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.
Prokineticins, comprising prokineticin-1 (also called EG-VEGF) and prokineticin-2 (also called Bv8), are secreted bioactive proteins, structurally related to a class of venom-like proteins.1,2 Prokineticins exert their biological activities by stimulating 2 closely related G protein–coupled receptors (GPCRs), prokineticin receptor-1 (PKR1), and PKR2.3,4 Prokineticin-2 is the most potent agonist for both receptors under physiological condition.4,5 PKR2, and to a lesser extent PKR1, are also expressed in the brain, whereas PKR1 is mostly expressed in peripheral organs, including spleen, prostate, pancreas, heart, monocytes, and leukocytes.5,6 Prokineticins are involved in regulating diverse biological processes that include gastrointestinal motility,1 hematopoiesis,7 angiogenesis,8 monocyte differentiation9 and macrophage activation,10 pain sensitization,11,12 circadian rhythms,13,14 coordination of circadian behavior and physiology by suprachiasmatic nucleus,15 olfactory bulb activation,16,17 and neuronal survival.18 However, their roles and putative involvement in signaling pathways regulating heart function had not been elucidated until recently. We have shown that in cultured coronary endothelial cells, prokineticin-2 via PKR1 induces vessel-like formation. In cardiomyocytes, prokineticin-2 via PKR1 protects cardiomyocytes against hypoxia induced apoptosis.19 Moreover, transient PKR1 gene transfer reduces mortality and preserves left ventricular function by promoting angiogenesis and cardiomyocyte survival in the coronary ligation mouse model for myocardial infarction.19
See accompanying article on page 803
Coronary vasculogenesis requires cells to delaminate from the epicardium, undergo epithelial-mesenchymal transformation, migrate toward the capillary plexus within the myocardium, and differentiate into endothelial or smooth muscle cells.20 Recently, the postnatal and adult epicardium have been found to be potential sources of vascular progenitors that can be differentiated into smooth muscle or endothelial cells on stimulation with growth factors that do not use GPCR signaling.21 The well-studied cardiac role of GPCRs via Gq signaling is to promote cardiac hypertrophy22,23 or protect cardiomyocytes against hypoxic insult.24,25 However, none of the GPCRs have been shown to be involved in epicardial-derived progenitor cell (EPDC) differentiation to promote neovascularization.
In this study, we showed that PKR1 signaling may affect vascularization as well as heart function via a cardiac cell communication, using coculture model, EPDCs and heart explants and transgenic mice model. We verified the role of PKR1 in EPDC differentiation and vascularization in hearts and epicardial explants of PKR1-null mutants.
Materials and Methods
Generation of Transgenic and PKR1-Null Mutant Mice
Transgenic (TG) mice overexpressing full-length PKR1 in the heart22 and the germ line null allele mice for PKR1 were generated as previously described (supplemental Figure II and supplemental Table I, available online at http://atvb.ahajournals.org). All animal experimentation was performed in accordance with institutional guidelines of the French Animal Care Committee, with European regulation-approved protocols.
Analysis of Gene Expression by RT-PCR
Total RNA from adult mice hearts was isolated using TRI Reagent (Molecular Research Center) and treated with DNase using the RNase-Free DNase Set. Semiquantitative RT-PCR was performed on 0.5 to 5 μg of total RNA extracted from all hearts, using GAPDH as an internal control. The primers are shown in supplemental Table I.
Histomorphological and Electron Microscopy Analysis
Twelve-week-old mice hearts were dissected, cryosectioned (10 μm), and stained with Mallory tetrachrome. For electron microscopy, hearts were fixed by immersion in glutaraldehyde, postfixed with osmium tetroxide, and embedded in epoxy resin using routine methods.
Immunostaining and Western Blotting
Immunofluorescence and Western blot analyses were performed as previously described25 (for details see supplemental materials).
Echocardiography and Hemodynamic Assessment
Echocardiographic and hemodynamic analyses were performed on 12- to 16-week-old mice as previously described (for details see supplemental materials).
