Urotensin II Receptor Antagonist Research Articles

The effects of the urotensin-II receptor antagonist palosuran treatment on the corpora cavernosa of streptozotocin-induced diabetic rats

ObjectiveTo investigate the effects of treatment with palosuran, a urotensin receptor blocker, on molecular changes in the corpora cavernosa (CC) and erectile function in diabetic rats. MethodsStreptozotocin-induced diabetic rats were treated with palosuran 300 mg/kg per day for 6 weeks. Contraction of CC induced by potassium chloride, phenylephrine, and Nω-Nitro-L-arginine methyl ester and relaxation of CC induced by electric field stimulation (EFS) and sodium nitroprusside (SNP) (endothelium-dependent and endothelium-independent stimuli, respectively), and Y-27632 (Rho-kinase inhibitor) were examined in organ baths. Direct contraction or relaxation induced by palosuran and urotensin-II (U-II) were also evaluated. Expressions of nitric oxide synthetase (NOS) enzymes, RhoA, oxidative stress regulators, and U-II were analyzed by Western blotting or immunohistochemisty. ResultsInduction of diabetes in rats resulted in decreased relaxant response to SNP, decreased pD2 value of SNP, attenuated relaxant response to Y-27632 as well as the decreased RhoA expression in CC. Palosuran treatment of diabetic rats reversed all of these parameters; however, it further impaired the already weakened relaxation of diabetic CC in response to EFS. Although induction of diabetes did not change U-II expression in CC significantly, palosuran treatment reduced U-II expression in diabetic CC. The expression level of nNOS was lowered in diabetic CC; however palosuran treatment did not change the decreased nNOS expression. In vitro exposure of diabetic CC strips to palosuran produced a direct relaxant response. ConclusionPalosuran treatment did not affect the expression of NOS enzymes or reduced nitrergic conduction induced by EFS stimulation in diabetic CC. However, while directly triggering a relaxant response, it did not induce a prominent contraction either by decreasing U-II expression, or increasing the sensitivity of CC to NO which suggested that palosuran has the potential to support erectile function. Further and comprehensive studies are required to clarify this issue.

Urotensin II Enhances Advanced Aortic Atherosclerosis Formation and Delays Plaque Regression in Hyperlipidemic Rabbits.

Accumulated evidence shows that elevated urotensin II (UII) levels are associated with cardiovascular diseases. However, the role of UII in the initiation, progression, and regression of atherosclerosis remains to be verified. Different stages of atherosclerosis were induced in rabbits by a 0.3% high cholesterol diet (HCD) feeding, and either UII (5.4 μg/kg/h) or saline was chronically infused via osmotic mini-pumps. UII promoted atherosclerotic fatty streak formation in ovariectomized female rabbits (34% increase in gross lesion and 93% increase in microscopic lesion), and in male rabbits (39% increase in gross lesion). UII infusion significantly increased the plaque size of the carotid and subclavian arteries (69% increase over the control). In addition, UII infusion significantly enhanced the development of coronary lesions by increasing plaque size and lumen stenosis. Histopathological analysis revealed that aortic lesions in the UII group were characterized by increasing lesional macrophages, lipid deposition, and intra-plaque neovessel formation. UII infusion also significantly delayed the regression of atherosclerosis in rabbits by increasing the intra-plaque macrophage ratio. Furthermore, UII treatment led to a significant increase in NOX2 and HIF-1α/VEGF-A expression accompanied by increased reactive oxygen species levels in cultured macrophages. Tubule formation assays showed that UII exerted a pro-angiogenic effect in cultured endothelial cell lines and this effect was partly inhibited by urantide, a UII receptor antagonist. These findings suggest that UII can accelerate aortic and coronary plaque formation and enhance aortic plaque vulnerability, but delay the regression of atherosclerosis. The role of UII on angiogenesis in the lesion may be involved in complex plaque development.

