Ramona S. Scotland

Investigation of Vascular Responses in Endothelial Nitric Oxide Synthase/Cyclooxygenase-1 Double-Knockout Mice Key Role for Endothelium-Derived Hyperpolarizing Factor in the Regulation of Blood Pressure in Vivo

Background—Endothelium-dependent dilatation is mediated by 3 principal vasodilators: nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). To determine the relative contribution of these factors in endothelium-dependent relaxation, we have generated mice in which the enzymes required for endothelial NO and PGI2 production, endothelial NO synthase (eNOS) and cyclooxygenase-1 (COX-1), respectively, have been disrupted (eNOS−/− and COX-1−/− mice). Methods and Results—In female mice, the absence of eNOS and COX-1 had no effect on mean arterial blood pressure (BP), whereas BP was significantly elevated in eNOS−/−/COX-1−/− males compared with wild-type controls. Additionally, endothelium-dependent relaxation remained intact in the resistance vessels of female mice and was associated with vascular smooth muscle hyperpolarization; however, these responses were profoundly suppressed in arteries of male eNOS−/−/COX-1−/− animals. Similarly, the endothelium-dependent vasodilator bradykinin produced dose-dependent hypotension in female eNOS−/−/COX-1−/− animals in vivo but had no effect on BP in male mice. Conclusions—These studies indicate that EDHF is the predominant endothelium-derived relaxing factor in female mice, whereas NO and PGI2 are the predominant mediators in male mice. Moreover, the gender-specific prevalence of EDHF appears to underlie the protection of female eNOS−/−/COX-1−/− mice against hypertension.

Vanilloid receptor TRPV1, sensory C-fibers, and vascular autoregulation: a novel mechanism involved in myogenic constriction.

Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8±10.3% inhibition, n=8; P<0.05). Activation of C-fiber nerve endings by pressure was attributable to stimulation of neuronal vanilloid receptor, TRPV1, because blockers of this channel, capsazepine (71.9±11.1% inhibition, n=9; P<0.001) and ruthenium red (46.1±11.7% inhibition, n=4; P<0.05), suppressed the myogenic constriction. In addition, this C-fiber dependency is likely related to neuropeptide substance P release and activity because blockade of tachykinin NK1 receptors (66.3±13.7% inhibition, n=6; P<0.001), and not NK2 receptors (n=4, NS), almost abolished the myogenic response. Previous studies support a role for 20-hydroxyeicosatetraenoic acid (20-HETE) in myogenic constriction responses; herein, we show that 20-HETE–induced constriction of mesenteric resistance arteries is blocked by capsazepine. Together, these results suggest that elevation of intraluminal pressure is associated with generation of 20-HETE that, in turn, activates TRPV1 on C-fiber nerve endings resulting in depolarization of nerves and consequent vasoactive neuropeptide release. These findings identify a novel mechanism contributing to Bayliss’ myogenic constriction and highlights an alternative pathway that may be targeted in the therapeutics of vascular disease, such as hypertension, where enhanced myogenic constriction plays a role in the pathogenesis.

Natriuretic Peptide Receptor-C Regulates Coronary Blood Flow and Prevents Myocardial Ischemia/Reperfusion Injury Novel Cardioprotective Role for Endothelium-Derived C-Type Natriuretic Peptide

Background—Ischemia/reperfusion (I/R) injury complicates myocardial infarction and stroke by exacerbating tissue damage and increasing risk of mortality. We have recently identified C-type natriuretic peptide (CNP) as an endothelium-derived hyperpolarizing factor in the mesenteric resistance vasculature and described a novel signaling pathway involving activation of natriuretic peptide receptor C (NPR-C), which plays a pivotal role in the regulation of local blood flow. We tested the hypothesis that CNP/NPR-C signaling is a novel regulatory pathway governing coronary blood flow and protecting against I/R injury. Methods and Results—CNP and (Cys18)-atrial natriuretic factor (4-23) amide (cANF4–23) elicited dose-dependent decreases in coronary perfusion pressure (CPP) that were blocked by Ba2+ and ouabain in the isolated Langendorff rat heart. The endothelium-dependent vasodilator acetylcholine elicited the release of CNP from the coronary endothelium. CNP and cANF4–23 reduced infarct size after 25 minutes of global ischemia and 120 minutes of reperfusion, maintaining CPP and left ventricular pressure at preischemic values. The vasorelaxant and protective activity of CNP and cANF4–23 were enhanced in the absence of endothelium-derived nitric oxide. Conclusion—Endothelium-derived CNP is involved in the regulation of the coronary circulation, and NPR-C activation underlies the vasorelaxant activity of this peptide. Moreover, this newly defined pathway represents a protective mechanism against I/R injury and a novel target for therapeutic intervention in ischemic cardiovascular disorders.

