ABSTRACT
Obesity is a significant health concern that leads to impaired vascular function and subsequent abnormalities in various organs. The impact of obesity on ocular blood vessels, however, remains largely unclear. In this study, we examined the hypothesis that obesity induced by high-fat diet produces vascular endothelial dysfunction in the ophthalmic artery. Mice were subjected to a high-fat diet for 20 weeks, while age-matched controls were maintained on a standard diet. Reactivity of isolated ophthalmic artery segments was assessed in vitro. Reactive oxygen species (ROS) were quantified in cryosections by dihydroethidium (DHE) staining. Redox gene expression was determined in ophthalmic artery explants by real-time PCR. Furthermore, the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), the receptor for advanced glycation end products (RAGE), and of the lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) was determined in cryosections using immunofluorescence microscopy. Ophthalmic artery segments from mice on a high-fat diet exhibited impaired vasodilation responses to the endothelium-dependent vasodilator acetylcholine, while endothelium-independent responses to nitroprusside remained preserved. DHE staining intensity in the vascular wall was notably stronger in mice on a high-fat diet. Messenger RNA expression for NOX2 was elevated in the ophthalmic artery of mice subjected to high fat diet. Likewise, immunostainings revealed increased expression of NOX2 and of RAGE, but not of LOX-1. These findings suggest that a high-fat diet triggers endothelial dysfunction by inducing oxidative stress in the ophthalmic artery via involvement of RAGE and NOX2.
Subject(s)
Diet, High-Fat , Ophthalmic Artery , Vascular Diseases , Animals , Mice , Diet, High-Fat/adverse effects , Endothelium, Vascular/metabolism , Obesity , Ophthalmic Artery/metabolism , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , Scavenger Receptors, Class E/genetics , Scavenger Receptors, Class E/metabolism , Vascular Diseases/metabolism , VasodilationABSTRACT
The voltage-gated potassium channel 1.6 (Kv1.6) plays a vital role in ocular neurovascular beds and exerts its modulatory functions via interaction with other proteins. However, the interactome and their potential roles remain unknown. Here, the global proteome landscape of the ophthalmic artery (OA) and neuroretina was mapped, followed by the determination of Kv1.6 interactome and validation of its functionality and cellular localization. Microfluorimetric analysis of intracellular [K+] and Western blot validated the native functionality and cellular expression of the recombinant Kv1.6 channel protein. A total of 54, 9 and 28 Kv1.6-interacting proteins were identified in the mouse OA and, retina of mouse and rat, respectively. The Kv1.6-protein partners in the OA, namely actin cytoplasmic 2, alpha-2-macroglobulin and apolipoprotein A-I, were implicated in the maintenance of blood vessel integrity by regulating integrin-mediated adhesion to extracellular matrix and Ca2+ flux. Many retinal protein interactors, particularly the ADP/ATP translocase 2 and cytoskeleton protein tubulin, were involved in endoplasmic reticulum stress response and cell viability. Three common interactors were found in all samples comprising heat shock cognate 71 kDa protein, Ig heavy constant gamma 1 and Kv1.6 channel. This foremost in-depth investigation enriched and identified the elusive Kv1.6 channel and, elucidated its complex interactome.
Subject(s)
Potassium Channels, Voltage-Gated , Mice , Rats , Animals , Potassium Channels, Voltage-Gated/genetics , Potassium Channels/metabolism , Proteome/metabolism , Ophthalmic Artery/chemistry , Ophthalmic Artery/metabolism , Cytoplasm/metabolismABSTRACT
OBJECTIVE: The aim of this study was to examine the diagnostic value of ophthalmic artery Doppler indices in predicting preeclampsia along with other markers in the third trimester of pregnancy. METHODS: Normotensive pregnancies were included during 28-32 weeks of gestation to undergo uterine and ophthalmic artery Doppler ultrasound. Maternal and fetal characteristics were documented at the visit between the 28 and 32 weeks of gestation, and pregnancy-associated plasma protein A (PAPP-A) values in the first trimester were collected to be integrated into a multiparametric prediction model. RESULTS: Of 795 included participants, 48 cases progressed to preeclampsia. All assessed ophthalmic Doppler parameters including first and second peak systolic velocities (PSVs), second to first peak ratio (PR), and pulsatility index (PI), were statistically different in patients who developed preeclampsia later on. The average PR (sensitivity: 100% [95% CI, 0.81-1.00]; specificity: 90% [95% CI, 0.86-0.93]) and PI between the eyes, PAPP-A multiple of median and uterine artery PI were determined to be the most important predictors of PE, which were subsequently integrated into a multiple regression model (sensitivity: 94% [95% CI, 0.70-1.00]; specificity: 93% [95% CI, 0.89-0.96]). CONCLUSION: This study provided a screening method for individuals at higher risk of progressing to preeclampsia in the third trimester of pregnancy.
