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* UMR 7175/CNRS/Universite Strasbourg I, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France,
Institut de Génétique et de Biologie Moléculaire, Illkirch France,
Center of Cardiovascular Medicine, University Wurzburg, Wurzburg, Germany.
L'institut du Thorax, Inserm U533, Nantes, France; and
|| Department of Pharmacology and Toxicology, Kyushu University, Fukuoka, Japan
1Correspondence: UMR 7175/CNRS/Universite Strasbourg I, Ecole Supérieure de Biotechnologie de Strasbourg, Bld. Sébastien Brandt BP. 10413, F-67412 Illkirch, France. E-mail: nebigil{at}esbs.u-strasbg.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: PKR1 • MI
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Prokineticin-1, also called endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has been postulated to be a potent angiogenic factor based on its functional similarity to VEGF (2)
. Prokineticin-1 shares 58% identity with Bv8/prokineticin-2 (3
, 4)
. Recently, the human prokineticin-1, prokineticin-2 long isoform (PK2L) and short isoform have been shown to be expressed in the heart (3
, 5
, 6)
but very little is known about their structure and possible cardiac function. Prokineticins are involved in regulating various biological processes that include gastrointestinal motility (3)
, pain sensitization (7)
, angiogenesis (2
, 8)
, circadian rhythms (9)
, olfactory bulb activation (10)
, hematopoiesis (11)
, monocyte differentiation (12)
, and macrophage activation (13)
. They exert their biological activities by stimulating two highly similar receptors. PKR1 and PKR2, that belong to the Gq protein coupled receptor (GqPCR) family (1
, 4
, 14)
. Both PKR1 and PKR2 are mainly expressed in human and rat endocrine tissues, including thyroid, pituitary, and adrenal glands, as well as in the ovary and testis PKR2 and to a lesser extent PKR1 are expressed in the brain (1
, 4
, 14)
. PKR1, originally called GPR73 (15)
, is mainly expressed spleen, prostrate, pancreas, monocytes leukocytes (1
, 4
, 14)
, and in human heart (15)
. Prokineticin receptors mediate diverse biological processes; however, their roles and putative involvement in signaling pathway(s) regulating heart function have not yet been elucidated.
Prokineticins may have beneficial effects on cardiac repair possibly by inducing angiogenesis (16)
or regenerating cardiomyocytes. Prokineticins utilize two Gq-coupled receptors for their biological effects. It was shown that Gq/G11 signaling is an essential pathway to regulate cardiac development and hypertrophy (17)
. The serotonin-2B, Gq-coupled receptor, is involved in development of hypertrophy and protection of cardiomyocytes (17
18
19
20)
. Moreover, Gq/G13 contributes to vessel formation (21)
, as observed in VEGF knockout mice (22)
. Prokineticins induce differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage and activate differentiation and macrophage migration (12
, 13)
. In summary, PKR1 signaling could be important in regulating cardiac cell function.
Accordingly, the aim of this study was to investigate whether PKR1 signaling prevents ventricular remodeling after myocardial infarction using both in vivo and in vitro systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cells were maintained in DMEM supplemented with 10% FCS, penicillin G (100 U/ml), and streptomycin (100 µg/ml), under 5% CO2 at 37°C. H5V endothelial cells derived from mouse heart were a kind gift of Dr. Annunciata Vecchi (Istituto Clinico Humanitas, Rozzano, Italy). H5V cells were maintained in DMEM supplemented with 10% heat inactivated FCS, penicillin G (100 U/ml), and streptomycin (100 µg/ml), glutamine (1 mM) under 5% CO2 at 37°C.
Analysis of gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA from adult mouse hearts (n=4) or from cultured cells was isolated by the guanidinium thiocyanate-phenol chloroform method. Semiquantitative RT-PCR was performed on different concentration of total RNA (0.5–5 µg), using the GAPDH as an internal control as described previously (18
19
20)
. Each reaction was performed in duplicate and the values were averaged to calculate the relative expression levels. All PCR-amplified fragments were sequenced to confirm their identity. Each experiment was repeated four times (Table 1
).
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RNA in situ hybridization
In situ hybridization on cryosectioned adult mouse heart and aorta was performed with digoxigenin-labeled antisense or sense riboprobes synthesized from the 3' untranslated region of mouse PKR1 cDNA as described previously (23)
.
