Vascular Endothelial Growth Factor Plasmid Research Articles

Novel nanofibrous membrane-supporting stem cell sheets for plasmid delivery and cell activation to accelerate wound healing.

The integration of biomaterials with cells for high overall performances is vitally important in tissue engineering, as scaffold‐free cell sheet lacks enough mechanical performance and cell viability while cell‐free scaffold possesses limited biological functions. In this study, we propose a new strategy to strengthen cell sheets and enhance cell activity for accelerating wound healing based on a novel sandwich structure of cell sheet‐plasmid@membrane‐cell sheet (CpMC). Specifically, the CpMC contains two adipose‐derived stem cell (ADSC) sheets on outer surfaces and an electrospun gelatin/chitosan nanofibrous membrane (NFM) encapsulating vascular endothelial growth factor (VEGF) plasmids in between. The physicochemical properties of NFM including swelling, stiffness, strength, elasticity, and biodegradation can be tailored by simply adjusting the ratio between gelatin and chitosan to be 7:3 which is optimal for most effectively supporting ADSCs adhesion and proliferation. The swelling/biodegradation of NFM mediates the sustained release of encapsulated VEGF plasmids into adjacent ADSCs, and NFM assists VEGF plasmids to promote the differentiation of ADSCs into endothelial, epidermal, and fibroblast cells, in support of the neoangiogenesis and regeneration of cutaneous tissues within 2 weeks. The proposed membrane‐supporting cell sheet strategy provides a new route to tissue engineering, and the developed CpMC demonstrates a high potential for clinical translation.

Preparation of Double-Layer Nanoparticles for Inhibitory Efficiency in Vascular Restenosis via Balloon Infusion

ABSTRACTThe effect of double-layer nanoparticles (DNPs) on vascular restenosis was investigated in this study, which were prepared by combining vascular endothelial growth factor (VEGF) and paclitaxel (PTX). Different blank NPs (Blank), VEGF NPs, PTX-NPs single-layer NPs (PTX-NPs), and DNPs were prepared with VEGF, PTX, polylactic-polyglycolic acid (PLGA), and polyvinyl alcohol (PVA), so as to analyze the cytotoxicity of NPs. Besides, different NPs were compared in terms of the characterization, encapsulation efficiency (EE), in vitro release (IVR), and cell proliferation inhibition ability. 30 adult New Zealand white rabbits were selected to construct a balloon injury model (BIM). Then, they were divided into 5 groups according to different treatment NPs, including a model group (BIM), a Blank group (BIM + Blank NPs); a PTX-NP group (BIM + PTX-NPs), a VEGF-NPs group (BIM + VEGF-NPs), and DNPs group (BIM + DNPs), with 6 rabbits in each group. There was a comparison on the histology, arterial stenosis, hyperplasia index (HI), and intimal thickness (IT) of arterial vessels from different groups, and immunohistochemistry was employed to analyze the changes of VEGF and C-reactive protein (CRP) protein expression levels in arterial tissues of different groups. The results showed that the cell survival rate (SR) was 88.69 ±3.17% and 90.64 ± 1.86% in turn when PLGA and PVA were both 100 ¿tg/mL. The polydispersity index (PDI), minimal Zeta potential (ZP), and average particle size (APS) of DNPs were 0.16 ±0.02, -8.45 ±0.07, and 242.26 ±2.58 nm, respectively. The drug loading (DL) of PTX and plasmid VEGF 165 in DNPs were 29.33% and 4.93%, respectively. The release of PTX from DNPs on the 30th day was close to 50%, and the cumulative release of deoxyribonucleic acid (DNA) was close to 75%. There was no obvious difference in the cell SR within 7 days after DNPs were added into Chinese Hamster ovary (CHO) cells and vascular smooth muscle cells (VSMC) compared with the control group (P > 0.05). The arterial stenosis, HI, and IT of the DNPs group were greatly lower than those of the model group (P < 0.05). The expression of VEGF protein in arterial tissues of DNPs group was higher in contrast to the model group (P < 0.01), while the expression of CRP protein was lower than that of the model group (P < 0.05). It showed that the DNPs combined with VEGF and paclitaxel administered by balloon infusion could obviously inhibit the vascular restenosis by increasing the expression of VEGF and reducing the expression of CRP, which provided a reference for the clinical prevention and treatment of postoperative vascular restenosis.

