The Role of PER‐1, ET‐1 Axis in Regulating Endothelial Cell Function

Abstract

ObjectiveResults from major epidemiological studies give evidence to the essential role that dietary salt plays in the development of hypertension. Contrastingly, the mechanisms that mediate individual susceptibility to salt‐sensitive hypertension are unknown. Blood pressure (BP) and sodium reabsorption by the kidneys are regulated by the circadian clock. Consistent with these observations, when mice with global knockout (KO) of the circadian protein period 1 (PER1) are exposed to a high salt diet, they demonstrate increased endothelin‐1 (ET‐1) levels, impaired natriuresis, and hypertension. However, the cumulative sodium balance of PER1 KO mice is not different from control mice. Thus, we hypothesize that PER1 might mediate endothelial cell dysfunction and susceptibility to salt sensitive hypertension.Materials and MethodsHuman Umbilical Vein Endothelial Cells (HUVEC) were transfected with siRNA targeting PER1 or non‐targeting siRNA controls. Endothelial cell function was assessed by measurement of nitric oxide bioavailability, proliferation, and migration. Nitric Oxide (NO) was quantified by DAF fluorescence, proliferation by EZQuant™, and migration by measuring velocity and distance travelled by single cells using time lapse imaging.ResultsNitric oxide bioavailability was increased with PER1 knockdown (Mean fluorescence 14433 ± 1344 vs. 7423 ± 472, p<0.005). Migration velocity and distance were decreased with PER1 siRNA compared to non‐target controls, though not statistically significant (Mean distance 118.9 uM ± 11.39 vs. 138 uM ± 10.88, p=0.2) and (Mean velocity 42.3 uM/sec ± 3.98 vs. 46 uM/sec ± 3.6, p=0.14).There were no differences in proliferation at 24, 48, or 72 hours (Fold change in cell numbers of PER1 siRNA treated cells compared to non‐target controls were 0.87 ± .05 at 24 hours, 0.87 ± .09 at 48 hours, and 0.90 ± .06 at 72 hours, p=NS).ConclusionsOur results suggest that knock down of PER1 increases NO bioavailability and decreases migration of endothelial cells. ET‐1 can increase NO bioavailability via ETB receptors. Experiments are ongoing to delineate the role of ET‐1/ETA and ET‐B in mediating the effect of PER1 on endothelial cell function.