Sex differences in resident immune cell phenotype underlie more efficient acute inflammatory responses in female mice

Females are protected against mortality arising from severe sepsis; however, the precise mechanisms that confer this survival advantage in females over males are unclear. Resident leukocytes in resting tissues have a significant influence on circulating cytokine levels and recruitment of blood leukocytes during acute inflammatory responses. Whether the phenotype of resident leukocytes is distinct in females is unknown. In the present study, we show that the numbers of leukocytes occupying the naive peritoneal and pleural cavities is higher in female than in male mice and rats, comprising more T and B lymphocytes and macrophages. The altered immune cell composition of the female peritoneum is controlled by elevated tissue chemokine expression. Female resident macrophages also exhibit greater TLR expression and enhanced phagocytosis and NADPH oxidase-mediated bacterial killing. However, macrophage-derived cytokine production is diminished by proportionally more resident immunomodulatory CD4+ T lymphocytes. Ovarian hormones regulate macrophage phenotype, function, and numbers, but have no significant impact on T-lymphocyte populations in females. We have identified a fundamental sex difference in phenotype of resident leukocytes. We propose that the distinct resident leukocyte population in females allows aggressive recognition and elimination of diverse infectious stimuli without recruitment of circulating neutrophils or excessive cytokine production.

A Role of Matrix Metalloproteinase-8 in Atherosclerosis

Rationale: Atherosclerotic lesions express matrix metalloproteinase (MMP)8, which possesses proteolytic activity on matrix proteins particularly fibrillar collagens and on nonmatrix proteins such as angiotensin (Ang) I. 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.

Vascular natriuretic peptide receptor-linked particulate guanylate cyclases are modulated by nitric oxide-cyclic GMP signalling.

The sensitivity of the particulate guanylate cyclase–cyclic guanosine‐3′,5′‐monophosphate (cGMP) system to atrial (ANP) and C‐type (CNP) natriuretic peptides was investigated in aortae and mesenteric small arteries from wild‐type (WT) and endothelial nitric oxide synthase (eNOS) knockout (KO) mice. ANP and CNP produced concentration‐dependent relaxations of mouse aorta that were significantly attenuated by the natriuretic peptide receptor (NPR)‐A/B antagonist HS‐142‐1 (10−5 M). Both ANP and CNP were more potent in aortae from eNOS KO mice compared to WT. The potency of ANP and CNP in aortae from WT animals was increased in the presence of the NOS inhibitor, NG‐nitro‐L‐arginine (3 × 10−4 M) and the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolol[4,3,a]quinoxalin‐1‐one (5 × 10−6 M). In contrast, the potency of ANP and CNP in aortae from eNOS KO animals was reduced following pretreatment of tissues with supramaximal concentrations of the NO‐donor, glyceryl trinitrate (3 × 10−5 M, 30 min) or ANP (10−7 M, 30 min). Responses to acetylcholine in aortae from WT mice (dependent on the release of endothelium‐derived NO) were significantly reduced following pretreatment of tissues with GTN (3 × 10−5 M, 30 min) and ANP (10−7 M, 30 min). CNP and the NO‐donor, spermine‐NONOate caused concentration‐dependent relaxations of mesenteric small arteries from WT animals that were significantly increased in eNOS KO mice compared to WT. ANP was unable to significantly relax mesenteric arteries from WT or eNOS KO animals. In conclusion, both NPR‐A‐ and NPR‐B‐linked pGC pathways are modulated by NO–cGMP in murine aorta and mesenteric small arteries and crossdesensitisation occurs between NPR subtypes. The biological activity of endothelium‐derived NO is also influenced by the ambient concentration of NO and natriuretic peptides. Such an autoregulatory pathway may represent an important physiological homeostatic mechanism and link the paracrine activity of NO and CNP with the endocrine functions of ANP and BNP in the regulation of vascular tone and blood pressure.