Gene activated scaffolds incorporating star-shaped polypeptide-pDNA nanomedicines accelerate bone tissue regeneration in vivo.

Increasingly, tissue engineering strategies such as the use of biomaterial scaffolds augmented with specific biological cues are being investigated to accelerate the regenerative process. For example, significant clinical challenges still exist in efficiently healing large bone defects which are above a critical size. Herein, we describe a cell-free, biocompatible and bioresorbable scaffold incorporating a novel star-polypeptide biomaterial as a gene vector. This gene-loaded scaffold can accelerate bone tissue repair in vivo in comparison to a scaffold alone at just four weeks post implantation in a critical sized bone defect. This is achieved via the in situ transfection of autologous host cells which migrate into the implanted collagen-based scaffold via gene-loaded, star-shaped poly(l-lysine) polypeptides (star-PLLs). In vitro, we demonstrate that star-PLL nanomaterials designed with 64 short poly(l-lysine) arms can be used to functionalise a range of collagen based scaffolds with a dual therapeutic cargo (pDual) of the bone-morphogenetic protein-2 plasmid (pBMP-2) and vascular endothelial growth factor plasmid (pVEGF). The versatility of this polymeric vector is highlighted in its ability to transfect Mesenchymal Stem Cells (MSCs) with both osteogenic and angiogenic transgenes in a 3D environment from a range of scaffolds with various macromolecular compositions. In vivo, we demonstrate that a bone-mimetic, collagen-hydroxyapatite scaffold functionalized with star-PLLs containing either 32- or 64- poly(l-lysine) arms can be used to successfully deliver this pDual cargo to autologous host cells. At the very early timepoint of just 4 weeks, we demonstrate the 64-star-PLL-pDual functionalised scaffold as a particularly efficient platform to accelerate bone tissue regeneration, with a 6-fold increase in new bone formation compared to a scaffold alone. Overall, this article describes for the first time the incorporation of novel star-polypeptide biomaterials carrying two therapeutic genes into a cell free scaffold which supports accelerated bone tissue formation in vivo.

Enhancement of sciatic nerve regeneration with dual delivery of vascular endothelial growth factor and nerve growth factor genes

BackgroundPeripheral nerve injury is one common clinical disease worldwide, in which sciatic nerve is anatomically the most challenging to regenerate given its length and large cross-sectional area. For the present, autologous nerve grafting remains to be the most ideal strategy when treating with sciatic nerve injury. However, this method sacrifices healthy nerves and requires highly intensive surgery, still calling for other advanced alternatives for nerve grafting.ResultsIn this study, we utilized previously well-established gene delivery system to dually deliver plasmid DNA (pDNA) encoding vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), exploring therapeutics for sciatic nerve injury. Low-molecular-weight branched polyethylenimine (bPEI) was constructed as the backbone structure of gene vectors, and it was further crosslinked to synthesize degradable polycations via the conjugation of dialdehydes. Potential synergistic effect between VEGF and NGF proteins were observed on rat sciatic nerve crush injury model in this study.ConclusionsWe concluded that dual delivery of plasmid VEGF and NGF as gene therapy could enhance sciatic nerve regeneration.

Mechanism of GPR119 in regulating lipid metabolism and anti-atherosclerosis by hypoxia-inducible factor-1α/ vascular endothelial growth factor pathway

