Miles A. Tanner

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells

OBJECTIVE Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. METHODS Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2 = 10 mmHg) or normoxia (pO2 = 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. RESULTS Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol . micrograms-1 protein, hypoxia vs. normoxia respectively, n = 8, P < 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol . microgram-1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol . micrograms-1 protein (n = 6, p < 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. CONCLUSIONS These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide.

Induction of angiotensin II subtype 2 receptor-mediated blood pressure regulation in synthetic diet-fed rats.

Objective Chronic feeding of a purified synthetic diet induces renin–angiotensin system-dependent moderate high blood pressure in normal Sprague–Dawley rats. The present study was designed to characterize the angiotensin II (Ang II) receptor type 2 (AT2)-specific mechanism of blood pressure regulation in these rats. Methods The effect of the AT2 receptor antagonist PD123319 (PD) on blood pressure was examined in vivo in synthetic diet-fed rats. Ang II-dependent contraction of aortic rings prepared from the synthetic diet-fed rats was also investigated. Results After 8 weeks of feeding the synthetic diet, the mean arterial pressure (MAP) was significantly elevated above levels measured in control rats (117 ± 2 versus 102 ± 3 mmHg, P < 0.05). Intravenous administration of PD to conscious hypertensive rats elicited an immediate dose-dependent increase in MAP that was sustained for approximately 7.4 min with 3 mg/kg PD. The angiotensin converting enzyme inhibitor captopril, but not the Ang II type 1 receptor blocker losartan, significantly attenuated the effect of PD on blood pressure. PD did not increase the plasma level of catecholamines. The PD-dependent blood pressure increase was not observed in normotensive control rats. Aortic ring assays revealed that functional activation of the AT2 receptor occurs only in the hypertensive rats, and this AT2 response is abolished by indomethacin (5 μmol/l) but not by Nω-nitro-L-arginine methyl ester (100 μmol/l). Conclusion These results clearly demonstrate that AT2 receptor-mediated blood pressure regulation is functional in this experimental model of hypertension. Furthermore, cyclooxygenase metabolites might be the key mediators for the AT2 receptor-mediated blood pressure-lowering action.

Restenosis is associated with decreased coronary artery nitric oxide synthase

The purpose of the present study was to test the hypothesis that restenosis is associated with decreased constitutive nitric oxide synthase activity. Male miniswine with moderately elevated serum cholesterol levels underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries, followed 2 weeks later by repeat injury on the same coronary segments. After 4 weeks, the coronary arteries were either immediately frozen in liquid nitrogen or pressure-perfusion fixed and prepared for histologic examination. Constitutive nitric oxide synthase activity was quantified using a fibroblast reporter cell method, while constitutive nitric oxide synthase protein was compared between balloon-injured and non-balloon-injured arteries using Western blot analysis. Immunohistochemical studies were performed using a specific antibody against constitutive nitric oxide synthase protein. Following balloon injury, there was decreased constitutive nitric oxide synthase activity in balloon-injured coronary arteries, compared to distal non-balloon-injured segments from the same artery. Histological examination demonstrated an intact endothelium. Specific antibody staining revealed that there was less constitutive nitric oxide synthase protein reactivity by immunohistochemical analysis. Western analysis confirmed less constitutive nitric oxide synthase protein. The data are consistent with the hypothesis that restenosis is associated with decreased endothelial cell nitric oxide production. The data suggest this is secondary to a decreased amount of constitutive nitric oxide synthase enzyme in the endothelium. A deficiency in constitutive nitric oxide synthase enzyme may contribute to the impaired second messenger and paracrine functions of the endothelium observed during restenosis following balloon injury, including abnormal vasomotion, extracellular matrix formation, and platelet aggregation.

Heterogeneity in Kv7 Channel Function in the Cerebral and Coronary Circulation

Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study aimed to examine the hypotheses that (i) Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity and (ii) regional differences exist in the activity of these channels.

Effects of Angiotensin II on Canine and Porcine Coronary Epicardial and Resistance Arteries

Coronary resistance arteriolar diameter importantly regulates myocardial blood flow, and is influenced by circulating neurohumoral agents. Angiotensin II (A-II) is a circulating polypeptide that is chronically elevated in heart failure and serves as a potent peripheral vasoconstrictor agent. However, its effects on isolated coronary resistance arterioles is relatively unknown. We compared the vasomotor effects of A-II on coronary epicardial and resistance arterioles in vitro from both the canine and porcine heart in order to determine the effects of A-II in different vascular beds and species. Epicardial rings were studied under isometric recording conditions, while resistance arterioles (50-150 microns) were studied in vitro using a video imaging system to record diameter. A-II, whether applied to passively distended or preconstricted porcine resistance arterioles, did not cause vasoconstriction when applied as a bolus or as cumulative doses. In preconstricted canine resistance arterioles, A-II elicited dose-dependent vasodilation (EC50 = 0.2 nM). In passively distended canine arterioles, high concentrations of A-II (0.1 microM) applied as a bolus elicited transient vasoconstriction in 28% of the vessels studied. In large epicardial rings, A-II was a weak vasoconstrictor, with greater potency in canine arteries compared to porcine arteries. In canine arteries, vasoconstriction to A-II was augmented after incubation with indomethacin. In contrast to the findings in canine arteries, the A-II vasoconstrictor response in porcine coronary arteries was decreased after incubation with indomethacin or removal of the endothelium. Thus, A-II elicits the release of a vasodilator prostanoid in epicardial canine coronary arteries and a vasoconstrictor prostanoid in porcine vessels which modulate the vasomotor action of A-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharides and cytokines downregulate the angiotensin II type 2 receptor in rat cardiac fibroblasts.

