Thrombospondin-1 in vascular development, vascular function, and vascular disease.

Angiogenesis is vital to developmental, regenerative and repair processes. It is normally regulated by a balanced production of pro- and anti-angiogenic factors. Alterations in this balance under pathological conditions are generally mediated through up-regulation of pro-angiogenic and/or downregulation of anti-angiogenic factors, leading to growth of new and abnormal blood vessels. The pathological manifestation of many diseases including cancer, ocular and vascular diseases are dependent on the growth of these new and abnormal blood vessels. Thrompospondin-1 (TSP1) was the first endogenous angiogenesis inhibitor identified and its anti-angiogenic and anti-inflammatory activities have been the subject of many studies. Studies examining the role TSP1 plays in pathogenesis of various ocular diseases and vascular dysfunctions are limited. Here we will discuss the recent studies focused on delineating the role TSP1 plays in ocular vascular development and homeostasis, and pathophysiology of various ocular and vascular diseases with a significant clinical relevance to human health.

Artesunate mitigates choroidal neovascularization and scar formation.

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.

Cytochrome P450 1B1 Expression Regulates Intracellular Iron Levels and Oxidative Stress in the Retinal Endothelium.

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.

AhR and CYP1B1 Control Oxygen Effects on Bone Marrow Progenitor Cells: The Enrichment of Multiple Olfactory Receptors as Potential Microbiome Sensors.

Polycyclic aromatic hydrocarbon (PAH) pollutants and microbiome products converge on the aryl hydrocarbon receptor (AhR) to redirect selective rapid adherence of isolated bone marrow (BM) cells. In young adult mice, Cyp1b1-deficiency and AhR activation by PAH, particularly when prolonged by Cyp1a1 deletion, produce matching gene stimulations in these BM cells. Vascular expression of Cyp1b1 lowers reactive oxygen species (ROS), suppressing NF-κB/RelA signaling. PAH and allelic selectivity support a non-canonical AhR participation, possibly through RelA. Genes stimulated by Cyp1b1 deficiency were further resolved according to the effects of Cyp1b1 and Cyp1a1 dual deletions (DKO). The adherent BM cells show a cluster of novel stimulations, including select developmental markers; multiple re-purposed olfactory receptors (OLFR); and α-Defensin, a microbial disruptor. Each one connects to an enhanced specific expression of the catalytic RNA Pol2 A subunit, among 12 different subunits. Mesenchymal progenitor BMS2 cells retain these features. Cyp1b1-deficiency removes lymphocytes from adherent assemblies as BM-derived mesenchymal stromal cells (BM-MSC) expand. Cyp1b1 effects were cell-type specific. In vivo, BM-MSC Cyp1b1 expression mediated PAH suppression of lymphocyte progenitors. In vitro, OP9-MSC sustained these progenitors, while Csf1 induced monocyte progenitor expansion to macrophages. Targeted Cyp1b1 deletion (Cdh5-Cre; Cyp1b1fl/fl) established endothelium control of ROS that directs AhR-mediated suppression of B cell progenitors. Monocyte Cyp1b1 deletion (Lyz2-Cre; Cyp1b1fl/fl) selectively attenuated M1 polarization of expanded macrophages, but did not enhance effects on basal M2 polarization. Thus, specific sources of Cyp1b1 link to AhR and to an OLFR network to provide BM inflammatory modulation via diverse microbiome products.

Retinopathy of prematurity shows alterations in Vegfa164 isoform expression.

