II induced increases of 159 +/- 49% (mean +/- SD) and 108 +/- 33%, respectively, above control values in the number of cells migrating over a fixed distance in 4 days. The addition of IGFBP-1 caused a 19-21% inhibition of IGF-I- or IGF-II-stimulated migration, whereas the addition of IGFBP-2 inhibited the effect of both by greater than 60%. The addition of IGFBP-2 alone had no effect, whereas IGFBP-1 addition was associated with a 42 +/- 12% increase. In contrast, [Trp221]IGFBP-1 in which the RGD sequence was changed to WGD, thus eliminating its capacity to bind to the alpha 5 beta 1 integrin, inhibited IGF-I-stimulated migration by 67 +/- 17%. An IGF analog that has a reduced affinity for IGFBP-2-stimulated migration equally well as IGF-I alone even in the presence of IGFBP-2. Likewise, the addition of insulin, which cannot bind to IGFBPs, at supraphysiological concentrations that are adequate to activate the IGF-I receptor resulted in a similar increase in migration. In summary, IGF-I and -II stimulate smooth muscle cell migration after wounding. This migratory response is modulated by IGFBPs. Both IGFBP-1 and IGFBP-2 appear to neutralize the effects of the IGFs by inhibiting their interaction with IGF receptors, but IGFBP-1 also has a direct stimulatory effect that requires an intact RGD integrin recognition sequence.

Insulin-like growth factor (IGF)-binding proteins inhibit the smooth muscle cell migration responses to IGF-I and IGF-II.

Smooth muscle cells have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the role of IGF-binding proteins (IGFBPs) in this process has not been determined. As IGFBPs are synthesized and secreted by smooth muscle cells and bind to IGFs with high affinity, this study was undertaken to determine the ability of IGFBP-1 and -2 to modulate cellular migration in response to IGF-I and -II. Confluent monolayers of porcine vascular smooth muscle cells were wounded with a razor blade. After wounding, IGF-I and -II induced increases of 159 +/- 49% (mean +/- SD) and 108 +/- 33%, respectively, above control values in the number of cells migrating over a fixed distance in 4 days. The addition of IGFBP-1 caused a 19-21% inhibition of IGF-I- or IGF-II-stimulated migration, whereas the addition of IGFBP-2 inhibited the effect of both by greater than 60%. The addition of IGFBP-2 alone had no effect, whereas IGFBP-1 addition was associated with a 42 +/- 12% increase. In contrast, [Trp221]IGFBP-1 in which the RGD sequence was changed to WGD, thus eliminating its capacity to bind to the alpha 5 beta 1 integrin, inhibited IGF-I-stimulated migration by 67 +/- 17%. An IGF analog that has a reduced affinity for IGFBP-2-stimulated migration equally well as IGF-I alone even in the presence of IGFBP-2. Likewise, the addition of insulin, which cannot bind to IGFBPs, at supraphysiological concentrations that are adequate to activate the IGF-I receptor resulted in a similar increase in migration. In summary, IGF-I and -II stimulate smooth muscle cell migration after wounding. This migratory response is modulated by IGFBPs. Both IGFBP-1 and IGFBP-2 appear to neutralize the effects of the IGFs by inhibiting their interaction with IGF receptors, but IGFBP-1 also has a direct stimulatory effect that requires an intact RGD integrin recognition sequence.

Effects of fluvastatin on growth of porcine and human vascular smooth muscle cells in vitro

