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Veins have emerged as the origin of all other endothelial cell subtypes needed to expand vascular networks during developmental and pathological neoangiogenesis. Here, we uncover the role of the angioneurin Fibronectin Leucine Rich Transmembrane protein (FLRT) 2 in central nervous system (CNS) vascular development in the mouse. Early postnatal FLRT2 deletion reveals specific defects in retinal veins, impacting endothelial cell proliferation, sprouting and polarity that result in reduced tip cells at the vascular front. FLRT2 interacts with VE-cadherin and together with the endocytic adaptor protein Numb contribute to the modulation of adherens junction morphology in both retina and cerebral cortex in vivo. Utilizing expansion microscopy, we visualize the altered dynamic distribution of VE-cadherin in tissue of FLRT2 endothelial mutants. Additionally, FLRT2 in cortical vessels regulates the crosstalk between adherens and tight junctions, influencing blood-brain barrier development. Our findings position FLRT2 as a vein-specific regulator of CNS vascular development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Diabetes elevates endothelin-1 (ET-1) in the vitreous and enhances constriction of retinal venules to this peptide. However, mechanisms contributing to ET-1-induced constriction of retinal venules are incompletely understood. We examined roles of sodium-hydrogen exchanger 1 (NHE1), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and extracellular calcium (Ca 2+ ) in retinal venular constriction to ET-1 and the impact of diabetes on these signaling molecules. Retinal venules were isolated from control pigs and pigs with streptozocin-induced diabetes for in vitro studies. ET-1-induced vasoconstriction was abolished in the absence of extracellular Ca 2+ and sensitive to c-Jun N-terminal kinase (JNK) inhibitor SP600125 but unaffected by extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 kinase inhibitor SB203580, or broad-spectrum PKC inhibitor Gö 6983. Diabetes (after 2 weeks) enhanced venular constriction to ET-1, which was insensitive to PD98059 and Gö 6983 but was prevented by NHE1 inhibitor cariporide, SB203580, and SP600125. In conclusion, extracellular Ca 2+ entry and activation of JNK, independent of ERK and PKC, mediate constriction of retinal venules to ET-1. Diabetes activates p38 MAPK and NHE1, which cause enhanced venular constriction to ET-1. Treatments targeting these vascular molecules may lessen retinal complications in early diabetes., (© 2021 by the American Diabetes Association.)

Objectives: Low or high sympatho-adrenal-medullary axis (SAM) and hypothalamic-pituitary-adrenal axis (HPA) dysregulation reflect chronic stress. Retinal vessel dynamics may relate to SAM, HPA activity and stroke risk. Our objectives were therefore to assess the relationships between retinal vessel, SAM and HPA responses, and to determine stroke risk., Methods: A prospective bi-ethnic gender cohort ( n = 275, 45 ± 9 years) was included. Urine/serum/saliva samples for SAM [norepinephrine:creatinine ratio (u-NE)] and HPA [adrenocorticotrophic hormone (ACTH), cortisol] were obtained at baseline, three-year follow up and upon flicker light-induced provocation. Diastolic ocular perfusion pressure was measured as a marker of hypo-perfusion. Retinal arterial narrowing and venous widening calibres were quantified from digital images in the mydriatic eye. A validated stress and stroke risk score was applied., Results: An interaction term was fitted for venous dilation in u-NE tertiles (p ≤ 0.05) and not in u-NE median/quartiles/quintiles. Independent of race or gender, tertile 1 (low u-NE) had a 112% increase in u-NE, decreases in cortisol, and no changes in ACTH over three years (positive feedback). Tertile 3 (high u-NE) contradictorily had decreases in u-NE and cortisol, and increases in ACTH (negative feedback). In tertile 1, reduced arterial dilation, and faster arterial vasoconstriction and narrowing were related to higher SAM activity and hypo-perfusion ( p ≤ 0.05), whereas delayed venous dilation, recovery and widening were related to cortisol hypo-secretion ( p ≤ 0.05). In tertile 1, delayed venous recovery responses predicted stress and stroke risk [odds ratio 4.8 (1.2-19.6); p = 0.03]. These associations were not found in u-NE tertiles 2 and 3., Conclusions: In response to low norepinephrine, a reflex increase in SAM activity occurred, enhancing arterial vasoconstriction and hypo-perfusion. Concomitant HPA dysregulation attenuated retinal vein vasoactivity and tone, reflecting delayed vein recovery responses and non-adaptation to stress. These constrained vein recovery responses are indicative of increased chronic stress and stroke risk.

Purpose: We studied the topographic distribution of contractile protein in different orders of the human macular microvasculature to further understanding of the sites for capillary blood flow regulation., Methods: Nine donor eyes from eight donors were cannulated at the central retinal artery and perfusion labeled for alpha smooth muscle actin (αSMA) and filamentous actin (F-actin). Confocal images were collected from the macula region, viewed, projected, and converted to a 255 grayscale for measurements. The mean intensity was measured for macular arterioles, venules, and capillary segments. The diameter of each vessel segment measured was recorded. The normalized mean intensity values from all images were ranked according to vessel types and size with a total of nine categories., Results: F-actin was present throughout the macular microvasculature whereas αSMA labeling showed variations. Overall, αSMA has a more prominent presence in the macular arterioles than in the macular capillaries and venules, and αSMA strongly labeled the smaller macular arterioles. Some capillaries also labeled positive for αSMA, including some of the capillaries in the innermost capillary ring surrounding the foveola. It was weakly present in the capillaries on the venous side and larger venules. In the larger macular arterioles closer to 100 μm in diameter, αSMA labeling was weakly present and not as ubiquitous as in the smaller arterioles., Conclusions: Nonuniform distribution of contractile proteins in the different types, orders, and sizes of macular microvasculature indicates that these vessels may have different contractile capability and roles in macular flow regulation.

