Chronic social defeat stress causes retinal vascular dysfunction.

PURPOSE: The roles of vascular dysfunction and chronic stress have been extensively discussed in the pathophysiology of glaucoma. Our aim was to test whether chronic stress causes retinal vascular dysfunction and therewith induces retinal ganglion cells (RGCs) loss. METHODS: Twelve mice underwent chronic social defeat (CSD) stress, while 12 mice received control treatment only. Intraocular pressure (IOP) was measured with a rebound tonometer. Blood plasma corticosterone concentration and adrenal gland weight were used to assess stress levels. Brn-3a staining in retinas and PPD staining in optic nerve cross sections were conducted to assess the survival of RGCs and axons respectively. The ET-1 and α-SMA levels were determined in retina. Retinal vascular autoregulation, functional response to various vasoactive agents and vascular mechanics were measured using video microscopy. RESULTS: No significant difference in IOP levels was observed during and after CSD between CSD mice and controls. CSD stress caused hypercortisolemia 2 days post-CSD. However, increased corticosterone levels went back to normal 8 months after CSD. CSD-exposed mice developed adrenal hyperplasia 3 days post-CSD, which was normalized by 8 months. RGC and axon survival were similar between CSD mice and controls. However, CSD stress caused irreversible, impaired autoregulation and vascular dysfunction of retinal arterioles in CSD mice. In addition, impaired maximal dilator capacity of retinal arterioles was observed 8 months post-CSD rather than 3 days post-CSD. Remarkably, ET-1 levels were increased 3 days post-CSD while α-SMA levels were decreased 8 months post-CSD. CONCLUSIONS: We found that CSD stress does not cause IOP elevation, nor loss of RGCs and their axons. However, it strikingly causes irreversible impaired autoregulation and endothelial function in murine retinal arterioles. In addition, CSD changed vascular mechanics on a long-term basis. Increased ET-1 levels and loss of pericytes in retina vessels may involve in this process.

Effect of insulin treatment on pulsatility ratio and resistance index of the retinal artery in patients with type 2 diabetes.

This study aimed to evaluate whether long-term insulin treatment is associated with abnormalities in retinal circulation in type 2 diabetic patients. We evaluated 19 eyes of nondiabetic individuals and 68 eyes of type 2 diabetic patients. The eyes of diabetic patients were classified into two groups according to the presence or absence of long-term insulin therapy. We used a Doppler optical coherence tomography flowmeter to measure diameter, velocity, and blood flow in the major temporal retinal artery. The pulsatility ratio (PR) and resistance index (RI), indices of vascular rigidity, were calculated from the blood velocity profile. PR and RI were significantly elevated in type 2 diabetic patients compared with nondiabetic subjects (P < 0.05). In type 2 diabetes patients, PR and RI were significantly higher in patients receiving long-term insulin treatment than in those without (P < 0.01). There was a significant difference in velocity (P < 0.05), but not diameter and blood flow, between nondiabetic subjects and type 2 diabetes patients. No significant difference in diameter, velocity, or blood flow was observed between the groups with and without long-term insulin treatment. Long-term insulin treatment can affect PR and RI, which might be associated with vascular rigidity of the retinal artery in patients with type 2 diabetes.

Hydrogen sulfide dilates the isolated retinal artery mainly via the activation of myosin phosphatase.

AIMS: Hydrogen sulfide (H2S) is shown in ocular tissues and suggested to involve in the regulation of retinal circulation. However, the mechanism of H2S-induced relaxation on retinal artery is not clarified yet. Herein, we aimed to evaluate the role of several calcium (Ca2+) signaling and Ca2+ sensitization mechanisms in the relaxing effect of H2S donor, NaHS, on retinal arteries. MATERIALS AND METHODS: Relaxing effects of NaHS (10-5-3 × 10-3M) were determined on precontracted retinal arteries in Ca2+ free medium as well as in the presence of the inhibitors of Ca2+ signaling and Ca2+ sensitization mechanisms. Additively, Ca2+ sensitivity of the contractile apparatus were evaluated by CaCl2-induced contractions in the presence of NaHS (3 × 10-3M). Functional experiments were furtherly assessed by protein and/or mRNA expressions, as appropriate. KEY FINDINGS: The relaxations to NaHS were preserved in Ca2+ free medium while NaHS pretreatment decreased the responsiveness to CaCl2. The inhibitors of plasmalemmal Ca2+-ATPase, sarcoplasmic-endoplasmic reticulum Ca2+-ATPase, Na+-Ca2+ ion-exchanger and myosin light chain kinase (MLCK) unchanged the relaxations to NaHS. Likewise, Ca2+ sensitization mechanisms including, rho kinase, protein kinase C and tyrosine kinase were unlikely to mediate the relaxation to NaHS in retinal artery. Whereas, a marked reduction was determined in NaHS-induced relaxations in the presence of MLCP inhibitor, calyculin A. Supportively, NaHS pretreatment significantly reduced phosphorylation of MYPT1-subunit of MLCP. SIGNIFICANCE: The relaxing effect of NaHS in retinal artery is likely to be related to the activation of MLCP and partly, to decrement in Ca2+ sensitivity of contractile apparatus.

