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.

Objective: We asked whether Tbx1 is expressed in multipotent CPCs and, if so, what role it may play in them.

Methods and Results: We used clonal analysis of Tbx1-expressing cells and loss and gain of function models, in vivo and in vitro, to define the role of Tbx1 in CPCs. We found that Tbx1 is expressed in multipotent heart progenitors that, in clonal assays, can give rise to 3 heart lineages expressing endothelial, smooth muscle and cardiomyocyte markers. In multipotent cells, Tbx1 stimulates proliferation, explaining why Tbx1^–/– embryos have reduced proliferation in the second heart field. In this population, Tbx1 is expressed while cells are undifferentiated and it disappears with the onset of muscle markers. Loss of Tbx1 results in premature differentiation, whereas gain results in reduced differentiation in vivo. We found that Tbx1 binds serum response factor, a master regulator of muscle differentiation, and negatively regulates its level.

Conclusions: The Tbx1 protein marks CPCs, supports their proliferation, and inhibits their differentiation. We propose that Tbx1 is a key regulator of CPC homeostasis as it modulates positively their proliferation and negatively their differentiation.

Objectives: We tested whether the lack of Ang II type 1a receptor (AT_1aR) or Ang II type 1b receptor (AT_1bR) would decrease cellular effects induced by aldosterone.

Methods and Results: We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT_1aR, AT_1bR, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-B phosphorylation after a 20-minute stimulation. Small interference RNA for AT_1aR downregulated phosphorylation of ERK1/2, JNK, and NF-B after aldosterone stimulation compared to controls. Downregulation of AT_1bR or MR only abolished the activation of NF-B. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT_1aR knockout mice.

Conclusion: We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT_1aR and AT_1bR. Our data suggest that aldosterone augments AT₁R-dependent activation of ERK1/2, JNK, and NF-B in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT₁R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

Objective: Here we tested the hypothesis that adult neural stem cells (NSCs) can differentiate into vascular lineage, as well as neural lineage, in the process of collaborative organogenesis.

Methods and Results: NSCs, clonally isolated from mouse brain, were shown to develop endothelial and smooth muscle phenotypes in vitro. To elucidate whether NSCs can simultaneously differentiate into vascular and neural cells in vivo, genetically labeled NSCs were administered to mice with unilateral sciatic nerve crush injury or operatively induced brain and myocardial ischemia. Two weeks later, necropsy examination disclosed recruitment of the labeled NSCs to sites of injury differentiating into vascular cells (endothelial cells and vascular smooth muscle cells) and Schwann cells in regenerating nerve. Similarly, NSC-derived vascular cells/astrocytes and endothelial cells were identified in ischemic brain tissue and capillaries in myocardium 2 weeks following transplantation, respectively.

Conclusions: These findings, concurrent vasculogenesis and neurogenesis from a common stem cell, suggest that certain somatic stem cells are capable of differentiating into not only somatic cells of identity but also into vascular cells for tissue regeneration.

Objective: To test the hypothesis that SS may downregulate angiotensin type 1 receptors (AT₁Rs). Angiotensin II has been shown to be proinflammatory and to promote atherosclerosis.

Methods and Results: Using immunohistochemistry, we found a pronounced expression of AT₁R in the inner, atheroprone regions of the aortic arch of C57BL/6 and endothelial NO synthase–deficient (eNOS^–/–) mice but not eNOS-overexpressing mice. In human umbilical vein endothelial cells (HUVECs), laminar SS (15 dyn/cm²) induced a biphasic decrease in AT₁R protein expression characterized by a first reduction at 1 hour (31±4% of static control, P<0.01), partial recovery at 3 hours (65±9%), and a second more prolonged decline at 6, 12, and 24 hours (48±9%, 36±9%, 33±5%, respectively, P<0.05). One and 24 hours of SS significantly reduced fluorescent angiotensin binding compared to static HUVECs. Shear-induced downregulation of AT₁R was abolished by treatment with protein kinase A and G inhibitors or N^G-nitro-l-arginine methyl ester (L-NAME). Fittingly, stimulating static HUVECs with an NO donor decreased AT₁R protein levels. RT-PCR revealed a significant (P<0.05) decrease of AT₁R mRNA in HUVECs exposed to SS during 3 (6±2% of static control), 6 (4±1%), 12 (4±1%), and 24 hours (15±4%), suggesting a transcriptional downregulation of AT₁R at length. Finally, angiotensin-induced vascular cell adhesion molecule was abated in HUVECs exposed to SS and in the outer aortic arch of mice.

