Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP's mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.
Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population, and affects those with type 1 and type 2 diabetes mellitus. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31+ vascular endothelial cells obtained from human PDR fibrovascular membranes (FVM) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVM and angiogenic tufts in the retina of mice with oxygen-induced retinopathy (OIR), suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in OIR, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.
Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.
Vascular endothelial cell growth factor A (VEGF) is a biologically and therapeutically important growth factor because it promotes angiogenesis in response to hypoxia, which underlies a wide variety of both physiological and pathological settings. We report here that both VEGF receptor 2 (VEGFR2)-positive and -negative cells depended on VEGF to endure hypoxia. VEGF enhanced the viability of platelet-derived growth factor receptor α (PDGFRα)-positive and VEGFR2-negative cells by enabling indirect activation of PDGFRα, thereby reducing the level of p53. We conclude that the breadth of VEGF's influence extends beyond VEGFR-positive cells and propose a plausible mechanistic explanation of this phenomenon.
The role of lymphatics in atherosclerosis is not yet understood. Here, we investigate lymphatic growth dynamics and marker expression in atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. The prolymphangiogenic growth factor, VEGF-C, was elevated in atherosclerotic aortic walls. Despite increased VEGF-C, we found that adventitial lymphatics regress during the course of formation of atherosclerosis (P < 0.01). Similar to lymphatic regression, the number of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1(+)) macrophages decreased in the aortic adventitia of apoE(-/-) mice with atherosclerosis (P < 0.01). Intimal lymphatics in the atherosclerotic lesions exhibited an atypical phenotype, with the expression of podoplanin and VEGF receptor 3 (VEGFR-3) but not of LYVE-1 and prospero homeobox protein 1. In the aortas of atherosclerotic animals, we found markedly increased soluble VEGFR-2. We hypothesized that the elevated soluble VEGFR-2 that was found in the aortas of apoE(-/-) mice with atherosclerosis binds to and diminishes the activity of VEGF-C. This trapping mechanism explains, despite increased VEGF-C in the atherosclerotic aortas, how adventitial lymphatics regress. Lymphatic regression impedes the drainage of lipids, growth factors, inflammatory cytokines, and immune cells. Insufficient lymphatic drainage could thus exacerbate atherosclerosis formation. Our study contributes new insights to previously unknown dynamic changes of adventitial lymphatics. Targeting soluble VEGFR-2 in atherosclerosis may provide a new strategy for the liberation of endogenous VEGF-C and the prevention of lymphatic regression.-Taher, M., Nakao, S., Zandi, S., Melhorn, M. I., Hayes, K. C., Hafezi-Moghadam, A. Phenotypic transformation of intimal and adventitial lymphatics in atherosclerosis: a regulatory role for soluble VEGF receptor 2.
The endothelial barrier maintains vascular and tissue homeostasis and modulates many physiological processes, such as angiogenesis. Vascular barrier integrity can be disrupted by a variety of soluble permeability factors, and changes in barrier function can exacerbate tissue damage during disease progression. Understanding endothelial barrier function is critical for vascular homeostasis. Many of the signaling pathways promoting vascular permeability can also be triggered during disease, resulting in prolonged or uncontrolled vascular leak. It is believed that recovery of the normal vasculature requires diminishing this hyperpermeable state. Although the molecular mechanisms governing vascular leak have been studied over the last few decades, recent advances have identified new therapeutic targets that have begun to show preclinical and clinical promise. These approaches have been successfully applied to an increasing number of disease conditions. New perspectives regarding how vascular leak impacts the progression of various diseases are highlighted in this review.
Management of neoangiogenesis remains a high-value therapeutic goal. A recently uncovered association between the DNA damage repair pathway and pathological angiogenesis could open previously unexplored possibilities for intervention. An attractive and novel target is the Eyes absent (EYA) tyrosine phosphatase, which plays a critical role in the repair versus apoptosis decision after DNA damage. This study examines the role of EYA in the postnatal development of the retinal vasculature and under conditions of ischemia-reperfusion encountered in proliferative retinopathies. We find that the ability of the EYA proteins to promote endothelial cell (EC) migration contributes to a delay in postnatal development of the retinal vasculature when Eya3 is deleted specifically in ECs. By using genetic and chemical biology tools, we show that EYA contributes to pathological angiogenesis in a model of oxygen-induced retinopathy. Both in vivo and in vitro, loss of EYA tyrosine phosphatase activity leads to defective assembly of γ-H2AX foci and thus to DNA damage repair in ECs under oxidative stress. These data reveal the potential utility of EYA tyrosine phosphatase inhibitors as therapeutic agents in inhibiting pathological neovascularization with a range of clinical applications.