Purpose: To investigate a sequential chronological change in tip cells during the development of the retinal vasculature in newborn mice., Materials and Methods: Newborn C57BL/6 mice were used for this study. To elucidate the patterns in the developing retinal vasculature, histology, and immunohistochemistry-antiplatelet endothelial cell adhesion molecule-1, anticollagen type IV, isolectin IB 4 -were performed on sections of mouse retina on postnatal days (P)-4, -8, and -12. Staining patterns of isolectin IB 4 -stained arterial and venous tip cells were compared in retinal wholemounts, in which the numbers and characteristics of tip cells were compared between arteries and veins on P-4, -6, and -8. In addition, vascular densities and branching patterns were compared between arterial and venous vascular forefront areas., Results: Tip cells in the superficial vascular plexus were observed until P-8. The number of tip cells was highest on P-6, decreasing dramatically from P-6 to P-8 (P-4, 165.2 ± 10.1, n = 17; P-6, 183.8 ± 19.4, n = 15; P8, 21.4 ± 6.4, n = 15) (p < 0.05, respectively, t-test). There was a greater number of tip cells in veins versus arteries on P-4 and P-6 (P-4, 91.0 ± 9.2 veins versus 74.2 ± 10.4 arteries; P-6, 104.0 ± 10.2 veins versus 79.8 ± 11.3 arteries) (p < 0.05, respectively). Arterial tip cells had thinner and longer sprouts compared with venous tip cells (basal thickness: 15.7 ± 8.7 veins versus 9.9 ± 3.5 μm arteries) (length, 20.3 ± 9.1 veins versus 37.1 ± 13.2 μm arteries on P-4) (p < 0.05, respectively). Vessel areas and densities of vascular branch points were significantly higher around veins compared to arteries (vessel areas: 58.9 ± 1.2% veins versus 40.8 ± 1.9% arteries; vascular branch points, 1371.9 ± 136.7/mm 2 veins versus 1046.7 ± 175.5/mm 2 arteries) (p < 0.05, respectively)., Conclusion: The number of tip cells increased to a greater extent in the superficial vascular plexus of veins versus arteries until P-6. Consequently, there are more vessel areas and vascular branch points near retinal veins versus arteries. Arterial tip cells are longer and thinner than the shorter and thicker venous tip cells.

Purpose: To determine whether the degree of venous tortuosity is significantly correlated with the aqueous vascular endothelial growth factor (VEGF) concentration in eyes with a central retinal vein occlusion (CRVO)., Methods: We reviewed the medical records of 32 eyes of 32 patients who had macular edema due to a CRVO. All of the patients were examined at the Nagoya University Hospital and were scheduled to receive an intravitreal injection of bevacizumab (IVB) or ranibizumab (IVR) within 12 weeks of the onset of the CRVO to treat the macular edema. Aqueous humor was collected just before the IVB or IVR, and the VEGF concentration was determined by enzyme-linked immunosorbent assay (ELISA). The venous tortuosity index was calculated by dividing the length of the retinal veins by the chord length of the same segment. The correlation between the mean tortuosity index of the inferotemporal and supratemporal branches of the retinal vein and the aqueous VEGF concentration was determined., Results: The mean aqueous VEGF concentration was 384 ± 312 pg/ml with a range of 90 to 1077 pg/ml. The degree of venous tortuosity was significantly correlated with the VEGF concentration in the aqueous. (r = 0.49, P = 0.004), with the foveal thickness (r = 0.40, P = 0.02), and with the best-corrected visual acuity (r = 0.38, P = 0.03)., Conclusions: The significant correlation between the aqueous VEGF concentration and the venous tortuosity indicates that the degree of retinal venous tortuosity can be used to identify eyes that are at a high risk of developing neovascularization.

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca(2+)/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2(+/-) ) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

Purpose: To determine whether there are differences in retinal vascular oxygen saturation measurements, estimated using a hyperspectral fundus camera, between normal eyes and treated eyes of subjects with asymmetrical primary open-angle glaucoma (POAG)., Methods: A noninvasive hyperspectral fundus camera was used to acquire spectral images of the retina at wavelengths between 556 and 650 nm in 2-nm increments. In total, 14 normal eyes and both eyes of 11 treated POAG subjects were imaged and analyzed using algorithms that use the spectral variation of the optical densities of blood vessels to estimate the oxygen saturation of blood within the retinal vasculature. In the treated POAG group, each of the eyes were categorized, based on the mean deviation of the Humphrey visual-field analyzer result, as either more-advanced or less-advanced, glaucomatous eyes. Unpaired t-tests (two-tailed) with Welch's correction were used to compare the mean oxygen saturation between the normal subjects and the treated POAG subgroups., Results: In less-advanced and more-advanced-treated POAG eyes, mean retinal venular oxygen saturations (48.2±21.6% and 42.6±18.8%, respectively) were significantly higher than in normal eyes (27.9±9.9%; P=0.03 and 0.01, respectively). Arteriolar oxygen saturation was not significantly different between normal eyes and treated POAG eyes., Conclusions: The increased oxygen saturation of the retinal venules in advanced-treated POAG eyes may indicate reduced metabolic consumption of oxygen in the inner retinal tissues.

Purpose: To investigate the effect of intravitreal injections of the vascular endothelial growth factor inhibitor ranibizumab on retinal oxygenation in patients with central retinal vein occlusion (CRVO)., Methods: Retinal oxygen saturation in patients with CRVO was analysed using the Oxymap Retinal Oximeter P3, before and during 6 months of treatment with intravitreal injections of ranibizumab., Results: At presentation, retinal venous oxygen saturation was lower in eyes with CRVO than in the healthy fellow eyes (32±13% vs 59±10%, respectively, p=0.001) whereas retinal arterial saturation was higher in eyes with CRVO than in the fellow eyes (95%±8% and 91%±3%, p=0.04). Mean visual acuity increased from 51±24 letters ETDRS at baseline to 66±24 and 69±20 letters ETRDS, respectively, at 3 months and 6 months treatment (mean±SD, p<0.0001, repeated measures analysis of variance) and central retinal thickness was reduced from 697±139 µm to 368±113 µm and 340±96 µm, respectively, from baseline to 3 months and 6 months treatment (p<0.0001). Venous saturation increased during treatment (from 35.5%±13.8% at baseline to 43.1%±10.8% and 43.5%±13.7% after 3 months and 6 months treatment, respectively, p=0.012), while no significant change was found in arterial saturation (p=0.24)., Conclusions: Retinal venous oxygen saturation was markedly reduced in untreated CRVO and was roughly halfway normalised during intravitreal ranibizumab treatment. Retinal artery oxygen saturation was not reduced in CRVO., Trial Registration Number: NCT01360385., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRβ blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the retinal oxygen saturation trend with onset of diabetes and increasing severity of diabetic retinopathy by comparing diabetic groups with and without retinopathy to controls., Methods: A fundus camera-based dual-wavelength snapshot oximeter imaged retinas of healthy subjects and patients with and without diabetic retinopathy. The images were analyzed to determine oxygen saturation in major retinal arteries and veins, which is inversely proportional to optical density ratio., Results: Control retinal oxygen saturation (n = 14) in arteries was 92.3 ± 4.2% and in veins, 57.2 ± 6.0%. Retinal oxygen saturation for diabetic patients with no signs of diabetic retinopathy (NDR, n = 45) in arteries was 96.3 ± 8.6% (P = 0.662) and in veins, 58.7 ± 7.5% (P = 0.998). Retinal oxygen saturation for diabetics with mild to moderate nonproliferative diabetic retinopathy (NPDR, n = 23) in arteries was 97.7 ± 5.8% (P = 0.590) and in veins, 61.1 ± 7.6% (P = 0.658). Retinal oxygen saturation for diabetics with severe NPDR (n = 12) in arteries was 102 ± 10.2% (P = 0.023) and in veins, 66.8 ± 8.4% (P < 0.001). Retinal oxygen saturation for patients with proliferative diabetic retinopathy (PDR, n = 13) in arteries was 103.6 ± 8.7% (P = 0.003) and in veins, 66.6 ± 10.2% (P = 0.026). Retinal oxygen saturation for all diabetics with retinopathy combined (all DR, n = 48) in arteries was 100.4 ± 7.6% (P = 0.004) and in veins, 64.2 ± 8.4% (P = 0.007)., Conclusions: A trend of increasing retinal oxygen saturation was found from controls to NDR group to increasing levels of diabetic retinopathy, though significance was only reached for the comparison of controls to severe-NPDR, PDR, and all-DR groups.

