Exposure to platelet-derived growth factor modulates the porcine aortic smooth muscle cell response to somatomedin-C

Platelet-derived growth factor (PDGF) is a potent mitogen for smooth muscle cells, but to detect maximal stimulation by PDGF, the cells must be incubated with plasma. Somatomedin-C (Sm-C), a peptide growth factor that is present in plasma, has been shown to interact with PDGF synergistically to stimulate DNA synthesis in cultured fibroblasts. These studies were designed to test the hypothesis that PDGF interacted with Sm-C to stimulate smooth muscle cell replication and to compare the response of this cell type to that of fibroblasts. When PDGF or Sm-C was added individually to smooth muscle cell cultures, each growth factor induced only minimal increases in [3H]thymidine incorporation into DNA (i.e. PDGF, 3,400 +/- 1,120 to 54,900 +/- 1,550 cpm; Sm-C, 3,400 +/- 1,120 to 10,950 +/- 980 cpm). In contrast, addition of increasing concentrations of Sm-C to cultures that were continuously exposed to PDGF in the presence of Sm-C-deficient plasma resulted in a synergistic increase in [3H]thymidine incorporation (3,400 +/- 1,120 to 54,500 +/- 1,800 cpm; P less than 0.001). To determine if Sm-C was required for smooth muscle cell replication, cultures were sequentially exposed to PDGF, followed by Sm-C-deficient plasma. The rate at which the non-Sm-C-exposed cells synthesized DNA was retarded compared to that of cells exposed to Sm-C-containing plasma; however, 68% nuclear labeling was present after 44 h of incubation. To exclude the possibility that some cellular secretory product was substituting for Sm-C, the medium was changed every 2 h and replaced by fresh Sm-C-deficient medium. Using these test conditions, exposure to PDGF and Sm-C-deficient plasma induced only 11% labelling. Readdition of pure Sm-C to this medium restored nuclear labeling to 82% at 44 h. Other variables that appeared to modulate the cellular response to Sm-C were culture density and simultaneous PDGF exposure. Sm-C and PDGF both appear to be potent mitogens for porcine aortic smooth muscle cells, and when added together to quiescent cultures, their effects are synergistic. Smooth muscle cells appear to require Sm-C to initiate DNA synthesis, and in its absence produce a Sm-like factor that can partially compensate for Sm-C deficiency and allow replication.     

Stimulation of Na-dependent phosphate transport by platelet-derived growth factor in rat aortic smooth muscle cells

We investigated the effect of platelet-derived growth factor B homodimer (PDGF-BB) on inorganic phosphate (Pi) transport activity, which has been reported to be involved in the mechanism of atherosclerosis, in A-10 rat aortic vascular smooth muscle cells (VSMCs). PDGF-BB time- and dose-dependently stimulated Pi transport in A-10 cells. Using northern blot analysis, the PDGF-BB-enhanced Pi transporter (PiT) in A-10 cells was identified as Pit-1 (Glvr-1), a member of the type III Na-dependent PiT. An inhibitor of PDGF beta-receptor tyrosine kinase suppressed PDGF-BB-induced Pi transport. Both a protein kinase C (PKC) inhibitor calphostin C and PKC down regulation suppressed the stimulatory effect of PDGF-BB on Pi transport. On the other hand, inhibition of mitogen-activated protein (MAP) kinases by selective inhibitors did not affect Pi transport. Ly294002, a phosphatidylinositol (PI) 3-kinase inhibitor, partially attenuated PDGF-BB-induced Pi transport. A selective inhibitor of S(6) kinase, rapamycin, reduced this effect of PDGF-BB, while Akt kinase inhibitor did not. In summary, these results indicated that PDGF-BB is a potent and selective stimulator of Pi transport in VSMCs. The mechanism responsible for this effect is not mediated by MAP kinase, but involves activation of PKC, PI 3-kinase and S(6) kinase.     

Two platelet-derived growth factor receptors in vascular smooth muscle cells.

Distinct genes encode alpha and beta PDGF receptors which differ in their abilities to be triggered by three dimeric forms of the PDGF molecules. By use of a strategy involving introduction of expression vectors for alpha and beta PDGF receptor cDNA into the cells originally lacking these receptors, we demonstrated that each receptor was able to couple independently with mitogenic signal transduction pathways inherently present in these cells. Moreover, both receptors were capable of inducing a readily detectable chemotactic response. The vascular smooth muscle cells which express both types of PDGF receptors are mitogenic and chemotactic for PDGFs. Moreover, the alpha receptor is the preferred receptor for platelet PDGF-AB as well as the PDGF-AA isoform which is ubiquitously produced in many cells forming atherosclerotic plaques including macrophages, endothelial cells and even arterial smooth muscle cells. Our results indicated that the availability of specific PDGF isoforms and the relative expression of each receptor gene product appear to be major determinants of the PDGF response. An understanding of the mechanisms by which the expression of PDGF and their receptors on vascular smooth muscle cells are regulated will give greater insights as to how these gene products are involved in atherosclerosis.     