Immunohistochemistry
Adult cardiac mouse tissues was cryosectioned and used for immunohistochemistry (18
19
20)
. The hearts were sectioned at four equivalent levels from the base to the apex. Briefly, for immunostaining, sections (10 µm thick) were fixed and incubated with primary antibodies against, rat anti-PECAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-18916), active caspase-3 (Chemicon International, Temecula, CA, USA, AB-3623), rabbit polyclonal VEGF neutralizing antibody (abcam, ab9953) and mouse PKR1. A monoclonal PKR1 antibody was raised against the N-terminal- peptide of the mouse PKR1 (DYDMPLDEEEDVT) and its selectivity was tested on COS cells expressing GFP-PKR1. Fluorescent signals were visualized and photographed with an Olympus BX41 microscope (Olympus America Inc., Melville, NY, USA) equipped with a digital camera. Signal intensity was quantified on digitalized images and calculated as the product of averaged pixel intensity per area. Densitometric analysis was carried out using Molecular Dynamics Image Quant software. Some of the fluorescent signals were obtained with confocal microscope and images were controlled by LEICA software. The laser was chosen according to the sample and compared pixel intensity and pixel distribution. These data were analyzed using the pixel data from which background intensity was subtracted.
Assessment of formation of capillary-like structures by endothelial cells
Twenty-four-well culture plates were coated with Matrigel (BD Biosciences, Bedford, MA, USA) according to the manufacturer's instructions and as described previously (24)
. H5V cells were trypsin-harvested and seeded onto the coated plates at 105 cells per well in the serum free assay medium with or without prokineticin-2 (1–15 nM) and incubated at 37°C for 24 h. Tube formation as two dimensional branched structures was observed using an inverted phase contrast microscope (Leica), and the number of branching points was quantified in five random fields from each sample. Each experiment was repeated four times. Images were captured at a magnification of 10x with a digital microscope camera system.
Detection of apoptosis
Apoptosis was detected by the TdT-mediated dUTP nick end-labeling (TUNEL) assay (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer's protocol. Mouse heart samples were obtained 3 and 7 days after coronary ligation. Twenty different heart sections (n=3 for each group) at the ischemic border zone were analyzed by fluorescence microscopy. Slides were counterstained with DAPI. Cells were scored for TUNEL-positive nuclei per total number of cells detected by DAPI. Alternatively active caspase-3 staining was performed on fixed heart sections. Generally depressed mitochondrial respiratory complex activity promotes release of cytochrome c from mitochondria to cytosol to induce caspase-3 activity and apoptosis. Frozen sections were fixed with 10% formalin for 5 min at room temperature and primary antiserum was rabbit IgG antiactive-caspase3 (Promega, Madison, WI, USA). Staining was visualized by using Cy3-conjugated goat anti-rabbit antibody. The percentage of apoptotic cells (TUNEL and active caspase-3 positive) was evaluated by viewing each field at 40x magnification respectively. The positive area of active-caspase3 or Tunel was analyzed by histological examination from 20 different mice heart sections (n=3 each group) at the ischemic border zone using NIH image.
To examine the antiapoptotic effect on cardiac cells in vitro, H9c2 cells or cardiomyocytes were seeded into eight chamber slides at a density of 10,000/cm2 and pretreated with prokineticin-2 (recombinant human prokineticin-2, Peprotech, France) for 24 h and then exposed to H2O2 (200 or 100 µM, respectively) treatment for 24 h. The concentration of the H2O2 were chosen which induces apoptosis by 
40% in the absence of serum and glucose in each cell types (25
, 26)
. TUNEL labeling (Roche Molecular Biochemicals) was performed on fixed cells. Slides were counterstained with DAPI. Cells were scored for TUNEL-positive nuclei per total number of cells detected by DAPI. The percentage of TUNEL-positive cells was evaluated by viewing each field at x63 magnification. Generally, 10 different microscopic fields containing 15–20 cells each were recorded for each sample.
Detection of Akt activation
Western blot analysis was performed as described previously (20)
. Proteins were extracted from H9c2 cells with SDS lysis buffer and separated on 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated overnight with the PhosphoPlus Akt (Ser-473) Antibody kit (Cell Signaling Technology, Danvers, MA, USA), and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG. Phosphorylated protein was visualized by enzyme-linked chemiluminescence (Amersham Biosciences, Indianapolis, IN, USA) and quantified by scanning laser densitometry, normalizing to total amounts of the corresponding proteins. This experiment was repeated three times.
