Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Control Release. Author manuscript; available in PMC 2013 June 28.
Published in final edited form as:
PMCID: PMC3372613

Therapeutic Angiogenesis Using Genetically Engineered Human Endothelial Cells


Cell therapy holds promise as a method for the treatment of ischemic disease. However, one significant challenge to the efficacy of cell therapy is poor cell survival in vivo. Here we describe a non-viral, gene therapy approach to improve the survival and engraftment of cells transplanted into ischemic tissue. We have developed biodegradable poly(β-amino esters) (PBAE) nanoparticles as vehicles to genetically modify human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF). VEGF transfection using these nanoparticles significantly enhanced VEGF expression in HUVECs, compared with a commercially-available transfection reagent. Transfection resulted in the upregulation of survival factors, and improved viability under simulated ischemic conditions. In a mouse model of hindlimb ischemia, VEGF nanoparticle transfection promoted engraftment of HUVECs into mouse vasculature as well as survival of transplanted HUVECs in ischemic tissues, leading to improved angiogenesis and ischemic limb salvage. This study demonstrates that biodegradable polymer nanoparticles may provide a safe and effective method for genetic engineering of endothelial cells to enhance therapeutic angiogenesis.

Keywords: Polymer nanoparticles, Genetic engineering, Endothelial cells, Angiogenesis, Ischemia

1. Introduction

Angiogenic cell therapy has been used to facilitate angiogenesis for treatment of ischemic diseases such as peripheral arterial disease or myocardial infarction [1-4]. Transplantation of endothelial cells (ECs) or endothelial progenitor cells (EPCs) have been reported to induce collateral blood vessel development in ischemic limb or myocardium [1-4], leading to improved limb survival or left ventricular function [1-4]. One challenge to angiogenic cell therapy is that ischemic regions are often exposed to hypoxic and inflammatory environments immediately after transplantation [5]. This environment can stimulate apoptosis, thereby limiting the efficacy of transplanted cells [5]. Indeed, significant levels of cell death have been observed after cell transplantation into ischemic tissue [6,7]. Several clinical studies have also reported that bone marrow-derived cell therapy provides little benefit to ischemic myocardium, in part due to poor cell survival in ischemic tissue [8,9].

Gene delivery has the potential to enhance the angiogenic efficacy of cell therapy by modifying cells with angiogenic or anti-apoptotic factors. For example, genetic modification of ECs or mesenchymal stem cells (MSCs) with the anti-apoptotic factor (Bcl-2) improved efficacy of cell therapy in a model of myocardial infarction or hindlimb ischemia [1,10]. Intramyocardial transplantation of MSCs transduced with adenovirus encoding vascular endothelial growth factor (VEGF) significantly enhanced angiogenesis and improved cardiac function of infarcted myocardium, compared with MSCs without VEGF modification [11]. However, most of studies have relied on viral vectors to deliver therapeutic genes to the cells, and thus raise the potential of safety concerns such as immunogenicity, mutagenesis, or toxicity [12]. Several studies have demonstrated the potential of non-viral vectors (polyethylenimine (PEI), FuGene, or Lipofectamine) for genetic modification [13-15], but the ability of those materials to transfect primary cells remains sub-optimal.

Poly(β-amino esters) (PBAE) are cationic polymers that hold promise as non-viral gene delivery for in vitro and in vivo application [16]. Conditions have been developed where PBAEs self-assemble with DNA to form stable nanoparticles that can effectively transfect a variety of cells, and biodegrade via hydrolytic cleavage of backbone ester groups [17]. High-throughput screening has identified lead PBAEs that are capable of transfecting several types of primary cells significantly higher than commercially available transfection reagents [18-21]. We reasoned that these biodegradable PBAEs could be used for ex vivo genetic engineering with angiogenic or anti-apoptotic factors to improve survival and engraftment of transplanted cells in vivo [22].

Here we develop human ECs for use in treating ischemic tissue in mice. Human umbilical vein endothelial cells (HUVECs) were transfected with VEGF using PBAEs, and then transplanted into ischemic limb tissue. We show that survival and engraftment of HUVECs in ischemic tissue is improved through polymer-mediated overexpression of angiogenic growth factors.

2. Materials and methods

2.1. Materials

Monomers were purchased from Sigma-Aldrich (St. Louis, MO), Scientific Polymer Products Inc (Ontario, NY), and Molecular Biosciences (Boulder, CO). Anhydrous dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich. Sodium acetate buffer solution (25 mM, pH 5.2) was prepared by diluting a 3 M stock solution (Sigma-Aldrich). VEGF165 plasmid was kindly provided by Professor Johnny Huard at University of Pittsburgh and amplified by Aldevron (Fargo, ND). The plasmid backbone is pBLAST49. pBLAST49-hVEGF165 is a human VEGF expression vector containing VEGF165 sequence, blasticidin resistance gene, hybrid EF1α/HTLV promoter, and a multiple cloning site.