BACKGROUND: Pathologic ocular neovascularization in retinopathy of prematurity (ROP) and other proliferative retinopathies are characterized by dysregulation of vascular endothelial growth factor-A (VEGF-A). A study of Vegfa isoform expression during oxygen-induced ischemic retinopathy (OIR) may enhance our understanding of Vegf dysregulation. METHODS: Following induction of OIR, immunohistochemistry and polymerase chain reaction (PCR) was performed on room air (RA) and OIR mice. RESULTS: Total Vegfa messenger RNA (mRNA) expression was stable in RA mice, but increased in OIR mice with a peak at postnatal day 17 (P17), before returning to RA levels. Vegfa164a expression was similar in both OIR and RA mice at P10 (Phase 1 OIR), but 2.4-fold higher in OIR mice compared to RA mice at P16 (Phase 2 OIR). At P10, Vegfa164b mRNA was similar in OIR vs RA mice, but was expressed 2.5-fold higher in OIR mice compared to RA mice at P16. At P10 and P16, Vegfr2/Vegfr1 expression was increased in OIR mice compared to RA mice. Increased activation of microglia was seen in OIR mice. CONCLUSIONS: Vegfa164a, Vegfa164b, and Vegfr1 were overexpressed in OIR mice, leading to abnormal signaling and angiogenesis. Further studies of mechanisms of Vegf dysregulation may lead to novel therapies for ROP and other proliferative retinopathies. IMPACT: Vegfa164 has two major isoforms, a proangiogenic, Vegfa164a, and an antiangiogenic, Vegfa164b, with opposing receptors, inhibitory Vegfr1, and stimulatory Vegfr2, but their role in OIR is unclear. In Phase 1 OIR, both isoforms and receptors are expressed similarly. In Phase 2 OIR, both isoforms are overexpressed, with an increased ratio of inhibitory Vegfr1. Modulation of angiogenesis by Vegf regulation enables pruning of excess angiogenesis during physiology, but results in ineffective angiogenesis during OIR. Knowledge of VEGF dysregulation may have novel therapeutic implications in the management of ROP and retinal proliferative diseases.

Inhibition of retinal neovascularization by a PEDF-derived nonapeptide in newborn mice subjected to oxygen-induced ischemic retinopathy.

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.

Promotion of adipogenesis by 15-(S)-hydroxyeicosatetraenoic acid.

Excess adipogenesis is a characteristic of obesity, which is associated with serious health problem, including type 2 diabetes. Here, to better understand the mechanisms for the development of adipocytes, we investigated the regulatory role of 15-(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in adipogenesis by treating 3T3-L1 murine preadipocytes and human bone marrow mesenchymal stem cells (hBMSCs) with 15(S)-HETE. In the 3T3-L1 study, 15(S)-HETE stimulated lipid accumulation and enhanced expression of adipogenic markers such as peroxisome proliferator-activated receptor gamma (PPARγ), yet reduced the activity of factor negatively controlling adipogenesis, such as the 5'-AMP-activated protein kinase. In the early stage of adipogenesis, 15(S)-HETE increased activation of protein kinase B and expression of CCAAT/enhancer-binding protein (C/EBP)ß and C/EBPδ. Finally, 15(S)-HETE promoted adipogenesis in hBMSCs, and resulted in increased lipid accumulation and expression of adipogenic markers. In conclusion, 15(S)-HETE functions as a natural PPARγ agonist and enhances adipogenesis. Our findings may provide the basis for the development of novel therapeutic measures to treat obesity.

Effect of intravitreal Rho kinase inhibitor ripasudil (K-115) on feline retinal microcirculation.

Ripasudil (K-115) is a novel Rho kinase inhibitor with a potent intraocular pressure-lowering effect. However, it is unclear whether ripasudil affects the retinal blood flow (RBF). We investigated the effect of ripasudil on feline retinal microcirculation. Ripasudil (5 µM, 50 µM or 5 mM; n = 5 each concentration) or vehicle (PBS; n = 5) was injected intravitreally. The vessel diameter (D) and blood velocity (V) were measured by laser Doppler velocimetry simultaneously in the first-order retinal arterioles and the RBF was calculated. The measurements started 5 min before the injection and were performed every 10 min for 120 min. After the intravitreal injection, the retinal circulatory parameters did not change significantly in PBS or 5 µM of ripasudil. The blood V and RBF increased significantly compared to baseline, whereas the vessel D did not change significantly in 50 µM and 5 mM of ripasudil. The V in 50 µM, and the V and RBF in 5 mM of ripasudil significantly increased compared to those in PBS. Intravitreal administration of ripasudil increased the blood V and RBF in cats, suggesting that ripasudil has the potential to improve the retinal blood flow.

Water-extracted Ampelopsis brevipedunculata downregulates IL-1ß,CCL5, and COX-2 expression via inhibition of PKC-mediated JNK/NF-κB signaling pathways in human monocytic cells.

