Coronary Arteriolar Dilation Research Articles

TNF-α Contributes to Endothelial Dysfunction by Upregulating Arginase in Ischemia/Reperfusion Injury

We tested whether tumor necrosis factor (TNF)-alpha increases arginase expression in endothelial cells as one of the primary mechanisms by which this inflammatory cytokine compromises endothelial function during ischemia-reperfusion (I/R) injury. Mouse hearts were subjected to 30 minutes of global ischemia followed by 90 minutes of reperfusion and their vasoactivity before and after I/R was examined in wild-type (WT), tumor necrosis factor knockout (TNF-/-), and TNF 1.6 (TNF++/++) mice. In WT mice, dilation to the endothelium-dependent vasodilator ACh was blunted in I/R compared with sham control. L-arginine or arginase inhibitor NOHA restored NO-mediated coronary arteriolar dilation in WT I/R mice. O2(-) production was reduced by eNOS inhibitor, L-NAME, or NOHA in WT I/R mice. In TNF-/- mice, I/R did not alter Ach-induced vasodilation and O2(-) production compared with sham mice. The increase in arginase expression that occurs during I/R in WT mice was absent in TNF-/- mice. Arginase expression was confined largely to the endothelium and independent of inflammatory cell invasion. Arginase activity was markedly lower in TNF-/-, but higher in WT I/R than that in WT sham mice. Our data demonstrate TNF-alpha upregulates expression of arginase in endothelial cells, which leads to O2(-) production then induces endothelial dysfunction in I/R injury.

Impaired flow‐induced dilation in coronary arterioles of dogs fed a low‐salt diet

Low-salt diet is considered to be helpful in prevention and treatment of hypertension. However, sodium restriction increases the production of angiotensin II and may induce counter-regulatory responses. To this end, we examined the effect of low-salt diet on superoxide formation and endothelial function in coronary arterioles. Dogs were fed with a low-salt diet (0.5% NaCl) for 2–3 weeks. Compared with normal dogs, plasma angiotensin II increased 3 fold and flow-induced dilation (FID) of coronary arterioles decreased by 60% in low-salt fed dogs. In normal dogs, FID of coronary arterioles is mainly a NO-mediated response. However, in low-salt fed dogs, the inhibition of NO synthesis (L-NAME; 300 μM) had no significant effect on FID but administration of apocynin (10 μM), a selective inhibitor of NADPH oxidase, which had no effect on FID in normal dogs, significantly enhanced FID. Western blot analysis revealed a two-fold increase in gp91phox in coronary arteries of low-salt fed dogs. Superoxide formation in the endothelial and smooth muscle layers of coronary arteries was assessed by dihydroethidium staining and confocal microscopy. In cannulated, pressurized coronary arteries, administration of apocynin resulted in a greater reduction of superoxide formation in dogs fed a low-salt diet. We conclude that long-term low-salt diet impairs endothelial function of coronary arterioles by decreasing NO bioavailability via an increased angiotensin-NADPH oxidase-dependent superoxide formation. (NIH HL-68813, HL-43023 and HL-083647)

Anti–LOX-1 Rescues Endothelial Function in Coronary Arterioles in Atherosclerotic ApoE Knockout Mice

We hypothesized that atherosclerosis inhibits NO-mediated endothelium-dependent dilation of coronary arterioles through interaction of ox-LDL with its receptor, LOX-1, through the production of O2ÿ- in endothelial cells. We assessed the role of ox-LDL in endothelial dysfunction in a murine model of atherosclerosis (ApoE KO mice). Coronary arterioles from WT control and ApoE KO mice were isolated and pressurized without flow. Although dilation of vessels to endothelium-independent vasodilator SNP was not altered between ApoE KO and WT mice, dilation to the endothelium-dependent agonist, ACh was reduced in ApoE KO versus WT mice. Impaired vasodilation to ACh in ApoE KO mice is partially restored by NAD(P)H oxidase inhibitor, apocynin or DPI. Messenger RNA expression for NAD(P)H oxidases was higher in ApoE KO mice than that in WT and anti-LOX-1 treated ApoE KO mice. Anti-LOX-1, given in vivo, restored NO-mediated coronary arteriolar dilation in ApoE KO mice, but did not affect the endothelium-dependent vasodilation in controls. These results suggest that ox-LDL impairs endothelium-dependent NO-mediated dilation of coronary arterioles by activation of a signaling cascade involving LOX-1 and NAD(P)H oxidase expression.