Conclusions: Our results demonstrate that SS may convey some of its atheroprotective effects through downregulation of AT₁R in endothelial cells.

Objective: To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca²⁺ release in persistent AF.

Methods: We studied cell shortening, membrane currents, and [Ca²⁺]_i in right atrial myocytes isolated from sheep with persistent AF (duration 129±39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca²⁺]_i transients were examined in confocal imaging.

Results: Myocyte shortening and underlying [Ca²⁺]_i transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca²⁺]_i. SR Ca²⁺ content was not different. Calculated fractional SR Ca²⁺ release was reduced in AF (by 20.6%, P<0.05). Peak Ca²⁺ current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca²⁺ release in AF was significantly reduced with increased areas of delayed Ca²⁺ release. Propagation between RyR was unaffected but Ca²⁺ release at subsarcolemmal sites was reduced. Rate of Ca²⁺ extrusion by Na⁺/Ca²⁺ exchanger was increased.

Conclusions: In persistent AF, reduced SR Ca²⁺ release despite preserved SR Ca²⁺ content is a major factor in contractile dysfunction. Fewer Ca²⁺ channel–RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na⁺/Ca²⁺ exchanger, underlie the reduction in Ca²⁺ release.

Objective: We assessed the hypothesis that UCP2 participates in central cardiovascular regulation by maintaining reactive oxygen species homeostasis in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that maintain vasomotor tone located. We also elucidated the molecular mechanisms that underlie transcriptional upregulation of UCP2 in response to oxidative stress in RVLM.

Methods and Results: In Sprague–Dawley rats, transcriptional upregulation of UCP2 in RVLM by rosiglitazone, an activator of its transcription factor peroxisome proliferator-activated receptor (PPAR), reduced mitochondrial hydrogen peroxide level in RVLM and systemic arterial pressure. Oxidative stress induced by microinjection of angiotensin II into RVLM augmented UCP2 mRNA or protein expression in RVLM, which was antagonized by comicroinjection of NADPH oxidase inhibitor (diphenyleneiodonium chloride), superoxide dismutase mimetic (tempol), or p38 mitogen-activated protein kinase inhibitor (SB203580) but not by extracellular signal-regulated kinase 1/2 inhibitor (U0126). Angiotensin II also induced phosphorylation of the PPAR coactivator, PPAR coactivator (PGC)-1, and an increase in formation of PGC-1/PPAR complexes in a p38 mitogen-activated protein kinase–dependent manner. Intracerebroventricular infusion of angiotensin II promoted an increase in mitochondrial hydrogen peroxide production in RVLM and chronic pressor response, which was potentiated by gene knockdown of UCP2 but blunted by rosiglitazone.

Conclusions: These results suggest that transcriptional upregulation of mitochondrial UCP2 in response to an elevation in superoxide plays an active role in feedback regulation of reactive oxygen species production in RVLM and neurogenic hypertension associated with chronic oxidative stress.

Objective: To examine the mechanism of IGFBP-3–mediated repair following vascular injury.

Methods and Results: We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3–expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3–mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway.

Conclusion: These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

Objective: Here, we investigated the impact of obesity on inflammation in the periadventitial adipose tissue and on lesion formation after vascular injury.

Methods and Results: High-fat, high-sucrose feeding induced inflammatory changes and decreased adiponectin expression in the periadventitial adipose tissue, which was associated with enhanced neointima formation after endovascular injury. Removal of periadventitial fat markedly enhanced neointima formation after injury, which was attenuated by transplantation of subcutaneous adipose tissue from mice fed on regular chow. Adiponectin-deficient mice showed markedly enhanced lesion formation, which was reversed by local delivery, but not systemic administration, of recombinant adiponectin to the periadventitial area. The conditioned medium from subcutaneous fat attenuated increased cell number of smooth muscle cells in response to platelet derived growth factor-BB.