Subject(s)
Pre-Eclampsia , Pregnancy , Female , Humans , Pre-Eclampsia/diagnostic imaging , Pregnancy-Associated Plasma Protein-A , Ophthalmic Artery/diagnostic imaging , Ophthalmic Artery/metabolism , Ultrasonography, Prenatal/methods , Uterine Artery/diagnostic imaging , Pregnancy Trimester, First , Ultrasonography, Doppler/methods , Biomarkers , Pulsatile FlowABSTRACT
Purpose: Obstructive sleep apnea recently has been associated with a higher frequency of ischemic optic neuropathies. Intermittent hypoxia (IH) has been proposed as a major component of obstructive sleep apnea cardiovascular consequences. However, there currently are no pathophysiologic data regarding the effect of IH on the ocular vascular system. Thus, we assessed the impact of chronic IH exposure on the morphology and vascular reactivity of the rat ophthalmic artery (OA). Methods: Rats were exposed to 14 days of IH or normoxia (NX). Ophthalmic artery reactivity was studied using wire myography in rats treated or not with tempol (1 mM/day). Expression of endothelin-1 (ET-1) and its receptors, and of the three nitric oxide synthase (NOS) isoform genes was quantified using quantitative polymerase chain reaction (qPCR) in the retina and optic nerve. Structural alterations (optical and electron microscopy) and superoxide anion production were studied in OA sections. Results: Superoxide ion expression in the OA wall was increased by 23% after IH exposure. Ophthalmic artery contractile response to 3.10-8 M ET-1 was increased by 18.6% and nitric oxide-mediated relaxation was significantly delayed in IH compared to NX rats. In the absence of nitric oxide, cytochrome P450 blockade increased relaxation to acetylcholine in IH rats and delayed it in NX rats. Tempol treatment abolished the IH-induced changes in OA reactivity. Conclusions: These results strongly suggest that chronic IH induces oxidative stress in the rat OA, associated with endothelial dysfunction through alterations of nitric oxide and endothelium-derived hyperpolarising factors (EDHF) pathways.
Subject(s)
Endothelin-1/metabolism , Hypoxia/physiopathology , Ophthalmic Artery/physiopathology , Oxidative Stress , Receptor, Endothelin A/metabolism , Animals , Chronic Disease , Cyclic N-Oxides/pharmacology , Hypoxia/metabolism , Male , Muscle, Smooth, Vascular/physiology , Myography , Nitric Oxide Synthase/genetics , Ophthalmic Artery/metabolism , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Spin Labels , Superoxides/metabolismABSTRACT
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.
Subject(s)
Butadienes/pharmacology , Endothelin-1/pharmacology , Ischemia/prevention & control , MAP Kinase Signaling System/drug effects , Nitriles/pharmacology , Ophthalmic Artery/physiopathology , Pyridones/pharmacology , Pyrimidinones/pharmacology , Retinal Artery/physiopathology , Animals , Disease Models, Animal , Immunohistochemistry , Ischemia/pathology , Ischemia/physiopathology , MAP Kinase Signaling System/physiology , Male , Myography , Ophthalmic Artery/metabolism , Ophthalmic Artery/pathology , Organ Culture Techniques , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Retinal Artery/metabolism , Retinal Artery/pathology , Swine , Up-Regulation , Vasoconstriction/drug effectsABSTRACT
Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.
