Constitutively Active Glycogen Synthase Kinase-3β Gene Transfer Sustains Apoptosis, Inhibits Proliferation of Vascular Smooth Muscle Cells, and Reduces Neointima Formation After Balloon Injury in Rats

Abstract

Objective— Glycogen synthase kinase (GSK)-3β is a crucial factor in many cellular signaling pathways and may play an important role in smooth muscle proliferation and apoptosis after angioplasty. Methods and Results— To investigate the effect of GSK-3β modulation on neointima formation, smooth muscle proliferation, and apoptosis after balloon injury in vivo, we delivered adenoviral vectors expressing the constitutively active form of GSK-3β (GSK-S9A: 9th serine switched to alanine) or a control gene into rat carotid arterial segments after balloon injury with a 2F Fogarty catheter. Viral infusion mixtures (5×10 8 pfu) were incubated in the arterial lumen for 20 minutes, and the effects of gene delivery were evaluated 3 days and 2 weeks after gene delivery with morphometry and immunohistochemical staining for proliferating and apoptotic cells. There were no significant differences in intimal, medial, and lumen areas at 3 days after the procedure. However, 2 weeks after gene delivery, the active GSK-3β gene transfer resulted in a significantly lower intima to media ratio (0.29±0.06 versus 0.86±0.09, P <0.01) and a greater lumen area (0.41±0.02 versus 0.31±0.01 mm 2 , P <0.01) compared with the control gene transfected group. This was attributable to a significant reduction in intimal area (0.05±0.01 versus 0.15±0.02 mm 2 , P <0.01), whereas the medial area was similar (0.17±0.01 versus 0.18±0.01 mm 2 , P =0.21). Proliferation index was significantly reduced both at 3 days and 2 weeks in the active GSK-3β gene transferred group (2.97±0.29% versus 5.71±0.50%, P <0.01). In addition, apoptotic index, which was not significantly different between the 2 groups at 3 days, was significantly higher in the active GSK-3β gene transferred group at 2 weeks (3.14±0.68% versus 22.7±1.63%, n=10, P <0.01, for control versus active GSK-3β gene transfer). Conclusions— In vivo delivery of the active GSK-3β gene inhibits smooth muscle proliferation, sustains apoptosis, and reduces neointima formation after balloon injury in rats and may be a future therapeutic target to limit neointima hyperplasia after angioplasty.

Celecoxib, a Cyclooxygenase-2 Inhibitor, Reduces Neointimal Hyperplasia Through Inhibition of Akt Signaling

“…14 -16 We showed previously that the modulation of GSK-3␤ is important in VSMC proliferation and survival and the development of neointimal hyperplasia in balloon-injured rat carotid arteries. 14 Here, we show for the first time that celecoxib inhibits proliferation and induces apoptosis of VSMCs and reduces neointimal hyperplasia after angioplasty. Furthermore, the importance of Akt signaling in these processes was indicated by the finding that DN-Akt gene transfer results in a significant reduction of VSMC proliferation and neointimal growth and that constitutively active myr-Akt gene transfer reverses the favorable effects observed with the administration of celecoxib.…”

“…Western blot analysis was performed as described previously. 14 The primary antibodies used were anti-phospho-Akt (Ser473) (1:500 dilution, Cell Signaling Technology), anti-total-Akt (1:500 dilution, Cell Signaling Technology), anti-phospho-GSK-3␤ (Ser9) (1:750 dilution, Cell Signaling Technology), and anti-␣-tubulin (1:4000 dilution, Oncogene). For in vivo studies, 3 carotid arteries were pooled in each group.…”

“…14 The primary antibodies used were anti-PC10, for proliferating cell nuclear antigen (PCNA 1:200, Dako), anti-HA (1:200, Santa Cruz), anti-cleaved caspase-3 (1:200, Cell Signaling Technology), and anti-ED1 for macrophages (1:200, Serotec). The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick endlabeling (TUNEL) assay was performed with minor modification to a previously described method, 14 using an Apoptag kit (Intergen Co). DAB was use as a chromogen.…”

“…14 -16 Furthermore, we recently reported that constitutively active (the dephosphorylated form) GSK-3␤ gene transfer inhibits the proliferation of vascular smooth muscle cells (VSMCs) and reduces neointimal hyperplasia after balloon injury in rat carotid arteries. 14 Thus, we reasoned that celecoxib-mediated suppression of Akt could inhibit SMC proliferation and neointimal hyperplasia in response to arterial injury after angioplasty or stent implantation.…”

A Novel Approach against Vascular Intimal Hyperplasia Through the Suppression of Girdin

“…[4][5][6] Accumulating evidence suggests that the PI3K-Akt pathway and its downstream components also play essential roles in vascular remodeling. [7][8][9][10][11] However, the underlying molecular mechanisms are not completely understood.…”