The purpose of this study was to investigate regional differences in oxygen saturation of blood in first degree retinal vessels using a novel non-flash hyperspectral retinal camera (Photon etc Inc). Nine healthy individuals (mean age 24.4 ± 3.6 yrs, 5 males) were imaged at 548, 569, 586, 600, 605 and 610 nm wavelengths. Optical density values were extracted with the aid of Image-J software for blood oxygen saturation (SO2) determination. Arteriolar and venular SO2 were measured at three locations (ranging 1-3 optic nerve head radii) from the disc margin along the vessels in the superior and inferior temporal quadrants. Retinal SO2 was significantly higher in the superior temporal arteriole and venule as compared to the inferior temporal vessels (p = 0.033 and p = 0.032 for arterioles and venules, respectively). SO2 was not significantly different between the three measurement sites for any of the given vessels imaged (p > 0.05). In conclusion, greater SO2 values were found in the superior temporal first degree retinal arterioles and venules in young healthy individuals than in the equivalent inferior vessels. However, there were no detectable differences in retinal SO2 along each of the major vessels, a finding that is consistent with the concept of these vessels not contributing primarily to gas exchange. Moreover, the SO2 was consistently higher in the arterioles than in the equivalent venules (p < 0.0001)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To determine the ability of an intravitreal injection of tenecteplase (TNK) to penetrate an intraretinal venous thrombus and its effectiveness in thrombolysis in a porcine model of branch retinal vein occlusion (BRVO)., Methods: Six pigs (group 1) were anesthetized, and a BRVO was induced photothrombotically in the left eye; immediately afterward, fluorescence-conjugated TNK (100 μg) was injected into both eyes, with enucleation at 24 hours. Retinal penetration was assessed on frozen sections by epifluorescence microscopy. A further six pigs (group 2) were anesthetized; BRVO was induced in both eyes, and TNK was injected into the vitreous in the left eye. Both eyes were harvested a week later. The area of the lasered site and an area away from the burn were dissected and processed in epoxy resin and stained for light or transmission electron microscopy. The percentage blockage, clot volume, cytostructure, and extent of thrombolysis by TNK were assessed., Results: TNK penetrated the veins in both eyes of group 1 pigs, with more intense staining in the eyes with the occlusion. In group 2 eyes, thrombolysis was significant in the eyes injected with TNK (P = 0.03); blockage was seen in all six untreated eyes and one treated eye. Clot volume was significantly higher in untreated eyes (P = 0.028). Percentage blockage varied from 8.5% to 83.9%. Damage by TNK to the neural retina was not seen. There was no significant difference in cytostructure between treated and untreated eyes (P = 0.357)., Conclusions: TNK was able to penetrate the retinal veins with and without an occlusion and effect lysis of BRVO, and did not cause damage to the retinal tissue. Intravitreal TNK may be useful as an acute treatment for RVOs of recent onset.

Introduction: C-reactive protein (CRP) levels are associated with cardiovascular disease and systemic inflammation. We assessed whether CRP-associated loci were associated with serum CRP and retinal markers of microvascular disease, in Asian populations., Methods: Genome-wide association analysis (GWAS) for serum CRP was performed in East-Asian Chinese (N = 2,434) and Malays (N = 2,542) and South-Asian Indians (N = 2,538) from Singapore. Leveraging on GWAS data, we assessed, in silico, association levels among the Singaporean datasets for 22 recently identified CRP-associated loci. At loci where directional inconsistencies were observed, quantification of inter-ethnic linkage disequilibrium (LD) difference was determined. Next, we assessed association for a variant at CRP and retinal vessel traits [central retinal artery equivalent (CRAE) and central retinal vein equivalent (CRVE)] in a total of 24,132 subjects of East-Asian, South-Asian and European ancestry., Results: Serum CRP was associated with SNPs in/near APOE, CRP, HNF1A and LEPR (p-values ≤4.7×10(-8)) after meta-analysis of Singaporean populations. Using a candidate-SNP approach, we further replicated SNPs at 4 additional loci that had been recently identified to be associated with serum CRP (IL6R, GCKR, IL6 and IL1F10) (p-values ≤0.009), in the Singaporean datasets. SNPs from these 8 loci explained 4.05% of variance in serum CRP. Two SNPs (rs2847281 and rs6901250) were detected to be significant (p-value ≤0.036) but with opposite effect directions in the Singaporean populations as compared to original European studies. At these loci we did not detect significant inter-population LD differences. We further did not observe a significant association between CRP variant and CRVE or CRAE levels after meta-analysis of all Singaporean and European datasets (p-value >0.058)., Conclusions: Common variants associated with serum CRP, first detected in primarily European studies, are also associated with CRP levels in East-Asian and South-Asian populations. We did not find a causal link between CRP and retinal measures of microvascular disease.

Purpose: By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin., Methods: Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies., Results: Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells., Conclusions: Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

Objective: To evaluate the association between vitreous fluid levels of inflammatory factors and macular edema in patients with branch retinal vein occlusion (BRVO)., Methods: In 39 patients with BRVO and macular edema and 21 individuals with idiopathic macular hole (MH) serving as controls, vitreous fluid samples were obtained during vitreoretinal surgery, and the levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor 2 (sVEGFR-2), soluble intercellular adhesion molecule 1 (sICAM-1), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), pentraxin 3 (PTX3), and pigment epithelium-derived factor (PEDF) were measured by enzyme-linked immunosorbent assay. Macular edema was examined by optical coherence tomography., Results: Vitreous fluid levels of sVEGFR-2, VEGF, sICAM-1, IL-6, MCP-1, and PTX3 were significantly higher in the patients with BRVO than in those with MH; however, the PEDF level was significantly lower in the BRVO group. Vitreous fluid levels of all 7 factors were significantly correlated with the retinal thickness at the central fovea. There were also significant correlations of sVEGFR-2 with sICAM-1, IL-6, MCP-1, and PTX3 but no correlation with VEGF. However, there were significant correlations of VEGF with sICAM-1, IL-6, MCP-1, and PEDF in the BRVO group., Conclusions: Vitreous fluid levels of sVEGFR-2, VEGF, sICAM-1, IL-6, MCP-1, PTX3, and PEDF are strongly correlated with retinal vascular permeability and the severity of macular edema in patients with BRVO. These findings may be useful for understanding macular edema and developing new treatments for BRVO.