Objective To investigate the effect and mechanism of G protein coupled receptor 119 (GPR119) in regulating lipid metabolism. Methods (1) Macrophage THP-1 was induced by oxidized low density lipoprotein (oxLDL) to the formation of lipid foam cells, protein expression of GPR119, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) were detected by Western blotting. (2) Constructing GPR119 over-expressed and low-expressed plasmids, the plasmids were transfected into THP-1 cells which induced by oxLDL. The lipid content in macrophages was observed by oil red O staining. Cholesterol efflux was detected by liquid scintillation counter. The mRNA and protein expressions of HIF-1α, VEGF were detected by reverse transcription PCR and Western blotting. (3) Constructing GPR119, HIF-1α, and VEGF over-expressed plasmids, then co-transfection of GPR119 and HIF-1α/VEGF plasmids. The lipid content in macrophages was observed by oil red O staining. Cholesterol efflux was detected by liquid scintillation counter. Results Compared with the control group, the lipid droplets were densely distributed in macrophages, with a large number and volume. The protein expression of GPR119 was significantly decreased and HIF-1α, VEGF were significantly increased in macrophages induced by oxLDL (P<0.05). After over-expression of GPR119, the lipid droplets were sparsely distributed and the number was significantly reduced in macrophages, the lipid droplets were mostly located in the area around the cells. The cholesterol efflux was significantly increased (P<0.01). The mRNA and protein expressions of HIF-1α and VEGF were significantly decreased (P<0.01). On the contrary, in the GPR119 inhibition group, the lipid droplets were densely distributed in macrophages, with a large number and volume. The lipid droplets even covered the nuclei. The cholesterol efflux was significantly reduced (P<0.05). The mRNA and protein expression of HIF-1α, VEGF were significantly increased (P<0.05). After GPR119 were co-expressed with HIF-1α and VEGF, the number of lipid droplets was increased, lipid droplets were dense and bulky in oxLDL-induce macrophages. The cholesterol efflux was inhibited. Conclusion GPR119 can regulate lipid metabolism and possibly by down-regulating the expression of HIF-1α and VEGF. Key words: GPR119; Lipid metabolism; Atherosclerosis; HIF-1α; VEGF

Reduction-Responsive Nucleic Acid Delivery Systems To Prevent In-Stent Restenosis in Rabbits.

Cardiovascular and cerebrovascular ischemic diseases seriously affect human health. Endovascular stent placement is an effective treatment but always leads to in-stent restenosis (ISR). Gene-eluting stent, which combines gene therapy with stent implantation, is a potential method to prevent ISR. In this study, an efficient gene-eluting stent was designed on the basis of one new nucleic acid delivery system to decrease the possibility of ISR. The reduction-responsive branched nucleic acid vector (SKP) with low cytotoxicity was first synthesized via ring-opening reaction. The impressive in vitro transfection performances of SKP were proved using luciferase reporter, enhanced green fluorescent protein plasmid, and vascular endothelial growth factor plasmid (pVEGF). Subsequently, SKP/pVEGF complexes were coated on the surfaces of pretreated clinical stents to construct gene-eluting stents (S-SKP/pVEGF). Antirestenosis performance of S-SKP/pVEGF was evaluated via implanting stents into rabbit aortas. S-SKP/pVEGF could lead to the localized upregulation of VEGF proteins, improve the progress of re-endothelialization, and inhibit the development of ISR in vivo. Such efficient pVEGF-eluting stent with responsive nucleic acid delivery systems is very promising to prevent in-stent restenosis of cerebrovascular diseases.

Biodegradable Carriers for Delivery of VEGF Plasmid DNA for the Treatment of Critical Limb Ischemia.

The safe and efficient delivery of therapeutic nucleic acid is a prerequisite for an effective DNA therapy. In this study, we condensed the low molecular weight polyethylenimine (PEI, 1.8k Da) with 2,6-pyridinedicarboxaldehyde (PDA), both of which are degradable in vivo, to synthesize a biodegradable polycationic material (PDAPEI) to deliver vascular endothelial growth factor (VEGF) plasmid DNA (pDNA). Particle size and zeta potential of this novel degradable PEI derivatives-pDNA nanoparticle were investigated and in vitro cytotoxicity was estimated on human umbilical vein endothelial cells (HUVECs). Using pDNA-encoding VEGF-A and green fluorescence protein (GFP), we also checked transfection efficiency of the vector (PDAPEI) and found its excellent performance at 40 w/w ratio. We successfully established peripheral ischemia animal model on C57/BL6J mice to evaluate the therapeutic effect of PDAPEI/pVEGF-A polyplex system on ischemic disease and a conclusion was made that PDAPEI is a promising gene vector in the treatment of peripheral ischemic artery disease (PAD).

Bilayered Nanoparticles with Sequential Release of VEGF Gene and Paclitaxel for Restenosis Inhibition in Atherosclerosis.

