Fina Lovren

MicroRNA-145 Targeted Therapy Reduces Atherosclerosis

Background— MicroRNA are essential posttranscriptional modulators of gene expression implicated in various chronic diseases. Because microRNA-145 is highly expressed in vascular smooth muscle cells (VSMC) and regulates VSMC fate and plasticity, we hypothesized that it may be a novel regulator of atherosclerosis and plaque stability. Methods and Results— Apolipoprotein E knockout mice (ApoE−/−) mice were treated with either a microRNA-145 lentivirus under the control of the smooth muscle cell (SMC)-specific promoter SM22&agr; or a SM22&agr; control lentivirus before commencing the Western diet for 12 weeks. The SMC-targeted microRNA-145 treatment markedly reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries. It also significantly increased fibrous cap area, reduced necrotic core area, and increased plaque collagen content. Cellular plaque composition analyses revealed significantly less macrophages in ApoE−/− mice treated with the SMC-specific microRNA-145. These mice also demonstrated marked increases in calponin levels and &agr;-smooth muscle actin–positive SMC areas in their atherosclerotic lesions. Furthermore, lentiviral delivery of microRNA-145 resulted in reduced KLF4 and elevated myocardin expression in aortas from ApoE−/− mice, consistent with an effect of microRNA-145 to promote a contractile phenotype in VSMC. Conclusions— VSMC-specific overexpression of microRNA-145 is a novel in vivo therapeutic target to limit atherosclerotic plaque morphology and cellular composition, shifting the balance toward plaque stability vs plaque rupture.

Adiponectin primes human monocytes into alternative anti-inflammatory M2 macrophages

Altered macrophage kinetics is a pivotal mechanism of visceral obesity-induced inflammation and cardiometabolic risk. Because monocytes can differentiate into either proatherogenic M1 macrophages or anti-inflammatory M2 macrophages, approaches that limit M1 while promoting M2 differentiation represent a unique therapeutic strategy. We hypothesized that adiponectin may prime human monocytes toward the M2 phenotype. Adiponectin promoted the alternative activation of human monocytes into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Adiponectin-treated cells displayed increased M2 markers, including the mannose receptor (MR) and alternative macrophage activation-associated CC chemokine-1. Incubation of M1 macrophages with adiponectin-treated M2-derived culture supernatant resulted in a pronounced inhibition of tumor necrosis factor-alpha and monocyte chemotactic protein-1 secretion. Activation of human monocytes into M2 macrophages by adiponectin was mediated, in addition to AMP-activated protein kinase and peroxisome proliferator-activated receptor (PPAR)-gamma, via PPAR-alpha. Furthermore, macrophages isolated from adiponectin knockout mice demonstrated diminished levels of M2 markers such as MR, which were restored with adiponectin treatment. We report a novel immunoregulatory mechanism through which adiponectin primes human monocyte differentiation into anti-inflammatory M2 macrophages. Conditions associated with low adiponectin levels, such as visceral obesity and insulin resistance, may promote atherosclerosis, in part through aberrant macrophage kinetics.

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. Angiotensin converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(1-7) with A779. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.

Acetylcholine‐induced relaxation of peripheral arteries isolated from mice lacking endothelial nitric oxide synthase

Acetycholine‐mediated relaxations in phenylephrine‐contracted aortas, femoral and mesenteric resistance arteries were studied in vessels from endothelial nitric oxide synthase knock‐out (eNOS −/−) and the corresponding wild‐type strain (eNOS +/+) C57BL6/SV19 mice. Aortas from eNOS (+/+) mice relaxed to acetylcholine in an endothelium‐dependent NG‐nitro‐L‐arginine (L‐NOARG) sensitive manner. Aortas from eNOS (−/−) mice did not relax to acetylcholine but demonstrated enhanced sensitivity to both authentic NO and sodium nitroprusside. Relaxation to acetylcholine in femoral arteries was partially inhibited by L‐NOARG in vessels from eNOS (+/+) mice, but relaxation in eNOS (−/−) mice was insensitive to a combination of L‐NOARG and indomethacin and the guanylyl cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). The L‐NOARG/ODQ/indomethacin‐insensitive relaxation to acetylcholine in femoral arteries was inhibited in the presence of elevated (30 mM) extracellular KCl. In mesenteric resistance vessels from eNOS (+/+) mice, the acetylcholine‐mediated relaxation response was completely inhibited by a combination of indomethacin and L‐NOARG or by 30 mM KCl alone. In contrast, in mesenteric arteries from eNOS (−/−) mice, the acetylcholine‐relaxation response was insensitive to a combination of L‐NOARG and indomethacin, but was inhibited in the presence of 30 mM KCl. These data indicate arteries from eNOS (−/−) mice demonstrate a supersensitivity to exogenous NO, and that acetylcholine‐induced vasorelaxation of femoral and mesenteric vessels from eNOS (−/−) mice is mediated by an endothelium‐derived factor that has properties of an EDHF but is neither NO nor prostacyclin. Furthermore, in mesenteric vessels, there is an upregulation of the role of EDHF in the absence of NO.

