VEGF promotes cardiac stem cells differentiation into vascular endothelial cells via the PI3K/Akt signaling pathway

Abstract

Recent research suggested that cardiac stem cells (CSCs) may have the clinical application for cardiac repair. However, their characteristics and the regulatory mechanisms of their growth and differentiation have not been fully investigated. Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. But the homing role of VEGF for CSCs is unclear. In this report, CSCs were isolated, purified, and expanded in vitro from rat heart. VEGF, SU5416 (VEGF receptor blocker), and Wortmannin (PI3K/Akt signaling pathway inhibitor) were used for differentiation into vascular endothelial cells (VECs). Real-time qPCR was selected to confirm the role of PI3K/Akt signaling pathway in VECs differentiation from rat CSCs. The result of real-time qPCR demonstrated that PI3K/Akt signaling pathway plays an important role in rat CSCs differentiated into VECs. So, our research provides a theoretical basis and experimental evidence for therapeutic application of rat CSCs to treat cardiac repair.

Keywords::

• cardiac stem cells
• PI3K/Akt signaling pathway
• rat
• vascular endothelial cells

Introduction

In recent years, cardiac disease has emerged as the leading cause of death worldwide, particularly in developed countries. The exact prevalence of heart failure is cardinal symptom and largely unknown but is growing because new treatments have increased the life expectancy of these patients. Whereas heart failure is often ischemic in origin, the origin of heart failure is more varied, including a spectrum of cardiomyopathies and arrhythmias. A novel emerging treatment for heart failure is cellular cardiomyoplasty, whereby stem cells are delivered to the dysfunctional myocardium.(Mishra et al. 2011, Yi et al. 2010) Due to its unique characteristics, stem cell transplantation is considered a potentially effective treatment for heart failure. Both embryonic stem cells and adult stem cells (ASCs) are being studied for clinical applications, and some types of autologous cells (skeletal myoblasts, bone marrow stem cells, mesenchymal stem cells, and endothelial progenitor cells) are being tested in clinical trials (Otsuru et al. 2012, Perez-Simon et al. 2011, Trounson et al. 2011, Bai et al. 2013).

Cardiac stem cells (CSCs) isolated from the myocardium have self-renewal capabilities in vitro and the ability to differentiate into all three major cardiac cell types: cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells (VECs) in vivo. VECs take part not only in vascularization during embryonic development, but also in postnatal vascularization and reparative processes post-trauma. Therefore, VECs hold extensive prospects for vascular tissue engineering and possible clinical application in heart failure. The expression of vascular endothelial growth factor (VEGF) increases in response to myocardial ischemia and promotes vascular repair. VEGF is widely recognized to be a potential therapeutic target for regulating angiogenesis (Freedman and Isner 2001, Zachary and Morgan 2011). However, whether VEGF is involved in CSCs differentiation remains unknown.

The Phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or Akt) signal transduction cascade has been investigated extensively for its roles in angiogenesis. Initial studies implicated both PI3K and Akt in prevention of apoptosis. However, more recent evidence has also associated this pathway with regulation of angiogenesis and cell cycle. Here, we discuss PI3K/Akt-mediated signal transduction in differentiation of VECs from rat CSCs.

Material and methods

Isolation, culture, and purification of rat CSCs

Rat heart tissues were obtained from 20 to 30 neonatal rats. All animal experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at Hebei Medical University. All operations were conducted aseptically. The heart tissues were washed 3 times with phosphate buffer saline (PBS) containing 100 IU/ml penicillin and 100 μg/ml streptomycin to remove pericardium and capillaries. The heart tissue pieces were digested with 200 U/ml collagenase Type II and collagenase Type I (both from Invitrogen) in Hank's Balanced Salt Solution for 60 min at 37°C, followed by a 10-min treatment with 0.125% trypsin. Enzymatic activity was then neutralized with the same volume of fetal bovine serum (GIBCO, USA). The cell suspension containing the majority of c-kit⁺ CSCs was obtained after being purified through flow cytometry, the antibodies (c-kit and Flk 1) were used in the cell purification.

Surface marker expression analysis of rat CSCs

Cultures of rat CSCs were seeded on glass coverslips coated with poly-L-lysine. When cultures reached 60% confluence they were washed three times with PBS, then fixed with 4% paraformaldehyde for 15 min at room temperature, and again washed three times (5 min per wash) with PBS. Cells were permeabilized by incubating with 0.125% Triton X-100 for 10 min. Rat CSCs were then washed three times with PBS and incubated with 4% bovine serum albumin at room temperature for 30 min. The cells were then incubated with primary antibodies in a humidified chamber at 4°C overnight. After three washes with PBS for 5 min each, the cells were incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibodies at room temperature for 1 h, and were rinsed three times with PBS for 5 min each. Finally, nuclei were labeled by incubation with 4,6 diamidino-2-phenylindole (Sigma, USA). The cells were examined by a phase contrast fluorescence microscope (Olympus, Japan).

