[Local shRNA modulation of phosphatidylinositol 3-kinase signaling pathway reduces intimal hyperplasia in vein grafts: experiment with rats].

OBJECTIVE: To investigate whether knockdown Pik3cb p110beta subunit by shRNA in autologous vein grafts can reduce intimal hyperplasia. METHODS: 180 adult SD rats underwent carotid artery bypass graft surgery by using the autologous branch of jugular vein, and they were randomly divided into 6 equal groups: Group A (with the jugular vein grafts treated with 25% Pluronic F-127 only), Group B (with the graft treated with the plasmid encoding shRNA targeting Pik3cb p110beta subunit, pU6-Pik3cb-shRNA-1), Group C (with the graft treated with the plasmid encoding shRNA targeting Pik3cb p110beta subunit, pU6-Pik3cb-shRNA-2), Group D (with the graft treated with the half pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2), and Group E (with the graft treated with the pGenesil-1 scramble shRNA), and Group F (with the jugular vein grafts treated with wortmannin). Specimens of jugular vein graft were harvested 1, 3, 7, 14, and 28 days after surgery to assess the neointimal hyperplasia. Another 18 rats were randomly divided into 6 equal groups as mentioned above to be used in a parallel experiment: 72 h after surgery specimens of jugular vein graft were harvested to undergo fluorescence quantitative real-time PCR and Western blotting to detect the mRNA expression of P13K p110beta subunit and the protein expression of phosphorylated Akt - phospho-Akt (Thr 308) and phospho-Akt (Ser473)-, and mTOR (Ser 2448). And another 9 rats received jugular vein grafts treated with the pGenesil-1 scramble shRNA, and on the postoperative days 1, 2, and 3 respectively 3 rats were killed to undergo fluorescence staining to detect the transfection efficacy. RESULTS: The transfection rate of the plasmid pGenesil-1 was 60% in the vascular smooth muscle cells and 90% in the endothelial cells. The thickness of tunica intima 28 days after the surgery of the pU6-Pik3cb-shRNA-1, pU6-Pik3cb-shRNA-2, 1/2 (shRNA1 + shRNA2), and wortmannin groups were (34.6 +/- 2.7) microm, (39.4 +/- 2.5) microm, (36.7 +/- 2.9) microm, and (40.6 +/- 3.1) microm respectively, all significantly lower than that of the control group (61.8 +/- 4.3 microm, P < 0.05). The expression levels of phospho-Akt (Thr 308), phospho-Akt (Ser 473), and mTOR of the shRNA intervention and wortmannin groups were all down-regulated. CONCLUSION: Knockdown of Pik3cb in interposition carotid artery branch of jugular vein grafts reduces intimal hyperplasia with the possible mechanism of downregulation of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in VASC growth and survival. Modulation of the phosphatidylinositol 3-kinase pathway through knockdown Pik3cb may represent a novel therapy to prevent vein graft intimal hyperplasia after bypass grafting.

[Origin of neointimal cells in autologous vein graft in rat model].

OBJECTIVE: To investigate the potential cell sources of neointimal cells in autologous vein graft in rat model. METHODS: Vein graft neointimal cell origins were investigated using a model of vein-to-artery interposition modal. Slides were stained with hematoxylin and eosin, immunohistochemical staining was also performed with primary antibodies alpha-smooth actin or CD34. RESULTS: Neointimal thickening was greater at the proximal ends (65.2 +/- 4.6) microm and, to a lesser extent, distal ends (64.7 +/- 5.3) microm, in comparison to the middle of the graft (63.5 +/- 5.6) microm. Vein-originating cells survived and make a contribution to neointimal formation within the vein graft, mostly adjacent to the lumen, suggesting an intimate association with endothelial cells, donor arterial smooth muscle cells or circulating progenitor cells. CONCLUSIONS: Vein graft neointimal cells arise predominantly from vein-derived endothelial cells, donor arteria smooth muscle cells or circulating progenitor cells. It suggests clinical relevance of stenosis-inhibiting therapies directed at the vein graft or early system pharmacologic administration.

Berberine inhibits tumor necrosis factor-α-induced expression of inflammatory molecules and activation of nuclear factor-κB via the activation of AMPK in vascular endothelial cells.

Berberine, which is a well­known drug used in traditional medicine, has been demonstrated to exert diverse pharmacological effects, including anti­inflammatory effects. However, whether berberine can affect the production of inflammatory molecules in vascular endothelial cells remains to be elucidated. Therefore, the present study aimed to determine the effects of berberine, and the underlying molecular mechanisms of these effects. The effect of berberine on tumor necrosis factor (TNF)­α­induced inflammatory molecule expression was examined in cultured human aortic endothelial cells (HAECs). The HAECs were stimulated with TNF­α and incubated with or without berberine. The activation of nuclear factor (NF)­κB and adenosine monophosphate­activated protein kinase (AMPK) were analyzed using western blotting, and the protein secretion of intercellular adhesion molecule (ICAM)­1 and monocyte chemoattractant protein (MCP)­1 was measured using ELISA kits. The mRNA expression levels of ICAM­1 and MCP­1 were analyzed using reverse transcription­quantitative polymerase chain reaction. The results of the present study demonstrated that berberine significantly inhibited the TNF­α­induced expression of ICAM­1 and MCP­1, as well as the activation of NF­κB in the HAECs. These effects were attenuated following co­treatment with AMPK inhibitor compound C, or specific small interfering RNAs. In conclusion, the results of the present study indicated that berberine inhibits the TNF­α­induced expression of ICAM­1 and MCP­1, and the activation of NF­κB in HAECs in vitro, possibly through the AMPK­dependent pathway.

Berberine suppresses in vitro migration of human aortic smooth muscle cells through the inhibitions of MMP-2/9, u-PA, AP-1, and NF-κB.

Berberine, a type of isoquinoline alkaloid isolated from Chinese medicinal herbs, has been reported to have various pharmacological activities. Studies have demonstrated that berberine has beneficial effects on vascular remodeling and alleviates restenosis after vascular injury. However, its mechanism of action on vascular smooth muscle cell migration is not fully understood. We therefore investigated the effect of berberine on human aortic smooth muscle cell (HASMC) migration. Boyden chamber assay was performed to show that berberine inhibited HASMC migration dosedependently. Real-time PCR and Western blotting analyses showed that levels of matrix metalloproteinase (MMP)-2, MMP-9, and urokinase-type plasminogen activator (u-PA) were reduced by berberine at both the mRNA and protein levels. Western blotting assay further confirmed that activities of c-Fos, c-Jun, and NF-κB were significantly attenuated. These results suggest that berberine effectively inhibited HASMC migration, possibly by down-regulating MMP-2, MMP-9, and u-PA; and interrupting AP-1 and NF-κB mediated signaling pathways.