2.2. Poly(β-amino esters) (PBAE) synthesis

Acrylate-terminated C32 polymer (C32-Ac) was prepared by polymerizing butanediol diacrylate (C) with aminopentanol (32) using a 1.2:1.0 diacrylate to amine monomer molar ratio at 90°C for 24 hours (Fig. 1) [20]. Subsequently, amine-terminated C32 polymers were generated by reacting C32-Ac with diamine monomer (122) in DMSO (Fig. 1). End-capping reactions were performed overnight at room temperature using a 1.6-fold molar excess of amine over acrylate end groups.

Fig. 1
Synthesis of end-modified poly(β-amino esters). Acrylate-terminated C32 polymer (C32-Ac) was first synthesized and then end-modified with amine group (122).

2.3. Biophysical characterization

Particle size and zeta potential of the polyplexes were measured by using a ZetaPALS dynamic light scattering detector (Brookhaven Instruments Corp., Holtsville, NY) [18]. EGFP-N1 plasmid (Elim Biopharmaceuticals, Hayward, CA) was used for complexation. Samples were prepared for biophysical characterization in the same manner and at the same concentrations, as they were for transfection using 30:1 polymer/plasmid weight ratio. After a 10-min complexation, 200 μl of the polymer/DNA solution (n=3) was added to 1.4 ml of growth medium containing 12% serum to reach a final volume of 1.6 ml. Particle size and zeta potential was measured 1 h after complexation. Effective diameters were calculated from the autocorrelation function using the MAS option of the BIC particle-sizing software assuming a log normal distribution. The solution viscosity and refractive index were assumed equal to those of pure water at 25°C. Average electrophoretic mobilities were measured using BIC PALS zeta potential analysis software, and zeta potential was calculated using the Smoluchowsky model for aqueous suspensions.

2.4. Transfection

Human umbilical vein endothelial cells (HUVECs, Lonza, Walkersville, MD) were grown in Endothelial Growth Medium-2 (EGM-2) (Lonza) supplemented with the SingleQuot kit (Lonza) at 37°C and 5% CO2. For viability test, HUVECs were seeded onto 96-well plates (15,000 cells per well) and transfected with VEGF plasmid (300 ng per well) in EGM-2 medium using optimized PBAE transfection conditions (1:30 (w/w) of plasmid to polymer ratio) [18]. The polyplexes were formulated by mixing PBAE with the plasmids in sodium acetate buffer and waiting 10 min for the complexes to form. The polyplexes were then added to HUVECs under EGM-2 medium with 12% fetal bovine serum (FBS). Following 2 hours of incubation, the medium with the polyplexes was replaced with fresh EGM-2 medium. Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) and polyethyleneimine (PEI), commercially available transfection reagents, were used for control transfection. Two days after transfection, cell viability (n=4) was measured using the CellTiter 96® Aqueous One Solution Cell Proliferation assay kit (Promega, Madison, WI). This kit employs a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity test. The tetrazolium compound contained in this kit is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. This conversion is presumably accomplished by nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide produced by dehydrogenase enzymes in metabolically active cells. Cellular metabolic activity was determined by measuring optical absorbance at 490 nm using a Victor3 Multilabel plate counter (Perkin-Elmer Life Sciences, Boston, MA). For ELISA or real-time PCR analysis, HUVECs were seeded onto 6-well plates (3.0 × 105 cells per well) and transfected with VEGF plasmid (12 μg of plasmid per well, 1:30 (w/w) of plasmid to polymer ratio).

2.5. Enzyme-linked immunosorbent assay (ELISA)

Two days after transfection, the level of VEGF expression in HUVECs (n=3) was measured using an ELISA kit for human VEGF (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction. To obtain total proteins, cells were lysed in CelLytic™ MT (Sigma) containing a protease inhibitor cocktail (Sigma). After centrifugation of lysates, the supernatants were retrieved and used for protein samples. Total protein concentration in samples was determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL). The VEGF expression was presented as the amount of VEGF per mg of total protein. The human VEGF expression was also examined in mouse ischemic limb muscle (n=3) retrieved at 2 days after HUVEC injection.

