Chen Huei Leo

Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes.

BACKGROUND AND PURPOSE To investigate whether diabetes affects either or both nitric oxide (NO)‐mediated and endothelium‐derived hyperpolarizing factor (EDHF)‐type relaxation in endothelium‐dependent relaxation of mesenteric arteries from streptozotocin‐induced diabetic rats.

Localization of relaxin receptors in arteries and veins, and region-specific increases in compliance and bradykinin-mediated relaxation after in vivo serelaxin treatment

Relaxin is a potent vasodilator of small resistance arteries and modifies arterial compliance in some systemic vascular beds, yet receptors for relaxin, such as RXFP1, have only been localized to vascular smooth muscle. This study first aimed to localize RXFP1 in rat arteries and veins from different organ beds and determine whether receptors are present in endothelial cells. We then tested the hypothesis that region‐specific vascular effects of relaxin may be influenced by the cellular localization of RXFP1 within different blood vessels. The aorta, vena cava, mesenteric artery, and vein had significantly higher (P<0.05) RXFP1 immunostaining in endothelial cells compared with vascular smooth muscle, whereas the femoral artery and vein and small pulmonary arteries had higher (P<0.01) RXFP1 immunostaining in the vascular smooth muscle. Male rats were treated subcutaneously with recombinant human relaxin‐2 (serelaxin; 4 μg/h) for 5 d; vasodilation and compliance in mesenteric and femoral arteries and veins were compared with placebo controls. Serelaxin significantly (P=0.04) reduced wall stiffness and increased volume compliance in mesenteric arteries but not in the other vessels examined. This was associated with changes in geometrical properties, and not compositional changes in the extracellular matrix. Serelaxin treatment had no effect on acetylcholine‐mediated relaxation but significantly (P< 0.001) enhanced bradykinin (BK)‐mediated relaxation in mesenteric arteries, involving enhanced nitric oxide but not endothelium‐derived hyperpolarization or vasodilatory prostanoids. In conclusion, there is differential distribution of RXFP1 on endothelial and smooth muscle across the vasculature. In rats, mesenteric arteries exhibit the greatest functional response to chronic serelaxin treatment.—Jelinic, M., Leo, C‐H., Post Uiterweer, E. P., Sandow, S. L., Gooi, J. H., Wlodek, M. E., Conrad, K. P., Parkington, H., Tare, M., Parry, L. J. Localization of relaxin receptors in arteries and veins, and region‐specific increases in compliance and bradykinin‐mediated relaxation after in vivo serelaxin treatment. FASEB J. 28, 275–287 (2014). www.fasebj.org

Acute Intravenous Injection of Serelaxin (Recombinant Human Relaxin-2) Causes Rapid and Sustained Bradykinin-Mediated Vasorelaxation

Background A recent clinical trial (RELAXin in Acute Heart Failure [RELAX‐AHF]) demonstrated that 48 hours of continuous intravenous infusion of the vasorelaxant peptide serelaxin (recombinant human relaxin‐2) to patients with acute heart failure reduced cardiovascular mortality at 180 days. The persistence of a vasorelaxant response as a potential mechanism for this long‐term benefit and the vascular effects of a bolus intravenous injection of serelaxin have not been examined. This study investigates changes in resistance artery reactivity and passive mechanical wall properties following an intravenous serelaxin injection and whether these vascular effects persist in the absence of detectable circulating serelaxin. Methods and Results Male rats were injected with 13.3 μg/kg serelaxin into the tail vein; mesenteric arteries were assessed 3 and 24 hours after treatment by using wire‐myography. Serelaxin increased basal nitric oxide synthase activity and reduced maximal contraction to endothelin‐1 at 3 hours after administration. Serelaxin treatment also selectively enhanced bradykinin‐mediated endothelium‐dependent relaxation. This effect was sustained for 24 hours in the absence of circulating serelaxin. Serelaxin‐mediated augmentation of bradykinin‐evoked relaxation involved endothelium‐derived hyperpolarization after 3 hours and prostacyclin‐mediated relaxation after 24 hours. Furthermore, upregulation of inducible nitric oxide synthase, phosphorylation of protein kinase B at Ser473 and endothelial nitric oxide synthase at Ser1177 was observed at 24 hours after serelaxin injection. There were no effects of serelaxin on passive arterial wall stiffness. Conclusion Our data show that a bolus intravenous injection of serelaxin modulates endothelial vasodilator function 3 hours after administration, an effect that was sustained for 24 hours. The prolonged bradykinin‐mediated vasorelaxation is principally mediated through prostacyclin.