The effects of fluvastatin, a new fully synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on growth and cell-cycle kinetics of porcine and human vascular smooth muscle cells (SMC) were studied by growth curves and flow cytometric determination of cellcycle distribution. Growth curves were obtained from counting after 2, 4, 7, 9, 11, and 14 days of incubation in Dulbecco's minimum essential medium and 10% fetal calf serum (FCS). For cell-cycle phase determination, cells were synchronized in Go by 48 hours of incubation in serum-free medium, then stimulated by incubation in 10% FCS, with or without fluvastatin. There was a concentration-dependent decrease in the proliferation of human and porcine SMC when cells were incubated in the presence of fluvastatin. The reduction in the number of cells was significant with 10−5 M and 10−4 M fluvastatin. The G/S-phase transition of human and porcine vascuar SMC was reduced to 50% of controls by 10−4M fluvastatin, as revealed by cell-cycle analysis. The effects of fluvastatin on growth kinetics and cell-cycle distribution could be completely reversed by the addition of 1 mM mevalonolactone, indicating that the fluvastatin effects are due to specific inhibition of HMG-CoA reductase. The addition of low density lipoprotein as a source of cholesterol failed to support SMC growth and phase transition. Addition of squalene or cholesterol to the culture medium also failed to normalize cell growth. It is concluded that nonsterol products synthesized from mevalonate are necessary for the growth of SMC. HMG-CoA reductase inhibitors, such as fluvastatin, block the synthesis of these nonsterol precursors in human and porcine vascular SMC in vitro and are therefore growth inhibitory.

Effects of flwastatin on growth of porcine and human vascular smooth muscle cells in vitro

The effects of fluvastatin, a new fully synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on growth and cell-cycle kinetics of porcine and human vascular smooth muscle cells (SMC) were studied by growth curves and flow cytometric determination of cellcycle distribution. Growth curves were obtained from counting after 2, 4, 7, 9, 11, and 14 days of incubation in Dulbecco's minimum essential medium and 10% fetal calf serum (FCS). For cell-cycle phase determination, cells were synchronized in G o by 48 hours of incubation in serum-free medium, then stimulated by incubation in 10% FCS, with or without fluvastatin. There was a concentration-dependent decrease in the proliferation of human and porcine SMC when cells were incubated in the presence of fluvastatin. The reduction in the number of cells was significant with 10 −5 M and 10 −4 M fluvastatin. The G/S-phase transition of human and porcine vascuar SMC was reduced to 50% of controls by 10 −4 M fluvastatin, as revealed by cell-cycle analysis. The effects of fluvastatin on growth kinetics and cell-cycle distribution could be completely reversed by the addition of 1 mM mevalonolactone, indicating that the fluvastatin effects are due to specific inhibition of HMG-CoA reductase. The addition of low density lipoprotein as a source of cholesterol failed to support SMC growth and phase transition. Addition of squalene or cholesterol to the culture medium also failed to normalize cell growth. It is concluded that nonsterol products synthesized from mevalonate are necessary for the growth of SMC. HMG-CoA reductase inhibitors, such as fluvastatin, block the synthesis of these nonsterol precursors in human and porcine vascular SMC in vitro and are therefore growth inhibitory.

Mevalonate is essential for growth of porcine and human vascular smooth muscle cells in vitro.

The effects of mevalonate depletion on growth and cell cycle kinetics of porcine and human vascular smooth muscle cells (SMC) were studied by growth curves and flow cytometric determination of cell cycle distribution. Porcine and human SMC were growth arrested by serum depletion for 48 h. Subsequently, they were stimulated to maximal growth and DNA synthesis by addition of serum. There was a concentration dependent decrease in the proliferation of human and porcine SMC, when cells were incubated in the presence of fluvastatin, a new, fully synthetic HMGCoA-reductase inhibitor. The reduction of cell number was significant with 10(-5) M and 10(-4)M fluvastatin. The highest concentration induced cell loss after prolonged incubation (> 4 days). The G/S-phase transition of human and porcine vascular SMC was reduced to 50% of controls by 10(-4) M fluvastatin as revealed by cell cycle analysis. The effects of fluvastatin on growth kinetics and cell cycle distribution could be completely reversed by the addition of 1 mM mevalonolactone. This indicates that the inhibitory effect of fluvastatin is caused by the inhibition of HMGCoA-reductase and depletion of mevalonate rather than being unspecific. Addition of LDL to supply cholesterol failed to support cell growth and transition of smooth muscle cells from G(zero)/G1-phase to S-phase, even though LDL was taken up by the cells as shown by confocal fluorescence microscopy. Neither did the addition of squalene or cholesterol to the culture medium normalize cell growth. It is concluded that nonsterol products that are synthesized from mevalonate are necessary for growth of smooth muscle cells. HMGCoA-reductase inhibitors like fluvastatin block the synthesis of these nonsterol precursors in human and porcine vascular SMC in vitro and are therefore growth inhibitory.

Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury.

Smooth muscle cell proliferation and extracellular matrix accumulation are the principal mechanisms leading to vascular restenosis. We have previously demonstrated the growth-inhibitory effect of antisense oligomers targeting the c-myc proto-oncogene in human smooth muscle cells. The goal of this study was to investigate whether c-myc antisense oligomers reduce neointimal formation in balloon-denuded porcine coronary arteries. First, type I collagen synthesis, which reflects synthetic function, was markedly reduced following c-myc antisense oligomers in porcine vascular smooth muscle cells independent of the growth inhibition. These effects in vitro provided the rationale for assessing c-myc antisense oligomers in the prevention of neointima in vivo. Second, the efficiency of single transcatheter delivery of oligomers into denuded porcine coronary arteries was determined. Despite rapid plasma clearance following local delivery, oligomers persisted at the site of injection for at least 3 days, exceeding by severalfold their concentration in peripheral organs. Third, morphometric analyses were carried out in balloon-denuded coronary arteries at 1 month after transcatheter c-myc antisense oligomer administration. Maximal neointimal area was reduced from 0.80 +/- 0.17 mm2 in the control group (n = 12) to 0.24 +/- 0.06 mm2 in the antisense-treated group (n = 13, P < .01). Likewise, a significant reduction in maximal neointimal thickness was observed in the antisense-treated group (P < .01). These changes in vascular remodeling following denuding injury resulted in an increase in residual lumen from 64 +/- 6% in the control group to 81 +/- 5% in the antisense-treated group (P < .05). (1) Single transcatheter administration allowed for endoluminal delivery of oligomers to the site of coronary arterial injury. (2) C-myc antisense oligomers reduced the formation of neointima in denuded coronary arteries, implying a therapeutic potential of this approach for the prevention of coronary restenosis. (3) It is postulated that the c-myc proto-oncogene is involved in the process of vascular remodeling, regulating smooth muscle cell proliferation and extracellular matrix synthesis.

Effects of cytokines on insulin-like growth factor-binding protein secretion by muscle cells in vitro.

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mouse myocytes (BC3H-I) and porcine vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an [125I]IGF-I binding capacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP-4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulatory effect was markedly suppressed by either TGF-beta 1 (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.

Effects of cytokines on insulin-like growth factor-binding protein secretion by muscle cells in vitro

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mouse myocytes (BC3H-I) and porcine vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an [125I]IGF-I binding capacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP-4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulatory effect was markedly suppressed by either TGF-beta 1 (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.

Role of the lipoxygenase pathway in angiotensin II-induced vascular smooth muscle cell hypertrophy.

The 12-lipoxygenase pathway is a key mediator of angiotensin II (Ang II)-induced effects in the adrenal cortex. We also recently demonstrated that Ang II increases 12- and 15-lipoxygenase product levels in vascular smooth muscle cells. However, the relation between lipoxygenase activation and Ang II-induced vascular smooth muscle cell hypertrophy is not known. We studied the effects of Ang II and 12-lipoxygenase products on both total cell protein content and the levels of the matrix protein fibronectin in quiescent porcine aortic smooth muscle cells. Ang II-induced increases in cellular protein content were attenuated by the specific 12-lipoxygenase inhibitor baicalein; in contrast, the cyclooxygenase inhibitor ibuprofen had no effect. Direct addition of the 12-lipoxygenase product 12-S-hydroxyeicosatetraenoic acid increased total cell protein content. We have recently shown that porcine vascular smooth muscle cell growth is potentiated in high glucose (25 mmol/L) culture conditions. We observed that both Ang II and 12-S-hydroxyeicosatetraenoic acid induced a greater increase in protein content in cells cultured for two passages in high glucose. Furthermore, Ang II and 12-S-hydroxyeicosatetraenoic acid also markedly increased fibronectin levels in cells cultured in high glucose. These results suggest that 12-lipoxygenase activation plays a key role in Ang II-induced vascular smooth muscle cell hypertrophy. Furthermore, both Ang II and lipoxygenase effects are enhanced in cells cultured under hyperglycemic conditions.