Subject(s)
Myosins/physiology , Neovascularization, Pathologic , Neural Tube/physiopathology , Ophthalmic Artery/physiopathology , Pigmentation , Pseudopodia/pathology , Vitreous Body/pathology , Animals , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Ophthalmic Artery/metabolism , Pseudopodia/metabolism , Vitreous Body/blood supply , Vitreous Body/metabolismABSTRACT
PURPOSE: The transient middle cerebral artery occlusion (MCAO) model of stroke is one of the most commonly used models to study focal cerebral ischemia. This procedure also results in the simultaneous occlusion of the ophthalmic artery that supplies the retina. Retinal cell death is seen days after reperfusion and leads to functional deficits; however, the mechanism responsible for this injury has not been investigated. Given that the eye may have a unique ocular immune response to an ischemic challenge, this study examined the inflammatory response to retinal ischemia in the MCAO model. METHODS: Young male C57B/6 mice were subjected to 90-min transient MCAO and were euthanized at several time points up to 7 days. Transcription of inflammatory cytokines was measured with quantitative real-time PCR, and immune cell activation (e.g., phagocytosis) and migration were assessed with ophthalmoscopy and flow cytometry. RESULTS: Observation of the affected eye revealed symptoms consistent with Horner's syndrome. Light ophthalmoscopy confirmed the reduced blood flow of the retinal arteries during occlusion. CX3CR1-GFP reporter mice were then employed to evaluate the extent of the ocular microglia and monocyte activation. A significant increase in green fluorescent protein (GFP)-positive macrophages was seen throughout the ischemic area compared to the sham and contralateral control eyes. RT-PCR revealed enhanced expression of the monocyte chemotactic molecule CCL2 early after reperfusion followed by a delayed increase in the proinflammatory cytokine TNF-α. Further analysis of peripheral leukocyte recruitment by flow cytometry determined that monocytes and neutrophils were the predominant immune cells to infiltrate at 72 h. A transient reduction in retinal microglia numbers was also observed, demonstrating the ischemic sensitivity of these cells. Blood-eye barrier permeability to small and large tracer molecules was increased by 72 h. Retinal microglia exhibited enhanced phagocytic activity following MCAO; however, infiltrating myeloid cells were significantly more efficient at phagocytizing material at all time points. Immune homeostasis in the affected eye was largely restored by 7 days. CONCLUSIONS: This work demonstrates that there is a robust inflammatory response in the eye following MCAO, which may contribute to a worsening of retinal injury and visual impairment. These results mirror what has been observed in the brain after MCAO, suggesting a conserved inflammatory signaling response to ischemia in the central nervous system. Imaging of the eye may therefore serve as a useful non-invasive prognostic indicator of brain injury after MCAO. Future studies are needed to determine whether this inflammatory response is a potential target for therapeutic manipulation in retinal ischemia.
Subject(s)
Arterial Occlusive Diseases/metabolism , Biomarkers/metabolism , Cytokines/metabolism , Infarction, Middle Cerebral Artery/metabolism , Inflammation Mediators/metabolism , Ophthalmic Artery/metabolism , Stroke/metabolism , Animals , Arterial Occlusive Diseases/genetics , Blood-Retinal Barrier/physiology , Capillary Permeability/physiology , Cytokines/genetics , Disease Models, Animal , Flow Cytometry , Infarction, Middle Cerebral Artery/genetics , Male , Mice , Mice, Inbred C57BL , Phagocytosis/physiology , Real-Time Polymerase Chain Reaction , Stroke/geneticsABSTRACT
Cerebral vasculature is often the target of stroke studies. However, the vasculature supplying the eye might also be affected by ischemia. The aim of the present study was to investigate if the transient global cerebral ischemia (GCI) enhances vascular effect of endothelin-1 (ET-1) and 5-hydroxytryptamine/serotonin (5-HT) on the ophthalmic artery in rats, leading to delayed retinal damage. This was preformed using myography on the ophthalmic artery, coupled with immunohistochemistry and electroretinogram (ERG) to assess the ischemic consequences on the retina. Results showed a significant increase of ET-1 mediated vasoconstriction at 48 hours post ischemia. The retina did not exhibit any morphological changes throughout the study. However, we found an increase of GFAP and vimentin expression at 72 hours and 7 days after ischemia, indicating Müller cell mediated gliosis. ERG revealed significantly decreased function at 72 hours, but recovered almost completely after 7 days. In conclusion, we propose that the increased contractile response via ET-1 receptors in the ophthalmic artery after 48 hours may elicit negative retinal consequences due to a second ischemic period. This may exacerbate retinal damage after ischemia as illustrated by the decreased retinal function and Müller cell activation. The ophthalmic artery and ET-1 mediated vasoconstriction may be a valid and novel therapeutic target after longer periods of ischemic insults.
