Downregulation of the ERK 1 and 2 mitogen activated protein kinases using antisense oligonucleotides inhibits proliferation of porcine vascular smooth muscle cells.

The current model of the arterial response to injury suggests that proliferation of vascular smooth muscle cells is a central event. Mitogen activated protein kinases are part of the final common pathway of intracellular signalling involved in cell division and thus constitute an attractive target in attempting to inhibit this proliferation. We hypothesised that antisense oligonucleotides to mitogen activated protein kinase would inhibit serum induced smooth muscle cell proliferation by downregulating the protein. Porcine vascular smooth muscle cells were cultured and an antisense oligonucleotide sequence against the ERK family of mitogen activated protein kinases (AMK1) was introduced by liposomal transfection. Sense oligonucleotides and a random sequence were used as controls. Proliferation was inhibited by AMK1 versus the sense controls, as assessed by tritiated thymidine incorporation (P<0.01). Immunoblots revealed downregulation of the target protein by AMK1 by 63% versus the sense control (P<0.05). In conclusion, antisense oligonucleotides specifically inhibited proliferation and downregulated the target protein. This is consistent with a central role for mitogen activated protein kinases in vascular smooth muscle cell proliferation in the porcine model. In addition, the data suggest a possible role for antisense oligonucleotides in the modulation of the arterial injury response.

Expression of intercellular adhesion molecules in human saphenous veins: effects of inflammatory cytokines and neointima formation in culture.

Atherosclerosis causes occlusions in as many as 50% of human saphenous vein coronary artery bypass grafts. Monocyte infiltration is an early step in saphenous vein-graft atherosclerosis, however, comparatively little is known of its underlying mechanisms. As a first approach, we sought to define the occurrence, location and regulation of leukocyte adhesion molecules in human saphenous vein before and after surgical preparation for grafting, during neointima formation in culture and on stimulation with inflammatory cytokines. We compared the distribution of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and platelet endothelial cell adhesion molecule (PECAM-1 or CD-31) in endothelial cells and smooth muscle cells (SMCs), using immunocytochemistry. ICAM-1 was expressed on endothelial cells before culture and on both endothelial cells and medial or neointimal SMCs after culturing vein for 14 days in 30% foetal bovine serum or after culturing for 24 h with TNF-alpha. Relative tissue levels of ICAM-1 measured by Western blotting were significantly elevated by culturing freshly-isolated (0.02+/-0.01 to 0.18+/-0.03) and surgically-prepared (0.02+/-0.01 to 0.14+/-0.03; n=6) veins or following TNF-alpha treatment of surgically-prepared veins (0.04+/-0.01 to 0.32+/-0.11, n=7). VCAM-1 was undetectable before or after culturing but was strongly upregulated on endothelial cells by incubation with the cytokines TNF-alpha, IL-1alpha or interferon-gamma. PECAM-1 was expressed constitutively on endothelial cells. We conclude that human saphenous vein expresses several adhesion molecules capable of mediating monocyte migration. The increased expression of ICAM-1 in SMC after culturing or cytokine treatment and of VCAM-1 in endothelial cells suggests that interactions with beta1 and beta2 integrins are important pathways for stimulated monocyte ingress into human saphenous vein grafts.

Increased secretion of basement membrane-degrading metalloproteinases in pig saphenous vein into carotid artery interposition grafts.

Late saphenous vein bypass graft failure in humans involves medial and neointimal thickening as the result of migration and proliferation of vascular smooth muscle cells (SMCs). Recent work on angioplasty indicates that basement membrane-degrading metalloproteinases (MMPs) cooperate with growth factors to mediate SMC migration and proliferation. We sought evidence here for a similar role in experimental vein grafts in pigs. Tissue levels and secretion of MMP-2 and MMP-9 were compared by quantitative zymography in veins and grafts removed 2 to 168 days after implantation. Pro and active forms of MMP-2 were present in veins, but levels were increased in vein grafts after 7 days (4- and 6-fold, respectively) and 28 days (3-fold for both), returning to values in veins after 168 days. MMP-9 was not detected in veins, was increased in grafts after 2 days, further increased after 7 days (6-fold) and 28 days (15-fold), and declined to undetectable levels by 168 days. Immunocytochemistry detected increased expression of MMP-2 and MMP-9 with the same time course. MMP-2 was widely distributed, whereas MMP-9 was concentrated in highly proliferative SMCs at the superficial layers of the thickened neointima. We conclude that increased production of the basement membrane-degrading MMP-2 and MMP-9 spanned the period of neointima formation and SMC proliferation in experimental vein grafts. MMPs therefore constitute new therapeutic targets for reducing late vein graft failure.

