Postnatal blocking of interferon-gamma function prevented atherosclerotic plaque formation in apolipoprotein E-knockout mice.

It is unknown whether interferon-gamma has a positive or negative impact on atherosclerotic plaque formation. Thus, we examined the effects of postnatal interferon-gamma function blocking on plaque formation in apolipoprotein E-knockout (apoEKO) mice by overexpressing a soluble mutant of interferon-gamma receptor (sIFNgammaR), an interferon-gamma inhibitory protein. Mice were fed a Western-type diet from 8 weeks of age. sIFNgammaR or mock plasmid (control) was injected into the thigh muscle at 8 and 10 weeks' age, because serum sIFNgammaR protein was transiently increased with a peak at 2 days after a single sIFNgammaR gene transfer and remained elevated for 2 weeks. At 12 weeks' age, control apoEKO mice showed marked atherosclerotic plaques from the ascending aorta to the aortic arch. The plaques in the aortic root had massive lipid cores and macrophage infiltration with thin fibrous cap and few smooth muscle cells, demonstrating low plaque stability. In contrast, the luminal plaque area was remarkably reduced in sIFNgammaR-treated apoEKO mice. sIFNgammaR treatment not only reduced lipid core areas and macrophage infiltration but also increased smooth muscle cell count and fibrotic area, suggesting improved plaque stability. In controls, interleukin-1beta, monocyte chemoattractant protein-1, and vascular cell adhesion molecules-1 were remarkably upregulated in the aortic wall. These changes were significantly reversed by sIFNgammaR. sIFNgammaR treatment had no effects on serum cholesterol levels. In conclusion, sIFNgammaR treatment prevented plaque formation in apoEKO mice by inhibiting inflammatory changes in the arterial wall. The present study provides insight into a new strategy for preventing atherosclerosis.

Inhibition of intrinsic interferon-gamma function prevents neointima formation after balloon injury.

It is still controversial whether intrinsic interferon (IFN)-gamma promotes or attenuates vascular remodeling in hyperproliferative vascular disorders, such as neointima formation after balloon injury. Thus, we investigated whether inhibition of intrinsic IFN-gamma function prevents neointima formation. For this purpose, naked DNA plasmid encoding a soluble mutant of IFN-gamma receptor alpha-subunit (sIFNgammaR; an IFN-gamma inhibitory protein) or mock plasmid was injected into the thigh muscle of male Wistar rats 2 days before balloon injury (day -2). sIFNgammaR gene transfer significantly elevated serum levels of sIFNgammaR protein for 2 weeks. In mock-treated rats, balloon injury induced smooth muscle cell proliferation in the neointima with a peak at day 7 and produced thick neointima at day 14. sIFNgammaR treatment reduced the number of proliferating intimal smooth muscle cells by 50% at day 7 and attenuated neointima formation with a 45% reduction of the intima/media area ratio at day 14. In mock-treated rats, at day 7, balloon injury induced phosphorylation of signal transducer and activator of transcription-1 and upregulations of IFN regulatory factor-1 (a transcription factor mediating IFN-gamma signal). Balloon injury also upregulated the key molecules of neointima formation, such as intercellular adhesion molecule-1 and platelet-derived growth factor beta-receptor. These changes were suppressed by sIFNgammaR treatment. In conclusion, it is suggested that intrinsic IFN-gamma promotes neointima formation probably through IFN regulatory factor-1/intercellular adhesion molecule-1-mediated and platelet-derived growth factor-mediated mechanisms. Thus, inhibition of IFN-gamma signaling may be a new therapeutic target for prevention of neointima formation of hyperproliferative vascular disorders.

Inhibition of STAT3 prevents neointima formation by inhibiting proliferation and promoting apoptosis of neointimal smooth muscle cells.

In cultured vascular smooth muscle cells (SMCs), STAT3 mediates proliferation signal by directly activating transcription of early growth response genes. Recently, we have found that balloon injury transiently induces JAK2 and STAT3 expressions and activations with a peak at day 7 in rat carotid artery. However, the specific role of STAT3 in neointima formation remains unknown. Adenoviral vector encoding a dominant negative STAT3 (AxCAdnSTAT3) or beta-galactosidase (control) was overexpressed in a balloon-injured artery to inhibit endogenous STAT3 activation selectively. In controls, neointima became evident after day 4, and reached a maximum at day 14. The number of bromodeoxyuridine (BrdU)-positive proliferating or TUNEL-positive apoptotic neointimal SMCs peaked at day 7, decreasing to lower levels by day 14. AxCAdnSTAT3 not only abrogated STAT3 phosphorylation but also decreased BrdU labeling index by 60% and increased TUNEL index by 35% at day 7 versus controls, resulting in the 40% reduction in the intima/media area ratio at day 14. At day 7, in controls, vascular injury upregulated antiapoptotic mediator Mcl-i and Bcl-xL expression by 8-fold to 5-fold, respectively, versus sham, whereas proapoptotic Bax slightly increased by 1.5-fold versus sham. AxCAdnSTAT3 reversed the upregulated Mcl-1 and Bcl-xL levels by 70% and 37%,respectively, while having no affect on Bax expression. In conclusion, the STAT3-mediated pathway plays an important role in neointima formation through enhanced vascular SMC accumulation by promoting cell proliferation and survival.

Rho-kinase inhibition reduces neointima formation after vascular injury by enhancing Bax expression and apoptosis.

Recently, we have shown that a specific Rho-kinase inhibitor, Y27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), prevents neointima formation after vascular injury associated with increased terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL)+ smooth muscle cells. Because the mechanism of the action of Y27632 remains unclear, we investigated the expression changes in Bcl family proteins, apoptosis regulators of smooth muscle cells, in the rat carotid artery after balloon injury (BI). Y27632 (BI + Y group) or saline (BI group) was administered peritoneally from Day 1 to Day 14 after BI. Y27632 markedly prevented neointima formation at Day 14. In the BI group, TUNEL+ smooth muscle cells were transiently increased in the neointima, but not in the media, with a peak at Day 7, returning to a lower level by Day 14. Y27632 significantly increased TUNEL+ smooth muscle cells at Days 7 and 14. Smooth muscle cell apoptosis was confirmed by electron microscopic examination. At Day 14, although proapoptotic Bax was slightly, but not significantly, increased in the BI group, it was significantly upregulated in the BI + Y group. Antiapoptotic Bcl-xL was upregulated in the BI group, and the upregulated Bcl-xL was not affected by Y27632. These findings indicate that Rho-kinase inhibition induces neointimal smooth muscle cell apoptosis through Bax upregulation, resulting in reduced neointima formation.