1. Transforming growth factors-beta (TGF-beta) are multifunctional proteins that regulate cell growth, differentiation, migration and extracellular matrix production and have an important role in ...embryonic development and tissue remodelling. 2. The diverse biological actions of TGF-beta are elicited following their interaction with type I and type II TGF-beta receptors, both of which are transmembrane serine/threonine kinases, suggesting an important role for protein phosphorylation in the mechanism of action of these cytokines on the growth of cells and their extracellular environment. 3. Alterations in TGF-beta gene expression and action in various cell types associated with the cardiovascular system may contribute to the pathophysiology of a number of diseases, such as hypertension, atherosclerosis and restenosis, as well as the development of cardiac abnormalities.
Vascular smooth muscle cells (VSMCs) are known to produce activins, bone morphogenetic proteins and transforming growth factor-βs (TGF-βs). To determine whether these TGF-β superfamily members exert ...autocrine effects on VSMCs we examined whether specific type I receptors (ALKs) for such peptides were expressed by the cells. RNA from both quiescent or growth-factor-activated VSMCs was reverse transcribed then cDNAs encoding ALK-2, ALK-3, ALK-5, and ALK-6 were amplified and characterised using specific PCR primers. All four ALK mRNAs were abundantly expressed. The ALK-5 fragment harbored a deletion of 12 nucleotides, removing 4 extracellular amino acids (Gly-Pro-Ser-Val) adjacent to its transmembrane domain. This deletion, which may arise from anomalous splicing of heteronuclear RNA, is likely to influence formation of the ALK-5:type II receptor complex through conformational changes associated with removal of a putative hinge region containing proline. Our finding that multiple ALKs are expressed by VSMCs would account for the multiplicity of effects TGF-β peptides exert on these cells.
1. Over the past decade major advances in molecular cell biology have greatly increased our understanding of the way in which many growth factor genes are expressed and regulated. This knowledge is ...currently being translated into investigations of the cardiovascular system. 2. Two growth factor families appear to play particularly important roles, the fibroblast growth factors and the transforming growth factors-beta. These are multifunctional growth factors capable of remodelling the vasculature through their effects on cell migration, proliferation and matrix formation. 3. An understanding of their regulation, properties and nature of their receptors is providing novel insights into the physiology and pathobiology of the vasculature. It is also providing highly specific targets for future therapy.
To investigate whether the differences in the growth properties between vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats depend ...on the type and nature of the growth factor.
The growth characteristics of VSMC from SHR and WKY rats were compared in the presence of different growth factors. These were related to growth factor receptor expression on the VSMC.
Growth rates, cell densities at which VSMC become quiescent and 3H-thymidine incorporation into DNA were examined in VSMC from SHR and WKY rats following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) or the three isoforms of platelet-derived growth factor (PDGF-AA, PDGF-AB and PDGF-BB). Receptor expression (receptor binding and Northern analysis) for these growth factors was also examined on VSMC from the two strains.
VSMC from SHR exposed to the individual growth factors exhibited growth rates higher than those from WKY rats. For EGF, bFGF, PDGF-AB and PDGF-BB there was no difference in the sensitivity of VSMC to stimulation of 3H-thymidine incorporation between the strains. In contrast, VSMC from SHR exhibited significantly increased sensitivity to the mitogenic effects of PDGF-AA. VSMC from SHR exposed to PDGF-BB or EGF proliferated and then became quiescent at densities approximately 50 and 200% higher, respectively, than those from WKY rats. In contrast, in the presence of bFGF, proliferating VSMC from WKY rats became quiescent at densities approximately 20% higher than those from SHR. Scatchard analysis of 125I-labelled growth factor binding to VSMC from the two strains revealed similar receptor numbers and dissociation constants for all growth factors. Steady-state messenger RNA levels for the different receptors were also similar and could not account for the enhanced growth rates of VSMC from SHR.
The rate and magnitude of the proliferative response elicited in VSMC cultures from SHR and WKY rats is critically dependent on the nature of the growth factor stimulus.