2.6. Quantitative real-time polymerase chain reaction (TaqMan method)

The mRNA expression of several molecules in HUVECs 2 days after transfection was examined using quantitative real-time polymerase chain reaction (PCR). Total RNA was isolated using the RNeasy Mini kit (Qiagen, Chatsworth, CA) from the cells of each group (n=4 per group). The RNA concentration was determined by measuring absorbance at 260 nm using a spectrophotometer. A reverse transcription reaction for cDNA synthesis was performed with 1 μg of pure total RNA using SuperScriptTM III reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA). TaqMan® Fast Universal PCR Master Mix (Applied Biosystems) and synthesized cDNA were used for reaction. The expression of several molecules was quantitated using TaqMan® Gene Expression Assays (Applied Biosystems) for targets (PI3K: Hs00180679_m1, Akt-1: Hs00920503_m1, KDR/Flk-1: Hs00176676_m1, Tie-2: Hs00176096_m1, and VCAM-1: Hs01003372_m1) and endogenous reference (Glyceraldehydes 3-phosphate dehydrogenase (GAPDH): Hs02758991_g1). The expression level of target genes was determined by the comparative Ct method, whereby the target was normalized to the endogenous reference (GAPDH).

2.7. HUVEC culture under hypoxic and serum-deprived conditions

Two days after VEGF transfection, HUVECs were cultured under hypoxic and serum-deprived condition, a simulated ischemic condition [23]. EGM-2 was changed to Endothelial Basal Medium-2 (EBM-2) without FBS and growth factors, and then transfected HUVECs were further cultured for 2 days in a hypoxic incubator (MCO-18M, Sanyo, Japan) with air condition of 1% oxygen and 5% CO2 at 37°C. Two days after culture, cell viability (n=4) was measured using the CellTiter 96® Aqueous One Solution assay kit. The viability of transfected HUVECs was converted to percent viability by comparison to that of HUVECs (without transfection) cultured under normal condition with normoxia (20% oxygen) and complete growth medium (EGM-2) for the comparable time period.

2.8. Hindlimb ischemia mouse model

All procedures for surgery were approved by “Committee on Animal Care” of Massachusetts Institute of Technology. Hindlimb ischemia was induced in a mouse model as previously described [24]. Female athymic mice at 6 weeks of age (18-20 g body weight, Taconic, Hudson, NY) were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg). The femoral artery and its branches were ligated through a skin incision with 5-0 silk suture (Ethicon, Somerville, NJ). The external iliac artery and all of the above arteries were then ligated. The femoral artery was excised from its proximal origin as a branch of the external iliac artery to the distal point where it bifurcates into the saphenous and popliteal arteries. All animals received humane care in compliance with “the Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.9. Cell transplantation

Immediately after arterial dissection, athymic mice were randomly divided into four experimental groups. The control group received an injection of only phosphate buffered saline (PBS, n=10). HUVECs (1.0 × 106 cells per mouse; no transfection, LIPO-VEGF, and C32-122-VEGF) were suspended with 100 μl of EGM-2 medium and injected intramuscularly into two sites of the gracilis muscle in the medial thigh using 29-gauge tuberculin syringes (n=10 per group). Physiological status of ischemic limbs was followed up to 4 weeks after injection treatments.

2.10. Triphenyltetrazolium chloride (TTC) staining

To identify the morphological differences among ischemic muscles in each group, the retrieved muscles (4 weeks after cell transplantation) were stained with TTC. The muscles were cut into 2-mm slices and then the slices were incubated for 30 min in 2% TTC (Sigma) solution and fixed for 30 min in 10% (v/v) buffered formaldehyde.

2.11. Histology

Ischemic and normal limb muscles were harvested at 4 weeks after cell transplantation. Specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated with a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4-μm sections and the sections were stained with hematoxylin & eosin (H&E) to examine the muscle degeneration and tissue inflammation. Masson’s trichrome collagen staining was performed to examine tissue fibrosis in the ischemic regions.

