Biology subjects

Best, A. K.

Publications and source records attributed to Best, A. K..

5 recordsLinked to original sources

bioRxiv2026

Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here, we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing, but found a minimal number of differentially expressed genes between phospho-dead mutants and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

physiology↗

bioRxiv2025

A connexin 43 targeting peptide prevents blood vessel neointima formation

The gap junction protein connexin 43 (Cx43) is associated with human pathological vascular smooth muscle cell (SMC) proliferation and neointima formation. We previously identified mitogen-activated protein kinase (MAPK) phosphorylation of Cx43 results in binding with the cell cycle protein cyclin E, facilitating neointima formation in mice. However, the specific nature of these interactions and their relevance to human disease have not been elucidated. Using an ex vivo human saphenous vein model of neointima formation, we identified increased MAPK-phosphorylated Cx43 and cyclin E in explant tissues. We used peptide arrays to define a cyclin E-Cx43 binding region and generated CycliCx, a stearate-linked Cx43 phospho-mimetic peptide. In human coronary artery SMC, CycliCx inhibits platelet-derived growth factor-BB (PDGF-{beta})-induced changes in Cx43 trafficking and interactions with cyclin E, and stimulation of proliferation. RNAseq analysis identified CycliCx significantly inhibits PDGF-{beta}-induced proliferative pathways in SMC by limiting PDGF-{beta}-induced early G1/S phase cell cycle progression transcripts. Finally, we show CycliCx limits neointima formation in mice in vivo and in ex vivo human saphenous vein explants. Our data provide strong evidence for selective targeting of Cx43 as a viable therapeutic strategy for preventing neointimal formation in humans.

physiology↗

bioRxiv2025

Injury Induced Connexin 43 Expression Regulates Endothelial Wound Healing

Endothelial cell (EC) injury is a major contributing factor to vascular surgical failure. As such, understanding the mechanisms of endothelial healing is essential to the development of vascular therapeutics and procedures. Gap junctions formed by connexin 43 (Cx43) are implicated in regulating skin wound healing, but their role in endothelial healing is unknown. Secondary analysis of RNAseq data from in vivo injured mouse aortas (GEO: GSE115618), identified significant Cx43 upregulation in EC post-injury. We developed a novel in vivo model of EC injury using mouse carotid artery ligation to test the role of Cx43. We identified that EC immediately adjacent to the wound edge upregulate Cx43 protein expression, predominantly at cell-cell junctions. We show significantly delayed EC healing in a mouse model of inducible EC-specific Cx43 deletion (EC-Cx43 KO) at 24 hr post ligation. Single cell RNAseq analysis of 10,829 cells from 18 hr injured EC-WT and EC-Cx43 KO carotids revealed a Cx43-associated reduction in enrichment of EC pathways associated with migration, proliferation, and ERK/MAPK signaling pathways. Finally, the importance of Cx43 phosphorylation on EC healing was tested in mice with single-point alanine mutations (phospho-null) in known phosphorylation sites that alter Cx43 channel assembly and opening. Mice containing alanine mutations at ERK phosphorylated Cx43 serines (Cx43S255/262/279/282A) reduces healing rates similar to EC-Cx43 KO. These data suggest that EC injury-induced Cx43 upregulation, and subsequent Cx43 gap junction-mediated cell-to-cell communication are required for normal EC migration during wound healing after vascular injury. New and NoteworthyThese findings demonstrate for the first time that mechanical injury to large artery endothelium induces the expression of gap junction protein Cx43. This upregulation improves migratory and proliferative capacity of endothelial cells at the wound edge, facilitating timely wound closure. This phenomenon is dependent on appropriate gap junction function and turnover.

physiology↗

bioRxiv2021

Endothelial alpha globin is a nitrite reductase

Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (2{beta}2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1{Delta}/{Delta}) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/{Delta}36-39). In EC Hba1{Delta}/{Delta} or Hba1WT/{Delta}36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of the full alpha globin protein from the endothelium in the EC Hba1{Delta}/{Delta} model was associated with decreased exercise capacity and decreased intracellular nitrite utilization in hypoxic conditions. These effects were not seen in Hba1WT/{Delta}36-39 animals. Hypoxia induced vasodilation was decreased by 60% in isolated thoracodorsal arteries from EC Hba1{Delta}/{Delta}, while infusion of erythrocytes only partially rescued the dilatory response. Lastly, unlike other models where blood pressure is decreased, EC Hba1{Delta}/{Delta} blood pressure was not altered in response to hypoxia. Overall, we conclude that alpha globin in the resistance artery endothelium can act as a nitrite reductase to provide a local vasodilatory response to hypoxia.

physiology↗

bioRxiv2019

Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium

In BriefInterleukine-1 beta (IL-1{beta}) has been identified as a critical factor that contributes to the inflammatory response in cardiovascular disease (e.g., atherosclerosis). Pannexin 1 (Panx1) channel activity in endothelial cells regulates localized inflammatory cell recruitment. In response to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular calcium to control the production and release of cytokines including IL-1{beta}.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC=\"FIGDIR/small/750323v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@59e72borg.highwire.dtl.DTLVardef@57bd5corg.highwire.dtl.DTLVardef@50ed73org.highwire.dtl.DTLVardef@1151f56_HPS_FORMAT_FIGEXP M_FIG C_FIG AbstractThe proinflammatory cytokine IL-1{beta} is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1{beta} expression and release are tightly controlled by changes in intracellular Ca2+. In addition, purinergic signaling through ATP release has also been reported to promote IL-1{beta} production. Despite this, the mechanisms that control IL-1{beta} synthesis and expression have not been identified. The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, especially during inflammation. However, resolution of purinergic signaling occurs quickly due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that TNF signaling induced NF{kappa}{beta}-associated Panx1 transcription. Genetic inhibition of Panx1 reduced the expression and secretion of IL-1{beta}. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL1-{beta} expression was not altered after direct ATP stimulation, following degradation of ATP by apyrase, or after pharmacological blockade of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, control IL-1{beta} production. Because Panx1 forms a large pore channel, we hypothesized it may act to passively diffuse Ca2+ into the cell upon opening to regulate IL-1{beta}. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated intracellular Ca2+. Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in intracellular Ca2+, and IL-1{beta} transcription. Furthermore, we found that the Ca2+-sensitive NF{kappa}{beta}-p65 protein failed to localize to the nucleus after genetic or pharmacological block of Panx1. Taken together, our study provides the first evidence that intracellular Ca2+ regulation via the Panx1 channel induces a feed-forward effect on NF{kappa}{beta} to regulate IL-1{beta} synthesis and release in endothelium during inflammation.

cell biology↗