External stenting reduces long-term medial and neointimal thickening and platelet derived growth factor expression in a pig model of arteriovenous bypass grafting.

Bypass of stenotic coronary arteries with autologous saphenous vein is an established treatment for ischemic heart disease. However, its long-term clinical success is limited. Late vein graft failure is the result of medial and intimal thickening consequent upon medial vascular smooth muscle cell migration, proliferation and extracellular matrix deposition, followed later by superimposed atherosclerosis. These changes directly compromise graft blood flow and provoke thrombosis. Vein graft wall thickening may represent an adaptation imposed by arterial hemodynamic factors, and these factors have been shown to promote vascular smooth muscle cell migration and proliferation through activation of key mediators including platelet-derived growth factor (PDGF). Many pharmacological interventions aimed at preventing these long-term changes have proven unsuccessful in clinical evaluation. We recently demonstrated in a pig saphenous vein graft model that application of an external polyester stent to the outside of carotid interposition vein grafts reduced intimal hyperplasia and total wall thickness 1 month after implantation. However, it is not known whether the benefits of the stent are maintained in the longer term or what mechanisms underlie its effect. The present study therefore compared morphological changes and PDGF expression in stented grafts and contralateral unstented grafts in the same pigs, 6 months after graft implantation. Reduced medial thickening, neointima formation, and cell proliferation were sustained in externally stented grafts, and these effects were associated with a significant reduction in PDGF expression.

Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries.

Basement membrane-degrading metalloproteinases (gelatinases) appear necessary for vascular smooth muscle cell migration and proliferation in culture and for intimal migration of cells after balloon injury to the rat carotid artery. We investigated in the present study the secretion of gelatinases from pig carotid artery tissue after balloon injury. Segments of injured artery and segments proximal and distal to the area of injury were removed 3, 7, and 21 days after balloon dilatation. Medial explants from these segments were then cultured for 3 days, and the serum-free conditioned media were subjected to gelatin zymography. Production of 72- and 95-kD gelatinases was quantified by densitometry. Balloon-injured segments secreted significantly more 72- and 95-kD gelatinase than did paired distal segments at all time points. Release of both gelatinase activities was increased at 3 and 7 days relative to segments from uninjured arteries but declined again by 21 days after balloon injury. Similar results were found for gelatinase levels in extracts of arterial tissue. Consistent with the protein secretion data, in situ hybridization demonstrated that the mRNAs for both gelatinases were upregulated after balloon injury. Expression was prominent in medial smooth muscle cells, particularly around foci of necrosis, and in neointimal cells 3 and 7 days after balloon injury; 72-kD gelatinase mRNA persisted after 21 days and was prominent in regrown endothelial cells. The upregulation of gelatinase activity paralleled the time course of smooth muscle cell migration and proliferation in this model. We conclude that increased gelatinase production occurs in response to balloon injury and may play a role in permitting migration and proliferation of vascular smooth muscle cells.

Extracellular matrix degrading metalloproteinases in the pathogenesis of arteriosclerosis.

We review the importance of extracellular matrix remodelling to the processes of vascular smooth muscle cell migration and proliferation that contribute to morphogenesis of the atherosclerotic plaque. In particular, the role of the matrix degrading metalloproteinase (MMP) family is discussed. This family of neutral, ZN(2+)-requiring enzymes are capable, in principle, of degrading all matrix proteins. Their activity is tightly controlled, however, at the level of synthesis of the inactive zymogens, activation by limited proteolysis and binding to endogenous inhibitor proteins (TIMPs). Direct evidence is presented for the involvement of MMPs in proliferation and outgrowth of vascular smooth muscle cells from explants of rabbit aorta in vitro. This was obtained using two structurally-unrelated inhibitors of MMPs, Ro 31-4724 and Ro 31-7467, both of which inhibited proliferation of cells in a concentration-dependent manner, Ro 31-4724 also inhibited outgrowth. Rabbit aortic smooth muscle cells were further shown to release MMPs, namely a 95 and a 72 kDa gelatinases that were inhibited by Ro 31-4724 and Ro 31-7467. The evidence suggests that degradation of basement membrane by gelatinase is required for proliferation and outgrowth of these cells. The implications of these findings for the pathogenesis and treatment of atherosclerosis are also discussed.