Protective effect of urotensin II receptor antagonist urantide and exercise training on doxorubicin-induced cardiotoxicity

Doxorubicin (DOX) has a wide antitumor spectrum, but its adverse cardiotoxicity may lead to heart failure. Urotensin II (UII) is the most potent vasoconstrictor in mammals. It plays a role by activating the UII receptor (UT), the orphan G protein-coupled receptor (GPR14), collectively referred to as the UII/UT system. In the new version of "Chinese expert consensus on cardiac rehabilitation of chronic heart failure," it is pointed out that exercise rehabilitation is the cornerstone of cardiac rehabilitation. In this study, in vitro and in vivo assessments were performed using DOX-treated H9C2 cells and rats. It was found that the UT antagonist Urantide and exercise training improved DOX-induced cardiac insufficiency, reduced DOX-induced cardiomyocyte apoptosis, improved the structural disorder of myocardial fibers, and inhibited DOX-induced myocardial fibrosis. Further studies showed that Urantide alleviated DOX-induced cardiotoxicity by downregulating the expression levels of the p38 mitogen-activated protein kinase signaling pathway.

Commentary for “antagonizing urotensin receptor is a novel therapeutic strategy for glucocorticoid‐induced skeletal muscle atrophy”

In 1950, the discovery of the glucocorticoid cortisone was awarded the Nobel Prize in Physiology and Medicine. Since then, glucocorticoids have been a mainstay in clinical practice. Endogenous glucocorticoids like cortisone are natural hormones released in response to stress or circadian stimuli, whereas synthetic glucocorticoids like dexamethasone are used as potent anti-inflammatory drugs. While synthetic glucocorticoids are the mainstay treatments for many clinical conditions, their chronic daily intake also induces a plethora of concerning adverse effects, including muscle loss. The debilitating effects of chronic daily glucocorticoids in muscle are generally evident quite rapidly and compromise muscle mass, structure and strength in the long term. Glucocorticoid-induced muscle wasting correlates with increased protein catabolism, muscle mass decrease and inhibition of endogenous regenerative potential.1, 2 The neuropeptide urotensin II (UII) and its receptor, UII receptor, are expressed in a variety of tissues, including the skeletal muscle. Impaired regulation of UII and UII receptor have been associated with various pathologies.3 Of relevance for this study, prior studies had reported on the correlation between UII and muscle atrophy and on the finding that UII promotes loss of muscle weight and strength.4 In the study published here, Yin et al., show a novel approach in which a UII receptor antagonist rescues the glucocorticoid-induced skeletal muscle atrophy in mice. The study reports a potentially clinically relevant effect of Palosuran, a potent and selective antagonist of the UII receptor, in preventing dexamethasone-induced muscle atrophy. This experimental study has a huge potential and, if confirmed in clinical studies, could alleviate the muscle wasting experienced by the many people who take glucocorticoids regularly. Though this is the first report using this drug to counteract muscle atrophy, Palosuran has been largely investigated in the context of kidney function, particularly diabetic nephropathy. Experimental studies in rodents have reported on the potential role of UII receptor antagonists in alleviating renal complications in diabetes.5 However, initial clinical studies in humans have provided mixed results with this drug in the treatment of diabetic nephropathy.6, 7 In the study presented here, the Authors investigate a novel context for this drug and detail a large set of compelling experiments to elucidate the muscle atrophy counteraction by Palosuran. Importantly, their pharmacological experiments were complemented by analyses in a transgenic mouse with body-wide germline knockout of the UII receptor gene. Taken together, the findings reported in this study show that UII antagonism improves muscle mass and function in the context of dexamethasone-induced muscle wasting. Several limitations and open questions need further discussion. While inhibition of the UII axis through UII receptor antagonists has shown promising results in experimental animals, a potent and safe antagonist that can be used in humans is yet to be found. While the results by Yin et al, are promising, additional mechanistic and clinical studies are needed to support the novel indication of Palosuran for glucocorticoid-induced muscle loss. For instance, the experiments presented here should be investigated in disease models where chronic glucocorticoid intake is the mainstay treatment. Moreover, many tissues in the body express the UII receptor and therefore a thorough evaluation of toxicity and off-target effects of UII antagonist regimens is warranted. Additionally, it should also be evaluated if Palosuran alters or interacts with the anti-inflammatory action of glucocorticoid drugs. The results presented in this study depict a rather convincing effect of Palosuran on counteracting the loss of muscle mass and strength. However, the study did not address the effects of UII signalling and its counteraction on individual myofiber types, particularly type 2A, 2B and 2X. Moreover, the effects of UII axis activation versus inhibition on muscle bioenergetics remain unknown. Nevertheless, the rich dataset presented in this study supports the novel idea of repurposing UII antagonists to treat or prevent glucocorticoid-induced muscle atrophy. This is a significant advancement not only to potentially reduce the iatrogenic burden of synthetic glucocorticoids but also to further decipher their multifaceted action in health and disease. Drs. Prabakaran, Panta and Quattrocelli contributed to the preparation and collection of original literatures and figures and the writing and editing of manuscript. Drs. Prabakaran, Panta and Quattrocelli were responsible for the structural designs, scientific quality and writing. Not applicable. This work was supported by the following grant support: NIH grants DK121875 (MQ), HL158531 (MQ); CCHMC grants Trustee Award (MQ), Heart Institute Translational Grant (MQ). The authors declare no conflict of interest. The paper was handled by editors and has undergone a rigorous peer-review process. Dr. Mattia Quattrocelli was not involved in the journal's review of/or decisions related to this manuscript. Not applicable. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Urotensin II Induces Cardiac Fibrosis through the TGF-β/Smad Signaling Pathway during the Development of Cardiac Hypertrophy.