Exercise Training Restores Coronary Arteriolar Dilation to NOS Activation Distal to Coronary Artery Occlusion

Exercise training has been shown to restore vasodilation to nitric oxide synthase (NOS) activation in arterioles distal to coronary artery occlusion. Because reactive oxygen species are generated during NOS uncoupling and the production of vasodilator H2O2 is increased during exercise in patients with coronary disease, we proposed that H2O2 may contribute to the restoration of vasodilation in porcine coronary occlusion model. Left circumflex (LCX) coronary artery of miniature swine was progressively occluded for 8 weeks followed by exercise training (EX; 5 days/wk treadmill) or sedentary (SED) protocols for 12 weeks. Arterioles were isolated from distal LCX and nonoccluded left anterior descending (LAD) artery for in vitro study. Vasodilation to NOS activators adenosine and ionomycin was impaired in SED LCX, but not LAD, arterioles. This impairment was restored by L-arginine. NO production induced by adenosine was also reduced in SED LCX arterioles. EX had no effect on LAD arterioles but improved NO production and restored dilation of LCX arterioles. NOS blockade (L-NAME) inhibited vasodilation to NOS activators in LAD (SED & EX) arterioles but was ineffective in SED LCX arterioles. In EX LCX arterioles, vasodilation to NOS activators was slightly inhibited by L-NAME but abolished by catalase. H2O2 production was markedly increased by adenosine in EX LCX arterioles. This study demonstrates that endothelium-dependent NO-mediated dilation is impaired in SED LCX arterioles and that EX training restores the impaired function. It appears that H2O2, in addition to NO, contributes significantly to EX-induced restoration of endothelium-dependent dilation of coronary arterioles distal to occlusion.

C‐reactive protein inhibits endothelium‐dependent prostacyclin synthase‐mediated dilation of coronary arterioles: role of peroxynitrite

We have recently shown that C-reactive protein (CRP), a biomarker of cardiovascular disease, inhibits nitric oxide (NO)-mediated vasodilation via NAD(P)H oxidase-dependent superoxide (O2−) production. Here, we examined whether CRP can influence the response to another endothelium-derived vasodilator – prostacyclin (PGI2). Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. Endothelium-dependent agonist arachidonic acid (AA) stimulated PGI2 release and dilation. PGI2 synthase (PGI2S) inhibitor trans-2-phenyl cyclopropylamine blocked dilation to AA but not to endothelium-dependent NO-mediated agonist serotonin. Luminal administration of CRP (pathophysiologic level of 7 μg/ml, 60 min) attenuated dilations to serotonin and AA but not to exogenous PGI2. CRP also reduced basal NO level, caused tyrosine nitration of PGI2S, and inhibited PGI2 release. Peroxynitrite scavenger urate prevented inhibitory actions of CRP on PGI2S and PGI2 release/dilation but not on serotonin-induced dilation. Moreover, NO synthase inhibitor L-NAME or O2− scavenger TEMPOL prevented the detrimental effect of CRP on AA-induced dilation. Our data show that CRP inhibits endothelium-dependent PGI2S-mediated dilation of coronary arterioles. This vascular dysfunction is a result of PGI2S nitration by peroxynitrite derived from basal NO and CRP-induced O2− production. (HL-71761 to LK)

Impaired forskolin‐mediated dilation in coronary arterioles of hypercholesterolemic swine: role of voltage‐dependent K + channels

Impaired forskolin‐mediated dilation in coronary arterioles of hypercholesterolemic swine: role of voltage‐dependent K ⁺ channels

