Vascular Endothelial Growth Factor Gene Transfection in Endocardial Endothelial Cells Promote Capillary Growth <em>in Vitro</em>

Research Article

J Cardiovasc Disord. 2014;1(1): 4.

Vascular Endothelial Growth Factor Gene Transfection in Endocardial Endothelial Cells Promote Capillary Growth in Vitro

Kuruvilla L1, Santhosh Kumar TR2, Panicker SR2, Kartha CC1*

1Division of Cellular & Molecular Cardiology, sree chitra tirunal institute for Medical Sciences and Technology, Trivandrum, India

2Cancer Biology, Rajiv Gandhi Center for Biotechnology, Trivandrum, India

*Corresponding author: Kartha CC, Disease Biology & Molecular Medicine, Rajiv Gandhi Center for Biotechnology, Trivandrum - 695014, Kerala, India

Received: August 16, 2014; Accepted: September 11, 2014; Published: September 15, 2014


Background: Different types of cells have been employed both in in vitro and in vivo studies for revascularization of ischemic myocardium. Whether Endocardial Endothelial Cells (EEC) lying adjacent to the myocardium can be used in gene therapy for successful revascularization of ischemic myocardium remains unexplored. Here we present evidence for the first time that EECs can be made to take up Vascular Endothelial Growth Factor (VEGF) gene with consequent VEGF protein synthesis to promote angiogenesis in vitro.

Methods and Results: Immortalized EECs (iEEC) with stable h TERT tranfection were transfected with VEGF gene. The iEECs, Rat Aortic Endothelial Cells (RAECs) and VEGF transfected cells were seeded in various combinations onto matrigel and the number of tubes counted using tube formation assay. Cocultures of RAECs and VEGF transfected EECs gave the maximum number of tubes (24.6 ± 3.7) and also the maximum tube length (334 ± 45 μm) within the shortest period of time. iEECs alone did not show any tube formation whereas iEECs with VEGF gene formed a few incomplete tubes.

Conclusion: These results confirm that VEGF gene transfection in EECs can promote capillary growth in vitro.

Keywords: Endocardium; Endothelium; VEGF; Angiogenesis


Significant number of patients with coronary artery disease failed to have complete revascularization even after Percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Grafting (CABG), resulting in refractory angina [1]. A combination of surgical laser Transmyocardial Revascularization (TMR) and therapeutic angiogenesis offers an attractive possibility for successful revascularization of ischemic myocardium. Following TMR, new capillary formation has been observed in the area of the channels which is a result of up-regulation of VEGF [2,3,4]. Different strategies for delivering these angiogenic factors has been attempted, such as direct intra coronary and intra venous delivery, percutaneous endocardial delivery catheters using fluoroscopic guidance or electromagnetic three-dimensional navigation system for therapeutic myocardial angiogenesis. EECs that line the cardiac cavities auto regulate cardiac performance by direct interaction with superfusing blood [5] and via various cardioactive mediators [6].We hypothesized that if the VEGF gene is directly delivered to the endocardial endothelial cells and is able to secrete VEGF protein in required levels, this may augment collateral blood vessel development constituting endogenous bypass conduits around the damaged areas in the myocardium. We tested this hypothesis in an in vitro system with EECs and aortic endothelial cells.

Materials and Methods

All fine chemicals including Medium199, FBS and all supplements were procured from Sigma-Aldrich, St. Louis, USA. MEM with D-valine was obtained from Pan Biotech, Germany. Matrigel basement membrane matrix (7.8 mg/ml) was purchased from BD biosciences, San Jose, CA, USA. Rabbit polyclonal anti-mouse VEGF and monoclonal anti-mouse Hsc70 antibodies were from Santa Cruz Biotechnology, Inc. Santa Cruz, CA, and USA. The pWZL Blast VEGF plasmid was procured from Addgene, plasmid 10909.