VECs differentiation of rat CSCs

For differentiation into VECs, rat CSCs were incubated in VECs medium containing 20 ng/ml VEGF₁₆₅ (Peprotech, USA), as Group A; Group B was set up by adding 5 μM SU5416 (VEGF receptor blocker) from Group A; Group C was set up by adding 100 nM Wortmannin (PI3K/Akt signaling pathway inhibitor) from Group A. While control cells were continued to be cultured in complete medium without any inducer. The medium was refreshed every 3 days; 1 week later, it was detected for the expression of the CD31 and CD144, the endothelial specific marker via immunofluorescence.

PI3K/Akt signaling pathway analysis in differentiation of VECs

After 8 days, RNA was extracted with Trizol reagent (Invitrogen, USA) to detect the expression of PI3K/Akt signaling pathway members and special genes of VECs using real-time qPCR in induced VECs. The members of PI3K/Akt signaling pathway include VEGFR2, PI3K, Akt, and endothelial nitric oxide synthase (eNOS). Special genes of VECs include CD31 and CD144. Information of gene-specific primer pairs is listed in Table I.

Table I. Primer sequences used in real-time qPCR.

Gene	Primer Sequence	Tm (°C)	Cycle	Fragment size (bp)
c-kit	F 5’ CCTGAAGGCAACACCTGCTA 3’
	R 5’ 3’ GACGTGCATCCCTGTACCTT	55	30	95
GATA 4	F 5’ 3’ TGAATGGTATCAACCGGCCC
	R 5’ 3’ TAGTCTGGCAGTTGGCACAG	60	30	86
CD31	F 5’ 3’ GGTAATAGCCCCGGTGGATG
	R 5’ 3’ TTCTTCGTGGAAGGGTCTGC	55	30	160
VEGFR-2	F 5’ 3’ CCCGCACGAATGATATCCCA
	R 5’ 3’ TCCTGCAGTGCATAACCTGG	58	30	136
eNOS	F 5’ 3’ GCTCTTTCGGAAGGCGTTTG
	R 5’ 3’ AAGGCCTCATGCTCTAGGGA	60	30	78
CD144	F 5’ 3’ AGCAAGAGTGCGCTTGAGAT
	R 5’ 3’ GTCGTAGCTTGTGGTGTCCA	60	30	84
PI3K	F 5’ 3’ ACTGCACATCTACGGCTACG
	R 5’ 3’ GTCGGAGGAATTGGTGCTCA	60	30	72
Akt	F 5’ 3’ ACCATGAACGACGTAGCCATT
	R 5’ 3’ TTGTTGAGTGGGGACTCGC	55	30	160
GAPDH	F 5’ 3’ CTCATGGCCTACATGGCCTC
	R 5’ 3’ CTCATGGCCTACATGGCCTC	60	30	70

Statistical analysis

Statistical analyses of the data were performed with a one-way ANOVA followed by the Tukey–Kramer honestly significant difference test for the three sets of results. A P-value of less than 0.05 was considered significant. Statistical analyses were done with a JMP^® Statistical Discovery Software (SAS Institute, Cary, NC).

Results

Morphological features and Phenotypic Characterization of rat CSCs

Primary cells isolated from heart tissue adhered to plates at 48 h and began to proliferate 72 h later. The cells proliferated quickly, displaying obviously karyokinesis and different morphologies including fusiform, triangular and irregular shape after purification (Figure 1A). Rat CSCs at various passages were positive for the c-kit, GATA 4, Nkx2.5, and Flk 1 cell surface markers but cTnT and CD31 were weakly expressed (Figure 2). We did not observe a significant difference in the prevalence of c-kit positive cells at different passages (p > 0.05).

Figure 1. Morphology of rat cardiac stem cell and vascular endothelial cells. (A) Rat primary c-kit+ cardiac stem cells after purification through FCM. (B) Vascular endothelial differentiation of rat c-kit+ cardiac stem cells, cells converted from fusiform and triangular to roundness in shapes after day 6.

Figure 2. Detection of rat c-kit+ cardiac stem cells markers by immunofluorescence staining. The results show that rat c-kit+ cardiac stem cells at various passages were positive for the c-kit, GATA 4, Nkx2.5 and Flk 1 cell surface markers but cTnT and CD31 were weakly expressed.