Complete reendothelialization followed by inhibition of smooth muscle cell (SMC) proliferation is considered as an effective therapeutic option to prevent restenosis. We have designed poly(lactide-co-glycolide)-loaded bilayered nanoparticles (NPs) with the ability to sequentially release vascular endothelial growth factor (VEGF)-encoding plasmids from the outer layer and paclitaxel (PTX) from the core to promote endothelial regeneration as well as prevent restenosis. Comparing with conventional NPs, which release VEGF plasmid and PTX simultaneously, we expect that the bilayered NPs could release the VEGF plasmid more rapidly, followed by a delayed release of PTX, resulting in an efficient VEGF gene transfection, which ideally could promote reendothelialization and inhibit excessive SMC growth. Indeed, in the present study, we have observed efficient gene transfection using a model plasmid as well as cell growth attenuation in vitro using Chinese hamster ovary cells. Therapeutic efficacy of the bilayered NPs on restenosis was further evaluated in vivo using a rabbit model of atherosclerosis. The bilayered NPs were administered locally via balloon angioplasty to the injured aortic wall through perfusion. Twenty-eight days after the NP administration, rabbits treated with the bilayered NPs exhibited rapid reendothelialization and inhibition of restenosis, as demonstrated by histological analysis. Increased level of VEGF and decreased level of C-reactive protein, a biological marker that is closely related to atherosclerosis, were also observed from animals treated with the bilayered NPs, implicating ameliorated atherosclerosis. Our results suggest that the VEGF plasmid-/PTX-loaded bilayered NPs exert a beneficial impact on atherosclerotic restenosis by sequentially releasing VEGF and PTX in vivo.

Exogenous vascular endothelial growth factor delivery prior to endothelial precursor cell transplantation in orthotopic liver transplantation-induced hepatic ischemia/reperfusion injury.

Vascular endothelial growth factor (VEGF) promotes angiogenesis in vivo. We hypothesized that exogenous delivery of VEGF prior to bone marrow-derived endothelial precursor cell (EPC) transplantation may improve orthotopic liver transplantation (OLT)-induced hepatic ischemia/reperfusion injury (HIRI). OLT between Sprague Dawley donor rats and inbred LEW Wistar recipient rats was performed in 6 experimental groups to comparatively assess the effects of the VEGF gene: an untreated normal control group, a surgical control group, a liposomal control group, a VEGF group receiving only the liposome-encapsulated VEGF plasmid, an EPC group receiving only EPCs, and an EPC+VEGF group receiving the liposome-encapsulated VEGF plasmid followed by EPCs. VEGF plasmid delivery to liver tissue, endogenous VEGF, and vascular endothelial growth factor receptor (VEGFR) expression, liver transaminase levels, hepatocellular injury levels, apoptosis, apoptotic biomarkers, hepatotrophic mitogens, angiogenesis, and nitric oxide synthase (NOS) activity were assayed after OLT. Exogenous VEGF gene delivery prior to EPC transplantation significantly increased endogenous VEGF and VEGFR expression, significantly reduced liver transaminase levels, significantly reduced hepatocellular injury levels, significantly reduced hepatic apoptosis levels, and significantly reduced several apoptotic biomarkers (ie, B cell lymphoma 2-associated X protein/B cell lymphoma 2 ratio, caspase 3 activity, and heat shock protein 70 expression) in post-OLT-induced HIRI. Moreover, VEGF gene delivery prior to EPC transplantation significantly increased hepatotrophic mitogen expression (ie, epidermal growth factor, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, and transforming growth factor α), angiogenesis, and NOS activity in post-OLT-induced HIRI. In conclusion, exogenous liposomal delivery of the VEGF gene prior to bone marrow-derived EPC transplantation may be an effective strategy in decreasing OLT-induced HIRI. Liver Transplantation 23 804-812 2017 AASLD.