Inhibition of proliferation, but not of Ca2+ mobilization, by cyclic AMP and GMP in rabbit aortic smooth-muscle cells.

The effects on cellular proliferation and Ca2+ mobilization of analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP) and of agents that elevate the intracellular concentrations of cyclic nucleotides were compared in closely similar preparations of first-passage rabbit aortic vascular smooth-muscle cells. Proliferation induced by foetal-bovine serum was inhibited by 78% by 1 mM-8-bromo cAMP and by 42% by 1 mM-8-bromo cGMP. In the presence of 100 microM-isobutylmethylxanthine, 100 microM-forskolin increased intracellular cAMP concentration 5-fold and inhibited proliferation by 87%, but did not affect cGMP concentration or cell viability (ATP concentration). Similarly in the presence of 100 microM-isobutylmethylxanthine, 1 mM-SIN-1 (3-morpholinosydnonimine) elevated cGMP concentration 4-fold and inhibited proliferation by 48%, but did not affect cAMP or ATP concentration. Isobutylmethylxanthine (1 mM) elevated cAMP concentration by 3-fold and cGMP concentration by 20-fold and inhibited proliferation by 81%. Concentrations of 8-bromo cAMP, 8-bromo cGMP, forskolin or SIN-1 that inhibited proliferation did not affect the elevation of intracellular free Ca2+ concentration caused by 2% (v/v) foetal-bovine serum, 100 nM-5-hydroxytryptamine or 10 nM-angiotensin II. The results demonstrate that elevation of intracellular cAMP and cGMP concentrations both independently inhibit vascular smooth-muscle cell proliferation, but these effects on proliferation are not mediated by inhibition of Ca2+ mobilization.

Involvement of extracellular-matrix-degrading metalloproteinases in rabbit aortic smooth-muscle cell proliferation.

We investigated the influence of two structurally unrelated inhibitors of matrix-degrading metalloproteinases, Ro 31-4724 and Ro 31-7467, on the primary proliferation of smooth-muscle cells from rabbit aortic explants. Both agents inhibited proliferation in a concentration-dependent manner, but did not affect cell viability. Smooth-muscle cells grown out from explants secreted 95 kDa and 72 kDa gelatinase enzymes that were also inhibited in a concentration-dependent manner by Ro 31-4724 and Ro 31-7467. Interstitial collagenase and stromelysin were not detected. We conclude that metalloproteinases are likely to be involved in the initiation of smooth-muscle proliferation.

Inhibition of vascular smooth muscle cell proliferation by endothelium-dependent vasodilators.

This review considers the hypothesis that the endothelium-derived vasodilator agents, prostacyclin and nitric oxide, also function physiologically to inhibit vascular smooth muscle cell (VSMC) proliferation. The underlying biochemical mechanisms are also discussed. Prostacyclin and other agents that increase intracellular cAMP concentration are potent and effective inhibitors of the proliferation of isolated VSMC in culture. Such agents inhibit the initiation of proliferation in quiescent cells and the proliferation of logarithmically growing cells from a variety of sources, including man. The data implicate prostacyclin as an important regulator of VSMC proliferation, although there is little direct in vivo evidence. Nitric oxide-releasing drugs (and atriopeptins which increase intracellular cGMP concentration by a different mechanism) also inhibit proliferation of cultured VSMC. The effects are, however, partial and obtained at higher concentrations than those required for vasodilatation. Even allowing for the instability of the agents under tissue culture conditions, cGMP-elevating agents appear to be poorer at inhibiting proliferation than cAMP-elevating agents, despite similar or greater vasodilator potency. These data imply that nitric oxide is less likely than prostacyclin to be a physiological regulator of VSMC proliferation, although definitive experiments in vivo are again lacking. It also follows that nitrovasodilators are less attractive as therapy for VSMC proliferation than prostacyclin analogues or other cAMP-elevating agents, such as phosphodiesterase inhibitors. By analogy with the mechanisms of vasodilatation, inhibition of calcium mobilization and the subsequent activation of protein kinase C are considered as possible mechanisms underlying inhibition of proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)