Epicardial Explants From Neonatal and Adult Hearts
Cardiac explants and EPDC cultures were performed as previously described (for details see supplemental materials). For BrdU staining, P1 mice received 100 μL (i.p.) of BrdU (20 μg/mL) 4 hours before the hearts were excised for the explant experiments.
Assessment of Formation of Capillary-Like Structures by EPDCs
EPDC cells were seeded onto the 24-well culture plates coated with Matrigel (BD Biosciences) at 105 cells per well in the serum free assay medium with or without prokineticin-2 (5 to 10 nmol/L) and incubated at 37°C for 24 hour, then the formation of vessel-like structures as an in vitro model of angiogenesis were assessed as previously described.19 Images were captured at a magnification of 10× with a digital microscope camera system.
Coculture with EPDCs and H9c2 Cardiomyoblasts
Coculture model were generated as previously described (for details see supplemental materials).
Data are expressed as mean±SEM. Multigroup comparisons were performed using 1-way ANOVA with post hoc correction. Comparisons between 2 groups were made using unpaired Student t test. For all analyses, P<0.05 was considered significant utilizing Microsoft Excel 2000 for Windows.
Phenotype Analyses of TG Hearts
Histomorphological analyses revealed no sign of hypertrophy in TG heart (Figure 1A and 1B). Heart to body or lung to body weight ratios in TG mice was similar to non-TG (NTG) mice (n=5, Figure 1C). TG mice did not show any significant cardiac functional changes, as detected by echocardiographic or hemodynamic analyses (supplemental Table II). In response to isoproterenol (20 ng/g), βadrenergic stimulation, TG mice exhibit increased dp/dt max and decreased contractile time (n=6, P<0.05, Figure 1D), indicating improved myocardial contractile function. Semithin sections of TG hearts revealed an increased extracellular matrix filled with capillary vessels (Figure 1E). Ultrastractural analysis with electron microscopy showed that TG hearts displayed (n=3, Figure 1F) no spontaneous defects, normal cardiomyocytes, sometimes with enlarged gap junctions (Figure 1G) and increased capillary formation (Figure 1E).
Cardiomyocyte-PKR1 Signaling Induces Vascular Remodeling in TG Heart
To investigate the potential angiogenic activity of cardiomyocyte-PKR1 signaling and its impact on the vasculature, blood vessels were quantified on 12-week-old TG and NTG hearts. The density of PECAM-1+ capillary vessels (Figure 2A) was increased by 30% and the number of α-SMA+ arterioles (Figure 2B) was increased by 12% in TG hearts (n=4, P<0.01). The 1.5-fold increase in size was obtained in TG arteries with <30 μm of diameter (n=4, P<0.05; supplemental Figure IIIA).
Induction of EPDCs in TG Heart
An increased number of Ki67+ proliferating cells was observed at the epicardial-subepicardial area of TG hearts (Figure 2C). Staining of cryosectioned hearts with antibody for epicardin (an EPDC specific marker) revealed a 2.2-fold increase in epicardin levels in the TG epicardium-subepicardium (n=4, P<0.05, Figure 2D). Interestingly, some of the cell populations were found dual epicardin+/α-SMA+ or epicardin+/PECAM-1+ in the epicardium-subepicardial area of TG hearts (n=5, P<0.05), but not in the NTG hearts (supplemental Figure III).
Cardiomyocyte-PKR1 Signaling Promotes EPDC Differentiation in Coculture System
To test whether cardiomyocyte-PKR1 signaling induces EPDC proliferation and differentiation in a paracrine fashion, H9c2 cells overexpressing PKR1 were cocultured with EPDCs.26 Note that H9c2 cardioblast cell line was used instead of isolated mouse cardiomyocytes to eliminate the possibility of endothelial cell contamination during the isolation process. Figure 3A shows cardiomyocyte specific antibody (MHC-MF-20) stained H9c2 and epicardin stained EPDCs, confirming a coculture model (n=4, left panel). Cocultured EPDCs with H9c2 infected with Adenovirus control vector (Adv-Control) showed a few specific staining for smooth muscle cells (Figure 3A middle panel). However, coculturing EPDCs with H9c2 infected with adenovirus carrying PKR1 cDNA (H9c2-Adv-PKR1 cells) at the ratio 1:2 exhibit significantly increased PECAM-1+ and α-SMA+ cells by a factor of ≈3.0 and ≈2.0, respectively (Figure 3A right panel). These in vitro data suggested that excessive cardiomyocyte-PKR1 signaling in the coculture condition induced EPDCs to express both endothelial and smooth muscle lineage-specific proteins, mimicking in vivo TG model.