Myocardial fibrosis is an important pathological phenomenon of cardiac remodeling that is induced by hypertension, myocardial ischemia, valvular heart disease, hypertrophic cardiomyopathy, and other heart diseases and can progress to heart failure. Urotensin II (UII) is regarded as a cardiovascular autacoid/hormone that is not only the most potent vasoconstrictor in mammals but also involved in cardiac remodeling. However, the molecular mechanisms responsible for UII-induced cardiac fibrosis have not yet been fully elucidated. Therefore, we aimed to investigate the effect of UII on myocardial fibrosis in cardiac hypertrophy and the mechanism of UII-induced cardiac fibrosis. Cardiac tissue from mice subjected to Transverse aortic constriction (TAC) was collected. Cardiac hypertrophy, myocardial fibrosis, and the expression of UII protein were assessed using echocardiography and pathological and molecular biological analyses. The effect of UII on fibrosis was evaluated in UII-treated mice and isolated rat primary cardiac fibroblasts, and the results indicated that UII induced significant myocardial fibrosis and increases in the proliferation and fibrotic responses both in mice and cultured fibroblasts. Mechanistically, UII treatment induced activation of the TGF-β/Smad signaling pathway, which was suppressed by the UII receptor antagonist. In conclusion, UII plays critical roles in cardiac fibrosis by modulating the TGF-β/Smads signaling pathway, which may be a promising therapeutic target in hypertrophic cardiomyopathy and related problems, such as cardiac remodeling and heart failure.

Identification of new target proteins of a Urotensin-II receptor antagonist using transcriptome-based drug repositioning approach

Drug repositioning research using transcriptome data has recently attracted attention. In this study, we attempted to identify new target proteins of the urotensin-II receptor antagonist, KR-37524 (4-(3-bromo-4-(piperidin-4-yloxy)benzyl)-N-(3-(dimethylamino)phenyl)piperazine-1-carboxamide dihydrochloride), using a transcriptome-based drug repositioning approach. To do this, we obtained KR-37524-induced gene expression profile changes in four cell lines (A375, A549, MCF7, and PC3), and compared them with the approved drug-induced gene expression profile changes available in the LINCS L1000 database to identify approved drugs with similar gene expression profile changes. Here, the similarity between the two gene expression profile changes was calculated using the connectivity score. We then selected proteins that are known targets of the top three approved drugs with the highest connectivity score in each cell line (12 drugs in total) as potential targets of KR-37524. Seven potential target proteins were experimentally confirmed using an in vitro binding assay. Through this analysis, we identified that neurologically regulated serotonin transporter proteins are new target proteins of KR-37524. These results indicate that the transcriptome-based drug repositioning approach can be used to identify new target proteins of a given compound, and we provide a standalone software developed in this study that will serve as a useful tool for drug repositioning.

Identification of novel Urotensin-II receptor antagonists with potent inhibition of U-II induced pressor response in mice

