Over-expression Of Vascular Endothelial Growth Factor 165(VEGF₁₆₅ Protects The Cardiomyocytes Against Doxorubicin-induced Apoptosis

[Background]Doxorubicin (Dox, Adriamycin) is one of the original anthracyclines isolated in the early 1960s from the pigment-producing bacterium Streptomyces peucetius, together with daunorubicin (DNR). Till today, anthracyclines remain some of the most effective anticancer drugs and play a major role in cancer chemotherapy. Doxorubicin is widely used in the treatment of neoplastic diseases such as cancers in bladder, breast, liver, lung, ovary, pancreas, prostate, thyroid and uterus and it is used in virtually all the first-line chemotherapy regimens for lymphomas. The mechanisms of action of doxorubicin in tumor cells that have been suggested include such as intercalation into DNA, leading to inhibition of synthesis of macromolecules; DNA cross-linking; DNA binding and alkylation; initiation of DNA damage via inhibition of topoisomerase II; direct membrane effects and so on.However, doxorubicin is a double-edged sword because its use can cause cardiomyopathy that leads to a form of congestive heart failure that is usually refractory to common medications. Furthermore, with the use of higher doses of doxorubicin and its combined use with the other agents, the incidence of cardiomyopathy has greatly increased. The mechanism responsible for Dox-induced cardiomyopathy remains unclear; several hypotheses have been proposed to explain doxorubicin-induced myocardial damage:(1) the iron and free-radical hypothesis. Increased oxidative stress and antioxidant deficit have been suggested to play an important role in doxorubicin-induced cardiomyopathy and congestive heart failure. (2) The metabolic hypothesis. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca2+ movements. All of which impair cardiac contraction and relaxation. (3) The "unifying" hypothesis. The two above possible mechanisms of cardiac damage that have been proposed may happen sequentially and work together. (4) Apoptosis hypothesis. It is becoming increasingly clear that apoptosis of myocardial cells plays a critical role in the onset of cardiomyopathy; loss of cardiomyocytes could initiate or exacerbate heart failure. Dox-induced apoptosis in cardiomyocytes is more likely attributable to hydrogen peroxide production. Thus, inhibition of doxorubicin-induced cardiomyocytes apoptosis may provide new opportunities for the prevention and treatment of doxorubicin-induced heart failure. Apoptosis depends on the activation of caspases. The two main pathways stimulating caspases are:(Ⅰ) the extrinsic pathway mediated by formation of a cytosolic complex between the cell surface receptor Fas, FADD protein and subsequent activation of initiator caspases (e.g. caspase-8); and (Ⅱ) the intrinsic pathway involving the release of mitochondrial cytochrome c (a 13-kDa heme-containing protein) leading to caspase-9 activation through formation of the apoptosome complex between apoptosis activating factor (Apaf-1), cytochrome c, and pro-caspase-9.All of these hypotheses seem very different from that underlying Dox's antitumor activity. This has raised hopes that treatment protocols can be designed that protect the heart without diminishing the drug's antitumor activity. Therefore, a variety of approaches to preventing this cardiotoxicity have been tried, including the use of Dox analogues, alternative drug-delivery methods, antioxidants, and the iron chelator dexrazoxane; but so far, the ability of these treatments to protect the heart from Dox-induced damage has been limited. So more research is in dire need to explore the more effective and less limited way to prevent the Dox-induced cardiotoxicity.Vascular endothelial growth factor (VEGF) is a sub-family of the platelet-derived growth factor family, which is involved in vasculogenesis, angiogenesis, and lymphangiogenesis during embryonic and postnatal development. The first member VEGF-A (also called VEGF), was cloned in 1989 based on the pioneering studies of an unknown angiogenesis and vascular permeability factor in the 1970s and 1980s. VEGF has subsequently been implicated in a variety of functions such as endochondral bone formation, tissue regeneration, haematopoietic stem cell survival and pathological processes such as neoplastic, inflammatory, and ischemic diseases. Of all the numerous alternatively spliced isoforms of human VEGF-A, VEGF165 (i.e. 165 residues beyond the signal sequence) is the most abundant and mitogenic VEGF isoform. VEGF family stimulate cellular responses by binding to their receptors (the VEGFRs) on the cell surface, VEGFR-2 (KDR/Flk) appears to mediate almost all of the known cellular responses to VEGF and is mainly expressed on vascular endothelial cells. Within the cell, the VEGFR-2 protein is translated as a 150 kDa protein without significant glycosylation. It is then processed, by a series of glycosylations, to a mature 230 kDa form that is expressed on the cell surface. We have previously shown that VEGF165 significantly attenuated oxidative stress-induced cardiomyocytes apoptosis. Friehs et al. and Ruixing et al. have respectively reported that intrapericardial administration of recombinant VEGF protein or intramyocardial injection of VEGF165 cDNA can reduce myocardial apoptosis in a model of left ventricular hypertrophy and in a model of acute myocardial infarction, separately. There is also evidence