PB1347 - Pulsative shear stress and AMPK differentially contribute to vascular endothelial protection from obesity-associated lipotoxicity

Background: Dyslipidemia associated with high levels of plasma free fatty acids critically contributes to metabolic disorders in prediabetes, whereas hyperglycemia is a hallmark of diabetes. High glucose (HG) or palmitate (PA) are thought to exert acute toxic effects on vascular endothelial cells (EC).

Aims: We studied long-term effects of HG or PA on apoptosis, inflammation, and monolayer integrity of human umbilical vein endothelial cells (HUVEC) in vitro under static conditions or shear stress, and AMP-activated protein kinase (AMPK) contribution to EC protection from lipotoxicity.

Methods: Effects of HG (30 mM) or PA (0.8 mM) were assessed by flow cytometry to analyze ICAM-1 exposure (inflammatory marker); phosphatidylserine (FS) by AnnexinV (procoagulation marker); active caspase-3 (apoptotic marker), or PI-positive cells (necrotic markers). EC monolayers were assessed by phase contrast microscopy. Shear stress was created using an Ibidi Pump system at 5-10 dyne/cm2. Knockdown of catalytic α-subunits of AMPK in HUVEC was by lentivirus-assisted shRNAs technique. AMPK expression/activity was analyzed by Western blots.

Results: HG did not significantly affect EC monolayers and axpression FS, ICAM-I or active caspase-3, having only little level PI-positive cells. In contrast, PA significantly increased all these parameters (1.5-fold for FS and PI, 3-fold for ICAM-1, 2.5-fold for activated caspase-3, p< 0.01, n=5), resulting in EC monolayer disintegration. Effects of PA were negated by shear stress regardless of AMPK knockdown. AMPK transiently protected EC monolayers and viability from lipotoxicity in static conditions. AMPKα1 isoform was critical for EC protection. PA decreased endothelial AMPK in static conditions.

Conclusion(s): Prediabetic hyper/dyslipidemia is more deleterious to vascular EC than isolated hyperglycemia. The effects of PA are likely proinflammatory than proapoptotic. AMPKα1 is critical for transient EC protection from lipotoxicity in static environment, whereas shear stress provides long-lasting AMPK-independent protection. Microcirculation is critical to prevent endothelial dysfunction in obese/diabetic patients.
Supported by RSF grant #23-75-00027