ABSTRACT
Angiogenesis, although required during eye development, has a causative effect in many ocular diseases. Aberrant neovascularization contributes to the progression of neovascular age-related macular degeneration (nAMD), a vision-threaten disease in aging Americans. Since increased amounts of vascular endothelial growth factor (VEGF) drives neovascularization during the pathogenesis of nAMD the standard of care are anti-VEGF therapies attempt to disrupt this vicious cycle. These current anti-VEGF therapies try to maintain vascular homeostasis while abating aberrant neovascularization but regrettably don't prevent fibrosis or scar formation. In addition, some patients demonstrate an incomplete response to anti-VEGF therapy as demonstrated by progressive vision loss. Here, we show choroidal endothelial cells (ChEC) incubated with artesunate demonstrated decreased migration and inflammatory and fibrotic factor expression, which corresponded with decreased sprouting in a choroid/retinal pigment epithelium (RPE) explant sprouting angiogenesis assay. To assess the efficacy of artesunate to curtail neovascularization in vivo, we utilized laser photocoagulation-induced rupture of the Bruch's membrane to induce choroidal neovascularization (CNV). Artesunate significantly inhibited CNV and the accompanying fibrotic scar, perhaps due in part to its ability to inhibit mononuclear phagocyte (MP) recruitment. Thus, artesunate shows promise in inhibiting both CNV and fibrosis.
Subject(s)
Choroidal Neovascularization , Vascular Endothelial Growth Factor A , Humans , Animals , Mice , Vascular Endothelial Growth Factor A/metabolism , Artesunate/therapeutic use , Cicatrix/prevention & control , Cicatrix/pathology , Endothelial Cells/metabolism , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/prevention & control , Choroidal Neovascularization/etiology , Vascular Endothelial Growth Factors , Disease Models, Animal , Mice, Inbred C57BLABSTRACT
Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17ß-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1-/-) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1-/- retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1-/- retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1-/- mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis.
Subject(s)
Estrogen Receptor alpha , Hepcidins , Animals , Mice , Cytochrome P-450 CYP1B1/genetics , Cytochrome P-450 CYP1B1/metabolism , Endothelial Cells/metabolism , Endothelium/metabolism , Estrogen Receptor alpha/metabolism , Hepcidins/genetics , Hepcidins/metabolism , Iron/metabolism , Oxidative Stress/physiology , Retina/metabolism , Intracellular Space/metabolismABSTRACT
Retinopathy of prematurity (ROP) is a growing cause of lifelong blindness and visual defects as improved neonatal care worldwide increases survival in very-low-birthweight preterm newborns. Advancing ROP is managed by laser surgery or a single intravitreal injection of anti-VEGF, typically at 33-36 weeks gestational age. While newer methods of scanning and telemedicine improve monitoring ROP, the above interventions are more difficult to deliver in developing countries. There is also concern as to laser-induced detachment and adverse developmental effects in newborns of anti-VEGF treatment, spurring a search for alternative means of mitigating ROP. Pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor appears late in gestation, is undetected in 25-28 week vitreous, but present at full term. Its absence may contribute to ROP upon transition from high-to-ambient oxygen environment or with intermittent hypoxia. We recently described antiangiogenic PEDF-derived small peptides which inhibit choroidal neovascularization, and suggested that their target may be laminin receptor, 67LR. The latter has been implicated in oxygen-induced ischemic retinopathy (OIR). Here we examined the effect of a nonapeptide, PEDF 336, in a newborn mouse OIR model. Neovascularization was significantly decreased in a dose-responsive manner by single intravitreal (IVT) injections of 1.25-7.5 µg/eye (1.0-6.0 nmol/eye). By contrast, anti-mouse VEGFA164 was only effective at 25 ng/eye, with limited dose-response. Combination of anti-VEGFA164 with PEDF 336 gave only the poorer anti-VEGF response while abrogating the robust inhibition seen with peptide-alone, suggesting a need for VEGF in sensitizing the endothelium to the peptide. VEGF stimulated 67LR presentation on endothelial cells, which was decreased in the presence of PEDF 336. Mouse and rabbit eyes showed no histopathology or inflammation after IVT peptide injection. Thus, PEDF 336 is a potential ROP therapeutic, but is not expected to be beneficial in combination with anti-VEGF.
