Roles of KLF4 and AMPK in the inhibition of glycolysis by pulsatile shear stress in endothelial cells

Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2+/+ mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2−/−). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK.

Platelet Dysfunction During Mechanical Circulatory Support

Objective: Mechanical circulatory support has emerged as lifesaving therapy for patients with advanced heart failure. However, mechanical circulatory support remains limited by a paradoxical coagulopathy accompanied by both thrombosis and bleeding. While mechanisms of mechanical circulatory support thrombosis are increasingly defined, mechanical circulatory support-related bleeding, as related to shear-mediated alteration of platelet function, remains poorly understood. We tested the hypothesis that platelet exposure to elevated shear stress, while a defined prothrombotic activator of platelets, coordinately induces downregulation of key platelet adhesion receptors GPIb (glycocalicin)-IX-V, α IIb β 3 , and P-selectin, thus decreasing platelet functional responsiveness to physiological stimuli. Approach and Results: Human gel-filtered platelets were exposed to continuous or pulsatile shear stress in vitro. Surface expression of platelet receptors and platelet-derived microparticle generation were quantified by flow cytometry. Shedding of receptor soluble forms were assessed via ELISA, and platelet aggregation was measured by optical aggregometry. We demonstrate that platelet exposure to elevated shear stress led to a downregulation of GPIb and α IIb β 3 receptors on platelets with a progressive increase in the generation of platelet-derived microparticles expressing elevated levels of α IIb β 3 and GPIb on their surface. No shear-mediated shedding of GPIb and β 3 subunit soluble fragments was detected. Soluble P-selectin was extensively shed from platelets, while surface expression of P-selectin on platelets and microparticles was not significantly altered by shear. Shear-mediated downregulation of GPIb, α IIb β 3 , and P-selectin on platelets was associated with an evident decrease of platelet aggregatory response induced by ADP and TRAP 6 (thrombin receptor activating peptide 6). Conclusions: Our data clearly indicate that accumulation of shear stress, consistent with supraphysiologic conditions characterizing device-supported circulation (1) induces adequate platelet degranulation, yet (2) causes downregulation of primary platelet adhesion receptors via ejection of receptor-enriched platelet-derived microparticles, thus mechanistically limiting platelet activation and the aggregatory response.

The Ocular Pulse Decreases Aqueous Humour Outflow Resistance by Stimulating Nitric Oxide Production

Intraocular pressure (IOP) is not static, but rather oscillates by 2-3 mmHg due to cardiac pulsations in ocular blood volume known as the ocular pulse. The ocular pulse induces pulsatile shear stress in Schlemm's canal (SC). We hypothesize that the ocular pulse modulates outflow facility by stimulating shear-induced nitric oxide (NO) production by SC cells. We confirmed that living mice exhibit an ocular pulse with a peak-to-peak (pk-pk) amplitude of 0.5 mmHg under anaesthesia. Using iPerfusion, we measured outflow facility (flow/pressure) during alternating periods of steady or pulsatile IOP in both eyes of 16 cadaveric C57BL/6 mice (13-14 weeks). Eyes were retained in situ, with an applied mean pressure of 8 mmHg and 1.0 mmHg pk-pk pressure amplitude at 10 Hz to mimic the murine heart rate. One eye of each cadaver was perfused with 100 µM L-NAME to inhibit nitric oxide synthase, while the contralateral eye was perfused with vehicle. During the pulsatile period in the vehicle-treated eye, outflow facility increased by 16 [12, 20] % (p<0.001) relative to the facility measured during the preceding and subsequent steady periods. This effect was partly inhibited by L-NAME, where pressure pulsations increased outflow facility by 8% [4, 12] (p < 0.001). Thus, the ocular pulse causes an immediate increase in outflow facility in mice, with roughly one-half of the facility increase attributable to NO production. These studies reveal a dynamic component to outflow function that responds instantly to the ocular pulse and may be important for outflow regulation and IOP homeostasis.

Endothelial pulsatile shear stress is a backstop for COVID-19

There has not been any means to inhibit replication of the SARS-CoV-2 virus responsible for the rapid, deadly spread of the COVID-19 pandemic and an effective, safe, tested across diverse populations vaccine still requires extensive investigation. This review deals with the repurpose of a wellness technology initially fabricated for combating physical inactivity by increasing muscular activity. Its action increases pulsatile shear stress (PSS) to the endothelium such that the bioavailability of nitric oxide (NO) and other mediators are increased throughout the body. In vitro evidence indicates that NO inhibits SARS-CoV-2 virus replication but there are no publications of NO delivery to the virus in vivo. It will be shown that increased PSS has potential in vivo to exert anti-viral properties of NO as well as to benefit endothelial manifestations of COVID-19 thereby serving as a safe and effective backstop.

