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.

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.

Novel anti-angiogenic PEDF-derived small peptides mitigate choroidal neovascularization.

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.

Bim Expression Promotes the Clearance of Mononuclear Phagocytes during Choroidal Neovascularization, Mitigating Scar Formation in Mice.

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.

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.

Targeted Thrombospondin-1 Expression in Ocular Vascular Development and Neovascularization.

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.

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.

Bcl-2 Expression in Pericytes and Astrocytes Impacts Vascular Development and Homeostasis.

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-2Flox/Flox) and Pdgfrb-cre (Bcl-2PC mice) or Gfap-cre (Bcl-2AC mice). Bcl-2PC and Bcl-2AC mice demonstrated increased retinal vascular cell apoptosis, reduced numbers of pericytes and endothelial cells and fewer arteries and veins in the retina. Bcl-2PC mice also demonstrated delayed advancement of the superficial retinal vascular layer and aberrant vascularization of the deep vascular plexus and central retina. Although pathologic neovascularization in oxygen-induced ischemic retinopathy (OIR) was not affected by lack of expression of Bcl-2 in either pericytes or astrocytes, laser-induced choroidal neovascularization (CNV) was significantly reduced in Bcl-2PC mice compared to littermate controls. Together these studies begin to reveal how cell autonomous modulation of apoptosis in vascular cells impacts development and homeostasis.

Resveratrol inhibits uveal melanoma tumor growth via early mitochondrial dysfunction.

PURPOSE: To test the efficacy of resveratrol, a nontoxic plant product, in the treatment of uveal melanoma. METHODS: The effect of oral administration and peritumor injection of resveratrol was tested on tumor growth in two animal models of uveal melanoma. The mechanism of resveratrol action on uveal melanoma cells was studied in vitro in a cell-viability assay: with JC-1 dye, to measure mitochondrial membrane potential; by Western blot analysis, to analyze the cellular redistribution of cytochrome c and Smac/diablo; and in a fluorescence assay with specific substrates, to measure activation of different caspases. RESULTS: Resveratrol treatment inhibited tumor growth in animal models of uveal melanoma. Since oral administration resulted in relatively low bioavailability of resveratrol, the effect of increased local levels was tested by peritumor injection of the drug. This method resulted in tumor cell death and tumor regression. In vitro experiments with multiple uveal melanoma cell lines demonstrate that resveratrol causes a decrease in cell viability, resulting at least in part from an increase in apoptosis through a mitochondrial pathway. An early event in drug action is the direct targeting of mitochondria by resveratrol, which leads to a decrease in mitochondrial membrane potential and the eventual activation of caspase-3. CONCLUSION: These data suggest that resveratrol can inhibit tumor growth and can induce apoptosis via the intrinsic mitochondrial pathway and that by further increasing bioavailability of resveratrol the potency of the drug can be increased, leading to tumor regression. The nontoxic nature of the drug at levels needed for therapy make resveratrol an attractive candidate for the treatment of uveal melanoma.

Resveratrol inhibits tumor growth of human neuroblastoma and mediates apoptosis by directly targeting mitochondria.

PURPOSE: Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Because resveratrol, a natural plant product, has been reported to have limited toxicity at chemotherapeutic levels, we investigated its efficacy in the treatment of neuroblastoma as well as its underlying mechanism of action. EXPERIMENTAL DESIGN: Resveratrol was tested in mouse xenograft models of human neuroblastoma and in vitro using human cell lines. RESULTS: Resveratrol inhibited the outgrowth of tumors by as much as 80%. The bioavailability of the drug in serum was in the low micromolar range (2-10 micromol/L) and no accumulation was observed in tumor tissue. When resveratrol levels were increased by peritumor injection, rapid tumor regression occurred. Resveratrol decreased tumor cell viability in vitro by 75% to 90%, resulting from an inhibition of cell proliferation and an induction of apoptosis. Loss of mitochondrial membrane potential was an early response to resveratrol. In addition, resveratrol treatment of isolated mitochondria also led to depolarization, suggesting that the drug may target mitochondria directly. Following depolarization, resveratrol caused the release of cytochrome c and Smac/Diablo from the mitochondria and subsequently the activation of caspase-9 (4- to 8-fold) and caspase-3 (4- to 6-fold). CONCLUSIONS: These studies indicate that, despite low bioavailability, resveratrol is effective at inhibiting tumor growth. Elevated levels of resveratrol enhance its antitumor potency leading to tumor regression, associated with widespread tumor cell death, the underlying mechanism of which involves the direct activation of the mitochondrial intrinsic apoptotic pathway.

