Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1β secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40+ Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C-dependent ATP release that caused P2X7-dependent production of TNF-α and IL-1β by macrophages. P2X7-/- mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TNF-α, IL-1β, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X7 pathway.

Chronic hyperglycemia during diabetes leads to increased production of reactive oxygen species (ROS) and increased oxidative stress (OS). Here we investigated whether changes in the metabolic state can be used as a marker of OS progression in kidneys. We examined redox states of kidneys from diabetic mice, Akita(/+) and Akita(/+);TSP1(-/-) mice (Akita mice lacking thrombospondin-1, TSP1) with increasing duration of diabetes. OS as measured by mitochondrial redox ratio (NADH/FAD) was detectable shortly after the onset of diabetes and further increased with the duration of diabetes. Thus, cryo fluorescence redox imaging was used as a quantitative marker of OS progression in kidneys from diabetic mice and demonstrated that alterations in the oxidative state of kidneys occur during the early stages of diabetes.

We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR). Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE) dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose) on caspase-14 expression in human RPE (ARPE-19) cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose). We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS) to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER). These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.

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

To evaluate the effects of deionised water, blood, phosphate-buffered saline (PBS) and a new anti-corrosive solution based on methoxy propyl amine (MOPA) on the cyclic fatigue resistance of endodontic NiTi rotary instruments under in vitro conditions.

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.

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.

Astrocytes (AC) are the most abundant cells in the central nervous system. In the retina, astrocytes play important roles in the development and integrity of the retinal neurovasculature. Astrocytes dysfunction contributes to pathogenesis of a variety of neurovascular diseases including diabetic retinopathy. Recent studies have demonstrated the expression of Cyp1b1 in the neurovascular cells of the central nervous system including AC. We recently showed retinal AC constitutively express Cyp1b1, and global Cyp1b1-deficiency (Cyp1b1-/-) attenuates retinal ischemia-mediated neovascularization in vivo and the pro-angiogenic activity of retinal vascular cells in vitro. We also demonstrated that Cyp1b1 expression is a key regulator of retinal AC function. However, the underlying mechanisms involved need further investigation. Here we determined changes in the transcriptome profiles of Cyp1b1+/+ and Cyp1b1-/- retinal AC by RNA sequencing. We identified 585 differentially expressed genes, whose pathway enrichment analysis revealed the most significant pathways impacted in Cyp1b1-/- AC. These genes included those of axon guidance, extracellular matrix proteins and their receptors, cancer, cell adhesion molecules, TGF-β signaling, and the focal adhesion modulation. The expression of a selected set of differentially expressed genes was confirmed by RT-qPCR analysis. To our knowledge, this is the first report of RNAseq investigation of the retinal AC transcriptome and the molecular pathways impacted by Cyp1b1 expression. These results demonstrated an important role for Cyp1b1 expression in the regulation of various retinal AC functions, which are important in neurovascular development and integrity.

Human cytomegalovirus (HCMV) is the leading infectious cause of vision loss among congenitally infected children. Retinal pericytes play an essential role in maintaining retinal vascular and endothelial cell proliferation. However, the role of retinal pericytes in ocular HCMV pathogenesis is unknown.

Supplemental oxygen, which is routinely administered to preterm infants with pulmonary insufficiency, contributes to bronchopulmonary dysplasia (BPD) in these infants. Hyperoxia also contributes to the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults. The mechanisms of oxygen-mediated pulmonary toxicity are not completely understood. Recent studies have suggested an important role for cytochrome P450 (CYP)1A1/1A2 in the protection against hyperoxic lung injury. The role of CYP1B1 in oxygen-mediated pulmonary toxicity has not been studied. In this investigation, we tested the hypothesis that CYP1B1 plays a mechanistic role in oxygen toxicity in pulmonary cells in vitro. In human bronchial epithelial cell line BEAS-2B, hyperoxic treatment for 1-3 days led to decreased cell viability by about 50-80%. Hyperoxic cytotoxicity was accompanied by an increase in levels of reactive oxygen species (ROS) by up to 110%, and an increase of TUNEL-positive cells by up to 4.8-fold. Western blot analysis showed hyperoxia to significantly down-regulate CYP1B1 protein level. Also, there was a decrease of CYP1B1 mRNA by up to 38% and Cyp1b1 promoter activity by up to 65%. On the other hand, CYP1B1 siRNA appeared to rescue the cell viability under hyperoxia stress, and overexpression of CYP1B1 significantly attenuated hyperoxic cytotoxicity after 48 h of incubation. In immortalized lung endothelial cells derived from Cyp1b1-null and wild-type mice, hyperoxia increased caspase 3/7 activities in a time-dependent manner, but endothelial cells lacking the Cyp1b1 gene showed significantly decreased caspase 3/7 activities after 48 and 72 h of incubation, implying that CYP1B1 might promote apoptosis in wild type lung endothelial cells under hyperoxic stress. In conclusion, our results support the hypothesis that CYP1B1 plays a mechanistic role in pulmonary oxygen toxicity, and CYP1B1-mediated apoptosis could be one of the mechanisms of oxygen toxicity. Thus, CYP1B1 could be a novel target for preventative and/or therapeutic interventions against BPD in infants and ALI/ARDS in adults.