Abstract 195: Chronic Enhancement of Endogenous Nitric Oxide Production via Whole Body Periodic Acceleration (WBPA) Induces Cardioprotection in 15 Months Diabetic Mice

Background: Diabetes mellitus (DM) is a major risk factor for the development of cardiovascular disease. Over time, diabetic patients develop cardiomyopathy, referred to as diabetic cardiomyopathy (DCM). The etiology of DCM in part relates to the duration of hyperglycemia. We have previously shown that type 1 diabetic mice (T1D) have abnormally elevated cardiomyocyte diastolic [Ca 2+ ] d , sodium ([Na + ] d ) after 3 months of diabetes. Whole body periodic acceleration (WBPA) is the motion of the supine body headward to footward in a sinusoidal fashion to induce pulsatile shear stress, increasing expression and phosphorylation of endothelial-derived nitric oxide synthase (eNOS, p-eNOS). We have previously shown that WBPA decreases [Ca 2+ ] d , sodium ([Na + ] d in various models of cardiomyopathy. We hypothesized that WBPA might reverse ion dyshomeostasis in the long term (15-months) hyperglycemia (glucose>250mg/dl) mice. Methods: 15 C57BL/6J (CONT) and diabetic mice (T1D) were randomized to receive WBPA (480 rpm, 1 hr daily for 20 days). Diastolic [Ca 2+ ] d , ([Na + ] d ) (selective microelectrodes), and ROS production (fluorescent) were determined in isolated cardiomyocytes at day 0 and at upon completion of the treatment (day 20). Results: Hyperglycemia produced an increase in [Ca 2+ ] d , [Na + ] d and ROS production in cardiomyocytes. 20 days of WBPA treatment in established DCM significantly decreased [Ca 2+ ] d , [Na + ] d , and ROS production toward normal values. Conclusions: These findings suggest that WBPA may be a therapeutic strategy to reverse ion dyshomeostasis and oxidative stress in a very established(15mos) DCM

Abstract 355: NOX Deficiency Alters Endothelial Response to Blood Flow--Induced Shear Stress

Increased levels of Reactive oxygen species (ROS) lead to vascular complications like atherosclerosis. Important producers of ROS are the family of NADPH oxidases (NOXes). NOX subtypes have recently been shown to be responsive to blood flow-induced shear stress. Shear stress is also responsible for the focal nature of atherosclerosis by regulating expression of the athero-protective transcription factors KLF2 and Nrf2. These transcription factors inhibit pro-inflammatory and promote antioxidant gene expression. High, pulsatile blood flow in straight vessel segments induces KLF2 and Nrf2. In contrast, disturbed flow at vessel bends and bifurcations inhibits them, thus rendering the vessel wall susceptible to systemic risk factors for vascular disease. Several studies indicate that ROS might influence KLF2/Nrf2 expression and/or activation. In this study we set out to determine the interrelationship between shear stress-induced NOX subtypes and KLF-2/Nrf2 activation. HUVEC were exposed to 1.5 Pascal pulsatile shear stress (athero-protective) for 6 days in IBIDI flow chambers and compared to static conditions (athero-prone). NOX 1 and NOX 5 were expressed at undetectable levels for PCR analyses in static HUVEC. NOX 2 and NOX 4 were expressed in static HUVEC. Upon exposure to shear stress, NOX 2 expression decreased to undetectable levels while NOX 4 was reduced by 50%. This shift in NOX 2/NOX 4 balance resulted in a ROS shift from O 2- to H 2 O 2 , as determined by DHE and CMDCF staining. Subsequently, we silenced NOX 2 and 4 by transducing HUVEC with lenti-viral shRNAs, using non-targeting shRNA and non-transduced cells as controls. Silencing did not affect static or shear stress-induced KLF2 and Nrf2 expression, activation, or target gene expression. However, we did observe an Nrf2-independent effect of NOX silencing on antioxidant expression, which is currently under further investigation. Our preliminary results show a shift in NOX/ROS balance under shear stress, without affecting KLF2 or Nrf2. Together with the anti-oxidant activities of KLF2 and Nrf2, this shift in oxidant balance might be causative in the quiescent, anti-atherogenic effect of high, pulsatile shear stress on ECs.