Heparin sensitive and resistant vascular smooth muscle cells: biology and role in restenosis.

Vascular smooth muscle cells (VSMC)s are characterized by their acute growth inhibition by heparin and heparan sulfates; however, recently the isolation of VSMCs which display greatly diminished sensitivity to the antiproliferative action of heparin have been reported. These heparin resistant (HR) VSMCs have been derived through multiple passage of normal rat VSMCs in culture media containing high heparin doses, by transformation of VSMCs with oncogene-containing vectors, or have been isolated from vascular tissues of spontaneously hypertensive rats, healthy humans, or humans with restenosis where their presence is not limited to sites of injury. Initial characterizations of HR VSMCs are reviewed, and here we propose a definition of HR VSMCs. To date the mechanisms underlying heparin insensitivity remain elusive. Further study of HR VSMCs may expand our understanding of cell growth regulation by heparin, establish whether HR VSMCs contribute to the reported failure of heparin to combat restenosis in humans, and identify cellular mechanisms driving certain vascular proliferative diseases.

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells.

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, there data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways.

Heparin inhibits mitogen-activated protein kinase activation in intact rat vascular smooth muscle cells.

Heparin is potently antiproliferative for vascular smooth muscle cells in vivo and in vitro, inhibiting early proto-oncogene expression and blocking proliferation in the G1 phase of the cell cycle. The mitogen-activated protein kinase (MAPK) family of serine- and threonine-specific kinases is activated in response to a wide range of mitogenic and other factors and is a key intermediate in cell signaling. We found that heparin inhibits activation of MAPK in response to fetal calf serum and phorbol 12-myristate 13-acetate, but not epidermal growth factor, revealing heparin-sensitive and -insensitive pathways of MAPK activation. This report tentatively links suppression of early proto-oncogene expression and inhibition of cellular proliferation by heparin with inhibition of a mitogenically relevant kinase in living cells.

Protamine and protamine-insulins exacerbate the vascular response to injury.

Endothelial cells and smooth muscle cells produce heparinlike compounds that are growth inhibitory for vascular smooth muscle cells, and it has been suggested that these compounds play a regulatory role that is perturbed with vascular injury. Indeed, exogenous heparin preparations effectively suppress smooth muscle cell proliferation following injury imposed on vascular endothelium. We now report that protamine, an agent that binds heparin and negates its anticoagulant properties, has potent stimulatory effects on vascular smooth muscle cell proliferation. The administration of protamine, alone or as part of commonly used insulin preparations, stimulated the proliferation of cultured smooth muscle cells, exacerbated vascular smooth muscle cell proliferative lesions in laboratory rats, and interfered with the growth-inhibitory effects of heparin in culture and in vivo. These results confirm the importance of endogenous heparinlike compounds in arterial homeostasis and may require reconsideration of protamine use following vascular reparative procedures and in diabetics.

Isolation of heparin-insensitive aortic smooth muscle cells. Growth and differentiation.

Previous work has shown heparin and heparan sulfates to be potent inhibitors of vascular smooth muscle cell (VSMC) growth. This laboratory has previously isolated a VSMC line insensitive to the antiproliferative action of heparin by subjecting VSMCs that grew out from rat aortic medial explants to continuous passage in media containing heparin at 200 micrograms/mL. In the present study, we have isolated two additional heparin-resistant (HR) cell lines and have used the HR cells to investigate cellular mechanisms responsible for the potent antiproliferative activity of heparin. In contrast to normal heparin-sensitive VSMCs, the HR cells were smaller, displayed elongated processes, and possessed altered growth characteristics; however, both HR and normal cells bound and internalized comparable amounts of heparin. Immunohistochemical detection of smooth muscle cell-specific actin in growth-arrested cells showed staining of nearly all normal VSMCs and of a much smaller percentage of HR cells; heparin treatment caused a marked increase in the percentage of HR cells expressing smooth muscle cell alpha-actin, indicating that the antiproliferative and differentiation-promoting actions of heparin are independent. Proteins from control VSMCs and HR cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and heparin affinity chromatography. Several proteins were expressed preferentially by either HR cells or normal VSMCs, with the most significant difference being the secretion of a high-affinity, heparin-binding protein (M(r), 38,000) by control VSMCs but not by HR cells. We conclude that the aortic VSMC population may give rise to HR cells under selective conditions and that their unique characteristics, such as alterations in their ability to produce heparin-binding proteins, will prove useful in deciphering the cellular mechanisms involved in heparin's regulation of VSMC growth and differentiation.

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.

Antiproliferative effects of novel, nonanticoagulant heparin derivatives on vascular smooth muscle cells in vitro and in vivo.