Subject(s)
Endothelin-1/metabolism , Ischemic Attack, Transient/pathology , Ophthalmic Artery/metabolism , Ophthalmic Artery/physiology , Retina/metabolism , Retina/pathology , Vasoconstriction , Animals , Chickens , Electroretinography , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Male , Night Vision , Rats, Wistar , Vimentin/metabolismABSTRACT
Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) modulate vascular tone. In view of their therapeutic potential for ocular diseases, we examined the effect of exogenous CO and H2S on tone of isolated rabbit ophthalmic artery and their interaction with endogenous and exogenous NO. Ophthalmic artery segments mounted on a wire myograph were challenged with cumulative concentrations of phenylephrine (PE) in the presence or absence of NG-nitro-L-arginine (LNNA) to inhibit production of NO, the CO-releasing molecules CORMs or the H2S-donor GYY4137. The maximal vasoconstriction elicited by PE reached 20-30% of that induced by KCl but was dramatically increased by incubation with LNNA. GYY4137 significantly raised PE-mediated vasoconstriction, but it did not change the response to PE in the presence of LNNA or the relaxation to sodium nitroprusside (SNP). CORMs concentration-dependently inhibited PE-induced constriction, an effect that was synergistic with endogenous NO (reduced by LNNA), but insensitive to blockade of guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-α]quinoxalin-1-one (ODQ). In vascular tissues cyclic GMP (cGMP) levels seemed reduced by GYY4137 (not significantly), but were not changed by CORM. These data indicate that CO is able per se to relax isolated ophthalmic artery and to synergize with NO, while H2S counteracts the effect of endogenous NO. CO does not stimulate cGMP production in our system, while H2S may reduce cGMP production stimulated by endogenous NO. These findings provide new insights into the complexities of gas interactions in the control of ophthalmic vascular tone, highlighting potential pharmacological targets for ocular diseases.
Subject(s)
Carbon Monoxide/pharmacology , Hydrogen Sulfide/pharmacology , Muscle Tonus , Nitric Oxide/pharmacology , Ophthalmic Artery/drug effects , Animals , Cyclic GMP/biosynthesis , Ophthalmic Artery/metabolism , Ophthalmic Artery/physiology , RabbitsABSTRACT
PURPOSE: We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal. METHODS: Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R(-/-)) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy. RESULTS: In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R(-/-) mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R(-/-) mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R(-/-) mice, neither in endothelium-intact nor in endothelium-removed arteries. CONCLUSIONS: These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation and endothelium-independent vasoconstriction.
Subject(s)
Endothelium, Vascular/metabolism , Gene Expression Regulation , Ophthalmic Artery/metabolism , RNA, Messenger/genetics , Receptor, Muscarinic M3/genetics , Animals , Disease Models, Animal , Endothelium, Vascular/surgery , Mice , Mice, Knockout , Ophthalmic Artery/physiopathology , Ophthalmic Artery/surgery , Real-Time Polymerase Chain Reaction , Receptor, Muscarinic M3/biosynthesis , Retinal Diseases/genetics , Retinal Diseases/metabolism , Retinal Diseases/physiopathology , VasoconstrictionABSTRACT
PURPOSE: To evaluate the regulatory cross-talk of the vascular and neural networks in the cornea. METHODS: b-FGF micropellets (80 ng) were implanted in the temporal side of the cornea of healthy C57Bl/6 mice. On day 7, blood vessels (hemangiogenesis) and nerves were observed by immunofluorescence staining of corneal flat mounts. The next group of mice underwent either trigeminal stereotactic electrolysis (TSE), or sham operation, to ablate the ophthalmic branch of the trigeminal nerve. Blood vessel growth was detected by immunohistochemistry for PECAM-1 (CD31) following surgery. In another set of mice following TSE or sham operation, corneas were harvested for ELISA (VEGFR3 and pigment epithelium-derived factor [PEDF]) and for quantitative RT-PCR (VEGFR3, PEDF, and CD45). PEDF, VEGFR3, beta-3 tubulin, CD45, CD11b, and F4/80 expression in the cornea were evaluated using immunostaining. RESULTS: No nerves were detected in the areas subject to corneal neovascularization, whereas they persisted in the areas that were neovessel-free. Conversely, 7 days after denervation, significant angiogenesis was detected in the cornea, and this was associated with a significant decrease in VEGFR3 (57.5% reduction, P = 0.001) and PEDF protein expression (64% reduction, P < 0.001). Immunostaining also showed reduced expression of VEGFR3 in the corneal epithelial layer. Finally, an inflammatory cell infiltrate, including macrophages, was observed. CONCLUSION: Our data suggest that sensory nerves and neovessels inhibit each other in the cornea. When vessel growth is stimulated, nerves disappear and, conversely, denervation induces angiogenesis. This phenomenon, here described in the eye, may have far-reaching implications in understanding angiogenesis.
