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The Effect of Von Hippel-Lindau Gene Transfer on Human Vascular Smooth Muscle Cell Proliferation and Apoptosis

Posted on: Tuesday, 22 February 2005, 03:00 CST

Von Hippel-Lindau (VHL) gene is a tumor suppressor gene that plays a genome "gatekeeper" role and controls several downstream effector genes. We have previously demonstrated that both in vivo and in vitro adenovirus-mediated gene transfer of tumor suppressor genes into the vascular endothelium is effective in decreasing neointimal hyperplasia and abnormal cell proliferation. The degree of apoptosis induced by these genes is critical in mediating the in vivo responses to gene therapy and the maintenance of the crucial balance between cell death and viability. Since VHL gene is known to regulate vascular endothelial growth factor (VEGF) as well as other angiogenic factors, it may exhibit a greater potential in the attenuation of vascular disorders in comparison to other tumor suppressor genes. This study focused on whether adenovirus-mediated VHL gene transfer into human vascular smooth muscle cells has an effect on cell proliferation and induction of apoptosis. Human aortic smooth muscle cells (HASMC) were grown as monolayers and transfected with varying titers of adenovirus containing the VHL cDNA (AdVHL). The negative controls were adenovirus containing green fluorescent protein (AdGFP), vector alone (AdNull), and virus-free infection medium. Adenovirus encoding wild-type p53 (Adp53) was used as positive control. Cell viability and proliferation were determined by using trypan blue exclusion and MTS-based CellTiter 96 AQ Proliferation Assay. Apoptosis was evaluated by TUNEL assay, morphologic changes, and nucleosomal DNA degradation. Following AdVHL transfection HASMCs demonstrated a dose-dependent decrease in viability as compared to negative controls (p<0.05). AdVHL- transfected cells exhibited a decrease in their proliferative ability by 40.21 1.66 (SEM)%. In cultures transfected with the positive control, Adp53, the cell viability as well as proliferation was highly reduced (p< 0.001). AdGFP and AdNuII did not increase HASMC apoptosis above baseline levels. The cells exposed to adenoviruses expressing tumor suppressor genes underwent apoptosis, with Adp53 demonstrating a very high magnitude of cell death (75.27 3.52 [SEM]%). AdVHL expression caused 45.36 2.55 (5EM)% apoptosis in HASMC. Recombinant adenovirus-mediated VHL expression is efficacious in limiting vascular smooth muscle cell growth in vitro. Overexpression of VHL suppresses HASMC proliferation and regulates apoptosis. Further experiments are indicated to examine whether VHL may be a useful adjunct in limiting myointimal hyperplasia.

Introduction

Von Hippel-Lindau (VHL) disease is an inherited tumor susceptibility syndrome featuring a variety of benign and malignant tumors of highly vascular nature. The gene responsible for this syndrome has been found to be a tumor suppressor gene that plays a critical genome "gatekeeper" role. It controls several downstream effector genes.1-3 Deletions, single or multiple mutations in the VHL gene, lead to the VHL syndrome,4 characterized by widespread occurrence of vascular tumors. Alteration in the VHL gene has been demonstrated both in vitro and in vivo, to upregulate vascular endothelial growth factor (VEGF), which is thought to play a role in the development of these tumors.5-7 Indeed, Seth and coworkers8 have demonstrated that VHL arrests cell cycle by regulation of p27^sup Kip1^ in cancer cells at both the mRNA and protein levels. Because VHL is a tumor suppressor gene, and since gene therapy using tumor suppressor genes has been demonstrated to be highly effective in limiting abnormal cell proliferation including intimai hyperplasia,9- 13 we explored the effect of adenovirus-mediated VHL expression in vascular smooth muscle cells. Since VHL is known to regulate VEGF and is involved with other vascular, as well as angiogenic, factors, it may exhibit greater potential in the attenuation of vascular disorders.

This study investigates the hypothesis that gene transfer of VHL gene into vascular smooth muscle cells could cause alteration in viability, proliferative ability, and programmed cell death, in an in vitro model. In order to examine the effect of VHL gene transfer, an adenoviral vector expressing human VHL gene was utilized for transfection of human aortic smooth muscle cells.