Increased Cyclooxygenase-2 Expression and Prostaglandin-Mediated Dilation in Coronary Arterioles of Patients With Diabetes Mellitus

Based on findings of experimental models of diabetes mellitus (DM) showing increased expression of vascular cyclooxygenase-2 (COX-2), we hypothesized that in patients with DM changes in COX-2-dependent prostaglandin synthesis affect vasomotor responses of coronary arterioles. Arterioles were dissected from the right atrial appendages obtained at the time of cardiac surgery of patient with DM(+) or without documented diabetes DM(-). Isolated arterioles (89+/-15 microm in diameter) were cannulated and pressurized (at 80 mm Hg), and changes in diameter were measured with video microscopy. After spontaneous tone developed [DM(-): 32+/-7%; DM(+): 37+/-5%; P=NS], arteriolar responses to bradykinin were investigated. Dilations to bradykinin (0.1 nmol/L to 1 micromol/L) were significantly (P<0.05) greater in DM(+) than DM(-) patients (10 nmol/L: 77+/-10% versus 38+/-14%). In both groups, dilations were similar to the NO-donor, sodium nitroprusside. In arterioles of DM(+), but not those of DM(-), patients' bradykinin-induced dilations were reduced by the nonselective COX inhibitor indomethacin or by the selective COX-2 inhibitor NS-398 (DM(+) at 10 nmol/L: to 20+/-4% and 29+/-7%, respectively). Correspondingly, a marked COX-2 immunostaining was detected in coronary arterioles of DM(+), but not in those of DM(-) patients. We conclude that in coronary arterioles of diabetic patients bradykinin induces enhanced COX-2-derived prostaglandin-mediated dilation. These findings are the first to show that in humans diabetes mellitus increases COX-2 expression and dilator prostaglandin synthesis in coronary arterioles, which may serve to increase dilator capacity and maintain adequate perfusion of cardiac tissues.

The levosimendan metabolite OR‐1896 elicits vasodilation by activating the KATP and BKCa channels in rat isolated arterioles

We characterized the vasoactive effects of OR‐1896, the long‐lived metabolite of the inodilator levosimendan, in coronary and skeletal muscle microvessels. The effect of OR‐1896 on isolated, pressurized (80 mmHg) rat coronary and gracilis muscle arteriole (∼150 μm) diameters was investigated by videomicroscopy. OR‐1896 elicited concentration‐dependent (1 nM–10 μM) dilations in coronary (maximal dilation: 66±6%, relative to that in Ca2+‐free solutions; pD2: 7.16±0.42) and gracilis muscle arterioles (maximal dilation: 73±4%; pD2: 6.71±0.42), these dilations proving comparable to those induced by levosimendan (1 nM–10 μM) in coronary (maximal dilation: 83±6%; pD2: 7.06±0.14) and gracilis muscle arterioles (maximal dilation: 73±12%; pD2: 7.05±0.1). The maximal dilations in response to OR‐1896 were significantly (P&lt;0.05) attenuated by the nonselective K+ channel inhibitor tetraethylammonium (1 mM) in coronary (to 34±9%) and gracilis muscle arterioles (to 28±6%). Glibenclamide (5 or 10 μM), a selective ATP‐sensitive K+ channel (KATP) blocker, elicited a greater reduction of OR‐1896‐induced dilations in skeletal muscle arterioles than in coronary microvessels. Conversely, the selective inhibition of the large conductance Ca2+‐activated K+ channels (BKCa) with iberiotoxin (100 nM) significantly reduced the OR‐1896‐induced maximal dilation in coronary arterioles (to 21±6%), but was ineffective in skeletal muscle arterioles (72±8%). Accordingly, OR‐1896 elicits a substantial vasodilation in coronary and skeletal muscle arterioles, by activating primarily BKCa and KATP channels, respectively, and it is suggested that OR‐1896 contributes to the long‐term hemodynamic effects of levosimendan. British Journal of Pharmacology (2006) 148, 696–702. doi:10.1038/sj.bjp.0706781