demonstrating that when cardiomyocytes co-cultured with bone marrow stem cells (BMSCs), adipose tissue-derived stem cells (ASCs) or Mesenchymal stem cell (MSC), VEGF secreted by these different types of stem cells might prevent cardiomyocytes apoptosis induced by hypoxia or H2O2. Therefore, we hypothesized that VEGF165 will protect the cardiomyocytes from Dox-induced apoptosis.Replication-deficient adenoviruses are useful vectors for the transfer of therapeutic transgenes to tissues and cells. Several characteristics of the adenovirus make it suitable as a vector for gene transfer:it can be produced in high titers, it transfects a variety of dividing and nondividing cells efficiently, and it is episomal. Thus, we employed Ad5 (serotype 5 adenovirus)-VEGF to explore the possibility of VEGF to protect the cardiomyocytes against doxorubicin-induced apoptosis.[Methods]1. Construct the replication-deficient adenoviruses encoding human VEGF165 (Ad5-VEGF165). Then they were added into H9c2 cells (embryonic rat heart-derived myoblasts) at a Multiplicity of Infection (MOI, plaque-forming unit (PFU)/cell) of 20:1. Ad5-GFP (green fluorescence protein) served as vector control.2μM of Doxorubicin was added into cell culture medium 24 hours after transfection and treated cells also for 24 hours. Untransfected H9c2 cells were treated with Dox (2μM) for 24 hours, then all groups of cells were harvested for indicated experiments.2. To confirm the efficiency of Ad5-VEGF165,we analyzed the gene expression of transfected H9c2 cells by detecting target proteins using Western blot assay. H9c2 cells and culture medium were harvested, respectively,24h after transfection. Whole-cell lysates and whole culture medium concentrates were analyzed by polyclonal antibodies of VEGF165. H9c2 cells and HUVECs (Human umbilical vein endothelial cells, as positive control) lysates were also analyzed by the antibodies of Flk-1, which is the receptor of VEGF.3. To detect the featured H9c2 cells apoptosis, Flow cytometric analysis of Annexin V staining was applied. Both treated and untreated cells were collected, washed with PBS, suspended in Annexin V binding buffer (10mM HEPES, pH 7.4,2.5mM CaC12,140mM NaCl), stained with Annexin V-FITC, and analyzed by flow cytometry using a FACScalibur flow cytometer.4. To test the apoptosis-associated proteins change, all the 4 groups of cells, i.e control group, Dox group, GFP+Dox group, and VEGF+Dox group were harvested, Cytoplasmic proteins were prepared and immunoblots were performed to evaluate the expression levels of cleaved-caspase-3, cleaved-caspase-8, FADD, Bcl-2, phospho-Akt (Ser473), and total-Akt (t-Akt).5. Electrophoretic mobility shift assay (EMSA) was also applied to examine the NF-κB binding activity which mediates between Akt and Bcl-2 signals. 6. Statistical analysis. Results are expressed as means±standard errors. For tests of significance between the groups, one-way analysis of variance (ANOVA) and Student-Newman-Keuls test were performed. A value of p< 0.05 was considered to be significant.[Results]1. VEGF165 proteins were detected 24h after gene transfection as a secreted pattern. VEGF receptor, Flk-1, was detected in H9c2 cells. The expression pattern is the same as that in HUVECs (positive control).2. The proportion of apoptotic H9c2 cells (i.e. Annexin V staining positive cells) were significantly increased by 315% following Dox treatment, however, pre-treatment with Ad5-VEGF165 decreased the Dox-induced apoptotic cells by 53%. VEGF treatment alone group showed no significant difference compared with control group. Dox treatment significantly increased cleaved caspase-3 levels by 168% compared with control. The levels of cleaved-caspase-3 after treatment with GFP+ Dox were not significantly different compared with Dox treatment alone. Treatment of cardiomyocytes with VEGF165+Dox significantly attenuated the increase in cleaved caspase-3 levels by 33% compared with Dox alone.3. As the important molecules that play crucial role in the death receptor-mediated apoptosis, the levels of FADD and cleaved caspase-8 were significantly increased by 234% and 106%, respectively, after Dox treatment. In contrast, treatment with Ad5-VEGF165 resulted in an attenuated cleaved caspase-8 and FADD levels, respectively, by 47% and 40% compared with Dox alone. Ad5-GFP+Dox alone did not significantly alter FADD/cleaved-caspase 8 protein levels compared with Dox alone.4. Ad5-VEGF165 transfection significantly increased the level of Bcl-2 by 236% compared with Dox-treated cells. Also treatment of cardiomyocytes with VEGF165 +Dox significantly increased the NF-κB DNA binding activity by 143% compared with Dox treatment alone cells. In concert, Ad5-VEGF165 administration significantly increased the levels of phospho-Akt (Ser473) by 153% compared with Dox-treated cells.[Conclusions]1. Over-expression of VEGF165 attenuated the Dox-induced apoptosis in H9c2 cardiomyocytes.2. Over-expression of VEGF165 attenuated the Dox-increased cleaved caspase-8 and FADD levels in H9c2 cardiomyocytes, which indicated that the protective mechanisms of VEGF165 may involve the death receptor-mediated apoptotic signaling pathway.3. Over-expression of VEGF165 upregulated the prosurvival Akt/NF-κB/Bcl-2 signaling pathway in Dox-treated H9c2 cardiomyocytes, which reveals to us that the mitochondrial-mediated apoptotic signaling pathway was also involved in the protective mechanisms of VEGF165.