Angiogenic cell therapy has therapeutic potential for a variety of diseases. Recently, it has been reported that cell therapy could be improved through genetic engineering of the cells to overexpress genes encoding for angiogenic growth factors or anti-apoptotic factors [1
]. Although this combined therapeutic strategy holds promise for the treatment of ischemic diseases, additional improvements to gene delivery are necessary to maximize the potential of this therapy. Viral vector-based delivery systems have demonstrated high efficiency of gene transfer, but they could raise serious safety risks of immunogenicity and genetic mutation which may hinder their clinical application [12
]. Viral gene delivery is also plagued by production/manufacturing challenges and other limitations including nucleic acid cargo capacity [12
]. Non-viral vectors may provide a safe approach to overcome current limitations of viral vectors. Numerous cationic polymers and lipids have been tested as vehicles for non-viral gene delivery including poly(L-lysine), PEI, DOPE, and others [26
]. Those materials may confer several advantages including ease of preparation, purification and chemical modification as well as the stability. The transient nature of non-viral genetic modification may provide for additional safety advantages. While significant strides have been made in improving non-viral gene delivery, the efficacy is often sub-optimal, particularly for primary cells in the presence of serum [27
We have developed end-modified PBAEs, easy-to-synthesize biodegradable polymers, that are able to deliver DNA to primary human cells [20
]. Several end-modified PBAEs (C32-103, C32-117, C32-118, and C32-122) significantly enhance DNA delivery to various types of human stem cells (human MSCs, human embryonic stem cells, and human adipose-derived stem cells), showing a 2- to 5-fold higher transfection efficiency than commercially-available lipid reagent, Lipofectamine 2000 [18
In the presence of 12% serum, end-modification of C32 polymer (C32-122) shows smaller particle size, more neutral zeta potentials, and improved stability [18
]. VEGF transfection using end-modified PBAE (C32-122) significantly enhanced VEGF expression in HUVECs, compared with unmodified PBAE (C32), PEI, and Lipofectamine 2000 (). In addition, C32-122 did not show significant cytotoxicity in gene transfer to HUVECs (). Compared to Lipofectamine 2000, C32-122 was well tolerated in HUVECs for transfection with the DNA doses tested in our study.
Enhanced expression of endogenous VEGF might stimulate anti-apoptosis signal transduction in ECs. The expression of VEGF-dependent signaling molecules (PI3K and Akt-1) was enhanced by VEGF transfection with C32-122 (). It is known that PI3K and Akt-1 signal transduction activated by VEGF could inhibit EC apoptosis [28
]. VEGF is known to regulate the expression of endothelial receptors (KDR/Flk-1 for VEGF and Tie-2 for angiopoietin) and the signaling mediated by these receptors [30
]. The expression of the endothelial receptors increased in HUVECs modified with C32-122-VEGF (). The activation of those receptors may improve proliferation and angiogenic potential of ECs or EPCs [31
]. These favorable changes in gene expression pattern led to improvement of the viability of HUVECs under a simulated ischemic condition with hypoxia (1% oxygen) and serum deprivation (). Pre-transfection of C32-122-VEGF also promoted the survival of transplanted HUVECs in ischemic tissue at early stage of transplantation ().
Genetic engineering using C32-122-VEGF facilitated engraftment of transplanted human ECs into mouse capillary network. It has been reported that enhanced expression of endogenous VEGF following transfection may increase expression of adhesion molecules (VCAM-1 and ICAM-1) [33
]. Our study has shown that the expression of VCAM-1 was upregulated in HUVECs following transfection with C32-122-VEGF (). The enhanced expression of adhesion molecules in ECs may facilitate EC mobilization and incorporation into host vasculature [33
]. Indeed, the incorporation of transplanted HUVECs into mouse vasculature was significantly enhanced by pre-transfection of C32-122-VEGF (). Together with improved cell survival, efficient engraftment into host tissue greatly improved angiogenesis in ischemia regions (). It has also been reported that overexpression of adhesion molecules (E-selectin, VCAM-1, or ICAM-1) in ECs can facilitate interaction between transplanted ECs and host EPCs, leading to enhanced neovascularization through recruitment of EPCs [35
]. Enhanced blood vessel formation in ischemic tissue inhibited tissue necrosis and fibrosis caused by ischemic event () and ultimately improved ischemic limb salvage ().
Combinations of molecules with different actions on angiogenesis may maximize the benefits of PBAE-engineered cell therapy. For examples, the combination of stromal-derived factor (SDF)-1α and VEGF significantly increased EPC-mediated angiogenesis in ischemic limb tissue [36
]. Since SDF-1α is a chemokine for EPCs and VEGF increases the expression of SDF-1α receptors (e.g., CXCR4) on EPCs, this combination could enhance the efficacy of EPC therapy by promoting EPC recruitment to ischemic sites [36
]. In other studies, bone marrow cell-based VEGF gene therapy combined with other angiogenic/myogenic factors (basic fibroblast growth factor or insulin-like growth factor-1) greatly enhanced angiogenesis and left ventricle function of ischemic myocardium [37
]. PBAE-engineered cell therapy with multiple genes or combination with protein delivery could exhibit synergistic effects on therapeutic angiogenesis following cell transplantation.
Although this study has reported successful outcome of genetically engineered cell transplantation for therapeutic angiogenesis, the use of mature ECs needs be replaced with more potent cells such as EPCs. It is obvious that mature ECs (e.g., HUVECs used in this study) are good cell sources in experimental approach for demonstrating the effectiveness of VEGF transfection using PBAE nanoparticles. However, fully matured cells have some disadvantages for clinical application. Despite mature ECs such as HUVECs share some endothelial features with EPCs, they have a limited proliferative capacity, compared with EPCs. Thus, the use of mature ECs may lead to marginal therapeutic efficacy for treating ischemic diseases. In addition, ECs should be obtained from biopsy of autologous blood vessels, whereas EPCs can be easily isolated from autologous cell sources including bone marrow or peripheral blood, which are relatively more acceptable for patients with ischemia. This demonstrates the feasibility of EPCs for clinical setting. Indeed, recent clinical studies have focused on the application of CD34- or CD133-positive EPCs in ischemia treatments [39
]. In this regard, genetic engineering using PBAE nanoparticles needs to be further evaluated for EPC transplantation for more clinically relevant therapeutic angiogenesis in future study.
This study suggests that genetically engineered ECs modified with non-viral DNA delivery systems may provide an effective system to promote therapeutic angiogenesis for the salvage of ischemic tissue. Genetic engineering of angiogenic cells could be applied for treatment of other types of ischemic diseases such as myocardial or cerebral ischemia. Since angiogenesis is an important process for most types of tissue regeneration, this technology may have utility in the repair of vascularized massive tissues including bone, skeletal muscle, or adipose tissue.