Upregulation of Arginase by H2O2Impairs Endothelium-Dependent Nitric Oxide-Mediated Dilation of Coronary Arterioles

Overproduction of reactive oxygen species such as hydrogen peroxide (H2O2) has been implicated in various cardiovascular diseases. However, mechanism(s) underlying coronary vascular dysfunction induced by H2O2 is unclear. We studied the effect of H2O2 on dilation of coronary arterioles to endothelium-dependent and endothelium-independent agonists. Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. All vessels developed basal tone and dilated dose-dependently to activators of nitric oxide (NO) synthase (adenosine and ionomycin), cyclooxygenase (arachidonic acid), and cytochrome P450 monooxygenase (bradykinin). Intraluminal incubation of vessels with H2O2 (100 micromol/L, 60 minutes) did not alter basal tone but inhibited vasodilations to adenosine and ionomycin in a manner similar as that by NO synthase inhibitor L-NAME. H2O2 affected neither endothelium-dependent responses to arachidonic acid and bradykinin nor endothelium-independent dilation to sodium nitroprusside. The inhibited adenosine response was not reversed by removal of H2O2 but was restored by excess L-arginine. Inhibition of L-arginine consuming enzyme arginase by alpha-difluoromethylornithine or N(omega)-hydroxy-nor-L-arginine also restored vasodilation. Administering deferoxamine, an inhibitor of hydroxyl radical production, prevented the H2O2-induced impairment of vasodilation to adenosine. Western blot and reverse-transcription polymerase chain reaction results indicated that arginase I was upregulated after treating vessels with H2O2. H2O2 specifically impairs endothelium-dependent NO-mediated dilation of coronary microvessels by reducing L-arginine availability through upregulation of arginase. The formation of hydroxyl radicals from H2O2 may contribute to this process.

Translocation of Ultrafine Particles

We read with great interest the article by Geiser et al. (2005) on the mechanism of translocation of ultrafine particles (UFPs) across cellular membranes in vivo in rats following inhalation and in vitro using porcine pulmonary macrophages and human red blood cells.

A New Insight Into the Pathogenesis of Coronary Vasospasm

See related article, pages 682–689 To elucidate the control mechanisms of coronary vascular tone is one of the central interests in cardiac pathophysiology, because ischemic heart disease is one of the major causes of death in many countries. Although the coronary vascular tone is finally determined by the contractile state of smooth muscle, exo-smooth muscle mechanisms including autonomic nerves, endothelial cells, and blood cells have been shown indispensable for the control. Particular types of receptors, ion channels, and intracellular signal-cascades exist in coronary smooth muscle cells and mediate the controls by exo-smooth muscle elements (Figure). For example, stimulation of α1-adrenergic receptor, which is physiologically achieved by noradrenaline (NA) released from sympathetic presynapses, increases the intracellular Ca2+ (Ca2+i) via phosphatidylinositol (PI)-turnover and also probably via opening of receptor-activated Ca2+-permeable TRP channel. Membrane depolarization does so via opening voltage-dependent Ca2+ channels. The augmentation of Ca2+i results in contraction of the smooth muscle cells and thus constriction of the coronary artery.1 Activation of β2-adrenergic receptor, in turn, inhibits the coronary smooth muscle contraction through protein-kinase A pathway.2 The contraction is also suppressed by protein-kinase G activated by a dilating factor, nitric oxide (NO), which is produced in the endothelial cells. The mechanism for control of vascular tone . Activation of voltage-gated Ca2+ channel at the presynapse of sympathetic nerve increases intracellular Ca2+, which triggers a release of norepinephrine (NE). NE stimulates adrenergic α1 receptor (α1R) in the smooth muscle cells and drives PI-turnover as described in this scheme. The intracellular Ca2+, which flows into the cells via smooth-muscle’s Ca2+ channel or moves from endoplasmic reticulum (ER), contracts the cells. The constriction of smooth muscle cells is prevented by activation …