Alternatively, in vivo Akt activity was detected by immunostaining of cryosectioned hearts with p-Akt and total Akt antibody followed by CY3 conjugated anti-rabbit IgG. Ten random microscopic fields in the left ventricle were examined and the active Akt was quantified as number of p-Akt-positive cells/high-power fields, and normalized by the total Akt positive cells. The samples were then incubated with a cardiomyocyte specific monoclonal antibody MF-20 for 1h at 37°C followed by fluorescein (Vector MOM immunodetection kit) conjugated goat anti-mouse IgG and analyzed by fluorescence microscopy.
Production of adenoviruses
Recombinant adenovirus for PKR1 was produced by the method of He et al. (27)
. After confirming the sequence, a PCR fragment for PKR1 was inserted into the pShuttle-CMV and the resulting linearized recombinant plasmid was transfected into HEK293 cells. After 15 days, the cells were harvested and freeze-thawing procedures were repeated three times. The supernatant of freeze-thawed cells containing recombinant virus were then amplified three times. Titer of virus was determined as described previously (27)
. The adenovirus construct encoding dominant-negative Akt (d3A-Akt) with a K179A/T308A/S473A mutation was a kind gift of Dr. Kenneth Walsh and used on isolated cardiomyocytes (28)
. Cardiomyocytes were plated in medium containing 10% FCS overnight and then incubated with adenovirus vector at a multiplicity of infection of 10 in medium containing 2% FCS as described before (20)
.
siRNA transfection
Transfection was performed by using siPORT Amine transfection reagent (Ambion, Austin, TX, USA) with 10nM siRNA for mouse PKR1 (Ambion siRNA #181827), 30nM siRNA for rat PKR1 (Ambion siRNA #58105) according to the manufacturer's instructions (Ambion). Negative control transfection was performed by using negative control siRNA (Ambion). Forty-eight hours after transfection 65% of PKR-1 level was reduced as detected by semiquantitative RT-PCR normalized by expression of GAPDH.
Animals, surgical procedures, and intramyocardial gene delivery
Male C57BL/6J (Charles River) mice at 12 wk of age (20–25 g) were used to produce myocardial infarction (MI) by ligation of the left anterior descending coronary artery as described previously (29)
. Adenovirus DNA encoding mouse PKR1 (Adv-PKR1; 5.5x108pfu/ml) was injected subepicardially with a 29-gauge needle into the ischemic area of the left ventricular wall immediately after coronary ligation (n=19), and infection efficiency was analyzed by GFP signaling using Adeno-GFP-infected hearts as described previously (30)
. Control animals (n=28) received an equivalent volume of either sterile saline, or nonrecombinant adenovirus (Adv-control). Titer-matched stocks of Adv-PKR1 and Adv-Control were used for intracardiac injections. We found that at this concentration, adenoviruses do not cause significant inflammatory responses in mouse heart. All procedures confirmed to the guiding principles of the American Physiological Society and European Union animal care committee and were approved by the institutional animal research committee.
Electron Microscopy
Hearts were fixed by immersion in glutaraldehyde, postfixed with osmium tetroxide and embedded in epoxy resin using routine methods (20)
. Mallory tetrachrome staining was used for the histological analyses. Myocardial injury was determined by the percentage of necrosis/fibrosis area against the total area of myocardium utilizing National Institutes of Health image software.
Hemodynamic Assessment
Cardiac hemodynamics were performed 5 wk after MI. Mice were anesthetized with 5% isoflurane, intubated and mechanically ventilated as described previously (29)
. Body temperature was maintained at 37°C. A 1.4-French high-fidelity Millar pressure catheter (Millar Instruments, Houston, TX, USA) was inserted via the right carotid artery into the left ventricle. After LV function and heart rate had stabilized, LV pressure, end-diastolic pressure, maximal positive (+dP/dt) or negative (-dP/dt) pressure development were recorded. Calibration of the Millar catheter was verified before and after each measurement.
RNA isolation, PCR, and protein analyses on human samples
The experimental protocol complied with the Declaration of the World Medical Association proclaimed in Helsinki. Cardiac samples from explanted hearts were obtained from 13 patients with end-stage heart failure who underwent a cardiac transplantation. Additional cardiac samples (n=5) obtained from explanted nonfailing hearts were used as controls (Table 2
) (see http://www.anatomy.usyd.edu.au/mru/researchexamples/cardiac/heartsamples.html). Immunostaining on cryosectioned heart samples were performed utilizing PKR1 (Life span, LS-A31S2), prokineticin-1 (R&D Systems, Minneapolis, MN, USA MAB 2100) and prokineticin-2 (Abnova Corporation, Taipei, Taiwan; H0060675) antibodies as primary antibody and CY3 as secondary antibody. Fluorescent signals were obtained with confocal microscope and images were controlled and analyzed by LEICA software. The laser was chosen according to the sample and compared pixel intensity and pixel distribution. These data were analyzed using the pixel data from which background intensity was subtracted.
