Optimal angiogenic and lymphangiogenic gene therapy requires knowledge of the best growth factors for each purpose. We studied the therapeutic potential of human vascular endothelial growth factor (VEGF) family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D as well as a VEGFR-3–specific mutant (VEGF-C ^156S ) using adenoviral gene transfer in rabbit hindlimb skeletal muscle. The significance of proteolytic processing of VEGF-D was explored using adenoviruses encoding either full-length or mature (ΔNΔC) VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands, VEGF-A and VEGF-D ^ΔNΔC , induced the strongest angiogenesis and vascular permeability effects as assessed by capillary vessel and perfusion measurements, modified Miles assay, and MRI. The most significant feature of angiogenesis induced by both VEGF-A and VEGF-D ^ΔNΔC was a remarkable enlargement of microvessels with efficient recruitment of pericytes suggesting formation of arterioles or venules. VEGF-A also moderately increased capillary density and created glomeruloid bodies, clusters of tortuous vessels, whereas VEGF-D ^ΔNΔC –induced angiogenesis was more diffuse. Vascular smooth muscle cell proliferation occurred in regions with increased plasma protein extravasation, indicating that arteriogenesis may be promoted by VEGF-A and VEGF-D ^ΔNΔC . Full-length VEGF-C and VEGF-D induced predominantly and the selective VEGFR-3 ligand VEGF-C ^156S exclusively lymphangiogenesis. Unlike angiogenesis, lymphangiogenesis was not dependent on nitric oxide. The VEGFR-1 ligand VEGF-B did not promote either angiogenesis or lymphangiogenesis. Finally, we found a positive correlation between capillary size and vascular permeability. This study compares, for the first time, angiogenesis and lymphangiogenesis induced by gene transfer of different human VEGFs, and shows that VEGF-D is the most potent member when delivered via an adenoviral vector into skeletal muscle.

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.

Cardiopoietic cells, produced through cardiogenic conditioning of patients’ mesenchymal stem cells, have shown preliminary efficacy. The Congestive Heart Failure Cardiopoietic Regenerative Therapy (CHART-1) trial aimed to validate cardiopoiesis-based biotherapy in a larger heart failure cohort. This multinational, randomized, double-blind, sham-controlled study was conducted in 39 hospitals. Patients with symptomatic ischaemic heart failure on guideline-directed therapy (n = 484) were screened; n = 348 underwent bone marrow harvest and mesenchymal stem cell expansion. Those achieving > 24 million mesenchymal stem cells (n = 315) were randomized to cardiopoietic cells delivered endomyocardially with a retention-enhanced catheter (n = 157) or sham procedure (n = 158). Procedures were performed as randomized in 271 patients (n = 120 cardiopoietic cells, n = 151 sham). The primary efficacy endpoint was a Finkelstein–Schoenfeld hierarchical composite (all-cause mortality, worsening heart failure, Minnesota Living with Heart Failure Questionnaire score, 6-min walk distance, left ventricular end-systolic volume, and ejection fraction) at 39 weeks. The primary outcome was neutral (Mann–Whitney estimator 0.54, 95% confidence interval [CI] 0.47–0.61 [value > 0.5 favours cell treatment], P = 0.27). Exploratory analyses suggested a benefit of cell treatment on the primary composite in patients with baseline left ventricular end-diastolic volume 200–370 mL (60% of patients) (Mann–Whitney estimator 0.61, 95% CI 0.52–0.70, P = 0.015). No difference was observed in serious adverse events. One (0.9%) cardiopoietic cell patient and 9 (5.4%) sham patients experienced aborted or sudden cardiac death. The primary endpoint was neutral, with safety demonstrated across the cohort. Further evaluation of cardiopoietic cell therapy in patients with elevated end-diastolic volume is warranted.

Estimation of right ventricular (RV) performance still remains technically challenging due to its anatomical and functional distinctiveness. The current guidelines for the echocardiographic quantification of RV function recommend using multiple indices to describe the RV in a thorough and comprehensive manner, such as RV index of myocardial performance, tricuspid annular plane systolic excursion, fractional area change, Doppler tissue imaging-derived tricuspid lateral annular systolic velocity (S’-wave), three-dimensional RV ejection fraction (3D RVEF), longitudinal strain (RVLS)/strain rate by speckle-tracking echocardiography (STE). Among these, the last one mentioned here is an innovative and a particularly promising tool that yields more precise information about complex regional and global RV mechanics. STE was initially designed to evaluate left ventricular function, but recently it has been introduced to assess RV performance, which is difficult due to its unique structure and physiology. Many studies have shown that both free wall and 6-segment RVLS present a stronger correlation with the RVEF assessed by cardiac magnetic resonance than conventional parameters and seem to be more sensitive in detecting myocardial dysfunction at an earlier, subclinical stage. Estimation of right ventricular (RV) performance still remains technically challenging due to its anatomical and functional distinctiveness. The current guidelines for the echocardiographic quantification of RV function recommend using multiple indices to describe the RV in a thorough and comprehensive manner, such as RV index of myocardial performance, tricuspid annular plane systolic excursion, fractional area change, Doppler tissue imaging-derived tricuspid lateral annular systolic velocity (S’-wave), three-dimensional RV ejection fraction (3D RVEF), longitudinal strain (RVLS)/strain rate by speckle-tracking echocardiography (STE). Among these, the last one mentioned here is an innovative and a particularly promising tool that yields more precise information about complex regional and global RV mechanics. STE was initially designed to evaluate left ventricular function, but recently it has been introduced to assess RV performance, which is difficult due to its unique structure and physiology. Many studies have shown that both free wall and 6-segment RVLS present a stronger correlation with the RVEF assessed by cardiac magnetic resonance than conventional parameters and seem to be more sensitive in detecting myocardial dysfunction at an earlier, subclinical stage.

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.