Endothelium‐dependent dilation in coronary arterioles following chronic nitric oxide synthase inhibition

This study tested the hypothesis that chronic inhibition of NOS with NG-nitro-L-arginine methyl ester (L-NAME) decreases endothelium-dependent dilation due to decreased NO release. Vasomotor reactivity of coronary arterioles isolated from 8 swine that were chronically administered L-NAME (8.3 ± 0.5 mg/kg/d) in their water for 30–90 days and 7 controls was examined. Arterioles isolated from the left ventricular apex were mounted on glass micropipettes and allowed to develop spontaneous tone. Dilation was produced by increasing doses of endothelium-dependent bradykinin (BK) and –independent sodium nitroprusside (SNP), performed in the presence and absence of L-NAME [300μM] in the bath. The relative contribution of NO release to the dilation was assessed by comparing dilation between arterioles with and without acute L-NAME treatment. BK and SNP dilation was similar in both groups. However, the relative contribution of NO release to BK-induced dilation (3 x 10−10 M, BK) was significantly decreased in the arterioles from pigs chronically treated with L-NAME (24±12 %) compared to arterioles from control pigs (62±11 %). These results suggest that endothelium-dependent dilation is maintained in coronary arterioles following chronic L-NAME inhibition of NOS activity, perhaps by compensatory increases in the release of non-NOS mediators. (NIH Grant #s RR-18276 and HL-52490).

Impaired nitric oxide‐mediated coronary arteriolar dilation in hypertrophic cardiomyopathy: role of angiotensin‐II and oxidative stress

Coronary ischemia is commonly observed during exercise in patients with hypertrophic cardiomyopathy (HCM). To determine if alterations in adenosine and nitric oxide− (NO) mediated pathways contribute to the coronary dysfunction observed in HCM, coronary arterioles from HCM transgenic mice (TG) were isolated, cannulated and pressurized for in vitro study. Vasodilation to adenosine (mediated by NO and KATP channels) and A23187 (calcium ionophore; mediated by NO) were reduced in TG, with no change in dilation to pinacidil (KATP channel agonist). The NO synthase inhibitor L-NAME eliminated differences between TG and control in response to adenosine and A23187, suggesting an impairment of NO-mediated dilation in TG. In the presence of the superoxide anion (O2−) scavenger TEMPOL or the NAD(P)H oxidase inhibitor apocynin, impaired vasodilations were restored. Lucigenin-chemiluminescence and dihydroethidium fluorescent signal for O2- was significantly higher in arterioles from TG. Furthermore, ACE protein and mRNA expression and the release of angiotensin-II were increased in arterioles isolated from the TG heart. Given that angiotensin-II is known to increase O2− production via NAD(P)H oxidase, our results suggest that upregulation of vascular renin-angiotensin system in the TG heart increases O2− production and impairs NO-mediated dilation via NAD(P)H-oxidase dependent mechanisms.

TNF‐alpha Produces Endothelial Dysfunction in Diabetes

Diabetes is leading risk factor for ischemic heart disease, and recent evidence suggests that inflammation is involved in the pathophysiological sequelae of acute myocardial infarction. We hypothesized that the inflammatory cytokine, tumor necrosis factor alpha (TNF) produces endothelial dysfunction in Type II diabetes. To test this, endothelium-dependent (acetylcholine, ACh) and –independent vasodilation (sodium nitroprusside, SNP) of isolated, pressurized (60 cmH2O) coronary arterioles (50–100 μm) from genetically modified mice with Type II diabetes (db/db) and the lean control heterozygotes (Db/db) were determined. In Db/db mice, SNP and ACh induced dose-dependent vasodilation, and dilation to ACh was blocked by the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 μM) (P<0.05). In db/db, dilation to ACh was blunted compared to Db/db (P<0.05), but SNP produced comparable dilation. Plasma concentration of TNF was significantly increased in db/db mice vs Db/db, 6.89±0.3 vs 1.14±0.2, respectively (P<0.05). Immunostaining result show that TNF in mouse heart is localized predominantly in vascular smooth muscle cells rather than in cardiac muscle and endothelial cells. Neutralizing antibodies to TNF (72 hr treatment; 0.625 mg/kg, i.p.) restored NO-mediated coronary arteriolar dilation in db/db mice. In conclusion, an increase in circulating and/or the local vascular TNF in Type II diabetes induces endothelial dysfunction in db/db mice.