Subject(s)
Cornea/blood supply , Cornea/innervation , Corneal Neovascularization/physiopathology , Nerve Degeneration/physiopathology , Signal Transduction/physiology , Trigeminal Nerve/physiopathology , Animals , Corneal Neovascularization/metabolism , Denervation , Disease Models, Animal , Electrolysis , Enzyme-Linked Immunosorbent Assay , Eye Proteins/genetics , Eye Proteins/metabolism , Fluorescent Antibody Technique, Indirect , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Nerve Degeneration/metabolism , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Ophthalmic Artery/metabolism , Ophthalmic Artery/physiopathology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , Serpins/metabolism , Trigeminal Nerve/metabolism , Tubulin/metabolism , Vascular Endothelial Growth Factor Receptor-3/genetics , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
PURPOSE: To characterize the vitreous and plasma pharmacokinetics of topotecan after ophthalmic artery infusion (OAI) subsequent to superselective artery catheterization and to compare it with periocular injection (POI). METHODS: The ophthalmic artery of 4 pigs was catheterized and 1 mg of topotecan infused over a period of 30 minutes. The contralateral eye was subsequently used for administering topotecan by POI. Serial vitreous specimens were obtained by microdialysis and plasma samples collected and assayed for total and lactone topotecan. RESULTS: Maximum total topotecan concentration in the vitreous (median, range) was significantly higher after OAI compared with POI (131.8 ng/mL [112.9-138.7] vs. 13.6 ng/mL [5.5-15.3], respectively; P < 0.005). Median vitreous exposure calculated as area under the curve for total topotecan attained after OAI was significantly higher than after POI (299.8 ng·hour/mL [247.6-347.2] and 48.9 ng·hour/mL [11.8-63.4], respectively; P < 0.05). The vitreous to plasma exposure ratio was 29 after OAI and 3.4 after POI. Systemic exposure for total topotecan was low after both modalities of administration, with a trend to be lower after OAI compared with POI (10.6 ng·hour/mL [6.8-13.4] vs. 18.7 ng·hour/mL [6.3-21.7]; P = 0.54). CONCLUSION: Superselective OAI resulted in significantly higher vitreous concentrations and exposure and a trend toward lower systemic exposure than POI.
Subject(s)
Ophthalmic Artery/metabolism , Topoisomerase I Inhibitors/pharmacokinetics , Topotecan/pharmacokinetics , Vitreous Body/metabolism , Animals , Area Under Curve , Biological Availability , Catheterization , Chromatography, High Pressure Liquid , Infusions, Intra-Arterial , Injections, Intraocular , Sus scrofaABSTRACT
PURPOSE: The hyaloid vascular system (HVS) is a transient network nourishing developing eyes and has been widely used as a natural model to study blood vessel regression. Failure of its regression in humans leads to several blinding diseases. Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a recently defined lymphatic marker that is also expressed by a subpopulation of macrophages. To date, there is no report on its expression in the HVS. This study was conducted to investigate whether LYVE-1 is expressed in the HVS and how it is associated with the vascular structure and macrophage phenotype. METHODS: Normal C57BL/6 mouse eyeballs were sampled from embryonic day (E) 10.5 to postnatal (P) and adult stages for immunofluorescent microscopic studies with antibodies against LYVE-1, CD31 (panendothelial cell marker), and F4/80 (macrophage marker). Additionally, Angiopoietin-2 (Ang-2) knockout mice with abnormally persistent HVS were examined. RESULTS: The LYVE-1 expression was detected on normal HVS between E12.5 and P14. The LYVE-1(+) cells were F4/80(+) but CD31(-), indicating a macrophage lineage. Additionally, LYVE-1(+) cells bud on CD31(+) vessels and constitute an integral part of the network in both normal developing and Ang-2 knockout mice. CONCLUSIONS: This study provides the first evidence that the HVS contains a LYVE-1(+) cellular component in both physiological and pathologic conditions. This novel finding not only provides a new concept in defining the embryogenesis and pathogenesis of the HVS, it also leads to a completely natural model in which to study the functions of the LYVE-1 pathway, an important topic for lymphatic research as well.
