The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function.

AIMS: Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function. METHODS AND RESULTS: Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tß4) and Tß4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1. CONCLUSION: The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

A Prokineticin-Driven Epigenetic Switch Regulates Human Epicardial Cell Stemness and Fate.

Epicardial adipose tissues (EATs) and vascular tissues may both belong to the mesoepithelial lineage that develops from epicardium-derived progenitor cells (EPDCs) in developing and injured hearts. Very little is known of the molecular mechanisms of EPDC contribution in EAT development and neovascularization in adult heart, which the topic remains a subject of intense therapeutic interest and scientific debate. Here we studied the epigenetic control of stemness and anti-adipogenic and pro-vasculogenic fate of human EPDCs (hEPDCs), through investigating an angiogenic hormone, prokineticin-2 (PK2) signaling via its receptor PKR1. We found that hEPDCs spontaneously undergoes epithelial-to-mesenchymal transformation (EMT), and are not predestined for the vascular lineages. However, PK2 via a histone demethylase KDM6A inhibits EMT, and induces asymmetric division, leading to self-renewal and formation of vascular and epithelial/endothelial precursors with angiogenic potential capable of differentiating into vascular smooth muscle and endothelial cells. PK2 upregulates and activates KDM6A to inhibit repressive histone H3K27me3 marks on promoters of vascular genes (Flk-1 and SM22α) involved in vascular lineage commitment and maturation. In PK2-mediated anti-adipogenic signaling, KDM6A stabilizes and increases cytoplasmic ß-catenin levels to repress peroxisome proliferator-activated receptor-γ expression and activity. Our findings offer additional molecular targets to manipulate hEPDCs-involved tissue repair/regeneration in cardiometabolic and ischemic heart diseases. Stem Cells 2018;36:1589-1602.

Prokineticin receptor-1-dependent paracrine and autocrine pathways control cardiac tcf21+ fibroblast progenitor cell transformation into adipocytes and vascular cells.

Cardiac fat tissue volume and vascular dysfunction are strongly associated, accounting for overall body mass. Despite its pathophysiological significance, the origin and autocrine/paracrine pathways that regulate cardiac fat tissue and vascular network formation are unclear. We hypothesize that adipocytes and vasculogenic cells in adult mice hearts may share a common cardiac cells that could transform into adipocytes or vascular lineages, depending on the paracrine and autocrine stimuli. In this study utilizing transgenic mice overexpressing prokineticin receptor (PKR1) in cardiomyocytes, and tcf21ERT-creTM-derived cardiac fibroblast progenitor (CFP)-specific PKR1 knockout mice (PKR1 tcf-/-), as well as FACS-isolated CFPs, we showed that adipogenesis and vasculogenesis share a common CFPs originating from the tcf21+ epithelial lineage. We found that prokineticin-2 is a cardiomyocyte secretome that controls CFP transformation into adipocytes and vasculogenic cells in vivo and in vitro. Upon HFD exposure, PKR1 tcf-/- mice displayed excessive fat deposition in the atrioventricular groove, perivascular area, and pericardium, which was accompanied by an impaired vascular network and cardiac dysfunction. This study contributes to the cardio-obesity field by demonstrating that PKR1 via autocrine/paracrine pathways controls CFP-vasculogenic- and CFP-adipocyte-transformation in adult heart. Our study may open up new possibilities for the treatment of metabolic cardiac diseases and atherosclerosis.

Prokineticin receptor-1 signaling promotes Epicardial to Mesenchymal Transition during heart development.

The epicardium plays an essential role in coronary artery formation and myocardial development. However, signals controlling the developing epicardium and epicardial-mesenchymal transition (EMT) in the normal and diseased adult heart are studied less rigorously. Here we investigated the role of angiogenic hormone, prokineticin-2 and its receptor PKR1 in the epicardium of developing and adult heart. Genetic ablation of PKR1 in epicardium leads to partial embryonic and postnatal lethality with abnormal heart development. Cardiac developmental defects are manifested in the adult stage as ischemic cardiomyopathy with systolic dysfunction. We discovered that PKR1 regulates epicardial-mesenchymal transition (EMT) for epicardial-derived progenitor cell (EPDC), formation. This event affects at least three consequential steps during heart development: (i) EPDC and cardiomyocyte proliferation involved in thickening of an outer compact ventricular chamber wall, (ii) rhythmicity, (iii) formation of coronary circulation. In isolated embryonic EPDCs, overexpression or activation of PKR1 alters cell morphology and EMT markers via activating Akt signaling. Lack of PKR1 signal in epicardium leads to defective heart development and underlies the origin of congenital heart disease in adult mice. Our mice provide genetic models for congenital dysfunction of the heart and should facilitate studies of both pathogenesis and therapy of cardiac disorders in humans.