Methods

Recombinant Adenoviruses

A recombinant adenovirus containing VHL cDNA (AdVHL) was constructed by homologous recombination using methods described previously.8 In brief, human VHL cDNA was cloned into an adenovirus shuttle vector. Shuttle vector was cotransfected with the adenoviral genome. Adenoviral plaques were screened for the presence of VHL sequences by polymerase chain reaction (PCR) using a set of primers, which were 5'CAGGTCATCTTCTGCAATCGCAGTC-3' and 3'GGATCAGTTCGGACTCTTAATGTCCT-5'. The control adenoviruses used in this study were the following: (1) AdNull, identical to AdVHL except that it was devoid of any VHL cDNA; (2) AdGFP, adenovirus with cDNA for green fluorescent protein; and (3) Adp53, recombinant adenovirus containing human wild type p53. AdGFP was used instead of Adβgal for reporting transfection efficiencies and durability, as cells bearing this recombinant virus could be directly visualized under the microscope by virtue of the green fluorescent protein incorporated into them. Aliquots of these recombinant viruses kindly provided by Dr. Seth (Human Gene Therapy Institute, Des Moines, Iowa) were utilized to propagate them. The viruses were propagated in 293 (ATCC CRL1573) adenovirus-transformed human embryonic kidney cells, purified by 2 cesium chloride density centrifugations, and stored at -70C until use.

Cell Culture

Primary cultures of human aortic vascular smooth muscle cells (HASMC), obtained from Clonetics (Walkersville, MD) were grown in SMGM2 media, provided by the cell vendor (Clonetics). Cells from the 3rd through 5th passage were used for all experiments and controls. The cells of the same passage number were used for treatment and the corresponding control. Fetal kidney cells (293 cells) (American Type Culture Collection, Manassas, VA) were cultured in DMEM (GIBCO BRL Gaithersburg, MD) containing 10% FBS as previously described.9-11

Transfection

The cells were plated in 6-well cell culture treated dishes (Falcon, Franklin Lakes, NJ) and grown as monolayers. After the cultures reached 50% confluence, the medium was replaced with serum- free medium (for synchronizing the cells in culture). After 48 hours, cell viability was assayed. Subconfluent cultures at 2.5 10^sup 5^ cells/ well were used for transfection experiments. The old media was aspirated from the cultures and the monolayers were rinsed with phosphatebuffered saline (GIBCO BRL Gaithersburg, MD). The cells were transfected with varying titers of AdVHL, 1-500 pfu (plaque-forming units)/cell. The monolayers were gently rocked for 60-90 minutes at 37C, 5% CO2. Complete media was added and the cultures were further incubated at 37C, 5% CO2 for 48 hours. The negative controls used were AdNull, AdGFP, and infection medium without virus. GFP was utilized as transfection reporter gene. Since our prior work has demonstrated the efficacy of Adp53 in controlling smooth muscle cells (SMC) proliferation as well as regulation of apoptosis, Adp53 was used as positive control.11

The expression of GFP was visualized by observing the green fluorescence under Zeiss Axiophot fluorescence microscope (Carl Zeiss Inc, Thornwood, NY).

Cellular Assays

After 48 hours of transfection, cell viability was assessed by Trypan blue exclusion. Cells were trypsinized and harvested from culture dishes and resuspended in phosphate-buffered saline. Equal volumes of cell suspension were mixed with 0.4% trypan blue dye solution (Sigma, St. Louis, MO), and viable cell number for each treatment group was enumerated manually by using a hemacytometer. The cells excluding trypan blue represented the fraction of viable cells. The viable cell counts were made by 2 independent observers, blinded to the treatments. The interobserver and intraobserver variability was < 3%. For each experiment, there were triplicate wells for treatment and controls. All assays were performed 3 times on each well. In addition, the entire experiment was independently performed for a total of 3 times.

Cell proliferation was determined by using the commercially available CellTiter 96 AQ Proliferation Assay (Promega, Madison, WI), an assay that incorporates a colorimetric method.14-17 Briefly, cells grown in flat-bottomed, tissue culture-treated, 96-well plates (Falcon, Franklin Lakes, NJ), with 500 cells/well, were transfected with AdVHL and controls at varying titers. After 48 hours, 20 L, of the solution containing a tetrazolium compound, (3-[4,5- dimethylthiazol-2-yl]5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]2H- tetrazolium) inner salt (MTS) and an electron-coupling reagent, phenazine methosulfate (PMS), were added to 100 L of culture medium in each well. Cultures were incubated in humidified incubators at 37C 5% CO2 for 4 hours. Cell proliferation was assayed colorimetrically at 490 nm on a microplate reader (Packard, Meriden, CT) by virtue of the red color intensity of the MTS formazan product formed by cellular degradation of MTS. The MTS/PMS assay is designed for the measurement of cellular oxidative activity as a fun\ction of mitochondrial activity in living cells.18 It measures the mitochondrial dehydrogenase activity. The bioreduced formazan product is proportional to the cell number as well as the metabolic capacity of the cells. The oxidative activity was calculated as the percentage of the absorbance for untransfected cell cultures and expressed as percentage cells. The experiment was performed 4 times on separate days with 5 replicates for each concentration.