Conclusions: Our findings suggest that periadventitial fat may protect against neointimal formation after angioplasty under physiological conditions and that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular disease accelerated by obesity.

Objective: The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure.

Methods and Results: Using -myosin heavy chain peptide (MyHC-)–loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88^–/– mice with similar distributions of heart-infiltrating cell subsets and comparable CD4⁺ T-cell responses. Injection of complete Freund’s adjuvant (CFA) or MyHC-/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88^–/– mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow–derived cells was critical for development of cardiac fibrosis during progression to heart failure.

Conclusions: Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

Objective: We studied whether MMP8 plays a role in atherogenesis.

Methods and Results: In atherosclerosis-prone apolipoprotein E–deficient mice, inactivating MMP8 resulted in a substantial reduction in atherosclerotic lesion formation. Immunohistochemical examinations showed that atherosclerotic lesions in MMP8-deficient mice had significantly fewer macrophages but increased collagen content. In line with results of in vitro assays showing that Ang I cleavage by MMP8 generated Ang II, MMP8 knockout mice had lower Ang II levels and lower blood pressure. In addition, we found that products of Ang I cleavage by MMP8 increased vascular cell adhesion molecule (VCAM)-1 expression and that MMP8-deficient mice had reduced VCAM-1 expression in atherosclerotic lesions. Intravital microscopy analysis showed that leukocyte rolling and adhesion on vascular endothelium was reduced in MMP8 knockout mice. Furthermore, we detected an association between MMP8 gene variation and extent of coronary atherosclerosis in patients with coronary artery disease. A relationship among MMP8 gene variation, plasma VCAM-1 level, and atherosclerosis progression was also observed in a population-based, prospective study.

Conclusions: These results indicate that MMP8 is an important player in atherosclerosis.

Objective: Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Na_v1.5).

Methods and Results: HEK293 cells stably expressing Na_v1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD⁺ on arrhythmic risk was evaluated in wild-type or SCN5A^+/– mouse heart. A280V GPD1-L caused a 2.48±0.17-fold increase in intracellular NADH level (P<0.001). NADH application or cotransfection with A280V GPD1-L resulted in decreased I_Na (0.48±0.09 or 0.19±0.04 of control group, respectively; P<0.01), which was reversed by NAD⁺, chelerythrine, or superoxide dismutase. NAD⁺ antagonism of the Na⁺ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase (PK)A inhibitor, PKAI_6–22. The effects of NADH and NAD⁺ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia in wild-type mouse hearts. Extracellular application of NAD⁺ to SCN5A^+/– mouse hearts ameliorated the risk of ventricular tachycardia.

Conclusions: Our results show that Na_v1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and I_Na. This effect required protein kinase C activation and was mediated by oxidative stress. NAD⁺ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Na_v1.5 by altering the oxidized to reduced NAD(H) balance.

Objective: We have now investigated the physiological function of p300 in adult hearts.

Methods and Results: We analyzed transgenic mice exhibiting cardiac-specific overexpression of a dominant-negative p300 mutant lacking the C/H3 domain (p300C/H3 transgenic [TG] mice). p300C/H3 significantly inhibited p300-induced activation of GATA- and myocyte enhancer factor 2-dependent promoters in cultured ventricular myocytes, and p300C/H3-TG mice showed cardiac dysfunction that was lethal by 20 weeks of age. The numbers of mitochondria in p300C/H3-TG myocytes were markedly increased, but the mitochondria were diminished in size. Moreover, cardiac mitochondrial gene expression, mitochondrial membrane potential and ATP contents were all significantly disrupted in p300C/H3-TG hearts, suggesting that mitochondrial dysfunction contributes to the progression of the observed cardiomyopathy. Transcription of peroxisome proliferator-activated receptor coactivator (PGC)-1, a master regulator of mitochondrial gene expression, and its target genes was significantly downregulated in p300C/H3-TG mice, and p300C/H3 directly repressed myocyte enhancer factor 2C-dependent PGC-1 promoter activity and disrupted the transcriptional activity of PGC-1 in cultured ventricular myocytes. In addition, myocytes showing features of autophagy were observed in p300C/H3-TG hearts.