Vascular actions of relaxin: nitric oxide and beyond

The peptide hormone relaxin regulates the essential maternal haemodynamic adaptations in early pregnancy through direct actions on the renal and systemic vasculature. These vascular actions of relaxin occur mainly through endothelium‐derived NO‐mediated vasodilator pathways and improvements in arterial compliance in small resistance‐size arteries. This work catalysed a plethora of studies which revealed quite heterogeneous responses across the different regions of the vasculature, and also uncovered NO‐independent mechanisms of relaxin action. In this review, we first describe the role of endogenous relaxin in maintaining normal vascular function, largely referring to work in pregnant and male relaxin‐deficient animals. We then discuss the diversity of mechanisms mediating relaxin action in different vascular beds, including the involvement of prostanoids, VEGF, endothelium‐derived hyperpolarisation and antioxidant activity in addition to the classic NO‐mediated vasodilatory pathway. We conclude the review with current perspectives on the vascular remodelling capabilities of relaxin.

Serelaxin: A Novel Therapeutic for Vascular Diseases

Vascular dysfunction is an important hallmark of cardiovascular disease. It is characterized by increased sensitivity to vasoconstrictors, decreases in the endothelium-derived vasodilators nitric oxide (NO) and prostacyclin (PGI2), and endothelium-derived hyperpolarization (EDH). Serelaxin (recombinant human relaxin) has gained considerable attention as a new vasoactive drug, largely through its beneficial therapeutic effects in acute heart failure. In this review we first describe the contribution of endogenous relaxin to vascular homeostasis. We then provide a comprehensive overview of the novel mechanisms of serelaxin action in blood vessels that differentiate it from other vasodilator drugs and explain how this peptide could be used more widely as a therapeutic to alleviate vascular dysfunction in several cardiovascular diseases.

Increased superoxide production and altered nitric oxide-mediated relaxation in the aorta of young but not old male relaxin-deficient mice

The vascular effects of exogenous relaxin (Rln) treatment are well established and include decreased myogenic reactivity and enhanced relaxation responses to vasodilators in small resistance arteries. These vascular responses are reduced in older animals, suggesting that Rln is less effective in mediating arterial function with aging. The present study investigated the role of endogenous Rln in the aorta and the possibility that vascular dysfunction occurs more rapidly with aging in Rln-deficient (Rln(-/-)) mice. We compared vascular function and underlying vasodilatory pathways in the aorta of male wild-type (Rln(+/+)) and Rln(-/-) mice at 4 and 16 mo of age using wire myography. Superoxide production, but not nitrotyrosine or NADPH oxidase expression, was significantly increased in the aorta of young Rln(-/-) mice, whereas endothelial nitric oxide (NO) synthase and basal NO availability were both significantly decreased compared with Rln(+/+) mice. In the presence of the cyclooxygenase inhibitor indomethacin, sensitivity to ACh was significantly decreased in young Rln(-/-) mice, demonstrating altered NO-mediated relaxation that was normalized in the presence of a membrane-permeable SOD or ROS scavenger. These vascular phenotypes were not exacerbated in old Rln(-/-) mice and, in most cases, did not differ significantly from old Rln(+/+) mice. Despite the vascular phenotypes in Rln(-/-) mice, endothelium-dependent and -independent vasodilation were not adversely affected. Our data show a role for endogenous Rln in reducing superoxide production and maintaining NO availability in the aorta but also demonstrate that Rln deficiency does not compromise vascular function in this artery or exacerbate endothelial dysfunction associated with aging.

Time-dependent activation of prostacyclin and nitric oxide pathways during continuous i.v. infusion of serelaxin (recombinant human H2 relaxin)

In the RELAX‐AHF trial, a 48 h i.v. serelaxin infusion reduced systemic vascular resistance in patients with acute heart failure. Consistent with preclinical studies, serelaxin augments endothelial vasodilator function in rat mesenteric arteries. Little is known about the contribution of endothelium‐derived relaxing factors after a longer duration of continuous serelaxin treatment. Here we have assessed vascular reactivity and mechanistic pathways in mesenteric arteries and veins and the aorta after 48 or 72 h continuous i.v. infusion of serelaxin.

Endothelium-dependent nitroxyl-mediated relaxation is resistant to superoxide anion scavenging and preserved in diabetic rat aorta

The aim of the study was to investigate whether diabetes-induced oxidant stress affects the contribution of nitroxyl (HNO) to endothelium-dependent relaxation in the rat aorta. Organ bath techniques were employed to determine vascular function of rat aorta. Pharmacological tools (3mM l-cysteine, 5mM 4-aminopyridine (4-AP), 200μM carboxy-PTIO and 100μM hydroxocobalamin, HXC) were used to distinguish between NO and HNO-mediated relaxation. Superoxide anion levels were determined by lucigenin-enhanced chemiluminescence. In the diabetic aorta, where there is increased superoxide anion production, responses to the endothelium-dependent relaxant ACh were not affected when the contribution of NO to relaxation was abolished by either HXC or carboxy-PTIO, indicating a preserved HNO-mediated relaxation. Conversely, when the contribution of HNO was inhibited with l-cysteine or 4-AP, the sensitivity and maximum relaxation to ACh was significantly decreased, suggesting that the contribution of NO was impaired by diabetes. Furthermore, whereas HNO appears to be derived from eNOS in normal aorta, in the diabetic aorta it may also arise from an eNOS-independent source, perhaps derived from nitrosothiol stores. Similarly, exposure to the superoxide anion generator, pyrogallol (100μM) significantly reduced the sensitivity to the NO donor, DEANONOate and ACh-induced NO-mediated relaxation but had no effect on responses to the HNO donor, Angelis salt and ACh-induced HNO-mediated relaxation in the rat aorta. These findings demonstrate that NO-mediated relaxation is impaired during oxidative stress but the HNO component of relaxation is preserved under those conditions.

