Hypoxia and the Renin-Angiotensin System in Atherosclerosis

.................................................................................................................................................. 3 Background ............................................................................................................................................. 6 Aim ........................................................................................................................................................ 11 Material and methods ............................................................................................................................ 12 Histological examination of atherosclerotic plaques – and – comparative analysis of plaque morphology and serum markers of inflammation between patients receiving ARB treatment and controls .............................................................................................................................................. 12 Study population ............................................................................................................................ 12 Study design ................................................................................................................................... 12 Atherosclerotic plaques ................................................................................................................. 14 Serum samples ............................................................................................................................... 15 In vitro examination of the effects of hypoxia and ARB treatment on expression of AT1 and markers of inflammation in cell culture .......................................................................................................... 15 Cell culture – smooth muscle cells ................................................................................................ 16 Cell culture – monocyte derived macrophages ............................................................................. 16 Real time reverse transcriptase polymerase chain reaction (Real Time RT-PCR) ....................... 16 Enzyme-linked immunosorbent assay (ELISA) .............................................................................. 17 Western Immunoblotting ............................................................................................................... 18 Statistics ............................................................................................................................................ 19 Results ................................................................................................................................................... 20 Expression of AT1 co localizes with CD68 positive macrophages and HIF-1α expression in human carotid atherosclerotic plaques .......................................................................................................... 20 Significant correlation between CD68 and AT1 expression as well as HIF-1α and CD68 expression in human carotid atherosclerotic plaques .......................................................................................... 23 Similar plaque morphology and levels of serum markers of inflammation in ARB treated patients and controls ....................................................................................................................................... 24 SMC cell-culture experiments proved inconclusive as to the effects of hypoxia and ARB treatment on AT1 mRNA expression ................................................................................................................. 28 Expression of HIF-1α mRNA was reduced in SMCs exposed to hypoxia ........................................ 29 No change in secretion of cytokines, ACE or angiotensin II from SMCs exposed to hypoxia or Candesartan compared to controls..................................................................................................... 30 Macrophage cell-culture experiments proved inconclusive as to the effects of exposure to hypoxia, angiotensin II or Candesartan in AT1 mRNA expression .................................................................. 30 Expression of AT1 protein in macrophages verified with Western Immunoblotting......................... 31 Discussion ............................................................................................................................................. 32