The nuclear factor k[kappa]B signal transduction pathway : Its role in atherogenesis and intimal hyperplasia

Sammanfattning: Vascular inflammation is a hallmark of major cardiovascular diseases such as atherosclerosis, and also suggested as a critical component implicated in intimal hyperplasia. Given its central role in regulating expression of inflammatory genes, the nuclear factor kappaB (NF-kappaB) signal transduction pathway was postulated to play an important role in these pathological processes. The aim of the thesis was to elucidate the role of the NF-kappaB pathway in intimal hyperplasia using a rodent model of carotid artery balloon injury. Specific objectives were to characterize NF-kappaB activation upon vascular injury and to explore the molecular basis of the inflammatory response, specifically expression and functional relevance of neutrophil gelatinase-associated lipocalin (NGAL), telomerase (TERT) and leukotriene B4 (LTB4) in the vascular repair process. The present study demonstrates that angioplastic injury to the carotid artery elicited two phases of NF- kappaB activation characterized by an early activation in the arterial media and a late activation coupled with high levels of inhibitor of NF-kappaB kinase (IKK) activity in the intima. The early NF-kappaB activation is crucially involved in the acute inflammatory response in the media, but appears to be dispensable for intimal formation. The late NF-kappaB activation is critical to the development of intimal lesions, as interruption of IKKbeta by overexpressing an adenoviral-mediated dominant negative mutant of IKKbeta (dnIKKbeta) in the injured artery inhibited the late phase of NF-kappaB activation, resulting in downregulation of inflammatory gene expression combined with a reduction of intimal size. To elucidate the molecular mechanisms linking NF-kappaB mediated inflammation with vascular repair, NGAL and TERT expression were investigated. NGAL is a member of the lipocalin family, and was recently found associated with increased proteolytic activity in atherosclerotic lesions. TERT, an RNA reverse transcriptase plays a critical role in maintaining cell replication capacity and was shown involved in smooth muscle cell (SMC) proliferation in a genetic hypertension model. Therefore, NGAL and TERT might contribute to intimal hyperplasia by modulating proteolytic activity and maintaining the proliferative potential of intimal SMC, respectively. We demonstrate that both NGAL and TERT are highly induced in conjunction with IKKbeta/NF-kappaB activation in the intima postangioplasty and in the isolated intimal SMC in vitro, but suppressed by dnIKKbeta. These results for the first time reveal that NF-kappaB regulates NGAL and TERT expression in intimal SMC following vascular injury. Furthermore, in addition to mono- and homomeric forms, SMC produced NGAL could form a heterodimer with matrix metalloproteinase-9, and is associated with increased proteolytic activity in the intimal SMC, indicating that NGAL could be important in the regulation of proteolytic activity involved in vascular repair. Additionally, pharmacological inhibition of telomerase led to a dosedependent growth arrest of intimal SMC due to replicative senescence, suggesting that telomerase activity is indispensable for the proliferative capability of intimal cells. Finally, we assessed the direct effects of LTB4 on vascular SMC. LTB4 is derived from the 5- lipoxygenase metabolism of arachidonic acid, and exerts its action via cell surface receptors denoted BLT1 and BLT2. Our data reveal that SMC express functional BLT1, activation of which induces SMC migration and proliferation, and that up-regulation of BLT1 in SMC by proinflammatory cytokines occurs via activation of an IKKbeta/NF-kappaB dependent pathway. BLT1 was also expressed in human atherosclerotic lesions. Blockade of BLT1 significantly suppressed intimal hyperplasia after angioplastic injury in the rat as well as SMC migration in vitro. These results suggest that targeting BLT1 on SMC may represent a novel therapeutic strategy for restenosis after angioplasty. In summary, the present study demonstrates that NF-kappaB is an important transcription factor in the vascular inflammatory response and intimal hyperplasia, and that NGAL, TERT and BLT1 in vascular SMC are regulated by NF-kappaB in these pathological processes. Therefore, NF-kappaB could be a potential therapeutic target.