Abstract-Optimal angiogenic and lymphangiogenic gene therapy requires knowledge of the best growth factors for each purpose. We studied the therapeutic potential of human vascular

Background-New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options. Methods and Results-We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B 186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B 186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B 186 , whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. Key Words: angiogenesis Ⅲ gene therapy Ⅲ metabolism Ⅲ myocardial infarction S evere coronary heart disease is still a leading cause of death in developed countries in spite of improved management of risk factors and more effective treatments. It is estimated that approximately 5 million people in the United States and the European Union have ischemic heart disease; however, a steadily increasing number of patients fall into a category in which currently available revascularization techniques cannot be applied. This is especially true of elderly patients who have had multiple bypass and stenting operations. 1 It is estimated that these patients represent up to 3% to 5% of all patients in specialty cardiology clinics. Thus, there is a clear need to develop efficient, minimally invasive procedures for the treatment of these no-option patients. Clinical Perspective p 856Therapeutic vascular growth (ie, angiogenesis and arteriogenesis) with genes or proteins has been suggested as an alternative approach for the treatment of these patients. 2 Vascular endothelial growth factors (VEGFs) are potent inducers of vascular growth via binding to 3 tyrosine kinase receptors (VEGFRs). VEGFR-2 is the main regulator of angiogenesis, exerting its function via nitric oxide production, whereas the role of VEGFR-1 is far less defined. 3 VEGF-B 4 and placental growth factor (PlGF) 3 share structural and functional characteristics and bind to VEGFR-1, whereas VEGF-A 5 binds to both VEGFR-1 and VEGFR-2. 846 Circulation

Background— It is unclear what is the most efficient vector and growth factor for induction of therapeutic vascular growth in the heart. Furthermore, the histological nature of angiogenesis and potential side effects caused by different vascular endothelial growth factors (VEGFs) in myocardium have not been documented. Methods and Results— Adenoviruses (Ad) at 2 doses (2×10 11 and 2×10 12 viral particles) or naked plasmids (1 mg) encoding Lac Z control, VEGF-A 165 , or the mature, soluble form of VEGF-D (VEGF-D ΔNΔC ) were injected intramyocardially with the NOGA catheter system into domestic pigs. AdVEGF-D ΔNΔC gene transfer (GT) induced a dose-dependent myocardial protein production, as measured by ELISA, resulting in an efficient angiogenic effect 6 days after the injections. Also, AdVEGF-A 165 produced high gene transfer efficacy, as demonstrated with immunohistochemistry, leading to prominent angiogenesis effects. Despite the catheter-mediated approach, angiogenesis induced by both AdVEGFs was transmural, with maximal effects in the epicardium. Histologically, strongly enlarged α-smooth muscle actin–positive microvessels involving abundant cell proliferation were found in the transduced regions, whereas microvessel density did not change. Myocardial contrast echocardiography and microspheres showed marked increases in perfusion in the transduced areas. VEGF-D ΔNΔC but not matrix-bound VEGF-A 165 was detected in plasma after adenoviral GT. A modified Miles assay demonstrated myocardial edema resulting in pericardial effusion with the higher AdVEGF doses. All effects returned to baseline by 3 weeks. Naked plasmid–mediated GT did not induce detectable protein production or vascular effects. Conclusions— Like AdVEGF-A 165 , AdVEGF-D ΔNΔC GT using the NOGA system produces efficient transmural angiogenesis and increases myocardial perfusion. AdVEGF-D ΔNΔC could be useful for the induction of therapeutic vascular growth in the heart.

Background— For clinically relevant proangiogenic therapy, it would be essential that the growth of the whole vascular tree is promoted. Vascular endothelial growth factor (VEGF) is well known to induce angiogenesis, but its capability to promote growth of larger vessels is controversial. We hypothesized that blood flow remodels vascular growth during VEGF gene therapy and may contribute to the growth of large vessels. Methods and Results— Adenoviral (Ad) VEGF or LacZ control gene transfer was performed in rabbit hindlimb semimembranous muscles with or without ligation of the profound femoral artery (PFA). Contrast-enhanced ultrasound and dynamic susceptibility contrast MRI demonstrated dramatic 23- to 27-fold increases in perfusion index and a strong decrease in peripheral resistance 6 days after AdVEGF gene transfer in normal muscles. Enlargement by 20-fold, increased pericyte coverage, and decreased alkaline phosphatase and dipeptidyl peptidase IV activities suggested the transformation of capillaries toward an arterial phenotype. Increase in muscle perfusion was attenuated, and blood vessel growth was more variable, showing more sprouting angiogenesis and formation of blood lacunae after AdVEGF gene transfer in muscles with ligated PFA than in normal muscles. Three-dimensional ultrasound reconstructions and histology showed that the whole vascular tree, including large arteries and veins, was enlarged manifold by AdVEGF. Blood flow was normalized and enlarged collaterals persisted in operated limbs 14 days after AdVEGF treatment. Conclusions— This study shows that (1) blood flow modulates vessel growth during VEGF gene therapy and (2) VEGF overexpression promotes growth of arteries and veins and induces capillary arterialization leading to supraphysiological blood flow in target muscles.