Activation of JNK and xanthine oxidase by TNF-α impairs nitric oxide-mediated dilation of coronary arterioles

Elevated levels of tumor necrosis factor-alpha (TNF), a proinflammatory cytokine, are associated with coronary artery disease. However, it is unclear whether vasodilator function of coronary resistance arterioles is susceptible to TNF. Herein, we examined whether TNF can affect endothelium-dependent nitric oxide (NO)-mediated dilation of coronary arterioles to adenosine and whether inflammatory signaling pathways such as mitogen-activated protein kinases, ceramide sphingolipids, and oxidative stress are involved in the TNF-mediated effect. To eliminate confounding influences associated with in vivo preparations, coronary arterioles from porcine heart were isolated and pressurized without flow for in vitro study. Intraluminal treatment with TNF (1 ng/ml, 90 min) significantly attenuated the NO release and vasodilation to adenosine. This inhibitory effect was not observed in denuded vessels or in the presence of NO synthase inhibitor l-NMMA. Histochemical data showed that superoxide production and JNK phosphorylation in arteriolar endothelial cells was enhanced by TNF. Administration of superoxide scavenger or inhibitors of ceramide-activated protein kinase (dimethylaminopurine), JNK (SP600125 and dicumarol), and xanthine oxidase (allopurinol) reduced superoxide production as well as restored NO release and vasodilation to adenosine. Conversely, the effects of TNF were insensitive to inhibitors of p38 (SB203580), ERK (PD98059), NAD(P)H oxidase (apocynin), or mitochondrial respiratory chain (rotenone). These data indicate that TNF inhibits endothelium-dependent NO-mediated dilation of coronary arterioles by ceramide-induced activation of JNK and subsequent production of superoxide via xanthine oxidase. Because myocardial ischemia augments adenosine production and elevates TNF level, inhibiting adenosine-stimulated endothelial release of NO by TNF could contribute to inadequate regulation of coronary blood flow during the development of ischemic heart disease.

Role of Focal Adhesion Kinase in Flow-Induced Dilation of Coronary Arterioles

Flow-induced regulation of endothelial NO synthase (eNOS) depends on integrin signaling and tyrosine kinase activation. Integrins cluster in focal adhesion complexes, where the extracellular matrix is connected to the cytoskeleton and where focal adhesion kinase (FAK) is located. FAK plays a central role in integrin signaling and Src activation. Accordingly, we hypothesized that FAK plays an important role in flow-induced dilation (FID). To inactivate FAK-dependent signaling, anti-FAK, phosphospecific (Tyr397) antibody (FAKab), which binds against the FAK autophosphorylation site, was incorporated into endothelium of rat coronary arterioles using liposomal transfection. The responses to flow, acetylcholine (Ach), or the NO donor MAHAMANONOate (NOC-9) were observed before and after FAKab. In control and vehicles (denatured antibody or transfecting reagent alone), flow produced progressive dilation to a maximal value of 35% increase in diameter, which was inhibited by Nomega-nitro-L-arginine methyl ester (L-NAME). However, FAKab prevented FID (P<0.01 versus control). Combined treatment with FAKab and L-NAME did not produce inhibition greater than FAKab alone. FAKab did not blunt Ach- or NOC-9-induced dilation. Western analysis demonstrated that FAKab prevented flow-induced phosphorylation of FAK (pY397-FAK), Akt (pS473-Akt), and eNOS (pS1179-eNOS). Our study demonstrates the pivotal role of FAK in NO-mediated FID. Inhibition of FAK signaling with FAKab impaired FID and phosphorylation of Akt and eNOS. Our data suggest that the activation of FAK is central to the mechanotransduction of FID via regulation of activation of Akt and eNOS.