Conclusions: Collectively, our findings suggest that p300 is essential for the maintenance of mitochondrial integrity and for myocyte survival in the postnatal left ventricular myocardium.

Objective: To investigate the mechanisms for modulations of EC SDF-1 expression by homocysteine and shear stress.

Methods and Results: Homocysteine stimulation induced dose- and time-dependent SDF-1 expression and phosphorylation of mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. By using specific inhibitors, small interfering (si)RNA, and dominant negative mutants, we demonstrated that activation of JNK pathway is critical for the homocysteine-induced SDF-1 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that homocysteine increased Sp1- and AP-1–DNA binding activities in ECs. Inhibition of Sp1 and AP-1 activations by specific siRNA blocked the homocysteine-induced SDF-1 promoter activity and expression. Preshearing of ECs for 1 to 4 hours at 20 dyn/cm² inhibited the homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activation, and SDF-1 expression. The homocysteine-induced SDF-1 expression was suppressed by NO donor. Inhibitor or siRNA for endothelial NO synthase abolished the shear inhibition of SDF-1 expression.

Conclusions: Our findings serve to elucidate the molecular mechanisms underlying the homocysteine induction of SDF-1 expression in ECs and the shear stress protection against this induction.

Objective: Ca²⁺ promotes cell growth raising the possibility that changes in intracellular Ca²⁺ initiate division of c-kit–positive human cardiac progenitor cells (hCPCs) and determine their fate.

Methods and Results: Ca²⁺ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca²⁺. These findings were confirmed in the heart of transgenic mice in which enhanced green fluorescent protein was under the control of the c-kit promoter. Ca²⁺ oscillations in hCPCs were regulated by the release of Ca²⁺ from the endoplasmic reticulum through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the reuptake of Ca²⁺ by the sarco-/endoplasmic reticulum Ca²⁺ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs, whereas ryanodine receptors were not detected. Although Na⁺-Ca²⁺ exchanger, store-operated Ca²⁺ channels and plasma membrane Ca²⁺ pump were present and functional in hCPCs, they had no direct effects on Ca²⁺ oscillations. Conversely, Ca²⁺ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca²⁺ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and 5-bromodeoxyuridine incorporation. Induction of Ca²⁺ oscillations in hCPCs before their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny.

Conclusion: IP3R-mediated Ca²⁺ mobilization control hCPC growth and their regenerative potential.

Objective: We investigated whether CO-mediated vasoconstriction is mechanistically linked to enhanced reactive oxygen species production that masks vasodilatory pathways.

Methods and Results: Sprague–Dawley rat interlobar and interlobular arteries were examined in terms of superoxide (O₂^·–) generation and vascular reactivity in the absence and presence of antioxidants. Both authentic CO and the CO-releasing molecule (CORM)-3 constricted renal arteries and increased O₂^·– production in a dose-dependent manner. The antioxidants tempol, ebselen, and deferoxamine inhibited CO-induced O₂^·– production and converted CO from constrictor to dilator. CO-induced O₂^·– generation was found to involve the activity of multiple oxidases including nitric oxide synthase, NADPH oxidase, xanthine oxidase, and complex IV of the mitochondrial electron chain. Furthermore, inhibition of these enzymes converted CO from constrictor to dilator. Similarly, biliverdin and bilirubin inhibited CO-induced O₂^·– production and vasoconstriction, allowing for a vasodilatory response to CO to be expressed. CO-induced vasoconstriction was dependent on a non-thromboxane agonist of the thromboxane receptor, whereas vasodilatory mechanisms of CO relied on the activation of soluble guanylate cyclase and calcium-gated potassium channels.

Conclusions: CO-induced vasoconstriction involves the generation of reactive oxygen species, which, when negated, allows for the expression of vasodilatory pathways which are masked by the primary oxidative stress response to this gas.

Objective: Evaluate the β-adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy.

Methods and Results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (I_CNG). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal β-adrenergic stimulation was reduced by 54% in isolated hypertrophied hearts, along with a 32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of β-adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 µmol/L) and of both the PDE3 inhibitor cilostamide (1 µmol/L) and the PDE4 inhibitor Ro 201724 (10 µmol/L).

