Biology subjects

Dunaway, L. S.

Publications and source records attributed to Dunaway, L. S..

6 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

Arterial iron regulates vasodilation during anemia via endothelial holo-alpha globin

Iron deficiency is a highly prevalent nutrient deficiency and the most common cause of anemia. Although iron deficiency exacerbates cardiovascular disease, the direct impact of iron deficiency on the vasculature remains unstudied. We assessed iron levels across the vascular endothelium and found resistance artery endothelial cells to have the lowest iron stores suggesting they may be especially impacted by iron deficiency. Anemia has previously been shown to increase arterial NO signaling in patients, and we have previously shown endothelial -globin (Hb) scavenges nitric oxide (NO) in the resistance artery endothelium. We hypothesize iron regulates vascular function through downregulation of endothelial Hb. To test this, we used a novel model of iron deficiency anemia (IDA). In female mice, IDA increased NO signaling which was rescued to control levels by repletion of vascular iron with ferric dextran. Despite being similarly anemic and having a similar reduction in Hb protein, there were no changes in NO signaling across groups in male mice. We further measured whether Hb was in its heme-bound (holo-Hb) or heme-free (apo-Hb) state and found males did not fully lose holo-Hb. Using endothelial specific Hb knockout mice, we show loss of endothelial Hb is necessary for increased NO signaling in IDA and for the rescue of NO signaling by ferric dextran in female mice. Altogether the data presented here demonstrate iron modulates endothelial NO signaling through the regulation of Hb.

physiology↗

bioRxiv2024

Nitrosation of CD36 regulates endothelial function and serum lipids

During obesity, endothelial cells (ECs) become lipid laden leading to endothelial dysfunction. We demonstrate endothelium downregulates caveolin-1 (Cav1) in mouse and human in response to obesity. Using an EC-specific Cav1 knockout mouse, we find mice are hyperlipidemic regardless of diet, but retain endothelial cell function. Whereas initially this was thought to be due to Cav1 mediate endocytosis, we find instead the mice have significantly increased nitric oxide (NO) in response to the lack of Cav1. The presence or absence of NO toggled inversely EC lipid content and plasma lipid in mice. We found the fatty acid translocase CD36 was directly nitrosated by endogenous NO at the same cysteines that are palmitoylated on CD36. The nitrosation of CD36 prevented its trafficking to the plasma membrane and decreased lipid uptake. The physiological effect of this mechanism was a reliance on NO for endothelial function. This work suggests that CD36 nitrosation occurs as a protective mechanism to prevent EC lipotoxicity and preserve function. TeaserNitric oxide regulates serum lipids and endothelial cell lipid content through nitrosation of CD36.

molecular biology↗

bioRxiv2023

Loss of endothelial cell heterogeneity in arteries after obesogenic diet

BackgroundIt is well recognized that obesity leads to arterial endothelial dysfunction and cardiovascular disease. However, the progression to endothelial dysfunction is not clear. Endothelial cells (ECs) adapt to the unique needs of their resident tissue and respond to systemic metabolic perturbations. We sought to better understand how obesity affects EC phenotypes in different tissues specifically focusing on mitochondrial gene expression. MethodsWe performed bulk RNA sequencing (RNA-seq) and single cell RNA-seq (scRNA-seq) on mesenteric and adipose ECs isolated from normal chow (NC) and high fat diet (HFD) fed mice. Differential gene expression, gene ontology pathway, and transcription factor analyses were performed. We further investigated our hypothesis in humans using published human adipose single nuclei RNA-seq (snRNA-seq) data. ResultsBulk RNA-seq revealed higher mitochondrial gene expression in adipose ECs compared to mesenteric ECs in both NC and HFD mice. We then performed scRNA-seq and categorized EC clusters as arterial, capillary, venous, or lymphatic. HFD decreased the number of differentially expressed genes between mesenteric and adipose ECs in all subtypes, but the largest effect was seen in arterial ECs. Further analysis of arterial ECs revealed genes coding for mitochondrial oxidative phosphorylation proteins were enriched in adipose compared to mesentery under NC conditions. In HFD mice, these genes were decreased in adipose ECs becoming similar to mesenteric ECs. Transcription factor analysis revealed C/EBP and PPAR{gamma}, both known to regulate lipid handling and metabolism, had high specificity scores in the NC adipose artery ECs. These findings were recapitulated in snRNA-seq data from human adipose. ConclusionsThese data suggest mesenteric and adipose arterial ECs metabolize lipids differently and the transcriptional phenotype of these two vascular beds converge in obesity, in part, due to downregulation of PPAR{gamma} and C/EBP in adipose artery ECs. This work lays the foundation for investigating vascular bed specific adaptations to obesity.

molecular biology↗

bioRxiv2023

Endothelial Histone Deacetylase 1 Activity Impairs Kidney Microvascular NO Signaling in Rats Fed a High Salt Diet

AimWe aimed to identify new mechanisms by which a high salt diet (HS) decreases NO production in kidney microvascular endothelial cells. Specifically, we hypothesized HS impairs NO signaling through a histone deacetylase 1 (HDAC1)-dependent mechanism. MethodsMale Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or high salt diet (4% NaCl) for two weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured. ResultsWe found that HS impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS fed rats to be sufficient to induce dysfunction suggesting a humoral factor, we termed Plasma Derived Endothelial-dysfunction Mediator (PDEM), mediates the endothelial dysfunction. The antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling. ConclusionWe conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.

physiology↗

bioRxiv2022

Amount of Pannexin 1 in smooth muscle cells regulates sympathetic nerve induced vasoconstriction

Pannexin 1 (Panx1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in SMCs influences a-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure. To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, over-expression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Cx43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone. These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.

physiology↗