Purpose: To examine the role of Roundabout 4 (Robo4) in retinal endothelial permeability and analyze the structural events that lead to barrier disruption., Materials and Methods: Small interfering RNA (siRNA) technology was used to knockdown Robo4 expression to study its effects on the permeability of human retinal vascular endothelial cells (HRVECs) in vitro. The endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the HRVEC monolayers. The impact of Robo4 siRNA on the expression of tight junction (TJ) proteins and the activation of LIM-kinase (LIMK)/cofilin pathway were measured by western blotting. The change of actin cytoskeleton was detected using indirect immunofluorescence., Results: Robo4 siRNA increased the permeability of HRVEC monolayers. The expression levels of TJ-associated proteins occludin and zonula occludens (ZO)-1 were suppressed in Robo4-depleted cells. In addition, there was rearrangement of F-actin in HRVECs. These processes were induced through increased activity in the LIMK/cofilin pathway which coincided with a disruption in the barrier properties of retinal endothelial monolayers., Conclusions: Knockdown of Robo4 expression in HRVECs induced endothelial hyperpermeability associated with the downregulation of ZO-1, occludin, and the rearrangement of F-actin and that LIM-kinase 1 (LIMK1)/cofilin signal transduction system may be involved in the modulating process.

Purpose: To study the variation in intravascular oxygen saturation (oximetry) during an acute retinal vein occlusion (RVO) using hyperspectral computed tomographic spectroscopy based oximetry measurements., Methods: Thirty rabbits were dilated and anesthetized for experiments. Baseline oximetry measurements were made using a custom-made hyperspectral computed tomographic imaging spectrometer coupled to a fundus camera. RVO were induced using argon green laser following an intravenous injection of Rose Bengal. RVO induction was confirmed by fluorescein angiography. Retinal oximetry measurements were repeated in arterial and venous branches one hour after RVO induction and up to 4 weeks afterwards. Comparison of retinal oximetry before and after vein occlusion was made using the Student T-test., Results: One hour after RVO induction, we observed statistically significant reductions in the intravascular oxygen saturation in temporal retinal arteries (85.1 ± 6.1% vs. 80.6 ± 6.6%; p<0.0001) and veins (71.4 ± 5.5% vs. 64.0 ± 4.7%; p<0.0001). This decrease was reversible in animals that spontaneously recannulated the vein occlusion. There were no statistically significant differences in oxygen saturation in the nasal control arteries and veins before and after temporal vein RVO induction., Conclusions: We demonstrate, for the first time, acute changes in the intravascular oxygen content of retinal vessels 1 hour after RVO. These changes are reversible upon spontaneous recannulation of retinal vessels. This study demonstrates that hyperspectral computer tomographic spectroscopy based oximetry can detect physiological variations in intravascular retinal oxygen saturation. The study also provides the first qualitative and quantitative evidence of the variation in retinal vascular oxygen content directly attributable to an acute retinal vein occlusion.

Purpose: To assess spectrophotometric oximetry across a broad range of arterial saturation levels and to study the effect of chronic systemic hypoxemia on retinal oxygen extraction., Methods: The study included 16 patients with Eisenmenger syndrome, a cyanotic cardiac defect, and 17 healthy volunteers. Oxygen saturation in selected major retinal arteries and veins was assessed using noninvasive spectrophotometric oximetry. Arterial blood gases were determined within 1 day of the ophthalmic examination in blood samples from the femoral artery., Results: The retinal arterial oxygen saturation of 81% ± 9% (mean ± SD) in patients with Eisenmenger syndrome was subnormal and demonstrated more interindividual variation than the 93% ± 3% observed in healthy subjects (P < 0.001). A comparable difference was found for the respective retinal venous oxygen saturations of 44% ± 12% and 59% ± 5% (P < 0.001). Fractional arteriovenous oxygen extraction was comparable between the two groups (37% ± 6% and 34% ± 5%, respectively; P = 0.29). Retinal and femoral artery oxygen saturation were correlated (ρ = 0.82; P < 0.001), the former approximating the latter at least as well as fingertip oximetry., Conclusions: When compared to arterial blood gas analysis of blood samples drawn by arterial puncture, the gold standard in the field, fundus oximetry was found to be in good overall agreement with the arterial blood samples. Blood flow measurements will be needed to determine whether the systemic hypoxia is completely compensated, as suggested by oxygen extraction being comparable between the two groups.

Purpose: To examine the intraocular distribution of bevacizumab at four weeks after intravitreal bevacizumab (IVB) injection and to investigate the effects of IVB on apelin and vascular endothelial growth factor (VEGF) in the central retinal vein occlusion (CRVO) of monkey eyes., Methods: Direct laser coagulation was performed on all branch retinal veins in the right eyes of six Rhesus monkeys to establish a CRVO model. The eyes of the first three monkeys were enucleated one week, two weeks, and 24 weeks after the establishment of the CRVO model; this was the CRVO group. Subsequently, IVB was injected into the eyes of the last three monkeys one week, two weeks, and 24 weeks after laser coagulation; this was the IVB group. The left eye of the first monkey was used as normal control. Immunohistochemistry and reverse-transcription PCR was used to examine the expression of apelin and VEGF. The penetration of bevacizumab into the retina and iris was investigated by fluorescence immunostaining., Results: Immunoreactivity for bevacizumab could be detected in the vessel walls of the iris and choroid on day 28 after injecting IVB: apelin and VEGF staining had been more prominent than normal in the CRVO eye, but these decreased following IVB injection. Expression of apelin mRNA (p<0.01) was lower in the IVB group than the CRVO group and did not vary significantly between groups., Conclusions: Bevacizumab could be detected in the iris and choroid after four weeks of intravitreal injection. Apelin may be partially suppressed by bevacizumab, and it may play a role in retinal neovascularization during the development of CRVO.

Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

Purpose: Retinal blood vessel diameter and arteriovenous ratio (AVR) are commonly used diagnostic parameters. Because vascular walls are typically not visible in funduscopy, clinical AVR estimation is based on the lumen rather than the entire vessel diameter. Here the authors used a transgenic mouse model to quantify AVR in vivo based on total vessel dimensions (wall and lumen)., Methods: Confocal scanning laser ophthalmoscopy (cSLO) and indocyanine green angiography of the retinal vasculature were performed in wild-type and transgenic mice expressing green fluorescent protein (GFP) under the transcriptional control of the smooth muscle type α-actin (αSMA) promoter. Spectral-domain-OCT and ERG were performed to control for integrity of retinal structure and function in vivo and histology to demonstrate the location of GFP expression., Results: Native cSLO imaging and angiography yielded only inner vessel diameters similar to those observed through clinical funduscopy. In αSMA-GFP mice, autofluorescence imaging of the GFP-marked vascular walls also allowed the determination of outer vessel diameters. The mean AVR based on either inner diameter (AVR(id) = 0.72 ± 0.08) or outer diameter (AVR(od) = 0.97 ± 0.09) measurements were significantly different (P < 0.01)., Conclusions: Transgenic αSMA-GFP expression in murine vessel wall components allowed quantification of retinal vessel outer diameters in vivo. Although arterioles and venules differ in lumen and vessel wall width, they share a common outer diameter, leading to an AVR(od) close to unity. Because vessel walls are primary targets in common hypertensive and metabolic diseases, αSMA-GFP transgenic mice may prove valuable in the detailed assessment of such disorders in vivo.

Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Retinal vein occlusion is an important cause of visual loss. Several ocular and systemic conditions have been reported for retinal vein occlusion. The pathogenesis of thrombus formation in the retinal vein, which results in retinal vein occlusion, is unclear. The aim of this study was to investigate the correlation between increased serum leptin levels and the occurrence of retinal vein occlusion (RVO). The study group consisted of 40 patients with RVO (58.1 +/- 6 years old; 17 males and 23 females): 15 patients with central RVO, 23 with branch RVO, and 2 with hemispheric RVO. The patients who had any ocular or systemic pathology were not included in the study. The control group consisted of 40 healthy individuals of similar gender, age, date and type of health survey, and geographic region. The blood samples of the RVO patients (n = 40) and controls (n = 40) were obtained antecubitally. Leptin levels were measured by an enzyme-linked immunosorbent assay (ELISA) method, and Student's t-test was used to determine differences between the groups. The mean serum leptin levels were 12.5 +/- 1.64 ng/ml in patients with RVO and 8.4 +/- 1.22 ng/ml in the control subjects; namely, the mean serum leptin levels were significantly higher in the patients with RVO (p < 0.001). These results suggest that leptin may be involved in the pathogenesis of venous thrombosis in the retina probably through its effects on homeostasis of the vessel wall.

Background and Objective: To evaluate the difference in arteriovenous passage time calculated from retinal angiograms using indocyanine green and fluorescein sodium dye in healthy individuals., Patients and Methods: Examinations of eight healthy individuals (mean age +/- standard deviation: 27.3 +/- 4.2 years) who had four consecutive ophthalmic visits that were 2 weeks apart were analyzed. Indocyanine green and fluorescein angiography were performed sequentially (with the indocyanine green angiography occurring first) using scanning laser ophthalmoscopy. Arteriovenous passage times were calculated offline using digital angiogram video analysis., Results: Mean arteriovenous passage times calculated from indocyanine green angiography (1.417 +/- 0.136 seconds) recordings were significantly shorter than fluorescein sodium times (2.539 +/- 0.421 seconds) (P < .0001) and intra-group variability was similar. There were no significant correlations between indocyanine green and fluorescein sodium arteriovenous passage times., Conclusion: Arteriovenous passage times calculated using indocyanine green and fluorescein angiography are not interchangeable.

Methionine is an important amino acid involved in protein synthesis and transmethylation reactions. It is also the precursor of homocysteine and cysteine, two important risk factors for cardiovascular diseases. As homocysteine research has gained impulsion, the evaluation of plasma methionine concentrations has acquired importance. Methionine measurement generally has been performed by HPLC after o-phthalaldehyde derivatization. Its separation from other amino acids is time-consuming. We set up a new specific capillary electrophoresis method in which analyte derivatization was avoided by sample concentration before analysis. Methionine was detected by UV absorbance at 204 nm with a detection limit of 0.5 micromol/L. By a capillary with an effective length of 50 cm filled with 125 mmol/L Tris phosphate buffer at pH 2.3, the separation occurred in less than 14 min. Precision tests indicated a good test repeatability for both migration times (coefficient of variation [CV]<0.3%) and areas (CV<2.0%). Moreover, a good reproducibility of intraassay and interassay tests was obtained (CV<2.9% and CV<3.5%, respectively). The Passing-Bablok regression and the Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference HPLC assay are similar. Assay performance was evaluated measuring methionine concentrations in retinal venous occlusive disease.

Purpose: To measure hemoglobin oxygen saturation (SO(2)) in retinal vessels and to test the reproducibility and sensitivity of an automatic spectrophotometric oximeter., Methods: Specialized software automatically identifies the retinal blood vessels on fundus images, which are obtained with four different wavelengths of light. The software calculates optical density ratios (ODRs) for each vessel. The reproducibility was evaluated by analyzing five repeated measurements of the same vessels. A linear relationship between SO(2) and ODR was assumed and a linear model derived. After calibration, reproducibility and sensitivity were calculated in terms of SO(2). Systemic hyperoxia (n = 16) was induced in healthy volunteers by changing the O(2) concentration in inhaled air from 21% to 100%., Results: The automatic software enhanced reproducibility, and the mean SD for repeated measurements was 3.7% for arterioles and 5.3% venules, in terms of percentage of SO(2) (five repeats, 10 individuals). The model derived for calibration was SO(2) = 125 - 142 . ODR. The arterial SO(2) measured 96% +/- 9% (mean +/- SD) during normoxia and 101% +/- 8% during hyperoxia (n = 16). The difference between normoxia and hyperoxia was significant (P = 0.0027, paired t-test). Corresponding numbers for venules were 55% +/- 14% and 78% +/- 15% (P < 0.0001). SO(2) is displayed as a pseudocolor map drawn on fundus images., Conclusions: The retinal oximeter is reliable, easy to use, and sensitive to changes in SO(2) when concentration of O(2) in inhaled air is changed.

Aim: To determine if recombinant tissue plasminogen activator (rtPA) injected into the vitreous cavity can penetrate the retinal vessels of porcine eyes with or without vascular occlusion., Methods: Eight eyes (group I) of four pigs underwent clamping of the optic nerve flush with the globe for 90 minutes. One hour after reperfusion, one eye of each pig was injected with 75 microg of rtPA, and the fellow eye was injected with balanced salt solution (BSS). Eyes were processed for immunohistochemistry. Four additional eyes (group II) of two pigs were subjected to the same injections, but without optic nerve clamping., Results: After reperfusion, the clinical picture was similar to that of a central retinal vein occlusion. Immunoperoxidase staining showed rtPA only in the retinal veins but not the retinal arteries in all eyes injected with rtPA in both groups I and II. Those eyes also showed intense rtPA staining at the level of the internal limiting membrane (ILM). No staining was seen at the level of the ILM or inside the retinal vessels in the BSS injected eyes. Immunofluorescence staining showed intense staining at the level of the ILM, but not inside the retinal vessels in the rtPA-injected eyes., Conclusions: rtPA may penetrate the retinal veins, but not the arteries of porcine eyes with and without vascular occlusion. The ILM may play a part in preventing rtPA penetration.