Subject(s)
Animals, Newborn , Bevacizumab/administration & dosage , Eye Proteins/metabolism , Ischemia/drug therapy , Nerve Growth Factors/metabolism , Retinal Neovascularization/drug therapy , Serpins/metabolism , Animals , Disease Models, Animal , Female , Intravitreal Injections , Ischemia/metabolism , Ischemia/pathology , Male , Mice , Mice, Inbred C57BL , Oxygen/toxicity , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Vascular Endothelial Growth Factor A/antagonists & inhibitorsABSTRACT
Endoplasmic reticulum (ER) stress is recognized as a contributing factor to various ocular neurovascular pathologies including retinitis pigmentosa, glaucoma, and diabetic retinopathy (DR). ER stress in particular is implicated in the development of DR, which is significantly influenced by inflammation driven retinal vascular degeneration and dysfunction. Ultimately, loss of vision occurs if left untreated. However, the identity of the target cells and their temporal involvement in diabetes-mediated dysfunction need further investigation. Early diabetes-induced stress in photoreceptor cells is proposed as the driver of inflammatory mediated neurovascular changes during diabetes. Although tunicamycin induced ER stress results in photoreceptor loss, its consequences for retinal vascular degeneration and retinal ganglion (RGC) and pigment epithelium (RPE) cell loss remains unclear. Here we show intravitreal delivery of tunicamycin primarily induced ER stress in photoreceptor cells resulting in their loss by apoptosis. This was concomitant with induced expression of the unfolded protein response marker CHOP in these cells. We also demonstrated significant degeneration of retinal capillaries following the loss of photoreceptor cells with minimal impact on loss of RGC and RPE cells. However, activation of retinal microglial and Muller cells were noticeable. Thus, our data support the notion that ER stress mediated dysfunction and/or loss of photoreceptor cells in response to inflammation and oxidative stress could precede retinal vascular and neuronal dysfunction and degeneration.
Subject(s)
Anti-Bacterial Agents/pharmacology , Photoreceptor Cells, Vertebrate/pathology , Retinal Degeneration/pathology , Retinal Ganglion Cells/pathology , Retinal Pigment Epithelium/pathology , Retinal Vessels/pathology , Tunicamycin/pharmacology , Animals , Atrophy , Capillaries/pathology , Endoplasmic Reticulum Stress/drug effects , Female , Fluorescent Antibody Technique, Indirect , In Situ Nick-End Labeling , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , N-Methylaspartate/pharmacology , Oxidative Stress , Photoreceptor Cells, Vertebrate/metabolism , Retinal Degeneration/metabolism , Retinal Ganglion Cells/metabolism , Retinal Pigment Epithelium/metabolism , Zonula Occludens-1 Protein/metabolismABSTRACT
Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 µg). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 µM, PEDF 336 (100 µM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Choroidal Neovascularization/prevention & control , Eye Proteins/therapeutic use , Nerve Growth Factors/therapeutic use , Oligopeptides/therapeutic use , Serpins/therapeutic use , Administration, Ophthalmic , Angiogenesis Inhibitors/chemistry , Animals , Apoptosis , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Disease Models, Animal , Drug Carriers , Electroretinography , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Eye Proteins/chemistry , Mice , Mice, Inbred C57BL , Nerve Growth Factors/chemistry , Oligopeptides/chemistry , Ophthalmic Solutions , Prodrugs , Rabbits , Rats , Serpins/chemistryABSTRACT
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.
