Distinct roles of E2F proteins in vascular smooth muscle cell proliferation and intimal hyperplasia.

Intimal hyperplasia (IH) and restenosis limit the long-term utility of bypass surgery and angioplasty due to pathological proliferation and migration of vascular smooth muscle cells (VSMCs) into the intima of treated vessels. Consequently, much attention has been focused on developing inhibitory agents that reduce this pathogenic process. The E2F transcription factors are key cell cycle regulators that play important roles in modulating cell proliferation and cell fate. Nonselective E2F inhibitors have thus been extensively evaluated for this purpose. Surprisingly, these E2F inhibitors have failed to reduce IH. These findings prompted us to evaluate the roles of different E2Fs during IH to determine how selective targeting of E2F isoforms impacts VSMC proliferation. Importantly, we show that E2F3 promotes proliferation of VSMCs leading to increased IH, whereas E2F4 inhibits this pathological response. Furthermore, we use RNA probes to show that selective inhibition of E2F3, not global inhibition of E2F activity, significantly reduces VSMC proliferation and limits IH in murine bypass grafts.

Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms.

OBJECTIVE: Because tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of vein graft neointimal hyperplasia, we sought to determine mechanisms by which TNF could induce proliferative and migratory responses in smooth muscle cells (SMCs). METHODS AND RESULTS: In rabbit jugulocarotid interposition vein grafts, SMCs expressed TNF as early as four days postoperatively. In rabbit aortic SMCs, TNF and platelet-derived growth factor (PDGF) elicited comparable migration (1.7-fold/basal), and their effects were partially additive. In contrast, while TNF failed to promote SMC [(3)H]thymidine incorporation alone, it doubled the [(3)H]thymidine incorporation observed with PDGF alone. To gain mechanistic insight into these phenomena, we found that TNF and PDGF each activated p38(mapk) equivalently in SMCs, but that PDGF was two to three times more efficacious than TNF in activating SMC extracellular signal-regulated kinases (ERK) 1 and 2 and phosphoinositide 3-kinase. However, only TNF activated NF kappa B. SMC [(3)H]thymidine incorporation that depended on TNF, but not PDGF, was abolished by overexpression of a dominant-negative I kappa B alpha mutant. Inhibition of ERK activation by U0126 reduced SMC migration stimulated only by PDGF (by 35%, P<0.05), but not by TNF. Inhibition of phosphoinositide 3-kinase by LY294002, however, significantly reduced both TNF- and PDGF-stimulated chemotaxis (by 38-54%, P<0.05). In contrast, both U0126 and LY294002 abolished SMC [(3)H]thymidine incorporation induced by either TNF, PDGF, or both agonists. CONCLUSIONS: In primary rabbit SMCs, TNF promotes migration and mitogenesis through signaling mechanisms that are both distinct from and overlapping with those employed by PDGF. TNF-induced SMC mitogenesis requires complementary co-stimulation with other growth factors.

Systemic basic fibroblast growth factor induces favorable histological changes in the corpus cavernosum of hypercholesterolemic rabbits.

PURPOSE: Hypercholesterolemia causes erectile dysfunction that is associated with abnormalities in vascular smooth muscle and endothelial cells. We determined the effects of basic fibroblast growth factor (bFGF) on corporeal tissue in hypercholesterolemic rabbits. MATERIALS AND METHODS: A total of 16 New Zealand White rabbits were fed a 1% cholesterol diet for 6 weeks and were randomly divided into 3 groups. Group 1 (5 rabbits) received 2.5 microg recombinant bFGF intravenously once and again 3 weeks later. Group 2 (6 rabbits) received 2.5 microg bFGF intravenously once and placebo 3 weeks later. Group 3 (5 rabbits) received placebo intravenously each time. Rabbits were continuously fed a 1% cholesterol diet and sacrificed 3 weeks after the last treatment. Smooth muscle, endothelial cell and collagen content were assessed by immunohistochemistry and histochemical staining of corporeal tissue. Vascular endothelial growth factor (VEGF) protein and mRNA expression were assessed by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. RESULTS: Corporeal smooth muscle content was greater in groups 1 and 2 (35.24% +/- 4.25% and 24.79% +/- 3.39%, p <0.01) vs group 3 (19.68% +/- 2.94%, vs groups 1 and 2 p <0.001 and <0.05, respectively). Endothelial cell and collagen content were similar among the groups. VEGF protein was increased in group 1 vs group 2 (97.90 +/- 26.00 vs 57.03 +/- 14.99 pg/ml, p <0.01) and vs group 3 (39.93 +/- 15.08, p <0.01). There was no statistical difference between groups 2 and 3. VEGF mRNA expression was similar among the groups. CONCLUSIONS: Systemic bFGF increases smooth muscle content and VEGF protein in hypercholesterolemic rabbit corporeal tissue.

Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia.

The activation of vascular smooth muscle cells (SMCs) in neointimal hyperplasia involves signaling through receptor tyrosine kinases as well as G protein-coupled receptors. Overexpression of G protein-coupled receptor kinase-2 (GRK2) in SMCs can attenuate mitogenic signaling and proliferation in response to not only several G protein-coupled receptor agonists, but also platelet-derived growth factor (PDGF). To test whether overexpression of GRK2 could inhibit other SMC responses implicated in neointimal hyperplasia, we assessed SMC chemotaxis and mitogenic signaling evoked by PDGF and G(q)-coupled receptor agonists. To test the effects of GRK2 overexpression on neointimal hyperplasia in vivo, we employed a rabbit autologous vein graft model system. GRK2 overexpression reduced PDGF-promoted SMC chemotaxis by 85% (P<0.01), but had no effect on chemotaxis promoted by epidermal growth factor (EGF). Congruently, GRK2 overexpression reduced by approximately 50% (P<0.05) the [(3)H]thymidine incorporation induced by combinations of PDGF and Gq-coupled receptor agonists, but had no effect on that induced by PDGF plus EGF. PDGF-, but not EGF-promoted phosphoinositide 3-kinase activity in SMCs was also inhibited by GRK2 overexpression. In rabbit vein grafts, we achieved GRK2 overexpression in medial SMCs, reduced cell proliferation during the first week after graft implantation, and reduced steady state neointimal thickness by 29% (P<0.01), without affecting medial thickness or potentiating SMC apoptosis. Because of its ability to dampen chemotactic and mitogenic signaling through PDGF and Gq-coupled receptors, GRK2 overexpression in SMCs may be a useful therapeutic approach for neointimal hyperplasia.

Neointimal hyperplasia rapidly reaches steady state in a novel murine vein graft model.

OBJECTIVE: Neointimal hyperplasia remains a principal cause of vein graft failure. Genetic contributions to vein graft neointimal hyperplasia could be well studied in the mouse; however, surgical approaches to vein bypass surgery in the mouse have yet to replicate approaches commonly employed in human patients. Consequently, the goal of this study was to develop a murine interposition vein graft model that reproduces characteristics of human vein graft disease. METHOD: Using C57BL/6J mice, we excised inferior venae cavae (IVCs) from donor mice and grafted them, with end-to-side anastomosis, into the carotid circulation of recipients. IVC grafts were harvested from 3 to 56 days postoperatively, and analyzed for the development of neointima and media. RESULTS: Thickening of both the vein graft neointima and media progressed rapidly between postoperative weeks 1 and 4, and reached steady state levels by approximately week four, with a graft-wall thickness of 91 +/- 4 microm (14 cell layers), a lumen area of 0.56 mm(2), an average neointima-media ratio of 0.4 to 0.6, and a predominance of alpha-smooth muscle actin-staining cells. Comprising predominately smooth muscle actin-expressing cells, the neointima was 50% thicker in the proximal than in the distal third of the grafts (P <.001), but proximal and distal vein graft anastomoses were widely patent. CONCLUSIONS: In syngeneic murine carotid interposition IVC grafts implanted with end-to-side anastomoses, moderate, nonocclusive neointimal hyperplasia reaches steady state after the fourth postoperative week. This neointimal hyperplasia, like that of human grafts, predominates near vein graft anastomoses. This vein graft model should facilitate genetic analyses of the pathogenesis of neointimal hyperplasia.