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.     

Synergistic roles of platelet-derived growth factor-BB and interleukin-1beta in phenotypic modulation of human aortic smooth muscle cells

The phenotype of smooth muscle cells (SMCs) plays an important role in vascular function in health and disease. We investigated the mechanism of modulation of SMC phenotype (from contractile to synthetic) induced by the synergistic action of a growth factor (platelet-derived growth factor, PDGF-BB) and a cytokine (interleukin, IL-1beta). Human aortic SMCs grown on polymerized collagen showed high expression levels of contractile markers (smooth muscle alpha-actin, myosin heavy chain, and calponin). These levels were not significantly affected by PDGF-BB and IL-1beta individually, but decreased markedly after the combined usage of PDGF-BB and IL-1beta. PDGF/IL-1beta costimulation also induced a sustained phosphorylation of Akt and p70 ribosomal S6 kinase (p70S6K). The effects of PDGF/IL-1beta costimulation on contractile marker expression and Akt and p70S6K phosphorylation were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 and by adenovirus expressing a dominant-negative Akt, and they were mimicked by constitutively active Akt. PDGF-BB/IL-1beta induced a sustained phosphorylation of PDGF receptor (PDGFR)-beta and its association with IL-1 receptor (IL-1R1). Such activation and association of receptors were blocked by a PDGFR-beta neutralizing antibody (AF385), an IL-1R1 antagonist (IL-1ra), as well as a specific inhibitor of PDGFR-beta phosphorylation (AG1295); these agents also eliminated the PDGF-BB/IL-1beta-induced signaling and phenotypic modulation. PDGF-BB/IL-1beta inhibited the polymerized collagen-induced serum response factor DNA binding activity in the nucleus, and this effect was mediated by the PDGFR-beta/IL-1R1 association and phosphatidylinositol 3-kinase/Akt/p70S6K pathway. Our findings provide insights into the mechanism of SMC phenotypic modulation from contractile to synthetic, e.g., in atherosclerosis.     

Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor.

Smooth muscle cells use fibronectin to bind to type I and type III collagens but bind to type V collagen by a trypsin-resistant intrinsic glycoconjugate. The binding site on type V collagen is located in the alpha A chain. By using collagen-coated filters in a modified Boyden chamber assay for chemotaxis, it was observed that the platelet-derived growth factor was chemotactic for smooth muscle cells but that several other growth factors were inactive. We suggest that the migration of smooth muscle cells from the media to the intima of a blood vessel, which leads to the formation of an atherosclerotic plaque, may be the result of a chemotactic migration of cells responsive to the platelet-derived growth factor.     

Sex steroids modulate human aortic smooth muscle cell matrix protein deposition and matrix metalloproteinase expression

Large artery stiffening increases cardiovascular risk and promotes isolated systolic hypertension which is more prevalent in elderly women than men. Variation in sex steroid levels between males and females and throughout life may modulate arterial stiffness. We hypothesized that sex steroids directly influence expression of important structural proteins which determine arterial biomechanical properties. Human aortic smooth muscle cells were incubated with physiological concentrations of 17beta-estradiol, progesterone, 17beta-estradiol and progesterone, or testosterone for 4 weeks. Collagen, elastin, and fibrillin-1 deposition was examined (histochemistry/immunohistochemistry). Gene and protein expression of 2 important matrix metalloproteinases (MMPs), MMPs 2 and 3, regulating matrix turnover was assessed. All sex steroids reduced collagen deposition relative to control (100%). However, the reduction was greater with female sex steroids than testosterone (control, 100%; 17beta-estradiol plus progesterone, 20+/-2%; testosterone 74+/-12%, P<0.001). Female sex steroids increased elastin deposition compared with control (control, 100%; 17beta-estradiol, 540+/-60%; progesterone, 290+/-40%; 17beta-estradiol plus progesterone, 400+/-80%, all P<0.01). The elastin/collagen ratio was >11-fold higher in the presence of 17beta-estradiol and progesterone compared with testosterone. Fibrillin-1 deposition was doubled in the presence of female sex steroids (17beta-estradiol plus progesterone) compared with testosterone (P<0.01). MMP-2 gene and protein expression was unaffected by any sex steroid. Testosterone increased both gene and protein expression of MMP-3 relative to both control and female sex steroids (P<0.01). This may contribute to degradation of elastic matrix proteins. In conclusion, female sex steroids promote an elastic matrix profile, which likely contributes to variation in large artery stiffness observed between sexes and with changes in hormonal status across the lifespan.     

