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 H
O
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.
Induced pluripotent stem cells (iPSC)‐based therapies have been hailed as the future of regenerative medicine because of their potential to provide treatment options for most degenerative diseases. A ...key promise of iPSC‐based therapies is the possibility of an autologous transplant that may engraft better in the longer‐term due to its compatibility with the patient's immune system. Despite over a decade of research, clinical translation of autologous iPSC‐based therapies has been slow—partly due to a lacking pre‐defined regulatory path. Here, we outline regulatory considerations for developing an autologous iPSC‐based product and challenges associated with the clinical manufacturing of autologous iPSCs and their derivatives. These challenges include donor tissue source, reprogramming methods, heterogeneity of differentiated cells, controls for the manufacturing process, and preclinical considerations. A robust manufacturing process with appropriate quality controls and well‐informed, prospectively designed preclinical studies provide a path toward successful approval of autologous iPSC‐based therapies.
Regulatory considerations for developing a phase I investigational new drug application for an autologous induced pluripotent stem cells‐based therapy.
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
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.
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.
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.
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.
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.
Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder caused by a missense substitution in CTRP5. Distinctive clinical features include sub-retinal pigment epithelium (RPE) ...deposits, choroidal neovascularization, and RPE atrophy. In induced pluripotent stem cells-derived RPE from L-ORD patients (L-ORD-iRPE), we show that the dominant pathogenic CTRP5 variant leads to reduced CTRP5 secretion. In silico modeling suggests lower binding of mutant CTRP5 to adiponectin receptor 1 (ADIPOR1). Downstream of ADIPOR1 sustained activation of AMPK renders it insensitive to changes in AMP/ATP ratio resulting in defective lipid metabolism, reduced Neuroprotectin D1(NPD1) secretion, lower mitochondrial respiration, and reduced ATP production. These metabolic defects result in accumulation of sub-RPE deposits and leave L-ORD-iRPE susceptible to dedifferentiation. Gene augmentation of L-ORD-iRPE with WT CTRP5 or modulation of AMPK, by metformin, re-sensitize L-ORD-iRPE to changes in cellular energy status alleviating the disease cellular phenotypes. Our data suggests a mechanism for the dominant behavior of CTRP5 mutation and provides potential treatment strategies for L-ORD patients.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment ...epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak - with a structure similar to the human macula - this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.
Neovascular or wet age-related macular degeneration (nAMD) causes vision loss due to inflammatory and vascular endothelial growth factor (VEGF)-driven neovascularization processes in the choroid. Due ...to the excess in VEGF levels associated with nAMD, anti-VEGF therapies are utilized for treatment. Unfortunately, not all patients have a sufficient response to such therapies, leaving few if any other treatment options for these patients. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator found in endothelial cells that participates in modulating barrier function, angiogenesis, and inflammation. S1P, through its receptor (S1PR1) in endothelial cells, prevents illegitimate sprouting angiogenesis during vascular development. In the present paper, we show that, in choroidal endothelial cells, S1PR1 is the most abundantly expressed S1P receptor and agonism of S1PR1-prevented choroidal endothelial cell capillary morphogenesis in culture. Given that nAMD pathogenesis draws from enhanced inflammation and angiogenesis as well as a loss of barrier function, we assessed the impact of S1PR agonism on choroidal neovascularization in vivo. Using laser photocoagulation rupture of Bruch's membrane to induce choroidal neovascularization, we show that S1PR non-selective (FTY720) and S1PR1 selective (CYM5442) agonists significantly inhibit choroidal neovascularization in this model. Thus, utilizing S1PR agonists to temper choroidal neovascularization presents an additional novel use for these agonists presently in clinical use for multiple sclerosis as well as other inflammatory diseases.
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.
e12266
Here, we report the isolation of RPE cells from wild‐type and transgenic mice retina. We demonstrate that lack of thrompospondin‐1 or pigment epithelium‐derived factor impacts the proliferation, migration, adhesion, oxidative state, and phagocytic activity of these cells.
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