RGS5 channels intracellular signaling pathways to feature growth arrest of vascular smooth muscle cells
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PROVIDER: PRJNA728477 | ENA |
REPOSITORIES: ENA
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Project description:Members of the regulator of G-protein signaling (RGS) family terminate the activity of specific Ga subunits. As such, RGS5 acts as an inhibitor of Gαq/11 and Gαi/o activity in vascular smooth muscle cells (VSMCs), which regulate the arterial tone and blood pressure. While Rgs5 shows a variable expression in different types of mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 altered the blood pressure. To study the impact of RGS5 on the VSMC phenotype and signaling, Rgs5 expression was silenced by siRNA in 3D spheroids supporting a contractile VSMC phenotype. Loss of RGS5 elevated the baseline calcium level and the agonist-induced Gαq/11-mediated release of calcium but inhibited RhoA activity. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promotes their resting state as evidenced by gene expression array-based analyses and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation and migration, which was mimicked by selectively inhibiting Gαi/o but not Gαq/11 activity. Collectively, the heterogeneous expression level of Rgs5 suggests artery type-specific functions comprising the inhibition of acute agonist-induced Gαq/11/calcium-mediated VSMC contraction as well as the support of a resting VSMC phenotype with low ERK1/2 activity by chronically suppressing the activity of Gαi/o.
2021-07-19 | GSE174130 | GEO
Project description:Background: Dysregulation of TRPV4-mediated signaling has been associated with inflammation and tissue fibrosis, both of which are key features in the pathophysiology of lymphatic system diseases; however, the expression and functional roles of lymphatic TRPV4 channels remain largely unexplored. Methods: We generated a single-cell RNA sequencing dataset from microdissected mouse collecting lymphatic vessels to characterize the expression of Trpv4. Using a novel Trpv4fx/fx mouse strain and the Cre-lines Prox1-CreERT2 and LysM-Cre we assessed the role of TRPV4 channels in lymphatic endothelial cells (LECs) and peri-lymphatic myeloid cells, respectively. Confocal microscopy and extensive functional experimentation on isolated and pressurized lymphatics, including measurements of intracellular calcium activity, were employed to validate our scRNAseq findings and to elucidate the underlying mechanisms. Clinical significance was assessed using biopsies from breast cancer-related lymphedema (BCRL) patients. Results: We characterized the single-cell transcriptome of collecting lymphatic vessels and surrounding tissues. Trpv4 was highly enriched in LECs and in a subset of Lyve1+ macrophages displaying a tissue-resident profile. In clinical samples, BCRL was associated with increased infiltration of macrophages co-expressing LYVE1 and TRPV4. Pharmacological activation of TRPV4 channels led to contractile dysregulation in isolated collecting lymphatics. The response was multi-phasic, including initial vasospasm and subsequent vasodilation and inhibition of contractions, which was associated with the activation of thromboxane A2 receptors (TXA2Rs) in lymphatic muscle cells (LMCs) by secreted prostanoids from TRPV4+ myeloid cells, and increased nitric oxide (and perhaps other vasodilatory prostanoids) from LECs. The TXA2R-mediated vasospasm resulted from increased mobilization of calcium from intracellular stores through IP3 receptors and store-operated calcium entry. Conclusions: Our results uncovered a novel mechanism of lymphatic contractile dysregulation mediated by the crosstalk between TRPV4-expressing myeloid cells, including LYVE1+ macrophages, and LMCs or LECs. These findings highlight potentially important roles of TRPV4 channels in lymphatic dysfunction associated with inflammation, including secondary lymphedema.