C-Reactive Protein Inhibits Endothelium-Dependent NO-Mediated Dilation in Coronary Arterioles by Activating p38 Kinase and NAD(P)H Oxidase

Elevated levels of C-reactive protein (CRP), a proinflammatory marker, are associated with reduced systemic endothelium-dependent NO-mediated dilation in patients with coronary artery disease; however, the direct effect of CRP on coronary microvascular reactivity remains unknown. Herein, we examined whether CRP can modulate endothelium-dependent NO-mediated dilation of coronary arterioles and whether proinflammatory signaling pathways such as stress-activated protein kinases (p38 and c-Jun N-terminal kinase [JNK]) and oxidative stress are involved in the CRP-mediated effect. Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. Intraluminal treatment with a clinically relevant concentration of CRP (7 microg/mL; 1 hour) significantly attenuated the NO release and vasodilation to serotonin. Further incubation with the NO precursor l-arginine (3 mmol/L) partially restored serotonin-induced vasodilation. In the presence of superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), NAD(P)H oxidase inhibitor apocynin, or p38 kinase (an upstream activator of NAD(P)H oxidase) inhibitor SB203850, but not xanthine oxidase inhibitor allopurinol or JNK inhibitor SP600125, the detrimental effect of CRP on serotonin-induced dilation was prevented. Dihydroethidium staining showed that CRP produced SB203850- and TEMPOL-sensitive superoxide production in the arteriolar endothelium. CRP treatment of coronary arterioles significantly increased NAD(P)H oxidase activity. CRP inhibits endothelium-dependent NO-mediated dilation in coronary arterioles by producing superoxide from NAD(P)H oxidase via p38 kinase activation. By impairing endothelium-dependent NO-mediated vasoreactivity, CRP could facilitate the initiation of numerous cardiovascular diseases.

Upregulation of Vascular Arginase in Hypertension Decreases Nitric Oxide–Mediated Dilation of Coronary Arterioles

One characteristic of hypertension is a decreased endothelium-dependent nitric oxide (NO)-mediated vasodilation; however, the underlying mechanism is complex. In endothelial cells (ECs), L-arginine is the substrate for both NO synthase (NOS) and arginase. Because arginase has recently been shown to modulate NO-mediated dilation of coronary arterioles by reducing l-arginine availability, we hypothesized that upregulation of vascular arginase in hypertension contributes to decreased NO-mediated vasodilation. To test this hypothesis, hypertension (mean arterial blood pressure >150 mm Hg) was maintained for 8 weeks in pigs by aortic coarctation. Coronary arterioles from normotensive (NT) and hypertensive (HT) pigs were isolated and pressurized for in vitro study. NT vessels dilated dose-dependently to adenosine (partially mediated by endothelial release of NO) and sodium nitroprusside (endothelium-independent vasodilator). Conversely, HT vessels exhibited reduced dilation to adenosine but dilated normally to sodium nitroprusside. Adenosine-stimulated NO release was increased approximately 3-fold in NT vessels but was reduced in HT vessels. Moreover, arginase activity was 2-fold higher in HT vessels. Inhibition of arginase activity by N(omega)-hydroxy-nor-l-arginine or incubation with l-arginine partially restored NO release and dilation to adenosine in HT vessels. Immunohistochemistry showed that arginase expression was increased but NOS expression was decreased in arteriolar ECs of HT vessels. These results suggest that NO-mediated dilation of coronary arterioles is inhibited in hypertension by an increase in arginase activity in EC, which limits l-arginine availability to NOS for NO production. The inability of arginase blockade or l-arginine supplementation to completely restore vasodilation may be related to downregulation of endothelial NOS expression.

