| Abstrakt: |
Introduction:Flow disturbances are hypothesized to play a role in progressive pulmonary vein stenosis (PVS). In atherosclerosis literature, upregulation of KLF2 and KLF4, key mechanotranscription factors, are protective under laminar shear stress conditions, while downregulation of KLF2 /4 in low and disturbed flow states leads to proliferation and adverse vascular remodeling. However, the regulation of KLF2 /4 expression in pulmonary vein endothelial cells (PVECs) by shear stress conditions is unknown.Hypothesis:KLF2/4 expression in isolated PVECs is differentially regulated by low, high and disturbed shear stress conditions.Methods:Primary porcine PVECs were cultured for 48hrs using Ibidi flow chambers to investigate the effects of low and high laminar flow and disturbed (oscillating) flow using qPCR and immunofluorescence(IF) methods. Experimental variables were static, low unidirectional (LUF) (2dyn/cm2), high unidirectional (HUF) (10dyn/cm2) laminar flow and disturbed high oscillatory flow ( HOF) (10dyn/cm2, 0.5 Hz).Results:In PVECs, KLF2/4 expression was upregulated in HUF compared to static conditions (9 fold and 8 fold respectively (p<0.01). KLF4 expression increased with HOF (3.6 fold, p<0.05) but not LUF , while KLF2 expression was modestly increased in both HOF (3.5 fold, p=0.06) and LUF (2 fold, p<0.05) when compared to static conditions. Only HUF was associated with increased eNOS expression (3 fold; p<0.01) and decreased Endothelin-1 expression (5 fold; p<0.01). As upregulation of KLF2 and KLF4 expression is associated with endothelial quiescence, we investigated Ki67 expression in PVECs using IF. At 48hrs, Ki67 positive cells were significantly increased only under LUF conditions compared to static, HUF and HOF conditions (24.5% vs 4.2% vs 6.0% vs 2.8%, p<0.05).Conclusions:This is the first study to demonstrate shear stress mediated regulation of KLF2 and KLF4 expression in PVECs. HUF is a potent stimulus for KLF2/4 and protective gene expression patterns in contrast to LUF. In addition, LUF is associated with increased Ki67-positive PVECs. These data demonstrate a role for shear stress in PVECs homeostasis and provides insights into how altered flow patterns in PVS may contribute to neointimal formation. |