Neoangiogenesis of human mesenchymal stem cells transfected with peptide-loaded and gene-coated PLGA nanoparticles

Several factors are involved in angiogenesis. To form new blood vessels, we fabricated vehicles carrying an angiogenesis-related peptide (apelin) and gene (vascular endothelial growth factor (VEGF)165) that were internalized by human mesenchymal stem cells (hMSCs). These non-toxic poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) easily entered hMSCs without cytotoxicity. The negatively charged outer surface of PLGA NPs can be easily complexed with highly positively charged polyethylenimine (PEI) to deliver genes into cells. PLGA NPs complexed with PEI could be coated with negatively charged VEGF plasmid DNA and loaded with apelin. The physical characteristics of these PLGA NPs were determined by size distribution, gel retardation, and morphological analyses. Transfection of VEGF-coated apelin-loaded PLGA NPs resulted in the differentiation of hMSCs into endothelial cells and vascular formation in Matrigel in vitro. Following injection of hMSCs transfected with these PLGA NPs into an ischemic hind limb mouse model, these cells differentiated into endothelial cells and accelerated neovascularization.

A chitosan-graft-PEI-eprosartan conjugate for cardiomyocyte-targeted VEGF plasmid delivery in myocardial ischemia gene therapy

A multifunctional copolymer conjugate chitosan-graft-polyethyleneimine-eprosartan (CPE) containing low-molecular-weight chitosan backbone and polyethyleneimine (PEI) arms with eprosartan (ES) conjugated via an amide bond was fabricated as a cardiomyocyte-targeted vascular endothelial growth factor (VEGF) plasmid delivery nanovector. Here, ES was utilised to specifically bind to overexpressed angiotensin II type 1 receptor (AT1R) of cardiomyocytes and strengthen endosomal buffering capacity of CPE. The self-assembled CPE/VEGF complexes exhibited desirable and homogenous particle size, moderate positive charges, advanced gene condensation and protection profiles in vitro. Flow cytometry and ELISA analyses confirmed that ES-targeted function and ES-enhanced buffering capacity induced specific cellular uptake and high transfection of CPE/VEGF complexes in AT1R-overexpressed H9C2 cells. In vivo investigation on myocardial ischemia rat model revealed that CPE/VEGF complexes possessed strong therapeutic effect. These findings suggested that CPE could be an ideal cardiomyocyte-targeted nanovector for effective transfer of VEGF plasmid, and a multifunctional CPE/VEGF delivery system with cardiomyocyte-specific targetability, enhanced endosomal buffering capacity and strong therapeutic effect might be a new promising strategy for effective myocardial ischemia gene therapy.

Magnetic nanosphere-guided site-specific delivery of vascular endothelial growth factor gene attenuates restenosis in rabbit balloon-injured artery

New and efficient strategies to protect endothelium or to enhance endothelial regrowth are important for treatment of restenosis after percutaneous transluminal angioplasty. Magnetic DNA microspheres are used to accelerate vascular endothelial growth factor (VEGF) re-endothelialization and to attenuate intimal hyperplasia in balloon-injured artery. This study aimed to assess DNA-gelatin magnetic nanospheres containing VEGF expression plasmids in vascular restenosis attenuation. Ninety-six rabbits underwent balloon injury and were randomly divided for gene transfer with naked VEGF plasmids (NAK group), magnetic VEGF microspheres (MIC group), and LacZ (CON group: naked LacZ plasmid and LacZ nanosphere subgroups). Serum and tissue VEGF levels were measured. Also, the ratios of intima area to media area were determined to assess neointima formation. Microsphere gene delivery through the artery by a magnet resulted in VEGF overexpression in transfected arterial segments. Tissue VEGF integral optical densities were significantly increased in MIC rabbits compared with NAK animals. Serum VEGF was below detection in all animals. X-Gal staining showed higher transfection efficiency in the CON group. The impact of neointimal thickening was evaluated by light microscopy as the ratio of intima area to media area in cross sections. Significant differences in the ratio of intima area to media area were obtained between the NAK group (0.12 ± 0.02, 0.41 ± 0.03, 0.61 ± 0.05, and 0.72 ± 0.04 at 1, 2, 3, and 4 weeks, respectively) and the MIC group (0.06 ± 0.03, 0.20 ± 0.05, 0.25 ± 0.04, and 0.26 ± 0.03 at 1, 2, 3, and 4 weeks, respectively) at 2, 3, and 4 weeks (P < .05). Intra-arterial VEGF gene delivery by magnetic microspheres significantly increased DNA stability, transfection efficiency, and targeting specificity, resulting in exogenous VEGF overexpression and attenuated intimal hyperplasia in balloon-injured artery.