Phenotypic delineation of the retinal arterial macroaneurysms with supravalvular pulmonic stenosis syndrome.

Retinal arterial macroaneurysms with supravalvular pulmonic stenosis (RAMSVPS), also known as Familial Retinal Arterial Macroaneurysms (FRAM) syndrome, is a very rare multisystem disorder. Here, we present a case series comprising ophthalmologic and systemic evaluation of patients homozygous for RAMSVPS syndrome causative IGFBP7 variant. New clinical details on 22 previously published and 8 previously unpublished patients are described. Age at first presentation ranged from 1 to 34 years. The classical feature of macroaneurysms and vascular beading involving the retinal arteries was universal. Follow up extending up to 14 years after initial diagnosis revealed recurrent episodes of bleeding and leakage from macroaneurysms in 55% and 59% of patients, respectively. The majority of patients who underwent echocardiography (18/23) showed evidence of heart involvement, most characteristically pulmonary (valvular or supravalvular) stenosis, often requiring surgical correction (12/18). Four patients died in the course of the study from complications of pulmonary stenosis, cerebral hemorrhage, and cardiac complications. Liver involvement (usually cirrhosis) was observed in eight patients. Cerebral vascular involvement was observed in one patient, and stroke was observed in two. We conclude that RAMSVPS is a recognizable syndrome characterized by a high burden of ocular and systemic morbidity, and risk of premature death. Recommendations are proposed for early detection and management of these complications.

Topographic Distribution of Contractile Protein in the Human Macular Microvasculature.

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.

Short-Time Ocular Ischemia Induces Vascular Endothelial Dysfunction and Ganglion Cell Loss in the Pig Retina.

Visual impairment and blindness are often caused by retinal ischemia-reperfusion (I/R) injury. We aimed to characterize a new model of I/R in pigs, in which the intraocular pathways were not manipulated by invasive methods on the ocular system. After 12 min of ischemia followed by 20 h of reperfusion, reactivity of retinal arterioles was measured in vitro by video microscopy. Dihydroethidium (DHE) staining, qPCR, immunohistochemistry, quantification of neurons in the retinal ganglion cell layer, and histological examination was performed. Retinal arterioles of I/R-treated pigs displayed marked attenuation in response to the endothelium-dependent vasodilator, bradykinin, compared to sham-treated pigs. DHE staining intensity and messenger RNA levels for HIF-1α, VEGF-A, NOX2, and iNOS were elevated in retinal arterioles following I/R. Immunoreactivity to HIF-1α, VEGF-A, NOX2, and iNOS was enhanced in retinal arteriole endothelium after I/R. Moreover, I/R evoked a substantial decrease in Brn3a-positive retinal ganglion cells and noticeable retinal thickening. In conclusion, the results of the present study demonstrate that short-time ocular ischemia impairs endothelial function and integrity of retinal blood vessels and induces structural changes in the retina. HIF-1α, VEGF-A, iNOS, and NOX2-derived reactive oxygen species appear to be involved in the pathophysiology.

Retinal arteriole reactivity in mice lacking the endothelial nitric oxide synthase (eNOS) gene.

