Background: Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit. Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma. Here, we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier (BRB).

Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice, a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells. Capillary density, mitochondrial volume, and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software. Dynamin-related protein (DRP-1), mitofusin-2 (MFN-2) and optic atrophy-1 (OPA-1) expression were assessed by western blot analysis of enriched endothelial cells. Mitochondrial structure was evaluated by transmission electron microscopy (TEM) and oxygen consumption rate was monitored by Seahorse analysis. The integrity of the BRB was evaluated by quantifying Evans blue leakage.

Results: Our data demonstrate that two and three weeks after ocular hypertension induction, the total mitochondria volume in endothelial cells decreased from 0.140±0.002 μm³ from non-injured retinas to 0.108±0.005 and 0.093±0.007 μm³, respectively in glaucomatous eyes (mean ± S.E.M, ANOVA, P<0.001; N=6/group). Frequency distribution showed a substantial increase of smaller mitochondria complexes (<0.5 μm³) in endothelial cells from glaucomatous retinas. Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas, while MFN-2 and OPA-1 expression was not affected. Structural alteration in endothelial cell mitochondria was confirmed by TEM, which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas (n=3–6/group). In addition, this model did not trigger changes in the density of the vascular network, suggesting that mitochondrial fragmentation was not due to endothelial cell loss.

Conclusions: This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria, contributing to vascular dysfunction in glaucoma.

Background: Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit. Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma. Here, we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier (BRB).

Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice, a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells. Capillary density, mitochondrial volume, and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software. Dynamin-related protein (DRP-1), mitofusin-2 (MFN-2) and optic atrophy-1 (OPA-1) expression were assessed by western blot analysis of enriched endothelial cells. Mitochondrial structure was evaluated by transmission electron microscopy (TEM) and oxygen consumption rate was monitored by Seahorse analysis. The integrity of the BRB was evaluated by quantifying Evans blue leakage.

Results: Our data demonstrate that two and three weeks after ocular hypertension induction, the total mitochondria volume in endothelial cells decreased from 0.140±0.002 μm³ from non-injured retinas to 0.108±0.005 and 0.093±0.007 μm³, respectively in glaucomatous eyes (mean ± S.E.M, ANOVA, P<0.001; N=6/group). Frequency distribution showed a substantial increase of smaller mitochondria complexes (<0.5 μm³) in endothelial cells from glaucomatous retinas. Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas, while MFN-2 and OPA-1 expression was not affected. Structural alteration in endothelial cell mitochondria was confirmed by TEM, which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas (n=3–6/group). In addition, this model did not trigger changes in the density of the vascular network, suggesting that mitochondrial fragmentation was not due to endothelial cell loss.

Conclusions: This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria, contributing to vascular dysfunction in glaucoma.

Background: Retinopathy of prematurity (ROP) is the major cause of blindness in children, mainly caused by the retinal neovascularization (NV). Mounting of evidences shown that macrophage plays a pivotal role in the regulation of angiogenesis in ROP. Numerous studies confirmed that the deletion of macrophage significantly reduce the neovascularized areas in the oxygen-induced retinopathy (OIR) model. We have been studied the effect of lymphocyte derived-microparticles (LMPs) over ten years. LMPs are extracellular vesicles derived from apoptotic human CEM T lymphocytes. Our previous studies demonstrated that LMPs possess strong anti-angiogenic effect. Recently we observed that LMPs are capable to switch the phenotype of macrophage, thus to suppress the choroidal neovascularization (CNV). However, the role of LMPs on macrophage in ROP has not been clarified. Thus, my project is to disclose the relationship between LMPs and macrophage in ROP using the OIR model. Hypothesis: LMPs may inhibit retinal NV in the OIR model through targeting at macrophage by affecting the migration of macrophage, thus to inhibit pathological angiogenesis in ROP.

Methods: Cell culture [RAW 264.7 and bone marrow-derived macrophage (BMDM)] for cell migration and viability assay. Generate the OIR model for in vivo detection of macrophage recruitment. Quantification of retinal NV, immunohistostaining of the macrophage in vivo, ex vivo retinal explants for cell migration and qPCR.

Results: LMPs do not affect RAW 264.7 and BMDM cell viability (P>0.05). LMPs significantly decrease the BMDM cell migration indirectly (P<0.05). I successfully generate the OIR model and confirm that more macrophages infiltrate during retinal angiogenesis with counting the F4/80 immunostaining in the retinal flat mount. LMPs exert inhibiting effect on retinal angiogenesis through decreasing the migration of macrophages in vivo.