Bcl-2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl-2 (Bcl-2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl-2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl-2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl-2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl-2 allele (Bcl-2Flox/Flox) and VE-cadherin-cre (Bcl-2EC mice). Bcl-2EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl-2 deficient mice. Bcl-2EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl-2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl-2 function in the endothelium.

Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM) protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim(-/-)) mice. Bim(-/-) endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim(-/-) endothelial cells with Bim(-/-) pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.

Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).

Quantitatively determining physiological parameters at a microscopic level in the retina furthers the understanding of the molecular pathways of blinding diseases, such as diabetic retinopathy and glaucoma. An essential parameter, which has yet to be quantified noninvasively, is the retinal oxygen metabolic rate (rMRO2). Quantifying rMRO2 is challenging because two parameters, the blood flow rate and hemoglobin oxygen saturation (sO2), must be measured together. We combined photoacoustic ophthalmoscopy (PAOM) with spectral domain-optical coherence tomography (SD-OCT) to tackle this challenge, in which PAOM measured the sO2 and SD-OCT mapped the blood flow rate. We tested the integrated system on normal wild-type rats, in which the measured rMRO2 was 297.86 ± 70.23 nl/minute. This quantitative method may shed new light on both fundamental research and clinical care in ophthalmology in the future.

Silica nanoparticle supported imidazolium ionic liquid [SNImIL] was synthesized and utilized as a biocompatible additive for studying the thermal reversibility of human carbonic anhydrase II (HCA II). For this purpose, we prepared additive by modification of nanoparticles through the grafting of ionic liquids on the surface of nanoparticles (SNImIL). The SNImIL were fully characterized by Fourier transform infrared spectroscopy, scanning electron microscopy and thermo gravimetric analysis. The characterization of HCA II was investigated by various techniques including UV-vis and ANS fluorescence spectrophotometry, differential scanning calorimetry, and docking study. SNImIL induced disaggregation, enhanced protein stability and increased thermal reversibility of HCA II by up to 42% at pH 7.75.

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.

A significant proportion of children that survive hypoxic-ischemic encephalopathy (HIE) develop visual impairment. These visual deficits are generally attributed to injuries that occur in the primary visual cortex and other visual processing systems. Recent studies suggested that neuronal damage might also occur in the retina. An important structure affecting the viability of retinal neurons is the vasculature. However, the effects of HIE on the retinal neurovasculature have not been systemically evaluated. Here we investigated whether exposure of postnatal day 9 (P9) neonatal mice to HIE is sufficient to induce neurovascular damage in the retina. We demonstrate that the blood vessels on the surface of the retina, from mice subjected to HIE, were abnormally enlarged with signs of degeneration. The intermediate and deep vascular layers in these retinas failed to form normally, particularly in the periphery. All the vascular damages observed here were irreversible in nature up to 100 days post HIE. We also observed loss of retinal neurons, together with changes in both astrocytes and Müller cells mainly in the inner retina at the periphery. Collectively, our findings suggest that HIE results in profound alterations in the retinal vasculature, indicating the importance of developing therapeutic strategies to protect neurovascular dysfunction not only in the brain but also in the retina for infants exposed to HIE.

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.

To validate the ability of visible-light optical coherence tomography (vis-OCT) in imaging the full Schlemm's canal (SC) and its surrounding limbal vascular network in mice in vivo through a compound circumlimbal scan.