Dysfunctional vascular endothelial nitric oxide synthase (eNOS) has been proposed to play a main pathophysiological role in various ocular diseases. The aim of the present study was to test the hypothesis that the chronic lack of eNOS impairs endothelium-dependent vasodilation in retinal arterioles. The relevance of eNOS for mediating vascular responses was studied in retinal vascular preparations from eNOS-deficient mice (eNOS-/-) and wild-type controls in vitro. Changes in luminal diameter in response to vasoactive agents were measured by videomicroscopy. The thromboxane mimetic, U46619, induced similar concentration-dependent constriction of retinal arterioles in eNOS-/- and wild-type mice. Responses to the endothelium-independent vasodilator, nitroprusside, did not differ between both mouse genotypes, either. In contrast, responses to the endothelium-dependent vasodilator, acetylcholine, were blunted in eNOS-/- mice. Non-isoform-selective blockade of either nitric oxide synthase (NOS) or cyclooxygenase (COX) alone did not affect responses to acetylcholine. However, combined blockade of both enzyme families markedly attenuated cholinergic vasodilation. Also, combined blockade of COX and neuronal NOS (nNOS) blunted acetylcholine-induced vasodilation, while combined COX and inducible NOS (iNOS) inhibition had no effect. Simultaneous NOS and COX-1 blockade did not affect cholinergic vasodilation, whereas combined NOS and COX-2 inhibition markedly reduced vasodilation to acetylcholine. These findings are the first to demonstrate that the chronic lack of eNOS is associated with moderate endothelial dysfunction in retinal arterioles. However, eNOS-deficiency is partially compensated by nNOS and COX-2 metabolites, which are reciprocally regulated.

Inactive matrix Gla protein is a novel circulating biomarker predicting retinal arteriolar narrowing in humans.

Active matrix Gla protein (MGP), a potent inhibitor of calcification in large arteries, protects against macrovascular complications. Recent studies suggested that active MGP helps maintaining the integrity of the renal and myocardial microcirculation, but its role in preserving the retinal microcirculation remains unknown. In 935 randomly recruited Flemish participants (mean age, 40.9 years; 50.3% women), we measured plasma desphospho-uncarboxylated MGP (dp-ucMGP), a marker of poor vitamin K status using an ELISA-based assay at baseline (1996-2010) and retinal microvascular diameters using IVAN software (Vasculomatic ala Nicola, version 1.1) including the central retinal arteriolar (CRAE) and venular (CRVE) equivalent and the arteriole-to-venule ratio (AVR) at follow-up (2008-2015). CRAE (P = 0.005) and AVR (P = 0.080) at follow-up decreased across tertiles of the dp-ucMGP distribution. In unadjusted models, for a doubling of dp-ucMGP at baseline, CRAE and AVR at follow-up respectively decreased by 1.40 µm (95% confidence interval [CI], 0.32 to 2.48; P = 0.011) and 0.006 (CI, 0.001 to 0.011; P = 0.016). In multivariable-adjusted models accounting for sex, baseline characteristics and follow-up duration, these estimates were -1.03 µm (CI, -1.96 to -0.11; P = 0.028) and -0.007 (CI, -0.011 to -0.002; P = 0.007). Additional adjustment for changes from baseline to follow-up in major baseline characteristics yielded as estimates -0.91 µm (CI, -1.82 to -0.01; P = 0.048) and -0.006 (95% CI, -0.011 to -0.001; P = 0.014), respectively. Circulating inactive dp-ucMGP is a long-term predictor of smaller retinal arteriolar diameter in the general population. Our observations highlight the possibility that vitamin K supplementation might promote retinal health.

Search for the Source of the Retinal Relaxing Factor.