Conclusions: LMPs have the negative effect on retinal angiogenesis via reducing the infiltrated macrophages to the neovascularized areas in the OIR model.

Background: Retinopathy of prematurity (ROP) is the major cause of blindness in children, mainly caused by the retinal neovascularization (NV). Mounting of evidences shown that macrophage plays a pivotal role in the regulation of angiogenesis in ROP. Numerous studies confirmed that the deletion of macrophage significantly reduce the neovascularized areas in the oxygen-induced retinopathy (OIR) model. We have been studied the effect of lymphocyte derived-microparticles (LMPs) over ten years. LMPs are extracellular vesicles derived from apoptotic human CEM T lymphocytes. Our previous studies demonstrated that LMPs possess strong anti-angiogenic effect. Recently we observed that LMPs are capable to switch the phenotype of macrophage, thus to suppress the choroidal neovascularization (CNV). However, the role of LMPs on macrophage in ROP has not been clarified. Thus, my project is to disclose the relationship between LMPs and macrophage in ROP using the OIR model. Hypothesis: LMPs may inhibit retinal NV in the OIR model through targeting at macrophage by affecting the migration of macrophage, thus to inhibit pathological angiogenesis in ROP.

Methods: Cell culture [RAW 264.7 and bone marrow-derived macrophage (BMDM)] for cell migration and viability assay. Generate the OIR model for in vivo detection of macrophage recruitment. Quantification of retinal NV, immunohistostaining of the macrophage in vivo, ex vivo retinal explants for cell migration and qPCR.

Results: LMPs do not affect RAW 264.7 and BMDM cell viability (P>0.05). LMPs significantly decrease the BMDM cell migration indirectly (P<0.05). I successfully generate the OIR model and confirm that more macrophages infiltrate during retinal angiogenesis with counting the F4/80 immunostaining in the retinal flat mount. LMPs exert inhibiting effect on retinal angiogenesis through decreasing the migration of macrophages in vivo.

Conclusions: LMPs have the negative effect on retinal angiogenesis via reducing the infiltrated macrophages to the neovascularized areas in the OIR model.

Background: The aim of this project is to develop a new standardized and cost-efficient method to compare optical coherence tomography (OCT) scans to their corresponding paraffin embedded histopathology sections in post-mortem eyes. This correlation will clarify the interpretation of OCT images, and it will also enable direct immunohistochemical characterization of features observed on OCT.

Methods: Study design: donor eyes were obtained from two separate eye banks. In order to minimize post-mortem change like retinal detachment and vitreous opacification, the eyes were fixed in a previously tested fixative solution. Time between death and fixation has been kept under 6 hours. Methods: Using a customized imaging device, nine post-mortem eyes were imaged with a SD-OCT machine. Subsequently, an 8mm trephine was used to isolate a portion of the posterior pole including the macular area and the optic nerve head for histopathological analysis. Paraffin embedded cross sections of the retina were obtained and visually compared to each OCT image (b-scans).

Results: To facilitate the correlation of OCT images to their histopathological sections, three principle aspects were controlled during tissue processing: rotation, tilt and location. Using markings as well as anatomical landmarks, serial histopathological sections in an orientation comparable to OCT b-scans were obtained, thereby facilitating image pairing.

Conclusions: Compared to other well-established methods using resin and electron microscopy, our standardized Methods allowed us to successfully compare OCT b-scans to serial retinal cross sections of a wider macular area at a lower cost. Our novel approach allows us to translate features observed on OCT images into well-established histopathological images, providing the clinician with additional tools to obtain difficult diagnoses with more confidence.

Background: The aim of this project is to develop a new standardized and cost-efficient method to compare optical coherence tomography (OCT) scans to their corresponding paraffin embedded histopathology sections in post-mortem eyes. This correlation will clarify the interpretation of OCT images, and it will also enable direct immunohistochemical characterization of features observed on OCT.

Methods: Study design: donor eyes were obtained from two separate eye banks. In order to minimize post-mortem change like retinal detachment and vitreous opacification, the eyes were fixed in a previously tested fixative solution. Time between death and fixation has been kept under 6 hours. Methods: Using a customized imaging device, nine post-mortem eyes were imaged with a SD-OCT machine. Subsequently, an 8mm trephine was used to isolate a portion of the posterior pole including the macular area and the optic nerve head for histopathological analysis. Paraffin embedded cross sections of the retina were obtained and visually compared to each OCT image (b-scans).