Porcine vascular smooth muscle cells immortalized with SV40 ori-defective DNA: characteristics of cell growth and collagen synthesis.

A cell line derived from medial smooth muscle cells (SMC) was established from the porcine coronary artery by transfection with ori-defective simian virus 40 plasmid DNA (SV40 DNA). The characteristics of transfected cells (SV40-SMC) such as cell growth, collagen and non-collagen syntheses were investigated. SV40-SMC expressed SV40 large T antigen, c-myc and c-myb encoded proteins in the nuclei. SV40-SMC demonstrated a 'hills and valleys'-like arrangement in overconfluence and actin filaments upon immunofluorescent staining. Under electron microscopic observation, SV40-SMC had larger amounts of synthetic organelles and smaller amounts of filament bundles than those of SMC. SV40-SMC demonstrated three times higher growth activity and 4.4 times greater cellular density than SMC. Smooth muscle cells did not grow in media containing 5% plasma derived serum (PDS) instead of normal serum, whereas SV40-SMC proliferated in this medium. SV40-SMC did not grow in soft agar gel, while HeLa S3 cells, a cell line of human cervical carcinoma, formed colonies in this gel. By immunofluorescent (IF) staining, collagen phenotypes I, III, IV and V were detected in both SV40-SMC and SMC. However, protein synthesis including collagen and non-collagen was higher in SV40-SMC than in the control sample. It was concluded that SV40-SMC were a continuous cell line for vascular SMC regarding morphological characteristics, and demonstrated a higher growth activity, with increased collagen and non-collagen syntheses. This cell line is useful for the investigation of atherogenesis in relation to a proliferation of SMC and an accumulation of extracellular matrices in vascular intima.

Proliferative synergy of ANG II and EGF in porcine aortic vascular smooth muscle cells

To test growth effects of angiotensin II (ANG II) in porcine vascular smooth muscle cells (VSMC) and potential ANG II synergy with epidermal growth factor (EGF), we exposed subconfluent, near-quiescent porcine aortic VSMC to ANG II, EGF, or ANG II + EGF (each 10(-9) M) in Dulbecco's modified Eagle's-Ham's F-12 medium with insulin + 0.4% fetal calf serum (FCS) selected for minimal ANG II-degrading capacity. Cell number and DNA and protein synthesis (by [3H]-thymidine and [35S]methionine incorporation, respectively) were determined serially over 1-6 days. ANG II alone induced an early 20% increase and then a plateau in cell number over the 0.4% FCS control (P < 0.01; n = 8), thus without sustained increase in proliferation rate. Yet ANG II alone did not increase fractional DNA or protein synthesis (each as cpm/10(3) cells) and, by flow cytometry, reduced S phase fraction without increase in cell size. EGF alone induced brisk DNA synthesis yet minimal cell division over days 0-4, thus late-cycle arrest. ANG II + EGF, despite no increase in fractional DNA or protein synthesis rates over EGF alone, induced significant indomethacin-resistant dose-dependent (P < 0.001) increase in cell proliferation rate over EGF alone with a median effective dose of 5 x 10(-10) M ANG II, thus proliferative synergy. We propose that 1) ANG II induces a subpopulation of cells arrested in or beyond S phase to proceed through mitosis but does not influence G1 traversal or S phase entry and 2) ANG II + EGF achieve proliferative synergy by complementary actions at sequential cell cycle loci, with EGF supporting progression from G0/G1 to S phase and ANG II inducing completion of mitosis by cells already in or beyond S phase ("late-cycle completion").

