Biology subjects

Phillips, S. A.

Publications and source records attributed to Phillips, S. A..

3 recordsLinked to original sources

bioRxiv2026

Flow-sensitive K+ channels link flow to piezo1/PI3K/Akt1 pathway

BackgroundEndothelial response to flow is key to vascular function in health and disease. Our earlier studies demonstrated that endothelial Kir2.1 is essential for flow-induced Akt1/eNOS signaling and for flow-induced vasodilation (FIV) but the mechanistic integration between Kir and other flow signaling pathways remained poorly understood. MethodsWe use a combination of electrophysiological recordings in real time of flow exposure, Ca2+ imaging, pressure myography of resistance arteries, and echocardiography. ResultsWe demonstrate that Kir2.1 is essential for flow-induced PI3K phosphorylation, whereas expression of myristoylated Akt1, which bypasses PI3K-dependent membrane recruitment, restores flow-induced Akt1/eNOS phosphorylation in Kir2.1-deficient endothelium. It also restores FIV in Kir2.1-deficient mesenteric arteries. We further demonstrate that Kir2.1 is essential for flow-induced Ca{superscript 2} influx mediated by Piezo1 and TRPV4 channels, whereas Ca{superscript 2} influx induced by pharmacological activation of these channels is Kir2.1 independent. Deficiency of Piezo1 does not affect endothelial Kir2.1 channels. We also discover that flow activation of endothelial Kir2.1 requires Syndecan1, thus creating a link between glycocalyx and downstream effects. Physiologically, we find that endothelial Kir2.1 is suppressed by infusion of Angiotensin-II and by advanced aging, resulting in significant impairment of FIV. In both cases, FIV is fully restored by endothelium-specific over-expression of Kir2.1. ConclusionsOur study reveals that Kir2.1 serves as a mechanistic linker between endothelial glycocalyx to Piezo1-mediated Ca2+ influx and downstream signaling suggesting a new integrated model of endothelial mechanotransduction. A functional loss of endothelial Kir2.1 is shown to play a significant role in FIV impairment in Angiotensin-induced hypertension and aging.

physiology↗

bioRxiv2025

Endothelial Nucleoporin93 (Nup93) Maintains Vascular Function via Sun1-Dependent Regulation of RhoA-eNOS Signaling

As the innermost lining of blood vessels, endothelial cells (ECs) regulate blood flow, maintain vascular tone, and limit inflammation for vessel health. EC-derived nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), is a vasodilator essential for improving blood flow and vascular homeostasis. The RhoA/ROCK pathway regulates eNOS levels, where overactivation decreases eNOS expression and downstream NO production. As such, RhoA/ROCK hyperactivity and increased pMLC have been identified as major contributors to age-associated vasoconstriction and hypertension. Intriguingly, recent studies identify Sun1, a key component of the linker of nucleoskeleton and cytoskeleton (LINC) complex, as a major regulator of RhoA/ROCK activity. Moreover, endothelial aging deteriorates nuclear pore complexes (NPCs) (i.e. nucleoporin [Nup93]) and impairs nucleocytoplasmic transport, thereby insinuating a role for nuclear envelope components in vessel homeostasis. Here, we show that targeted loss of endothelial Nup93 in adult mice significantly reduces eNOS expression and NO bioavailability for consequent defects in NO-dependent vasodilatory responses. In vitro knockdown of Nup93 in primary human ECs also decreases both eNOS expression and NO production. Mechanistically, we find that loss of Nup93 significantly reduces endothelial Sun1 levels for a concomitant increase in RhoA activity. Indeed, restoring Sun1 protein levels in Nup93-deficient ECs mitigates RhoA activity to rescue both eNOS expression and NO production. Taken together, we demonstrate endothelial Nup93, through Sun1 stabilization, as a novel regulator of eNOS-NO signaling and vessel reactivity, contributing to the growing importance of nuclear membrane components in EC and vascular biology.

cell biology↗

bioRxiv2021

Mesowestern Blot: Simultaneous Analysis of Hundreds of Sub-Microliter Lysates

Western blotting is a widely-used technique for molecular-weight-resolved analysis of proteins and their post-translational modifications, but has been refractory to affordable scale-up. Here, we report the Mesowestern blot, which uses a 3D-printable gel-casting mold to enable affordable, high-throughput Western blotting with standard sample preparation and small (<1 uL) sample sizes. The casted polyacrylamide gel contains 336, 0.5 uL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution range. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder as well as cell lysate (RIPA buffer) demonstrate protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semi-dry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is approximately 2 orders of magnitude, with a limit of detection (for {beta}-actin) of around 30 ng of total protein from mammalian cell lysates (~30-3000 cells). Because the gel mold is 3D-printable, users have significant design freedom for custom layouts, and there are few barriers to adoption by the typical cell and molecular biology laboratory already performing Western blots.

biochemistry↗