Urotensin II (U-II) has been found to be one of the most potent vasoconstrictor (Ames et al., 1999; Bohm et al., 2002) reported till date. U-II exerts its response via activation of a G-protein coupled receptor, Urotensin II receptor(UT). Binding of U-II to UT leads to an instant increase in the inositol phosphate turnover and intracellular Ca2+. Such an instant Ca2+ release and potent vasoconstriction exerted by U-II is expected to have an important role in the progression of cardiac diseases.We have previously shown that UT antagonist DS37001789 prevents U-II induced blood pressure elevation in mice (Nishi et al., 2019) in a dose dependent manner, with potent efficacy at 30 and 100 mg/kg. Further to this, we have also shown that DS37001789 ameliorates mortality in pressure-overload mice with heart failure (Nishi et al., 2020). We therefore conducted an extensive structure-activity relationship studies to identify molecules with superior efficacy. In the present manuscript, we report the identification of two potent, non-peptide small molecule antagonists of Urotensin II receptor (UT), RCI-0879 and RCI-0298 which blocked the action of U-II, both in vitro and in vivo. These molecules were found to be very potent in in vitro Ca2+ and radioligand binding assays using human and mouse UT over-expressing CHO cells. RCI-0879 and RCI-0298 also exhibited superior efficacy in in vivo mouse pressor response model using C57BL/6 mice, compared to our initial molecules (Nishi et al., 2019) and demonstrated ED50 values of 3.2 mg/kg and 6.8 mg/kg respectively. Our findings reported herewith, further strengthen our concept and belief in UT antagonization as a potential therapeutic approach for the management of chronic heart failure.

Potential Therapeutic Value of Urotensin II Receptor Antagonist in Chronic Kidney Disease and Associated Comorbidities.

Chronic kidney disease (CKD) remains a common disorder, leading to growing health and economic burden without curative treatment. In diabetic patients, CKD may result from a combination of metabolic and nonmetabolic-related factors, with mortality mainly driven by cardiovascular events. The marked overactivity of the urotensinergic system in diabetic patients implicates this vasoactive peptide as a possible contributor to the pathogenesis of renal as well as heart failure. Previous preclinical studies with urotensin II (UII) antagonists in chronic kidney disease were based on simple end points that did not reflect the complex etiology of the disease. Given this, our studies revisited the therapeutic value of UII antagonism in CKD and extensively characterized 1-({[6-{4-chloro-3-[3-(dimethylamino)propoxy]phenyl}-5-(2-methylphenyl)pyridin-2-yl]carbonyl}amino) cyclohexanecarboxylic acid hydrochloride (SAR101099), a potent, selective, and orally long-acting UII receptor competitive antagonist, inhibiting not only UII but also urotensin-related peptide activities. SR101099 treatment more than halved proteinurea and albumin/creatinine ratio in spontaneously hypertensive stroke-prone (SHR-SP) rats fed with salt/fat diet and Dahl-salt-sensitive rats, respectively, and it halved albuminuria in streptozotocin-induced diabetes rats. Importantly, these effects were accompanied by a decrease in mortality of 50% in SHR-SP and of 35% in the Dahl salt-sensitive rats. SAR101099 was also active on CKD-related cardiovascular pathologies and partly preserved contractile reserve in models of heart failure induced by myocardial infarction or ischemia/reperfusion in rats and pigs, respectively. SAR101099 exhibited a good safety/tolerability profile at all tested doses in clinical phase-I studies. Together, these data suggest that CKD patient selection considering comorbidities together with new stratification modalities should unveil the urotensin antagonists' therapeutic potential. SIGNIFICANCE STATEMENT: Chronic kidney disease (CKD) is a pathology with growing health and economic burden, without curative treatment. For years, the impact of urotensin II receptor (UT) antagonism to treat CKD may have been compromised by available tools or models to deeper characterize the urotensinergic system. New potent, selective, orally long-acting cross-species UT antagonist such as SAR101099 exerting reno- and cardioprotective effects could offer novel therapeutic opportunities. Its preclinical and clinical results suggest that UT antagonism remains an attractive target in CKD on top of current standard of care.

DeepHIT: a deep learning framework for prediction of hERG-induced cardiotoxicity.

Blockade of the human ether-à-go-go-related gene (hERG) channel by small compounds causes a prolonged QT interval that can lead to severe cardiotoxicity and is a major cause of the many failures in drug development. Thus, evaluating the hERG-blocking activity of small compounds is important for successful drug development. To this end, various computational prediction tools have been developed, but their prediction performances in terms of sensitivity and negative predictive value (NPV) need to be improved to reduce false negative predictions. We propose a computational framework, DeepHIT, which predicts hERG blockers and non-blockers for input compounds. For the development of DeepHIT, we generated a large-scale gold-standard dataset, which includes 6632 hERG blockers and 7808 hERG non-blockers. DeepHIT is designed to contain three deep learning models to improve sensitivity and NPV, which, in turn, produce fewer false negative predictions. DeepHIT outperforms currently available tools in terms of accuracy (0.773), MCC (0.476), sensitivity (0.833) and NPV (0.643) on an external test dataset. We also developed an in silico chemical transformation module that generates virtual compounds from a seed compound, based on the known chemical transformation patterns. As a proof-of-concept study, we identified novel urotensin II receptor (UT) antagonists without hERG-blocking activity derived from a seed compound of a previously reported UT antagonist (KR-36676) with a strong hERG-blocking activity. In summary, DeepHIT will serve as a useful tool to predict hERG-induced cardiotoxicity of small compounds in the early stages of drug discovery and development. https://bitbucket.org/krictai/deephit and https://bitbucket.org/krictai/chemtrans. Supplementary data are available at Bioinformatics online.