VECs differentiation of rat CSCs

After the induction with osteoblast VECs, rat CSCs had apparent changes in appearance. From the 4th day after the induction, rat CSCs in Group A changed from fusiform to t roundness or irregularity, until the cells were very closely located and showed cobblestone shape (Figure 1B). But there were no obvious morphological changes in Group B, Group C, and control. Immunofluorescence results showed that CD31 and CD144, markers of VECs, were expressed in the differentiated cells (Figure 3).

Figure 3. Identification surface markers of vascular endothelial cells, the expression of CD31 (FITC, green) and CD144 (Cy5, red) were detected using immunofluorescence.

PI3K/Akt signaling pathway analysis in VECs differentiation

Real-time qPCR was used to detect the gene expression of members of PI3K/Akt signaling pathway, including VEGFR2, PI3K, Akt, and eNOS. The result demonstrated that after incubation with VEGF, the specific genes, including VEGFR2, PI3K, Akt, eNOS, CD31, and CD144, markers of PI3K/Akt signaling pathway and VECs, were expressed and gene expression level showed a time-lapse increase in Group A. However, the markers of rat CSCs, including c-Kit, GATA 4, and Nkx2.5, the gene expression level showed a time-lapse decrease (Figure 4). There are no significant differences in Group B, Group C, and control group.

Figure 4. Real time qPCR analyses of PI3K/Akt cell signaling pathway members and vascular endothelial cell markers in endothelial cell differentiation. The result indicated that after endothelial cell induction the specific genes, including PI3K/Akt cell signaling pathway members (PI3K and Akt), and endothelial cell markers (CD31, VEGFR-2 and CD144) were detected, and gene expression level showed a time-lapse increase, cardiac stem cells markers (c-kit and GATA 4) showed a time-lapse gradually decrease.

Discussion

VEGF is an endothelial cell-specific mitogen which induces angiogenesis and vascular permeability in vivo. Previous evidence suggested that VEGF is an important angiogenesis factor during heart form and embryonic development (Millauer et al. 1993, Ferrara 2001). The discovery of the existence of multiple VEGF isoforms raised several questions regarding their relative significance (Plate and Warnke 1997). Instead, the VEGF₁₆₅ is potentially the most important isoform and was found, similar to native VEGF, to be a soluble heparin-binding endothelial cell mitogen (Leung et al. 1989). Subsequent studies confirmed that native VEGF from numerous sources corresponds to VEGF₁₆₅ (Houck et al. 1992). Therefore, VEGF₁₆₅ was selected to induce rat CSCs and activate PI3K/Akt signaling pathway for differentiation into VECs in our research. Although originally identified as a factor that induces vascular permeability, VEGF exhibits multiple biological activities in VECs, including the enhancement of VECs survival. VEGF effects on cell survival have been shown to be mediated by VEGFR2/PI3K/Akt signaling pathway (Gerber et al. 1998, Fujio and Walsh 1999). And VEGF enhances endothelial cell migration and capillary-like structure formation in vitro and these activities of VEGF are PI3K-Akt-dependent.

PI3K pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis (Ma et al. 2009). Previous studies demonstrated that VEGF-induced increase in NO release from endothelial cells is attenuated by PI3K inhibitors (Papapetropoulos et al. 1997). Subsequently, it was demonstrated that VEGF stimulates Akt-mediated eNOS phosphorylation at Ser1177, leading to an increase in eNOS activity (Fulton et al. 1999, Dimmeler et al. 1999). It was also reported that production of NO in response to fluid shear stress in cultured endothelial cells was controlled by Akt-dependent phosphorylation of eNOS, (Dimmeler et al. 1999) and another study demonstrated that shear stress induces eNOS phosphorylation predominantly through a protein kinase A-dependent, Akt-independent mechanism (Boo 2006). In addition to its role in angiogenesis, Akt has also been involved as a general regulator of organ growth. Studies in Drosophila have demonstrated that components of the insulin/IGF signaling pathway including Akt are involved in the regulation of organ growth and body size in response to nutritional input (Verdu et al. 1999, Weinkove and Leevers 2000). In brief, PI3K/Akt signaling pathway also regulates cardiovascular homeostasis and vessel integrity at least in part by controlling NO synthesis. And this pathway is activated by a variety of stimuli in endothelial cells and regulates multiple critical steps in angiogenesis, including endothelial cell survival, migration, and capillary structure formation. Therefore, real-time qPCR was used to confirm the function of PIAK/Akt signaling pathway in VECs differentiation from CSCs.

In this study, CSCs were isolated and purified from rat heart tissue, and their morphology and surface marker expression were investigated. We also demonstrated that VGEF can regulate rat CSCs to differentiate into VECs via stimulating PI3K/Akt signaling pathway. The present study has important bearing on the potential application of CSCs as an ASC source for tissue engineering.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.