Purpose: To demonstrate spectroscopic retinal oximetry measurements on arteries and veins in swine using intravitreal illumination. Retinal arterial and venous saturations are measured for a range of inspired O2 levels after pars plana vitrectomy., Methods: Pars plana vitrectomy and intravitreal manipulations were performed on two female American Yorkshire domestic swine. Light from a scanning monochromator was coupled into a fiberoptic intraocular illuminator inserted into the vitreous. The retinal vessels were illuminated obliquely, minimizing vessel glints. Multispectral images of the retinal vasculature were obtained as the swine's arterial blood oxygen saturation was decreased from 100% to 67% in decrements of approximately 10%. Retinal vessel spectra were used to calculate oxygen saturation in selected arteries and veins. Arterial oxygen saturations were calibrated using blood gas analysis on blood drawn from a Swan-Ganz catheter placed in the femoral artery., Results: Oblique illumination of retinal vessels using an intravitreal fiberoptic illuminator provided a substantial reduction in the central vessel glint usually seen in fundus images, thus simplifying the analysis of spectral data. The vessel shadows were displaced from the vessel image simplifying the light paths in the eye. Using a full spectral analysis simplified by the light path reductions, we calculated retinal vessel saturations. The reduction of glint allowed for increased accuracy in measuring retinal vessel spectral optical density. Abnormally low retinal venous oxygen saturations were observed shortly after pars plana vitrectomy., Conclusions: Retinal oximetry using intravitreal illumination has been demonstrated. As a research tool, intravitreal illumination addresses several difficulties encountered when performing retinal oximetry with transcorneal illumination.

Purpose: To measure the oxygen saturation (SO(2)) in retinal arterioles and venules in patients with glaucomatous optic neuropathy., Methods: We examined SO(2) in retinal arterioles and venules simultaneously by imaging spectrometry. Oxygen saturation was evaluated according to the difference of the extinction spectra of haemoglobin and oxyhaemoglobin. The arterio-venous difference (avD) was calculated by (SO(2art) - SO(2ven)). The optic nerve head topography was estimated by Heidelberg retinal tomography and the visual field using the Octopus G1. We examined one eye in each of 58 healthy persons (mean age 58.6 +/- 10.7 years; mean rim area 1.52 +/- 0.33 mm(2); mean defect 0.65 +/- 1.31 dB; mean intraocular pressure [IOP] 18.5 +/- 2.7 mmHg), 49 patients with normal-tension primary open-angle glaucoma (NTG) (mean age 63.0 +/- 8.5 years; mean rim area 0.89 +/- 0.34 mm(2); mean defect 5.4 +/- 4.1 dB; mean IOP 19.2 +/- 2.9 mmHg), and 45 patients with high-tension primary open-angle glaucoma (POAG) (mean age 62.6 +/- 10.3 years; mean rim area 0.97 +/- 0.47 mm(2); mean defect 7.1 +/- 6.4 dB; mean IOP 31.6 +/- 10.8 mmHg)., Results: The intraclass correlation coefficients of the SO(2) measurement were 0.82 (arteriole) and 0.59 (venule). In normal eyes, the SO(2art), SO(2ven) and avD were 92.3 +/- 3.4%, 55.7 +/- 6.8% and 36.6 +/- 7.0%, respectively. Equivalent data were 89.7 +/- 5.4%, 56.0 +/- 8.3% and 33.7 +/- 10.6%, respectively, in NTG eyes and 91.4 +/- 4.0%, 58.3 +/- 10.5% and 33.1 +/- 11.5%, respectively, in POAG eyes. Over all examined eyes, the arteriolar SO(2) and the retinal arterio-venous difference correlated significantly with the rim area., Conclusion: Eyes with NTG showed significantly decreased arteriolar SO(2). These changes were not seen in POAG patients.

'Vascular endothelial growth factor-A (VEGF-A) blockade has been recently validated as an effective strategy for the inhibition of new blood vessel growth in cancer and ocular pathologies. However, several studies have also shown that anti-VEGF therapy may not be as effective in the treatment of established unwanted blood vessels, suggesting they may become less dependent on VEGF-A for survival. The VEGF-A dependence of vessels may be related to the presence of vascular mural cells (pericytes or smooth muscle cells). Mural cell recruitment to the growing endothelial tube is regulated by platelet-derived growth factor-B (PDGF-B) signaling, and interference with this pathway causes disruption of endothelial cell-mural cell interactions and loss of mural cells. We have investigated the basis of blood vessel dependence on VEGF-A in models of corneal and choroidal neovascularization using a combination of reagents (an anti-VEGF aptamer and an anti-PDGFR-beta antibody) to inhibit both the VEGF-A and PDGF-B signaling pathways. We demonstrate that neovessels become refractory to VEGF-A deprivation over time. We also show that inhibition of both VEGF-A and PDGF-B signaling is more effective than blocking VEGF-A alone at causing vessel regression in multiple models of neovascular growth. These findings provide insight into blood vessel growth factor dependency and validate a combination therapy strategy for enhancing the current treatments for ocular angiogenic disease.

The phosphorescence lifetime imaging system previously used to image oxygen in the retina of the cat eye was modified to allow imaging of phosphorescence lifetimes in the much smaller mouse eye. Following the lead of Shonat and coworkers, a frequency domain approach was used in which the excitation light source was modulated in a 50% on: 50% off square wave while the gate of the intensified CCD camera was similarly modulated but delayed with respect to the excitation. These were analyzed by fitting the intensity at each pixel to a sinusoid. The phase of the phosphorescence relative to the excitation was determined and from the phase shift and frequency, the phosphorescence lifetime was calculated. The Stern-Volmer relationship was then used to calculate the oxygen pressure at each pixel of the image array. High resolution maps of phosphorescence lifetime and oxygen pressure in the retina of the mouse eye have been attained. The retinal veins draining into the optic head appear as large, highly phosphorescent vessels against a lower phosphorescence background with a network of smaller vessels. The oxygen pressure in the retinal veins is typically from 20 to 30 mm Hg while the background has somewhat higher oxygen pressures. Experiments are underway to resolve the oxygen in the choroid from that in the retina. The arteries on the retinal surface can be observed, but their small diameter, relatively high oxygen pressures (> 90 mm Hg), and surrounding tissue with much lower oxygen pressures, makes accurate determination of the oxygen pressure a challenge.

