Objective Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48 h of chronic hypoxia (CH). CH couples endothelial transient receptor potential subfamily V member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium‐dependent hyperpolarization (EDH) pathway does not present in control animals. TRVPV4 channel activity has been shown to be regulated by membrane cholesterol. Hence, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelium‐dependent vasodilation. Methods Male Sprague–Dawley rats were exposed to ambient atmospheric (atm.) pressure or 48‐h of hypoxia (0.5 atm). Vasodilation to acetylcholine (ACh) was determined using pressure myography in gracilis arteries. EC membrane cholesterol was depleted using methyl‐β‐cyclodextrin (MβCD) and supplemented with MβCD‐cholesterol. Results Inhibiting TRPV4 did not affect ACh‐induced vasodilation in normoxic controls. However, TRPV4 inhibition reduced resting diameter in control arteries suggesting basal activity. TRPV4 contributes to ACh‐induced vasodilation in these arteries when EC membrane cholesterol is depleted. Inhibiting TRPV4 attenuated ACh‐induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion in arteries from CH animals abolished the contribution of TRPV4 to ACh‐induced vasodilation. Conclusion Endothelial cell membrane cholesterol impedes the contribution of TRPV4 channels in EDH‐mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.

The endothelium contains morphologically similar cells throughout the vasculature, but individual cells along the length of a single vascular tree or in different regional circulations function dissimilarly. Observations made in large arteries are extrapolated to explain the function of endothelial cells (EC) in the resistance vasculature, only a fraction of these observations are consistent between artery sizes. To what extent endothelial (EC) and vascular smooth muscle (VSMC) cells from different arteriolar segments of the same tissue differ phenotypically at the single-cell level remains unknown. Therefore, single-cell RNA-seq (10x Genomics) was performed using a 10X Genomics Chromium system. Cells were enzymatically digested from large (>300 µm) and small (<150 µm) mesenteric arteries from 9 adult male Sprague-Dawley rats, pooled to create six samples (3 rats/sample, 3 samples/group). After normalized integration, the dataset was integrated and scaled before unsupervised cell clustering and cluster visualization using UMAP plots. Differential gene expression analysis allowed us to infer the biological identity of the different clusters. Analysis revealed 630 and 641 differentially-expressed genes (DEG) between conduit and resistance arteries for EC and VSMC, respectively. Gene ontology analysis (GO-Biological Processes, GOBP) of scRNA-seq data discovered 562 and 270 pathways for EC and VSMC, respectively, that differed between large and small arteries. We identified eight and seven unique EC and VSMC subpopulations, respectively, with DEG genes and pathways identified for each cluster. These results and this dataset allow the discovery and support novel hypotheses needed to identify mechanisms that determine the phenotypic heterogeneity between conduit and resistance arteries.

Numerous studies show that ion channel activity is sensitive to the level of membrane cholesterol, with cholesterol most often suppressing channel activity. Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48‐hr of exposure to chronic hypoxia (CH). Moreover, CH exposure also couples endothelial Transient Receptor Potential Subfamily V Member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium‐dependent hyperpolarization (EDH)‐pathway not present in control animals. TRPV4 appears to provide activator Ca2+ for endothelial large‐conductance Ca2+‐activation K+ channels in this setting. Taken together, this work suggests that changes in endothelial cell membrane cholesterol mediate this CH‐induced increase in ion channel functionality. Interestingly, both BK and TRPV4 channels contain Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs that bind cholesterol through protein‐sterol interactions. It is essential to separate the effects of CH from the influence of EC membrane cholesterol changes related to hypoxic exposure. Thus, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelial‐dependent vasodilation. To test this hypothesis, vasodilation responses to acetylcholine (ACh) were determined in gracilis arteries from male Sprague‐Dawley rats using standard pressure myography. Rats were exposed to either ambient atmospheric (atm.) pressure or 48‐hrs of hypoxia (0.5 atm). Arteries were pretreated with N‐nitro‐L‐arginine and indomethacin. EC membrane cholesterol was depleted using MβCD and supplemented with MβCD‐cholesterol. In arteries from normoxic animals (a replete cholesterol state), inhibition of TRPV4 did not affect ACh‐induced vasodilation. However, TRPV4 channels contribute to ACh‐induced vasodilation in these arteries when EC membrane cholesterol is depleted with MβCD. In contrast, inhibition of TRPV4 channels attenuated ACh‐induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion with MβCD‐cholesterol in arteries from CH animals abolished the contribution of TRPV4 channels to ACh‐induced vasodilation. In conclusion, our results demonstrate that EC membrane cholesterol impedes the contribution of TRPV4 channels in EDH‐mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.