Antagonist Urantide Research Articles

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.

Liver injury mediated by the UII and its receptor (UT) system is possibly associated with the activation of autophagy-related and apoptosis-resisted pathways of Kupffer cells in acute liver failure

The system of urotensin II (UII) and its receptor (UT) (or: UII/UT system) mediates hepatic immune inflamed injury in acute liver failure (ALF) with autophagy inhibition. However, it is unknown whether the system has an effect on liver autophagy in ALF. In this study, we attempted to explore hepatic autophagy response in ALF through blocking the UII/UT signal. Autophagy-related genes were examined in the liver tissues of lipopolysaccharide (LPS)/d-galactosamine (D-GalN)-induced ALF after pretreatment of UT receptor specific antagonist urantide. And then, the levels of autophagy- and apoptosis-related genes were assayed in LPS-stimulated KCs via urantide pretreatment. We found that the expressions of hepatic autophagy related genes, including Beclin-1, Atg5, Atg7, LC3 and p62 mRNA, and LC3 II and p62 protein, were significantly downregulated in LPS/D-GalN-induced ALF mice; but they were not affected by pretreatment of urantide, a special UT receptor antagonist. To probe inflammatory mechanisms of the UII/UT system, we further investigated the effect of the system on Kupffer cells (KCs), the innate immune cells in liver. We found that urantide pretreatment significantly inhibited production of inflammatory injury molecules including TRAF6 and ROS in LPS-stimulated KCs. LPS stimulation induced LC3 and p62 mRNA and LC3 II and p62 protein expression in KCs. After urantide pretreatment, LC3 and p62 mRNA and LC3 II protein were downregulated, while p62 protein was upregulated in LPS-stimulated KCs. In addition, antiapoptotic protein Bcl-2 inhibition and proapoptotic protein cleaved caspase-3 increase were observed in LPS-stimulated KCs, and the effects were enhanced after urantide pretreatment in the study. We conclude that liver injury mediated by the UII/UT system is possibly associated with the activation of autophagy-related and apoptosis-resisted pathways of KCs in ALF.

Urantide Improves Cardiac Function, Modulates Systemic Cytokine Response, and Increases Survival in A Murine Model of Endotoxic Shock.

Urotensin II is a potent vasoactive peptide activating the the G protein-coupled urotensin II receptor UT, and is involved in systemic inflammation and cardiovascular functions. The aim of our work was to study the impact of the UT antagonist urantide on survival, systemic inflammation, and cardiac function during endotoxic shock. C57Bl/6 mice were intraperitoneally injected with lipopolysaccharide (LPS) and then randomized to be injected either by urantide or NaCl 0.9% 3, 6, and 9 h (H3, H6, H9) after LPS. The effect of urantide on the survival rate, the levels of cytokines in plasma at H6, H9, H12, the expression level of nuclear factor-kappa B (NF-κB-p65) in liver and kidney (at H12), and the cardiac function by trans-thoracic echocardiography from H0 to H9 was evaluated. Urantide treatment improved survival (88.9% vs. 30% on day 6, P < 0.05). This was associated with changes in cytokine expression: a decrease in IL-6 (2,485 [2,280-2,751] pg/mL vs. 3,330 [3,119-3,680] pg/mL, P < 0.01) at H6, in IL-3 (1.0 [0.40-2.0] pg/mL vs. 5.8 [3.0-7.7] pg/mL, P < 0.01), and IL-1β (651 [491-1,135] pg/mL vs. 1,601 [906-3,010] pg/mL, P < 0.05) at H12 after LPS administration. Urantide decreased the proportion of cytosolic NF-κB-p65 in liver (1.3 [0.9-1.9] vs. 3.2 [2.3-4], P < 0.01) and kidney (0.3 [0.3-0.4] vs. 0.6 [0.5-1.1], P < 0.01). Urantide improved cardiac function (left ventricular fractional shortening: 24.8 [21.5-38.9] vs. 12.0 [8.7-17.6] %, P < 0.01 and cardiac output: 30.3 [25.9-39.8] vs. 15.1 [13.0-16.9] mL/min, P < 0.0001). These results show a beneficial curative role of UT antagonism on cytokine response (especially IL-3), cardiac dysfunction, and survival during endotoxic shock in mice, highlighting a potential new therapeutic target for septic patients.