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Total RNA was isolated using TRIZOLReagent (Life Technologies, Gaithersburg, MD, USA) and treated with DNase using the RNase-Free DNase Set and the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA quality was assessed using an Agilent 2100 bioanalyzer and RT-PCR with primers for 
-actin. The experiments were performed as triplicate PCR reactions. Mean gene expression ratios were calculated for each control, nonfailing heart samples and end-stage failing heart samples as described previously (31)
.
Statistic analysis
Data are expressed as mean ± SEM. Multigroup comparisons were performed using one-way ANOVA with post hoc correction. Comparisons between two groups were made using unpaired Student's t test. For all analyses, P < 0.05 was considered significant utilizing Microsoft Excel 2000 for Windows.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that both prokineticin-1, prokineticin-2 and their receptor PKR1 were expressed in mouse heart and testis. PKR1 and its ligands were also expressed in H9c2 cardioblast cell line derived from rat heart as well as in H5V capillary endothelial cells derived from mouse heart (Fig. 1A
third and fourth panels and histograms, respectively).
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In situ hybridization analysis with a digoxigenin-labeled riboprobe on cryosectioned mouse heart and aorta samples shows a PKR1 specific staining (brown color), indicating that PKR1 is expressed in aortic endothelial cells, but not in smooth muscle cells of aorta (Fig. 1B
, first panel). PKR1 is also expressed in the ventricular wall of the heart (Fig. 1B
, second panel). Control sense riboprobes did not show any staining in this tissue (Fig. 1B
, third panel). Immunohistochemical analysis was also performed with a monoclonal N-terminal specific anti-mouse PKR1 antibody, which confirmed the presence of PKR1 protein in (Fig. 1B
, last panel) myocardium and epicardium in agreement with the PKR1 transcript expression profile. Together these data show that PKR1 and its ligands are postnatally expressed in cardiac tissues and cells, and might be functional in the heart.
Prokineticin-2 via PKR1 induces vessel-like formation in capillary endothelial cells derived from mouse heart
To evaluate the angiogenic effect of prokineticin signaling on heart, a capillary endothelial cell line derived from mouse heart (H5V) were plated on Matrigel and treated with various concentrations of prokineticin-2 (5–15nM) or its vehicle for 24 h, and the formation of vessel-like structures as an in vitro model of angiogenesis was quantified. Prokineticin-2 treatment resulted in a 7-fold increase of vessel-like structures compared to vehicle alone (n=4, P<0.01; Fig. 2
A middle image, right histogram). Moreover, 3 fold overexpression of PKR1 in Adv-PKR1 infected H5V cells increased vessel-like structures in an 8 fold compared to Adv-control vector (Fig. 2B
; n=4, P<0.01). This data indicate that PKR1 overexpression initiates angiogenic signaling in the absence of exogenous ligands. The prokineticin-2-mediated angiogenic effect was completely abolished in the presence of a neutralizing antibody (1:200 dilution) against PKR1 (Fig. 2A
, right panel). This antibody was not able to inhibit PDGF-induced tube like formation (data not shown), indicating a PKR1-specific inhibition. Moreover prokineticin-2 was not able to induce vessel-like formation in the siRNA-PKR1-transfected cells in which the PKR1 expression was reduced by 65%, confirming PKR1-mediated angiogenic signaling (n=4, P>0.05). Note that prokineticin-2 effect was not altered in negative control iRNA (nciRNA) transfected cells (Fig. 2B
).
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Since VEGF is a key angiogenic stimulator involved in many GPCR-mediated angiogenic reactions (31)
, we investigated whether PKR1 signaling may promote capillary induction by inducing VEGF expression. We performed semiquantitative RT-PCR analysis on RNA extracts and Western blot analysis on cell lysate from H5V cells treated with prokineticin-2 or its vehicle. Prokineticin-2 treatment for 24 h changed neither VEGFA 164 and 120 transcripts nor VEGF protein levels (Fig. 2C
). Moreover, in the presence of a neutralizing antibody (4 µg/ml) against VEGF, the prokineticin-2-mediated angiogenic effect remained unaltered, indicating that involvement of VEGF is unlikely in angiogenesis promoting effect of PKR1 signaling (Fig. 2D
).