同型半胱氨酸对人血管平滑肌细胞基质金属蛋白酶-9和基质金属蛋白酶抑制物-1表达的影响

目的探讨同型半胱氨酸(Hcy)对人血管平滑肌细胞(VSMCs)基质金属蛋白酶(MMP)-9和基质金属蛋白酶抑制物(TIMP)-1表达的影响。方法体外培养人VSMCs,分别应用逆转录聚合酶链反应(RT-PCR)法和Westernblot蛋白印迹法检测MMP-9mRNA、TIMP-1mRNA和蛋白表达。结果人VSMCs在不同浓度Hcy(100、200、500、1000μmol/L)孵育24h后MMP-9mRNA和蛋白的表达均显著高于对照组,并提示Hcy呈剂量依赖性诱导人VSMCsMMP-9的表达;但对TIMP-1mRNA和蛋白的表达无影响。结论Hcy可呈剂量依赖性诱导人VSMCsMMP-9的mRNA表达,而对TIMP-1mRNA的表达无明显影响,表明Hcy是在转录水平调控MMP-9的基因表达,这一效应可能是Hcy导致动脉粥样硬化的分子机制之一。     

Prostaglandin E1 inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor

The effect of prostaglandins (PG) on initiation of DNA synthesis in arterial smooth muscle cells (SMC) stimulated with platelet-derived growth factor (PDGF) was examined. A concentration of 10 ng/ml PGE1 inhibited DNA synthesis, measured as autoradiographically labeled nuclei, by about 70%. Similar results were obtained with PGE2 and PGD2 but at concentrations 10-20 times higher, whereas PGF2 alpha lacked effect. The inhibitory action of the prostaglandins was restricted to the first 6 h of the lag phase. Treatment with PGE1 also raised the intracellular concentration of cyclic AMP, indicating that the inhibition may be mediated via changes in the levels of cyclic nucleotides.     

Release of active tissue factor by human arterial smooth muscle cells

Tissue factor (TF), the initiator of coagulation, is thought to function predominantly at the cell surface. Recent data have suggested that active TF is present extracellularly in atherosclerotic plaques, the arterial wall, and the blood. This study was conducted to determine whether smooth muscle cells (SMCs), a major source of arterial TF, could generate extracellular TF. Active TF accumulated in the medium of cultured human SMCs, representing approximately 10% of that measured in the underlying cells at 24 hours. Platelet-derived growth factor, phorbol ester, and tumor necrosis factor-alpha caused approximately 3-fold increases in TF activity in the medium. Release of TF into the medium was dependent on the presence of the TF transmembrane domain but not the cytoplasmic domain. Antibodies to TF precipitated most of the activity from the culture medium, whereas antibodies to the beta(1)-integrin subunit precipitated approximately 33% of the activity. Treatment with detergent or phosphatidylserine:phosphatidylcholine did not increase activity, suggesting that all TF released by SMCs was in the appropriate lipid milieu and not encrypted. Western blotting showed that the medium contained full-length TF protein. Fluorescent cytometry showed that extracellular TF was present largely in particles < or =200 nm, which had a density of 1.10 g/mL. We hypothesize that active extracellular TF found in the injured arterial wall and atherosclerotic plaques derives, in part, from SMC microparticles.     

Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells

Loss-of-function mutations in the human ERG1 potassium channel (hERG1) frequently underlie the long QT2 (LQT2) syndrome. The role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous, loss-of-function LQT2 syndrome mutation. The hERG N629D mutation was introduced into the orthologous mouse gene, mERG, by homologous recombination in mouse embryonic stem cells. Intact homozygous embryos showed abrupt cessation of the heart beat. N629D/N629D embryos die in utero by embryonic day 11.5. Their developmental defects include altered looping architecture, poorly developed bulbus cordis, and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of I(Kr) function, depolarized resting potential, prolonged action potential duration (LQT), failure to repolarize, and propensity to oscillatory arrhythmias. N629D/N629D myocytes manifest calcium oscillations and increased sarcoplasmic reticulum Ca(+2) content. Although the N629D/N629D protein is synthesized, it is mainly located intracellularly, whereas +/+ mERG protein is mainly in plasmalemma. N629D/N629D embryos show robust apoptosis in craniofacial regions, particularly in the first branchial arch and, to a lesser extent, in the cardiac outflow tract. Because deletion of Hand2 produces apoptosis, in similar regions and with a similar final developmental phenotype, Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. In conclusion, loss of I(Kr) function in N629D/N629D cardiovascular system leads to defects in cardiac ontogeny in the first branchial arch, outflow tract, and the right ventricle.     