PKR1 Signaling Upregulates its own Ligand
We have shown that in human end-stage failing heart samples, both PKR1 and prokineticin-2 were downregulated, implicating a possible prokineticin-2/PKR1 loop in heart.19 Next prokineticin-2 levels were determined in the TG and NTG hearts. Prokineticin-2 transcript and protein levels were increased by a factor of 1.85±0.35 and 1.64±0.08, respectively, in the TG hearts (n=4, P<0.05, Figure 3B and histogram). Double immunostaining on cryosectioned hearts revealed that prokineticin-2 protein was localized in the epicardin+ cells in TG epicardium-subepicardium area (Figure 3B). To further investigate whether PKR1 signaling upregulates its own ligand expression, H9c2 cells were infected with different doses (1 or 10 MOI) of Adv-PKR119 or Adv-control (Adv-C) vector, and then the prokineticin-2 and PKR1 levels were determined by immunostaining analysis. Figure 3C shows that PKR1 upregulates prokineticin-2 levels in dose-dependent manner in the H9c2 cells, as observed in TG hearts (n=4, P<0.01).
Prokineticin-2 Promotes Neonatal EPDC Proliferation and Differentiation In Vitro
To assess a direct effect of prokineticin-2 on epicardium, we used explant cultures from wild-type hearts of postnatal day 1 (P1) neonates21 and tested the ability of prokineticin-2 to induce outgrowth in addition to any differentiation phenotype (Figure 4A), because PKR1 is expressed in epicardium19 as well as in EPDCs (supplemental Figure IVA). Untreated cardiac explants demonstrated 16±6% proliferating cells as detected by BrdU staining. Treatment of the epicardial explants with prokineticin-2 (5 to 10 nmol/L) stimulated extensive outgrowth of cells in dose-dependent manner (n=5, P<0.01, Figure 4B and 4D). All the emerging epithelial cells were positive for epicardin (purple, Figure 4C), but not positive for other stem cell markers27 such as Sca-1 or c-kit (supplemental Figure IVB).
Prokineticin-2 (5 nmol/L) promoted EPDC differentiation into PECAM-1+ endothelial (red) and α-SMA+ smooth muscle cells (green) (n=4, P<0.01, Figure 4E). Note that untreated neonatal EPDCs demonstrated few detectable smooth muscle cells. However, no fibroblast specific procollagen-1+ cells were detected (supplemental Figure IVB). Figure 4F shows that prokineticin-2–induced PECAM-1+ cells also express flk-1 (a VEGF receptor-2), another endothelial specific marker (n=3, Figure 4F). Endothelial cells differentiated from EPDCs by prokineticin-2 is able to promote vessel-like formation on Matrigel (n=3, Figure 4G), demonstrating that these differentiated cells carry not only endothelial-linage specific markers but also functional characteristics of endothelial cells.
Prokineticin-2 Promotes Adult EPDC Differentiation In Vitro
The postulated vasculogenic role of PKR1 was tested on adult EPDCs derived from 3-week (supplemental Figure VA) and 12-week-old (supplemental Figure VB) mice hearts. Prokineticin-2 treatment on the adult explants promoted significant outgrowth and differentiation of EPDCs into smooth muscle and endothelial cells as confirmed by α-SMA and PECAM-1 staining, respectively (supplemental Figure VA and VB). No fibroblast specific procollagen-1+cells were detected. This suggests that both 3-week-old and 12-week-old adult EPDCs are capable of response to prokineticin-2 to proliferate and differentiate into both smooth muscle and endothelial cells but not into fibroblasts.
