Kyoji Urayama

Prokineticin Receptor-1 Induces Neovascularization and Epicardial-Derived Progenitor Cell Differentiation

Objective—Identification of novel factors that contribute to myocardial repair and collateral vessel growth hold promise for treatment of heart diseases. We have shown that transient prokineticin receptor-1 (PKR1) gene transfer protects the heart against myocardial infarction in a mouse model. Here, we investigated the role of excessive PKR1 signaling in heart. Methods and Results—Transgenic mice overexpressing PKR1 in cardiomyocytes displayed no spontaneous abnormalities in cardiomyocytes but showed an increased number of epicardial-derived progenitor cells (EPDCs), capillary density, and coronary arterioles. Coculturing EPDCs with H9c2 cardiomyoblasts overexpressing PKR1 promotes EPDC differentiation into endothelial and smooth muscle cells, mimicking our transgenic model. Overexpressing PKR1 in H9c2 cardiomyoblasts or in transgenic hearts upregulated prokineticin-2 levels. Exogenous prokineticin-2 induces significant outgrowth from neonatal and adult epicardial explants, promoting EPDC differentiation. These prokineticin-2 effects were abolished in cardiac explants from mice with PKR1-null mutation. Reduced capillary density and prokineticin-2 levels in PKR1-null mutant hearts supports the hypothesis of an autocrine/paracrine loop between PKR1 and prokineticin-2. Conclusion—Cardiomyocyte-PKR1 signaling upregulates its own ligand prokineticin-2 that acts as a paracrine factor, triggering EPDCs proliferation/differentiation. This study provides a novel insight for possible therapeutic strategies aiming at restoring pluripotency of adult EPDCs to promote neovasculogenesis by induction of cardiomyocyte PKR1 signaling.

The prokineticin receptor-1 (GPR73) promotes cardiomyocyte survival and angiogenesis

Prokineticins are potent angiogenic factors that bind to two G protein‐coupled receptors to initiate their biological effects. We hypothesize that prokineticin receptor‐1 (PKR1/GPR73) signaling may contribute to cardiomyocyte survival or repair in myo‐cardial infarction. Since we showed that prokineticin‐2 and PKR1 are expressed in adult mouse heart and cardiac cells, we investigated the role of prokineticin‐2 on capillary endothelial cell and cardiomyocyte function. In cultured cardiac endothelial cells, prokineti‐cin‐2 or overexpression of PKR1 induces vessel‐like formation without increasing VEGF levels. In cardiomy‐ocytes and H9c2 cells, prokineticin‐2 or overexpressing PKR1 activates Akt to protect cardiomyocytes against oxidative stress. The survival and angiogenesis promoting effects of prokineticin‐2 in cardiac cells were com‐pletely reversed by siRNA‐PKR1, indicating PKR1 involvement. We thus, further investigated whether intramyocardial gene transfer of DNA encoding PKR1 may rescue the myocardium against myocardial infarction in mouse model. Transient PKR1 gene transfer after coronary ligation reduces mortality and preserves left ventricular function by promoting neovascularization and protecting cardiomyocytes without altering VEGF levels. In human end‐stage failing heart samples, reduced PKR1 and prokineticin‐2 transcripts and pro‐tein levels implicate a more important role for proki‐neticin‐2/PKR1 signaling in heart. Our results suggest that PKR1 may represent a novel therapeutic target to limit myocardial injury following ischemic events.—Urayama K., Guilini, C., Messaddeq, N., Hu, K., Steenman, M., Kurose, H., Ert, G., Nebigil C. G. The prokineticin receptor‐1 (GPR73) promotes cardiomyo‐cyte survival and angiogenesis FASEB J. 21, 2980–2993 (2007)

Divergent roles of prokineticin receptors in the endothelial cells: angiogenesis and fenestration