Conclusions: β-Adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of β-adrenergic cAMP signals in cardiac hypertrophy.

Objective: To test the hypothesis that inhibition of aldehyde removal by aldose reductase (AR), which metabolizes both free and phospholipid aldehydes, exacerbates atherosclerotic lesion formation.

Methods and Results: In atherosclerotic lesions of apolipoprotein (apo)E-null mice, AR protein was located in macrophage-rich regions and its abundance increased with lesion progression. Treatment of apoE-null mice with AR inhibitors sorbinil or tolrestat increased early lesion formation but did not affect the formation of advanced lesions. Early lesions of AR^–/–/apoE^–/– mice maintained on high-fat diet were significantly larger when compared with age-matched AR^+/+/apoE^–/– mice. The increase in lesion area attributable to deletion of the AR gene was seen in both male and female mice. Pharmacological inhibition or genetic ablation of AR also increased the lesion formation in male mice made diabetic by streptozotocin treatment. Lesions in AR^–/–/apoE^–/– mice exhibited increased collagen and macrophage content and a decrease in smooth muscle cells. AR^–/–/apoE^–/– mice displayed a greater accumulation of the AR substrate 4-hydroxy trans-2-nonenal (HNE) in the plasma and protein-HNE adducts in arterial lesions than AR^+/+/apoE^–/– mice.

Conclusions: These observations indicate that AR is upregulated in atherosclerotic lesions and it protects against early stages of atherogenesis by removing toxic aldehydes generated in oxidized lipids.

Objective: We aimed to investigate whether these small vessels are specialized in terms of structure and calcium signaling.

Methods and Results: Using in situ confocal imaging we have studied the ultrastructure, Ca signaling and coordination of contraction in precapillary arterioles in ureter and vas deferens. We have compared the data to that from a small mesenteric artery. In the precapillary arteriole, 1 myocyte covers a 10-µm length, and contraction of this single cell can decrease the diameter of this segment. In the mesenteric artery, more than 20 myocytes are required for this. In the precapillary arteriole, Ca signals arise solely from Ca release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca release and not via ryanodine receptors. Agonist-induced Ca signals do not require Ca entry into the cell, do not spread or synchronize with neighboring cells, and are unaffected by endothelial stimulation, thereby allowing local control. This contrasts with the mesenteric artery, where Ca entry and ryanodine receptors are important and stimulation of the endothelium inhibits myocyte Ca signals and contraction.

Conclusions: These data reveal the structural and signaling specializations underlying how blood flow is locally regulated, provide new insight into control of microcirculation, and provide a framework to explain its vulnerability to disease.

Objective: To investigate the effects of H on cardiac contractile function in human failing myocardium, and to clarify the role of K_ATP channels during this response.

Methods and Results: In isolated left ventricular trabeculae of nonfailing hearts, H (produced by Fe³⁺-nitrilotriacetic acid and H₂O₂) induced substantial systolic and diastolic dysfunction, whereas in failing myocardium, stunning was virtually absent. Although in failing myocardium, protein expression of sarcolemmal K_ATP channels (Kir6.2/SUR2) was 2-fold upregulated, their blockade with HMR-1098 did not impair contractile function in the presence of H. In contrast, when blocking mitochondrial K_ATP channels during H exposure (with 5-HD), failing myocardium developed contractile dysfunction to a degree that was comparable to H-induced stunning in nonfailing myocardium without K_ATP channel blockade.

Conclusions: Human failing left ventricular myocardium is resistant to H-induced stunning, and this resistance is related to endogenous activation of putative mitochondrial K_ATP channels. Given that certain sulfonylurea drugs that also block mitochondrial K_ATP channels (eg, glibenclamide) are frequently used for the treatment of diabetes, our results imply that in patients with heart failure and diabetes, these drugs may impair left ventricular function during ischemia/reperfusion.

Objective: To investigate the role of Gli3 in ischemic tissue repair.