Subject(s)
Angiogenesis Inhibitors/physiology , Choroidal Neovascularization/physiopathology , Eye Proteins/physiology , Macular Degeneration/physiopathology , Nerve Growth Factors/physiology , Serpins/physiology , Thrombospondins/physiology , Angiogenesis Inhibitors/therapeutic use , Angiostatins/therapeutic use , Choroidal Neovascularization/drug therapy , Endostatins/therapeutic use , Humans , Macular Degeneration/drug therapy , Molecular Targeted Therapy/methodsABSTRACT
Endoglin (Eng) is an auxiliary receptor for transforming growth factor-ß (TGFß), with important roles in vascular function. TGFß regulates angiogenesis through balancing the pro-proliferative and pro-differentiation signaling pathways of endothelial cells (EC). However, the contribution of endoglin to these TGFß activities, and more specifically modulation of EC phenotype, remains elusive. Mutations in endoglin cause hereditary hemorrhagic telangiectasia-1 in humans. The Eng+/- mice are viable and exhibit some of the vascular defects seen in humans with endoglin haploinsufficiency. In the present study we show that haploinsufficiency of endoglin results in attenuation of retinal neovascularization during oxygen-induced ischemic retinopathy. Although the importance of endoglin expression in angiogenesis and vascular development has been demonstrated, the underlying mechanisms remain obscure. To gain detailed insight into the cell autonomous regulatory mechanisms that affect angiogenic properties of EC, we prepared retinal EC from Eng+/+ and Eng+/- Immorto mice. The Eng+/- EC were more adherent, less migratory, and failed to undergo capillary morphogenesis. Aortic sprouting angiogenesis was similarly attenuated in aortas from Eng+/- mice. In addition, Eng+/- EC expressed increased levels of VEGF but reduced expression of endothelial NO synthase and NO production. Mechanistically, these changes were consistent with sustained activation of mitogen-activated protein kinase (MAPK) pathways, and aberrant Smad-dependent signaling pathways in Eng+/- EC. Taken together, our results underscore the importance of endoglin in both canonical and non-canonical TGFß signaling pathways modulating both the activation and quiescence of the endothelium during angiogenesis.
Subject(s)
Endothelium, Vascular/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Intracellular Signaling Peptides and Proteins/physiology , Neovascularization, Physiologic/genetics , Transforming Growth Factor beta/metabolism , Animals , Cell Adhesion/genetics , Cell Differentiation/genetics , Cell Movement/genetics , Endoglin , Gene Expression Regulation/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Mutant Strains , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Sequence Deletion/genetics , Signal Transduction/genetics , Smad Proteins/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
PURPOSE: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells. METHODS: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry. RESULTS: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection. CONCLUSIONS: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.
Subject(s)
Astrocytes/physiology , Bone Morphogenetic Protein 7/physiology , Ependymoglial Cells/physiology , Gliosis/etiology , Activin Receptors, Type I/metabolism , Animals , Astrocytes/drug effects , Astrocytes/pathology , Bone Morphogenetic Protein 4/pharmacology , Bone Morphogenetic Protein 4/physiology , Bone Morphogenetic Protein 7/administration & dosage , Bone Morphogenetic Protein 7/pharmacology , Bone Morphogenetic Protein Receptors, Type I/metabolism , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Ependymoglial Cells/drug effects , Ependymoglial Cells/pathology , Glial Fibrillary Acidic Protein , Gliosis/pathology , Gliosis/physiopathology , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Intravitreal Injections , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retinal Neurons/drug effects , Retinal Neurons/pathology , Retinal Neurons/physiology , Signal Transduction/drug effects , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
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.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Choroidal Neovascularization/drug therapy , Delayed-Action Preparations/administration & dosage , Neovascularization, Pathologic/drug therapy , Powders/administration & dosage , Stilbenes/administration & dosage , Vitis/chemistry , Animals , Bruch Membrane/drug effects , Bruch Membrane/metabolism , Cell Adhesion Molecules/metabolism , Choroidal Neovascularization/metabolism , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Lasers , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence/methods , Neovascularization, Pathologic/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Resveratrol , Tumor Suppressor Protein p53/metabolismABSTRACT
Purpose: Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid. Methods: Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid. Results: We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD. Conclusion: The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.
