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Rationale: Vascular wall stretch is the major stimulus for the myogenic response of small arteries to pressure. The molecular mechanisms are elusive, but recent findings suggest that G protein-coupled receptors can elicit a stretch response., Objective: To determine whether angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction., Methods and Results: We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, transient receptor potential channel 6 (TRPC6) channels, or several subtypes of the voltage-gated K+ (Kv7) gene family (KCNQ3, 4, or 5). We identified a mechanosensing mechanism in isolated mesenteric arteries and in the renal circulation that relies on coupling of the AT1R subtype a to a Gq/11 protein as a critical event to accomplish the myogenic response. Arterial mechanoactivation occurs after pharmacological block of AT1R and in the absence of angiotensinogen or TRPC6 channels. Activation of AT1R subtype a by osmotically induced membrane stretch suppresses an XE991-sensitive Kv channel current in patch-clamped vascular smooth muscle cells, and similar concentrations of XE991 enhance mesenteric and renal myogenic tone. Although XE991-sensitive KCNQ3, 4, and 5 channels are expressed in vascular smooth muscle cells, XE991-sensitive K+ current and myogenic contractions persist in arteries deficient in these channels., Conclusions: Our results provide definitive evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand-independent, mechanoactivation of AT1R subtype a. The AT1R subtype a signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4, or 5 to enact vascular smooth muscle cell activation and elevated vascular resistance., (© 2014 American Heart Association, Inc.)

Rationale: Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation., Objective: This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells., Methods and Results: We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs., Conclusions: These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.

Background: Atherosclerosis is a multifactorial disease in which inflammatory processes play an important role. Inflammation underlies lesion evolution at all stages, from establishment to plaque rupture and thrombosis. Costimulatory molecules of the tumor necrosis factor superfamily such as CD40/CD40L and OX40/OX40L have been implicated in atherosclerosis., Methods and Results: This study shows that the tumor necrosis factor superfamily members CD137 and CD137 ligand (CD137L), which play a major role in several autoimmune diseases, may constitute a pathogenic pair in atherogenesis. We detected CD137 protein in human atherosclerotic lesions not only on T cells but also on endothelial cells and showed that CD137 in cultured endothelial cells and smooth muscle cells was induced by proinflammatory cytokines implicated in atherosclerosis. Activation of CD137 by CD137L induced adhesion molecule expression on endothelial cells and reduced smooth muscle cell proliferation. In addition, treatment of atherosclerosis-prone apolipoprotein E-deficient mice with a CD137 agonist caused increased inflammation. T-cell infiltration, mainly of CD8(+) cells, and expression of the murine major histocompatibility complex class II molecule I-A(b) increased significantly in atherosclerotic lesions, as did the aortic expression of proinflammatory cytokines., Conclusions: Taken together, these observations suggest that CD137-CD137L interactions in the vasculature may contribute to the progression of atherosclerosis via augmented leukocyte recruitment, increased inflammation, and development of a more disease-prone phenotype.

Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid omega-hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell-specific promoter SM22alpha (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). Beta-galactosidase expression was detected in Ad-SM22-nLacZ-transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1-transduced A7r5 and PAC1 cells. Ad-SM22-4A1-transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79+/-1.77 versus 0.97+/-0.57 nmol 12-hydroxy lauric acid/10(6) cells per h). Smooth muscle-specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04+/-0.29 and 13.43+/-2.84 ng/mg protein in Ad-SM22-nLacZ-transduced and Ad-SM22-4A1-transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63+/-3.66 mm in Ad-SM22-4A1-transduced vessels versus 1.79+/-0.89 mm in Ad-SM22-nLacZ-transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle-specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.

Renal arteries (RAs) dilate in response to hypoxia, whereas the pulmonary arteries (PAs) constrict. In the PA, O2 tension is detected by an unidentified redox sensor, which controls K+ channel function and thus smooth muscle cell (SMC) membrane potential and cytosolic calcium. Mitochondria are important regulators of cellular redox status and are candidate vascular O2 sensors. Mitochondria-derived activated oxygen species (AOS), like H2O2, can diffuse to the cytoplasm and cause vasodilatation by activating sarcolemmal K+ channels. We hypothesize that mitochondrial diversity between vascular beds explains the opposing responses to hypoxia in PAs versus RAs. The effects of hypoxia and proximal electron transport chain (pETC) inhibitors (rotenone and antimycin A) were compared in rat isolated arteries, vascular SMCs, and perfused organs. Hypoxia and pETC inhibitors decrease production of AOS and outward K+ current and constrict PAs while increasing AOS production and outward K+ current and dilating RAs. At baseline, lung mitochondria have lower respiratory rates and higher rates of AOS and H2O2 production. Similarly, production of AOS and H2O2 is greater in PA versus RA rings. SMC mitochondrial membrane potential is more depolarized in PAs versus RAs. These differences relate in part to the lower expression of proximal ETC components and greater expression of mitochondrial manganese superoxide dismutase in PAs versus RAs. Differential regulation of a tonically produced, mitochondria-derived, vasodilating factor, possibly H2O2, can explain the opposing effects of hypoxia on the PAs versus RAs. We conclude that the PA and RA have different mitochondria.