Prokineticin-2 Effects on EPDCs Are Mediated by PKR1
Similar experiments were performed on PKR1-null mutant neonate cardiac explants. Prokineticin-2 in the PKR1-null mutant explants was not able to induce proliferation as detected by BrdU staining (Figure 5A and 5C). Moreover, prokineticin-2 (5 or 10 nmol/L) induced differentiation of endothelial and smooth muscle cells were significantly reduced in the PKR1-null mutant EPDCs compared to wild one (Figure 5B), indicating PKR1-mediated effects.
PKR1-Null Mutants Exhibit Reduced Capillary Network and Prokineticin-2 Levels
RT-PCR analyses revealed that both short and long isoform of prokineticin-2 were downregulated in the PKR1-null mutant hearts (n=4, P<0.01, Figure 5E). Interestingly, PKR1-null mutant hearts exhibit reduced capillary density in their subepicardium by 22% (n=4, P<0.01, Figure 5D). Note that reduced capillary numbers in PKR1-null mutant heart were significant up to the age of 3 weeks. At this age, mutant hearts exhibit an elevation in hypoxia-inducible factor (HIF-1 alpha) expression by 22% over the wild hearts (Figure 5F).
For the first time we showed that cardiomyocyte PKR1 signaling upregulates its own ligand, prokineticin-2 as a paracrine factor, to induce proliferation and differentiation of EPDCs into endothelial and smooth muscle cells thereby promoting neovascularization (supplemental Figure VII).
Cardiac-PKR1 signaling increases not only angiogenesis but also vasculogenesis in TG mice without inducing major cardiac pathology as observed for FGF-1 overexpressing TG heart.28 Surprisingly, PKR1-TG mice did not exhibit spontaneous cardiomyopathy as expected for the Gq protein–mediated hypertrophic cardiomyopathy.23 Previously, a survival promoting role of Gq protein signaling has also been shown in cardiomyocytes.24 Moreover, we have recently shown that PKR1 signaling stimulates survival pathway to protect cardiomyocytes against ischemic insult.19 All together these data clearly show that PKR1 signaling is involved in cardiomyocyte survival rather than hypertrophic growth.
It appears that the increased angiogenesis and vasculogenesis in TG hearts is not the result of acute cardiac dysfunction, because neither obvious hypertrophic cardiomyopathy nor dilated cardiomyopathy were found in the TG mice. Interestingly, TG hearts exhibited improved myocardial contractile function to isoproterenol, which could be resulted from increased myocardial perfusion attributable to myocardial neovascularization. Indeed, our morphometric studies indicate that increased capillary density and arteriole numbers in TG hearts first appears within 3-week-old mice (supplemental Figure VIA and VIB) and remains augmented even after 12 weeks, maintaining the growth status of the capillary arterial system. These findings show an alteration of the genetically determined circuit plan in TG mouse model and strongly support the hypothesis that cardiac-prokineticin-2/PKR1 is a regulator of the neovasculogenesis during postnatal life.
What is the mechanism of increased capillary density and number of arterioles in the TG hearts? Some of the cell populations was found dual epicardin+/α-SMA+ or epicardin+/PECAM-1+ in the epicardial-subepicardal area of adult TG hearts, representing a subset of vascular precursors which can give a rise to smooth muscle and endothelial cells. The number of Ki67+ cells in TG hearts and BrdU+/epicardin+ cells on treatment of EPDCs with prokineticin-2 were significantly increased, indicating that after proliferating/migrating epicardin+ cells in TG hearts may serve as vascular precursors to induce neovascularization, because proliferation, migration, and differentiation are well known effects of prokineticin signaling in astrocytes,29 enteric neural crest cells,30 and neuronal progenitors.31
Our coculture model supports the TG phenotype that the cardiomyocyte-PKR1 mediated paracrine signaling is sufficient to restore the pluripotency of EPDCs. The first target of cardiomyocyte-PKR1-mediated paracrine regulation is prokineticin-2 itself, for the following reasons: (1) in the TG hearts, prokineticin-2 levels were upregulated; (2) overexpressing PKR1 in H9c2 cells upregulates prokineticin-2 levels in PKR1-level-dependent manner; (3) ablation of PKR1 in mice downregulates the prokineticin-2 levels; (4) prokineticin-2 via PKR1 plays a direct role on proliferation, migration and differentiation of EPDCs.