Prokineticin-2 via PKR1 signaling prevents H9c2 cells and cardiomyocytes from apoptosis against hypoxic insult
To evaluate the effect of prokineticin-2 on cardiomyocyte survival, first H9c2 cells were utilized and apoptosis was induced by oxidative stress (200 µM H2O2). The H9c2 line of cardioblasts derived from rat heart is known to undergo apoptotic cell death following hypoxia, and is widely used as an in vitro model of ischemic injury to evaluate cardioprotective properties of agents such as erythropoietin (25)
. Representative TUNEL (green) and DAPI (blue) stained sections are shown in Fig. 3
A, demonstrating fewer apoptotic cells in the prokineticin-2 pretreated or Adv-PKR1 infected cells (Fig. 3A
). Quantitative analysis (see Materials and Methods) revealed that H9c2 cells subjected to H2O2 (200 µM) for 24 h in glucose-free medium, displayed 38 ± 5% apoptosis (n=4, Fig. 3B
). H9c2 cells pretreated with prokineticin-2 (5 nM) for 24 h or infected with Adv-PKR1 exhibited a significant decrease (12±4.6; 8±3.2%, respectively, n=4, P<0.01) in TUNEL positive cells (Fig. 3B
). The decrease in apoptotic cells was completely prevented in the presence of PKR1 neutralizing antibody. These results were confirmed by utilizing siRNA for PKR1. In the siRNA-PKR1-transfected, but not in negative control for siRNA transfected cells prokineticin-2 was not able to protect cardiomyocytes against ischemic injury (43±6.6%, n=4), indicating the involvement of PKR1 (Fig. 3A, B
).
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Prokineticins stimulate two major cell survival pathways, the PI3K/Akt and ERK1/2 MAPK (2
, 8
, 33)
. To investigate the potential roles of these pathways in prokineticin-2-mediated cytoprotection, H9c2 cells were incubated with various cell-permeable inhibitors of these pathways in the presence and absence of prokineticin-2 (5 nM). Under these conditions, LY-294002 (10 µM), a specific inhibitor of PI3K, and thus Akt (34)
, compromised the ability of prokineticin-2 to protect H9c2 cell survival after hypoxia, resulting in about three times more apoptotic cells than observed in cells treated with prokineticin-2 alone. PD98059 (50 µM), a specific inhibitor of MEK1/2, and thus ERK (35)
, and SB-203580 (10 µM) (36)
a specific inhibitor of p38, did not reduce the cytoprotective effects of prokineticin-2 (Fig. 3B
). Note that LY294002 or PD98059 compounds alone did not significantly alter survival of H9c2 cells in the absence of prokineticin-2.
Since inhibition of the PI3K/Akt, but not the ERK or p38 pathway compromised the cytoprotective effects of prokineticin-2, the ability of prokineticin-2 to activate this pathway was evaluated and quantified by Western blot analysis and densitometry, using antibodies specific for Akt kinase at the residues (Ser-473) that are phosphorylated upon activation. The relative level of phospho-Akt was time-dependent and reached a maximum value of 2.5-fold over control (n=3, P<0.05) after 10 min of exposure to 5nM prokineticin-2 without altering total Akt level (Fig. 3C
).
Next, we performed similar experiments on primary cardiomyocytes isolated from new-born mice to confirm the protective role of prokineticin/PKR1/Akt signaling as observed in H9c2 cells (Fig. 3D
, top). Similar results were obtained when serum and glucose depleted primary cultured mouse cardiomyocytes were subjected to 100 µM H2O2 (43±8%; n=3, Fig. 3D
). Pretreatment of cardiomyocytes with prokineticin-2 or Adv-PKR1 infection significantly inhibited apoptosis (13±5 and 10±2%, respectively, n=3, P<0.01). Accordingly, a dominant-negative d3A-Akt with K179A/T308A/S473A mutations reversed the prokineticin-2-protective effect in cardiomyocytes (Fig. 3D
) as observed with LY-294002 at a similar efficiency in H9c2 cells (Fig. 3B
), indicating involvement of Akt in the PKR1 survival promoting effect in cardiomyocytes as well.
PKR1 gene therapy reduces scar area and restores cardiac function after myocardial infarction
Because of the effects of PKR1 on cardiac cells in vitro, we hypothesized that PKR1 gene therapy may rescue cardiac muscle after myocardial infarction (MI) in vivo. We chose a mouse model of MI caused by coronary ligation. In these experiments, a recombinant adenovirus carrying PKR1 cDNA (Adv-PKR1) was injected into the infarcted area at the time of coronary ligation. Three days after transient Adv-PKR1 injection in the ventricular wall of the heart, PKR1 levels were significantly increased (3-fold, n=3, P<0.01) as compared to nonrecombinant (Adv-control vehicle) injected hearts (Fig. 4
A, right panel). PKR1 expression remained elevated in the Adv-PKR1 injected hearts after 7 days (Fig. 4A
, left panel). Furthermore, we observed that treatment with PKR1 significantly decreased MI-related mortality of the mice (Fig. 4B
). The survival rate 14 days after PKR1 gene transfer (n=19, Low-rank test, P<0.05) was 16 ± 4% higher than in the vehicle-treated (Adv-control vehicle and saline injected) MI groups (n=28).
