Abstract Rationale Despite the pulmonary vasculature being exposed to constant flow, there is limited understanding of the effects of shear stress on the pulmonary endothelium and most in vitro work is performed under static conditions. Pathologic shear stress, through high cardiac output, contributes to several forms of pulmonary hypertension. By understanding critical cellular mechanisms associated with physiologic flow, in comparison to static conditions, we can better unravel pathologic flow conditions. We hypothesize that under physiologic flow states, laminar shear stress strengthens classic endothelial mechanisms, including junctional stability and barrier function, through the flow sensitive transcription factors Krϋppel like factor 2 and 4 (KLF2 and KLF4) as key regulators of junctional proteins. Methods Human pulmonary artery endothelial cells (HPAECs) were exposed to physiologic laminar shear stress (16 dynes/cm2) versus static conditions for 20 hours via the ibidi Pump System and processed for immunofluorescence (IF), light microscopy (LM) and western blot. HPAECs were also plated in electrical cell impedance sensing (ECIS) plates and exposed to flow (16 dynes/cm2). KLF2 expression was manipulated using siRNA (Dharmacon) while KLF4 was pharmacologically inhibited using Kenpaullone (Tocris) prior to experimental flow conditions. Experiments were also carried out with integrin-β4 (ITGβ4) knockdown, a junctional protein under KLF4 transcriptional regulation. Results We observe an increase in barrier function as measured by transendothelial electrical resistance via ECIS with the addition of physiologic shear stress. The heightened barrier function is associated with alignment of cells in the direction of flow seen under LM. Under IF imaging there is alignment of F-actin fibers with flow and strong, continuous VE-cadherin staining of the cell periphery. Inhibition of KLF4 with Kenpaullone inhibits flow induced barrier function and leads to a reduction in ITGβ4 protein expression. Interestingly, when either KLF2 or ITGβ4 are silenced, endothelial barrier function is degraded and there is no longer alignment in the direction of flow with increased intracellular gaps as evidenced by F-actin and VE-Cadherin staining. Conclusions HPAECs demonstrate enhanced barrier function when exposed to physiologic laminar shear stress, which is at least partially mediated through KLF2 and KLF4. These flow sensitive transcription factors are integral to the normal physiologic processes such as endothelial barrier function through regulation of key junctional proteins, including ITGβ4. The role of KLF2 and KLF4 in regulating junctional proteins under physiologic flow is a critical step to understanding changes occurring during pathologic flow in conditions such as high cardiac output pulmonary hypertension. This abstract is funded by: T32 HL007829, R56 HL167875
Holbert et al. (Fri,) studied this question.