Murilo Rodrigues

Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.

Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cβ and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy.

Hypoxic retinal Müller cells promote vascular permeability by HIF-1–dependent up-regulation of angiopoietin-like 4

Significance Ischemic retinopathies include a diverse group of diseases in which immature retinal vasculature or damage to mature retinal vessels leads to retinal ischemia. The anticipated rise in the worldwide prevalence of diabetes will result in a concurrent increase in the number of patients with vision impairment from diabetic eye disease, the most common cause of ischemic retinopathy. We set out to identify novel hypoxia-inducible genes that promote vascular permeability and may therefore play a role in the pathogenesis of diabetic eye disease. We demonstrate that angiopoietin-like 4 (ANGPTL4) is up-regulated by the transcriptional enhancer, hypoxia-inducible factor-1 in hypoxic retinal Müller cells, and can promote vascular permeability. Our findings suggest that ANGPTL4 may be a potential therapeutic target for ischemic retinopathies. Vision loss from ischemic retinopathies commonly results from the accumulation of fluid in the inner retina [macular edema (ME)]. Although the precise events that lead to the development of ME remain under debate, growing evidence supports a role for an ischemia-induced hyperpermeability state regulated, in part, by VEGF. Monthly treatment with anti-VEGF therapies is effective for the treatment of ME but results in a major improvement in vision in a minority of patients, underscoring the need to identify additional therapeutic targets. Using the oxygen-induced retinopathy mouse model for ischemic retinopathy, we provide evidence showing that hypoxic Müller cells promote vascular permeability by stabilizing hypoxia-inducible factor-1α (HIF-1α) and secreting angiogenic cytokines. Blocking HIF-1α translation with digoxin inhibits the promotion of endothelial cell permeability in vitro and retinal edema in vivo. Interestingly, Müller cells require HIF—but not VEGF—to promote vascular permeability, suggesting that other HIF-dependent factors may contribute to the development of ME. Using gene expression analysis, we identify angiopoietin-like 4 (ANGPTL4) as a cytokine up-regulated by HIF-1 in hypoxic Müller cells in vitro and the ischemic inner retina in vivo. ANGPTL4 is critical and sufficient to promote vessel permeability by hypoxic Müller cells. Immunohistochemical analysis of retinal tissue from patients with diabetic eye disease shows that HIF-1α and ANGPTL4 localize to ischemic Müller cells. Our results suggest that ANGPTL4 may play an important role in promoting vessel permeability in ischemic retinopathies and could be an important target for the treatment of ME.

VEGF Secreted by Hypoxic Müller Cells Induces MMP-2 Expression and Activity in Endothelial Cells to Promote Retinal Neovascularization in Proliferative Diabetic Retinopathy

In proliferative diabetic retinopathy (PDR), retinal ischemia promotes neovascularization (NV), which can lead to profound vision loss in diabetic patients. Treatment for PDR, panretinal photocoagulation, is inherently destructive and has significant visual consequences. Therapies targeting vascular endothelial growth factor (VEGF) have transformed the treatment of diabetic eye disease but have proven inadequate for treating NV, prompting exploration for additional therapeutic options for PDR patients. In this regard, extracellular proteolysis is an early and sustained activity strictly required for NV. Extracellular proteolysis in NV is facilitated by the dysregulated activity of matrix metalloproteinases (MMPs). Here, we set out to better understand the regulation of MMPs by ischemia in PDR. We demonstrate that accumulation of hypoxia-inducible factor-1α in Müller cells induces the expression of VEGF, which, in turn, promotes increased MMP-2 expression and activity in neighboring endothelial cells (ECs). MMP-2 expression was detected in ECs in retinal NV tissue from PDR patients, whereas MMP-2 protein levels were elevated in the aqueous of PDR patients compared with controls. Our findings demonstrate a complex interplay among hypoxic Müller cells, secreted angiogenic factors, and neighboring ECs in the regulation of MMP-2 in retinal NV and identify MMP-2 as a target for the treatment of PDR.

Angiopoietin-like 4 is a potent angiogenic factor and a novel therapeutic target for patients with proliferative diabetic retinopathy.