EPDCs are characterized as the stem cell–derived multipotential vascular progenitors.32 EPDCs can either form endothelial cells, in response to a combination of myocardial vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) signaling, or differentiate into smooth muscle cells, on exposure to platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and bone morphogenetic protein-2 (BMP-2).33 However, thymosin β4 appears to be a necessary and perhaps sufficient signaling factor for adult epicardial progenitor mobilization and differentiation into the endothelial and smooth muscle cells to induce neovascularization.21 Given the contribution of epicardial cells to the endothelial and smooth muscle cells of coronary blood vessels in PKR1-TG heart, our studies with the explants and EPDCs derived from neonatal, 3-, and 12-week-old hearts confirmed a vascular development role for PKR1 signaling during the postnatal life. The epicardium does not become refractory to prokineticin/PKR1 signaling with age. During development EPDCs give a rise to vascular precursors and to adventitial fibroblasts. Prokineticin/PKR1 signaling can reprogram adult EPDCs to adopt a subset of embryonic cell fates (endothelial and smooth muscle cells at the expense of fibroblast), suggesting that PKR1 signaling inherent to EPDCs may determine distinct lineage choice decision.
Prokineticin-2 actions were mediated by PKR1 in EPDCs, because prokineticin-2–induced proliferation and differentiation were reduced in the PKR1-null mutant EPDCs. Surprisingly, the number of capillary vessels was reduced in the PKR1-null mutant hearts. Unexpectedly, we found that abnormal capillary density at the early age of PKR1-null mutant heart was compensated at 12 weeks after their birth. Decreased angiogenesis in PKR1-null mutant heart during the early postnatal life may create an ischemic condition that increases HIF-1 alpha expressions to transcriptionaly regulate the expression of angiogenic growth factors as a compensatory mechanism. Although the survival of PKR1-null mutant mice would tend to negate our hypothesis that PKR1 signaling mechanism is physiologically critical for the cardiac vasculature, it is clear that after 3 weeks of age, compensatory mechanisms are triggered to overcome this defect, explaining the survival rate. PKR1 might be involved in postnatal de novo vascularization rather than vasculogenesis during the embryogenesis. Previously, PKR1-null mutant mice have been shown to display loss of prokineticin-2–mediated macrophage migration and differentiation,10 or a reduction of nociceptive and thermal effects resulting from reduced capsaicin-induced activation of the vanilloid receptor.11 However, these two groups did not examine whether these mice exhibited any cardiovascular defects, because there was no embryonic lethality attributable to cardiovascular defect. It has been shown that PKR1-null mutants displayed reduced prokineticin-2 levels in inflammatory response,11 supporting our data that PKR1 signaling regulates its own ligand expression. All together our data suggest the existence of ligand/receptor feedback loops. The autocrine and paracrine loops are established when soluble factors secreted by cells bind and stimulate receptors on their own surfaces or neighboring cell surfaces. This is the first demonstration of GPCR/ligand paracrine regulation.
Our studies provided first evidence that cardiac PKR1-mediated signaling is involved in cell communication, thereby inducing neovascularization.
This paper is dedicated to the memory of Dr A.F. Nebigil. We thank all the members of the phenotyping facilities of the MCI for performing functional analyses.
Sources of Funding
K.U. is supported by fellowship from Japan Society for the Promotion of Science.
This work was supported by grants from the Centre National de la Recherché Scientific and CNRS/ATIP young scientist award, Foundation France (005326), Association pour la Recherché sur le Cancer (3619ARC), and Foundation pour la Recherché Médicale (C7100000).
K.U. and C.G. contributed equally to this work.
Original received October 10, 2007; final version accepted February 18, 2008.
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