2.12. Immunohistochemistry

Tissue sections were prepared with ischemic limb muscles harvested 2 days and 4 weeks after cell transplantation. To detect transplanted human cells, the sections (from samples day 2) were immunofluorescently stained with anti-human nuclear antigen (HNA, Chemicon, Temecula, CA). Ten sections from two muscle samples of each mouse were randomly selected for the counts (3 mice per each group). For staining of capillaries in the ischemic regions, the sections (from samples week 4) were immunofluorescently stained with anti-von Willebrand Factor (vWF, Abcam, Cambridge, UK). The staining signals for HNA and vWF were visualized with rhodamine- and FITC-conjugated secondary antibodies (Invitrogen), respectively. The sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). To quantify the number of HUVECs incorporated into vasculature, HNA- and vWF-stained cells were counted in ten sections from two muscle samples of each mouse (5 mice per each group). Microvessels for quantitative analysis were defined as mouse endothelial cell antigen (MECA)-positive microvessels. Tissue sections (from samples week 4) were immunohistochemically stained using antibodies against MECA (Chemicon). The staining signal was visualized with avidin-biotin complex immunoperoxidase (Vectastain® ABC kit, Vector Laboratories) and 3,3′-diaminobenzidine substrate solution kit (Vector Laboratories). Microvessels in ischemic areas were counted in ten sections from two muscle samples of each mouse (8 mice per each group) under a light microscope. Negative control staining was performed without primary antibody incubation.

2.13. Statistical analysis

Quantitative data are expressed as mean ± standard deviation. Statistical analysis was performed by the analysis of variance (ANOVA) using a Bonferroni test. A value of p<0.05 was considered statistically significant.

3. Results

3.1. Polymer nanoparticle synthesis

PBAEs were synthesized by the conjugate addition of amine monomers to diacrylates. Acrylate-terminated C32 (C32-Ac) was first synthesized by mixing butanediol diacrylate (C) with aminopentanol (32) in a 1.2:1.0 molar ratio (Fig. 1). C32-Ac was subsequently reacted with diamine monomers (122) to yield end-modified C32 polymers (C32-122) (Fig. 1). The nanoparticle complexation of polymer (C32-122) and plasmid DNA was confirmed by gel retardation assay (Fig. 2a). A transmission electron microscopic image showed the nanoparticle complexes of C32-122 and plasmid DNA with the size of around 200 nm (Fig. 2b). The particle size and zeta potential of PBAE nanoparticles were measured in 12% serum condition 1 hour after complexation with plasmid DNA. The particle size of C32 and C32-122 was 463 ± 35 nm and 219 ± 13 nm, respectively (Fig. 2c). Our previous study reported that the size of polymer-DNA nanoparticles increased over incubation time in 12% serum condition [18]. At 4 hours after complexation, the particle size of C32 and C32-122 increased to ~600 nm and ~400 nm, respectively [18]. The zeta potential of C32 and C32-122 was -1.91 ± 0.14 mV and -1.26 ± 0.19 mV, respectively (Fig. 2d).

Fig. 2
Characterization of the polyplexes of poly(β-amino esters) (C32 or C32-122) and DNA (EGFP plasmid). (a) Gel retardation analysis at 30 min after complexation. (b) Transmission electron microscopic image of the polyplexes. The scale bar indicates ...

3.2. Enhanced expression of VEGF and survival factors in vitro

Two days after VEGF transfection, VEGF expression was significantly enhanced (p<0.01) in HUVECs transfected with end-modified PBAE (C32-122), compared with no transfection, naked DNA transfection, or unmodified PBAE (C32) transfection (Fig. 3a). More importantly, C32-122 transfection (4.57 ± 0.37 ng/mg protein) significantly increased (p<0.01) VEGF expression, compared with commercially available reagents (PEI; 0.44 ± 0.03 ng/mg protein and Lipofectamine 2000; 0.71 ± 0.03 ng/mg protein) (Fig. 3a). Following VEGF transfection using C32-122, the expression of survival factors (anti-apoptotic factors, endothelial receptors, and adhesion molecules) was significantly enhanced in HUVECs (Figs. 3b-3f). Quantitative real-time polymerase chain reaction (PCR) analysis showed that the expression of anti-apoptotic factors (PI3K and Akt-1) was increased (p<0.01) in C32-122-transfected HUVECs, compared with no transfection or control transfection (C32 and LIPO) (Figs. 3b and 3c). C32-122-VEGF transfection also upregulated (p<0.01) the expression of endothelial receptors (KDR/Flk-1 and Tie-2) (Figs. 3d and 3e) and adhesion molecule (VCAM-1) (Fig. 3f). Expression of transgene (VEGF) in HUVECs was maintained up to 6 days after transfection (Fig. 3g). The duration of VEGF expression up to ~1 week could contribute to enhancing EC survival at early stage after transplantation into ischemic tissues.

Fig. 3
Enhanced expression of VEGF and survival factors in HUVECs 2 days after transfection. (a) ELISA for measurement of VEGF expression (n=3, *; p<0.01, compared to other groups). VEGF content was normalized to the amount of total protein. Real-time ...