Prevention of distal flap necrosis in a rat random skin flap model by gene electro transfer delivering VEGF(165) plasmid.

Background Therapeutic delivery of angiogenic growth factors is a promising approach for treating ischemia observed in skin flaps and chronic wounds.Several studies have demonstrated that vascular endothelial growth factor(VEGF) helps mitigate skin flap necrosis by facilitating angiogenesis. The present study aimed to demonstrate an electrically-mediated nonviral gene delivery approach using a non-invasive multi-electrode array (MEA) for effective treatment of ischemic skin flaps.Methods We used a standard random dorsal skin flap model in rats. The study aimed to determine the optimal treatment sites on the skin flap, optimal plasmid dose and timing of the treatment for preventing distal flap necrosis.Results We determined that two treatment sites on the ischemic flap with a plasmid dose of 50-100 μg per treatment site proved adequate to prevent 95% flap necrosis, and that this was significantly better than the no treatment or injection only group. A 2-day window was critical to deliver the VEGF to achieve flap survival and prevent necrosis. Histological examination demonstrated minimal electro transfer associated tissue damage.Conclusions Our results demonstrate that MEA can be used as a non-invasive physical gene delivery method for plasmid VEGF, resulting in a significant reduction of necrosis in ischemic wounds. We propose that this method could be translated into a potential therapeutic approach to deliver growth factors to prevent ischemia in cases of chronic wounds, burns and skin flap necrosis.

Topical Single-dose Vascular Endothelial Growth Factor has No Effect on Soft Tissue Healing.

Background:Vascular endothelial growth factor (VEGF) production in dermal wounds has been evaluated for evidence that it plays a probable role in wound healing. Events such as increased vascular permeability and concentration of inflammatory cells on the site of injury, produced by VEGF, were linked to tissue repair.Aim:The present study aimed to evaluate the effects of single-dose topical administration of VEGF on wound healing.Materials and Methods:A total of 30 male Wistar albino rats weighing 250-280 g were used in this study. In addition, 2-cm-long skin incisions were created over bilaterally exposed skin of the tibia region in each rat. VEGF plasmid 2 μg was administered locally into the right side wound bed of each animal. No other procedure besides skin closure was administered on the left side. To determine histologic assessments, skin samples were obtained from six anesthetized rats at each interval (4, 8, 12, 16 and 30 days) through excisional biopsy. The tissues were fixed in 10% neutral-buffered formalin for 1 week and then embedded in paraffin wax. Transverse sections of the embedded tissue 5-7 μm thick were stained with hematoxylin and eosin (H and E).Results:There was no significant difference regarding necrosis, epithelialization, inflammation, fibroblast activity, ulcerative formation, or hemorrhage between experimental and control groups. No statistically significant difference was found between the groups regarding granulation tissue formation and epidermal thickness.Conclusion:The administration method and dosage of VEGF is a major factor in terms of its effectiveness. The results of the present study did not evaluate the effectiveness of single-dose 2 μg topical administration of VEGF; however, various doses of VEGF plasmid should be tested in future studies in order to provide beneficial effects from topical administration of VEGF.

High Doses of Vascular Endothelial Growth Factor 165 Safely, but Transiently, Improve Myocardial Perfusion in No-Option Ischemic Disease