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We next analyzed morphological and functional changes in all surviving mice at the indicated times (1 wk and/or 5 wk) after MI. Representative staining of heart sections by Mallory tetrachrome can be seen in Fig. 4c
for Adv-vehicle-treated (Adv-c)- and PKR1-treated infarcted hearts, 1 or 5 wk after MI, demonstrating significantly less scar tissue (blue) after PKR1 treatment. The scar area was reduced in the Adv-PKR1 treated group (15±2% of left ventricle, n=4) as compared with saline (39±2%, n=3) or Adv-control vector (42±4%, n=4) treated groups (P<0.01).
PKR1 gene therapy reduces ultrastructural changes and improves cardiac function after myocardial infarction
Electron microscopic analysis of nonrecombinant Adv-control vehicle or saline-treated hearts (MI-vehicle) showed mitochondria with destroyed cristae, glycogen granule accumulation, and myocardial rupture with lysed fibers, increased collagen deposition and abnormal mitochondrial proliferation at the myocardial infarct area. Cardiomyocyte death was also observed, accompanied by diffuse interstitial fibrosis and infiltrated inflammatory cells (Fig. 5
A, left panels). In contrast, the PKR1 treated ischemic hearts displayed less collagen deposits, preserved myocardial fibers and mitochondrial structures, reduced apoptotic cells, and increased capillary vessels, accompanied with increased the number of intact mitochondria (Fig. 5A
, right panel). Note that the number of infiltrated cells in the scar area of PKR1 treated and Adv-control vector were analyzed after coronary ligation by counting macrophages and monocytes per infarcted field on electron micrographs at x20,000 magnification (10 microvessels area analyzed for each group n=4). We found that at this concentration, adenoviruses do not cause significant inflammatory responses in mouse heart.
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Hemodynamic measurements of all groups were performed by Millar catheterization 5 wk after myocardial infarction. Parameters were studied at baseline, including peak left ventricular (LV) pressure, LV end-diastolic pressure (LVEDP), heart rate, and LV +dP/dtmax and LV –dP/dtmin. Overall, MI mice had impaired LV function compared with sham controls (n=5), as shown in Fig. 5B
. LV systolic pressure and +dP/dtmax and –dP/dtmin was significantly increased, LV end-diastolic pressure was significantly reduced in PKR1-treated mice as compared to vehicle-treated MI hearts (Fig. 5B, P
<0.05). There were no significant differences in heart rate and left ventricular end-diastolic pressure between nontreated or vehicle-treated myocardial infarcted hearts. Together these findings show that PKR1 gene transfer during the setting of acute and chronic myocardial infarction resulted in an improvement of myocardial function and enhancement of LV performance.
Mechanism of cardioprotective effect of PKR1 gene transfer
Capillary density in the infarcted area was quantified by counting the numbers of endothelial cell-lined vessels after immunostaining with the endothelial specific marker PECAM-1, 1 wk (Fig. 6
A, left) and 5 wk after myocardial infarction. Quantitative analyses on heart sections revealed a significant increase in capillary density by PECAM-1 staining in Adv-PKR1 treated hearts as compared to vehicle treated hearts, both at 1 and 5 wk after coronary ligation (Fig. 6A
, right; n=4, P<0.05). Next, we investigated the possible involvement of VEGF in PKR1-mediated angiogenesis. VEGF-immunostaining (Fig. 6B
) or semiquantitative RT-PCR analyses (Fig. 6C
) revealed that neither VEGF protein levels nor VEGF A transcripts 164 and 188 were altered in PKR1-treated hearts as compared to vehicle-treated hearts (n=3, P>0.05).