3.3. Cell viability in vitro

VEGF transfection using PBAE (C32 and C32-122) did not cause significant cytotoxicity in HUVECs (Fig. 4a). However, similar conditions with 300 ngs of plasmid per 1.5 × 104 cells, Lipofectamine (1:1 ratio of plasmid to reagent) was more toxic to cells (p<0.01) than PBAE (1:30 ratio of plasmid to polymer) (Figs. 4a and 4b). When transfected HUVECs were cultured under a simulated ischemic condition with hypoxia (1% oxygen) and serum deprivation, the viability of C32-122-VEGF transfected HUVECs was much higher (p<0.01) than other HUVEC groups (Fig. 4c). The viability of HUVECs in C32-VEGF group is not significantly different (p>0.05) from that of HUVECs in no transfection group (Fig. 4c). The viability of LIPO-VEGF group is lower (p<0.05) than no transfection group, probably due to the cytotoxicity originated from Lipofectamine during transfection (Fig. 4c).

Fig. 4
Viability of transfected HUVECs. (a) HUVEC viability 2 days after transfection (n=4, *; p<0.01, compared to other groups). The viability of transfected HUVECs was normalized to that of HUVECs without transfection. (b) Viability of transfected ...

3.4. Enhancement of VEGF expression and cell survival in vivo

Genetically-engineered HUVECs were injected into mouse ischemic limb immediately after ischemia induction. The expression of human VEGF protein following HUVEC transplantation was enhanced by pre-transfection of C32-122-VEGF (Fig. 5a). Two days after cell injection, C32-122-VEGF group (620.8 ± 319.7 pg/mg protein) showed greater expression of human VEGF protein (p<0.05) in mouse ischemic muscle than no transfection group (82.0 ± 73.1 pg/mg protein), PEI-VEGF group (183.3 ± 116.2 pg/mg protein), or LIPO-VEGF group (204.6 ± 193.9 pg/mg protein) (Fig. 5a). Human nuclear antigen (HNA) staining of mouse ischemic muscle harvested at 2 days revealed that survival of transplanted HUVECs was improved by pre-transfection of C32-122-VEGF (Fig. 5d). The density of HNA-positive cells was higher (p<0.01) in C32-122 group than other control groups (Fig. 5b).

Fig. 5
Enhancement of survival and engraftment of genetically-modified HUVECs in mouse ischemic limb muscles. (a) ELISA for human VEGF detection in mouse ischemic muscles (n=5) retrieved at 2 days after HUVEC injection (#; p<0.05, compared to other groups). ...

3.5. Angiogenesis promoted by genetically-engineered endothelial cells

Transplantation of HUVECs transfected with C32-122-VEGF significantly enhanced angiogenesis in mouse ischemic limb. Double immunofluorescent staining of HNA and mouse von Willebrand Factor (vWF) (Fig. 5e) showed that incorporation of transplanted HUVECs into host capillaries was more extensive (p<0.05) in C32-122-VEGF group than other groups (Figs. 5c and 5e). Immunohistochemical staining for mouse endothelial cell antigen (MECA, Fig. 6a) revealed that injection of HUVECs engineered with C32-122-VEGF promoted microvessel formation in ischemic tissue, compared with injection of phosphate buffered saline (PBS) or control HUVECs (no transfection, PEI-VEGF, and LIPO-VEGF). The density of MECA-positive microvessels in the ischemic region was significantly higher (p<0.05) in C32-122-VEGF group (120 ± 35/mm2) than control groups (PBS; 40 ± 23/mm2, no transfection; 50 ± 20/mm2, PEI-VEGF; 60 ± 22/mm2, and LIPO-VEGF; 78 ± 25/mm2) (Fig. 6b).

Fig. 6
Enhanced angiogenesis by genetically-modified HUVECs in mouse ischemic limb muscle. (a) Immunohistochemical MECA staining of mouse ischemic muscle 4 weeks after injection. Scale bar indicates 100 μm. Negative control staining was performed without ...

3.6. Improvement of ischemic limb salvage by genetically-engineered endothelial cells

The therapeutic potential of genetically-engineered HUVECs was evaluated in terms of physiological status of ischemic limbs. The intramuscular transplantation of HUVECs engineered with C32-122-VEGF prevented necrosis of ischemic limbs. The injection of C32-122-VEGF-transfected HUVECs improved ischemic limb salvage (50%, 5 of 10), compared with either injection of PBS (10%, 1 of 10) or control HUVECs (no transfection; 20% (2 of 10), PEI-VEGF; 12.5% (1 of 8), and LIPO-VEGF; 30% (3 of 10), Fig. 7a). Ischemic muscle receiving HUVECs transfected with C32-122-VEGF has larger viable tissue area than other control groups, as confirmed by triphenyltetrazolium chloride (TTC) staining (Fig. 7a).