Subject(s)
Glycoproteins/metabolism , Lens, Crystalline/embryology , Ophthalmic Artery/embryology , Ophthalmic Artery/metabolism , Vitreous Body/embryology , Angiopoietin-2/metabolism , Animals , Antigens, Differentiation/metabolism , Female , Fluorescent Antibody Technique, Indirect , Lens, Crystalline/blood supply , Male , Membrane Transport Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Vitreous Body/blood supplyABSTRACT
PURPOSE: Alterations in FOXC1 dosage lead to a spectrum of highly penetrant, ocular anterior segment dysgenesis phenotypes. The most serious outcome is the development of glaucoma, which occurs in 50% to 75% of patients. Therefore, the need to identify specific pathways and genes that interact with FOXC1 to promote glaucoma is great. In this study, the authors investigated the loss of foxC1 in the zebrafish to characterize phenotypes and gene interactions that may impact glaucoma pathogenesis. METHODS: Morpholino knockdown in zebrafish, RNA and protein marker analyses, transgenic reporter lines, and angiography, along with histology and transmission electron microscopy, were used to study foxC1 function and gene interactions. RESULTS: Zebrafish foxC1 genes were expressed dynamically in the developing vasculature and periocular mesenchyme during development. Multiple ocular and vascular defects were found after the knockdown of foxC1. Defects in the hyaloid vasculature, arteriovenous malformations, and coarctation of the aorta were observed with maximal depletion of foxC1. Partial loss of foxC1 resulted in CNS and ocular hemorrhages, defects in intersegmental vessel patterning, and increased vascular permeability. To investigate the basis for these disruptions, the ultrastructure of foxC1-depleted hyaloid vascular cells was studied. These experiments, along with laminin-111 immunoreactivity, revealed disruptions in basement membrane integrity. Finally, codepletion of laminin alpha-1 and foxC1 uncovered a genetic interaction between these genes during development. CONCLUSIONS: Genetic interactions between FOXC1 and basement membrane components influence vascular stability and may impact glaucoma development and increase stroke risk in FOXC1 patients.
Subject(s)
Basement Membrane/blood supply , Endothelium, Vascular/metabolism , Forkhead Transcription Factors/physiology , Morphogenesis/physiology , Ophthalmic Artery/embryology , Zebrafish Proteins/physiology , Zebrafish/embryology , Animals , Basement Membrane/metabolism , Basement Membrane/pathology , Biomarkers/metabolism , Endothelium, Vascular/pathology , Eye/embryology , Fluorescein Angiography , Fluorescent Antibody Technique, Indirect , Gene Expression , Gene Knockout Techniques , Gene Silencing , In Situ Hybridization , Laminin/metabolism , Lens, Crystalline/blood supply , Microscopy, Confocal , Microscopy, Electron, Transmission , Ophthalmic Artery/metabolism , Ophthalmic Artery/pathology , Zebrafish Proteins/metabolismABSTRACT
PURPOSE: To determine whether constriction of proximal arterial vessels precedes involution of the distal hyaloid vasculature in the mouse, under normal conditions, and whether this vasoconstriction is less pronounced when the distal hyaloid network persists, as it does in oxygen-induced retinopathy (OIR). METHODS: Photomicrographs of the vasa hyaloidea propria were analysed from pre-term pups (1-2 days prior to birth), and on Days 1-11 post-birth. The OIR model involved exposing pups to approximately 90% O(2) from D1-5, followed by return to ambient air. At sampling times pups were anaesthetised and perfused with india ink. Retinal flatmounts were also incubated with FITC-lectin (BS-1, G. simplicifolia,); this labels all vessels, allowing identification of vessels not patent to the perfusate. RESULTS: Mean diameter of proximal hyaloid vessels in pre-term pups was 25.44 +/- 1.98 microm; +/- 1 SEM). Within 3-12 hrs of birth, significant vasoconstriction was evident (diameter:12.45 +/- 0.88 microm), and normal hyaloid regression subsequently occurred. Similar vasoconstriction occurred in the O(2)-treated group, but this was reversed upon return to room air, with significant dilation of proximal vessels by D7 (diameter: 31.75 +/- 11.99 microm) and distal hyaloid vessels subsequently became enlarged and tortuous. CONCLUSIONS: Under normal conditions, vasoconstriction of proximal hyaloid vessels occurs at birth, preceding attenuation of distal hyaloid vessels. Vasoconstriction also occurs in O(2)-treated pups during treatment, but upon return to room air, the remaining hyaloid vessels dilate proximally, and the distal vessels become dilated and tortuous. These observations support the contention that regression of the hyaloid network is dependent, in the first instance, on proximal arterial vasoconstriction.