Extended Intravitreal Rabbit Eye Residence of Nanoparticles Conjugated With Cationic Arginine Peptides for Intraocular Drug Delivery: In Vivo Imaging.

Purpose: Drug delivery by intravitreal injection remains problematic, small agents and macromolecules both clearing rapidly. Typical carriers use microparticles (>2 µm), with size-related liabilities, to slow diffusion. We recently described cationic nanoparticles (NP) where conjugated Arg peptides prolonged residence in rat eyes, through ionic interaction with vitreal poly-anions. Here we extended this strategy to in vivo tracking of NP-conjugate (NPC) clearance from rabbit eyes. Relating t1/2 to zeta potential, and varied dose, we estimated the limits of this charge-based delivery system. Methods: NPC carried covalently attached PEG8-2Arg or PEG8-3Arg pentapeptides, having known sequences from human eye proteins. Peptides were conjugated (61-64 per NPC); each NP/NPC also carried a cyanine7 tag (<0.5 dye/particle). In vivo imaging system (IVIS), after intravitreal injection, estimated NPC loss by 800-nm photon emission (745-nm excitation) at 1 to 3-week intervals following initial scan at day 10. Results: NPC of 2Arg-peptides or 3Arg-peptides showed clearance t1/2 of 7 days and 17 days respectively, unconjugated NP t1/2 was <<5 days. Doses of 90, 180, and 360 µg of PEG8-2Arg NPC were compared. The lower doses showed dose-proportional day-10 concentration, and similar clearance. Higher early loss was seen with a 360-µg dose, exceeding rabbit vitreal binding capacity. No inflammation was observed. Conclusions: This type of cationic NPC can safely increase residence t1/2 in a 1 to 3-week range, with dose <100 µg per mL vitreous. Human drug load may then range from 10 to 100 µg/eye, usefulness depending on individual drug potency and release rate, superimposed on extended intravitreal residence.

Mitochondria, calcium, and calpain are key mediators of resveratrol-induced apoptosis in breast cancer.

Resveratrol (RES), a natural plant polyphenol, has gained interest as a nontoxic chemopreventive agent capable of inducing tumor cell death in a variety of cancer types. However, the early molecular mechanisms of RES-induced apoptosis are not well defined. Using the human breast cancer cell lines MDA-MB-231 and MCF-7, we demonstrate that RES is antiproliferative and induces apoptosis in a concentration- and time-dependent manner. Preceding apoptosis, RES instigates a rapid dissipation of mitochondrial membrane potential by directly targeting mitochondria. This is followed by release of cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) into the cytoplasm and substantial increase in the activities of caspases-9 and -3 in MDA-MB-231 cells. In addition, live cell microscopy demonstrates that RES causes an early biphasic increase in the concentration of free intracellular calcium ([Ca2+]i), probably resulting from depletion of the endoplasmic reticulum stores in breast cancer cells. In caspase-3-deficient MCF-7 cells, apoptosis is mediated by the Ca2+-activated protease, calpain, leading to the degradation of plasma membrane Ca2+-ATPase isoform 1 and fodrin; the degradation is attenuated by buffering [Ca2+]i and blocked by calpain inhibitors. Mitochondrial permeability transition pore antagonists also blocked calpain activation. In vivo mouse xenograft studies demonstrate that RES treatment inhibits breast cancer growth with no systemic toxicities. Together, these results suggest a critical role for mitochondria not only in the intrinsic apoptotic pathway but also in the Ca2+ and calpain-dependent cell death initiated by RES. Thus, RES may prove useful as a nontoxic alternative for breast cancer treatment.

Calcitriol is a potent inhibitor of retinal neovascularization.

PURPOSE: Vitamin D compounds inhibit the growth of a variety of tumors in preclinical and clinical studies. Among the mechanisms suggested for this inhibition is antiangiogenesis. Retinal angiogenesis is the basis for vision loss in several major blinding diseases. The purpose of this study was to evaluate the antiangiogenic activity of calcitriol (1,25-dihydroxyvitamin D(3)) in vivo and its effect on retinal endothelial cell (EC) proliferation, migration, and capillary morphogenesis in vitro. METHODS: The mouse oxygen-induced ischemic retinopathy (OIR) model was used to assess the antiangiogenic activity of calcitriol. Ocular VEGF levels were determined by Western blot analysis of whole eye extracts from postnatal day (P) 15 mice during OIR. The effects of calcitriol on retinal EC proliferation, migration, and capillary morphogenesis were also assessed in vitro. RESULTS: Calcitriol-treated animals demonstrated a significant decrease in retinal neovascularization compared with control animals. This effect was dose dependent, and retinal neovascularization was significantly inhibited in calcitriol-treated mice. Although no deaths occurred, calcitriol administration was associated with increased serum calcium and a lack of increase in body weight in a dose-independent manner. The ocular level of VEGF was similar in control and calcitriol-treated animals. At a lower concentration of calcitriol, retinal EC capillary morphogenesis in solubilized basement membrane was inhibited without a significant inhibitory effect on EC proliferation and migration. The concentration of calcitriol required to inhibit retinal EC proliferation was significantly higher than that required to inhibit EC capillary morphogenesis. CONCLUSIONS: These data suggest calcitriol is a potent inhibitor of retinal neovascularization and may be of benefit in the treatment of a variety of eye diseases with a neovascular component.