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Since PKR1-treated mice hearts exhibit smaller scar areas and improved contractile function following MI, we assessed whether PKR1 may protect myocardium against apoptosis. Specifically, we carried out TUNEL and active caspase-3 labeling of LV sections 3 and 7 days after MI in mice that were treated with either Adv-PKR1 or Adv-control vector. No TUNEL or active caspase-3-positive cells were seen in sham (no MI, no PKR1) animals. We did not detect cardiomyocyte death 14 days after coronary ligation. Representative TUNEL (green) and active caspase-3 (red) stained LV sections are shown in Fig. 7
A, demonstrating fewer apoptotic cells in the PKR1-treated infarcted mouse heart 3 days after coronary ligation. Quantitative analyses by TUNEL assay at the border zone of the scar area of the heart sections 3 and 7 days after MI revealed a significant decrease in cell death in PKR1 treated hearts as compared to Adv-control vector treated hearts (n=3, P<0.001, Fig. 7A
, histogram). This is consistent with less apoptosis observed in H9c2 cardioblasts or primary cultured cardiomyocytes in vitro following hypoxic insult (Fig. 3A, B
).
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Since we found that the PI3K/Akt pathway was activated by PKR1 activation in H9c2 cells and involved in cardiomyocyte protection against hypoxic insult, we examined whether transient PKR1 gene transfer could activate Akt after coronary ligation. As shown in Fig. 7B
, both phosphorylated-Akt were found to be significantly increased in vivo in the PKR1-treated hearts compared to Adv-control vector injected hearts 1 wk after the coronary ligation. Akt was activated a modest 30% over Adv-control values (Fig. 7B
). Note that total Akt levels remained unaltered in all heart samples.
PKR1 and its ligands in control nonfailing and end-stage failing human explanted heart samples
Next we investigated the level of PKR1, and its ligands in explanted human end-stage failing and control nonfailing left ventricular heart samples. Confocal analysis of immunostained cryosections with the corresponding antibodies showed that prokineticin-2 and PKR1 protein levels were significantly decreased whereas prokineticin-1 levels were slightly increased in end-stage failing hearts compared to control nonfailing heart samples (Fig. 8
A; C, left panel) (P<0.005). Semiquantitative RT-PCR analysis on human left ventricular explanted normal heart samples (n=5) revealed that the predominantly expressed ligand was the short isoform of prokineticin-2. Interestingly, RT-PCR analysis on left ventricular end-stage human heart failure samples (n=13) revealed significantly decreased levels of PKR1 and prokineticin-2S/L ratio (P<0.005) (Fig. 8B
; C, right panel). However, prokineticin-1 transcription levels remained unchanged in end-stage failing hearts compared to control nonfailing heart samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for the prokineticins (1
, 4
, 14)
, induces tube-like formation in endothelial cells and protects cardiomyocytes against hypoxic insults. Our in vivo studies reveal that transient PKR1 gene transfer can protect the infarcted heart by inhibiting apoptosis and inducing angiogenesis.
Prokineticins-1 and -2 are powerful stimulators of angiogenesis (2
, 8)
. However which prokineticin receptor is involved in prokineticin-mediated cardiac angiogenesis has not been studied yet. The following observations suggest that PKR1 mediates angiogenic effects of prokineticin-2 in capillary endothelial cells. Neutralizing antibody against PKR1 completely abolished prokineticin-2 induced tube-like structures. Knocking out PKR1 by siRNA-PKR1 transfection in H5V cells completely prevented prokineticin-2 -induced angiogenesis. Overexpression of PKR1 in these cells induced vessel-like formation in the absence of exogenous ligands.
A number of GPCRs have been shown to play a role in angiogenesis (32)
. Activation of thrombin (37)
or angiotensin II receptors (38)
regulates microcapillary endothelial cell activation to induce angiogenesis indirectly by regulating VEGF transcription. Among various kinds of angiogenic growth factors, VEGF is thought to play an important role in the regulation of vessel formation in the heart that may be needed to maintain cardiac function (39)
. However, here we show that involment of VEGF seems to be unlikely in PKR1–mediated in vitro or in vivo angiogenesis, because VEGF protein and transcripts remained unaltered in the endothelial cells treated with prokineticin-2 for 24 h or in PKR1 transient transfected ischemic mouse hearts. Neutralizing antibody against VEGF did not alter prokineticin-mediated angiogenesis. How prokineticin/PKR1 signaling induces angiogenesis is currently under investigation.