Gene therapy can induce angiogenesis in ischemic tissues. The aim of this study was to assess safety, feasibility, and results, both clinical and on myocardial perfusion, of gene therapy in refractory angina. This was a phase I/II, prospective, temporal-controlled series, clinical trial. Thirteen patients were maintained for minimum 6 months under optimized clinical management, and then received intramyocardial injections of 2000 μg plasmid vascular endothelial growth factor 165 and were followed by single-photon emission computed tomography (SPECT), treadmill tests, Minnesota quality of life questionnaire (QOL), and New York Heart Association (NYHA) functional plus Canadian Cardiovascular Society (CCS) angina classifications. There were no deaths, early or late. During the optimized clinical treatment, we observed worsening of rest ischemia scores on SPECT (p<0.05). After treatment, there was a transitory increase in myocardial perfusion at the third-month SPECT under stress (pre-operative [pre-op] 18.38 ± 7.51 vs. 3 months 15.31 ± 7.30; p<0.01) and at the sixth month under rest (pre-op 13.23 ± 7.98 vs. 6 months: 16.92 ± 7.27; p<0.01). One year after, there were improvements in treadmill test steps (pre-op 2.46 ± 2.07 vs.12 months 4.15 ± 2.23; p<0.01) and oxygen consumption (pre-op 7.66 ± 4.47 vs.12 months 10.89 ± 4.65; p<0.05), QOL (pre-op 48.23 ± 18.35 vs.12 months 28.31 ± 18.14; p<0.01) scores, and CCS (pre-op 3 [3-3.5] vs.12 months 2 [1-2.5]; p<0.01) and NYHA (pre-op 3 [3-3] vs. 2 [2-2] vs. 12 months 2 [1-2]; p<0.01) classes. Gene therapy demonstrated to be feasible and safe in this advanced ischemic cardiomyopathy patient sample. There were improvements in clinical evaluation parameters, and a transitory increase in myocardial perfusion detectable by SPECT scintigraphy. NCT00744315 http://clinicaltrials.gov/

Synergistically Combined Gene Delivery for Enhanced VEGF Secretion and Antiapoptosis

With current pharmacological treatments, preventing the remodeling of the left ventricle and the progression to heart failure is a difficult task. Gene therapy is considered to provide a direct treatment to the long-term complications of ischemic heart diseases. Although current gene therapies that use single molecular targets seem potentially possible, they have not achieved success in the treatment of ischemic diseases. With an efficient polymeric gene carrier, PAM-ABP, we designed a synergistically combined gene-delivery strategy to enhance vascular endothelial growth factor (VEGF) secretion and to prolong its antiapoptotic effects. A hypoxia-inducible plasmid expressing both hypoxia-inducible heme oxygenase-1 (HO-1) and the Src homology domain-2 containing tyrosine phosphatase-1 microRNA (miSHP-1) as well as a hypoxia-responsive VEGF plasmid were combined in this study. The positive feedback circuit between HO-1 and VEGF and the negative regulatory role of SHP-1 in angiogenesis enhance VEGF secretion synergistically. The synergy in VEGF secretion as a consequence of the gene combination and prolonged HO-1 activity was confirmed in hypoxic cardiomyocytes and cardiomyocyte apoptosis under hypoxia and was decreased synergistically. These results suggest that the synergistic combination of VEGF, HO-1, and miSHP-1 may be promising for the clinical treatment of ischemic diseases.

High‐dose plasmid‐mediated VEGF gene transfer is safe in patients with severe ischemic heart disease (Genesis‐I). A phase I, open‐label, two‐year follow‐up trial

We aimed to assess safety and, secondarily, the efficacy of intramyocardial high-dose plasmid-vascular endothelial growth factor (VEGF) 165 (pVEGF165) gene transfer in no-option patients with coronary artery disease (CAD). Controlled trials of pVEGF165 in CAD have shown little benefit. One possible reason is shortness of dosage. We have shown in large mammalian models of chronic myocardial ischemia and acute myocardial infarction that intramyocardial pVEGF165 at doses significantly higher than those used in recent phase II trials is safe and efficacious on myocardial perfusion, left ventricular function, and infarct size limitation. Using an injection catheter, 10 patients with severe CAD not amenable for revascularization received 10 intramyocardial injections of 0.38 mg (total dose, 3.8 mg) pVEGF165 in zones exhibiting myocardial ischemia, as assessed by combined stress 99mTc-sestamibi single-photon emission computed tomography and stress echocardiography. No serious adverse events related to either VEGF or the injection procedure occurred over the 2-year follow-up. One patient suffered femoral artery thrombosis after a follow-up coronary angiography, successfully resolved with medical treatment. Six patients suffered uncomplicated coronary ischemic events during the second year follow-up. Angina functional class decreased from 2.6 ± 0.2 to 1.2 ± 0.3 (mean ± SEM, P < 0.05), quality of life increased from 56.9 ± 3.2 to 82.6 ± 2.4 (P < 0.05), the summed difference score of myocardial perfusion decreased from 13.4 ± 2 to 7.7 ± 1.8 (P < 0.04), and stress ejection fraction did not change (44.2 ± 3.6% to 47.8 ± 3.1%, P = NS). High-dose intramyocardial pVEGF165 is safe at 2 years follow-up in patients with severe CAD. The efficacy results observed must be taken cautiously given the uncontrolled, open-label study design.