ABSTRACT
Apoptosis plays an important role in development and remodeling of vasculature during organogenesis. Coordinated branching and remodeling of the retinal vascular tree is essential for normal retinal function. Bcl-2 family members, such as bim not only influence apoptosis, but also cell adhesive and migratory properties essential during vascular development. Here we examined the impact of bim deficiency on postnatal retinal vascularization, as well as retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) and laser-induced choroidal neovascularization. Loss of bim expression was associated with increased retinal vascular density in mature animals. This was mainly attributed to increased numbers of pericytes and endothelial cells. However, the initial spread of the superficial layer of retinal vasculature and, the appearance and density of the tip cells were similar in bim+/+ and bim-/- mice. In addition, hyaloid vessel regression was attenuated in the absence of bim. Furthermore, in the absence of bim retinal vessel obliteration and neovascularization did not occur during OIR. Instead, normal inner retinal vascularization proceeded independent of changes in oxygen levels. In contrast, choroidal neovascularization occurred equally well in bim+/+ and bim-/- mice. Together our data suggest bim expression may be responsible for the inherent sensitivity of the developing retinal vasculature to changes in oxygen levels, and promotes vessel obliteration in response to hyperoxia.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Hyperoxia/physiopathology , Membrane Proteins/metabolism , Neovascularization, Physiologic/physiology , Organogenesis/physiology , Proto-Oncogene Proteins/metabolism , Retina/embryology , Retinal Vessels/physiology , Animals , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Blotting, Western , Bromodeoxyuridine , DNA Primers/genetics , Immunohistochemistry , In Situ Nick-End Labeling , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Vascular Endothelial Growth Factor AABSTRACT
Inflammation is increasingly recognized as an important modulator in the pathogenesis of neovascular age-related macular degeneration (nAMD). Although significant progress has been made in delineating the pathways that contribute to the recruitment of inflammatory cells and their contribution to nAMD, we know little about what drives the resolution of these inflammatory responses. Gaining a better understanding of how immune cells are cleared in the choroid will give a novel insight into how sustained inflammation could influence the pathogenesis of nAMD. The pro-apoptotic Bcl-2 family member Bim is a master regulator of immune cell homeostasis. In its absence, immune cell lifespan and numbers increase. Most therapeutic regimes that squelch inflammation do so by enhancing immune cell apoptosis through enhanced Bim expression and activity. To test the hypothesis that Bim expression tempers inflammation during the pathogenesis of nAMD, we used the mouse laser-induced choroidal neovascularization (CNV) model in which inflammation acts as a facilitator of CNV. Here, we showed minimal to no change in the recruitment of F4/80-, CD80-, CD11b-, and Iba1-positive myeloid-derived mononuclear phagocytes to the site of laser photocoagulation in the absence of Bim expression. However, the resolution of these cells from the choroid of Bim-deficient (Bim -/-) mice was significantly diminished following laser photocoagulation. With time, we noted increased scar formation, demonstrated by collagen I staining, in Bim -/- mice with no change in the resolution of neovascularization compared to wild-type littermates. We also noted that mice lacking Bim expression in mononuclear phagocytes (BimFlox/Flox; Lyz2-Cre (BimMP) mice) had delayed resolution of F4/80-, CD80-, CD11b-, and Iba1-positive cells, while those lacking Bim expression in endothelial cells (BimFlox/Flox; Cad5-Cre (BimEC) mice) had delayed resolution of only CD11b- and Iba1-positive cells. Both BimMP and BimEC mice demonstrated increased scar formation, albeit to differing degrees. Thus, our studies show that resolving inflammation plays an important role in moderating scar formation in nAMD, and it is impacted by Bim expression in both the endothelium and mononuclear phagocyte lineages.
ABSTRACT
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.
Subject(s)
Choroidal Neovascularization , Fingolimod Hydrochloride , Choroid/metabolism , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/metabolism , Endothelial Cells/metabolism , Fingolimod Hydrochloride/pharmacology , Fingolimod Hydrochloride/therapeutic use , Humans , Inflammation/pathology , Phosphates , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth FactorsABSTRACT
Branching morphogenesis is a key developmental process during organogenesis, such that its disruption frequently leads to long-term consequences. The kidney and eye share many etiologies, perhaps, due to similar use of developmental branching morphogenesis and signaling pathways including cell death. Tipping the apoptotic balance towards apoptosis imparts a ureteric bud and retinal vascular branching phenotype similar to one that occurs in papillorenal syndrome. Here, to compare ureteric bud and retinal vascular branching in the context of decreased apoptosis, we investigated the impact of Bim, Bcl-2's rival force. In the metanephros, lack of Bim expression enhanced ureteric bud branching with increases in ureteric bud length, branch points, and branch end points. Unfortunately, enhanced ureteric bud branching also came with increased branching defects and other undesirable consequences. Although we did see increased nephron number and renal mass, we observed glomeruli collapse. Retinal vascular branching in the absence of Bim expression had similarities with the ureteric bud including increased vascular length, branching length, segment length, and branching interval. Thus, our studies emphasize the impact appropriate Bim expression has on the overall length and branching in both the ureteric bud and retinal vasculature.