The effects of urotensin II on migration and invasion are mediated by NADPH oxidase-derived reactive oxygen species through the c-Jun N-terminal kinase pathway in human hepatoma cells

AimsUrotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells. MethodsMigration was measured by wound healing assays and a Transwell® methodology, and invasion was analyzed using Matrigel® invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2′, 7′-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining. ResultsExposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125. ConclusionsExogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis.

IRF3 is an important molecule in the UII/UT system and mediates immune inflammatory injury in acute liver failure.

The urotensin II/urotensin receptor (UII/UT) system can mediate inflammatory liver injury in acute liver failure (ALF); however; the related mechanism is not clear. In this study, we confirmed that lipopolysaccharide/D-galactosamine (LPS/D-GalN) induced up-regulation of liver interferon regulatory factor 3 (IRF3) in ALF mice, whereas the UT antagonist urantide inhibited the up-regulated liver IRF3. LPS stimulation induced IRF3 transcription and nuclear translocation and promoted the secretion of interleukin-6 (IL-6), interferon (IFN)-β, and IFN-γ in Kupffer cells (KCs); these effects in LPS-stimulated KCs were inhibited by urantide. Knockdown of IRF3 using an adenovirus expressing an IRF3 shRNA inhibited IFN-β transcription and secretion as well as tumor necrosis factor (TNF)-α and IL-1β secretion from LPS-stimulated KCs; additionally, IL-10 transcription and secretion were promoted in response to LPS. However, LPS-stimulated TNF-α and IL-1β mRNA was not affected in the KCs. The IRF3 shRNA also did not have a significant effect on the NF-κB p65 subunit and p38MAPK protein phosphorylation levels in the nuclei of LPS-stimulated KCs. Therefore, IRF3 expression and activation depended on the signal transduction of the UII/UT system, and played important roles in UII/UT-mediated immune inflammatory injury in the liver but did not affect NF-κB and p38 MAPK activity.

Urotensin II contributes to collagen synthesis and up-regulates Egr-1 expression in cultured pulmonary arterial smooth muscle cells through the ERK1/2 pathway

AimThe objective of this study was to investigate the effects of urotensin II (UII) treatment on the proliferation and collagen synthesis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) and to explore whether these effects are mediated by mitogen-activated protein kinase (MAPK) signaling pathways and early growth response 1 (Egr-1). MethodsThe proliferation of cultured PASMCs stimulated with different doses of UII was detected by BrdU incorporation. The mRNA expression levels of procollagen I (procol I), procollagen III (procol III), extracellular regulated protein kinase 1/2 (ERK1/2), stress-stimulated protein kinase (Sapk), p38 MAPK (p38), and Egr-1 mRNA in cultured PASMCs after treatment with UII, the UII-specific antagonist urantide, and the ERK1/2 inhibitor PD98059 were detected by real-time polymerase chain reaction (PCR), and the protein expression levels of procol I, procol III, phosphorylated (p)-ERK1/2, p-Sapk, p-p38, and Egr-1 were detected by Western blotting. ResultsTreatment with UII increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.05). However, treatment with urantide and PD98059 inhibited the promoting effect of UII on PASMC proliferation (P<0.05). Real-time PCR analysis showed that UII up-regulated the expression of procol I, procol III, ERK1/2, Sapk, and Egr-1 mRNA (P<0.05), but not p38 mRNA. However, the up-regulating effect of UII was inhibited by PD98059 and urantide. Western blotting analysis showed that UII increased the synthesis of collagen I, collagen III, p-ERK1/2, p-Sapk, and Egr-1, and these effects also were inhibited by PD98059 and urantide (P<0.05). ConclusionsEgr-1 participates in the UII-mediated proliferation and collagen synthesis of cultured rat PASMCs via activation of the ERK1/2 signaling pathway.

