RNA Interference For HIF-1α Inhibits Foam Cells Formation And Effect Of Lovastatin On HIF-1α Expression In Vitro

Object:Oxidatively modified low-density lipoprotein(OX-LDL)emerged as a pathogenetic factor in atherosclerosis(AS).There is accumulating evidence for oxidative stress in vascular dysfunction/atherogenesis and a significant contribution of macrophages in OX-LDL uptake,foam cell formation,and disease progression.Foam cells are characteristic pathological cells in the lesions of AS.In the injury theory of R.Ross,AS can be considered to be a modified form of chronic inflammation induced by the interaction of several factors.OX-LDL plays a central role in AS.It is reported that OX-LDL triggers hypoxia-inducible factor-1α(HIF-1α)protein accumulation via redox-dependent mechanisms,suggesting a role of HIF-1αin AS and OX-LDL-induced pathogenesis.HIF-1αis essential for myeloid cell-mediated inflammation and is a key regulator for inflammatory cells function.Hypoxia-inducible factor-1(HIF-1)is a heterodimeric transcription factor composed ofαandβsubunits,of which HIF-1αis an essential determining factor in HIF-1 DNA binding activity and important regulator.Thus,suppression of HIF-1αwould be a powerful tool for exploring HIF-1αdependent processes and for interfering with hypoxia-induced pathophysiological events.Method:In essence,siRNA for HIF-1αwas found to be an appropriate tool for specific interference with HIF-1αrelated signal transduction and hypoxia-driven downstream events.In this study,a siRNA motif for RNA interference for HIF-1αwas selected and applied to the human monoblastic cell line(U937).To monitor suppression of HIF-1 pathway by HIF-1α-siRNA,we conducted large scale gene expression analysis of macrophages transfected by HIF-1α-siRNA after exposure to OX-LDL.Acorrding to the result of gene chip,we used Real-time PCR to test the expression of HIF-1αmRNA and 6 genes well related to development of AS.We used Western blot to test the expression of HIF-1αprotein and 4 genes.We also used Real-time PCR and Western blot to test the expression of HIF-1αafter treated by lovastatin in vitro.Results:Normal macrophages do not contain high levels of neutral lipids and are not colored with Oil red O,a dye specific for neutral lipids.Incubation of the U937 cells with 40 mg/L ox-LDL for 24 hours,resulted in the occurrence of a number of foam cells filled with large cytoplasmic lipid droplets.However,transfection of the U937 cells with HIF-1α-siRNAs led to a remarkable reduction of the number macrophages transformed into foam cell.We investigated the effects of HIF-1α-siRNAs on atherosclerosis-related genes by the differential gene expression analysis identified by Oligo Atherosclerosis Microarrays.The results showed:Seventy genes of 96 key genes involved in atherosclerosis were up-regulated by ox-LDL treatment and fifty-seven genes[such as intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule(VCAM-1),lectin-like ox-LDL receptor(LOX1),cyclooxygenase-2 (COX-2),interleukin-1(IL-1β),LDL receptor(LDLR)and so on were down-regulated by HIF-1α-siRNAs pretreatment.We used Real-time quantitative PCR experiments to verify the result of cDNA microarray analysis.The Ox-LDL-induced expression profile of ICAM-1,VCAM-1,LOX-1,COX-2,IL-1β, LDLR and HIF-1αmRNA was similar to that obtained after cDNA microarrays hybridization. The combined treatment of HIF-1α-siRNAs and ox-LDL reversed the effect of ox-LDL alone on the expression of most genes analyzed,confirming the results obtained from the microarrays.We used Western blot experiments to verify the result of cDNA microarray analysis.The Ox-LDL-induced expression profile of ICAM-1,VCAM-1,COX-2,IL-1βand HIF-1αprotein was similar to that obtained from cDNA microarrays hybridization.The combined treatment of HIF-1α-siRNAs and ox-LDL reversed the effect of ox-LDL alone on the expression of most genes analyzed,confirming the results obtained from the microarrays.The result showed:After treated with ox-LDL,the expression of HIF-1αwas markedly increased on U937 cells,the combined treatment of lovastatin and 40 mg/L ox-LDL,the expression of HIF-1αwas significantly decreased,and this inhibitory effect had dose dependant. The Western blot results were similar to Real-time PCR. Conclusion:Taken together,our observations demonstrate that the induction of HIF-1αby atherogenic factors may be a key step in coordinating the cellular events that result in atherosclerotic lesions.HIF-1α-siRNAs inhibit the transformation of the macrophages into foam cells through decreasing the expression of HIF-1α.Lovasatin could inhibit the expression of HIF-1α.Innovation:We first confirm induction of HIF-1αby atherogenic factors may be a key step in coordinating the cellular events that result in atherosclerotic lesions.Lovasatin could inhibit the expression of HIF-1α,which maybe one of mechanisms that prevent and cure AS.