Objective: We investigated the functional significance of ₂ subunits in myocytes of resistance-size (100 to 200 µm diameter) cerebral arteries.

Methods and Results: ₂-1 was the only ₂ isoform expressed in cerebral artery myocytes. Pregabalin, an ₂-1/-2 ligand, and an ₂-1 antibody, inhibited Ca_V1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of Ca_V1.2 ₁ and ₂-1 subunits were present in the arterial myocyte plasma membrane. ₂-1 knockdown using short hairpin RNA reduced plasma membrane-localized Ca_V1.2 ₁ subunits, caused a corresponding elevation in cytosolic Ca_V1.2 ₁ subunits, decreased intracellular Ca²⁺ concentration, inhibited pressure-induced vasoconstriction ("myogenic tone"), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total ₂-1 or Ca_V1.2 ₁ proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone.

Conclusions: ₂-1 is essential for plasma membrane expression of arterial myocyte Ca_V1.2 ₁ subunits. ₂-1 targeting can block Ca_V1.2 channels directly and inhibit surface expression of Ca_V1.2 ₁ subunits, leading to vasodilation. These data identify ₂-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.

Objective: Herein, we investigate the role of Ca²⁺/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo.

Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro.

Conclusions: These results elucidate a novel role for Ca²⁺/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca²⁺ and cGMP signaling cross-talk during cardiac hypertrophy.

Objective: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury.

Methods and Results: The in vivo administration (21 days) of nitro-oleic acid (OA-NO₂) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO₂ treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals,P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO₂ in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO₂–induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO₂ in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1^–/– mice (OA-NO₂–treated wild-type versus HO-1^–/– mice, P=0.016).

Conclusions: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury.

Objective: To study the role of IP-10 in cardiac repair and remodeling.

Methods and Results: We studied cardiac repair in IP-10-null and wild-type (WT) mice undergoing reperfused infarction protocols and examined the effects of IP-10 on cardiac fibroblast function. IP-10-deficient and WT animals had comparable acute infarct size. However, the absence of IP-10 resulted in a hypercellular early reparative response and delayed contraction of the scar. Infarcted IP-10^–/– hearts exhibited accentuated early dilation, followed by rapid wall thinning during infarct maturation associated with systolic dysfunction. Although IP-10-null and WT mice had comparable cytokine expression, the absence of IP-10 was associated with marked alterations in the cellular content of the infarct. IP-10^–/– infarcts had more intense infiltration with CD45⁺ leukocytes, Mac-2⁺ macrophages, and -smooth muscle actin (-SMA)⁺ myofibroblasts than WT infarcts but exhibited reduced recruitment of the subpopulations of leukocytes, T lymphocytes and -SMA⁺ cells that expressed CXCR3, the IP-10 receptor. IP-10 did not modulate cardiac fibroblast proliferation and apoptosis but significantly inhibited basic fibroblast growth factor-induced fibroblast migration. In addition, IP-10 enhanced growth factor-mediated wound contraction in fibroblast-populated collagen lattices.

Conclusions: Endogenous IP-10 is an essential inhibitory signal that regulates the cellular composition of the healing infarct and promotes wound contraction, attenuating adverse remodeling. IP-10-mediated actions may be due, at least in part, to direct effects on fibroblast migration and function.

Objective: To determine the functional role of dystroglycan in cardiac muscle and smooth muscle in the development of cardiomyopathy in muscular dystrophies.

Methods and Results: Using cre/lox–mediated gene targeting, we show here that loss of dystroglycan function in ventricular cardiac myocytes is sufficient to induce a progressive cardiomyopathy in mice characterized by focal cardiac fibrosis, increase in cardiac mass, and dilatation ultimately leading to heart failure. In contrast, disruption of dystroglycan in smooth muscle is not sufficient to induce cardiomyopathy. The specific loss of dystroglycan function in cardiac myocytes causes the accumulation of large, clustered patches of myocytes with membrane damage, which increase in number in response to exercise-induced cardiac stress, whereas exercised mice with normal dystroglycan expression accumulate membrane damage limited to individual myocytes.

Conclusions: Our findings suggest dystroglycan function as an extracellular matrix receptor in cardiac myocytes plays a primary role in limiting myocardial damage from spreading to neighboring cardiac myocytes, and loss of dystroglycan matrix receptor function in cardiac muscle cells is likely important in the development of cardiomyopathy in glycosylation-deficient muscular dystrophies.

Objective: We sought to investigate the effects of secretoneurin on therapeutic angiogenesis.

Methods and Results: We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells.

Conclusions: In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide–dependent mechanism.

Objective: Using the New Zealand white rabbit iliac model of stenting, we examined the effects of RSG on SESs, PESs, and bare metal stents endothelialization.

Methods and Results: Animals receiving SESs, PESs, or bare metal stents and either RSG (3 mg/kg per day) or placebo were euthanized at 28 days, and arteries were evaluated by scanning electron microscopy. Fourteen-day organ culture and Western blotting of iliac arteries and tissue culture experiments were conducted. Endothelialization was significantly reduced by RSG in SESs but not in PESs or bare metal stents. Organ culture revealed reduced vascular endothelial growth factor in SESs receiving RSG compared to RSG animals receiving bare metal stent or PESs. Quantitative polymerase chain reaction in human aortic endothelial cells (HAECs) revealed that sirolimus (but not paclitaxel) inhibited RSG-induced vascular endothelial growth factor transcription. Western blotting demonstrated that inhibition of molecular signaling in SES+RSG–treated arteries was similar to findings in HAECs treated with RSG and small interfering RNA to PPAR, suggesting that sirolimus inhibits PPAR. Transfection of HAECs with mTOR (mammalian target of rapamycin) short hairpin RNA and with Akt2 small interfering RNA significantly inhibited RSG-mediated transcriptional upregulation of heme oxygenase-1, a PPAR target gene. Chromatin immunoprecipitation assay demonstrated sirolimus interferes with binding of PPAR to its response elements in heme oxygenase-1 promoter.

Conclusions: mTOR/Akt2 is required for optimal PPAR activation. Patients who receive SESs during concomitant RSG treatment may be at risk for delayed stent healing.

Objective: The present study tested the hypothesis that insulin resistance is the underlying mediator for impaired NO-mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase (PTP)1B in db/db mice.

Methods and Results: The db/db mouse is morbidly obese, insulin-resistant, and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H oxidase 1 and its molecular regulators, Noxo1 and Noxa1.

Conclusions: Deletion of PTP1B improved both endothelium dependent and independent NO-mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity.

Objective: Transient receptor potential canonical (TRPC) channels are G protein–coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling.

Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1^–/– mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1^–/– mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1^–/– mice.

Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation.

Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm²) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled ¹⁵N-nitrite, whole body UVA irradiation (20 J/cm²) induced significant levels of ¹⁵N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R²=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (–28±7%).

Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.