Modulation of transforming growth factor‐beta 1 stimulated elastin and collagen production and proliferation in porcine vascular smooth muscle cells and skin fibroblasts by basic fibroblast growth factor, transforming growth factor‐α, and insulin‐like growth factor‐I

During tissue repair and development, matrix accumulation is modulated as multiple signals impinge on target cells. We have investigated the effects of combinations of the mitogenic cytokines, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGF-alpha), and insulin-like growth factor-1 (IGF-1) with transforming growth factor-beta 1 (TGF-beta 1) with respect to the production of two matrix components, elastin and type I collagen. Using specific enzyme-linked immunoassays for detection of secreted precursors in both vascular smooth muscle cells and skin fibroblasts from the domestic pig, production of these two fibrous proteins was shown to be strongly stimulated by TGF-beta 1. In the smooth muscle cell, both bFGF and TGF-alpha were potent antagonists of TGF-beta 1-mediated matrix production, whereas IGF-1 was only weakly additive with respect to elastin production. Antagonism was also evident to a lesser extent in skin fibroblasts. Reduced responsiveness to TGF-beta 1 did not appear to be due to a switch to a proliferative state, since TGF-beta 1 itself acted as a mitogen in confluent SMC, and TGF-alpha was only a weak mitogen in confluent fibroblast cultures. Although a predominant effect of TGF-beta is matrix accumulation, these findings suggest that this property will be significantly modified by the cytokine context.

Type V collagen represses the attachment, spread, and growth of porcine vascular smooth muscle cells in vitro

We attempted to clarify the effects of various purified extracellular components, including types I, III, IV, and V collagen and fibronectin on attachment, spread, growth, and DNA synthesis of porcine aortic smooth muscle cells (SMCs) in vitro. The number, area and shape index ( SI = 4π S L 2 ) of cells attached to different substrates were determined at various intervals of incubation. The cell number and [ 3H]thymidine incorporation into DNA were measured on the 1st and 6th days of culture. SMCs showed the largest number of attached cells on fibronectin, but the smallest number of attached cells on type V collagen. There was no evidence of effects of the serum in media on the attachment of SMCs to the substrates. The areas of attached SMCs were the largest on fibronectin and the smallest on type V collagen. The shape index of SMCs on fibronectin decreased relative to those on other substrates. On the 6th day in culture, the number and population doubling of SMCs on type V collagen were significantly fewer than those on other substrates. Both the incorporation rate of [ 3H]thymidine into DNA and the percentage of nuclei labeled with [ 3H]thymidine were significantly less in the SMCs on type V collagen on the 1st day than those on other substrates. SMCs on types I, III, and IV collagen showed intermediate levels of cell attachment, spread, and growth. These results suggest that attachment, spread, and growth of SMCs are affected mainly by solid phase purified extracellular components and are most strongly suppressed by type V collagen. When DNA synthesis of growth-arrested SMCs was reinitiated by the addition of serum, type V collagen most intensively inhibited the rate and amount of [ 3H]thymidine incorporation. Flow cytometric analysis demonstrated an increase in the proportion of cells in G 0 G 1 phase on type V collagen in comparison with that on other substrates. Thus, the antiproliferative effect of type V collagen may relate to inhibition of transition of SMCs from the G 0 G 1 into the S phase.

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells

Incubation of big endothelin-1 (big ET-1, 1–39) with the membrane fraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon, a metalloproteinase inhibitor. When the incubation was performed in the presence of N-ethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentation was abolished by phosphoramidon. The pH profile for IR-ET generation in the presence of NEM was apparently distinct from that observed in the absence of NEM. Reversephase HPLC of the incubation mixture with or without NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (1–21). When the cultured VSMCs were incubated with big ET-1, a conversion to the mature ET-1 was observed. This ET-1 generation from exogenously applied pig ET-1 was markedly inhibited by the addition of phosphoramidon, although the inhibitor did not influence the basal secretion of ET-1-like materials. These results suggest the presence of two types of metalloproteinases, which can generate ET-1, in VSMCs. The possibility that ET-1 functions in an autocrine manner to control the cardiovascular system warrants further attention.

