Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno-associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia-inducible factor-1alpha (HIF-1alpha). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF-1alpha markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF-1alpha overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF-1alpha treated muscles was significantly enhanced when compared to the controls and not further improved by co-expression of the arteriogenic growth factors angiopoietin-1 or platelet-derived growth factor-B. Our results show that AAV-mediated transduction of a stabilized form of HIF-1alpha can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF-1alpha should thus offer a potent alternative for pro-angiogenic gene therapy.

Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy. Whereas therapies targeting vascular endothelial growth factor A (VEGFA) can suppress leakage, side-effects include vascular rarefaction and geographic atrophy. By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 (VEGFR2)-induced vascular permeability, we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding. Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy, similarly sized lesions leak less in mutant mice, separating regulation of permeability from angiogenesis. Strikingly, vascular endothelial (VE)-cadherin phosphorylation at the Y685, but not Y658, residue is reduced when VEGFR2 pY949 signaling is impaired. These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage. Therapeutically, targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact.

UPARANT mitigates laser-induced CNV by inhibiting angiogenesis and inflammation through an action on transcription factors encoding angiogenesis and inflammatory genes. The finding that UPARANT is effective against CNV may help to establish uPAR and its membrane partners as putative targets in the treatment of AMD.

Our data delimitate specific temporal windows during CNV initiation, propagation, maturation, and even recovery in experimental CNV. We show that murine CNV undergoes hypoxia-associated sprouting angiogenesis, and demonstrate involvement of pericytes. Moreover, we have shown expression of HIF-1α to the retinal pigment epithelium surrounding the CNV lesions, together with VEGF upregulation, independently of the HSP response induced by the laser thermal insult.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a largeeyed animal model.

Oxidative stress plays an important role in the pathogenesis of ocular diseases, including hypertensive eye diseases. The beneficial effects of olive oil on cardiovascular diseases might rely on minor constituents. Currently, very little is known about the chemical composition and/or therapeutic effects of the cultivated olive tree’s counterpart, wild olive (also known in Spain as acebuche—ACE). Here, we aimed to analyze the antioxidant and retinoprotective effects of ACE oil on the eye of hypertensive mice made hypertensive via administration of NG-nitro-L-arginine-methyl-ester (L-NAME), which were subjected to a dietary supplementation with either ACE oil or extra virgin olive oil (EVOO) for comparison purposes. Deep analyses of major and minor compounds present in both oils was accompanied by blood pressure monitoring, morphometric analyses, as well as different determinations of oxidative stress-related parameters in retinal layers. Aside from its antihypertensive effect, an ACE oil-enriched diet reduced NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activity/gene/protein expression (with a major implication of NADPH oxidase (NOX)2 isoform) in the retinas of hypertensive mice. Supplementation with ACE oil in hypertensive animals also improved alterations in nitric oxide bioavailability and in antioxidant enzyme profile. Interestingly, our findings show that the use of ACE oil resulted in better outcomes, compared with reference EVOO, against hypertension-related oxidative retinal damage.

Understanding the mechanisms that underlie age-related macular degeneration (AMD) has led to the identification of key molecules. Hypoxia-inducible transcription factors (HIFs) have been associated with choroidal neovascularization and the progression of AMD into the neovascular clinical phenotype (nAMD). HIFs regulate the expression of multiple growth factors and cytokines involved in angiogenesis and inflammation, hallmarks of nAMD. This knowledge has propelled the development of a new group of therapeutic strategies focused on gene therapy. The present review provides an update on current gene therapies in ocular angiogenesis, particularly nAMD, from both basic and clinical perspectives.

Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.