The proliferation of vascular smooth muscle cells (VSMC) is strongly inhibited by whole heparin both in vitro and in vivo. To identify and characterize antiproliferative, but nonanticoagulant heparin derivatives, heparin fragments made by periodate treatment were produced and acylated with 2-, 4-, or 6-carbon chain lengths. In culture, the 4- and 6-carbon acylated compounds were more effective than whole heparin in inhibiting serum stimulated VSMC growth at equal mass or approximately equal mean molar concentrations. Further testing was performed in the rat carotid balloon injury model. Myointimal VSMC proliferation produced by balloon catheterization of rat carotid arteries was inhibited by the 4-carbon acylated compound as effectively as heparin at the same mass dose. Importantly, unlike heparin, the 4-carbon acylated compound had no anticoagulant effect in vivo. These experiments suggest nonanticoagulant, acylated heparin derivatives may have a pharmacologic role in preventing myointimal proliferative lesions that are responsible for failures of vascular surgeries and angioplasties.

Heparin inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells.

Heparin is a potent inhibitor of vascular smooth muscle cell (VSMC) growth. In this paper we show that heparin suppressed the induction of c-fos and c-myc mRNA in rat and calf VSMC. This effect of heparin is closely associated with its growth-inhibitory activity, as shown by isolating and characterizing a strain of rat VSMC that was resistant to heparin's antiproliferative effect; heparin did not suppress c-fos mRNA induction in these cells. Moreover, neither a nonantiproliferative heparin fragment or other glycosaminoglycans that lack growth-inhibitory activity repressed c-fos or c-myc mRNA levels. The effect of heparin on c-fos mRNA induction was selective for specific mitogens, as heparin inhibited c-fos mRNA induction in phorbol 12-myristate 13-acetate (TPA) stimulated but not epidermal growth factor (EGF) stimulated VSMC. The effect of heparin on gene expression is independent of ongoing protein synthesis, and inhibition of c-fos mRNA is at the transcriptional level. These results suggest that heparin may selectively inhibit a protein kinase C-dependent pathway for protooncogene induction and that this may be one mechanism used by heparin to inhibit cell proliferation.

Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells.

The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect.

Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway.

Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-fos and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase.

Regulation of cloned prolactin-inducible genes in pigeon crop.

Polyadenylated RNA from PRL-stimulated pigeon (Columba livia) crop was used as template to produce a cloned cDNA library in plasmids. The library was screened by differential hybridization against labeled nucleic acid populations representative of both unstimulated and PRL-stimulated crop tissue. By this method four independent clones coding for PRL-inducible mRNAs were identified. The regulation of these four genes ranged from modest (2- to 3-fold) to major (greater than 70-fold). A clone designated DA4 was complimentary to the most markedly stimulated crop mRNA. This mRNA encoded a polypeptide with a molecular weight of 35,500 which corresponds with the major induced protein synthesized in vivo. Messenger RNADA4 stimulation was dose dependent showing maximal induction by ovine PRL systemic injections in the 200 micrograms/day range. Above this dose PRL was less effective. The onset of mRNADA4 accumulation after a single PRL injection was rapid with statistically significant levels occurring by 3 h. Several lactogenic type hormones, but not an ungulate GH, were potent inducers of mRNADA4. The receptor responsible for mRNADA4 stimulation responds to mammalian lactogens (ovine PRL, human GH, human placental lactogen, bovine placental lactogen) and also can be blocked by an antibody to rabbit mammary gland PRL receptors. These results argue that regulation of pigeon crop gene expression (specifically mRNADA4 may be a relatively simple model of lactogenic hormone mechanisms.

Regulation of pigeon crop gene expression by prolactin.

The pigeon crop has long been known as a target tissue for PRL action. This study investigates the molecular events associated with PRL stimulation of the pigeon crop sac. Several experiments were performed comparing isolates from PRL-injected pigeons (200 micrograms/day, im, for 3 days) to saline-treated pigeons. Electrophoretic analysis of crop protein revealed a general pattern of similarity between PRL-stimulated and control isolates, but four major polypeptides were distinctly dissimilar. Two crop peptides (CP) migrating at molecular weight 16,400 and 29,900 (CP16, CP30) were selectively repressed by PRL treatment. In contrast, two products, CP25 and CP154, were dramatically induced in PRL-stimulated crops. Short (4-h) in vivo labeling using tritiated amino acids revealed that polypeptides comigrating with CP16 and CP30 were synthesized only in control crops. A product migrating with CP25 was selectively labeled in PRL-induced crop tissue. To investigate PRL's effect on mRNA populations, polyadenylated RNA was isolated from control or PRL-stimulated crops and translated in a reticulocyte lysate translation system. Gel electrophoresis and fluorography revealed PRL treatment to cause two major differences in the mRNA fractions: repression of a translatable mRNA coding for a product comigrating with CP16 and a large increase in presumptive CP25 mRNA (to 14% of the translatable message). Translation product profiles of mRNA isolated from crops treated locally revealed a dependence of CP25 mRNA induction on the dose of PRL (16-25 micrograms/day). These results show that PRL acts in a direct manner on the crop to regulate specific gene products. Control of two products results from alteration of mRNA levels.