The present study examines the effect of lipopolysaccharides and proinflammatory cytokines on the expression of the second isoform of the angiotensin II receptor (AT(2)), which may have a role in lowering collagen deposition in cardiac tissue. Cardiac fibroblasts express high levels of both angiotensin II type 1 (AT(1)) and type 2 receptors. Incubation with lipopolysaccharides for 24 h dose- and time-dependently decreased angiotensin II AT(2) receptor expression with no apparent difference in the affinity. Actinomycin D, cycloheximide, N(omega)-nitro-L-arginine methyl ester and the protein tyrosine kinase inhibitor herbimycin A, but not the protein kinase C inhibitors bisindolylmaleimide and calphostin C, abolished the inhibitory action of lipopolysaccharides. The cytokines interleukin-1beta and tissue necrosis factor-alpha mimicked the effect of lipopolysaccharides. All three compounds induced inducible nitric oxide synthase (iNOS). The nitric oxide donor sodium nitroprusside and the cGMP analog 8-bromoguanosine cyclic monophosphate downregulated angiotensin II AT (2) receptor expression. The findings are consistent with the pathway in which lipopolysaccharides or cytokines induce iNOS. The data suggest that lipopolysaccharide- or cytokine-dependent induction of iNOS and resultant production of nitric oxide leads to the production of cGMP, which in turn downregulates expression of the angiotensin II AT (2) receptor in cardiac fibroblasts.

Adiponectin Receptor Agonist, AdipoRon, Causes Vasorelaxation Predominantly Via a Direct Smooth Muscle Action.

AdipoRon, an adiponectin receptor agonist, was recently proposed for treating insulin resistance and hyperglycemia. As adiponectin is vasoprotective via NO‐mediated signaling, it was hypothesized that adipoRon similarly exerts potentially beneficial vasodilator effects. We therefore examined if adipoRon induces vasorelaxation and via what contributing mechanisms.

Phrenic long-term facilitation following intrapleural CTB-SAP-induced respiratory motor neuron death

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to progressive motor neuron degeneration and death by ventilatory failure. In a rat model of ALS (SOD1G93A), phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is enhanced greater than expected at disease end-stage but the mechanism is unknown. We suggest that one trigger for this enhancement is motor neuron death itself. Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kill respiratory motor neurons and mimic motor neuron death observed in SOD1G93A rats. This CTB-SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS. Here, we tested the hypothesis that phrenic motor neuron death is sufficient to enhance pLTF. pLTF was assessed in anesthetized, paralyzed and ventilated Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB-SAP (25 μg), or 2) un-conjugated CTB and SAP (control). CTB-SAP enhanced pLTF at 7 (CTB-SAP: 162 ± 18%, n = 8 vs. Control: 63 ± 3%; n = 8; p < 0.05), but not 28 days post-injection (CTB-SAP: 64 ± 10%, n = 10 vs. Control: 60 ± 13; n = 8; p > 0.05). Thus, pLTF at 7 (not 28) days post-CTB-SAP closely resembles pLTF in end-stage ALS rats, suggesting that processes unique to the early period of motor neuron death enhance pLTF. This project increases our understanding of respiratory plasticity and its implications for breathing in motor neuron disease.

Depletion of dendritic cells in perivascular adipose tissue improves arterial relaxation responses in type 2 diabetic mice

BACKGROUND Accumulation of multiple subtypes of immune cells in perivascular adipose tissue (PVAT) has been proposed to cause vascular inflammation and dysfunction in type 2 diabetes (T2DM). This study was designed to investigate specific roles for dendritic cells in PVAT in the development of vascular inflammation and impaired PVAT-mediated vasorelaxation in T2DM. METHODS AND RESULTS Studies were performed using db/db mice (model of T2DM) and their Db heterozygote (DbHET), lean and normoglycemic controls. Dendritic cell depletion was performed by cross-breeding DbHet with Flt3l-/- (null for ligand for FMS-kinase tyrosine kinase) mice. Using PCR, it was found that the majority of dendritic cells (CD11c+) were located in PVAT rather than the vascular wall. Flow cytometry similarly showed greater dendritic cell accumulation in adipose tissue from db/db mice than DbHET controls. Adipose tissue from db/db mice displayed increased mRNA levels of proinflammatory cytokines TNF-α and IL-6 and decreased mRNA levels of the anti-inflammatory mediator adiponectin, compared to DbHET mice. Depletion of dendritic cells in dbFlt3l-/dbFlt3l- (confirmed by flow cytometry) reduced TNF-α and IL-6 mRNA levels in diabetic adipose tissue without influencing adiponection expression. Moreover, in mesenteric arteries, dendritic cell depletion improved the ability of PVAT to augment acetylcholine-induced vasorelaxation and anti-contractile activity. CONCLUSIONS In a murine model of T2DM, dendritic cells accumulated predominantly in PVAT, as opposed to the vessel wall, per se. Accumulation of dendritic cells in PVAT was associated with overproduction of pro-inflammatory cytokines, which contributed to an impaired ability of PVAT to augment vasorelaxation and exert anti-contractile activity in T2DM.