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 receptor determines prognosis of bladder cancer regulating cell motility/invasion.

BackgroundNon Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC)/invasive have different gene profile and clinical course. NMIBC prognosis is not completely predictable, since the relapse rate is higher than 20%, even in the form of MIBC. The aim of this study is to evaluate if UTR expression can discriminate between NMIBC and MIBC and predict the risk of relapses in NMIBCs.MethodsWe have investigated upon urotensin-II (UII) receptor (UTR) expression in vivo in 159 patients affected by NMIBC. The biological role of UTR was also investigated in vitro. UTR expression was evaluated in a tissue-micro-array, consisting of normal, NMIBC and invasive bTCC samples.ResultsUTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC. The superagonist UPG84 induced growth suppression at nM concentrations on 3/4 cell lines. Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells.ConclusionsThe evaluation of UTR expression can discriminate between NMIBC at high and low risk of relapse. Moreover, our data suggest that UTR is involved in the regulation of motility, invasion and proliferation of bladder cancer cells. High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.

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.

Urotensin‐II receptor is over‐expressed in colon cancer cell lines and in colon carcinoma in humans

Urotensin (U)-II receptor (UTR) has been previously reported to be over-expressed in a number of tumours. Whether UTR-related pathway plays a role in colon carcinogenesis is unknown. We evaluated UTR protein and mRNA expression in human epithelial colon cancer cell lines and in normal colon tissue, adenomatous polyps and colon cancer. U-II protein expression was assessed in cancer cell lines. Moreover, we evaluated the effects of U-II(4-11) (an UTR agonist), antagonists and knockdown of UTR protein expression through a specific shRNA, on proliferation, invasion and motility of human colon cancer cells. Cancer cell lines expressed U-II protein and UTR protein and mRNA. By immunohistochemistry, UTR was expressed in 5-30% of epithelial cells in 45 normal controls, in 30-48% in 21 adenomatous polyps and in 65-90% in 48 colon adenocarcinomas. UTR mRNA expression was increased by threefold in adenomatous polyps and eightfold in colon cancer, compared with normal colon. U-II(4-11) induced a 20-40% increase in cell growth while the blockade of the receptor with specific antagonists caused growth inhibition of 20-40%. Moreover, the knock down of UTR with a shRNA or the inhibition of UTR with the antagonist urantide induced an approximately 50% inhibition of both motility and invasion. UTR appears to be involved in the regulation of colon cancer cell invasion and motility. These data suggest that UTR-related pathway may play a role in colon carcinogenesis and that UTR may function as a target for therapeutic intervention in colon cancer.

Effects of Urotensin II and Its Specific Receptor Antagonist Urantide on Rat Vascular Smooth Muscle Cells

We investigated the effects of urantide, a receptor antagonist of urotensin II (U-II), on the expression of U-II and its receptor GPR14 in rat vascular smooth muscle cells. Vascular smooth muscle cells from rat thoracic aorta were cultured by explant method. Subjects in this experiment were divided into eight groups: normal control group (group C), U-II group (group M), positive control group (Flu group) and urantide-treated groups (10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ mol/L). Cultured vascular smooth muscle cells in vitro were studied by immunocytochemistry, biochemistry, and flow cytometry. U-II (10⁻⁸ mol/L) promoted the proliferation of vascular smooth muscle cells at each time point, influenced cell cycle, increased proliferation index and S-phase cell fraction, and dramatically promoted the expression of U-II and GPR14. In the concentration range from 10⁻¹⁰ to 10⁻⁶ mol/L, urantide dramatically inhibited the proliferation of vascular smooth muscle cells and the protein expression of U-II and GPR14, especially at a concentration of 10⁻⁶ mol/L. U-II, binding with its receptor GPR14, promotes vascular smooth muscle cells proliferation and migration, which can be inhibited by urantide. This study provides an evidence for understanding the effects of U-II and its receptor GPR14 on vascular smooth muscle cells.