Subject(s)
Ophthalmic Artery/physiopathology , Vasoconstriction , Vitreous Body/blood supply , Animals , Animals, Newborn , Female , Fluorescein-5-isothiocyanate , Humans , Hypoxia/physiopathology , Infant, Newborn , Lectins/metabolism , Mice , Ophthalmic Artery/metabolism , Retinopathy of Prematurity/physiopathologyABSTRACT
Remodeling of the cerebral vasculature contributes to the pathogenesis of cerebral ischemia. Remodeling is caused by increased smooth muscle proliferation and may be due to an increase in the responsiveness of vascular cells to epidermal growth factor (EGF). Aldosterone is a risk factor for stroke, and the literature suggests it may play a role in increasing the expression of the receptor for EGF (EGFR). We hypothesized that mRNA for the EGF-stimulated pathway would be elevated in the vasculature of stroke-prone spontaneously hypertensive rats (SHRSP) and that this and experimental ischemic cerebral infract size would be reduced by aldosterone inhibition with spironolactone. We found that spironolactone treatment reduced the size of cerebral infarcts after middle cerebral artery occlusion in SHRSP (51.69 +/- 3.60 vs. 22.00 +/- 6.69% of hemisphere-infarcted SHRSP vs. SHRSP + spironolactone P < 0.05). Expression of EGF and EGFR mRNA was higher in cerebral vessels and aorta from adult SHRSP compared with Wistar-Kyoto rats. Only the expression of EGFR mRNA was elevated in the young SHRSP. Spironolactone reduced the EGFR mRNA expression in the aorta (1.09 +/- 0.25 vs. 0.56 +/- 0.11 phosphorimage units SHRSP vs. SHRSP + spironolactone P < 0.05) but had no effect on EGF mRNA. In vitro incubation of aorta with aldosterone +/- spironolactone produced similar results, suggesting a direct effect of aldosterone. Thus spironolactone may reduce the size of cerebral infarcts via a reduction in the expression of the EGFR mRNA, leading to reduced remodeling.
Subject(s)
Cerebral Infarction/prevention & control , ErbB Receptors/metabolism , RNA, Messenger/metabolism , Spironolactone/pharmacology , Stroke/prevention & control , Aldosterone/blood , Aldosterone/pharmacology , Animals , Aorta/drug effects , Aorta/metabolism , Basal Ganglia/blood supply , Basal Ganglia/drug effects , Basal Ganglia/pathology , Blood Pressure/drug effects , Body Weight/drug effects , Cerebral Cortex/blood supply , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Cerebral Infarction/etiology , Cerebral Infarction/pathology , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , ErbB Receptors/genetics , Genetic Predisposition to Disease , In Vitro Techniques , Infarction, Middle Cerebral Artery/complications , Male , Mineralocorticoid Receptor Antagonists/pharmacology , Ophthalmic Artery/chemistry , Ophthalmic Artery/metabolism , RNA, Messenger/analysis , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Stroke/etiologyABSTRACT
This review addresses whether the antiglaucoma agents beta-adrenergic antagonists and Ca2+ entry blockers cause vasoactive effects in the retinal and other ocular vasculatures, as they do in other tissues. The potent vasodilating effects of Ca2+ entry blockers on ocular vessels have recently been demonstrated in in vivo and in vitro studies, implying that the maintenance of ocular vascular tone relies almost exclusively on extracellular Ca2+. Ca2+ entry blockers may potentially play a role in relaxing the retinal, long posterior ciliary, and ophthalmociliary arteries to improve the ocular circulation in vascular diseases in which there is considerable vascular tone present. The beta-adrenergic antagonists are discussed with reference to their antihypertensive role, their effect on other vascular beds, and finally what is known of their effect in the ocular vasculature. The emerging evidence that particular selective beta-adrenergic antagonists, such as betaxolol, are also potent Ca2+ channel entry blockers in other vascular beds is presented. Betaxolol has been shown to induce vasodilatation in the retinal and other ocular vascular beds, although studies have shown that beta1-adrenergic receptors are sparse in these vascular beds. This implies that an alternative mechanism must be responsible for betaxolol-induced vasodilatation. Evidence is presented that betaxolol vasodilates via its potent Ca2+ channel entry blocking properties, and its potency and ability to vasodilate are compared with those of nimodipine and timolol, as well as with those of other Ca2+ channel entry blockers. Important areas for future research in this area are discussed.