Purpose/Aim of the study: the retinal relaxing factor (RRF) is an unidentified paracrine factor, which is continuously released from retinal tissue and causes smooth muscle cell relaxation. This study tried to identify the cellular source of the RRF. Furthermore, the possible RRF release by voltage-dependent sodium channel activation and the calcium-dependency of the RRF release were investigated. MATERIALS AND METHODS: mouse femoral arteries were mounted in myograph baths for in vitro isometric tension measurements. The vasorelaxing effect of chicken retinas, which contain no vascular cells, and of solutions incubated with MIO-M1 or primary Müller cell cultures were evaluated. The RRF release of other retinal cells was investigated by using cell type inhibitors. Concentration-response curves of veratridine, a voltage-dependent sodium channel activator, were constructed in the presence or absence of mouse retinal tissue to evaluate the RRF release. The calcium-dependency of the RRF release was investigated by evaluating the vasorelaxing effect of RRF-containing solutions made out of chicken retinas in the absence or presence of calcium. RESULTS: Chicken retinas induced vasorelaxation, whereas solutions incubated with Müller cell cultures did not. Moreover, the gliotoxin DL-α-aminoadipic acid, the microglia inhibitor minocycline, and the tetrodotoxin-resistant voltage-dependent sodium channel 1.8 inhibitor A-803467 could not reduce the RRF-induced relaxation. Concentration-response curves of veratridine were not enlarged in the presence of retinal tissue, and RRF-containing solutions made in the absence of calcium induced a substantial, but reduced vasorelaxation. CONCLUSIONS: the RRF is not released from vascular cells and probably neither from glial cells. The retinal cell type that does release the RRF remains unclear. Veratridine does not stimulate the RRF release in mice, and the RRF release in chickens is calcium-dependent as well as calcium-independent.

Aging decreases CO2 reactivity in the retinal artery, but not in the ocular choroidal vessels; a cross-sectional study.

BACKGROUND: The CO2 reactivity is often used to assess vascular function, but it is still unclear whether this reactivity is affected by aging. OBJECTIVE: To investigate the effects of aging on the CO2 reactivity in ocular and cerebral vessels, both of which are highly sensitive to hypercapnia, we compared the CO2 reactivity in the retinal artery (RA), retinal and choroidal vessels (RCV), optic nerve head (ONH), and middle cerebral artery (MCA) between young and middle-aged subjects. METHODS: We measured the CO2 reactivity in 14 young and 11 middle-aged males using laser-speckle flowgraphy during a 3-min inhalation of CO2-rich air. RESULTS: The CO2 reactivity in the RA and ONH were lower in the middle-aged group than in the young group, but no significant effect of age was observed in the RCV or MCA. The CO2 reactivity in the RA and ONH were correlated significantly with age, whereas those in the RCV or MCA were not. CONCLUSIONS: These findings suggest that there are regional differences in the effect of age on the CO2 reactivity among not only ocular and cerebral vessels, but also the retinal and choroidal vessels, even though these vessels are in neighboring areas.

Increased endothelin-1-mediated vasoconstriction after organ culture in rat and pig ocular arteries can be suppressed with MEK/ERK1/2 inhibitors.

PURPOSE: Even though retinal vascular changes following ischaemia have been poorly understood, the upregulation of vasoconstrictive endothelin-1 (ET-1) receptors (ETA /ETB ) following global cerebral ischaemia has been described. The aim of this study was to investigate whether or not the MEK/ERK1/2 pathway is involved in the observed upregulation and whether specific MEK/ERK1/2 inhibitors U0126 and trametinib can prevent it. METHODS: The aim was also to localize ETA and ETB receptors using immunohistochemistry in both fresh rat ophthalmic arteries and after 24-hr organ culture and study the receptors functionally using myography. Pig retinal arteries also underwent 24-hr organ culture to validate similar responses across species and the retinal vasculature. RESULTS: Results showed that following organ culture there is a significant increase in ET-1-mediated vasoconstriction, in particular via the ETB receptor. Furthermore, immunohistochemistry revealed a clear increase in pERK in the smooth muscle cells of rat ophthalmic artery. U0126 and trametinib were successful in attenuating the functional vasoconstriction in both rat and pig, as well as restoring immunofluorescence of pERK to fresh levels and counteracting ETB expression in the smooth muscle cells of the rat ophthalmic artery. CONCLUSION: This is the first study to show that the MEK/ERK1/2 pathway in responsible for the increase in functional vasoconstriction via ET-1 receptor in rat ophthalmic and pig retinal arteries. Furthermore, this study is the first to suggest a way of inhibiting and preventing such an increase. With these results, we suggest a novel approach in retinal ischaemia therapy.

The Hierarchy of Proinflammatory Cytokines in Ocular Inflammation.