Hydrogen peroxide induces endothelium-dependent and -independent coronary arteriolar dilation: role of cyclooxygenase and potassium channels.

Hydrogen peroxide, a relatively stable reactive oxygen species, is known to elicit vasodilation, but its underlying mechanism remains elusive. Here, we examined the role of endothelial nitric oxide (NO), prostaglandin, cytochrome P-450-derived metabolites, and smooth muscle potassium channels in coronary arteriolar dilation to abluminal H2O2. Pig subepicardial coronary arterioles (50-100 microm) were isolated and pressurized without flow for in vitro study. Arterioles developed basal tone and dilated dose dependently to H2O2 (1-100 microM). Disruption of th endothelium and inhibition of cyclooxygenase (COX) by indomethacin produced identical attenuation of vasodilation to H2O2. Conversely, the vasodilation to H2O2 was not affected by either the NO synthase inhibitor NG-nitro-l-arginine methyl ester or the cytochrome P-450 enzyme blocker miconazole. Inhibition of the COX-1, but not the COX-2 pathway, attenuated H2O2-induced dilation similarly to indomethacin. The production of prostaglandin E2 (PGE2), but not prostaglandin I2, from coronary arterioles was significantly increased by H2O2. Furthermore, inhibition of PGE2 receptors with AH-6809 attenuated vasodilation to H2O2 similar to that produced by indomethacin. In the absence of a functional endothelium, H2O2-induced dilation was attenuated, in an identical manner, by a depolarizing agent KCl and a calcium-activated potassium (KCa) channel inhibitor iberiotoxin. However, PGE2-induced dilation was not affected by iberiotoxin. The endothelium-independent dilation to H2O2 was also insensitive to the inhibition of guanylyl cyclase, lipoxygenase, ATP-sensitive potassium channels, and inward rectifier potassium channels. These results suggest that H2O2 induces endothelium-dependent vasodilation through COX-1-mediated release of PGE2 and also directly relaxes smooth muscle by hyperpolarization through KCa channel activation.

Ischemia-reperfusion selectively impairs nitric oxide-mediated dilation in coronary arterioles: counteracting role of arginase.

A reduction in L-arginine availability has been implicated in the impairment of endothelium-dependent nitric oxide (NO)-mediated vasodilation by ischemia-reperfusion (I/R). However, the mechanisms contributing to dysregulation of the L-arginine pool remain unknown. Because endothelial cells can metabolize L-arginine via two major enzymes, that is, NO synthase (NOS) and arginase, we hypothesized that up-regulation of arginase during I/R reduces L-arginine availability to NOS and thus impairs NO-mediated vasodilation. To test this hypothesis, a local I/R was produced in the porcine heart by occlusion of a small branch of left anterior descending artery for 30 min, followed by reperfusion for 90 min. Arterioles (60-110 microm) isolated from non-ischemic and ischemic regions of subepicardium were cannulated and pressurized without flow for in vitro study. Vessels from both regions developed similar levels of basal tone. Although the dilation of I/R vessels to endothelium-independent agonist sodium nitroprusside was not altered, the endothelium-dependent NO-mediated dilations to adenosine and serotonin were attenuated. I/R not only inhibited arteriolar production of NO but also increased arteriolar arginase activity. Arginase inhibitor alpha-difluoromethylornithine enhanced NO production/dilation in normal vessels and also restored the NO-mediated function in I/R vessels. Treating I/R vessels with L-arginine also restored vasodilations. Immunohistochemical data revealed that I/R up-regulated arginase but down-regulated NOS expression in the arteriolar endothelium. Pretreating the animals with protein synthesis inhibitor cycloheximide prevented I/R-induced arginase up-regulation and also preserved NO-mediated vascular function. These results suggest that one mechanism by which I/R inhibits NO-mediated arteriolar dilation is through increased arginase activity, which limits the availability of L-arginine to NOS for NO production. In addition, the inability of arginase blockade or L-arginine supplementation to completely restore vasodilatory function may be attributable to the down-regulation of endothelial NOS expression.