Regional and mechanistic differences in platelet-derived growth factor-isoform-induced alterations in cytosolic free calcium in porcine vascular smooth muscle cells

Three specific platelet-derived growth factor (PDGF) isoforms are thought to bind with differing affinities to two distinct PDGF receptors which undergo activation following dimerization. Recent evidence has been presented that marked differences exist between the ability of PDGF-AA versus PDGF-AB and PDGF-BB to stimulate alterations in second messengers in cultures of vascular smooth muscle cells (VSMC), a result which was thought to be due to low numbers of the A-type receptor in this cell type (Sachinidis, A., Locker, R., Vetter, W., Tatje, D., and Hoppe, J. (1990) J. Biol. Chem. 265, 10238-10243, 1990). In particular, PDGF-BB and PDGF-AB but not PDGF-AA could elicit alterations in cytosolic free calcium (Ca2+i). However, because these studies were performed on large cell populations using biochemical assays of PDGF activity, a minor PDGF-AA-Ca(2+)-responsive population of cells might go undetected. To test this possibility, VSMC were isolated from either thoracic or abdominal pig aorta, and alterations in Ca2+i were monitored using Multiparameter Digitized Video Microscopy following stimulation with PDGF isoforms alone, or either before or after exposure of VSMC to 5 mM EGTA. PDGF-AA-responsive cells were found to exist only in cultures of thoracic VSMC, caused oscillations in Ca2+i, represented 20% of the PDGF-BB-responsive cells, and were subsequently responsive to PDGF-BB. PDGF-BB elicited monophasic alterations in Ca2+i in both thoracic and abdominal VSMC. Prior addition of EGTA inhibited PDGF-AA but not PDGF-BB-induced alterations in Ca2+i. Addition of EGTA during PDGF-AA-induced Ca2+i oscillations inhibited subsequent oscillations in Ca2+i, while addition of EGTA at the peak of the PDGF-BB Ca2+ response resulted in a more rapid return of Ca2+i to prestimulation levels. These data suggest that regional differences in the distribution of PDGF-A- and B-type receptor exists in vivo, and that activation of the A- and B-type PDGF receptors results in distinct alterations in Ca2+i.

Cytosolic calcium transients differ between porcine coronary arterial and aortic smooth muscle cells in primary culture.

Using quin 2 microfluorometry of porcine vascular smooth muscle cells in primary culture at 25 degrees C, we investigated differences in cytosolic calcium transients between epicardial coronary artery and aorta. Both in coronary arterial and aortic smooth muscle cells, histamine induced transient and dose-dependent elevations of cytosolic calcium concentrations, with a similar time course and EC50 (coronary artery, 1.4 x 10(-7) M; aorta, 1.8 x 10(7) M). However, a transient and dose-dependent elevation of cytosolic calcium concentrations was induced by norepinephrine in aortic smooth muscle cells (EC50 = 2.5 x 10(-7) M) but not in coronary arterial smooth muscle cells. Isoproterenol, which produced no change in cytosolic calcium concentrations in aortic vascular smooth muscle cells, significantly and dose dependently decreased concentrations of calcium in coronary arterial smooth muscle cells (EC50 = 1.5 x 10(7) M). Dibutyryl cAMP decreased the concentration of cytosolic calcium both in the coronary arterial and aortic vascular smooth muscle cells with a similar time course and EC50 (coronary artery, 9.8 x 10(-6) M; aorta, 1.1 x 10(-5) M). Intracellular concentration of cAMP was increased in response to isoproterenol, as determined with radioimmunoassay of the coronary arterial smooth muscle cells but not in the aortic cells. Thus, the characteristics of receptors on the sarcolemma may play a key role in the regulation of responsiveness of vascular smooth muscle cells to various vasoactive substances. Aortic smooth muscle cells are alpha-receptor dominant, and activation results in a transient elevation of cytosolic calcium concentrations. The epicardial coronary arterial smooth muscle cells are beta-receptor dominant, and activation results in an increase in cAMP and a reduction of cytosolic calcium concentrations. These results may account for the poor contraction, or relaxation, of epicardial coronary artery induced by sympathetic stimulation and exogenously applied catecholamines.