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Project description:Voltage-gated potassium channels (VGKCs) comprise one of the largest, most diverse and complex families of ion channels. Approximately 70 genes encode the alpha subunits that form homomeric VGKC channels. In addition, these subunits can form functional heteromeric channels, thus exponentially increasing the diversity of VGKCs. The functional expression and physiological role of heteromeric K-channels have remained largely unexplored due to the lack of tools to probe their functions. Conotoxins have high affinity and specificity for heteromeric combinations of K-channels and show great promise for elucidating their functions. In this work, using conotoxin kM-RIIIJ as a pharmacological probe, we explore the expression and physiological functions of heteromeric Kv1.2 channels using constellation pharmacology. We report that heteromers of Kv1.2/1.1 are highly expressed in proprioceptive neurons found in the dorsal root ganglion (DRG). Inhibition of Kv1.2/1.1 heteromers leads to an influx of calcium ions, suggesting that these channels regulate neuronal excitability. We also present evidence that Kv1.2/1.1 heteromers counteract persistent sodium currents, and that inhibiting these channels leads to tonic firing of action potentials. Additionally, kM-RIIIJ induces impaired proprioception in mice, uncovering a previously unrecognized physiological role of heteromeric Kv1.2/1.1 channels in proprioceptive sensory neurons of the DRG.
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Project description:Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+]i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.
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Project description:Duchenne muscular dystrophy (DMD) is an irreversible muscle disease characterized by a progressive loss of muscle function, decreased ambulation, and ultimately death as a result of cardiac or respiratory failure. DMD is caused by the lack of dystrophin, a protein that is important for membrane stability and signaling in excitable cells. Although vascular smooth muscle cells (VSMCs) dysfunction occurs in many pathological conditions, little is known about vascular smooth muscle function in DMD. We have previously shown that striated muscle cells, as well as neurons isolated from dystrophic (mdx) mice have higher intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) concentrations and decreased cell viability in comparison with wild type (Wt). Experiments were carried out in isolated VSMCs from mdx (a murine model of DMD) and congenic C57BL/10SnJ Wt mice. We found elevated [Ca2+]i and [Na+]i in VSMCs from mdx mice compared to Wt. Exposure to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC3 and TRPC6 channel activator, induced a greater elevation of [Ca2+]i and [Na+]i in mdx than Wt VSMCs. The OAG induced increases in [Ca2+]i could be abolished by either removal of extracellular Ca2+ or by SAR7334, a blocker of TRPC3 and TRPC 6 channels in both genotypes. Mdx and Wt VSMCs were susceptible to muscle cell stretch-induced elevations of [Ca2+]i and [Na+]i which was completely inhibited by GsMTx-4, a mechanosensitive ion channel inhibitor. Western blots showed a significant upregulation of TRPC1 -3, -6 proteins in mdx VSMCs compare to age-matched Wt. The lack of dystrophin in mdx VSMCs produced a profound alteration of [Ca2+]i and [Na+]i homeostasis that appears to be mediated by TRPC channels. Moreover, we have been able to demonstrate pharmacologically that the enhanced stretch-induced elevation of intracellular [Ca2+] and concomitant cell damage in mdx VSMCs also appears to be mediated through TRPC1, -3 and -6 channel activation.
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Project description:Vascular smooth muscle cell (VSMC) subpopulations relevant to vascular disease and injury repair have been depicted in healthy vessels and atherosclerosis profiles. However, whether VSMC subpopulation associated with vascular homeostasis exists in the healthy artery and how are their nature and fate in vascular remodeling remains elusive. Here, using single-cell RNA-sequencing (scRNA-seq) to detect VSMC functional heterogeneity in an unbiased manner, we showed that VSMC subpopulations in healthy artery presented transcriptome diversity and that there was significant heterogeneity in differentiation state and development within each subpopulation. Notably, we detected an independent subpopulation of VSMCs that highly expressed regulator of G protein signaling 5 (Rgs5), upregulated the genes associated with inhibition of cell proliferation and construction of cytoskeleton compared with the general subpopulation, and mainly enriched in descending aorta. Additionally, the proportion of Rgs5+ VSMCs was markedly decreased or almost disappeared in the vascular tissues of neointimal formation, abdominal aortic aneurysm and atherosclerosis. Specific spatiotemporal characterization of Rgs5+ VSMC subpopulation suggested that this subpopulation was implicated in vascular homeostasis. Together, our analyses identify homeostasis-relevant transcriptional signatures of VSMC subpopulations in healthy artery, which may explain the regional vascular resistance to atherosclerosis at some extent.
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