Expression of the new G protein-coupled msr/apj receptor in the mouse embryo is restricted to the endothelial layer of the primary blood vessels and the newly forming heart (Mech. Dev. 84 (1999) 199). During development of the retinal vasculature, the msr/apj gene is not expressed throughout the vascular network, indicating a possible relationship between the localization of expression and the acquisition of arterial or venous identity (Mech. Dev. 110 (2002) 183). Here we first established that retinal expression of ephrin-B2 and its putative receptor EphB4 correlates with arterial and venous phenotype, respectively. Then we analyzed the expression pattern of msr/apj in the retinal vessels at various stages of postnatal development by in situ hybridization. In contrast to the expression of ephrin-B2 or EphB4, msr/apj transcripts can be detected as early as postnatal day P3. From P3 to P12, msr/apj expression in the vascular network is restricted to the venules and the associated capillaries. The msr/apj gene is thus an early and specific marker of the venous phenotype in the retinal vasculature.

Purpose: Infiltrating leukocytes have long been widely thought to be key mediators of ischemia-reperfusion injury. Recently, however, evidence suggests that platelets accumulating in postischemic tissues also contribute to ischemia-reperfusion injury because of their inflammatory properties and promotion of formation of thrombi. This study was designed to evaluate quantitatively the inhibitory effects of antithrombin (AT)-III on the interactions between blood cells and retinal endothelial cells in vivo after transient retinal ischemia., Methods: Transient retinal ischemia was induced for 60 minutes in male Long-Evans rats by ligation of the optic nerve. AT III (250 U/kg) was administered intravenously just after induction of ischemia. Leukocyte and platelet behavior in the retina was evaluated in vivo with a scanning laser ophthalmoscope. Expression of P-selectin and intracellular adhesion molecule (ICAM)-1 in the postischemic retina was investigated by reverse transcription-polymerase chain reaction and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically., Results: Administration of AT III significantly inhibited leukocyte rolling along the major retinal veins and subsequent accumulation of leukocytes in the postischemic retina. Furthermore, the maximum number of rolling and adherent platelets was reduced by 76% (P < 0.01) and 48% (P < 0.01), respectively, at 12 hours after reperfusion. Immunohistochemical studies also revealed the suppressive effect of AT III on expression of P-selectin and ICAM-1. Finally, histologic examination demonstrated the protective effects of AT III against retinal damage after transient retinal ischemia., Conclusions: This study demonstrates the inhibitory effects of AT III on leukocyte and platelet recruitment to the postischemic retina, which may account for the neuroprotective properties of this alpha-2 globulin against retinal ischemia-reperfusion injury.

Purpose: To study possible causes of the gradual venous occlusion that precedes the formation of venous loops and reduplications in diabetic retinopathy using histopathological techniques., Methods: Casts of the retinal vascular system from six eyes of five diabetic patients were used to identify venous loops and reduplications. Subsequently the lesions were studied by immunohistochemistry using antibodies against cellular and non-cellular components previously shown to be disturbed in the diabetic retina., Results: In all eyes the venous abnormalities identified on the casts were accompanied by abnormal wall partitions dividing the venous lumen. These partitions consisted of a double layer of flat cells displaying immunoreactivity to von Willebrand factor and actin (endothelial cells), but not to glial fibrillary acid protein or S-100 protein (glial cells), CD3, CD20, CD68, or neutrophil elastase (leucocytes). Neutrophile granulocytes adhering to the walls of larger retinal venules were unrelated to the venous partition and to capillary occlusion in the adjacent retinal areas., Conclusions: Venous loops and reduplications are associated with partitions of the larger retinal venules consisting of a double layer of endothelial cells anchored to a thin basement membrane. An elucidation of the factors distinguishing this endothelial cell proliferation from preretinal new vessel formation may be important for understanding the pathophysiology of proliferative diabetic retinopathy.

Purpose: To test a hypothesis on the physiological mechanism of the disappearance of macular edema after laser treatment. The hypothesis is based on the effect grid laser treatment has on retinal oxygenation and hemodynamics. It predicts that laser-induced reduction of macular edema is associated with shortening and narrowing of retinal vessels in patients with branch retinal vein occlusion (BRVO)., Methods: The study included 12 subjects, treated with argon laser photocoagulation for BRVO and macular edema. Fundus photographs taken at the time of diagnosis and again after laser treatment, were digitized, and diameter and segment length of retinal vessels was measured using NIH-Image program., Results: Macular edema disappeared or was dramatically reduced in all cases after laser treatment. The diameter of occluded venules constricted to 0.81+/-0.02 (mean +/- SD, P = 0.019) of the prelaser diameter and adjacent arterioles constricted to 0.78+/-0.01 (P = 0.008). The laser treatment also led to shortening of the affected vessels. The final segment length of the occluded venules was 0.95+/-0.17 (P = 0.005) of the length before treatment. The corresponding value for the adjacent arterioles is 0.95+/-0.14 (P = 0.008). Control arterioles and venules in the same fundus did not change in either length or width., Conclusions: These results do not reject the authors' hypothesis that the disappearance of macular edema in BRVO can be explained by the effect the laser photocoagulation has on retinal oxygenation. Increased oxygenation causes vessel constriction and shortening and lower intravascular pressure, which reduces edema formation according to Starling's law.

Objectives: To determine whether systemic hyperoxia can reverse retinal hypoxia and decrease vascular endothelial growth factor (VEGF) gene expression in ischemic nonhuman primate retina., Methods: Six eyes of 3 cynomolgus monkeys were studied. Retinal ischemia was induced via laser vein occlusion and confirmed with fluorescein angiography. Animals were randomly assigned to treatment with either 21% or 100% inhaled oxygen. Arterial PO2 was monitored while systemic acid-base status was maintained. An oxygen microelectrode on a micromanipulator was used to measure preretinal oxygen concentrations in ischemic and nonischemic retina in situ. RNA was isolated from fresh whole retinas, and VEGF messenger RNA levels were quantified with Northern blotting., Results: The preretinal PO2 in ischemic retina was less than the PO2 in nonischemic retina in animals breathing 21% oxygen (intervascular zone PO2, 14.3+/-0.53 vs 21.8+/-0.55 mm Hg; P=.002). After 8 hours of systemic hyperoxia (arterial PO2, 512+/-18 mm Hg), the preretinal PO2 in ischemic retina increased to 166.2+/-15.6 mm Hg (21.8% oxygen) and retinal VEGF messenger RNA levels were reduced by an average of 55%., Conclusions: These data demonstrate that systemic hyperoxia can lower retinal VEGF gene expression and reoxygenate ischemic adult primate retina.

The association between periodic acid Schiff staining and immunoreactivity to laminin, fibronectin, vitronectin, and type VI collagen was studied qualitatively and quantitatively in the retinal vascular bed from 7 eyes of 5 diabetic patients and from 5 eyes of 5 normal persons. In the retina from diabetic patients the number of arterioles showing immunoreactivity to vitronectin, the number of venules showing immunoreactivity to type VI collagen, and the number of both arterioles and venules showing immunoreactivity to laminin and fibronectin, was higher than in normals. There was no difference between the number of capillaries showing periodic acid Schiff staining and immunoreactivity to laminin, fibronectin, and vitronectin when comparing areas of vascular occlusion with adjacent control areas. However, the number of capillaries displaying immunoreactivity to type VI collagen was higher in control areas than in areas of vascular occlusion in diabetic patients and in normal controls. Staining with periodic acid Schiff correlated topographically with immunoreactivity to laminin and fibronectin, but not with immunoreactivity to vitronectin and type VI collagen. In areas of vascular occlusion there was seen no immunoreactivity or histological staining corresponding to the material accumulated to occlude the ghost vessels.