Fig. 7
Improvement of ischemic limb salvage by transplantation of genetically-modified HUVECs. (a) Physiological status of ischemic limbs 4 weeks after cell injection and TTC staining of ischemic muscles retrieved at 4 weeks after cell injection. (b) Histological ...

3.7. Reduced muscle degeneration following treatment with genetically-engineered endothelial cells

Histological examinations of the ischemic limbs retrieved at 4 weeks after transplantation confirmed that enhanced angiogenesis by transplantation of engineered HUVECs protected limb muscles against necrotic damage caused by ischemia. Hematoxylin and eosin (H&E) staining of the control group (PBS injection) showed muscle degeneration in the ischemic regions (Fig. 7b). In contrast, ischemic limb muscle injected with C32-122-VEGF-HUVECs was protected from necrosis (Fig. 7b). Masson’s trichrome staining indicated that fibrosis was markedly attenuated after transplantation of C32-122-VEGF-HUVECs into ischemic region (Fig. 7b).

4. Discussion

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 [1,10,11,13-15, 25]. 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 [18,19].

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 (Fig. 3a). In addition, C32-122 did not show significant cytotoxicity in gene transfer to HUVECs (Fig. 4a). 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 (Figs. 3b and 3c). It is known that PI3K and Akt-1 signal transduction activated by VEGF could inhibit EC apoptosis [28,29]. 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 [30,31]. The expression of the endothelial receptors increased in HUVECs modified with C32-122-VEGF (Figs. 3d and 3e). The activation of those receptors may improve proliferation and angiogenic potential of ECs or EPCs [31,32]. 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 (Fig. 4c). Pre-transfection of C32-122-VEGF also promoted the survival of transplanted HUVECs in ischemic tissue at early stage of transplantation (Figs. 5b and 5d).

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 (Fig. 3f). The enhanced expression of adhesion molecules in ECs may facilitate EC mobilization and incorporation into host vasculature [33,34]. Indeed, the incorporation of transplanted HUVECs into mouse vasculature was significantly enhanced by pre-transfection of C32-122-VEGF (Figs. 5c and 5e). Together with improved cell survival, efficient engraftment into host tissue greatly improved angiogenesis in ischemia regions (Figs. 6a and 6b). 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 (Fig. 7b) and ultimately improved ischemic limb salvage (Fig. 7a).

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 [37,38]. 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 [39,40]. 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.


This work was supported by grants (R01-EB000244-27 and R01-DE016516-03) from the National Institutes of Health. This work was also supported by the Basic Science Research Program (2010-0022037) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology and Yonsei University Research Fund (2010-7-0089), Republic of Korea.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


