Mechanisms of hydrogen sulfide induced vasodilation

Myogenic tone is an important regulator of blood flow and may contribute to peripheral vascular resistance and blood pressure. Myogenic tone is a luminal pressure-induced constriction of the vasculature that is mediated by a vascular smooth muscle cell (VSMC) plasma membrane potential (Em) depolarization and Ca2+ influx. Ca2+ sparks, which are ryanodine receptor (RyR) mediated Ca2+ release events, oppose myogenic tone by activating large-conductance Ca2+-activated K+ channels (BKCa) to hyperpolarize VSMC Em. The gaseous signaling molecules (gasotransmitters) NO and CO activate Ca2+ sparks to cause vasodilation. H2S, a third gasotransmitter produced by cystationine γ-lyase (CSE) in the vasculature, activates several K+ channels to promote VSMC Em hyperpolarization. We therefore sought to determine whether H2S inhibits the development of myogenic tone. We hypothesized that H2S opposes myogenic tone through the activation of Ca2+ sparks and subsequent BKCa channel activation. We observed that in small mesenteric arteries CSE-produced H2S reduced myogenic tone. We also found that RyR-mediated Ca2+ sparks and BKCa channel activity opposed tone in these arteries. We also observed that exogenous and endogenous H2S activates sparks and hyperpolarizes VSMC Em. Futhermore, exogenous H2S-mediated vasodilation, spark activation, and VSMC Em hyperpolarization required active endothelial BKCa channels and cytochrome P450 2C. Therefore, H2S seems to be an important regulator of myogenic tone in the mesenteric circulation. The mechanism by which H2S causes vasodilation in this bed is an unexpectedly complex pathway, with activation of endothelial BKCa channels and cytochrome P450 with subsequent activation of VSMC Ca2+ sparks. The effects of H2S here described may be an important mechanism by which this signaling molecule alters hemodynamic parameters in vivo

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function

Mechanisms of hydrogen sulfide induced vasodilation

Myogenic tone is an important regulator of blood flow and may contribute to peripheral vascular resistance and blood pressure. Myogenic tone is a luminal pressure-induced constriction of the vasculature that is mediated by a vascular smooth muscle cell (VSMC) plasma membrane potential (Em) depolarization and Ca2+ influx. Ca2+ sparks, which are ryanodine receptor (RyR) mediated Ca2+ release events, oppose myogenic tone by activating large-conductance Ca2+-activated K+ channels (BKCa) to hyperpolarize VSMC Em. The gaseous signaling molecules (gasotransmitters) NO and CO activate Ca2+ sparks to cause vasodilation. H2S, a third gasotransmitter produced by cystationine γ-lyase (CSE) in the vasculature, activates several K+ channels to promote VSMC Em hyperpolarization. We therefore sought to determine whether H2S inhibits the development of myogenic tone. We hypothesized that H2S opposes myogenic tone through the activation of Ca2+ sparks and subsequent BKCa channel activation. We observed that in small mesenteric arteries CSE-produced H2S reduced myogenic tone. We also found that RyR-mediated Ca2+ sparks and BKCa channel activity opposed tone in these arteries. We also observed that exogenous and endogenous H2S activates sparks and hyperpolarizes VSMC Em. Futhermore, exogenous H2S-mediated vasodilation, spark activation, and VSMC Em hyperpolarization required active endothelial BKCa channels and cytochrome P450 2C. Therefore, H2S seems to be an important regulator of myogenic tone in the mesenteric circulation. The mechanism by which H2S causes vasodilation in this bed is an unexpectedly complex pathway, with activation of endothelial BKCa channels and cytochrome P450 with subsequent activation of VSMC Ca2+ sparks. The effects of H2S here described may be an important mechanism by which this signaling molecule alters hemodynamic parameters in vivo.American Heart Association, National Institutes of HealthBiomedical SciencesDoctoralUniversity of New Mexico. Biomedical Sciences Graduate ProgramKanagy, NancyWalker, BenResta, TomGonzalez Bosc, LauraShuttleworth, Bil

Glycocalyx degradation and the endotheliopathy of viral infection.

The endothelial glycocalyx (EGX) contributes to the permeability barrier of vessels and regulates the coagulation cascade. EGX damage, which occurs in numerous disease states, including sepsis and trauma, results in endotheliopathy. While influenza and other viral infections are known to cause endothelial dysfunction, their effect on the EGX has not been described. We hypothesized that the H1N1 influenza virus would cause EGX degradation. Human umbilical vein endothelial cells (HUVECs) were exposed to varying multiplicities of infection (MOI) of the H1N1 strain of influenza virus for 24 hours. A dose-dependent effect was examined by using an MOI of 5 (n = 541), 15 (n = 714), 30 (n = 596), and 60 (n = 653) and compared to a control (n = 607). Cells were fixed and stained with FITC-labelled wheat germ agglutinin to quantify EGX. There was no difference in EGX intensity after exposure to H1N1 at an MOI of 5 compared to control (6.20 vs. 6.56 Arbitrary Units (AU), p = 0.50). EGX intensity was decreased at an MOI of 15 compared to control (5.36 vs. 6.56 AU, p<0.001). The degree of EGX degradation was worse at higher doses of the H1N1 virus; however, the decrease in EGX intensity was maximized at an MOI of 30. Injury at MOI of 60 was not worse than MOI of 30. (4.17 vs. 4.47 AU, p = 0.13). The H1N1 virus induces endothelial dysfunction by causing EGX degradation in a dose-dependent fashion. Further studies are needed to characterize the role of this EGX damage in causing clinically significant lung injury during acute viral infection

Arginine methylation of SMAD7 by PRMT1 in TGF-β–induced epithelial–mesenchymal transition and epithelial stem-cell generation

The epithelial-to-mesenchymal transdifferentiation (EMT) is crucial for tissue differentiation in development and drives essential steps in cancer and fibrosis. EMT is accompanied by reprogramming of gene expression and has been associated with the epithelial stem-cell state in normal and carcinoma cells. The cytokine transforming growth factor β (TGF-β) drives this program in cooperation with other signaling pathways and through TGF-β-activated SMAD3 as the major effector. TGF-β-induced SMAD3 activation is inhibited by SMAD7 and to a lesser extent by SMAD6, and SMAD6 and SMAD7 both inhibit SMAD1 and SMAD5 activation in response to the TGF-β-related bone morphogenetic proteins (BMPs). We previously reported that, in response to BMP, protein arginine methyltransferase 1 (PRMT1) methylates SMAD6 at the BMP receptor complex, thereby promoting its dissociation from the receptors and enabling BMP-induced SMAD1 and SMAD5 activation. We now provide evidence that PRMT1 also facilitates TGF-β signaling by methylating SMAD7, which complements SMAD6 methylation. We found that PRMT1 is required for TGF-β-induced SMAD3 activation, through a mechanism similar to that of BMP-induced SMAD6 methylation, and thus promotes the TGF-β-induced EMT and epithelial stem-cell generation. This critical mechanism positions PRMT1 as an essential mediator of TGF-β signaling that controls the EMT and epithelial cell stemness through SMAD7 methylation