Recent evidence indicates that the type II transforming growth factor-
β (TGF-
β) receptor (T
βRII) is a serine-threonine-tyrosine kinase. However, the significance of its tyrosine kinase is unclear. ...We investigated in vascular smooth muscle cells the effects of tyrosine kinase inhibition on the expression of TGF-
β receptor types I (ALK-5) and II (T
βRII) mRNA, induced by TGF-
β
1. TGF-
β
1 elevated ALK-5 mRNA levels 5-fold; essentially similar TGF-
β
1-dependent elevations were observed with growth factors, PDGF-BB and FGF-2. The tyrosine kinase inhibitor genistein abolished these TGF-
β
1 and growth factor responses. TGF-
β
1 also elevated T
βRII mRNA levels which were not inhibited by genistein. We conclude that tyrosine kinases participate in defining how cells respond to TGF-
β.
The effects of modulation of rabbit aortic smooth muscle cells (SMCs) from the ‘contractile’ phenotype on surface membrane receptors binding epidermal growth factor (EGF), basic fibroblast growth ...factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB), as well as their responsiveness to these growth factors was investigated in cell culture. Cells predominantly of the ‘contractile’ phenotype expressed low numbers of high affinity EGF and bFGF receptors (EGFr: 1.09 ± 0.18 fmol/10
6 cells; bFGFr: 0.32 ± 0.07 fmol/10
6 cells). Upon modulation from the ‘contractile’ phenotype, the expression of these cell surface receptors increased greatly: 8- and 11-fold with respect to EGF and bFGF receptors. Cell surface receptors binding
125I-PDGF-BB were largely unaltered. The elevated bFGF receptor number appeared dependent on SMC modulation from the ‘contractile’ phenotype and serum; the latter factor did not influence EGF receptor numbers. In both instances the increase in receptor numbers was independent of the proliferative status of the cells. Cells expressing high levels of the growth factor receptors also rapidly entered the cell cycle, proliferated, and exhibited growth factor-specific changes in shape in the presence of these growth factors. Because the effects on growth factor receptor numbers were observed in confluent cells, such alterations are likely to play a significant role in vessel remodelling following balloon catheter angioplasty, in atherosclerotic vessels and the vascular hypertrophy associated with hypertension.
We examined the mechanisms by which Ca2+ channel antagonists inhibit the growth of smooth muscle cells by determining their effect on epidermal growth factor (EGF)-stimulated (i) induction of the ...early signalling gene, c-fos, (ii) incorporation of 3Hthymidine into cells as a measure of DNA synthesis, and (iii) increase in cell number. Verapamil, diltiazem, and the dihydropyridines felodipine, MDL 72892 A-15 (MDL) and nisoldipine had no effect on EGF-stimulated c-fos mRNA induction. Furthermore, only small inhibitory effects were observed on EGF-stimulated increases in cell number; felodipine, MDL, and nisoldipine at 0.3 microM inhibited EGF-stimulated cell proliferation by 9, 11, and 15%, respectively. In contrast, the dihydropyridine Ca2+ channel antagonists were found to be potent inhibitors of 3Hthymidine incorporation suggesting that they inhibit DNA synthesis. However, further examination revealed that the potent effects of dihydropyridine Ca2+ channel antagonists on 3Hthymidine incorporation were due not to an effect on incorporation of 3Hthymidine into DNA, but to a marked inhibitory effect on the cellular uptake of 3Hthymidine. Thus, we conclude that the small antiproliferative effects of the dihydropyridine antagonists are predominantly due to their ability to inhibit the activity of the salvage pathway for thymidylate synthesis in human vascular smooth muscle cells.
1. This study examined and compared the growth characteristics of vascular smooth muscle cells (VSMC) isolated from 4 and 12 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats ...following stimulation with platelet-derived growth factor-BB (PDGF-BB). 2. Vascular smooth muscle cells from 4 week old SHR proliferated at a slower rate than VSMC isolated from 12 week old SHR (1.08 +/- 0.06/day vs 1.89 +/- 0.13/day respectively, P < 0.05). In contrast, there was no difference in the proliferation of VSMC from 4 and 12 week WKY rats (0.62 +/- 0.06/day vs 0.82 +/- 0.13/day, respectively, P > 0.05). 3. The cell density at which VSMC from 4 and 12 week old SHR become refractory to the mitogenic effects of PDGF-BB was similar and approximately two-fold greater than VSMC from age-matched WKY rats. 4. This study concludes that there is an age-dependent, differential and specific upregulation of growth rate mechanisms in VSMC from SHR which enhance proliferation in response to PDGF-BB.
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