PURPOSE: The concept of tissue-dependent cytokine hierarchy has been demonstrated in a number of diseases, but it has not been investigated in ophthalmic diseases. Here, we evaluated the functional hierarchy of interleukin-1ß (IL-1ß), IL-6, IL-17A, and tumor necrosis factor (TNF) in the induction of ocular inflammation. MATERIALS AND METHODS: We delivered adeno-associated virus (AAV) vectors expressing IL-1ß, IL-6, IL-17A, or TNF intravitreally in naïve C57/BL6 mice and compared and contrasted the inflammatory effects in the eye 5 weeks after AAV-mediated gene transfer. We also used an in vitro human system to test the effect of cytokines on barrier function. RESULTS: We found that IL-1ß had the highest ability to initiate ocular inflammation. The continuous overexpression of IL-1ß resulted in a significant upregulation of additional proinflammatory mediators in the eye. Using scanning laser ophthalmoscope and optical coherence tomography imaging techniques, we showed that a low dose of AAVIL-1ß was sufficient and was as pathogenic as a high dose of TNF in inducing vascular leakage, retinal degeneration, and cellular infiltration. Furthermore, only a marginal increase in IL-1ß was enough to cause cellular infiltration, thus confirming the highly pathogenic nature of IL-1ß in the eye. Contrary to our expectation, IL-6 or IL-17A had minimal or no effect in the eye. To examine the clinical relevance of our findings, we used an impedance assay to show that IL-1ß alone or TNF alone was able to cause primary human retinal endothelial cell barrier dysfunction in vitro. Again, IL-6 alone or IL-17A alone had no effect on barrier function; however, in the presence of IL-1ß or TNF, IL-17A but not IL-6 may provide additive proinflammatory effects. CONCLUSIONS: Our studies demonstrate the existence of a functional hierarchy of proinflammatory cytokines in the eye, and we show that IL-1ß is the most pathogenic when it is continuously expressed in the eye.

Severity of arterial defects in the retina correlates with the burden of intracerebral haemorrhage in COL4A1-related stroke.

Mutations in the α1 (COL4A1) or α2 (COL4A2) chains of collagen type IV, a major component of the vascular basement membrane, cause intracerebral haemorrhages with variable expressivity and reduced penetrance by mechanisms that remain poorly understood. Here we sought to investigate the cellular mechanisms of COL4A1-related intracerebral haemorrhage and identify a marker for haemorrhage risk stratification. A combination of histological, immunohistochemical, and electron microscopy analyses were used to analyse the brain parenchyma, cerebrovasculature, and retinal vessels of mice expressing the disease-causing COL4A1 p.G498V mutation. Mutant mice developed cerebral microhaemorrhages and macroscopic haemorrhages (macrohaemorrhages), the latter with reduced penetrance, mimicking the human disease. Microhaemorrhages that occurred in early postnatal life were associated with a transient, generalized increase in blood-brain barrier permeability at the level of capillaries. Macrohaemorrhages, which occurred later in life, originated from deep brain arteries with focal loss of smooth muscle cells. Similar smooth muscle cell loss was detected in retinal arteries, and a time-course analysis of arterial lesions showed that smooth muscle cells are recruited normally in arterial wall during development, but undergo progressive apoptosis-mediated degeneration. By assessing in parallel the extent of these retinal arterial lesions and the presence/absence of macrohaemorrhages, we found that the arterial lesion load in the retina is strongly correlated with the burden of macrohaemorrhages. We conclude that microhaemorrhages and macrohaemorrhages are driven by two distinct mechanisms. Moreover, smooth muscle cell degeneration is a critical factor underlying the partial penetrance of COL4A1-related macrohaemorrhages, and retinal imaging is a promising tool for identifying high-risk patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chronological Changes in Tip Cells during Sprouting Angiogenesis of Development of the Retinal Vasculature in Newborn Mice.

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 IB4-were performed on sections of mouse retina on postnatal days (P)-4, -8, and -12. Staining patterns of isolectin IB4-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/mm2 veins versus 1046.7 ± 175.5/mm2 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.

Dll4 and Notch signalling couples sprouting angiogenesis and artery formation.

Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

OBJECTIVE: Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2, and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2. Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels.

Acute and Chronic Hyperglycemia Elicit JIP1/JNK-Mediated Endothelial Vasodilator Dysfunction of Retinal Arterioles.

