Savalan Babapoor-Farrokhran

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.

KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma

Abstract Kaposi’s sarcoma (KS) is a vascular neoplasm caused by infection of endothelial or endothelial precursor cells with the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8). Research efforts have focused on defining the molecular events explaining how KSHV promotes pathological angiogenesis and KS tumor formation. mTOR/HIF-1 is a fundamental pathway driving these processes through the upregulation of angiogenic and inflammatory proteins, including VEGF, ANGPTL4, and ANGPT2. Interestingly, HIF-1 has also been implicated in the upregulation of metabolic genes associated with aerobic glycolysis and the growth of solid tumors. However, whether HIF-1 plays a role in regulating cell metabolism in KS remains unexplored. Here, we show that the HIF-1 metabolic effector, pyruvate kinase 2 (PKM2), is upregulated upon KSHV infection of endothelial cells and is necessary to maintain aerobic glycolysis in infected cells. We further demonstrate that PKM2 regulates KS angiogenic phenotype by acting as a coactivator of HIF-1 and increasing the levels of HIF-1 angiogenic factors, including VEGF. Indeed, inhibition of PKM2 expression blocked endothelial cell migration and differentiation and the angiogenic potential of KSHV-infected cells. We also investigated whether PKM2 regulates the angiogenic dysregulation induced by the KSHV-encoded G protein-coupled receptor (vGPCR), a viral oncogene that promotes Kaposi’s sarcomagenesis through the upregulation of HIF angiogenic factors. Interestingly, we found that PKM2 controls vGPCR-induced VEGF paracrine secretion and vGPCR oncogenesis. Our findings provide a molecular mechanism for how HIF-1 dysregulation fuels both angiogenesis and tumor metabolism in KS and support further investigations on therapeutic approaches targeting HIF-1 and PKM2 for KS treatment.

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.

Expression of angiogenic mediators in a patient with a retinal artery occlusion

The introduction of therapies targeting vascular endothelial growth factor (VEGF) has revolutionized the management of macular edema in the setting of ischemic retinal disease. Recent evidence further supports a role for anti-VEGF therapy for the treatment of retinal neovascularization (NV) in diabetic patients. These data have prompted clinicians to extend the use of antiVEGF therapies for the treatment of retinal NV for other ischemic retinal diseases. However, emerging data support a role for additional angiogenic factors in the promotion of retinal NV in ischemic retinal disease. Here we examine the expression of VEGF and other ischemia-driven angiogenic factors in the vitreous of a

Serum and tissue endothelin-1 are independent from intima-media thickness of peripheral arteries in patients with chronic kidney disease.

Aim We aimed to study the relationship of peripheral arteries’ atherosclerosis with serum and tissue endothelin-1 in chronic kidney disease patients. Methods Ninety patients were enrolled, including 35 patients with chronic kidney disease (case group), 31 patients with coronary artery diseases who were candidates for coronary artery bypass grafting (positive control group), and 24 living kidney donors (negative control group). Intima-media thickness of the common carotid and femoral arteries was determined by ultrasonography. Serum and tissue endothelin-1 were measured by ELISA method. Results The mean serum and tissue endothelin-1 levels in the donor group were significantly lower than other groups (p < 0.001 for both). The coronary artery bypass grafting group had higher carotid and femoral intima-media thickness than other groups (p < 0.001), and the chronic kidney disease group had higher carotid and femoral intima-media thickness than the donor group (p < 0.001). Regression analysis in all groups did not reveal any correlation between the carotid intima-media thickness/femoral intima-media thickness and the serum/tissue endothelin-1. There was a direct linear correlation between the carotid and femoral intima-media thickness (p < 0.001) in all groups. Conclusions Endothelin-1 level and intima-media thickness were higher in the chronic kidney disease patients and coronary artery bypass grafting candidates, without any correlation between endothelin-1 and peripheral arteries’ intima-media thickness of both groups. Perhaps endothelin-1 rises and remains high upon endothelial damage and initiation of atherosclerosis.

Lack of Evidence for Vasoactive and Inflammatory Mediators in the Promotion of Macular Edema Associated with Epiretinal Membranes

The development of symptoms in patients with epiretinal membranes (ERMs) often corresponds with the accumulation of interstitial fluid in the retina [i.e., the development of macular edema, (ME)]. To explore the potential value of pharmacologic therapeutic options to treat ME in patients with ERMs, we examine here the expression of vasoactive and inflammatory mediators in the vitreous of patients with idiopathic ERMs. We observed that vitreous concentrations of classic vasoactive factors (e.g., vascular endothelial growth factor) were similar in ERM patients with ME compared to controls. Using an array assessing the expression of 102 inflammatory cytokines we similarly did not observe a marked difference in cytokine expression in the vitreous of most ERM patients with ME compared to control patients. While the array data did implicate a group of inflammatory cytokines that were elevated in a subset of ERM patients who had severe ME (central subfield thickness ≥450 μm on spectral domain optical coherence tomography), expression of 3 of these inflammatory cytokines, all previously implicated in the promotion of ME in ischemic retinal disease, were not elevated by quantitative enzyme-linked immunosorbent assay. We conclude that therapies modulating vasoactive mediators or inflammatory cytokines may not affect ME in ERM patients.

Short-term effects of lovastatin therapy on proteinuria of type 2 diabetic nephropathy: A clinical trial study

Background: Diabetic nephropathy (DN) is characterized by albuminuria, hypertension, and a progressive decline in glomerular filtration rate. The 3-hydroxy-3-methylglutaryl coenzyme A is a well-known agent that is active in lowering total plasma and low-density lipoprotein cholesterol (LDL-C) levels in cases with hypercholesterolemia. Hence, in this study, proteinuria changes at the beginning and after the withdrawal of lovastatin in patients with type 2 DN (T2DN) were studied. Materials and Methods: Lovastatin was administered for thirty male patients with T2DN and then was withdrawn. Twenty-four hours, urine creatinine and protein levels were determined. Results: The mean levels of total cholesterol and LDL-C were reduced without any change in the triglyceride (TG) level while the high-density lipoprotein cholesterol (HDL-C) level was increased. There was a reverse linear correlation between the changes in the level of HDL-C and the changes in the level of 24 h urine protein after 90 days of lovastatin therapy (P = 0.007, r = −0.484). Conclusions: Short-term 3-month lovastatin therapy has no effect on proteinuria levels in patients with T2DN despite the antihyperlipidemic effects and reverse correlation of proteinuria with HDL-C.