Serelaxin (recombinant human relaxin-2) prevents high glucose-induced endothelial dysfunction by ameliorating prostacyclin production in the mouse aorta.

Diabetes-induced endothelial dysfunction is a critical initiating factor in the development of cardiovascular complications. Treatment with relaxin improves tumour necrosis factor α-induced endothelial dysfunction by enhancing endothelial nitric oxide synthase (eNOS) activity and restoring superoxide dismutase 1 protein in rat aortic rings ex vivo. It is, therefore, possible that relaxin treatment could alleviate endothelial dysfunction in diabetes. This study aimed to test the hypothesis that serelaxin (recombinant human relaxin-2) prevents high glucose-induced vascular dysfunction in the mouse aorta. Abdominal aortae were isolated from C57BL/6 male mice and incubated in M199 media for 3days with either normal glucose (5.5mM) or high glucose (30mM), and co-incubated with placebo (20mM sodium acetate) or 10nM serelaxin at 37°C in 5% CO2. Vascular function was analysed using wire-myography. High glucose significantly reduced the sensitivity to the endothelium-dependent agonist, acetylcholine (ACh) (pEC50; normal glucose=7.66±0.10 vs high glucose=7.29±0.10, n=11-12, P<0.05) and the contraction induced by NOS inhibitor, L-NAME (200μM) (normal glucose=59.9±8.3% vs high glucose=38.7±4.3%, n=6, P<0.05), but had no effect on the endothelium-independent agonist, sodium nitroprusside (SNP)-mediated relaxation. Treatment with serelaxin restored endothelial function (pEC50; 7.83±0.11, n=11) but not NO availability. The presence of the cyclooxygenase (COX) inhibitor, indomethacin (1μM) (pEC50; control=7.29±0.10 vs indo=7.74±0.18, n=6-12, P<0.05) and a superoxide dismutase mimetic, tempol (10μM) (pEC50; control=7.29±0.10 vs tempol=7.82±0.05, n=6-12, P<0.01) significantly improved sensitivity to ACh in high glucose treated aortae, but had no effect in serelaxin treated aortae. This suggests that high glucose incubation alters the superoxide and COX-sensitive pathway, which was normalized by co-incubation with serelaxin. Neither high glucose incubation nor serelaxin treatment had an effect on cyclooxygenase 1 and 2 (Ptgs1, Ptgs2), prostacyclin synthase (PTGIS) and receptor (Ptgir) as well as thromboxane A2 receptor (Tbxa2r) mRNA expression. Importantly, production of prostacyclin was significantly (P<0.05) attenuated in high glucose treated aortae, which was prevented by serelaxin treatment. Our data show that serelaxin treatment for 3 days restores high glucose-induced endothelial dysfunction by ameliorating vasodilator prostacyclin production and possibly through the reduction of superoxide in the mouse aorta.

Low intrinsic exercise capacity in rats predisposes to age-dependent cardiac remodeling independent of macrovascular function.

Rats selectively bred for low (LCR) or high (HCR) intrinsic running capacity simultaneously present with contrasting risk factors for cardiovascular and metabolic disease. However, the impact of these phenotypes on left ventricular (LV) morphology and microvascular function, and their progression with aging, remains unresolved. We tested the hypothesis that the LCR phenotype induces progressive age-dependent LV remodeling and impairments in microvascular function, glucose utilization, and β-adrenergic responsiveness, compared with HCR. Hearts and vessels isolated from female LCR (n = 22) or HCR (n = 26) were studied at 12 and 35 wk. Nonselected N:NIH founder rats (11 wk) were also investigated (n = 12). LCR had impaired glucose tolerance and elevated plasma insulin (but not glucose) and body-mass at 12 wk compared with HCR, with early LV remodeling. By 35 wk, LV prohypertrophic and glucose transporter GLUT4 gene expression were up- and downregulated, respectively. No differences in LV β-adrenoceptor expression or cAMP content between phenotypes were observed. Macrovascular endothelial function was predominantly nitric oxide (NO)-mediated in both phenotypes and remained intact in LCR for both age-groups. In contrast, mesenteric arteries microvascular endothelial function, which was impaired in LCR rats regardless of age. At 35 wk, endothelial-derived hyperpolarizing factor-mediated relaxation was impaired whereas the NO contribution to relaxation is intact. Furthermore, there was reduced β2-adrenoceptor responsiveness in both aorta and mesenteric LCR arteries. In conclusion, diminished intrinsic exercise capacity impairs systemic glucose tolerance and is accompanied by progressive development of LV remodeling. Impaired microvascular perfusion is a likely contributing factor to the cardiac phenotype.