PURPOSE: Hyperglycemia, a hallmark of diabetes mellitus, is associated with retinal inflammation and impairment of endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles. However, molecular mechanisms involved in this diminished endothelial vasodilator function remain unclear. We examined whether inflammatory stress-activated kinases, c-Jun N-terminal kinase (JNK) and p38, contribute to retinal arteriolar dysfunction during exposure to acute and chronic hyperglycemia. METHODS: Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks; chronic hyperglycemia, 471 ± 23 mg/dL) or age-matched control pigs (euglycemia, 79 ± 5 mg/dL), and then cannulated and pressurized for vasoreactivity study. For acute hyperglycemia study, vessels from nondiabetic pigs were exposed intraluminally to high glucose (25 mM ≈ 450 mg/dL) for 2 hours, and normal glucose (5 mM ≈ 90 mg/dL) served as the control. RESULTS: Endothelium-dependent vasodilation to bradykinin was reduced in a similar manner after exposure to acute or chronic hyperglycemia. Administration of NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) nearly abolished vasodilations either in control (euglycemia and normal glucose) or hyperglycemic (acute and chronic) vessels. Treatment of either acute or chronic hyperglycemic vessels with JNK inhibitor SP600125 or JNK-interacting protein-1 (JIP1) inhibitor BI-78D3, but not p38 inhibitor SB203580, preserved bradykinin-induced dilation in an L-NAME-sensitive manner. By contrast, endothelium-independent vasodilation to sodium nitroprusside was unaffected by acute or chronic hyperglycemia. CONCLUSIONS: Activation of JIP1/JNK signaling in retinal arterioles during exposure to acute or chronic hyperglycemia leads to selective impairment of endothelium-dependent NO-mediated dilation. Therapeutic targeting of the vascular JNK pathway may improve retinal endothelial vasodilator function during early diabetes.

Prostaglandin induced changes in the tone of porcine retinal arterioles in vitro involve other factors than calcium activity in perivascular cells.

The cellular basis for the regulation of retinal blood flow is unknown, but recently a new type of perivascular cell (PVC) with pericyte characteristics was identified in the retinal arterial vascular wall located immediately external to the vascular smooth muscle cells. A possible involvement of this cell type in the regulation of retinal vascular tone might be elucidated by studying differences in the response after the addition of compounds stimulating respectively relaxation and contraction. The effects of PGE2 and PGF2α on vascular tone and calcium activity in PVCs in porcine retinal arterioles were studied in a confocal myograph after the addition of the ryanodine receptor blocker ryanodine, the L-type Ca(2+) channel blocker nifedipine, the non-specific cation channel blocker LOE908, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) blocker CPA, and the inositol triphosphate receptor (IP3R) and transient receptor potential (TRP) ion channel blocker 2-APB. The Ca(2+) channel blockers nifedipine and LOE908 induced significant relaxation of retinal arterioles. After the addition of both PGE2 and PGF2α calcium activity in the PVCs was significantly reduced by both the SERCA inhibitor CPA and the IP3R antagonist 2-APB, but the changes in calcium activity were unrelated to the changes in tone induced by PGE2 and PGF2α. Changes in the tone of porcine retinal arterioles in vitro induced by PGE2 and PGF2α involve other factors than calcium activity in the perivascular cells.

4-Hydroxy-2-nonenal attenuates ß2-adrenoceptor-mediated vasodilation of rat retinal arterioles.

4-Hydroxy-2-nonenal (4-HNE) is a major reactive aldehyde formed by lipid peroxidation, and it plays an important role in the pathogenesis of several vascular diseases, including diabetes mellitus. In this study, we examined the effects of 4-HNE on the vasodilatory mechanisms of rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of 4-HNE significantly prevented the vasodilation of retinal arterioles induced by the ß2-adrenoceptor agonist salbutamol but not the nitric oxide donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3). Iberiotoxin, an inhibitor of large-conductance KCa (BKCa) channels, significantly reduced the salbutamol-induced vasodilation of retinal arterioles. The vasodilator effect of BMS-191011, a BKCa channel opener, on retinal arterioles was significantly attenuated by 4-HNE. These results suggest that 4-HNE attenuates retinal vasodilator responses to ß2-adrenoceptor agonists through the impairment of the BKCa pathway. The direct effect of 4-HNE on retinal blood vessels may, therefore, contribute to the retinal vascular dysfunction observed in patients with diabetes mellitus.

Oxygen saturation measurements of the retinal vasculature in treated asymmetrical primary open-angle glaucoma using hyperspectral imaging.

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.