Mounia Boulberdaa

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 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 (PKR1ad−/−) mutant mice, and using murine and human preadipocyte cell lines. Both PKR1−/− and PKR1ad−/− had excessive abdominal adipose tissue, but only PKR1−/− mice showed severe obesity and diabetes-like syndrome. PKR1ad−/−) 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.

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.

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-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 (PKR1tcf−/−), 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, PKR1tcf−/− 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 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)GFPcre 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 PKRG5‐/‐ mice are manifested in the adult stage as renal atrophy with glomerular defects, nephropathy, and uremia. PKR1Wf1‐/‐ 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. FASEB J. 30, 2733‐2740 (2016). www.fasebj.org

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 β‐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

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.