Mitra Farnoodian

NosA, a transcription factor important in Aspergillus fumigatus stress and developmental response, rescues the germination defect of a laeA deletion

Aspergillus fumigatus is an increasingly serious pathogen of immunocompromised patients, causing the often fatal disease invasive aspergillosis (IA). One A. fumigatus virulence determinant of IA is LaeA, a conserved virulence factor in pathogenic fungi. To further understand the role of LaeA in IA, the expression profile of ΔlaeA was compared to wild type, and several transcription factors were found significantly misregulated by LaeA loss. One of the transcription factors up-regulated over 4-fold in the ΔlaeA strain was Afu4g09710, similar in sequence to Aspergillus nidulans NosA, which is involved in sexual development. Here we assessed loss of nosA (ΔnosA) and overexpression of nosA (OE::nosA) on A. fumigatus in both a wild type and ΔlaeA background. Based on the multiple alterations of physiological development of single and double mutants, we suggest that NosA mediates the decreased radial growth and delayed conidial germination observed in ΔlaeA strains, the former in a light dependent manner. The ΔnosA mutant showed increased virulence in the Galleria mellonella larvae model of disseminated aspergillosis, potentially due to its increased growth and germination rate. Furthermore, the A. fumigatus nosA allele was able to partially remediate sexual development in an A. nidulans ΔnosA background. Likewise, the A. nidulans nosA allele was able to restore the menadione sensitivity defect of the A. fumigatus ΔnosA strain, suggesting conservation of function of the NosA protein in these two species.

Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion

The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors, and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promoted endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels show superior sensitivity and reproducibility over Matrigel when evaluating known inhibitors, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the US Environmental Protection Agency. The identified synthetic hydrogels should be suitable alternatives to Matrigel for common cell-culture applications.

Expression of pigment epithelium‐derived factor and thrombospondin‐1 regulate proliferation and migration of retinal pigment epithelial cells

Age‐related macular degeneration (AMD) is the leading cause of vision loss among elderly. Although the pathogenesis of AMD is associated with retinal pigmented epithelium (RPE) dysfunction and abnormal neovascularization the detailed mechanisms remain unresolved. RPE is a specialized monolayer of epithelial cells with important functions in ocular homeostasis. Pathological RPE damage contributes to major ocular conditions including retinal degeneration and irreversible loss of vision in AMD. RPE cells also assist in the maintenance of the ocular angiogenic balance by production of positive and negative regulatory factors including vascular endothelial growth factor (VEGF), thrombospondin‐1 (TSP1), and pigment epithelium‐derived factor (PEDF). The altered production of PEDF and TSP1, as endogenous inhibitors of angiogenesis and inflammation, by RPE cells have been linked to pathogenesis of AMD and choroidal and retinal neovascularization. However, lack of simple methods for isolation and culture of mouse RPE cells has resulted in limited knowledge regarding the cell autonomous role of TSP1 and PEDF in RPE cell function. Here, we describe a method for routine isolation and propagation of RPE cells from wild‐type, TSP1, and PEDF‐deficient mice, and have investigated their impact on RPE cell function. We showed that expression of TSP1 and PEDF significantly impacted RPE cell proliferation, migration, adhesion, oxidative state, and phagocytic activity with minimal effect on their basal rate of apoptosis. Together, our results indicated that the expression of PEDF and TSP1 by RPE cells play crucial roles not only in regulation of ocular vascular homeostasis but also have significant impact on their cellular function.

The Sustained Delivery of Resveratrol or a Defined Grape Powder Inhibits New Blood Vessel Formation in a Mouse Model of Choroidal Neovascularization

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch’s membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells.

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1−/−) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1−/− ChEC migration and capillary morphogenesis. TSP1−/− ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1−/− ChEC produced similar levels of VEGF, although TSP1−/− ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.

High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression

Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis.

Negative regulators of angiogenesis: important targets for treatment of exudative AMD

Angiogenesis contributes to the pathogenesis of many diseases including exudative age-related macular degeneration (AMD). It is normally kept in check by a tightly balanced production of pro- and anti-angiogenic factors. The up-regulation of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), is intimately linked to the pathogenesis of exudative AMD, and its antagonism has been effectively targeted for treatment. However, very little is known about potential changes in expression of anti-angiogenic factors and the role they play in choroidal vascular homeostasis and neovascularization associated with AMD. Here, we will discuss the important role of thrombospondins and pigment epithelium-derived factor, two major endogenous inhibitors of angiogenesis, in retinal and choroidal vascular homeostasis and their potential alterations during AMD and choroidal neovascularization (CNV). We will review the cell autonomous function of these proteins in retinal and choroidal vascular cells. We will also discuss the potential targeting of these molecules and use of their mimetic peptides for therapeutic development for exudative AMD.

