Inflammation and vascular smooth muscle cell (VSMC) phenotypic modulation appears to be important in cerebral aneurysm formation/progression yet molecular pathological mechanisms remain unknown. We investigated a potential direct role of Tumor Necrosis Factor-alpha (TNF-alpha) mediated via the transcription factor, KLF4, in inducing VSMC phenotypic modulation and inflammation in vitro and in vivo, which may be critical in the pathogenesis of cerebral aneurysms.
Methods:
Rat cerebral VSMCs were treated with TNF-alpha (5 and 50 ng/ml) for 24 hours. The expression of KLF4, VSMCs marker genes (SM-alpha-Actin, SM-22alpha and SM-MHC), the SRF co-activator, Myocardin, and inflammatory/ matrix remodeling genes (MMP-3, VCAM-1, iNOS, and MCP-1) was measured using qPCR. Cells were transfected with siRNA against KLF4 to examine the role of KLF4 as a direct mediator of phenotypic modulation. To investigate in vivo effects, pluronic gel (40% w/v) containing TNF-alpha (30-60 ug/ml) was applied to carotid vessels in 7-week-old rats for 6 and 24 hours. Carotid vessels were harvested and qPCR performed. In another experiment, aneurysm induction surgery was performed in rats using a standard model (unilateral carotid ligation/induction of hypertension). The circle of Willis cerebral vessels were harvested and qPCR performed comparing treated and control animals.
Results:
TNF-alpha treatment produced a dose-dependent decrease in SMC marker gene and myocardin expression while potently inducing expression of KLF4 and the aforementioned inflammatory/matrix remodeling genes (p<0.05 vs. baseline) demonstrating a profound direct role of TNF-alpha in inducing cerebral VSMC phenotypic modulation in vitro. This phenotypic modulation was markedly abrograted using siKLF4. In vivo, TNF-alpha applied to carotid vessels produced a marked increase in KLF4 expression at 6 hours and a subsequent decrease in SMC marker gene expression and increase in expression of inflammatory/matrix remodeling genes at 24 hours (p<0.05 treated vs. control). Similarly, increased expression of KLF4 and TNF-alpha was noted in the Circle of Willis in vivo two weeks after aneurysm induction surgery. Decreased marker gene expression along with increased inflammatory/matrix remodeling marker gene expression was also seen (all p<0.05 vs. control).
Conclusion:
TNF-alpha appears to potently induce phenotypic modulation of cerebral VSMCs producing a pro-inflammatory phenotype with elevation of matrix remodeling genes both in vitro and in vivo. KLF4 appears to be a critical mediator of this process. As inflammation (and specifically TNF-alpha) and matrix remodeling have been implicated in the pathogenesis of human cerebral aneurysms, the molecular mechanisms outlined herein may represent a novel target for future therapy.