Purpose: To study the role of oxygen distribution in the physiopathology of retinal vasoproliferative microangiopathies., Methods: Experimental retinal branch vein occlusion was induced in miniature pigs, using argon laser photocoagulation. The microvascular modifications, the retinal histologic features, and the retinal oxygen distribution were studied within 48 hours of the occlusion, and 3 weeks later. The retinal oxygen distribution modifications were also studied in ischemic retinal areas treated by scatter argon laser photocoagulation., Results: Impaired regulation and blood flow decrease in the vascular bed that was affected by a retinal branch vein occlusion and tissue hypoxia led to modifications of oxygen delivery resulted in a damage of the neuronal cells, whereas the abnormal permeability of the affected retinal vessel wall induced extracellular edema and disorganization of the inner retina. Three weeks after vein occlusion, retinal neovascularization occurred in ischemic/hypoxic retinas. The "critical PO2" (tissue oxygen partial pressure), which induces neovascularization in miniature pigs has not been determined. Photocoagulation of the ischemic retinal territories induced an increase of preretinal oxygen partial pressure, which is restored to the normal preretinal values., Conclusion: A retinal branch vein occlusion in miniature pigs represents a reproducible experimental model of retinal neovascularization. Oxygen partial pressure measurements of the ischemic retina confirmed the hypothesis that tissue hypoxia triggers neovascularization; laser photocoagulation should be applied over the whole ischemic retinal areas to eliminate hypoxia in the inner retina.

The pathogenesis of diabetic retinopathy includes the following components actual at a local level: diffuse retinal hypoxia leading to increase in the retina of anaerobic energy processes, development of local acidosis causing venous congestion, increased permeability of retinal capillaries and veins, impairment of the structure of vascular walls, development of foci of deep retinal hypoxia and of proliferative changes. These disorders develop in succession and mutually aggravate each other. A noticeable role belongs to deteriorated evacuation of the products of abnormal metabolism which depends on the existence of a solid barrier (pigmented epithelium, Bruch's membrane) between the retina and choroid and on the venous congestion and impaired liquid circulation in the vitreous body. A dichotomic type of division of the central retinal artery and absence of anastomoses between its branches facilitate the appearance of foci of retinal hypoxia. Local pathogenetic therapy of diabetic retinopathy should be aimed at removal of at least one out of three factors: hypoxia, acidosis, or venous congestion. This does not make less important the treatment of general metabolic disorders characteristic of diabetes mellitus.

The absolute volume of Weibel-Palade (WP) bodies, the storage organelles of von Willebrand factor (vWF), was estimated by a stereological method in a known volume of central retina from normal and 5-year diabetic dogs. The results showed that the volume of WP bodies present in the endothelium of the retinal vasculature varies with blood vessel type and in diabetes. In both diabetic and normal dogs the endothelium of the retinal veins contained a higher volume of WP bodies than that of the retinal arteries. In dogs which had been diabetic for a duration of 5 years the volume of WP bodies present in the endothelium of retinal veins was significantly greater than in the endothelium of veins from the control animals. However, there was no significant difference in the volume of WP bodies present in the endothelium of retinal arteries or capillaries between the two groups of animals.

Indocyanine green angiography with scanning laser ophthalmoscopy is a new supplementary method studying subretinal neovascular membranes. However, it is still controversial as to whether of not exudation of this dye occurs from regular choroidal vessels or such membranes. Fluorescence of the macula area, a parapapillary retinal vein, and of venous blood and plasma was therefore measured with a picture analysis system in five healthy young volunteers. Furthermore, the angiograms of 150 subretinal neovascular membranes were reviewed. Membrane fluorescence was measured in ten exemplary cases and compared with the background fluorescence. The results show that macula fluorescence is detectable much longer than the fluorescence of retinal vessels. However, a comparison with the fluorescence of blood and plasma samples revealed that this late fluorescence must be caused by retinal and choroidal capillaries with a significantly reduced hematocrit. It is not caused by apposition or exudation of indocyanine green. The same was true for the majority of subretinal neovascular membranes. Exudation was rarely detected in membranes that were regularly surrounded by exudates but could be induced by laser coagulation. These observations indicate that exudation occurs only after massive destruction of the endothelial barrier. The results are important for the interpretation of indocyanine green angiograms and for possible photodynamic therapy with this dye.

Angiotensin II (AII) binding sites were identified in cross-sections of the cat retinal and optic nerve vasculatures. The authors used 3H-AII and 125I-saralasin, an agonist and a high-affinity antagonist of AII receptors, respectively, to generate light microscopy autoradiograms in resin-embedded tissues. With both radioligands the presence of AII binding sites was confirmed in retinal arterioles but not in the veins or capillaries of the retina. Additionally the presence of such binding sites in the capillaries of the optic nerve head was shown. These results support the hypothesis that microvascular tone and perhaps autoregulatory responses of optic nerve capillaries might be influenced by vasoactive substances, such as AII, either leaking from the choroid or locally synthetized.

Recent studies have found basic fibroblast growth factor (bFGF), an angiogenic peptide, in retina and have suggested that bFGF is responsible for retinal vascular proliferation. To test the hypothesis that bFGF stimulates 3H-thymidine uptake in retinal vascular cells in vivo, we injected bFGF (100 ng) into the vitreous cavity of six cats at 0 hr and again at 24 hr. Eight control eyes received boiled bFGF or no injection. After 46 hr, 3H-thymidine was injected into the vitreous cavity of all eyes and 2 hr later the eyes were enucleated. Intense 3H-thymidine uptake was seen in eyes with bFGF (56 +/- 20 SD positive cells per section) but not in control eyes (7-10 positive cells per section (P less than 0.001). Trypsin digest preparations showed that the thymidine uptake was predominantly in the venular (89%) and capillary (10%) endothelium and not in arterioles (1%) (P less than 0.001). The data suggest that retinal venular endothelial cells respond preferentially to exogenous bFGF, and in part may explain their prominent role in the neovascular process. In a second group of experiments to test the hypothesis that retinal ischemia releases a diffusable factor similar to bFGF that can cause 3H-thymidine uptake in retinal vascular cells, we created branch retinal vein occlusion in six cat eyes. The fellow eyes received no injections. In the eyes with branch vein occlusion there was an intense 3H-thymidine uptake within the distribution of the occluded vein (84 +/- 77 SD positive cells per section), but none in the areas outside the occluded vein (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)