The anti-inflammatory and anti-hepatotoxic effects of Ampelopsis brevipedunculata (A.bre) have been well known in folk medicine. An ethanol-extract of A.bre has been reported to inhibit carbon tetrachloric acid induced hepatic injury, suggesting that extracted components from A.bre could potentially treat inflammatory disease. To test this hypothesis, in this study, we extracted polysaccharide components from leaves of A.bre and investigated the anti-inflammatory effects in PMA stimulated THP-1 cells. THP-1 cells activated by PMA in the presence or absence of A.bre demonstrated that a water-extract of A.bre inhibited the expression of pro-inflammatory cytokine IL-1ß and chemokine CCL-5 in a dose-dependent manner. In addition, A.bre suppressed production of cyclooxygenase (COX)-2 in THP-1 cells activated by PMA. Moreover, A.bre markedly down-regulated the expression of p-JNK1/3, whereas it did not inhibit production of the phosphorylated form of p38 and extracellular signal-regulated kinase in THP-1 cells treated by PMA. Particularly, A.bre inhibited the translocation of transcription factor NF-κB from the cytosol into the nucleus in PMA-stimulated THP-1 cells. Collectively, our data showed that water-extracted A.bre inhibited the protein kinase C-JNKs/NF-κB signaling pathways, resulting in the suppression of IL-1ß, CCL-5, and COX-2 expression. This study suggests that water extracted A.bre may be a therapeutic agent against inflammatory disease.

Thrombospondin-1, BIM and CFH polymorphisms and response to anti-VEGF treatment in neovascular age- related macular degeneration patients.

Age-related macular degeneration (AMD) is a vision threatening disease in older adults. Anti-VEGF treatment is effective for the majority of neovascular AMD (nAMD) patients, although approximately 30% of nAMD patients have an incomplete response for unknown reasons. Here we assessed the contribution of single nucleotide polymorphisms (SNPs) in key angioinflammatory regulatory genes in nAMD patients with an incomplete response compared to those responsive to anti-VEGF treatment. A total of 25 responsive and 30 nAMD patients with an incomplete response to anti-vascular endothelial growth factor (anti-VEGF) treatment were examined for known SNPs that impact the structure and function of thromobospondin-1 (TSP1), Bcl-2-interacting mediator of cell death (BIM) and complement factor H (CFH). Plasma levels of C-C motif chemokine ligand 2 (CCL2/MCP1), TSP1 and VEGF were assessed by ELISA. Patients responsive to anti-VEGF treatment showed a significant increase in the TSP1 rs2228262 AA allele and a trend for the BIM (rs724710) CT allele. Consistent with previous reports, 42% of the patients responsive to anti-VEGF expressed the CC allele for CFH rs1061170. Although the CFH TT allele had similarly low prevalence in both groups, the TC allele tended to be more prevalent in patients with an incomplete response. Patients with an incomplete response also had increased plasma CCL2/MCP1 levels, consistent with the role increased inflammation has in the pathogenesis of nAMD. Our studies point to new tools to assess the potential responsiveness of nAMD patients to anti-VEGF treatment and suggest the potential use of anti-CCL2 for treatment of nAMD patients with an incomplete response to anti-VEGF.

Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells.

The integrity of retinal endothelial cell (EC) is essential for establishing and maintaining the retinal blood barrier to ensure proper vision. Vitamin D is a hormone with known protective roles in EC function. The majority of vitamin D action is mediated through the vitamin D receptor (VDR). VDR is a nuclear receptor whose engagement by vitamin D impacts the expression of many genes with important roles in regulation of angiogenesis and inflammation. Although many studies have investigated vitamin D-VDR action in cardiovascular protection and tumor angiogenesis, its impact on retinal EC function and regulation of ocular angiogenesis and inflammation is exceedingly limited. We previously showed calcitriol, the active form of vitamin D, is a potent inhibitor of retinal neovascularization in vivo and retinal EC capillary morphogenesis in vitro. Here, using retinal EC prepared from wild-type (Vdr+/+) and VDR-deficient (Vdr-/-) mice, we show that retinal EC express VDR and its expression is induced by calcitriol. The lack of VDR expression had a significant impact on endothelial cell-cell and cell-matrix interactions. Vdr-/- retinal EC proliferated at a slower rate and were more adherent and less migratory. They also exhibited increased expression levels of inflammatory markers driven in part by sustained activation of STAT1 and NF-κB pathways and were more sensitive to oxidative challenge. These changes were attributed, in part, to down-regulation of endothelial nitric oxide synthetase, enhanced hepcidin expression, and increased intracellular iron levels. Taken together, our results indicate that VDR expression plays a fundamental role in maintaining the proper angiogenic and inflammatory state of retinal EC.