Vitamin D receptor expression is essential during retinal vascular development and attenuation of neovascularization by 1, 25(OH)2D3.

Vitamin D provides a significant benefit to human health, and its deficiency has been linked to a variety of diseases including cancer. Vitamin D exhibits anticancer effects perhaps through inhibition of angiogenesis. We previously showed that the active form of vitamin D (1, 25(OH)2D3; calcitriol) is a potent inhibitor of angiogenesis in mouse model of oxygen-induced ischemic retinopathy (OIR). Many of vitamin D's actions are mediated through vitamin D receptor (VDR). However, the role VDR expression plays in vascular development and inhibition of neovascularization by 1, 25(OH)2D3 remains unknown. Here using wild type (Vdr +/+) and Vdr-deficient (Vdr -/-) mice, we determined the impact of Vdr expression on postnatal development of retinal vasculature and retinal neovascularization during OIR. We observed no significant effect on postnatal retinal vascular development in Vdr -/- mice up to postnatal day 21 (P21) compared with Vdr +/+ mice. However, we observed an increase in density of pericytes (PC) and a decrease in density of endothelial cells (EC) in P42 Vdr -/- mice compared with Vdr +/+ mice, resulting in a significant decrease in the EC/PC ratio. Although we observed no significant impact on vessel obliteration and retinal neovascularization in Vdr -/- mice compared with Vdr +/+ mice during OIR, the VDR expression was essential for inhibition of retinal neovascularization by 1, 25(OH)2D3. In addition, the adverse impact of 1, 25(OH)2D3 treatment on the mouse bodyweight was also dependent on VDR expression. Thus, VDR expression plays a significant role during retinal vascular development, especially during maturation of retinal vasculature by promoting PC quiescence and EC survival, and inhibition of ischemia-mediated retinal neovascularization by 1, 25(OH)2D3.

Pilot Study of a "Large-Eye," Surgically Induced Dry Eye Rabbit Model by Selective Removal of the Harderian, Lacrimal, and Meibomian Glands.

BACKGROUND/AIMS: Establish a reliable rabbit dry eye (DE) model. METHODS: An interventional cohort study surgically removing glands contributing to the tear film. Eight rabbits were studied after removal of left lacrimal, Harderian, or both glands. Additional rabbits had Meibomian glands in the left eye thermally obstructed. All were followed for 10 weeks with phenol red thread (PRT) and slit-lamp examination with 2% fluorescein. We assessed corneal sensitivity using a Cochet-Bonnet esthesiometer. Outcome measures were severity/duration of reduced PRT, punctate epithelial erosions (PEE), and histologic evidence of corneal pannus. RESULTS: Fluorescein staining demonstrated signs of dryness including PEE in all of the interventional eyes. The subjective measurement of epithelial erosions correlated with decreased tear production. PRT measurements in the control eyes averaged 31.54 mm (±1.83) and 22.71 mm (±1.60) in the eight left eyes, without loss of corneal sensitivity. CONCLUSIONS: Surgical removal of either the Harderian or lacrimal gland results in statistically significant decreases in tear volume and the development of severe DE. Removal of both glands results in the occurrence of a DE of comparable severity/duration to removal of either the lacrimal or Harderian gland alone. Meibomian gland obstruction contributes less to the DE model.

ß2-Adrenergic Receptor Antagonism Attenuates CNV Through Inhibition of VEGF and IL-6 Expression.