β2-Adrenergic Receptor Antagonism Attenuates CNV Through Inhibition of VEGF and IL-6 Expression.

Purpose The role of β–adrenergic receptor (AR) signaling in neovascular ocular diseases has recently emerged. We have previously reported that intraperitoneal propranolol inhibits choroidal neovascularization (CNV) in vivo and β2-AR blockade reduces vascular endothelial growth factor (VEGF) expression in mouse retinal pigment epithelium and choroidal endothelial cells in culture. Here we tested the hypothesis that the β2-AR regulates CNV through modulation of VEGF and inflammatory cytokine expression. Methods Mice were subjected to laser burns, inducing CNV, and were treated with an intravitreal β2-AR antagonist. After 3 and 5 days, total eye interleukin-6 (IL-6) and VEGF protein levels were measured, respectively. After 14 days, CNV was measured on choroidal–scleral flatmounts. The effects of β-AR signaling on VEGF and IL-6 expression were investigated in various mouse retinal and human RPE cells by using specific β-AR agonists and antagonists. Results β2–Adrenergic receptor signaling increased Vegf mRNA expression by approximately 3- to 4-fold in mouse retinal microglia and pericytes in culture. β2–Adrenergic receptor signaling upregulated IL-6 mRNA expression between 10- and 60-fold in mouse retinal microglia, pericytes, RPE, and choroidal endothelial cells in culture. Intravitreal injection of β2-AR antagonist ICI 118,551 reduced CNV by 35% and decreased IL-6 protein levels by approximately 50%. In primary human RPE cells, β2-AR activation also stimulated VEGF and IL-6 mRNA expression by 2- and 10-fold, respectively. Conclusions Anti-VEGF therapy for CNV is highly effective; however, some patients are resistant to therapy while others undergo repeated, frequent treatments. β2–Adrenergic receptor signaling is a potential therapeutic target because of its angiogenic and inflammatory properties.

Negative regulators of angiogenesis, ocular vascular homeostasis, and pathogenesis and treatment of exudative AMD

Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is very tightly regulated and normally does not occur except during developmental and reparative processes. This tight regulation is maintained by a balanced production of positive and negative regulators, and alterations under pathological conditions such as retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration can lead to growth of new and abnormal blood vessels. Although the role of proangiogenic factors such as vascular endothelial growth factor has been extensively studied, little is known about the roles of negative regulators of angiogenesis in the pathogenesis of these diseases. Here, we will discuss the role of thrombospondin-1 (TSP1), one of the first known endogenous inhibitors of angiogenesis, in ocular vascular homeostasis, and how its alterations may contribute to the pathogenesis of age-related macular degeneration and choroidal neovascularization. We will also discuss its potential utility as a therapeutic target for treatment of ocular diseases with a neovascular component.

PEDF expression affects the oxidative and inflammatory state of choroidal endothelial cells

Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly population, and is associated with severe macular degeneration and choroidal neovascularization (CNV). Although the pathogenesis of AMD is associated with choroidal dysfunction and CNV, the detailed underlying mechanisms remain unresolved. Altered production of pigment epithelium-derived factor (PEDF), a neuroprotective and antiangiogenic factor, contributes to CNV. Furthermore, exogenous PEDF mitigates angiogenesis in preclinical CNV models. How PEDF expression affects choroidal endothelial cell (ChEC) function is unknown. Here we isolated ChECs from PEDF+/+ and PEDF-deficient (PEDF-/-) mice and determined the impact of PEDF expression on the proangiogenic and pro-inflammatory properties of ChECs. We showed that PEDF expression significantly affects the proliferation, migration, adhesion, and oxidative and inflammatory state of ChECs. The PEDF-/- ChECs were, however, more sensitive to H2O2 challenge and exhibited increased rate of apoptosis and oxidative stress. We also observed a significant increase in production of cytokines with a primary role in inflammation and angiogenesis including vascular endothelial growth factor (VEGF) and osteopontin, and a reprograming of chemokines and cytokines expression profiles in PEDF-/- ChECs. Collectively, our results indicate that PEDF expression has a significant impact on oxidative and inflammatory properties of ChECs, whose alteration could contribute to pathogenesis of chronic inflammatory diseases including exudative AMD.