ANGIOPOIETIN-LIKE 4 IN DIABETES

Role of ANGPTL4 in the development of macular edema and loss of vision in patients with diabetic retinopathy.

The goal of this NIH-NEI funded project is to elucidate the role of Angiopoietin-like 4 (ANGPTL4) in the promotion of pathological neovascularization and macular edema in diabetic retinopathy, the number one cause of blindness in the American working population. Our investigations use genetic, molecular biology and cell biology approaches as well as mouse models of ischemic and diabetic retinopathies.

Diabetes is one of the major contributors to disease and premature mortality worldwide. In the United States, more than 100 million adults live with diabetes or prediabetes, according to a report released in 2017 by the Centers for Disease Control and Prevention (CDC). If sugar levels are not managed appropriately, different chronic complications of the disease may occur as a result of oxidative stress, vascular damage, and immune dysfunction.

Diabetic retinopathy (DR) is a type of ischemic retinopathy and one of the most common complications in patients suffering from diabetes. Abnormal sugar levels initiate microvascular degeneration in the retina, leading to hypoxic stress and subsequent retinal neovascularization. Initial vascular damage causes leakage of fluids and blood, which produce edema and swelling of the macula, causing loss of central vision. This early stage of DR in which diabetic macular edema (DME) may be observed is called non-proliferative diabetic retinopathy (NPDR). Microvascular damage also causes hypoxic stress and leads to abnormal hypoxia-induced angiogenesis. These fragile new vessels often bleed into the vitreous, causing the development of floaters and even blockage of all vision. This more advanced stage of diabetic eye disease when the retina starts growing new blood vessels is called proliferative diabetic retinopathy (PDR). Unfortunately, the current treatments for NPDR and PDR (laser and anti-VEGF drugs) are only successful in half of the treated patients, underscoring the urgency to identify safe and effective therapeutic targets for the treatment of these patients.

We recently found that Angiopoietin-like 4 (ANGPTL4), a member of the family of Angiopoietin-like proteins (ANGPTLs) and a homolog of Angiopoietin 1 (ANGPT1) and 2 (ANGPT2), promotes the destabilization of the integrity of the vascular barrier, causing vascular hyperpermeability. These observations prompted us to investigate whether this factor could be involved in the induction of macular edema that is observed in DR. For this, we started a collaboration with Dr. Akrit Sodhi, from the Wilmer Eye Institute (Johns Hopkins Hospital, Baltimore, MD), who investigates the molecular pathogenesis of HIF-dependent retinal neovascularization. These collaborative efforts have yielded very exciting findings that suggest that ANGPTL4 may serve as an alternative therapy for patients with DR. Interestingly, we have found elevated ANGPTL4 levels in DR patients. We observed that ANGPTL4 is highly upregulated in hypoxic (glial) Müller cells and that this secretion causes the loss of vascular integrity of surrounding retinal capillaries. Our data shows that ANGPTL4 causes destabilization of endothelial cell junctions through the rapid activation of RhoA and the contraction of actomyosin fibers. An ANGPTL4-induced delayed activation of Rac1 and Cdc42 helps restore vascular integrity. Intraretinal coinjections of ANGPTL4 and VEGF in mice demonstrate that these factors cooperate in the induction of vascular hyperpermeability. Furthermore, we found that ANGPTL4 causes migration of retinal endothelial cells, promoting angiogenesis in the development of PDR, the most advanced stage of the disease.

We are currently studying the signal transduction pathways that are activated by ANGPTL4 to cause retinal vascular leakage and neovascularization. Our current investigations are focused on the identification of membrane receptors and intracellular effectors that are involved in the vasoactive functions of this important cytokine.

ANGIOPOIETIN-LIKE 4 IN HEAD AND NECK CANCER.

Role of ANGPTL4 in the lymphatic and hematogenous dissemination of cancer cells in head and neck squamous cell carcinoma.

The goal of this project is to elucidate the role of ANGPTL4 in the metastatic dissemination of head and neck squamous cell carcinoma, the tumor that accounts for 4% of all cancers in the United States and the 6th most common cancer worldwide. Our investigations use genetic, molecular biology and cell biology approaches as well as mouse models of head and neck cancer and tumor metastasis.

Head and neck squamous cell carcinoma (HNSCC) affects about 65,000 new individuals in the US each year. The etiology of HNSCC appears to be multifactorial and strongly related to diet, lifestyle (tobacco alone or in betel, and alcohol use) or viral infection (HPV). HNSCC prognosis highly depends on the stage of the tumor, the presence of distant metastasis originated from the circulation of cancer cells through vascular blood vessels (hematogenous spread) or lymphatic blood vessels (lymphatic spread), and the health status of the patient. In HNSCC, metastasis through the lymphatics and lymph node colonization are common, and represent a major factor for HNSCC poor prognosis.