Subject(s)
Adrenergic beta-Antagonists/therapeutic use , Calcium Channel Blockers/therapeutic use , Eye/blood supply , Glaucoma/physiopathology , Vasodilation/drug effects , Animals , Calcium Channels/drug effects , Calcium Channels/metabolism , Ciliary Arteries/drug effects , Ciliary Arteries/metabolism , Ciliary Arteries/physiopathology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Glaucoma/drug therapy , Glaucoma/metabolism , Humans , Microcirculation/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiopathology , Ophthalmic Artery/drug effects , Ophthalmic Artery/metabolism , Ophthalmic Artery/physiopathology , Retinal Artery/drug effects , Retinal Artery/metabolism , Retinal Artery/physiopathologyABSTRACT
PURPOSE: Endothelial cells modulate vascular tone by releasing the vasodilator nitric oxide (NO) or the vasoconstrictor endothelin-1. From one vascular bed to another and between vessels of different diameter, heterogeneities of endothelium-dependent regulatory mechanisms exist. Hence, the current study compared the effects of NO and endothelin-1 in the porcine ophthalmic artery and one of its branches, the ciliary artery. METHODS: Porcine eyes were obtained at the slaughterhouse. The ophthalmic and ciliary arteries were dissected free under a microscope and suspended in myograph systems (95% O2 and 5% CO2, 37 degrees C) for isometric tension recording. RESULTS: In both vessels, bradykinin stimulated the release of NO, but the sensitivity to bradykinin increased with decreasing vascular diameter. By contrast, the basal release of NO became less efficient in inhibiting contractions to serotonin and endothelin-1 in ciliary versus ophthalmic artery. Endothelin-1 induced potent contractions that were more pronounced in ciliary than in ophthalmic artery. Serotonin-induced contractions also were more efficient in ciliary artery but less than to those to endothelin-1. Contractions to serotonin were inhibited in both blood vessels by the 5-HT2 serotonergic antagonist ketanserin. CONCLUSIONS: Thus endothelium-derived vasoactive substances are potent regulators of porcine extraocular ophthalmic circulation. Their effects increase with decreasing vascular diameter, suggesting an important role of NO and endothelin-1 in the regulation of ophthalmic circulation. A dysfunction of these regulatory mechanisms could have implications about the pathogenesis of ophthalmic complications seen in diabetes, hypertension, and in certain forms of glaucoma associated with ocular vasospasms.
Subject(s)
Ciliary Body/blood supply , Endothelium, Vascular/metabolism , Ophthalmic Artery/metabolism , Animals , Arteries/metabolism , Blood Flow Velocity , Ciliary Body/physiology , Endothelins/pharmacology , Isometric Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Nitric Oxide/pharmacology , Ophthalmic Artery/physiology , Swine , Vasoconstrictor Agents/pharmacologyABSTRACT
The endothelium produces relaxing and contracting substances, among them nitric oxide and endothelin. The role of the endothelium was investigated in the regulation of vascular tone in isolated porcine ophthalmic arteries suspended in a myograph system for isometric tension recording. In quiescent arteries with endothelium, the inhibitor of nitric oxide formation from L-arginine, L-NG-monomethyl arginine (L-NMMA), evoked endothelium-dependent contractions which were reversed by L-, but not D-arginine. In arteries contracted with serotonin, bradykinin evoked potent endothelium-dependent relaxations; L-NMMA markedly reduced the sensitivity but not the maximal response. Acetylcholine caused relaxations in preparations with endothelium but contractions in those without endothelium; L-NMMA prevented the relaxations and unmasked the contractions. The relaxations to the nitrovasodilator SIN-1 were more pronounced in preparations without than with endothelium. In quiescent arteries, quick stretching evoked endothelium-dependent contractions which were prevented by indomethacin. Endothelin-1, serotonin, and norepinephrine evoked concentration-dependent contractions with pD2 values of 8.1 +/- 0.1, 6.9 +/- 0.1, and 5.9 +/- 0.1, respectively. Removal of the endothelium markedly augmented the contractions induced by serotonin but not endothelin and norepinephrine. Thus, the endothelium profoundly affects vascular tone of the porcine ophthalmic artery. Endothelium-derived nitric oxide is released both under basal conditions and after stimulation with acetylcholine and bradykinin. It may play an important protective role in the circulation against vasospasm.(ABSTRACT TRUNCATED AT 250 WORDS)