[1] Enis DR, Shepherd BR, Wang Y, Qasim A, Shanahan CM, Weissberg PL, Kashgarian M, Pober JS, Schechner JS. Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells. Proc. Natl. Acad. Sci. USA. 2005;102(2):425–430. [PubMed]
[2] Cho SW, Moon SH, Lee SH, Kang SW, Kim J, Lim JM, Kim HS, Kim BS, Chung HM. Improvement of postnatal neovascularization by human embryonic stem cell derived endothelial-like cell transplantation in a mouse model of hindlimb ischemia. Circulation. 2007;116(21):2409–2419. [PubMed]
[3] Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, Masaki H, Mori Y, Iba O, Tateishi E, Kosaki A, Shintani S, Murohara T, Imaizumi T, Iwasaka T. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation. 2001;104(9):1046–1052. [PubMed]
[4] Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 2001;7(4):430–436. [PubMed]
[5] Tang YL, Tang Y, Zhang YC, Qian K, Shen L, Phillips MI. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector. J. Am. Coll. Cardiol. 2005;46(7):1339–1350. [PubMed]
[6] Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002;105(1):93–98. [PubMed]
[7] Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J. Mol. Cell. Cardiol. 2001;33(5):907–921. [PubMed]
[8] Assmus B, Honold J, Schächinger V, Britten MB, Fischer-Rasokat U, Lehmann R, Teupe C, Pistorius K, Martin H, Abolmaali ND, Tonn T, Dimmeler S, Zeiher AM. Transcoronary transplantation of progenitor cells after myocardial infarction. N. Engl. J. Med. 2006;355(12):1222–1232. [PubMed]
[9] Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld JG, Smith HJ, Taraldsrud E, Grøgaard HK, Bjørnerheim R, Brekke M, Müller C, Hopp E, Ragnarsson A, Brinchmann JE, Forfang K. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N. Engl. J. Med. 2006;355(12):1199–1209. [PubMed]
[10] Li W, Ma N, Ong LL, Nesselmann C, Klopsch C, Ladilov Y, Furlani D, Piechaczek C, Moebius JM, Lützow K, Lendlein A, Stamm C, Li RK, Steinhoff G. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells. 2007;25(8):2118–2127. [PubMed]
[11] Matsumoto R, Omura T, Yoshiyama M, Hayashi T, Inamoto S, Koh KR, Ohta K, Izumi Y, Nakamura Y, Akioka K, Kitaura Y, Takeuchi K, Yoshikawa J. Vascular endothelial growth factor-expressing mesenchymal stem cell transplantation for the treatment of acute myocardial infarction. Arterioscler. Thromb. Vasc. Biol. 2005;25(6):1168–1173. [PubMed]
[12] Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 2003;4(5):346–358. [PubMed]
[13] Ye L, Haider H.Kh., Tan R, Toh W, Law PK, Tan W, Su L, Zhang W, Ge R, Zhang Y, Lim Y, Sim EK. Transplantation of nanoparticle transfected skeletal myoblasts overexpressing vascular endothelial growth factor-165 for cardiac repair. Circulation. 2007;116(11 Suppl):I113–I120. [PubMed]
[14] Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, Chiodoni C, Colombo MP, Versura P, D’Errico-Grigioni A, Ferri E, Baccarani M, Lemoli RM. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Stem Cells. 2006;24(2):454–461. [PubMed]
[15] Elmadbouh I, Haider H.Kh., Jiang S, Idris NM, Lu G, Ashraf M. Ex vivo delivered stromal cell-derived factor-1alpha promotes stem cell homing and induces angiomyogenesis in the infarcted myocardium. J. Mol. Cell. Cardiol. 2007;42(4):792–803. [PMC free article] [PubMed]
[16] Green JJ, Langer R, Anderson DG. A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc. Chem. Res. 2008;41(6):749–759. [PMC free article] [PubMed]
[17] Anderson DG, Akinc A, Hossain N, Langer R. Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters) Mol. Ther. 2005;11(3):426–434. [PubMed]
[18] Yang F, Green JJ, Dinio T, Keung L, Cho SW, Park H, Langer R, Anderson DG. Gene delivery to human adult and embryonic cell-derived stem cells using biodegradable nanoparticulate polymeric vectors. Gene Ther. 2009;16(4):533–546. [PMC free article] [PubMed]
[19] Green JJ, Zhou BY, Mitalipova MM, Beard C, Langer R, Jaenisch R, Anderson DG. Nanoparticles for gene transfer to human embryonic stem cell colonies. Nano Lett. 2008;8(10):3126–3130. [PubMed]
[20] Green JJ, Zugates GT, Tedford NC, Huang YH, Griffith LG, Lauffenburger DA, Sawicki JA, Langer R, Anderson DG. Combinatorial modification of degradable polymers enables transfection of human cells comparable to adenovirus. Adv. Mater. 2007;19(19):2836–2842.
[21] Green JJ, Shi J, Chiu E, Leshchiner ES, Langer R, Anderson DG. Biodegradable polymeric vectors for gene delivery to human endothelial cells. Bioconjug. Chem. 2006;17(5):1162–1169. [PubMed]
[22] Yang F, Cho SW, Son SM, Bogatyrev SR, Singh D, Green JJ, Mei Y, Park S, Bhang SH, Kim BS, Langer R, Anderson DG. Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles. Proc. Natl. Acad. Sci. USA. 2010;107(8):3317–3322. [PubMed]