Urotensin II receptor (UTR) exists in hyaline chondrocytes: A study of peripheral distribution of UTR in the African clawed frog, Xenopus laevis

Urotensin II (UII) and UII-related peptide (URP) exhibit diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response through UII receptor (UTR), which is expressed in the central nervous system and peripheral tissues of fish and mammals. In amphibians, only UII has been identified. As the first step toward elucidating the actions of UII and URP in amphibians, we cloned and characterized URP and UTR from the African clawed frog Xenopus laevis. Functional analysis showed that treatment of UII or URP with Chinese hamster ovary cells transfected with the cloned receptor increased the intracellular calcium concentration in a concentration-dependent manner, whereas the administration of the UTR antagonist urantide inhibited UII- or URP-induced Ca2+ mobilization. An immunohistochemical study showed that UTR was expressed in the splenocytes and leukocytes isolated from peripheral blood, suggesting that UII and URP are involved in the regulation of the immune system. UTR was also localized in the apical membrane of the distal tubule of the kidney and in the transitional epithelial cells of the urinary bladder. This result supports the view that the UII/URP–UTR system plays an important role in osmoregulation of amphibians. Interestingly, immunopositive labeling for UTR was first detected in the chondrocytes of various hyaline cartilages (the lung septa, interphalangeal joint and sternum). The expression of UTR was also observed in the costal cartilage, tracheal cartilages, and xiphoid process of the rat. These novel findings probably suggest that UII and URP mediate the formation of the cartilaginous matrix.

Presence of urotensin-II receptors at the cell nucleus: Specific tissue distribution and hypoxia-induced modulation

Urotensin II (UII) and its receptor UT, are widely expressed in the cardiovascular and central nervous system, where they exert regulatory actions under both physiological and pathological conditions. Our study, aimed at investigating the presence of functional nuclear UT in various rat and monkey tissues as well as in human cell lines, demonstrated for the first time by Western blot analysis and confocal immunofluorescence a tissue-specific nuclear expression of this receptor (heart and central nervous system). This nuclear UT was further characterized pharmacologically through radioligand binding studies using specific ligands of the urotensinergic system, as well as somatostatin. In 2D-gel experiments, we observed the presence of different post-translational modifications between membrane and nuclear UT receptors in brain extracts. Transcription initiation assays showed de novo RNA synthesis caused by UII and Urotensin-related peptide (URP) which were inhibited by an UT antagonist urantide. In hypoxic/ischemic conditions, UT receptors were differentially modulated in regard to subcellular localization. Thus, the unique regiospecificity of the nuclear UT receptor along with its particular modulation under hypoxic conditions could indicate a specific and complementary physiological role that could be correlated with pro-angiogenic and/or neuromodulatory actions of UII, both in the cardiovascular and central nervous system.

Abstract P148: Urotensin II Impairs Cardiac Functions and Proliferation of Cardiac Side Population Cells in Mice During Chronic Pressure Overload.