Apoptosis Assays

Multiple criteria were used to detect apoptosis. Internucleosomal DNA fragmentation was detected by DNA-laddering assay. After 48 hours of transfection, chromosomal DNA was extracted by phenol/ chloroform method from the HASMCs and analyzed by agarose gel electrophoresis to reveal the fragmentation pattern.

In addition, detection of apoptosis in situ was by Tdt-mediated dUTP nick end-labeling of free 3' OH DNA termini of fragmented DNA present in the apoptotic cells (TUNEL) using the ApopTag kit (Intergen, Purchase, NY) as per the manufacturer's instructions. Briefly, cells were transfected, and untransfected cells were harvested, washed with PBS, and fixed with 1% paraformaldehyde/PBS for 10 minutes at room temperature. The fixed cell suspension was applied on slides and air-dried. The slides were equilibrated in ApopTag equilibration buffer for 5 minutes, after which reaction buffer containing TdT enzyme and digoxigenin-dUTP was added to the slides. The slides were incubated in humidified chambers for 1 hour at 37C in dark. End-labeling was terminated by immersion in stop wash buffer for 20 minutes at 37C. Blocking solution containing antidigoxigenin antibody (sheep polyclonal) conjugated to fluorescein was applied and incubated for a further 30 minutes at 37C in humidifying chambers. The antibody solution was washed away with 3 changes of PBS for 5 minutes each. End-labeling was visualized after counterstaining with propidium iodide and observing the yellow-green fluorescence under Zeiss Axiophot fluorescence microscope (Carl Zeiss Inc, Thornwood, NY). Ten random fields per slide were examined under high magnification ( 1,000) and apoptotic cells were counted manually. In total, 1,000 cells were counted in each specimen. The incidence of apoptosis was also detected by observing the morphologic markings of programmed cell death including chromatin condensation and cell shrinkage under a phase contrast microscope. Cells with these features have been confirmed to be apoptotic by electron microscopic analysis in previous studies by us.19

All experiments were performed in triplicate and repeated 3 times on separate days. The apoptotic cell counts were made by 2 independent observers, blinded to the treatments. The interobserver and intraobserver variability was < 3%. The results are normalized to the number of viable cells.

Statistical Analysis

The data obtained were analyzed by Student's t test and Chi- square. Data obtained in the various treatment groups were analyzed by using oneway analysis of variance (ANOVA). Post-hoc Bonferroni multiple comparison test was employed for making comparisons between different treatment groups. A p value of < 0.05 was considered statistically significant. Statistical analyses were performed using Graphpad Instat 2.05a (Texasoft, Cedar Hill, CA) and StatView software (SAS Institute, Gary, NC).

Figure 1. HASMC viability at 48 hours posttransfection with AdVHL. All values are expressed as mean SEM of results obtained in triplicates wells from 3 different experiments.

Figure 2. HASMC proliferation after transfection varied with dose of virus. All values are expressed as mean SEM of results obtained in 5 wells from 4 different experiments.

Results

Cell Viability

Adenovirus-mediated VHL gene transfer caused loss of viability in HASMCs within 48 hours of transfection. This response was dose- dependent and ranged from 45% to 78% in varying concentrations of the AdVHL used. Viable cells were manually counted with trypan blue exclusion method. As depicted in Figure 1, the effect was more pronounced in cultures transfected with high concentrations of virus- containing media. HASMC transfected with the highest dose of 500 pfu/ cell AdVHL exhibited cell viability 45.47 2.97 (SEM) %, as compared to viability of untransfected cells, and demonstrated a significant decrease compared to the viability in cells transfected with AdGFP or AdNull (p<0.05). With a low multiplicity of infection (MOI) viz, 2, 5, and 10 pfu/cell, the viability in VHL-transfected cells was comparable to that in the negative controls. The maximum loss of viability was observed in cells transfected with the positive control, Adp53 500 pfu/cell (p < 0.001).