Purpose: The role of ß-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 ß2-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 ß2-AR regulates CNV through modulation of VEGF and inflammatory cytokine expression. Methods: Mice were subjected to laser burns, inducing CNV, and were treated with an intravitreal ß2-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 ß-AR signaling on VEGF and IL-6 expression were investigated in various mouse retinal and human RPE cells by using specific ß-AR agonists and antagonists. Results: ß2-Adrenergic receptor signaling increased Vegf mRNA expression by approximately 3- to 4-fold in mouse retinal microglia and pericytes in culture. ß2-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 ß2-AR antagonist ICI 118,551 reduced CNV by 35% and decreased IL-6 protein levels by approximately 50%. In primary human RPE cells, ß2-AR activation also stimulated VEGF and IL-6 mRNA expression by 2- and 10-fold, respectively. Conclusions: Anti-VEGF therapy for CNV is highly effective; however, some patients are resistant to therapy while others undergo repeated, frequent treatments. ß2-Adrenergic receptor signaling is a potential therapeutic target because of its angiogenic and inflammatory properties.

Resveratrol targeting of AKT and p53 in glioblastoma and glioblastoma stem-like cells to suppress growth and infiltration.

OBJECTIVE Glioblastoma multiforme (GBM) is an aggressive brain cancer with median survival of less than 2 years with current treatment. Glioblastomas exhibit extensive intratumoral and interpatient heterogeneity, suggesting that successful therapies should produce broad anticancer activities. Therefore, the natural nontoxic pleiotropic agent, resveratrol, was studied for antitumorigenic effects against GBM. METHODS Resveratrol's effects on cell proliferation, sphere-forming ability, and invasion were tested using multiple patient-derived GBM stem-like cell (GSC) lines and established U87 glioma cells, and changes in oncogenic AKT and tumor suppressive p53 were analyzed. Resveratrol was also tested in vivo against U87 glioma flank xenografts in mice by using multiple delivery methods, including direct tumor injection. Finally, resveratrol was delivered directly to brain tissue to determine toxicity and achievable drug concentrations in the brain parenchyma. RESULTS Resveratrol significantly inhibited proliferation in U87 glioma and multiple patient-derived GSC lines, demonstrating similar inhibitory concentrations across these phenotypically heterogeneous lines. Resveratrol also inhibited the sphere-forming ability suggesting anti-stem cell effects. Additionally, resveratrol blocked U87 glioma and GSC invasion in an in vitro Matrigel Transwell assay at doses similar to those mediating antiproliferative effects. In U87 glioma cells and GSCs, resveratrol reduced AKT phosphorylation and induced p53 expression and activation that led to transcription of downstream p53 target genes. Resveratrol administration via oral gavage or ad libitum in the water supply significantly suppressed GBM xenograft growth; intratumoral or peritumoral resveratrol injection further suppressed growth and approximated tumor regression. Intracranial resveratrol injection resulted in 100-fold higher local drug concentration compared with intravenous delivery, and with no apparent toxicity. CONCLUSIONS Resveratrol potently inhibited GBM and GSC growth and infiltration, acting partially via AKT deactivation and p53 induction, and suppressed glioblastoma growth in vivo. The ability of resveratrol to modulate AKT and p53, as well as reportedly many other antitumorigenic pathways, is attractive for therapy against a genetically heterogeneous tumor such as GBM. Although resveratrol exhibits low bioavailability when administered orally or intravenously, novel delivery methods such as direct injection (i.e., convection-enhanced delivery) could potentially be used to achieve and maintain therapeutic doses in the brain. Resveratrol's nontoxic nature and broad anti-GBM effects make it a compelling candidate to supplement current GBM therapies.

Mitochondria as the primary target of resveratrol-induced apoptosis in human retinoblastoma cells.

PURPOSE: To determine the molecular mechanisms by which resveratrol induces retinoblastoma tumor cell death. METHODS: After resveratrol treatment, Y79 tumor cell viability was measured using a fluorescence-based assay, and proapoptotic and antiproliferative effects were characterized by Hoechst stain and flow cytometry, respectively. Mitochondrial transmembrane potential (DeltaPsim) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), whereas the release of cytochrome c from mitochondria was assayed by immunoblotting and caspase activities were determined by monitoring the cleavage of fluorogenic peptide substrates. RESULTS: Resveratrol induced a dose- and time-dependent decrease in Y79 tumor cell viability and inhibited proliferation by inducing S-phase growth arrest and apoptotic cell death. Preceding cell death, resveratrol evoked a rapid dissipation of DeltaPsim. This was followed by the release of cytochrome c into the cytoplasm and a substantial increase in the activities of caspase-9 and caspase-3. Additionally, in a cell-free system, resveratrol directly induced the depolarization of isolated mitochondria. CONCLUSIONS: These results demonstrate that resveratrol, a nontoxic natural plant compound, inhibits Y79 cell proliferation and stimulates apoptosis through activation of the mitochondrial (intrinsic) apoptotic pathway and may warrant further exploration as an adjuvant to conventional anticancer therapies for retinoblastoma.