重组人结缔组织生长因子对人成骨细胞膜型基质金属蛋白酶1及基质金属蛋白酶2表达的影响

使用重组人结缔组织生长因子(recombinant human connective tissue growth factor,rCTGF)干预体外培养的人成骨细胞,发现rCTGF可呈时间-剂量依赖性地促进人成骨细胞膜型基质金属蛋白酶1及基质金属蛋白酶2的表达,200 ng/ml CTGF作用24～48 h达最大效果.rCTGF可明显增强p38丝裂原活化的蛋白激酶(p38-MAPK)磷酸化,p38-MAPK阻断剂SB 23058可阻断rCTGF上调膜型基质金属蛋白酶1及基质金属蛋白酶2的效应.

Abstract：

Human osteoblast was treated with recombinant human connective tissue growth factor (rCTGF). This experiment showed that rCTGF increased membrane type-1 matrix metalloproteinase and matrix metalloproteinase-2 protein expression in a dose- and time-depentent manner in human osteoblasts. rCTGF induced activation of p38 MAPK in human osteoblasts. p38 MAPK inhibitor SB23058 abrogated the effect of rCTGF on the expressions of membrane type-1 matrix metalloproteinase and matrix metalloproteinase-2 in human osteoblasts.     

Sphingosine 1-phosphate transactivates the platelet-derived growth factor beta receptor and epidermal growth factor receptor in vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid generated during vascular injury that regulates cell growth, differentiation, survival, and motility via endothelial differentiation gene (EDG) family G protein-coupled receptors. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), S1P-stimulated receptor tyrosine kinase transactivation has not been well studied. We show that platelet-derived growth factor beta receptor (PDGFbetaR) and EGFR are tyrosine phosphorylated in response to S1P in rat aortic vascular smooth muscle cells (VSMCs). S1P-stimulated transactivation of PDGFbetaR and EGFR was mediated via Gi-coupled EDG receptors. S1P-stimulated transactivation of EGFR and PDGFbetaR was dependent on Src, reactive oxygen species, and cholesterol-rich membranes. A phosphoinositide 3-kinase-Akt pathway was activated by S1P and blocked by AG1296 and AG1478. Activation of extracellular signal-regulated kinase (ERK) 1 and ERK2 pathway by S1P was blocked only by AG1478. In Chinese hamster ovary cells that expressed exogenous EDG-1, activation of Akt and ERK1/2 in response to S1P was observed and was enhanced by coexpression of PDGFbetaR or EGFR. S1P-mediated VSMC proliferation was shown to be secondary to transactivation, because it was suppressed by AG1296 and AG1478. These data establish S1P as an important stimulus for EGFR and PDGFbetaR activation in VSMCs that may have important implications for the vessel response to injury.     

Hammerhead ribozyme targeting human platelet-derived growth factor A-chain mRNA inhibited the proliferation of human vascular smooth muscle cells

Platelet-derived growth factor (PDGF) A-chain contributes to the pathogenesis of cardiovascular proliferative diseases, such as hypertensive vascular disease, atherosclerosis, and re-stenosis of an artery after angioplasty. To develop a ribozyme against human PDGF A-chain mRNA as a gene therapy for human arterial proliferative diseases, we designed and synthesized a 38-base hammerhead ribozyme to cleave human PDGF A-chain mRNA at the GUC sequence at nucleotide 591. In the presence of MgCl(2), synthetic hammerhead ribozyme to human PDGF A-chain mRNA cleaved the synthetic target RNA to two RNA fragments at a predicted size. Doses of 0.01-1.0 microM hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited angiotensin II (Ang II) and transforming growth factor (TGF)-beta(1)-induced DNA synthesis in vascular smooth muscle cells (VSMC) from human in a dose-dependent manner. One micromolor of hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited Ang II-induced PDGF A-chain mRNA and PDGF-AA protein expressions in VSMC from humans. These results indicate that the designed hammerhead ribozyme to human PDGF A-chain mRNA effectively inhibited growth of human VSMC by cleaving the PDGF A-chain mRNA and inhibiting the PDGF-AA protein expression in human VSMC. This suggests that the designed hammerhead ribozyme to PDGF A-chain mRNA is a feasible gene therapy for treating arterial proliferative diseases.