Prokineticins are secreted peptides that activate two G protein-coupled receptors: PKR1 and PKR2. Prokineticins induce angiogenesis and fenestration, but the cognate receptors involved in these functions are unknown. We hypothesized a role for prokineticin receptor signaling pathways and expression profiles in determining the selective effects of prokineticins on coronary endothelial cells (H5V). Activation of the PKR1/MAPK/Akt signaling pathway stimulates proliferation, migration, and angiogenesis in H5V cells, in which PKR1 predominates over PKR2. PKR1 was colocalized with Galpha(11) and was internalized following the stimulation of these cells with prokineticin-2. Knock down of PKR1 or Galpha(11) expression in H5V cells effectively inhibited prokineticin-2-induced vessel formation and MAPK/Akt activation, indicating a role for PKR1/Galpha(11) in this process. However, in conditions in which PKR2 predominated over PKR1, these cells displayed a fenestrated endothelial cell phenotype. H5V cells overexpressing PKR2 displayed large numbers of multivesicular bodies and caveolar clusters and a disruption of the distribution of zonula occluden-1 tight junction protein. Prokineticin-2 induced the colocalization of PKR2 with Galpha(12), and activated Galpha(12), which bound to zonula occluden-1 to trigger the degradation of this protein in these cells. Prokineticin-2 induced the formation of vessel-like structures by human aortic endothelial cells expressing only PKR1, and disorganized the tight junctions in human hepatic sinusoidal endothelial cells expressing only PKR2, confirming the divergent roles of these receptors. Our findings show the functional characteristics of coronary endothelial cells depend on the expression of PKR1 and PKR2 levels and the divergent signaling pathways used by these receptors.

Transgenic myocardial overexpression of prokineticin receptor-2 (GPR73b) induces hypertrophy and capillary vessel leakage

AIMS Prokineticins are small secreted bioactive molecules. They exert their biological activity by binding to two G protein-coupled receptors. Previously, we have shown that the overexpression of prokineticin receptor-1 (PKR1) in transgenic (TG) mouse hearts induced neovascularization. Since PKR1 and PKR2 are 85% identical and expressed in cardiovascular tissues, we hypothesized that PKR2 may also contribute to cardiomyocyte growth and vascularization. METHODS AND RESULTS We have generated TG mice overexpressing PKR2 in cardiomyocytes. TG mice exhibit increased hypertrophic gene expression and heart-to-body weight ratio accompanied by an increased length of cardiomyocytes at the age of 12 weeks. Increased left ventricular end-systolic and diastolic diameters without cardiac dysfunction at the age of 24 weeks indicate that TG mice have an eccentric hypertrophy with compensated cardiac function. Quantitative morphological analysis showed that TG hearts have a normal microvessel density and number of branch points. However, they exhibit increased abnormal endothelial cell shape and ultrastructure, changed cellular distribution of a tight junction protein zona occludens-1 (ZO-1), and vascular leakage in heart without a rise of angiogenic factor levels at early and late age. The application of media conditioned by H9c2 cardioblast cells overexpressing PKR2 significantly induced impaired ZO-1 localization in H5V endothelial cells, mimicking the TG model. CONCLUSION These findings provide the first genetic evidence that cardiomyocyte PKR2 signalling leads to eccentric hypertrophy in an autocrine regulation and impaired endothelial integrity in a paracrine regulation without inducing angiogenesis. These TG mice may provide a new genetic model for heart diseases.

Discovery and Cardioprotective Effects of the First Non-Peptide Agonists of the G Protein-Coupled Prokineticin Receptor-1

Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI.