Results: To facilitate the correlation of OCT images to their histopathological sections, three principle aspects were controlled during tissue processing: rotation, tilt and location. Using markings as well as anatomical landmarks, serial histopathological sections in an orientation comparable to OCT b-scans were obtained, thereby facilitating image pairing.

Conclusions: Compared to other well-established methods using resin and electron microscopy, our standardized Methods allowed us to successfully compare OCT b-scans to serial retinal cross sections of a wider macular area at a lower cost. Our novel approach allows us to translate features observed on OCT images into well-established histopathological images, providing the clinician with additional tools to obtain difficult diagnoses with more confidence.

Background: Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. The contribution of pericytes to microvascular deficits in glaucoma is currently unknown. To address this, we used two-photon excitation microscopy for longitudinal monitoring of retinal pericytes and capillaries in a mouse glaucoma model.

Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of albino mice expressing red fluorescent protein selectively in pericytes (NG2-DsRed). Minimally invasive, multiphoton imaging through the sclera of live NG2-DsRed mice was used to visualize pericytes and capillary diameter at one, two and three weeks after glaucoma induction. In vivo fluctuations in pericyte intracellular calcium were monitored with the calcium indicator Fluo-4. Ex vivo stereological analysis of retinal tissue prior to and after injection of microbeads was used to confirm our in vivo findings.

Results: Live two-photon imaging of NG2-DsRed retinas demonstrated that ocular hypertension induced progressive accumulation of intracellular calcium in pericytes. Calcium uptake correlated directly with the narrowing of capillaries in the superficial, inner, and outer vascular plexuses (capillary diameter: na?ve control =4.7±0.1 μm, glaucoma =4.0±0.1 μm, n=5–6 mice/group, Student’s t-test P<0.05). Frequency distribution analysis showed a substantial increase in the number of small-diameter capillaries (≤3 μm) and a decrease in larger-diameter microvessels (≥5–9 μm) at three weeks after induction of ocular hypertension (n=5–6 mice/group, Student’s t-test P<0.05).

Conclusions: Our data support two main conclusions. First, two-photon excitation microscopy is an effective strategy to monitor longitudinal changes in retinal pericytes and capillaries in live animals at glaucoma onset and progression. Second, ocular hypertension triggers rapid intracellular calcium increase in retinal pericytes leading to substantial capillary constriction. This study identifies retinal pericytes as important mediators of early microvascular dysfunction in glaucoma.

Background: Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. The contribution of pericytes to microvascular deficits in glaucoma is currently unknown. To address this, we used two-photon excitation microscopy for longitudinal monitoring of retinal pericytes and capillaries in a mouse glaucoma model.

Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of albino mice expressing red fluorescent protein selectively in pericytes (NG2-DsRed). Minimally invasive, multiphoton imaging through the sclera of live NG2-DsRed mice was used to visualize pericytes and capillary diameter at one, two and three weeks after glaucoma induction. In vivo fluctuations in pericyte intracellular calcium were monitored with the calcium indicator Fluo-4. Ex vivo stereological analysis of retinal tissue prior to and after injection of microbeads was used to confirm our in vivo findings.

Results: Live two-photon imaging of NG2-DsRed retinas demonstrated that ocular hypertension induced progressive accumulation of intracellular calcium in pericytes. Calcium uptake correlated directly with the narrowing of capillaries in the superficial, inner, and outer vascular plexuses (capillary diameter: na?ve control =4.7±0.1 μm, glaucoma =4.0±0.1 μm, n=5–6 mice/group, Student’s t-test P<0.05). Frequency distribution analysis showed a substantial increase in the number of small-diameter capillaries (≤3 μm) and a decrease in larger-diameter microvessels (≥5–9 μm) at three weeks after induction of ocular hypertension (n=5–6 mice/group, Student’s t-test P<0.05).

Conclusions: Our data support two main conclusions. First, two-photon excitation microscopy is an effective strategy to monitor longitudinal changes in retinal pericytes and capillaries in live animals at glaucoma onset and progression. Second, ocular hypertension triggers rapid intracellular calcium increase in retinal pericytes leading to substantial capillary constriction. This study identifies retinal pericytes as important mediators of early microvascular dysfunction in glaucoma.