Significance In proliferative diabetic retinopathy (PDR), the most vision-threatening sequela of diabetic eye disease, retinal ischemia leads to increased expression of angiogenic factors that promote neovascularization. Although therapies targeting the potent angiogenic mediator vascular endothelial growth factor have been remarkably successful for the treatment of diabetic macular edema, this approach has not proven sufficient to prevent the development of retinal neovascularization, implicating additional angiogenic factor(s) in PDR pathogenesis. We demonstrate here that angiopoietin-like 4 is a potent angiogenic mediator with markedly increased expression in the eyes of PDR patients. Our studies identify a novel therapeutic target for the treatment of ocular neovascular disease and may have broad implications for the treatment of other diseases dependent on pathologic angiogenesis. Diabetic eye disease is the most common cause of severe vision loss in the working-age population in the developed world, and proliferative diabetic retinopathy (PDR) is its most vision-threatening sequela. In PDR, retinal ischemia leads to the up-regulation of angiogenic factors that promote neovascularization. Therapies targeting vascular endothelial growth factor (VEGF) delay the development of neovascularization in some, but not all, diabetic patients, implicating additional factor(s) in PDR pathogenesis. Here we demonstrate that the angiogenic potential of aqueous fluid from PDR patients is independent of VEGF concentration, providing an opportunity to evaluate the contribution of other angiogenic factor(s) to PDR development. We identify angiopoietin-like 4 (ANGPTL4) as a potent angiogenic factor whose expression is up-regulated in hypoxic retinal Müller cells in vitro and the ischemic retina in vivo. Expression of ANGPTL4 was increased in the aqueous and vitreous of PDR patients, independent of VEGF levels, correlated with the presence of diabetic eye disease, and localized to areas of retinal neovascularization. Inhibition of ANGPTL4 expression reduced the angiogenic potential of hypoxic Müller cells; this effect was additive with inhibition of VEGF expression. An ANGPTL4 neutralizing antibody inhibited the angiogenic effect of aqueous fluid from PDR patients, including samples from patients with low VEGF levels or receiving anti-VEGF therapy. Collectively, our results suggest that targeting both ANGPTL4 and VEGF may be necessary for effective treatment or prevention of PDR and provide the foundation for studies evaluating aqueous ANGPTL4 as a biomarker to help guide individualized therapy for diabetic eye disease.

Hypoxia-inducible factor 1 upregulation of both VEGF and ANGPTL4 is required to promote the angiogenic phenotype in uveal melanoma

Purpose Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM). While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated. Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM. Experimental Design UM cells were examined for expression of HIF-1α, VEGF, and ANGPTL4. Their contribution to the angiogenic potential of UM cells was assessed using the endothelial cell tubule formation and directed in vivo angiogenesis assays. These results were corroborated in tissue from UM animal models and in tissue from patients with UM. Results Inhibition of VEGF partially reduced tubule formation promoted by conditioned medium from UM cells. Inhibition of ANGPTL4, which was highly expressed in hypoxic UM cells, a UM orthotopic transplant model, a UM tumor array, and vitreous samples from UM patients, inhibited the angiogenic potential of UM cells in vitro and in vivo; this effect was additive to VEGF inhibition. Conclusions Targeting both ANGPTL4 and VEGF may be required for the effective inhibition of angiogenesis in UM.

Ranibizumab in diabetic macular edema

By 2050 the prevalence of diabetes will more than triple globally, dramatically increasing the societal and financial burden of this disease worldwide. As a consequence of this growth, it is anticipated that there will be a concurrent rise in the numbers of patients with diabetic macular edema (DME), already among the most common causes of severe vision loss worldwide. Recent available therapies for DME target the secreted cytokine, vascular endothelial growth factor (VEGF). This review focuses on the treatment of DME using the first humanized monoclonal antibody targeting VEGF that has been Food and Drug Administration-approved for the use in the eye, ranibizumab (Lucentis(®)).

Expression Pattern of HIF-1α and VEGF Supports Circumferential Application of Scatter Laser for Proliferative Sickle Retinopathy.

Purpose Retinal vascular occlusions in sickle cell anemia patients cause tissue ischemia and the release of angiogenic mediators that promote the development of retinal neovascularization, initiating proliferative sickle retinopathy (PSR). Laser photocoagulation (LPC) has emerged as the most common treatment for PSR. Nonetheless, only two randomized controlled clinical trials have evaluated the use of LPC for PSR, and both failed to definitively demonstrate efficacy of this approach. This may be due to a lack of knowledge regarding the appropriate location for placement of laser coagulations in PSR eyes. To help address this question, we examined the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in PSR eyes. Methods The expression pattern of HIF-1α and VEGF in PSR (n = 5) and control (n = 3) eyes was examined by immunohistochemistry in different retinal regions defined by the presence or absence of retinal vessels. Results Hypoxia-inducible factor 1α and VEGF were expressed in the inner retina of 5/5 untreated PSR eyes adjacent to retinal neovascularization; expression of HIF-1α was not detected (and VEGF only lightly detected) in normal retinal and choroidal vasculature of 3/3 control eyes. Hypoxia-inducible factor 1α and VEGF were strongly expressed in retinal cells within avascular (nonperfused) retina, anterior to the boundary between perfused and nonperfused retina, as well as in posterior ischemic retina in the presence or absence of neovascular sea fans. Conclusions If the goal of LPC in PSR is to quench the expression of HIF-1–driven angiogenic mediators, our results support broad application of peripheral laser for its treatment.