Prokineticin receptor 1 is required for mesenchymal-epithelial transition in kidney development.

Identification of factors regulating renal development is important to understand the pathogenesis of congenital kidney diseases. Little is known about the molecular mechanism of renal development and functions triggered by the angiogenic hormone prokineticin-2 and its receptor, PKR1. Utilizing the Gata5 (G5)-Cre and Wilms tumor 1 (Wt1)(GFP)cre transgenic lines, we generated mutant mice with targeted PKR1 gene disruptions in nephron progenitors. These mutant mice exhibited partial embryonic and postnatal lethality. Kidney developmental defects in PKR(G5-/-) mice are manifested in the adult stage as renal atrophy with glomerular defects, nephropathy, and uremia. PKR1(Wt1-/-) embryos exhibit hypoplastic kidneys with premature glomeruli and necrotic nephrons as a result of impaired proliferation and increased apoptosis in Wt1(+) renal mesenchymal cells. PKR1 regulates renal mesenchymal-epithelial transition (MET) that is involved in formation of renal progenitors, regulating glomerulogenesis toward forming nephrons during kidney development. In the isolated embryonic Wt1(+) renal cells, overexpression or activation of PKR1 promotes MET defined by the transition from elongated cell to octagonal cell morphology, and alteration of the expression of MET markers via activating NFATc3 signaling. Together, these results establish PKR1 via NFATc3 as a crucial modifier of MET processing to the development of nephron. Our study should facilitate new therapeutic opportunities in human renal disorders.-Arora, H., Boulberdaa, M., Qureshi, R., Bitirim, V., Messadeq, N., Dolle, P., Nebigil, C. G. Prokineticin receptor 1 is required for mesenchymal-epithelial transition in kidney development.

A Role for the Long Noncoding RNA SENCR in Commitment and Function of Endothelial Cells.

Despite the increasing importance of long noncoding RNA in physiology and disease, their role in endothelial biology remains poorly understood. Growing evidence has highlighted them to be essential regulators of human embryonic stem cell differentiation. SENCR, a vascular-enriched long noncoding RNA, overlaps the Friend Leukemia Integration virus 1 (FLI1) gene, a regulator of endothelial development. Therefore, we wanted to test the hypothesis that SENCR may contribute to mesodermal and endothelial commitment as well as in endothelial function. We thus developed new differentiation protocols allowing generation of endothelial cells from human embryonic stem cells using both directed and hemogenic routes. The expression of SENCR was markedly regulated during endothelial commitment using both protocols. SENCR did not control the pluripotency of pluripotent cells; however its overexpression significantly potentiated early mesodermal and endothelial commitment. In human umbilical endothelial cell (HUVEC), SENCR induced proliferation, migration, and angiogenesis. SENCR expression was altered in vascular tissue and cells derived from patients with critical limb ischemia and premature coronary artery disease compared to controls. Here, we showed that SENCR contributes to the regulation of endothelial differentiation from pluripotent cells and controls the angiogenic capacity of HUVEC. These data give novel insight into the regulatory processes involved in endothelial development and function.

Impaired vascular function and repair in patients with premature coronary artery disease.