Cells and cell culture

Immortalized Endocardial Endothelial Cells (iEECs) and Rat Aortic Endothelial Cells (RAECs) were used for the experiments. The primary EECs and the iEECs with stable h TERT gene were obtained by the procedure described earlier [7]. RAECs were isolated from male Sprague-Dawley rats 2-3 months old according to the methods of McGuire PG and Orkin RW, 1987 [8]. Briefly, rat aortae were removed aseptically, cut into rings and placed in culture dishes containing Minimum Essential Medium with D-Valine and supplemented with 20% fetal bovine serum,75 μg/ml endothelial cell growth supplements. After 4-7 days, the aortic rings were removed; the cells harvested and used for experiments between passages 3 and 8. RAECs were characterized using immunocytochemical staining against factor VIII related antigen, by their cobblestone morphology and ability to grow in culture medium containing D-Valine.

All experiments had the approval of the Institutional Animal Ethics Committee.

Transfection of iEECs with the VEGF gene

A commercially available mammalian expression vector, pWZL Blast VEGF plasmid containing the mouse VEGF-A (VEGF 120) gene insert and blasticidin resistance gene was used. Briefly, iEECs at about ~80% confluence were transiently transfected with 10μg of plasmid DNA using Lipofectamine 2000 reagent as per manufacturer's instructions, using serum free OptiMEM medium. Six hours after transfection, the transfection complex was replaced with complete medium. Forty-eight hours after transfection, the cells were incubated in complete medium containing 0.5 μg/ml of blasticidin for the selection of transfected population. The selected clones of cells were trypsinised and seeded on to matrigel for tube formation assay. For obtaining the Condition Medium (CM), VEGF transfected cells were incubated for 48 hours in complete medium and the cell culture supernatant taken after removing cellular debris by centrifugation.

Western blot analysis

The cell lysates from VEGF transfected cells were subjected to 15% PAGE and proteins transferred to a nitrocellulose membrane. Lysates from primary EECs and iEECs served as controls in the analysis. Hsc 70 protein expression was taken as internal control. VEGF expression was detected using rabbit polyclonal anti-mouse VEGF antibody at a dilution of 1:200. The bands were developed using the chromogen, diaminobenzidine.

Experimental design for matrigel tube formation assay

The spontaneous formation of capillary-like structures by endothelial cells on Matrigel basement membrane matrix was used to assess the angiogenic potential. Matrigel was coated on 35 mm cell culture dishes and incubated at 37°C for 1 h to promote gelling. Five different groups were seeded onto matrigel for the in vitro angiogenesis assay to calculate the number of tubes and length of the tube, namelyone set each of iEECs, VEGF transfected iEECs and RAECs alone and another set of RAECs with VEGF-iEECs as co-culture and the last group of RAEC supplemented with CM. The cells were seeded in 1 ml suspensions containing 5.25 x 104 cells. Cellular organization into tubular structures was monitored every three hours, starting from 2 hours using an inverted phase-contrast photomicroscope and photographs taken at 400x magnifications by scoring a three branch point event as one tube [9]. At 11 hours the photomicrographs were taken at 100X so as to capture a larger number of cells per field. Images of the tubes were captured using a digital camera Evolution TM MP and the length of the tubes measured using Image-Pro® Plus 5.1 software after 24 hours. Each assay was performed in duplicate and the results expressed as mean of 3 independent experiments.

Statistical analysis

All results were expressed as the mean ± SD. Significance of differences between groups was determined with ANOVA followed by Tukey test. p< 0.05 was considered statistically significant.


VEGF gene is successfully transfected in iEECs

Western blot analysis was done to confirm whether iEECs transfected with the pWZL Blast VEGF plasmid had taken up the plasmid and were expressing VEGF protein. A band corresponding to the VEGF protein was observed at the 42 KDa position in VEGF transfected iEEC but not in primary EECs and iEECs which were used as controls, confirming successful VEGF transfection into iEECs with corresponding VEGF protein synthesis (Figure 1).