Selective cyclo-oxygenase-2 inhibition with celecoxib elevates blood pressure and promotes leukocyte adherence

Selective inhibitors of cyclo‐oxygenase‐2 have been shown to be effective anti‐inflammatory drugs with reduced gastrointestinal toxicity relative to conventional nonsteroidal anti‐inflammatory drugs (NSAIDs). In the present study, we examined the possibility that selective COX‐2 inhibition, by blocking prostacyclin synthesis, would increase blood pressure and cause leukocyte adherence and platelet aggregation. Normal rats and rats with hypertension induced by chronic administration of Nω‐nitro‐L‐arginine methylester were given celecoxib (10 mg kg−1) daily for 3 weeks. Celecoxib significantly elevated of blood pressure in both the normal and hypertensive rats (mean increase of >33 mm Hg after 3 weeks). In normal rats, celecoxib had no effect on serum 6‐keto prostaglandin (PG)F1α levels. Hypertensive rats exhibited a significant increase (82%) in serum 6‐keto PGF1α levels, and this was reduced to the levels of normal rats by treatment with celecoxib. Rats treated with celecoxib exhibited significant increases in weight gain (20%), plasma arginine‐vasopressin levels (148%) and plasma urea (69%) relative to vehicle‐treated controls. Plasma creatinine levels were unaffected by treatment with celecoxib, while plasma renin levels were significantly decreased (30%) relative to controls. Superfusion of mesenteric venules with celecoxib (3 μM) in vivo resulted in significant increases in leukocyte adherence to the endothelium in both normal and hypertensive rats. These studies suggest that suppression of COX‐2 significantly influences vascular and/or renal function, leading to elevated blood pressure and leukocyte adherence.

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide andnitroxyl ion

Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre‐contracted) were compared with those of endothelium‐derived NO (generated with acetylcholine), NO free radical (NO·; NO gas solution) and nitroxyl ion (NO−; from Angelis salt). The soluble guanylate cyclase inhibitor, ODQ (1H‐(1,2,4‐)oxadiazolo(4,3‐a)‐quinoxalin‐1‐one; 0.3, 1 and 10 μM), concentration‐dependently inhibited responses to all agents. 10 μM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration‐response curves for NO gas solution, Angelis salt and spermine NONOate. The NO· scavengers, carboxy‐PTIO, (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl‐imidazoline‐1‐oxyl‐3‐oxide; 100 μM) and hydroxocobalamin (100 μM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angelis salt. Hydroxocobalamin, but not carboxy‐PTIO, also inhibited responses to acetylcholine. The NO− inhibitor, L‐cysteine (3 mM), inhibited responses to Angelis salt, acetylcholine and the three NO donor drugs, but not NO gas solution. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO· and NO−. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium‐derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Adropin Is a Novel Regulator of Endothelial Function