Prokineticin Receptor-1 Is a New Regulator of Endothelial Insulin Uptake and Capillary Formation to Control Insulin Sensitivity and Cardiovascular and Kidney Functions

Background Reciprocal relationships between endothelial dysfunction and insulin resistance result in a vicious cycle of cardiovascular, renal, and metabolic disorders. The mechanisms underlying these impairments are unclear. The peptide hormones prokineticins exert their angiogenic function via prokineticin receptor‐1 (PKR1). We explored the extent to which endothelial PKR1 contributes to expansion of capillary network and the transcapillary passage of insulin into the heart, kidney, and adipose tissues, regulating organ functions and metabolism in a specific mice model. Methods and Results By combining cellular studies and studies in endothelium‐specific loss‐of‐function mouse model (ec‐PKR1−/−), we showed that a genetically induced PKR1 loss in the endothelial cells causes the impaired capillary formation and transendothelial insulin delivery, leading to insulin resistance and cardiovascular and renal disorders. Impaired insulin delivery in endothelial cells accompanied with defective expression and activation of endothelial nitric oxide synthase in the ec‐PKR1−/− aorta, consequently diminishing endothelium‐dependent relaxation. Despite having a lean body phenotype, ec‐PKR1−/− mice exhibited polyphagia, polydipsia, polyurinemia, and hyperinsulinemia, which are reminiscent of human lipodystrophy. High plasma free fatty acid levels and low leptin levels further contribute to the development of insulin resistance at the later age. Peripheral insulin resistance and ectopic lipid accumulation in mutant skeletal muscle, heart, and kidneys were accompanied by impaired insulin‐mediated Akt signaling in these organs. The ec‐PKR1−/− mice displayed myocardial fibrosis, low levels of capillary formation, and high rates of apoptosis, leading to diastolic dysfunction. Compact fibrotic glomeruli and high levels of phosphate excretion were found in mutant kidneys. PKR1 restoration in ec‐PKR1−/− mice reversed the decrease in capillary recruitment and insulin uptake and improved heart and kidney function and insulin resistance. Conclusions We show a novel role for endothelial PKR1 signaling in cardiac, renal, and metabolic functions by regulating transendothelial insulin uptake and endothelial cell proliferation. Targeting endothelial PKR1 may serve as a therapeutic strategy for ameliorating these disorders.

Genetic Inactivation of Prokineticin Receptor-1 Leads to Heart and Kidney Disorders

Objective—Prokineticins are potent angiogenic hormones that use 2 receptors, prokineticin receptor-1 (PKR1) and PKR2, with important therapeutic use in anticancer therapy. Observations of cardiac and renal toxicity in cancer patients treated with antiangiogenic compounds led us to explore how PKR1 signaling functioned in heart and kidney in vivo. Methods and Results—We generated mice with a conditional disruption of the PKR1 gene. We observed that PKR1 loss led to cardiomegaly, severe interstitial fibrosis, and cardiac dysfunction under stress conditions, accompanied by renal tubular dilation, reduced glomerular capillaries, urinary phosphate excretion, and proteinuria at later ages. Abnormal mitochondria and increased apoptosis were evident in both organs. Perturbation of capillary angiogenesis in both organs was restored at the adult stage potentially via upregulation of hypoxia-inducible factor-1 and proangiogenic factors. Compensatory mechanism could not revoke the epicardial and glomerular capillary networks, because of increased apoptosis and reduced progenitor cell numbers, consistent with an endogenous role of PKR1 signaling in stimulating epicardin+ progenitor cell proliferation and differentiation. Conclusion—Here, we showed for the first time that the loss of PKR1 causes renal and cardiac structural and functional changes because of deficits in survival signaling, mitochondrial, and progenitor cell functions in found both organs.