Urotensin II and urantide exert opposite effects on the cellular components of atherosclerotic plaque in hypercholesterolemic rabbits.

Increasing levels of plasma urotensin II (UII) are positively associated with atherosclerosis. In this study we investigated the role of macrophage-secreted UII in atherosclerosis progression, and evaluated the therapeutic value of urantide, a potent competitive UII receptor antagonist, in atherosclerosis treatment. Macrophage-specific human UII-transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet for 16 weeks to induce atherosclerosis. Immunohistochemical staining of the cellular components (macrophages and smooth muscle cells) of aortic atherosclerotic lesions revealed a significant increase (52%) in the macrophage-positive area in only male transgenic rabbits compared with that in the nontransgenic littermates. However, both male and female transgenic rabbits showed a significant decrease (45% in males and 31% in females) in the smooth muscle cell-positive area compared with that of their control littermates. The effects of macrophage-secreted UII on the plaque cellular components were independent of plasma lipid level. Meanwhile the wild-type rabbits were continuously subcutaneously infused with urantide (5.4 µg· kg−1· h−1) using osmotic mini-pumps. Infusion of urantide exerted effects opposite to those caused by UII, as it significantly decreased the macrophage-positive area in male wild-type rabbits compared with that of control rabbits. In cultured human umbilical vein endothelial cells, treatment with UII dose-dependently increased the expression of the adhesion molecules VCAM-1 and ICAM-1, and this effect was partially reversed by urantide. The current study provides direct evidence that macrophage-secreted UII plays a key role in atherogenesis. Targeting UII with urantide may promote plaque stability by decreasing macrophage-derived foam cell formation, which is an indicator of unstable plaque.

Urotensin II receptor antagonist reduces hepatic resistance and portal pressure through enhanced eNOS-dependent HSC vasodilatation in CCl4-induced cirrhotic rats.

Increased serum urotensin II (UII) levels in human cirrhotic populations have been recently shown, but the long-term effects of UII receptor antagonist on the cirrhosis have not been investigated. To investigate the therapeutic effects of urotensin II receptor (UT) antagonist palosuran on rats with carbon tetrachloride (CCl4)-induced cirrhosis, the hepatic and systemic hemodynamics, liver fibrosis, the metalloproteinase-13 (MMP-13)/tissue inhibitor of metalloproteinase-1 (TIMP-1) ratio, hepatic Rho-kinase activity, and the endothelial nitric oxide synthase (eNOS) activity are measured in CCl4-cirrhotic rats treated with palosuran or vehicle for 4 weeks. Primary hepatic stellate cells (HSCs) are used to investigate the changes in UII/UT expression and the in vitro effect of palosuran. Compared with the vehicle-treated cirrhotic rats, treatment with palosuran can reduce the portal pressure (PP), decrease the risk of liver fibrosis and the level of α smooth muscle actin, collagen-I (COL-I), and transforming growth factor β expression. However, treatment with palosuran can increase MMP-13/TIMP-1, pvasodilator-stimulated phosphoprotein (p-VASP), and p-eNOS expression. Moreover, in vitro UII/UT mRNA expression increases during HSC activation. MMP-13/TIMP-1, COL-I, and p-VASP are inhibited after palosuran treatment. Our data indicate that long-term administration of palosuran can decrease PP in cirrhosis, which results from decreased hepatic fibrosis and enhanced eNOS-dependent HSC vasodilatation.