Choroidal Mast Cells and Pathophysiology of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) remains a leading cause of vision loss in elderly patients. Its etiology and progression are, however, deeply intertwined with various cellular and molecular interactions within the retina and choroid. Among the key cellular players least studied are choroidal mast cells, with important roles in immune and allergic responses. Here, we will review what is known regarding the pathophysiology of AMD and expand on the recently proposed intricate roles of choroidal mast cells and their activation in outer retinal degeneration and AMD pathogenesis. We will focus on choroidal mast cell activation, the release of their bioactive mediators, and potential impact on ocular oxidative stress, inflammation, and overall retinal and choroidal health. We propose an important role for thrombospondin-1 (TSP1), a major ocular angioinflammatory factor, in regulation of choroidal mast cell homeostasis and activation in AMD pathogenesis. Drawing from limited studies, this review underscores the need for further comprehensive studies aimed at understanding the precise roles changes in TSP1 levels and choroidal mast cell activity play in pathophysiology of AMD. We will also propose potential therapeutic strategies targeting these regulatory pathways, and highlighting the promise they hold for curbing AMD progression through modulation of mast cell activity. In conclusion, the evolving understanding of the role of choroidal mast cells in AMD pathogenesis will not only offer deeper insights into the underlying mechanisms but will also offer opportunities for development of novel preventive strategies.

Adenosine Receptors Expression in Human Retina and Choroid with Age-related Macular Degeneration.

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.

Assessment of Choroidal Vasculature and Innate Immune Cells in the Eyes of Albino and Pigmented Mice.

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, mast cells, and vasculature in eyes of albino and pigmented mice using cell type-specific staining. We were able to visualize the choroidal arterial and venous systems. An arterial circle around the optic nerve was found in mice similar to the Zinn-Haller arterial circle that exists in humans and primates. Three different structural patterns of choriocapillaris were observed throughout the whole choroid: honeycomb-like, maze-like, and finger-like patterns. Choroidal mast cells were relatively few but dense around the optic nerve. Mast cell distribution in the middle and periphery was different among strains. Macrophages were found in all layers of the choroid. Thus, utilizing the simple and reliable methods described herein will allow the evaluation of transgenic and preclinical mouse models of ocular diseases that affect the choroid, including age-related macular degeneration (AMD), diabetic choroidopathy, and retinopathy of prematurity. These studies will advance our understanding of the pathophysiology, and molecular and cellular mechanisms that can be targeted therapeutically, in these diseases.

Bim Expression Modulates Branching Morphogenesis of the Epithelium and Endothelium.

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.

Fingolimod (FTY720), a Sphinogosine-1-Phosphate Receptor Agonist, Mitigates Choroidal Endothelial Proangiogenic Properties and Choroidal Neovascularization.

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.

Cytochrome P450 1B1: A Key Regulator of Ocular Iron Homeostasis and Oxidative Stress.

Cytochrome P450 (CYP) 1B1 belongs to the superfamily of heme-containing monooxygenases. Unlike other CYP enzymes, which are highly expressed in the liver, CYP1B1 is predominantly found in extrahepatic tissues, such as the brain, and ocular tissues including retina and trabecular meshwork. CYP1B1 metabolizes exogenous chemicals such as polycyclic aromatic hydrocarbons. CYP1B1 also metabolizes endogenous bioactive compounds including estradiol and arachidonic acid. These metabolites impact various cellular and physiological processes during development and pathological processes. We previously showed that CYP1B1 deficiency mitigates ischemia-mediated retinal neovascularization and drives the trabecular meshwork dysgenesis through increased levels of oxidative stress. However, the underlying mechanisms responsible for CYP1B1-deficiency-mediated increased oxidative stress remain largely unresolved. Iron is an essential element and utilized as a cofactor in a variety of enzymes. However, excess iron promotes the production of hydroxyl radicals, lipid peroxidation, increased oxidative stress, and cell damage. The retinal endothelium is recognized as a major component of the blood-retinal barrier, which controls ocular iron levels through the modulation of proteins involved in iron regulation present in retinal endothelial cells, as well as other ocular cell types including trabecular meshwork cells. We previously showed increased levels of reactive oxygen species and lipid peroxidation in the absence of CYP1B1, and in the retinal vasculature and trabecular meshwork, which was reversed by administration of antioxidant N-acetylcysteine. Here, we review the important role CYP1B1 expression and activity play in maintaining retinal redox homeostasis through the modulation of iron levels by retinal endothelial cells. The relationship between CYP1B1 expression and activity and iron levels has not been previously delineated. We review the potential significance of CYP1B1 expression, estrogen metabolism, and hepcidin-ferroportin regulatory axis in the local regulation of ocular iron levels.