Background: Sight-threatening diabetic macular edema (DME) is caused by increased microvascular permeability. While few direct vascular targeting strategies are available, VEGF pathway inhibition has shown to be effective in reducing retinal vascular leakage but is associated with non-negligible side effects. Thus, more options are needed. Vascular specific Activin-like kinase receptor type I (ALK1) pathway and its circulating ligand Bone morphogenetic protein-9 (BMP9) is known for its potent quiescent and stabilizing effect on the vasculature. However, little is known about this pathway in the context of microvascular permeability associated with diabetes. We hypothesize that BMP9/ALK1 pathway is inhibited in diabetic (DB) retinas leading to vascular destabilization and leakage and that its activation could re-establish proper vascular endothelial barrier functions (EBF).

Methods: The effect of hyperglycemia (i.e., HG >10 mM of D-glucose) on Alk1 signaling was evaluated in vitro by subjecting endothelial cells (EC) to increasing concentrations of D-glucose (5, 11, 25 mM) and in vivo using DB mice (Streptozotocin-induced diabetes). The contribution of Alk1 signaling on EBF was evaluated using Evans Blue permeation in inducible endothelial specific Alk1 KO mice. To evaluate the potential protective effects of BMP9/Alk1 signaling on EBF, BMP9 overexpression was achieved using adenoviral delivery in DB mice. Statistical-One-Way ANOVA or Student’s t-test was used.

Results: Endothelial tissue from DB mice showed a significant inhibition of BMP9/ALK1-canonical Smad1,5,8 quiescence signaling (DB n=5; CTL n=4; P<0.01), which was associated with reduced expression of target genes (JAG1, Id1,3, Hey1,2 & HES). Moreover, we showed that retinal hyperpermeability associated with diabetes was exacerbated in Alk1 heterozygote mice (n=4–9/group; P<0.0001). Finally, we demonstrated that activation of Alk1 signaling in ECs prevented vascular permeability induced by HG, both in vitro (n=3; P=0.009) and in vivo (n=4–9/group; P<0.0001).

Conclusions: Consistent with our hypothesis, vascular stability and quiescence induced by BMP9-ALK1 signaling is inhibited in the DB/HG endothelium which could be an important factor in vascular leakage leading to DME. Our results show that activation of this pathway could offer a therapeutically interesting future option to slow down the onset of DME.

Background: Sight-threatening diabetic macular edema (DME) is caused by increased microvascular permeability. While few direct vascular targeting strategies are available, VEGF pathway inhibition has shown to be effective in reducing retinal vascular leakage but is associated with non-negligible side effects. Thus, more options are needed. Vascular specific Activin-like kinase receptor type I (ALK1) pathway and its circulating ligand Bone morphogenetic protein-9 (BMP9) is known for its potent quiescent and stabilizing effect on the vasculature. However, little is known about this pathway in the context of microvascular permeability associated with diabetes. We hypothesize that BMP9/ALK1 pathway is inhibited in diabetic (DB) retinas leading to vascular destabilization and leakage and that its activation could re-establish proper vascular endothelial barrier functions (EBF).

Methods: The effect of hyperglycemia (i.e., HG >10 mM of D-glucose) on Alk1 signaling was evaluated in vitro by subjecting endothelial cells (EC) to increasing concentrations of D-glucose (5, 11, 25 mM) and in vivo using DB mice (Streptozotocin-induced diabetes). The contribution of Alk1 signaling on EBF was evaluated using Evans Blue permeation in inducible endothelial specific Alk1 KO mice. To evaluate the potential protective effects of BMP9/Alk1 signaling on EBF, BMP9 overexpression was achieved using adenoviral delivery in DB mice. Statistical-One-Way ANOVA or Student’s t-test was used.

Results: Endothelial tissue from DB mice showed a significant inhibition of BMP9/ALK1-canonical Smad1,5,8 quiescence signaling (DB n=5; CTL n=4; P<0.01), which was associated with reduced expression of target genes (JAG1, Id1,3, Hey1,2 & HES). Moreover, we showed that retinal hyperpermeability associated with diabetes was exacerbated in Alk1 heterozygote mice (n=4–9/group; P<0.0001). Finally, we demonstrated that activation of Alk1 signaling in ECs prevented vascular permeability induced by HG, both in vitro (n=3; P=0.009) and in vivo (n=4–9/group; P<0.0001).

Conclusions: Consistent with our hypothesis, vascular stability and quiescence induced by BMP9-ALK1 signaling is inhibited in the DB/HG endothelium which could be an important factor in vascular leakage leading to DME. Our results show that activation of this pathway could offer a therapeutically interesting future option to slow down the onset of DME.