Multiple-Dose Pharmacokinetics, Pharmacodynamics, and Safety of the Urotensin-II Receptor Antagonist Palosuran in Healthy Male Subjects

Purpose: To investigate the multiple-dose pharmacokinetics (PK), pharmacodynamics (PD), and tolerability of palosuran, a selective, potent antagonist of the human UT receptor. Methods: This was a double-blind, randomized, placebo-controlled study. Three dose levels were investigated for PKs, PDs, and safety in sequential groups of 8 subjects each. Results: The plasma concentration-time profile is characterized by rapid absorption and 2 peaks after drug administration. The apparent terminal half-life was approximately 25 h. Steady-state concentrations were reached after 4–5 days of dosing. The accumulation factor was approximately 2.5. With increasing doses, a more than dose proportional increase in AUCτ and C_max was observed. Urinary excretion of unchanged palosuran was below 3%. No consistent effect was found on any of the PD variables. Palosuran was well tolerated in multiple doses up to 500 mg b.i.d. Conclusion: Palosuran after multiple-dosing is a well-tolerated drug in healthy subjects, but this finding warrants further investigation in patients.

Urantide improves the structure and function of right ventricle as determined by echocardiography in monocrotaline-induced pulmonary hypertension rat model.

Urotensin II (UII) has been reported to playa key role in pulmonary arterial hypertension (PAH) development. Doppler echocardiography, a noninvasive and simple tool, is recommended for diagnosing PAH. This study was designed to investigate the effect of urantide, a UII receptor antagonist, on the structure and function of the right ventricle in PAH rat models by Doppler echocardiography. A total of 60 male rats were divided into two groups: early- and late-treatment groups. Rats in the urantide and MCT (monocrotaline) subgroups were injected with 10μg/kg urantide in the urantide group or an equal amount of normal saline in the MCT group 1week after PAH model construction in the early-treatment group and 4weeks after the construction in the late-treatment group. Rats in the control group received an equal volume of normal saline solution. PAH-related indexes were measured by echocardiography. PAH rat models exhibited higher right ventricular diastolic diameter and lower time to peak, ejection time, and peak flow velocity of pulmonary artery than controls (P < 0.05). However, compared with the MCT group, all abovementioned indexes were improved in the urantide group (P < 0.05). No significant differences in pulmonary artery diameter and left ventricular ejection fraction were noted among the groups. Compared with the MCT group, systolic pulmonary arterial pressure (SPAP) and mean pulmonary arterial pressure (mPAP) were significantly lower in the urantide group (P < 0.05). SPAP examined by echocardiography was correlated with mPAP by catheterization (P < 0.05). Urantide treatment improved right heart failure parameters in MCT-induced PAH rats, thus providing a potential new strategy for treating PAH.

A Novel Urotensin II Receptor Antagonist, KR-36996 Inhibits Smooth Muscle Proliferation through ERK/ROS Pathway.

Urotensin II (UII) is a mitogenic and hypertrophic agent that can induce the proliferation of vascular cells. UII inhibition has been considered as beneficial strategy for atherosclerosis and restenosis. However, currently there is no therapeutics clinically available for atherosclerosis or restenosis. In this study, we evaluated the effects of a newly synthesized UII receptor (UT) antagonist, KR-36996, on the proliferation of SMCs in vitro and neointima formation in vivo in comparison with GSK-1440115, a known potent UT antagonist. In primary human aortic SMCs (HASMCs), UII (50 nM) induced proliferation was significantly inhibited by KR-36996 at 1, 10, and 100 nM which showed greater potency (IC50: 3.5 nM) than GSK-1440115 (IC50: 82.3 nM). UII-induced proliferation of HASMC cells was inhibited by U0126, an ERK1/2 inhibitor, but not by SP600125 (inhibitor of JNK) or SB202190 (inhibitor of p38 MAPK). UII increased the phosphorylation level of ERK1/2. Such increase was significantly inhibited by KR-36996. UII-induced proliferation was also inhibited by trolox, a scavenger for reactive oxygen species (ROS). UII-induced ROS generation was also decreased by KR-36996 treatment. In a carotid artery ligation mouse model, intimal thickening was dramatically suppressed by oral treatment with KR-36996 (30 mg/kg) which showed better efficacy than GSK-1440115. These results suggest that KR-36996 is a better candidate than GSK-1440115 in preventing vascular proliferation in the pathogenesis of atherosclerosis and restenosis.