Subject(s)
Ureter , Endothelium , Epithelium , Morphogenesis , Proto-Oncogene Proteins c-bcl-2/metabolism , Ureter/metabolismABSTRACT
Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.
Subject(s)
Chromatin/chemistry , Endothelium, Vascular/metabolism , Histones/chemistry , Lysine/chemistry , Animals , Collagen/chemistry , Drug Combinations , Endothelial Cells/cytology , Female , Humans , Laminin/chemistry , Mice , Mice, Inbred C57BL , Molecular Chaperones/chemistry , Neovascularization, Pathologic , Proteoglycans/chemistry , Vascular Endothelial Growth Factor Receptor-1/chemistryABSTRACT
Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Endothelium, Vascular/metabolism , Neovascularization, Pathologic/metabolism , Oxidative Stress/physiology , Retinal Vessels/metabolism , Thrombospondins/biosynthesis , Animals , Antioxidants/pharmacology , Aryl Hydrocarbon Hydroxylases/genetics , Blotting, Western , Cell Movement , Cytochrome P-450 CYP1B1 , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Gene Expression , Gene Expression Regulation , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Lipid Peroxidation/drug effects , Mice , Mice, Mutant Strains , Microscopy, Fluorescence , Neovascularization, Pathologic/genetics , Oxidative Stress/drug effects , Phenotype , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Retinal Vessels/drug effects , Retinal Vessels/pathologyABSTRACT
Tight regulation of positive and negative regulators of angiogenesis is essential, particularly in the eye where their dysregulation can lead to vision loss. Thrombospondin-1 (TSP1) is a matricellular protein that negatively regulates angiogenesis and inflammation in the eye. It aids ocular vascular homeostasis such that its loss contributes to increased retinal vascular density and pathologic ocular neovascularization. Our previous studies demonstrated that mice globally lacking TSP1 expression had increased retinal vascular density, decreased hyperoxia-induced retinal vessel loss, and increased choroidal neovascularization. Here we determined the impact to the ocular vasculature of endothelial cell, pericyte, or astrocyte loss of TSP1 expression. Only lack of TSP1 expression in endothelial cells was sufficient to increase choroidal neovascularization with mice lacking expression in pericytes or astrocytes not demonstrating a significant impact. Although the global TSP1 knockout mice demonstrated increased retinal vascular density, individual cell type loss of TSP1 resulted in decreased retinal endothelial cell numbers before and/or after vascular maturation in a cell type specific fashion. Retinas from mice lacking TSP1 expression in endothelial cells, pericytes or astrocytes were not protected from retinal vessel regression in response to hyperoxia as we previously observed in the global knockout. Thus, modulation of TSP1 expression in individual cell types demonstrates a response that is unique to the role TSP1 plays in that cell type of interest, and their coordinated activity is critical for vision.
ABSTRACT
Adenosine receptors (AR) are widely expressed in a variety of tissues including the retina and brain. They are involved in adenosine-mediated immune responses underlying the onset and progression of neurodegenerative diseases. The expression of AR has been previously demonstrated in some retinal cells including endothelial cells and retinal pigment epithelial cells, but their expression in the choroid and choroidal cells remains unknown. Caffeine is a widely consumed AR antagonist that can influence inflammation and vascular cell function. It has established roles in the treatment of neonatal sleep apnea, acute migraine, and post lumbar puncture headache as well as the neurodegenerative diseases such as Parkinson and Alzheimer. More recently, AR antagonism with caffeine has been shown to protect preterm infants from ischemic retinopathy and retinal neovascularization. However, whether caffeine impacts the development and progression of ocular age-related diseases including neovascular age-related macular degermation remains unknown. Here, we examined the expression of AR in retinal and choroidal tissues and cells. We showed that antagonism of AR with caffeine or istradefylline decreased sprouting of thoracic aorta and choroid/retinal pigment epithelium explants in ex vivo cultures, consistent with caffeine's ability to inhibit endothelial cell migration in culture. In vivo studies also demonstrated the efficacy of caffeine in inhibition of choroidal neovascularization and mononuclear phagocyte recruitment to the laser lesion sites. Istradefylline, a specific AR 2A antagonist, also decreased choroidal neovascularization. Collectively, our studies demonstrate an important role for expression of AR in the choroid whose antagonism mitigate choroidal inflammatory and angiogenesis activities.
