Kallistatin stimulates vascular smooth muscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery

Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [(3)H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.     

Bradykinin B(1) receptor mediates inhibition of neointima formation in rat artery after balloon angioplasty

We evaluated the effects of the kallikrein-kinin system on the proliferation and migration of primary cultured vascular smooth muscle cells (VSMCs) in vitro and neointima formation in balloon-injured rat carotid arteries in vivo. In cultured rat VSMCs, tissue kallikrein inhibited cell proliferation, and this inhibitory effect was blocked by Sar-Tyr-Aca(epsilon)-Lys [D-betaNal(7), Ile(8)]-des-Arg(9)-bradykinin, a bradykinin B(1) receptor antagonist, and by icatibant, a bradykinin B(2) receptor antagonist. Platelet-derived growth factor significantly increased the expression of the B(1) receptor but not the B(2) receptor in VSMCs. Platelet-derived growth factor-induced cell migration was significantly attenuated by des-Arg(9)-bradykinin and to a lesser degree by bradykinin. Endogenous B(1) receptor mRNA increased in rat carotid arteries after balloon angioplasty. After local delivery of adenovirus carrying the human tissue kallikrein gene into the rat carotid artery, we observed a 54% reduction in the intima/media ratio at the injured site compared with the control ratio (n=7, P:<0.01). Administration of the B(1) receptor antagonist via minipumps blocked the protective effect of kallikrein and partially reversed the intima/media ratio toward the control ratio. Kallikrein gene delivery results in the regeneration of endothelium compared with the control groups, and the B(1) receptor antagonist abolished this effect. Nitrite/nitrate, cGMP, and cAMP levels in balloon-injured arteries significantly increased after kallikrein gene delivery, whereas the B(1) receptor antagonist abolished these increases (n=4 or 5, P:<0.05). These results indicate that the B(1) receptor contributes to the reduction of neointima formation via the promotion of reendothelialization and inhibition of VSMC proliferation and migration through NO-cGMP and cAMP signaling pathways. This study provides significant implications in treating restenosis after revascularization.     

Cell cycle regulator expression after coronary stenting in humans

Proliferation of vascular smooth muscle cells (VSMCs) is under the control of cell cycle regulator activity, which is induced by several growth factors. Recent attention has been drawn to treatments that target cell cycle regulators to prevent the proliferation of VSMCs after coronary angioplasty. However, histopathological evaluation of cell cycle regulator expression after human coronary stenting has not been sufficient. Thirty-one coronary arteries of 23 cadavers were examined. Time from stent implantation to patient death ranged from 0 to 235 days. Sections were stained with antibodies against platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), cyclin D1, p16, p21, and p27. Staining for macrophage colony stimulating factor receptor (MCSF-R) was conducted to detect dedifferentiated VSMCs. MCSF-R-positive cells were observed in neointima but decreased in the late stage. PDGF was detected in neointima and decreased gradually. Expression of cyclin D1 appeared to be associated with the proliferation of VSMCs, whereas p27 was downregulated with the proliferation of neointima and upregulated in the late stage. Our results suggest that one of the most promising methods for preventing excessive proliferation of neointima after stenting is to limit the decrease in p27 or the increase in cyclin D1.     

MicroRNA-24 attenuates vascular remodeling in diabetic rats through PI3K/Akt signaling pathway

Background and aimsThe vascular remodeling plays a crucial role in pathogenesis of diabetic cardiovascular complications. In this study, we intended to explore the effects and potential mechanisms of microRNA-24 (miR-24) on vascular remodeling under diabetic conditions.Methods and ResultsMiR-24 recombinant adenovirus (Ad-miR-24-GFP) was used to induce miR-24 overexpression either in carotid arteries or high glucose (HG)-induced vascular smooth muscle cells (VSMCs). Cell proliferation was analyzed using CCK-8 method. Cell migration was examined using wound-healing and transwell assay. mRNA and protein expressions of critical factors were, respectively, measured by real-time PCR and western blot as follows: qRT-PCR for the levels of miR-24, PIK3R1; western blot for the protein levels of PI3K (p85α), Akt, p-Akt, mTOR, p-mTOR, 4E-BP1, p-4E-BP1, p70s6k, p-p70s6k, MMP 2, MMP 9, collagen Ⅰ, as well as collagen Ⅲ. Carotid arteries in diabetic rats suffered balloon injury were harvested and examined by HE, immunohistochemical and Masson trichrome staining.The expression of miR-24 was decreased in HG-stimulated VSMCs and balloon-injured carotid arteries of diabetic rats, accompanied by increased mRNA expression of PIK3R1. The up-regulation of miR-24 suppressed VSMCs proliferation, migration, collagen deposition not only induced by HG in vitro, but also in balloon-injured diabetic rats, which were related to inactivation of PI3K/Akt signaling pathway.ConclusionThe up-regulation of miR-24 significantly attenuated vascular remodeling both in balloon-injured diabetic rats and HG-stimulated VSMCs via suppression of proliferation, migration and collagen deposition by acting on PIK3R1 gene that modulated the PI3K/Akt/mTOR axes.     

The role of c-jun in PDTC-sensitive flow-dependent restenosis after angioplasty and stenting

Restenosis after balloon angioplasty and stenting is exacerbated by low flow. Flow-dependent restenosis after angioplasty but not stenting is prevented by the antioxidant pyrrolidine dithiocarbamate (PDTC). c-jun may play a role in these events as AP-1 activity is both flow and redox sensitive. Carotid arteries of cholesterol fed rabbits underwent stenting or balloon injury in the presence of low or normal flow. c-jun mRNA expression was enhanced by low flow and injury (stent>balloon) and inhibited by the antioxidant PDTC irrespective of the injury type. The effect of locally delivered DZ13 (a DNAzyme specific for c-jun) or scrambled DZ13 (inactive DNAzyme) was assessed by histomorphometry at 28 days. Low flow significantly increased intimal hyperplasia in B and S relative to normal flow (P<0.05). The active DNAzyme DZ13 markedly reduced intimal hyperplasia (P<0.001) and increased lumen size (P<0.05) in balloon-injured but not in stented segments, and abrogated the effect of low flow on restenosis after angioplasty, similar to the morphological effects of PDTC. We conclude that c-jun expression is enhanced by low flow and by injury (stent>balloon) and markedly attenuated by PDTC, and that c-jun is an important mediator of flow-dependent restenosis in balloon-injured but not stented vessels.