Background— Adropin is a recently identified protein that has been implicated in the maintenance of energy homeostasis and insulin resistance. Because vascular function and insulin sensitivity are closely related, we hypothesized that adropin may also exert direct effects on the endothelium. Methods and Results— In vitro cell culture models were partnered with an in vivo murine injury model to determine the potential vascular effects of adropin. Adropin was expressed in human umbilical vein and coronary artery endothelial cells (ECs). Adropin-treated endothelial cells exhibited greater proliferation, migration and capillary-like tube formation and less permeability and tumor necrosis factor-&agr;-induced apoptosis. In keeping with a vascular protective effect, adropin stimulated Akt Ser473 and endothelial nitric oxide (NO) synthase Ser1177 phosphorylation. The former was abrogated in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002, whereas the latter was attenuated by LY294002 and by mitogen-activated protein kinase kinase 1 inhibition with PD98059. Together, these findings suggest that adropin regulates NO bioavailability and events via the phosphatidylinositol 3-kinase-Akt and extracellular signal regulated kinase 1/2 signaling pathways. Adropin markedly upregulated vascular endothelial growth factor receptor-2 (VEGFR2) transcript and protein levels, and in VEGFR2-silenced endothelial cells, adropin failed to induce phosphorylation of endothelial NO synthase, Akt, and extracellular signal regulated kinase 1/2, supporting VEGFR2 as an upstream target of adropin-mediated endothelial NO synthase activation. Last, adropin improved murine limb perfusion and elevated capillary density following induction of hindlimb ischemia. Conclusions— We report a potential endothelial protective role of adropin that is likely mediated via upregulation of endothelial NO synthase expression through the VEGFR2-phosphatidylinositol 3-kinase-Akt and VEGFR2-extracellular signal regulated kinase 1/2 pathways. Adropin represents a novel target to limit diseases characterized by endothelial dysfunction in addition to its favorable metabolic profile.

Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis.

Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser(1177) within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-alpha-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser(473)-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser(1177); these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.

Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality.

Sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. In the present study, we postulated that the adipokine adiponectin serves as a critical modulator of survival and endothelial activation in sepsis. To this aim, we evaluated both loss-of-function (adiponectin gene-deficient mice) and subsequent gain-of-function (recombinant adiponectin reconstitution) strategies in two well-established inflammatory models, cecal ligation perforation (CLP) and thioglyocollate-induced peritonitis. Adipoq(-/-) mice, subjected to CLP, exhibited a profound ( approximately 8-fold) reduction in survival compared with their wild-type Adipoq(+/+) littermates after 48 h. Furthermore, compared with wild-type controls, thioglycollate challenge resulted in a markedly greater influx of peritoneal neutrophils in Adipoq(-/-) mice accompanied by an excess production of key chemoattractant cytokines (IL-12p70, TNFalpha, MCP-1, and IL-6) and upregulation of aortic endothelial adhesion molecule VCAM-1 and ICAM-1 expressions. Importantly, all of these effects were blunted by recombinant total adiponectin administration given 3 days prior to thioglycollate challenge. The protective effects of adiponectin were ascribed largely to higher-order adiponectin oligomers, since administration of recombinant C39A trimeric adiponectin did not attenuate endothelial adhesion molecule expression in thioglycollate-challenged Adipoq(-/-) mice. These data suggest a critical role of adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis.

Vasorelaxant effects of a nitric oxide-releasing aspirin derivative in normotensive and hypertensive rats

Nonsteroidal anti‐inflammatory drugs have been reported to exacerbate hypertension and to interfere with the effectiveness of some anti‐hypertensive therapies. In this study, we tested the effects of a gastric‐sparing, nitric oxide‐releasing derivative of aspirin (NCX‐4016) on hypertension in rats. Hypertension was induced by administering L‐NAME in the drinking water (400 mg l−1). Groups of rats were treated daily with aspirin, NCX‐4016 or vehicle. NCX‐4016 significantly reduced blood pressure relative to the aspirin‐treated group over the 2‐week period of treatment. Aspirin and, to a lesser extent, NCX‐4016 suppressed whole blood thromboxane synthesis. In anaesthetized rats, acute intravenous administration of NCX‐4016 caused a significant fall in mean arterial pressure in hypertensive rats, but was devoid of such effects in normotensive controls. In vitro, NCX‐4016 relaxed phenylephrine‐pre‐contracted aortic rings obtained from both normotensive and hypertensive rats, and significantly reduced their responsiveness to the contractile effects of phenylephrine. These results suggest that NCX‐4016 reduces blood pressure in hypertensive rats, not simply through the direct vasodilatory actions of the nitric oxide released by this compound, but also through possible interference with the effects of endogenous pressor agents. These properties, added to its anti‐thrombotic effects, suggest that NCX‐4016 may be a safer alternative to aspirin for use by hypertensive patients.