In this study, we found a novel cardiomyocyte survival pathway that was induced by PKR1 activation. Prokineticin-2 treatment or overexpressing PKR1 in H9c2 cells as well as in cardiomyocytes prevents the apoptotic death induced by oxidative stress. It has been shown that extensive expression of Gq protein in cardiomyocytes induces hypertrophy or apoptosis (40)
. In contrast, increased Gq protein activity in cardiomyocytes has recently been shown to induce antiapoptotic signals by eliciting Endothelial Growth Factor Receptor (EGFR) phosphorylation and subsequent Akt activation (41)
. This is in agreement with other recent data, indicating Gq-coupled receptor activation in isolated cardiomyocytes is involved in promoting survival pathway by activating Akt signaling (20)
. Here we show that prokineticin-2 treatment of H9c2 cells activates Akt and prokineticin-2 was not able to inhibit apoptosis in the cardiomyocytes infected with dominant negative Akt. Moreover, PKR1 treated ischemic hearts exhibited increased phosphorylated Akt levels compared to vehicle-treated infarcted hearts. Given the known function of Akt (42)
, our in vitro and in vivo data are consistent with Akt having a central role in the effects of PKR1 signaling on protection of isolated cardiomyocytes and the heart in situ. Whether PKR1 receptor signaling transactivates EGFR in cardiomyocytes is currently under the investigation.
The survival and angiogenesis promoting effects of overexpression of PKR1 on cardiac cells in vitro, prompted us to study whether PKR1 gene therapy may rescue the heart against MI in the mouse model. PKR1 gene transfer during the setting of acute and chronic MI resulted in reduction of MI size improvement of LV performance,and consequently a reduction of mortality. Our morphological, ultrastructural and functional analyses revealed a reduced scar area and collagen deposition, associated with preserved myofibrils and mitochondrial structures in the PKR1 treated heart after myocardial infarction. Our data thus suggest that transient PKR1 gene transfer has beneficial effects on recovery of myocardial infarction.
Next, we investigated the mechanism of the cardioprotective effect of PKR1 signaling in infarcted heart. We found that transient gene therapy with PKR1 enhanced neovascularization after MI and prevented apoptosis. Together with the in vitro studies showing that PKR1 signaling produces a direct antiapoptotic effect in cultured cardiomyocytes and H9c2 cardioblasts, our in vivo results suggest that prevention of apoptosis is a possible mechanism of PKR1-mediated cardioprotective effects. A second mechanism to preserve myocardial function is to promote collateral vessel formation, in order to overcome insufficient tissue oxygenation. Therefore, PKR1-mediated angiogenesis may be important to maintain cardiomyocytes and successful cardioprotection, following the early phase of its antiapoptotic effect. Importantly, transient PKR1 transfection induces capillary network growth without increasing VEGF levels, consistent with the observation in cultured endothelium cells following prokineticin-2 treatment. A third mechanism could be that PKR1 signaling might induce infiltration of inflammatory cells, and thus indirectly increase capillary density. PKR1 signaling is involved in inflammation, monocyte activation, and macrophage differentiation (12)
, and PKR1 knockout mice exhibit lack of inflammation in response to prokineticin-2 stimulation (13
, 43)
. We therefore, investigated whether PKR1 transfection alters the infiltration of inflammatory cells. We compared the number of infiltrated cells in the scar area in the Adv-PKR1, Adv-control, and saline injected hearts, after myocardial infarction. We did not observe any significant differences among-these groups, arguing against an indirect effect of PKR1 through inflammatory response. Moreover, we observed a 30% increase of capillaries in the Adv-PKR1 vs. Adv-control injected ischemic hearts. These findings disprove adenoviral vectors triggering immune responses in the heart that can lead to angiogenesis.
Because the expression of angiogenic factors may vary in the decompensated state of heart failing, we investigate the expression levels of PKR1 and its ligands in human end-stage failing heart samples (explanted). We found that the reduced prokineticin-2 mRNA or protein levels are associated with reduced levels of its receptor PKR1 as compared to control nonfailing human heart tissues. It should be noted that desensitization to GPCR signaling as well as a decrease in levels of specific signaling components are accepted findings in heart failure in many model systems (44)
, and do not necessarily imply causality. One could assume that prokineticin pathology associated with reduced capillary density in patients with heart failure might lead to impaired contractility as observed for decreased VEGF expression (45)
. The difference of prokineticin-1 levels between failing and nonfailing human hearts was subtle; however, over the long term, the changes of prokineticin-2 and its receptor PKR1 may be relevant.
In summary, these data provide in vivo and in vitro evidence that PKR1 initiates angiogenesis, and abolishes apoptosis to prevent heart failure. Since GPCRs are the main drug targets for the treatment of cardiac and other cardiovascular disorders (46)
, PKR1 could be a novel therapeutic target for treatment of ischemic heart diseases.
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ACKNOWLEDGMENTS |
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Received for publication January 18, 2007. Accepted for publication March 8, 2007.
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