Expression and activation of vascular endothelial growth factor receptor 2 (VEGFR-2) by VEGF ligands are the main events in the stimulation of pathological angiogenesis. VEGFR-2 expression is generally low in the healthy adult blood vessels, but its expression is markedly increased in the pathological angiogenesis. In this report, we demonstrate that phosducin-like 3 (PDCL3), a recently identified chaperone protein involved in the regulation of VEGFR-2 expression, is required for angiogenesis in zebrafish and mouse. PDCL3 undergoes N-terminal methionine acetylation, and this modification affects PDCL3 expression and its interaction with VEGFR-2. Expression of PDCL3 is regulated by hypoxia, the known stimulator of angiogenesis. The mutant PDCL3 that is unable to undergo N-terminal methionine acetylation was refractory to the effect of hypoxia. The siRNA-mediated silencing of PDCL3 decreased VEGFR-2 expression resulting in a decrease in VEGF-induced VEGFR-2 phosphorylation, whereas PDCL3 over-expression increased VEGFR-2 protein. Furthermore, we show that PDCL3 protects VEGFR-2 from misfolding and aggregation. The data provide new insights for the chaperone function of PDCL3 in angiogenesis and the roles of hypoxia and N-terminal methionine acetylation in PDCL3 expression and its effect on VEGFR-2.
Pathologic ocular neovascularization commonly causes blindness. It is critical to identify the factors altered in pathologically proliferating versus normally quiescent vessels to develop effective targeted therapeutics. MicroRNAs regulate both physiological and pathological angiogenesis through modulating expression of gene targets at the posttranscriptional level. However, it is not completely understood if specific microRNAs are altered in pathologic ocular blood vessels, influencing vascular eye diseases. Here we investigated the potential role of a specific microRNA, miR-150, in regulating ocular neovascularization. We found that miR-150 was highly expressed in normal quiescent retinal blood vessels and significantly suppressed in pathologic neovessels in a mouse model of oxygen-induced proliferative retinopathy. MiR-150 substantially decreased endothelial cell function including cell proliferation, migration, and tubular formation and specifically suppressed the expression of multiple angiogenic regulators, CXCR4, DLL4, and FZD4, in endothelial cells. Intravitreal injection of miR-150 mimic significantly decreased pathologic retinal neovascularization in vivo in both wild-type and miR-150 knockout mice. Loss of miR-150 significantly promoted angiogenesis in aortic rings and choroidal explants ex vivo and laser-induced choroidal neovascularization in vivo. In conclusion, miR-150 is specifically enriched in quiescent normal vessels and functions as an endothelium-specific endogenous inhibitor of pathologic ocular neovascularization.
Pathologic ocular angiogenesis is a leading cause of blindness, influenced by both dysregulated lipid metabolism and inflammation. Retinoic-acid-receptor-related orphan receptor alpha (RORα) is a lipid-sensing nuclear receptor with diverse biologic function including regulation of lipid metabolism and inflammation; however, its role in pathologic retinal angiogenesis remains poorly understood. Using a mouse model of oxygen-induced proliferative retinopathy, we showed that RORα expression was significantly increased and genetic deficiency of RORα substantially suppressed pathologic retinal neovascularization. Loss of RORα led to decreased levels of proinflammatory cytokines and increased levels of antiinflammatory cytokines in retinopathy. RORα directly suppressed the gene transcription of suppressors of cytokine signaling 3 (SOCS3), a critical negative regulator of inflammation. Inhibition of SOCS3 abolished the antiinflammatory and vasoprotective effects of RORα deficiency in vitro and in vivo. Moreover, treatment with a RORα inverse agonist SR1001 effectively protected against pathologic neovascularization in both oxygen-induced retinopathy and another angiogenic model of very-low-density lipoprotein receptor (Vldlr)-deficient (Vldlr (-/-) ) mice with spontaneous subretinal neovascularization, whereas a RORα agonist worsened oxygen-induced retinopathy. Our data demonstrate that RORα is a novel regulator of pathologic retinal neovascularization, and RORα inhibition may represent a new way to treat ocular neovascularization.