Urotensin II (UII), a potent vasoactive “somatostatin-like” peptide, has been known to play a role in cardiovascular diseases including cardiac hypertrophy and heart failure. But its effect on cardiac side population cells (CSPs), one of somatic stem cells in the heart potentially participating in cardiac protection after injury, is unclear. The present study was therefore conducted to examine influences of UII on the differentiation, proliferation and function of CSPs. CSPs were isolated from neonatal rat hearts by fluorescence-activated cell sorting (FACS) and cultured in the absence or at the presence of UII. The expressions of alpha-cardiac myosin heavy chain, alpha-smooth muscle actin and Von Willebrand factor at mRNAs and protein levels were analyzed by reverse transcriptional PCR (RT-PCR) and immunofluoresence to evaluate the differentiation of CSPs into cardiomyocytes, smooth muscle cells and endothelial cells, respectively. The proliferation of CSPs was assessed by Luminescent Cell Viability Assay. The influence of UII on the proliferation of CSPs in vivo was also evaluated by FACS. Our results revealed that UII did inhibit the proliferation of CSPs through up-regulation of phosphorylated c-Jun N-terminal protein kinase (JNK), although it didn’t affect the differentiation of cultured CSPs into cardiaomyocytes, smooth muscle cells and endothelial cells. In vivo experiments also showed that injection of UII reduced the number of CSPs and impaired cardiac functions compared with vehicle injection in mice subjected to a chronic pressure overload, and an UII antagonist urantide induced a preserved cardiac function with an increased number of CSPs. These data indicate that UII reduces the number of CSPs by inhibiting the proliferation of CSPs possibly through increase of JNK phosphorylation, and blockage of UII may be a useful strategy for cardiac protection in the hypertensive.

Effects of urotensin II on functional activity of late endothelial progenitor cells

Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10−10, 10−9, 10−8, 10−7 and 10−6M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.

EGFR trans-activation by urotensin II receptor is mediated by β-arrestin recruitment and confers cardioprotection in pressure overload-induced cardiac hypertrophy

Urotensin II (UTII) and its seven trans-membrane receptor (UTR) are up-regulated in the heart under pathological conditions. Previous in vitro studies have shown that UTII trans-activates the epidermal growth factor receptor (EGFR), however, the role of such novel signalling pathway stimulated by UTII is currently unknown. In this study, we hypothesized that EGFR trans-activation by UTII might exert a protective effect in the overloaded heart. To test this hypothesis, we induced cardiac hypertrophy by transverse aortic constriction (TAC) in wild-type mice, and tested the effects of the UTII antagonist Urantide (UR) on cardiac function, structure, and EGFR trans-activation. After 7days of pressure overload, UR treatment induced a rapid and significant impairment of cardiac function compared to vehicle. In UR-treated TAC mice, cardiac dysfunction was associated with reduced phosphorylation levels of the EGFR and extracellular-regulated kinase (ERK), increased apoptotic cell death and fibrosis. In vitro UTR stimulation induced membrane translocation of β-arrestin 1/2, EGFR phosphorylation/internalization, and ERK activation in HEK293 cells. Furthermore, UTII administration lowered apoptotic cell death induced by serum deprivation, as shown by reduced TUNEL/Annexin V staining and caspase 3 activation. Interestingly, UTII-mediated EGFR trans-activation could be prevented by UR treatment or knockdown of β-arrestin 1/2. Our data show, for the first time in vivo, a new UTR signalling pathway which is mediated by EGFR trans-activation, dependent by β-arrestin 1/2, promoting cell survival and cardioprotection.

Immunomodulatory role of urotensins in teleost Channa punctatus

The present study, for the first time in ectothermic vertebrates, reports the immunoregulatory role of urotensins I and II (UI and UII). Urotensins decreased the phagocytosis and nitrite production by splenic phagocytes. On superoxide production, UI had stimulatory while UII showed inhibitory effect. UI exerted its effect on phagocytes through corticotrophin-releasing factor (CRF) receptor as its non-specific antagonist astressin completely blocked the effect of UI on phagocytosis, nitrite release and superoxide production. Among the antagonists for specific CRF receptor 1 and 2, only CRF receptor 1 antagonist NBI 27914 abolished the effect of urotensin I. On the other hand, UII mediated its effect through urotensin receptor (UT receptor) since its antagonist urantide antagonized the effect of UII on phagocytosis, superoxide and nitrite release. These findings provide the direct evidence on physiological role of UI and UII through CRF receptor 1 and UT receptor, respectively in control of fish immune responses.