Prokineticin receptor 1 (PKR1) signalling in cardiovascular and kidney functions

Prokineticins (PK1 and PK2) are peptide hormones that exert their biological activity via two common G-protein-coupled receptors: prokineticin receptor (PKR) 1 and 2. Their physiology was originally explored mostly in the context of angiogenic actions in the reproductive tract and gut motility. Since autocrine and paracrine loops have been established between PK2 and PKR1 in the heart, in this review we focus on the PK2/PKR1 signalling in the functions of the heart and kidney. PKR1 signalling is required for cardiomyocyte survival and angiogenesis. In the mouse model of myocardial infarction, intracardiac transient PKR1 transfection protects the structure and function of the heart. Gain- and loss-of-function studies reveal that PKR1 in mouse heart up-regulates its own ligand and PK2, which in turn acts as a paracrine signal and promotes epicardin-positive progenitor cell differentiation into a vasculogenic cell type. Transgenic mice over-expressing PKR1 in cardiomyocytes exhibit increased neovascularization. Loss of PKR1 causes structural and functional changes in the heart and kidney. In isolated epicardin-positive progenitor cells from the kidney, PK2, acting via PKR1, stimulates differentiation of these progenitor cells into endothelial and smooth muscle cells. Taken together, these data show that PK2/PKR1 is involved in postnatal cardiac and renal neovascularization. The knowledge gained from these studies should facilitate the discovery of therapeutic interventions in heart and kidney diseases targeting PKR1.

0365: Non-peptidic prokineticin receptor 1 agonist as a novel cardioprotective therapeutic

Objective Prokineticins are potent angiogenic peptides that bind to two G protein-coupled receptors to initiate their biological effects. We previously have shown that prokineticin receptor-1 (PKR1) signaling contributes to cardiomyocyte survival or repair in myocardial infarction. Here, we discovered the first non-peptidic PKR1 agonists and examined their effects in mice model of heart diseases. Methods and results Herein we identify a selective PKR1 agonist both in vitro and in vivo, utilizing GPCR structure-based virtual screening approach. High Throughput Docking was carried out by GOLD using homology model of PKR1. Asinex gold collection 3D chemical databese (250,000 compounds) was screened by the docking protocol. We provided a strategy with a high potential for in silico identifying one agonist hit. We present here IS20, the first synthetic PKR1 agonist that induces angiogenesis in the presence of PKR1 on the endothelial cells seeded on matrigel. IS20 reduced doxorubicin cytotoxicity in H9C2 cells. IS20 promotes mouse epicardial progenitor cell differentiation into endothelial cells. In vivo IS20 activates Akt in mice heart. IS20 treatment of mice after coronary ligation reduces mortality by 30%. Conclusion This study identifies a non-peptidic PKR1 agonist as therapeutic target holding promise for treatment of heart diseases.

0369: Proepicardial prokineticin receptor –1 (PKR1) as a developmental link between heart and kidney

Background Prokineticin receptor-1 (PKR1), signals play critical roles in heart and kidney functions. In particular, the systemic mutation of this receptor results in thinning of the myocardium and hypoplastic kidney. However, the molecular and cellular mechanisms controlled by PKR1 signaling in this process are unclear. Methods and Results Here, we analyze a tissue-restricted mutations of the PKR1 gene in the proepicardial lineages (Gata5 and Wt1), and we show that PKR1 signaling in the proepicardium and its derivatives is required for proper cardiac and renal morphogenesis. Neonatal mutant mice display impaired proliferation of epicardial-derived cells in their heart and kidneys. Moreover, we detect defective coronary and renal arteriogenesis associated with PKR1 deficiency. Epicardial development is dramatically impaired in mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived cells into coronary endothelial and smooth muscle cells. Abnormal mitochondria, lipid accumulation in mutant cardiomyocytes leads to lower contractile response to dobutamine. Impaired proliferation was observed in both Gata5 and WT1 but apoptosis was observed only WT1 lieage. Adult mutant hearts had abnormal rhythmicity and impaired systolic functions. Hypoplastic kidneys at the neonatal mutants were accompanied with deficient glomerular angiogenesis. Outgrown cell from kidney explants had a defective vasculogenic cell differentiation. Atrophy and dilated glomerular structure, abnormal mitochondria, lipid deposition and apoptosis were observed in the adult mutant kidney. Conclusions Our findings provide a mechanistic insight into the roles of PKR1 signaling in heart and kidney disorders controlling the maturation of epicardial-derived cell and differentiation in a cell autonomous fashion and affecting cellular communications in a paracrine fashion. Our mouse models recapitulate the complex human heart-kidney disorders.