[23] Cho SW, Goldberg M, Son SM, Xu Q, Yang F, Mei Y, Bogatyrev S, Langer R, Anderson DG. Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells. Adv. Funct. Mater. 2009;19(19):3112–3118. [PMC free article] [PubMed]
[24] Bhang SH, Cho SW, Lim JM, Kang JM, Lee TJ, Yang HS, Song YS, Park MH, Kim HS, Yoo KJ, Jang Y, Langer R, Anderson DG, Kim BS. Locally delivered growth factor enhances the angiogenic efficacy of adipose-derived stromal cells transplanted to ischemic limbs. Stem Cells. 2009;27(8):1976–1986. [PubMed]
[25] McGinn AN, Nam HY, Ou M, Hu N, Straub CM, Yockman JW, Bull DA, Kim SW. Bioreducible polymer-transfected skeletal myoblasts for VEGF delivery to acutely ischemic myocardium. Biomaterials. 2011;32(3):942–949. [PMC free article] [PubMed]
[26] Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat. Rev. Drug Discov. 2005;4(7):581–593. [PubMed]
[27] Merdan T, Kopecek J, Kissel T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv. Drug Deliv. Rev. 2002;54(5):715–758. [PubMed]
[28] Abid MR, Guo S, Minami T, Spokes KC, Ueki K, Skurk C, Walsh K, Aird WC. Vascular endothelial growth factor activates PI3K/Akt/forkhead signaling in endothelial cells. Arterioscler. Thromb. Vasc. Biol. 2004;24(2):294–300. [PubMed]
[29] Kumar P, Miller AI, Polverini PJ. p38 MAPK mediates gamma-irradiation-induced endothelial cell apoptosis, and vascular endothelial growth factor protects endothelial cells through the phosphoinositide 3-kinase-Akt-Bcl-2 pathway. J. Biol. Chem. 2004;279(41):43352–43360. [PubMed]
[30] Findley CM, Cudmore MJ, Ahmed A, Kontos CD. VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling. Arterioscler. Thromb. Vasc. Biol. 2007;27(12):2619–2626. [PubMed]
[31] Jia H, Bagherzadeh A, Bicknell R, Duchen MR, Liu D, Zachary I. Vascular endothelial growth factor (VEGF)-D and VEGF-A differentially regulate KDR-mediated signaling and biological function in vascular endothelial cells. J. Biol. Chem. 2004;279(34):36148–36157. [PubMed]
[32] Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, Rafii SS. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J. Exp. Med. 2001;193(9):1005–1014. [PMC free article] [PubMed]
[33] Yoon CH, Hur J, Oh IY, Park KW, Kim TY, Shin JH, Kim JH, Lee CS, Chung JK, Park YB, Kim HS. Intercellular adhesion molecule-1 is upregulated in ischemic muscle, which mediates trafficking of endothelial progenitor cells. Arterioscler. Thromb. Vasc. Biol. 2006;26(5):1066–1072. [PubMed]
[34] Matsuura K, Honda A, Nagai T, Fukushima N, Iwanaga K, Tokunaga M, Shimizu T, Okano T, Kasanuki H, Hagiwara N, Komuro I. Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. J. Clin. Invest. 2009;119(8):2204–2217. [PMC free article] [PubMed]
[35] Nishiwaki Y, Yoshida M, Iwaguro H, Masuda H, Nitta N, Asahara T, Isobe M. Endothelial E-selectin potentiates neovascularization via endothelial progenitor cell-dependent and -independent mechanisms. Arterioscler. Thromb. Vasc. Biol. 2007;27(3):512–518. [PubMed]
[36] Yu JX, Huang XF, Lv WM, Ye CS, Peng XZ, Zhang H, Xiao LB, Wang SM. Combination of stromal-derived factor-1α and vascular endothelial growth factor gene-modified endothelial progenitor cells is more effective for ischemic neovascularization. J. Vasc. Surg. 2009;50(3):608–616. [PubMed]
[37] Yau TM, Kim C, Li G, Zhang Y, Fazel S, Spiegelstein D, Weisel RD, Li RK. Enhanced angiogenesis with multimodal cell-based gene therapy. Ann. Thorac. Surg. 2007;83(3):1110–1119. [PubMed]
[38] Yau TM, Kim C, Li G, Zhang Y, Weisel RD, Li RK. Maximizing ventricular function with multimodal cell-based gene therapy. Circulation. 2005;112(9 Suppl):I123–I128. [PubMed]
[39] Kawamoto A, Katayama M, Handa N, Kinoshita M, Takano H, Horii M, Sadamoto K, Yokoyama A, Yamanaka T, Onodera R, Kuroda A, Baba R, Kaneko Y, Tsukie T, Kurimoto Y, Okada Y, Kihara Y, Morioka S, Fukushima M, Asahara T. Intramuscular transplantation of G-CSF-mobilized CD34(+) cells in patients with critical limb ischemia: a phase I/IIa, multicenter, single-blinded, dose-escalation clinical trial. Stem Cells. 2009;27(11):2857–2864. [PubMed]
[40] Babin-Ebell J, Sievers HH, Charitos EI, Klein HM, Jung F, Hellberg AK, Depping R, Sier HA, Marxsen J, Stoelting S, Kraatz EG, Wagner KF. Transmyocardial laser revascularization combined with intramyocardial endothelial progenitor cell transplantation in patients with intractable ischemic heart disease ineligible for conventional revascularization: preliminary results in a highly selected small patient cohort. Thorac. Cardiovasc. Surg. 2010;58(1):11–16. [PubMed]