Post-translational regulation of a hypoxia-responsive VEGF plasmid for the treatment of myocardial ischemia

Vascular endothelial growth factor (VEGF) gene therapy to promote therapeutic angiogenesis has been advanced as an alternative treatment for myocardial ischemia. The unregulated expression of VEGF and the use of viral vectors, however, have slowed the clinical development of angiogenic gene therapy. The development of clinically beneficial angiogenic gene therapy requires a disease-specific gene expression system and an efficient non-viral gene carrier. To address these requirements, we developed a new post-translationally regulated hypoxia-responsible VEGF plasmid, pβ-SP-ODD-VEGF, and a dendrimer-type bio-reducible polymer, PAM-ABP. The efficacy of VEGF gene therapy with the PAM-ABP/pβ-SP-ODD-VEGF was evaluated and compared to the RTP-VEGF plasmid, a previously constructed hypoxia-inducible plasmid, in an ischemia/reperfusion (I/R) rat model. Cine magnetic resonance imaging was used to analyze the ischemia/reperfusion rats treated with either the PAM-ABP/pβ-SP-ODD-VEGF or the PAM-ABP/RTP-VEGF. The PAM-ABP/pβ-SP-ODD-VEGF treatment more effectively protected cardiomyocytes against apoptosis, preserved left ventricular (LV) function, and prevented LV remodeling compared to the PAM-ABP/RTP-VEGF-treated rats. These results suggest that the pβ-SP-ODD-VEGF with PAM-ABP may be efficacious in the treatment of acute ischemic heart disease.

Post-translational regulated and hypoxia-responsible VEGF plasmid for efficient secretion

Gene therapy using angiogenic genes has emerged as a potentially viable alternative treatment strategy for myocardial ischemia. Non-specific expression of angiogenic genes, however, may result in side effects, including the growth of occult tumors. Regulation of gene expression may help to avoid the occurrence of these side effects. In this study, a plasmid expressing vascular endothelial growth factor (VEGF) was constructed with an oxygen dependent degradation (ODD) domain and a secretion signal peptide (SP) in order to stabilize the VEGF protein and facilitate the secretion of VEGF protein, specifically under hypoxic conditions. We found that this plasmid, pβ-SP-ODD-VEGF, expresses the SP-ODD-VEGF protein at increased levels under hypoxic conditions compared to normoxic conditions. Since the size of the ODD domain is almost the same as that of VEGF, the ODD-VEGF fusion protein may have lower secretion efficiency. To address this issue, a furin recognition site was located between the ODD domain and the VEGF site to facilitate elimination of the SP-ODD domain from the fusion protein before its secretion. This optimizes the likelihood that the VEGF secreted from the target cells will be wild-type VEGF. Treatment with a furin inhibitor reduced the secretion efficiency of the VEGF, indicating that furin digestion increases the secretion of VEGF. The secreted wild-type VEGF facilitated the growth of endothelial cells more efficiently under hypoxic conditions than normoxic conditions. These results suggest that this plasmid, pβ-SP-ODD-VEGF, warrants further study as a more efficient form of hypoxia-inducible gene therapy for the treatment of myocardial ischemia.

Promoting melanoma growth and metastasis by enhancing VEGF expression

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The present study was designed to determine the role of VEGF in tumor growth and metastasis. The sequences for the VEGF gene were cloned into expression plasmids and then transfected into melanoma B16 cells. Overexpression of VEGF transfected with expression plasmids or given exogenous VEGF and epidermal growth factor (EGF) significantly enhanced tumor cell proliferation, migration, and invasion. Tumor growth and metastasis of melanoma B16 cells transfected with VEGF plasmid were significantly promoted compared with those of cells administered with exogenous VEGF or EGF. These results indicated that VEGF can be an effective antiangiogenic strategy for melanoma.