Cell Proliferation

AdVHL-transfected HASMC exhibited an inhibition of cell proliferation. This decrease correlated with the viability observed in each culture and was dose-dependent. Proliferating HASMC cultures demonstrated a total cell number of 2.5 105 in the highest titer of AdVHL following transfection. In these cells, the absorbance at 490 nm varied from 1.747 0.041 (SEM) (absorbance units) for the maximum dose of 500 pfu/cell AdVHL to 2.414 0.102 (SEM) for the minimum dose of 1 pfu/cell used, as compared to 2.439 0.070 (SEM) in untransfected cells. Figure 2 illustrates the cellular proliferation in HASMC cultures at 48 hours posttransfection with various recombinant adenoviruses. For HASMC treated with the highest AdVHL titer, there was a total of 2.5 10s cells. At the lowest AdVHL concentration, the cell numbers were similar to those in negative controls 48 hours posttransfection (6.2 10^sup 5^ cells). With highest pfu of AdVHL there was significantly reduced proliferation as compared to both negative controls (p<0.0001). Adp53-transfected cells showed the highest reauction of proliferation among all treated cultures (p< 0.001).

Figure 3. Representative DNA-laddering assay.

When the percentage of oxidatively active/viable cells was calculated (percentage absorbance at 490 nm of untransfected cells) the data showed reduction in the cellular oxidative activity in transfected cultures of HASMC. These results correlate with those obtained from manual counts of viable cells. There was a 20% decrease in the smooth muscle cell oxidative activity in the maximum dose of AdVHL as compared to that in cells transfected with AdNuIl and AdGFP.

Apoptosis

The DNA-laddering assay showed characteristic 180-bp laddering pattern of chromosomal DNA of apoptotic cells in HASMCs transfected with AdVHL and Adp53 as illustrated in Figure 3. Such a characteristic fragmentation pattern of internucleosomal DNA was not observed in untransfected cultures or HASMCs treated with AdGFP and AdNull after 48 hours of transfection.

The incidence of apoptosis was confirmed in HASMCs treated with AdVHL and Adp53 by phase contrast microscopy as well as by TUNEL assay. There were vast numbers of Adp53-treated cells exhibiting cell shrinkage and chromatin condensation. Similarly, in AdVHL- treated HASMC cultures, these characteristics were detectable in a large number of cells. TUNEL assay was utilized to confirm the apoptosis as well as to quantify the apoptotic cell numbers (Figures 4, 5). Cells exposed to adenoviruses expressing tumor suppressor genes underwent apoptosis. The magnitude of apoptosis was very high in Adp53 cultures as compared to the rest of the HASMCs (p < 0.001). The HASMCs exposed to AdVHL demonstrated 45.36 2.55% apoptotic cells in the highest titer as compared to that of baseline levels of 3.00 1.33% apoptosis observed in the untransfected. Programmed cell death in the AdNuIl and AdGFP was similar to that seen in untransfected cells.

Discussion

This study was performed to investigate the effects of adenovirus- mediated VHL gene expression on 3 independent cellular processes of cell viability, proliferation, and apoptosis in human vascular smooth muscle cells. As was hypothesized, VHL overexpression led to decreased cell viability, growth inhibition, and upregulation of apoptosis.

Figure 4. HASMC apoptosis as detected by TUNEL at 48 hours following adenoviral transfection (50 pfu/cell) and in untransfected control. All values are expressed as mean SEM of results obtained in triplicates wells from 3 different experiments.

Figure 5. Representative photomicrographs of TUNEL assay for detection of apoptosis. TUNEL positive cells fluorescing cells. (Original magnification 1,000x.)

VHL syndrome, a pleomorphic condition, is an autosomal dominant disorder caused by deletions or mutations in a tumor suppressor gene mapped to human chromosome 3p25-26.20-22 It has been demonstrated that the gene-coding sequence contains 3 exons, and 2 isoforms of mRNA exist, reflecting the presence or absence of exon 2. The VHL disease itself is characterized clinically by vascular tumors.23 Indeed, the VHL protein is present in endothelial cells, fibroblasts, and pericytes that are all involved in active angiogenesis.24 Further data from Ye et al25 showed that the subcellular localization of VHL protein is regulated in a cell cycle- dependent manner. In addition, the VHL protein appears to have several distinct functions including the following: (1) down- regulation of hypoxia-inducible mRNAs; (2) proper assembly of the extracellular fibronectin matrix; and (3) regulation of exit from cell cycle.26

This study suggests that the adenoviral vector used here is highly efficient in the delivery of VHL gene into vascular smooth muscle cells as observed by virtue of GFP fluorescing from AdGFPtransfected cells. Following AdVHL transfection, these cells exhibit characteristic features of overexpression of VHL gene consistent with reports of AdVHL infection in other cell lines. Furthermore, previous studies into the mechanism of effect of VHL have shown that AdVHL infection resulted in Gl cell cycle arrest and growth inhibition of cancer cells.13 This was demonstrated to be associated with induction \of the cyclin-dependent kinase inhibitor (cdk) p27^sup Kip1^ and inhibition of cdk2 and cyclinBl-dependent cdc2 activities.13 Chen et al27 suggested that the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.