Background: Necrotic patches and hemorrhagic lesions develop in the renal tissue between day 4 and day 5 after transplantation of fully allogeneic DA rat kidneys to LEW recipients. These lesions are at least in part due to destruction and obstruction of blood vessels. Damage of graft endothelial cells and blood coagulation are likely to be mediated by intravascular graft leukocytes. However, this cell population has not been thoroughly characterized before., Methods: We perfused untreated control kidneys, renal isografts, and allografts on day 4 after transplantation with phosphate-buffered saline/ethylenediaminetetraacetic acid to harvest leukocytes from both the blood stream as well as from the marginal intravascular pool. The mRNA expression of typical products of activated monocytes was analyzed in reverse-transcriptase polymerase chain reaction experiments. Graft monocytes were purified and their immunophenotype was investigated by flow cytometry., Results: Allograft rejection led to a 10-fold increase in the number of intravascular graft leukocytes compared to isografts. A mean number of about 100x10(6) leukocytes was harvested from a single allogeneic kidney, about 73% of these cells were monocytes and most of them displayed an activated phenotype. Compared to isografts, intravascular allograft leukocytes displayed an increased expression of tumor necrosis factor-alpha, inducible NO synthase and tissue factor., Conclusions: Our study shows that large numbers of activated monocytes accumulate inside allograft vessels. As they express genes the products of which might damage the allograft by inducing cell death or thrombosis, we speculate that they directly participate in allograft destruction.

Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine D1-like receptors in atherosclerosis, the effects of dopamine and specific D1-like agonists SKF 38,393 and YM 435 on platelet-derived growth factor (PDGF) BB-mediated VSMC migration and proliferation were studied. We observed that cells stimulated by PDGF-BB (5 ng/mL), showed increased migration and proliferation. These effects were prevented by coincubation with dopamine, SKF 38,393, or YM 435 (1 to 10 mumol/L), and this prevention was reversed by Sch 23,390 (1 to 10 mumol/l), a specific D1-like antagonist. These actions are mimicked by forskolin (1 to 10 mumol/L), a direct activator of adenylate cyclase and 8-bromo-cAMP at 0.1 to 1 mmol/L and are blocked by a specific protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H 89), but not blocked by its negative control, N-[2-(N-formyl)-p-chlorociannamylamino)ethyl]-5-isoquinoline sulfonamide (H 85). PDGF-BB (5 ng/mL)-mediated activation of phospholipase D, protein kinase C, and mitogen activated protein kinase activity were significantly suppressed by coincubation with dopamine. These results suggest that vascular D1-like receptor agonists inhibit migration and proliferation of VSMC, possibly through protein kinase A activation and suppression of activated phospholipase D, protein kinase C, and mitogen-activated protein kinase activity.

The patch-clamp technique was used to examine the inhibition of delayed rectifier K+ channels by agents that release intracellular Ca2+. During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 mumol/L) present to inhibit delayed rectifier K+ current (IK(dr)) and Ca(2+)-activated Cl- current (ICl(Ca)), angiotensin II (Ang II) and caffeine increased Ca(2+)-activated K+ current (IK(Ca)) between -25 and 80 mV (n = 5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 mumol/L) present to inhibit IK(Ca) and ICl(Ca), Ang II and caffeine only caused inhibition of IK(dr). Block was achieved within 15 seconds of drug application and was reversible upon washout (n = 5). The effects of Ang II on IK(Ca) and IK(dr) were inhibited by the specific Ang II receptor antagonist losartan (1 mmol/L, n = 3). Intracellular BAPTA (10 mmol/L) also abolished the effects of Ang II and caffeine on both IK(Ca) and IK(dr). In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 mumol/L) caused little change in resting membrane potential; however, subsequent application of caffeine (10 mmol/L) caused a 26 +/- 2.9 mV depolarization from -54 +/- 3.1 to -28 +/- 1.7 mV (n = 6). 4-AP (10 mmol/L) blocked the caffeine-induced depolarization. When isolated cells were loaded with the Ca2+ indicator indo 1 (100 mumol/L), Ang II, caffeine, and 4-AP increased [Ca2+]i and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca2+]i that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca2+]i (n = 4). In inside-out patches, with 200 nmol/L ChTX in the patch pipette to block large-conductance Ca(2+)-activated K+ channels, a 45 +/- 7-picosiemen 4-AP-sensitive K+ channel was identified that was sensitive to cytoplasmic Ca2+ (n = 6). Increasing intracellular Ca2+ decreased channel opening probability [NxP(open), where N is the number of functional channels in a patch and P(open) is the opening probability] at all membrane potentials examined. At 0 mV, increasing Ca2+ from < 5 to 200 and 600 nmol/L free Ca2+ decreased NxP(open) by 52 +/- 3% and 73 +/- 7%, respectively (n = 6). The decrease in opening probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. The decrease in mean open time reflected significant decreases and increases in open and closed time constants, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)