BACKGROUND: Endothelial dysfunction is central to the pathogenesis of coronary artery disease, but the role of local and circulating endothelial progenitor cells in maintaining vascular health is poorly understood. We hypothesised that impaired local and circulating vascular repair mechanisms predispose to endothelial dysfunction and the premature onset of coronary artery disease. METHODS AND RESULTS: Patients with premature coronary artery disease (n = 16) and healthy age- and sex-matched controls (n = 16) underwent venous occlusion plethysmography with intra-arterial infusion of acetylcholine and sodium nitroprusside. Numbers of circulating endothelial progenitor cells were directly quantified in whole blood by flow cytometry. Endothelial cells were isolated from the blood vessel wall and from peripheral blood mononuclear cells, and expanded in vitro for phenotypic and functional characterisation and analysis of microRNA expression levels. A dose-dependent increase in forearm blood flow (p < 0.001) was attenuated in response to the endothelial-dependent vasodilator acetylcholine in patients compared with controls (p = 0.03). No differences in the number of circulating endothelial progenitor cells or in the phenotype, function or microRNA expression levels of endothelial outgrowth cells isolated from blood were observed in patients and controls. Conversely, local vessel wall endothelial cells from patients had significant impairments in proliferation, adhesion and migration, and significantly reduced expression levels of microRNAs known to regulate endothelial function (miRs -10 a, -let7b, -126 and -181 b) (p < 0.05 for all). CONCLUSION: Local vessel wall derived endothelial cells, rather than circulating endothelial progenitor cells and their progeny, are impaired in patients with vascular dysfunction and premature coronary artery disease.

Prokineticin receptor 1 as a novel suppressor of preadipocyte proliferation and differentiation to control obesity.

BACKGROUND: Adipocyte renewal from preadipocytes occurs throughout the lifetime and contributes to obesity. To date, little is known about the mechanisms that control preadipocyte proliferation and differentiation. Prokineticin-2 is an angiogenic and anorexigenic hormone that activate two G protein-coupled receptors (GPCRs): PKR1 and PKR2. Prokineticin-2 regulates food intake and energy metabolism via central mechanisms (PKR2). The peripheral effect of prokineticin-2 on adipocytes/preadipocytes has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: Since adipocytes and preadipocytes express mainly prokineticin receptor-1 (PKR1), here, we explored the role of PKR1 in adipose tissue expansion, generating PKR1-null (PKR1(-/-)) and adipocyte-specific (PKR1(ad-/-)) mutant mice, and using murine and human preadipocyte cell lines. Both PKR1(-/-) and PKR1(ad-/-) had excessive abdominal adipose tissue, but only PKR1(-/-) mice showed severe obesity and diabetes-like syndrome. PKR1(ad-/-)) mice had increased proliferating preadipocytes and newly formed adipocyte levels, leading to expansion of adipose tissue. Using PKR1-knockdown in 3T3-L1 preadipocytes, we show that PKR1 directly inhibits preadipocyte proliferation and differentiation. These PKR1 cell autonomous actions appear targeted at preadipocyte cell cycle regulatory pathways, through reducing cyclin D, E, cdk2, c-Myc levels. CONCLUSIONS/SIGNIFICANCE: These results suggest PKR1 to be a crucial player in the preadipocyte proliferation and differentiation. Our data should facilitate studies of both the pathogenesis and therapy of obesity in humans.

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.

Flavaglines alleviate doxorubicin cardiotoxicity: implication of Hsp27.

BACKGROUND: Despite its effectiveness in the treatment of various cancers, the use of doxorubicin is limited by a potentially fatal cardiomyopathy. Prevention of this cardiotoxicity remains a critical issue in clinical oncology. We hypothesized that flavaglines, a family of natural compounds that display potent neuroprotective effects, may also alleviate doxorubicin-induced cardiotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Our in vitro data established that a pretreatment with flavaglines significantly increased viability of doxorubicin-injured H9c2 cardiomyocytes as demonstrated by annexin V, TUNEL and active caspase-3 assays. We demonstrated also that phosphorylation of the small heat shock protein Hsp27 is involved in the mechanism by which flavaglines display their cardioprotective effect. Furthermore, knocking-down Hsp27 in H9c2 cardiomyocytes completely reversed this cardioprotection. Administration of our lead compound (FL3) to mice attenuated cardiomyocyte apoptosis and cardiac fibrosis, as reflected by a 50% decrease of mortality. CONCLUSIONS/SIGNIFICANCE: These results suggest a prophylactic potential of flavaglines to prevent doxorubicin-induced cardiac toxicity.

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.

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. CONCLUSIONS: 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.