The goal of this study was to elucidate the effects of VHL overexpression on proliferation in human vascular cells. Studies in tumor cell lines have shown a decrease in cell proliferation after VHL transfection. We demonstrated that adenovirus-mediated VHL gene transfer inhibits cell proliferation of HASMC in a dose-dependent manner, with the highest reduction in cell proliferation occurring at the maximum concentration of 500 pfu/cell AdVHL. The magnitude of loss of cell viability in AdVHL- and Adp53-infected HASMC is similar to that observed in cancer cells (Seth, personal communication).

The effect of adenovirus-mediated gene transfer of tumor suppressor genes is more pronounced when wild-type p53 is used for transfection, rather than AdVHL. In a previous investigation, we had studied the effect of AdVHL on human aortic endothelial cells. However, we did not observe a significant difference in the effects when a comparison between VHL-transfected endothelial cells and smooth muscle cells was made. This finding is confounding and raises the question of whether the similarity of effects of VHL gene transfer could adversely affect experiments in animal models. The dual effect may be counteractive. In such a situation, the adventitial route for gene transfer could be considered over the vascular endothelial route. In the current data set, some variability was encountered in the SEM in selected concentrations of the treatment groups. Additional experiments may be able to decrease this variability.

Apoptosis plays an important role in mediating the in vivo responses to adenovirus-mediated transfection with tumor suppressor genes. Work performed by Pal, Seth, and co-workers (unpublished data) in tumor cell lines with AdVHL, led us to hypothesize that apoptosis plays a major role in arresting cell growth and limiting proliferation in these cultures. The present investigation demonstrates that indeed gene transfer of AdVHL into HASMCs induces programmed cell death, and it is a factor in determining cell viability and proliferation in transfected vascular cell cultures. The direct effect of VHL gene transfer on the cell cycle regulatory elements, like cyclins among several others, remains to be determined. Which of the molecular mediators of apoptosis are involved in the decrease of cell numbers by the apoptotic pathway? Further mechanistic explorations to elucidate the signaling mechanisms of VHL in HASMC are warranted. The known importance of VHL in physiological and pathological situations, particularly neovascularization, and the role of pVHL in the elimination of misprocessed proteins such as those arising from various stresses,28 reveal the clinical implications of VHL therapy.

To further these preliminary data, studies are currently underway to investigate the effect of AdVHL expression on cell migration, and the effect of VHL gene transfer in co-cultures of endothelial and smooth muscle cells. Furthermore, we are examining a possible bystander effect of AdVHL-expressing cells on uninfected ones, on the expression of VHL, on the incorporation of the DNA, and on determining the time course of the effect of AdVHL. Further knowledge of the VHL gene's signaling mechanisms is needed, to determine whether AdVHL gene transfer is more beneficial to the vascular endothelium in preventing restenosis than other tumor suppressor genes or cell cycle check-point genes.

The results of this study suggest that recombinant adenovirus- mediated VHL gene expression is an effective method of inhibiting vascular cell growth in vitro, and these results provide evidence of regulation of apoptosis after VHL transfer. This is the first time that VHL gene transfer has been attempted in vascular smooth muscle cells. The tumor suppressor property of the VHL gene and its involvement in the regulation of angiogenic factors such as VEGF can be used clinically to limit proliferative processes such as intimai hyperplasia. Arrest of abnormal cell cycle progression may result in the preservation of normal phenotype and function in the vessel wall.

Acknowledgments

This investigation was supported by a grant from Maimonides Research and Development Foundation. We are grateful to Prem Seth, PhD, for providing us with recombinant adenovirus aliquots to propagate for this study. The authors thank Daisy Alapat, MD, for technical assistance.

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Theresa Jacob, PhD, Enrico Ascher, MD, Anil Hingorani, MD, and Shreedhar Kallakuri, MD, Brooklyn, NY

Vase Endovasc Surg 39:25-32, 2005

From the Division of Vascular Surgery, Department of Surgery, Maimonides Medical Center, Brooklyn, NY

Presented at the Annual Meeting of New England Society for Vascular Surgery, Providence, RI, September 19, 2001

Supported by a grant from Maimonides Research and Development Foundation

Correspondence: Enrico Ascher, MD, Director, Division of Vascular Surgery,Maimonides Medical Center, 4802 Tenth Avenue, Brooklyn, NY 11219

E-mail: eascher@maimonidesmed.org

2005 Westminster Publications, Inc, 708 Glen Cove Avenue, Glen Head, NY 11545, USA

Copyright Westminster Publications, Inc. Jan/Feb 2005

Source: Vascular and Endovascular Surgery

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