Expression of the angiogenic mediator, angiopoietin-like 4, in the eyes of patients with proliferative sickle retinopathy

The recent success of therapies directly targeting the angiogenic mediator, vascular endothelial growth factor (VEGF), for the treatment of proliferative diabetic retinopathy has encouraged clinicians to extend the use of anti-VEGF therapies for the treatment of another ischemic retinal vascular disease, proliferative sickle cell retinopathy (PSR), the most common cause of irreversible blindness in patients with sickle cell disease. However, results from case reports evaluating anti-VEGF therapies for PSR have been mixed. This highlights the need to identify alternative therapeutic targets for the treatment of retinal neovascularization in sickle cell patients. In this regard, angiopoietin-like 4 (ANGPTL4) is a novel angiogenic factor regulated by the transcription factor, hypoxia-inducible factor 1, the master regulator of angiogenic mediators (including VEGF) in ischemic retinal disease. In an effort to identify alternative targets for the treatment of sickle cell retinopathy, we have explored the expression of ANGPTL4 in the eyes of patients with PSR. To this end, we examined expression and localization of ANGPTL4 by immunohistochemistry in autopsy eyes from patients with known PSR (n = 5 patients). Complementary studies were performed using enzyme-linked immunosorbent assays in aqueous (n = 8; 7 patients, 2 samples from one eye of same patient) and vitreous (n = 3 patients) samples from a second group of patients with active PSR. We detected expression of ANGPTL4 in neovascular tissue and in the ischemic inner retina in PSR, but not control, eyes. We further observed elevated expression of ANGPTL4 in the aqueous and vitreous of PSR patients compared to controls. These results suggest that ANGPTL4 could contribute to the development of retinal neovascularization in sickle cell patients and could therefore be a therapeutic target for the treatment of PSR.

Hypoxia-Inducible Factor-Dependent Expression of Angiopoietin-Like 4 by Conjunctival Epithelial Cells Promotes the Angiogenic Phenotype of Pterygia

Purpose Disappointing results from clinical studies assessing the efficacy of therapies targeting vascular endothelial growth factor (VEGF) for the treatment of pterygia suggest that other angiogenic mediators may also play a role in its development. We therefore explore the relative contribution of VEGF, hypoxia-inducible factor (HIF)-1α (the transcription factor that regulates VEGF expression in ocular neovascular disease), and a second HIF-regulated mediator, angiopoietin-like 4 (ANGPTL4), to the angiogenic phenotype of pterygia. Methods Expression of HIF-1α, VEGF, and ANGPTL4 were examined in surgically excised pterygia, and in immortalized human (ih) and primary rabbit (pr) conjunctival epithelial cells (CjECs). Endothelial cell (EC) tubule formation assays using media conditioned by ihCjECs in the presence or absence of inducers/inhibitors of HIF-1 or RNA interference (RNAi) targeting VEGF, ANGPTL4, or both were used to assess their relative contribution to the angiogenic potential of these cells. Results HIF-1α and VEGF expression were detected in 6/6 surgically excised pterygia and localized to CjECs. Accumulation of HIF-1α in was confirmed in ihCjECs and prCjECs, including stratified prCjECs grown on collagen vitrigel, and resulted in expression of VEGF and the promotion of EC tubule formation; the latter effect was partially blocked using RNAi targeting VEGF mRNA expression. We demonstrate expression of a second HIF-regulated angiogenic mediator, ANGPTL4, in CjECs in culture and in surgically excised pterygia. RNAi targeting ANGPTL4 inhibited EC tubule formation and was additive to RNAi targeting VEGF. Conclusions Our results support the development of therapies targeting both ANGPTL4 and VEGF for the treatment of patients with pterygia.

Scleral penetration of an unusually aggressive case of a retinal hemangioblastoma

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited multisystem disorder characterized by the development of a variety of benign and malignant tumors. Ninety per cent of patients with VHL syndrome will manifest symptoms by age 65, but most already have clinically significant issues in their teens or twenties. Retinal vascularized lesions (hemangioblastomas) remain the most common manifestation of VHL disease, occurring in the majority of VHL patients, often as the first sign of the disease1. Patients affected by retinal hemangioblastomas present with loss of vision and may develop a painful secondary glaucoma with advanced lesions2. Since the risk of blindness is high in eyes with symptomatic retinal hemangioblastomas, diagnosis and treatment before symptoms develop is beneficial. Despite the considerable morbidity associated with these tumors, retinal and CNS hemangioblastomas are classified as benign tumors due to their generally limited local invasion and lack of metastatic spread3. Here we report an unusual case of a 56 year-old woman with known VHL disease and a history of bilateral retinal hemangioblastomas. This normally benign vascular tumor filled the vitreous cavity in one eye, and penetrated the scleral with extraocular extension, ultimately requiring enucleation.