ANGPTL4 mRNA and protein were found to be increased in HNSCC biopsies and to correlate with cancer cell migration and presence of lymph node metastasis. Interestingly, we observed that ANGPTL4 is upregulated both in malignant and in premalignant HNSCC-derived cell lines. Our research interest is to elucidate the molecular mechanisms by which ANGPTL4 promotes HNSCC dissemination. We are exploring the hypothesis that upregulation and secretion of ANGPTL4 in HNSCC not only increases the migratory capacity of cancer cells but it also remodels the vascular and lymphatic vasculature within and around tumor. For this purpose, we are using a series of premalignant and malignant head and neck cancer cell lines and several in vitro and in vivo angiogenesis assays. We are also using mouse models of HNSCC cancer metastasis in collaboration with another faculty member in our department, Dr. Abraham Schneider, who is an expert in the field.

Head and neck cancer presents an important therapeutic challenge that has proven relatively resistant to attempts to improve patient outcomes over the past several decades. We believe that our investigations on the role of ANGPTL4 in HNSCC are highly relevant as they may help identify novel molecular targets for the therapeutic management of patients suffering from this devastating disease.

ANGIOPOIETIN-LIKE 4 IN KAPOSI’S SARCOMA

Role of vGPCR-induced Angiopoietin-like 4 in the development of dysregulated angiogenesis and edema in Kaposi’s sarcoma lesions:

The goal of this project is to elucidate the role of ANGPTL4 in the promotion of aberrant angiogenesis and vascular leakage in Kaposi’s sarcoma (KS), a neovascular tumor that affects immunosuppressed individuals and one of the AIDS-defining diseases. Our investigations use genetic, molecular biology and cell biology approaches as well as mouse models of KS.

We have studied for many years the signal transduction pathways induced by the viral, constitutively-active G protein-coupled receptor (vGPCR) encoded by the human herpes virus 8 or KS herpesvirus (HHV8/KSHV). Infection with KSHV causes the development of KS, a vascular tumor that affects the oral cavity and visceral organs and that is characterized by proliferation of (stem) endothelial cells, formation of aberrant and leaky vascular slits, edema and inflammation. Interestingly, using a KS animal model, we found that vGPCR is the viral KSHV oncogene that initiates KS development. Therefore, we set out to investigate the molecular mechanisms whereby vGPCR causes endothelial cell-derived tumorigenesis and its potential use as a molecular target for KS therapy.

We have made fundamental contributions to the elucidation of the signal transduction cascades involved in vGPCR-induced neoplasia. We discovered that vGPCR induces KS initiation (in vitro and in vivo) through the activation of the TSC/mTOR/HIF pathway leading to the upregulation of HIF-responsive angiogenic and proliferative factors. We also found a Rac1-dependent activation of transcription factors (AP1, NFκB, NFAT) that induces the upregulation of cytokines and angiogenic molecules. All together, these vGPCR vasoactive factors induce aberrant angiogenesis, vessel hyperpermeability and inflammation in KS lesions, in an autocrine and paracrine fashion. Indeed, our findings propose that vGPCR tumorigenesis is a model of paracrine neoplasia, in which vGPCR secretions are a major responsible for tumor growth and maintenance. Our results represent also a new breakthrough in the field, as they provide an explanation of how a viral (lytic) oncogene, expressed in a minority of tumor cells, can induce tumor progression. Ultimately, our findings helped provide the foundation for clinical studies interrogating the use of inhibitors of mTOR/HIF for KS treatment. Indeed, several clinical trials are in place using mTOR inhibitors (sirolimus) or HIF (digoxin) for the management of HIV-related KS, and iatrogenic KS. In order to find out the most important cytokines and angiogenic factors secreted by vGPCR-expressing cells, we performed microarray analysis of vGPCR conditioned media. These experiments helped us identify ANGPTL4 as the highest upregulated angiogenic factor in vGPCR-expressing cells. We found that ANGPTL4 induces endothelial cell migration, helping in the recruitment of new endothelial and endothelial stem cells into the KS lesion, where they can be then infected with KSHV. We also found that ANGPTL4 acts as a potent inducer of the destabilization of the vascular barrier, promoting edema in the tumor. ANGPTL4 upregulation is a consequence of the activation of mTOR/HIF, which is observed both in KS-like tumors in mice and in human KS lesions. Activation of mTOR/HIF is responsible of many cellular and histopathological features observed in this tumor. As an example, we also found that HIF induces metabolic reprogramming of KS cancer cells. Interestingly, we observed that pyruvate kinase 2 (PKM2), a vGPCR-dependent HIF metabolic effector leads to a metabolic switch (aerobic glycolysis) in expressing cells and an upregulation of VEGF secretion within the tumor.

Collectively, our findings suggest that vGPCR, its secretions (ANGPTL4) and HIF could represent alternative therapeutic options for KS patients. We are currently investigating the molecular mechanisms by which ANGPTL4 cause abnormal angiogenesis and vascular leakage in KS