Methods and Results: Gli3-haploinsufficient (Gli3^+/–) mice and their wild-type littermates were physiologically similar in the absence of ischemia; however, histological assessments of capillary density and echocardiographic measurements of left ventricular ejection fractions were reduced in Gli3^+/– mice compared to wild-type mice after surgically induced myocardial infarction, and fibrosis was increased. Gli3-deficient mice also displayed reduced capillary density after induction of hindlimb ischemia and an impaired angiogenic response to vascular endothelial growth factor in the corneal angiogenesis model. In endothelial cells, adenovirus-mediated overexpression of Gli3 promoted migration (modified Boyden chamber), small interfering RNA–mediated downregulation of Gli3 delayed tube formation (Matrigel), and Western analyses identified increases in Akt phosphorylation, extracellular signal-regulated kinase (ERK)1/2 activation, and c-Fos expression; however, promoter–reporter assays indicated that Gli3 overexpression does not modulate Gli-dependent transcription. Furthermore, the induction of endothelial cell migration by Gli3 was dependent on Akt and ERK1/2 activation.

Conclusions: Collectively, these observations indicate that Gli3 contributes to vessel growth under both ischemic and nonischemic conditions and provide the first evidence that Gli3 regulates angiogenesis and endothelial cell activity in adult mammals.

Objective: The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension.

Methods and Results: Phosphorylation assays with PKC revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin’s spring elements showed that PKC targets the proline – glutamate – valine – lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKC (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKC significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKC decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKC phosphorylation.

Conclusions: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

Objective: We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension.

Methods and Results: Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho–extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells.

Conclusions: Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

Objective: Here, we determined from embryo to adult the cardiogenic proficiency of iPS programmed without c-MYC, a cardiogenicity-associated transcription factor.

Methods and Results: Transgenic expression of 3 human stemness factors SOX2, OCT4, and KLF4 here reset murine fibroblasts to the pluripotent ground state. Transduction without c-MYC reversed cellular ultrastructure into a primitive archetype and induced stem cell markers generating 3-germ layers, all qualifiers of acquired pluripotency. Three-factor induced iPS (3F-iPS) clones reproducibly demonstrated cardiac differentiation properties characterized by vigorous beating activity of embryoid bodies and robust expression of cardiac Mef2c, -actinin, connexin43, MLC2a, and troponin I. In vitro isolated iPS-derived cardiomyocytes demonstrated functional excitation-contraction coupling. Chimerism with 3F-iPS derived by morula-stage diploid aggregation was sustained during prenatal heart organogenesis and contributed in vivo to normal cardiac structure and overall performance in adult tumor-free offspring.

Conclusions: Thus, 3F-iPS bioengineered without c-MYC achieve highest stringency criteria for bona fide cardiogenesis enabling reprogrammed fibroblasts to yield de novo heart tissue compatible with native counterpart throughout embryological development and into adulthood.

Objective: To study the mechanism of PGE₂ induction of angiogenesis, we characterized its effect on fibroblast growth factor (FGF)-2 signaling in cultured endothelial cells and in ex vivo and in vivo assays of blood vessel formation.

Methods and Results: Using Western blotting assay, we demonstrated that PGE₂ induced upregulation of components of the FGF-2 pathway: FGF-2 protein, phosphorylation of FGF receptor type 1 (FGFR1), activation of FRS2 (FGFR substrate 2), phospholipase C, endothelial nitric oxide synthase, extracellular signal-regulated kinase 1/2, and the transcription factor STAT-3. Synergism between PGE₂ and FGF-2 promoted endothelial cell proliferation and robust angiogenesis in vivo, in rabbit cornea and Matrigel assays. The magnitude of the angiogenic response to PGE₂ was directly related to FGF-2 availability which determined the extent of FGFR1 activation. In fact, PGE₂ induction of angiogenesis in vitro was impaired in FGF-2^–/– endothelial cells and FGFR1 blockade abrogated PGE₂ action on the endothelium, preventing the activation of FGF-2 signaling.

Conclusion: We propose a model for the angiogenic switch based on the autocrine/paracrine FGF-2/FGFR1 activation by PGE₂ and FGF-2 synergistic interaction. The synergism between the PGE₂ and FGF-2 signaling pathways here described may explain the mechanism of action of drug combinations, the most notable being cyclooxygenase inhibitors with growth factors or growth factor receptor inhibitors.