ABSTRACT
INTRODUCTION: This study evaluated the effect of different pH values of 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4 on angiogenesis. METHODS: Endothelial cells were isolated from the mice molar teeth and placed in 42 Matrigel (Corning, NY)-coated wells, which were prepared and divided into 6 groups (n = 7). Synthetic tissue fluid was prepared and divided into 6 parts, and their pH values were adjusted to 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4. A 2-mL volume from each group was diluted in the growth medium at a ratio of 1:3 and used for tubulogenesis assay. Forty-two 6-week-old mice in 6 groups (n = 7) were used for choroidal neovascularization (CNV). A 2-µL volume from each group or saline (control) was delivered by intravitreal injection on the day of laser application and 1 week later. Data on the number of nodes, the total length of the branches, and CNV areas (µm2) were determined using ImageJ software (National Institutes of Health, Bethesda, MD) and analyzed with 1-way analysis of variance and post hoc Tukey tests. The correlation was assessed between the tested variables. RESULTS: The number of nodes decreased with changes in pH values as follows: 6.4 > 5.4 > 7.4 > 8.4 > 9.4 > 4.4. The total branch length decreased with pH value changes as follows: 6.4 > 4.4 > 6.4 > 7.4 > 8.4 > 9.4, and the CNV areas decreased with pH value changes as follows: 6.4 > 5.4 > 4.4 > 7.4 > 8.4 > 9.4. CONCLUSIONS: Moderately acidic pH values (5.4 and 6.4) enhanced angiogenesis, whereas moderately alkaline pH values (8.4 and 9.4) suppressed angiogenesis.
Subject(s)
Choroidal Neovascularization , Angiogenesis Inhibitors , Animals , Disease Models, Animal , Endothelial Cells , Hydrogen-Ion Concentration , Intravitreal Injections , Mice , Mice, Inbred C57BLABSTRACT
Diabetes associated complications, including diabetic retinopathy and loss of vision, are major health concerns. Detecting early retinal vascular changes during diabetes is not well documented, and only few studies have addressed this domain. The purpose of this study was to noninvasively evaluate temporal changes in retinal vasculature at very early stages of diabetes using fundus images from preclinical models of diabetes. Non-diabetic and Akita/+ male mice with different duration of diabetes were subjected to fundus imaging using a Micron III imaging system. The images were obtained from 4 weeks- (onset of diabetes), 8 weeks-, 16 weeks-, and 24 weeks-old male Akita/+ and non-diabetic mice. In total 104 fundus images were subjected to analysis for various feature extractions. A combination of Canny Edge Detector and Angiogenesis Analyzer plug-ins in ImageJ were utilized to quantify various retinal vascular changes in fundus images. Statistical analyses were conducted to determine significant differences in the various extracted features from fundus images of diabetic and non-diabetic animals. Our novel image analysis method led to extraction of over 20 features. These results indicated that some of these features were significantly changed with a short duration of diabetes, and others remained the same but changed after longer duration of diabetes. These patterns likely distinguish acute (protective) and chronic (damaging) associated changes with diabetes. We show that with a combination of various plugging one can extract over 20 features from retinal vasculature fundus images. These features change during diabetes, thus allowing the quantification of quality of retinal vascular architecture as biomarkers for disease progression. In addition, our method was able to identify unique differences among diabetic mice with different duration of diabetes. The ability to noninvasively detect temporal retinal vascular changes during diabetes could lead to identification of specific markers important in the development and progression of diabetes mediated-microvascular changes, evaluation of therapeutic interventions, and eventual reversal of these changes in order to stop or delay disease progression.