7, 8-Dihydroxyflavone, a TrkB receptor agonist, provides minimal protection against retinal vascular damage during oxygen-induced ischemic retinopathy.

Retinopathy of prematurity (ROP) is one of the main causes of blindness in children worldwide. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), play critical protective roles in the development and function of neurons and vasculature. Lack of BDNF expression results in increased endothelial cell apoptosis and reduced endothelial cell-cell contact. Premature babies who develop ROP tend to have lower serum BDNF levels. BDNF expression is also significantly lower in mouse retinas following exposure to hyperoxia compared to those reared in room air. Specifically, BDNF promotes angiogenic tube formation of endothelial cells (EC), and it is considered an EC survival factor required for stabilization of intramyocardial vessels. We hypothesized that the activation of TrkB receptor protects retinal vasculature in the mice during oxygen-induced ischemic retinopathy (OIR), a model of ROP. To test this hypothesis, we treated neonatal mice with 7,8-dihydroxyflavone (DHF) (5 mg/kg body weight), a TrkB receptor agonist. We examined its potential protective effects on retinal vessel obliteration and neovascularization, two hallmarks of ROP and OIR. We found that retinas from DHF treated postnatal day 8 (P8) and P12 mice have similar levels of vessel obliteration as retinas from age-matched control mice subjected to OIR. Similarly, DHF showed no significant effect on mitigation of retinal neovascularization during OIR in P17 mice. Collectively, our studies demonstrate that the TrkB receptor agonist DHF provides no significant protective effects during OIR.

Caffeine Inhibits Choroidal Neovascularization Through Mitigation of Inflammatory and Angiogenesis Activities.

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.

1,25(OH)2D3 regulates the proangiogenic activity of pericyte through VDR-mediated modulation of VEGF production and signaling of VEGF and PDGF receptors.

We have previously demonstrated that the active form of vitamin D (calcitriol; 1,25(OH)2D3) is a potent inhibitor of retinal neovascularization. However, the underlying molecular and cellular mechanisms involved remained poorly understood. Perivascular supporting cells including pericytes (PC) play important roles during angiogenesis, vascular maturation, and stabilization of blood vessels. How 1,25(OH)2D3 affects retinal PC proliferation and migration, and whether these effects are mediated through vitamin D receptor (VDR), are unknown. Here, we determined the impact of 1,25(OH)2D3 on retinal PC prepared from wild-type (Vdr+/+) and VDR-deficient (Vdr-/-) mice. Retinal PC expressed significantly higher VDR levels compared to retinal endothelial cells (EC). Unlike retinal EC, 1,25(OH)2D3 significantly decreased PC proliferation and migration and resulted in a G0/G1 cell cycle arrest. Although 1,25(OH)2D3 did not inhibit the proliferation of Vdr-/- PC, it did inhibit their migration. PC adhesion to various extracellular matrix (ECM) proteins and ECM production were also affected by incubation of PC with 1,25(OH)2D3. Vdr-/- PC were more adherent compared with Vdr+/+ cells. Mechanistically, incubation of Vdr+/+ PC with 1,25(OH)2D3 resulted in an increased expression of vascular endothelial growth factor (VEGF) and attenuation of signaling through VEGF-R2 and platelet-derived growth factor receptor-beta. Incubation with soluble VEGF-R1 (sFlt-1) partially reversed the effect of VEGF on Vdr+/+ PC. In addition, incubation of Vdr+/+ PC with VEGF or inhibition of VEGF-R2 increased VDR expression. Together, these results suggest an important role for retinal PC as a target for vitamin D and VDR action for attenuation of angiogenesis.