Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways.

Platelet-derived growth factor BB (PDGF BB) activation of the mitogen-activated protein kinases (MAPK), ERK1 and ERK2, has been shown to be necessary for mitogen-stimulated proliferation, but its role in regulating cell migration and its relationship to other chemotactic signaling events, such as CamKII activation, has not been defined. Using a modified Boyden chamber apparatus, we tested the effects of a selective inhibitor of the upstream activator of ERK1/2, MEK1, on PDGF-stimulated rat aortic vascular smooth muscle cells (VSMCs) alone and in combination with KN62, a selective inhibitor of CamKII. The MEK1 inhibitor, PD98059, caused a dose-dependent reduction in ERK2 activity that paralleled a decrease in migration up to 60%. This inhibition of migration was similar to that seen with KN62 and the combined effects of both inhibitors were non-additive. Although KN62 did not affect ERK2 activity in response to PDGF, PD98059 markedly inhibited PDGF-stimulated CamKII activity, suggesting that activation of CamKII by PDGF was dependent on ERK activity and that the effects of ERK inhibition on migration may be mediated through its ability to inhibit CamKII activity. To directly test this, VSMCs were infected with a recombinant adenovirus expressing constitutively activated CamKII. Infection reversed the inhibitory effects of KN62 on migration, but had no effect on the inhibition of migration seen with PD98059. These results suggest that while MAPK may act upstream of CamKII to control its activation in response to PDGF, it also regulates migration independently of CamKII activation.

The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin- dependent protein kinase II.

The migration of vascular smooth muscle cells (VSMCs) is thought to play a key role in the pathogenesis of many vascular diseases and is regulated by soluble growth factors/ chemoattractants as well as interactions with the extracellular matrix. We have studied the effects of antibodies to rat beta3 and human alphavbeta3 integrins on the migration of VSMCs. Both integrin antibodies as well as cyclic RGD peptides that bind to the vitronectin receptors alphavbeta3 and alphavbeta5 significantly inhibited PDGF-directed migration. This resulted in a reduction in the accumulation of inositol (1,4,5) trisphosphate and the activation of calcium/calmodulin-dependent protein kinase II (CamKII), an important regulatory event in VSMC migration identified previously. PDGF-directed VSMC migration in the presence of the anti-integrin antibodies and cyclic RGD peptides was restored when intracellular CamKII activity was elevated by either raising intracellular calcium levels with the ionophore, ionomycin, or infecting with a replication-defective recombinant adenovirus expressing a constitutively activated CamKII cDNA (AdCMV.CKIID3). Rescue of rat VSMCs was also observed in stably transfected cell lines expressing constitutively activated but not wild-type CamKII. These observations identify a key intermediate in the regulation of VSMC migration by outside-in signaling from the integrin alphavbeta3.

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

The effect of cellular age on zinc levels in untreated and zinc-treated human diploid fibroblasts.

Cellular aging is accompanied by increased cellular permeability to zinc(II). The intrinsic zinc content of human diploid fibroblast cells increases with cell age, so that it quadruples from early to late passage, on a Zn(II) per cell or per cell volume basis, but it remains constant on a Zn(II) per protein basis. When the cells are challenged with toxic concentrations (0.2 mM) of Zn(II), both the rate of zinc incorporation into the cells and the amount of zinc incorporated at equilibrium increases considerably with age (unless measured as zinc per protein). In terms of growth inhibition, Zn(II) is more toxic to the cell than Cu(II), Mn(II), or Mg(II).

The effect of aging on sister chromatid exchange.

The advent of the bromodeoxyuridine(BrdU)-differential staining techniques has greatly facilitated the detection of sister chromatid exchanges (SCE). These SCE have been demonstrated to be an accurate reflection of DNA damage both in vitro in cultured cells and in vivo in mouse and rate bone marrow and spleen cells. In this review, we examine the effect of cellular aging on both baseline and mutagen-induced SCE levels. In all systems examined, aging did not appear to significantly affect the baseline levels of SCE. However, in human fibroblast cultures we have found a significant decrease in the levels of mutagen-induced SCE as a function of both in vitro passage level (in vitro aging) and the age of the cell culture donor (in vivo aging). In addition we have found a similar decrease in mutagen-induced SCE levels in both mouse and rat bone marrow cells and mouse spleen cells where examinations were performed entirely in vivo. Diminished mutagen-induced SCE levels were obtained with a wide variety of agents including mitomycin-C, cyclophosphamide, adriamycin, ethyl methanesulfonate and N-acetyl-2-acetoxyamino-fluorene. These decreased SCE levels were accompanied by increased frequencies of chromosomal aberrations in the older cell populations. If SCE represents a form of DNA repair as has been suggested by several investigators, our finding would indicate impaired DNA repair occurring in old cells.

Isolation of axonally transported glycoproteins with goldfish visual system myelin.

[3H]fucose labeled glycoproteins which are rapidly transported in the goldfish optic nerve have been isolated with purified preparations of goldfish optic tectal myelin. SDS acrylamide gels of myelin proteins isolated one day after intraocular injection of fucose show a broad distribution of radioactivity among high molecular weight proteins. At progressively longer times after injection there is a shift in the distribution of radioactivity with the buildup of a peak of label with electrophoretic mobility corresponding to a molecular weight of approximately 50,000. There is no corresponding peak of radioactivity in gels of total tectal membranes. Gels of myelin labeled through intracranial injections of fucose also show no single prominent peak of radioactivity. Results are discussed with reference to possible mechanisms for the shift in radioactivity patterns on gels and the possible functional significance of the myelin-associated glycoproteins.