Objective: To elucidate the mechanism how HGF and its receptor c-Met reduces angiotensin II (Ang II)–induced inflammation.

Methods and Results: HGF reduced Ang II–induced vascular smooth muscle cell growth and inflammation by controlling translocation of SHIP2 (Src homology domain 2–containing inositol 5'-phosphatase 2), which led to Ang II–dependent degradation of epithelial growth factor receptor. Moreover, the present study also revealed a preventive effect of HGF on atherosclerotic change in an Ang II infusion and cuff HGF transgenic mouse model.

Conclusions: These data suggest that the HGF/c-Met system might regulate extrinsic factor signaling that maintains the homeostasis of organs.

Objective: To define the role of caveolin-1 in the mechanism of oxidant-induced pulmonary vascular hyperpermeability and edema formation.

Methods and Results: Using genetic approaches, we show that phosphorylation of caveolin-1 Tyr14 is required for increased pulmonary microvessel permeability induced by hydrogen peroxide (H₂O₂). Caveolin-1–deficient mice (cav-1^–/–) were resistant to H₂O₂-induced pulmonary vascular albumin hyperpermeability and edema formation. Furthermore, the vascular hyperpermeability response to H₂O₂ was completely rescued by expression of caveolin-1 in cav-1^–/– mouse lung microvessels but was not restored by the phosphorylation-defective caveolin-1 mutant. The increase in caveolin-1 phosphorylation induced by H₂O₂ was dose-dependently coupled to both increased ¹²⁵I-albumin transcytosis and decreased transendothelial electric resistance in pulmonary endothelial cells. Phosphorylation of caveolin-1 following H₂O₂ exposure resulted in the dissociation of vascular endothelial cadherin/β-catenin complexes and resultant endothelial barrier disruption.

Conclusions: Caveolin-1 phosphorylation–dependent signaling plays a crucial role in oxidative stress-induced pulmonary vascular hyperpermeability via transcellular and paracellular pathways. Thus, caveolin-1 phosphorylation may be an important therapeutic target for limiting oxidant-mediated vascular hyperpermeability, protein-rich edema formation, and acute lung injury.

Objective: Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis.

Methods and Results: UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice.

Conclusions: We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor-mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.

Objective: We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS).

Methods and Results: We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1–null mice (SPHK1^–/–), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier.

Conclusions: These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.

Objective: The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity.

Methods and Results: Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A (TFAM), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53^+/+ compared to p53^–/– mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content.

Conclusions: Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.

Objective: To determine the role of TFPI in the development of atherosclerosis, we bred mice which overexpress TFPI into the apolipoprotein E–deficient (apoE^–/–) background.

Methods and Results: On a high-fat diet, smooth muscle 22 (SM22)-TFPI/apoE^–/– mice were shown to have less aortic plaque burden compared to apoE^–/– mice. Unexpectedly, SM22-TFPI/apoE^–/– had lower plasma cholesterol levels compared to apoE^–/– mice. Furthermore, SM22-TFPI mice fed a high-fat diet had lower cholesterol levels than did wild-type mice. Because TFPI is associated with lipoproteins and its carboxyl terminus (TFPIct) has been shown to be a ligand for the very-low-density lipoprotein (VLDL) receptor, we hypothesized that TFPI overexpression may regulate lipoprotein distribution. We quantified VLDL binding and uptake in vitro in mouse aortic smooth muscle cells from SM22-TFPI and wild-type mice. Mouse aortic smooth muscle cells from SM22-TFPI mice demonstrated higher VLDL binding and internalization compared to those from wild-type mice. Because SM22-TFPI mice have increased circulating levels of TFPI antigen, we examined whether TFPIct may act to alter lipoprotein distribution. In vitro, TFPIct increased VLDL binding, uptake, and degradation in murine embryonic fibroblasts. Furthermore, this effect was blocked by heparinase treatment. In vivo, systemic administration of TFPIct reduced plasma cholesterol levels in apoE^–/– mice.

Conclusions: These studies suggest that overexpression of TFPI lowers plasma cholesterol through the interaction of its carboxyl terminus with lipoproteins and heparan sulfate proteoglycans.