A urotensin II receptor antagonist, KR36676, decreases vascular remodeling and inflammation in experimental pulmonary hypertension

The pathophysiological implications of binding of urotensin II (U-II) to urotensin II receptor (UT) in pulmonary arterial hypertension (PAH) have been proposed recently. Besides high expression of U-II in experimental models and patients with PAH, U-II has been shown to increase proliferation of pulmonary vascular smooth muscle cells and inflammatory responses, which were critical for PAH pathophysiology. However, the direct role of the urotensinergic system in the pathogenesis of PAH is yet to be understood. The aim of the present study was to determine whether a novel UT antagonist, KR36676, attenuates the pathophysiological progression of PAH in an animal model of PAH. PAH was induced by a single subcutaneous injection of monocrotaline (MCT, 60mg/kg) in rats. All the animals received KR36676 (30mg/kg/day) or vehicle by oral gavage. Three weeks after MCT-injection, changes in hemodynamic parameters, extent of right ventricular hypertrophy, fibrosis and pulmonary vascular remodeling, and degree of protein expression were determined. Oral administration of KR36676 effectively decreased the MCT-induced increase in right ventricular systolic pressure, hypertrophy and fibrosis. Furthermore, wall thickness of pulmonary arterioles, proliferation of pulmonary vascular cells, and inflammatory response significantly decreased in the KR36676-treated group following MCT injection compared to that in the MCT-treated vehicle group. These preventive effects of KR36676 are mediated, at least in part, by suppression of ERK1/2 and NF-κB signaling pathways. The novel UT antagonist, KR36676, effectively prevented MCT-induced PAH progression and pulmonary vascular remodeling in rat model. Our findings support the therapeutic efficacy of UT antagonist in PAH prevention and elucidate the possible underlying mechanisms of action.

Urotensin-ⅡReceptor Antagonist SB-710411 Protects Rat Heart against Ischemia-Reperfusion Injury via RhoA/ROCK Pathway.

AimSB-710411 is a rat selective urotensin-II (U-II) receptor antagonist, which can block U-II-induced contraction of the aorta and inhibit U-II-induced myocardial fibrosis in rats. However, the effect of SB-710411 on myocardial ischemia-reperfusion (I/R) injury is unclear. The present study was designed to investigate whether SB-710411 has a protective effect on myocardial I/R injury in rats and the possible mechanisms.Methods and ResultsMyocardial I/R injury was induced by occluding the left anterior descending coronary artery in adult male Sprague-Dawley rats. Hemodynamic parameters, electrocardiogram (ECG), infarct size, histological alteration, lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI), RhoA, and the protein expressions of U-II receptor (UTR), ROCK1 and ROCK2 were evaluated. Cardiac I/R injury significantly up-regulated the expressions of UTR, ROCK1 and ROCK2 proteins in rat myocardium. SB-710411 1.0 and 2.0 μg/kg significantly reduced cardiac I/R-induced the infarct size and histological damage in rat myocardium, markedly inhibited the changes of hemodynamic parameters and the increases of ST-segment in ECG, the serum LDH and CK-MB activities and cTnI level in rats subjected to myocardial I/R injury. Furthermore, SB-710411 obviously prevented myocardial I/R-increased RhoA activity and UTR, ROCK1 and ROCK2 protein expressions.ConclusionsOur results indicate that cardiac I/R injury increases myocardial UTR expression, and SB-710411 has a potent protective effect on myocardial I/R injury in rats. The cardioprotection may be associated with the inhibition of UTR-RhoA/ROCK pathway.

Use of Diverse Chemometric and Validation Methods to Accurately Predict Human Urotensin-II Receptor Antagonist Activity.

Despite being identified as the most potent receptor related to vasoconstriction, human urotensin-II receptor (hUT) has not been fully explored as a target for the treatment of cardiovascular diseases. In view of this and with an aim to identify precise structural requirements for binding of hUT antagonists, we endeavoured to develop, for the first time, multivariate QSAR models using chemometric methods like partial least squares (PLS) and feed-forward neural network (FFNN). A set of 48 pyrrolidine derivatives having hUT binding affinity was used for multivariate model development. The accuracy and predictability of the developed models was evaluated using crossvalidation. The PLS model showed good correlation between selected descriptors and Ki values (r(2) =0.745 and r(2) (CV) =0.773). However, the predictive performance of FFNN was better than the PLS technique with r(2) =0.810. The study clearly suggests the role of lipophilic and steric descriptors in the ligand-hUT interactions. The QSAR models generated can be successfully extended to predict the binding affinities and for the effective design of novel hUT antagonists.