Urotensin II is an autocrine/paracrine growth factor for aortic adventitia of rat

Urotensin II (UII) is a potent vasoconstrictive peptide; however, its significance in vascular adventitia has not been clearly elucidated. In this study, rat aortic adventitia showed mRNA expression and immunoreactivity of UII and its receptor (UT). Moreover, radioligand-binding assay showed that maximum binding capacity (Bmax) of [ 125I]-UII was higher in adventitia than in media (28.60 ± 1.94 vs. 20.21 ± 1.11 fmol/mg, P < 0.01), with no difference in binding affinity (dissociation constant [Kd] 4.27 ± 0.49 vs. 4.60 ± 0.40 nM, P > 0.05). Furthermore, in cultured adventitial fibroblasts, UII stimulated DNA synthesis, collagen synthesis and secretion in a concentration-dependent manner. These effects were inhibited by the UII receptor antagonist urantide (10 − 6 mol/l), Ca 2+ channel blocker nicardipine (10 − 5 mol/l), protein kinase C inhibitor H7 (10 − 6 mol/l), and mitogen-activated protein kinase inhibitor PD98059 (10 − 6 mol/l) but not the phosphatidyl inositol-3 kinase inhibitor wortmannin (10 − 7 mol/l). UII may act as an autocrine/paracrine factor through its receptor and the Ca 2+ channel, protein kinase C, and mitogen-activated protein kinase signal transduction pathways, in the pathogenesis of vascular remodeling by activating vascular adventitia.

Abstract 152: Urotensin-II Activates Rac-1 And ROS Production In Pulmonary Artery Smooth Muscle Cells Involving GPR14 And Gα I3 Proteins

Human urotensin II (hU-II) has recently been described as a potent vasoactive peptide associated with remodelling processes within the vasculature, thus contributing to various cardiovascular diseases including pulmonary hypertension. hU-II acts via the G-protein coupled receptor GPR14. However, the mechanisms mediating GPR14 signalling remain unclear. ROS have been shown to act as signalling molecules in vascular cells, and NADPH oxidases which are activated by Rac-1, are important sources of ROS generation. Therefore, we investigated whether hU-II and GPR14 can activate Rac-1 and NADPH oxidase-mediated ROS production in pulmonary artery smooth muscle cells (PASMC). Urotensin-II activated Rac-1 within 15 seconds and this response was abolished by treatment of PASMC with the GPR14 antagonist urantide. In line, urotensin-II induced a rapid increase in ROS levels within 5 minutes of application, which was prevented by urantide or transfection of a dominant-negative mutant of Rac-1 (RacT17N). Furthermore, in cells overexpressing GPR14 or constitutively active Rac-1 (RacG12V), hU-II-stimulated ROS generation was further enhanced. Interestingly, in the presence of Pertussis toxin, hU-II-induced Rac-1 activation and ROS generation were abolished, indicating coupling of GPR14 to Gα i proteins. Indeed, transfection of dominant-negative G α i3 completely diminished hU-II-stimulated ROS generation in PASMC. Similar effects were observed upon depletion of the NADPH oxidase subunit NOX4. Furthermore, hU-II, RacG12V and NOX4 stimulated proliferation of PASMC, whereas antioxidants, RacT17N or NOX4 depletion had opposite effects.These results show that hU-II is a potent activator of Rac-1 leading to rapid activation of NADPH oxidase and ROS generation involving GPR14 and Gα i3 proteins, and subsequent stimulation of PASMC proliferation. Thus, activation of NADPH oxidase by GPR14 and Rac-1 provides a novel mechanism how hU-II can promote vascular remodelling processes and may provide a novel therapeutic target for pulmonary vasculopathies associated with pulmonary hypertension.

The involvement of transforming growth factor-β1 secretion in Urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-β1 (TGF-β1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-β1, and the role of U II receptor UT in this process. The functional role of TGF-β1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-β1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-β1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-β1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-β1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-β1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.