Ovalbumin-induced neurogenic inflammation in the bladder of sensitized rats

We have developed and characterized a model of immediate hypersensitivity/inflammation of the urinary bladder in vivo induced by local application of ovalbumin (OA) in OA‐ sensitive female rats. Two parameters of the inflammatory response were assessed following OA challenge: plasma protein extravasation (PPE) and changes in smooth muscle reactivity. The former was estimated by measurement of Evans blue extravasation at 0.5, 2, 4, 8 and 24 h time point following in vivo challenge. Changes in reactivity were determined by measurement of isotonic tension responses of urinary bladder strips following OA challenge in vitro. Acute in vivo intravesical OA challenge (10 mg in 0.3 ml saline) in actively sensitized female Wistar rats caused a time‐dependent PPE in the urinary bladder which was biphasic with peak responses at 2–4 and 24 h. The PPE response to acute OA challenge, above base‐line, at 2 h was abolished by systemic capsaicin pretreatment (50 mg kg−1, s.c., 4 days before use) (P<0.05) whilst the response at 24 h was unaffected. The 2 h time point was then used for further studies. Degranulation of mast cells, achieved by pretreatment with compound 48/80 (5 mg kg−1, s.c. for 3 consecutive days), completely abolished the PPE response to OA challenge at the 2 h time point. The tachykinin NK1 receptor antagonist, SR 140333 (0.1 μmol kg−1, i.v.), abolished the 2 h PPE response whilst the tachykinin NK2 receptor antagonist MEN 11420 (0.1 μmol kg−1, i.v.) appeared to reduce the response by approximately 50% but this did not reach significance. The bradykinin B2 receptor antagonist, Hoe 140 (0.1 μmol kg−1, i.v.), similarly to SR 140333, blocked the 2 h PPE response to OA, whereas the selective B1 receptor antagonist B 9858 (0.1 μmol kg−1, i.v.) had no significant effect. Inhibition of cyclo‐oxygenase (COX) achieved by pretreatment with the COX inhibitor dexketoprofen (5.3 μmol kg−1, i.v.) also blocked the PPE response, whilst the leukotriene receptor antagonist ONO 1078 (1 μmol kg−1, i.v.) significantly reduced PPE by about 80%. In the rat isolated urinary bladder OA (1 mg ml−1) challenge produced a biphasic response with a rapidly achieved maximal contraction followed by a sustained contraction for approximately 25 min. In vitro capsaicin pretreatment (10 μM for 15 min) significantly attenuated the duration of the sustained contraction whilst having no effect on the maximum contractile response achieved. In vivo pretreatment of animals with compound 48/80 significantly attenuated (42%) the maximum contractile response. Combination of both treatments almost completely abolished the response. In vitro treatment with Hoe 140 (1 μM) had no significant effect on the response to OA and neither did ONO 1078 (1 μM). These results show that both the early inflammatory response and alterations in smooth muscle reactivity to OA challenge in actively sensitized animals are dependent on mast cell degranulation and the activation of sensory C‐fibres. Furthermore this model of allergic cystitis may be useful for investigating both the processes involved and potential novel therapies in the treatment of interstitial cystitis.

Endothelin alters the reactivity of vasa vasorum: mechanisms and implications for conduit vessel physiology and pathophysiology

The walls of certain large blood vessels are nourished by the vasa vasorum, a network of microvessels that penetrate the adventitia and media of the vessel wall. The purpose of this study was to characterize endothelin‐1 (ET‐1)‐mediated contraction of vasa and to investigate whether threshold concentrations of ET‐1 alters the sensitivity to constrictors. Arterial vasa were dissected from the walls of porcine thoracic aorta and mounted in a tension myograph. ET‐1 and ETB‐selective agonist, sarafotoxin 6c (S6c), produced concentration‐dependent contraction. ETA receptor antagonist, BQ123 (10 μM), caused a biphasic rightward shift of ET‐1 response curves. ETB receptor antagonist, BQ788 (1 μM), produced a rightward shift of response curves to ET‐1 and S6c of 5‐ and 80 fold respectively. ET‐1 responses were abolished in Ca2+‐free PSS but unaffected by selective depletion of intracellular Ca2+ stores. Nifedipine (10 μM), an L‐type Ca2+ channel blocker, attenuated ET‐1 responses by 44%. Inhibition of receptor‐operated Ca2+ channels or non‐selective cation entry using SKF 96365 (30 μM) and Ni2+ (1 mM) respectively, attenuated ET‐1 contractions by 60%. ET‐1 (1–3 nM) enhanced responses to noradrenaline (NA) (4 fold) but not to thromboxane A2‐mimetic, whilst K+ (10–20 mM) sensitized vasa to both types of constrictor. Therefore, ET‐1‐induced contraction of isolated vasa is mediated by ETA and ETB receptors and involves Ca2+ influx through L‐type and non‐L‐type Ca2+ channels. Furthermore elevation of basal tone of vasa vasorum alters the profile of contractile reactivity. These results suggest that ET‐1 may be an important regulator of vasa vasorum reactivity.

Distinct endothelial pathways underlie sexual dimorphism in vascular auto-regulation

BACKGROUND AND PURPOSE Pre‐menopausal females have a lower incidence of cardiovascular disease compared with age‐matched males, implying differences in the mechanisms and pathways regulating vasoactivity. In small arteries, myogenic tone (constriction in response to raised intraluminal pressure) is a major determinant of vascular resistance. Endothelium‐derived dilators, particularly NO, tonically moderate myogenic tone and, because the endothelium is an important target for female sex hormones, we investigated whether NO‐mediated moderation of myogenic tone differed between the sexes.

Toll-like receptors: Role in inflammation and therapeutic potential

Inflammation is an essential process in response to injury and infection. However, under certain circumstances dis‐regulation of this process can lead to pathologies such as rheumatoid arthritis, atherosclerosis, lupus, and is a contributory factor in the progression of many cancers. The Toll‐like family of receptors (TLRs) has major roles in the initiation of the inflammatory response and as such has attracted much focus for their potential as therapeutic targets. Here we review the role of TLRs in the inflammatory response and associated disease and examine how this important family of molecules might be targeted for therapeutic benefit.