Urotensin II Induces ER Stress and EMT and Increase Extracellular Matrix Production in Renal Tubular Epithelial Cell in Early Diabetic Mice

Background/Aims: Urotensin II (UII) and its receptor are highly expressed in the kidney tissue of patients with diabetic nephropathy (DN). The aim of this study is to examine the roles of UII in the induction of endoplasmic reticulum stress (ER stress) and Epithelial-mesenchymal transition (EMT) in DN in vivo and in vitro. Methods: Kidney tissues were collected from patients with DN. C57BL/6 mice and mice with UII receptor knock out were injected with two consecutive doses of streptozotocin to induce diabetes and were sacrificed at 3^th week for in vivo study. HK-2 cells in vitro were cultured and treated with UII. Markers of ER stress and EMT, fibronectin and type IV collagen were detected by immunohistochemistry, real time PCR and western blot. Results: We found that the expressions of protein of UII, GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were upregulated while E-cadherin protein was downregulated as shown by immunohistochemistry or western blot analysis in kidney of diabetic mice in comparison to normal control; moreover expressions of GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were inhibited while E-caherin expression was enhanced in kidney in diabetic mice with UII receptor knock out in comparison to C57BL/6 diabetic mice. In HK-2 cells, UII induced upregulation of GRP78, CHOP, ALPHA-SMA, fibroblast-specifc protein 1(FSP-1), fibronectin and type collagen and downregulation of E-cadherin. UII receptor antagonist can block UII-induced ER stress and EMT; moreover, 4-PBA can inhibit the mRNA expression of ALPHA-SMA and FSP1 induced by UII in HK-2 cells. Conclusions: We are the first to verify UII induces ER stress and EMT and increase extracellular matrix production in renal tubular epithelial cell in early diabetic mice. Moreover, UII may induce renal tubular epithelial EMT via triggering ER stress pathway in vitro, which might be the new pathogenic pathway for the development of renal fibrosis in DN.

Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis

Urotensin II (UII) exhibits diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response via the UII receptor (UTR) in mammals. However, in amphibians the function of the UII–UTR system remains unknown. In the present study, we investigated the potential immune function of UII using leukocytes isolated from the African clawed frog, Xenopus laevis. Stimulation of male frogs with lipopolysaccharide increased mRNA expression of UII and UTR in leukocytes, suggesting that inflammatory stimuli induce activation of the UII–UTR system. Migration assays showed that both UII and UII-related peptide enhanced migration of leukocytes in a dose-dependent manner, and that UII effect was inhibited by the UTR antagonist urantide. Inhibition of Rho kinase with Y-27632 abolished UII-induced migration, suggesting that it depends on the activation of RhoA/Rho kinase. Treatment of isolated leukocytes with UII increased the expression of several cytokine genes including tumor necrosis factor-α, interleukin-1β, and macrophage migration inhibitory factor, and the effects were abolished by urantide. These results suggest that in amphibian leukocytes the UII–UTR system is involved in the activation of leukocyte migration and cytokine gene expression in response to inflammatory stimuli.

Urotensin II promotes atherosclerosis in cholesterol-fed rabbits.

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of this study was to investigate whether UII affects the development of atherosclerosis in cholesterol-fed rabbits. UII was infused for 16 weeks through an osmotic mini-pump into male Japanese White rabbits fed on a high-cholesterol diet. Plasma lipids and body weight were measured every 4 weeks. Aortic atherosclerotic lesions along with cellular components, collagen fibers, matrix metalloproteinase-1 and -9 were examined. Moreover, vulnerability index of atherosclerotic plaques was evaluated. UII infusion significantly increased atherosclerotic lesions within the entire aorta by 21% over the control (P = 0.013). Atherosclerotic lesions were increased by 24% in the aortic arch (P = 0.005), 11% in the thoracic aorta (P = 0.054) and 18% in the abdominal aorta (P = 0.035). These increases occurred without changes in plasma levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides or body weight. Immunohistochemical staining revealed that macrophages and matrix metalloproteinase-9 were significantly enhanced by 2.2-fold and 1.6-fold in UII group. In vitro studies demonstrated that UII up-regulated the expression of vascular cell adhesion protein-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells, which was inhibited by the UII receptor antagonist urantide. In conclusion, our results showed that UII promotes the development of atherosclerotic lesions and destabilizes atherosclerotic plaques in cholesterol-fed rabbits.