Background-Reactive oxygen species (ROS) play a major role in vascular inflammation and pathophysiology of many vascular diseases such as atherosclerosis and injury-induced neointima formation after balloon angioplasty. Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor orchestrating antioxidant and cytoprotective responses on oxidative and electrophilic stress, and it has been shown to have antiinflammatory effects in vascular cells in vitro. We therefore postulated that Nrf2 gene transfer would have salutary effects on vascular inflammation after angioplasty. Methods and Results-Transduction of vascular smooth muscle cells (VSMCs) with Nrf2-expressing adenovirus increased the expression of several antioxidant enzymes including heme oxygenase-1 (HO-1) compared with ␤-galactosidase (AdLacZ)-transduced controls. Moreover, Nrf2 gene transfer also inhibited vascular smooth muscle cell (VSMC) proliferation, and the effect was partially reversed by the HO inhibitor Sn(IV) protoporphyrin. In vivo, adenoviral gene transfer effectively reduced oxidative stress determined by antibody staining against oxidized epitopes of LDL, as well as inhibited vascular inflammation assessed by the macrophage cell count and monocyte chemoattractant protein-1 (MCP-1) staining. However, the antiproliferative effects of Nrf2 in vivo were counterbalanced with diminished apoptosis in neointimal VSMCs, resulting in no change in neointimal hyperplasia. Conclusions-Nrf2

Objective Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. Approach and Results Two transgenic mouse strains with lymphatic insufficiency (sVEGFR3 and Chy) were crossed with atherosclerotic mice (LDLR−/−/ApoB100/100) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3 × LDLR−/−/ApoB100/100 mice and Chy × LDLR−/−/ApoB100/100 mice had higher plasma cholesterol levels compared to LDLR−/−/ApoB100/100 control mice during both normal chow diet (16.3 mmol/l and 13.7 mmol/l vs. 8.2 mmol/l, respectively) and Western-type high fat diet (e.g. after 2 weeks of fat diet 45.9 mmol/l and 42.6 mmol/l vs. 30.2 mmol/l, respectively). Cholesterol and triglyceride levels in very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3 × LDLR−/−/ApoB100/100 mice progressed faster than in control mice (e.g. intermediate cohort mice at 6 weeks 18.3% vs. 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3 × LDLR−/−/ApoB100/100 mice and Chy × LDLR−/−/ApoB100/100 mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% vs. 7.45% of podoplanin positive vessels, respectively). Conclusions We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels and enhanced atherogenesis.

AimsWe evaluated for the first time the effects of angiogenic and lymphangiogenic AdVEGF-DΔNΔC gene therapy in patients with refractory angina.Methods and resultsThirty patients were randomized to AdVEGF-DΔNΔC (AdVEGF-D) or placebo (control) groups. Electromechanical NOGA mapping and radiowater PET were used to identify hibernating viable myocardium where treatment was targeted. Safety, severity of symptoms, quality of life, lipoprotein(a) [Lp(a)] and routine clinical chemistry were measured. Myocardial perfusion reserve (MPR) was assessed with radiowater PET at baseline and after 3- and 12-months follow-up. Treatment was well tolerated. Myocardial perfusion reserve increased significantly in the treated area in the AdVEGF-D group compared with baseline (1.00 ± 0.36) at 3 months (1.31 ± 0.46, P = 0.045) and 12 months (1.44 ± 0.48, P = 0.009) whereas MPR in the reference area tended to decrease (2.05 ± 0.69, 1.76 ± 0.62, and 1.87 ± 0.69; baseline, 3 and 12 months, respectively, P = 0.551). Myocardial perfusion reserve in the control group showed no significant change from baseline to 3 and 12 months (1.26 ± 0.37, 1.57 ± 0.55, and 1.48 ± 0.48; respectively, P = 0.690). No major changes were found in clinical chemistry but anti-adenovirus antibodies increased in 54% of the treated patients compared with baseline. AdVEGF-D patients in the highest Lp(a) tertile at baseline showed the best response to therapy (MPR 0.94 ± 0.32 and 1.76 ± 0.41 baseline and 12 months, respectively, P = 0.023).ConclusionAdVEGF-DΔNΔC gene therapy was safe, feasible, and well tolerated. Myocardial perfusion increased at 1 year in the treated areas with impaired MPR at baseline. Plasma Lp(a) may be a potential biomarker to identify patients that may have the greatest benefit with this therapy.

Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67–4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.