Expression of Angiogenic MicroRNAs in Endothelial Progenitor Cells From Type 1 Diabetic Patients With and Without Diabetic Retinopathy.

PURPOSE: MicroRNA (miR) expression in endothelial progenitor cells (EPCs) in type 1 diabetes (DM1) and its relation with different stages of diabetic retinopathy (DR) have not been reported to date. Our aim was to analyze miR-222, miR-221, and miR-126 expression in EPCs from DM1 patients with and without DR. METHODS: We included 41 patients with DR, 35 without DR, and 38 controls. Blood was collected for flow cytometry and EPC culture. Total RNA was extracted and purified and real-time quantitative PCR was performed for miR expression in cultured EPCs. Relative changes in miR expression were analyzed with the 2-ΔΔCT method. RESULTS: Circulating EPCs were reduced and miR-126 expression was increased in DM1 compared to controls (0.030 [interquartile range [IQR], 0.020-0.050] vs. 0.060 [IQR, 0.030-0.110], P = 0.004; 1.740 [IQR, 0.890-4.120] vs. 0.990 [IQR, 0.487-3.015], P = 0.047 respectively) without differences between patients with and without DR. Patients with DR had higher expression of miR-221 than those without DR (1.405 [IQR, 0.820-2.867] vs. 0.915 [IQR, 0.507-1.292], P = 0.019) without differences among degrees of DR. Circulating EPCs were reduced in patients on statins (0.010 [IQR, 0.010-0.050] vs. 0.045 [IQR, 0.020-0.087], P = 0.008), and miR-221 expression increased in patients on angiotensin-converting enzyme (ACE) inhibitors/angiotensin receptor blocker (ARB) II (1.430 [IQR, 1.160-2.705] vs. 1.000 [IQR, 0.520-1.330], P = 0.021) compared to those without treatment. MicroRNA-126 expression was associated with body mass index (BMI; ρ = -0.267, P = 0.026) and diastolic blood pressure (ρ = -0.267, P = 0.034). MicroRNA-221 was associated with triglyceride concentration (ρ = 0.296, P = 0.012). CONCLUSIONS: Circulating EPCs were reduced and miR-126 expression was increased in DM1 compared to controls. Patients with DR had higher expression of miR-221 than those without DR. The identification of biomarkers of diabetic complications might be useful for monitoring disease progression and potential therapeutic targets.

Retinal detachment and proliferative vitreoretinopathy: central retinal artery blood velocities, intraocular pressure, and endothelin 1.

PURPOSE: To analyze if endothelin 1 may have an effect on central retinal artery (CRA) blood flow velocities and intraocular pressure (IOP) in retinal detachment. METHODS: Using radioimmunoassay, immunoreactive endothelin 1 levels were tested in both plasma and subretinal fluid specimens from patients with retinal detachment, while only plasma specimens from healthy subjects were tested. Central retinal artery Doppler sonography parameters and IOP were measured in eyes with retinal detachment, with and without proliferative vitreoretinopathy, their respective healthy fellow eyes, and normal eyes. RESULTS: Retinal detachment eyes had lower CRA peak systolic velocity and end-diastolic velocity, lower IOP, and higher plasma immunoreactive endothelin 1 levels than normal eyes (P < 0.0001). Eyes with proliferative vitreoretinopathy had lower CRA peak systolic velocity and end-diastolic velocity, higher resistivity index, lower IOP, higher plasma immunoreactive endothelin 1 levels, and higher subretinal fluid immunoreactive endothelin 1 than eyes without proliferative vitreoretinopathy (P < 0.0001). A statistically significant linear correlation was found among CRA parameters, IOP, and subretinal fluid immunoreactive endothelin 1 measurements. CONCLUSION: Endothelin 1 has shown a close relationship with IOP and CRA blood flow changes associated to retinal detachment as well as with proliferative vitreoretinopathy complications.

Circulating endothelial progenitor cells are reduced in rat oxygen-induced retinopathy despite a retinal SDF-1/CXCR4 and VEGF proangiogenic response.

AIMS: The purpose of this study is to investigate circulating endothelial progenitor cells (EPCs) and the signaling pathways involved in their recruitment in the ischemic retina of the 50/10 rat model of oxygen-induced retinopathy (OIR). MAIN METHODS: Within 12h after birth, litters of Sprague-Dawley rats and their mothers were exposed to alternating oxygen concentrations, followed by a room air exposition, to induce OIR. Retinopathy was quantified by ADPase stain in flat-mounted retinas and pre-ILM nuclei count in retinal sections. Semiquantitative real-time PCR and immunofluorescence were assessed in retinas to study stromal cell-derived factor 1 (SDF-1), its receptor CXCR4 and vascular endothelial growth factor (VEGF) expression. Circulating EPCs were evaluated by flow cytometry in peripheral blood. KEY FINDINGS: Our results showed increased immunolabelling of SDF-1 in endothelial cells and strong expression of CXCR4 in Müller cells in OIR retinas as compared to control retinas. We found increased levels of CXCR4 and VEGF mRNA in OIR retinas, especially during the vascular attenuation stage. The number of circulating EPCs was decreased in OIR rats as compared to control rats. SIGNIFICANCE: The decrease in circulating EPCs could be implied in vessel growth arrest during normal retinal development in OIR rats, while pro-angionenic signals released by Müller cells in the hypoxic retina could drive pathological neovascularization in the ischemic retina. These data warrant further studies to investigate new therapeutic approaches for ROP.

Preoperative duration of retinal detachment and subretinal immunoreactive endothelin-1: repercussion on logarithmic visual acuity.

PURPOSE: To analyze whether preoperative duration of primary rhegmatogenous retinal detachment (RD) influences endothelin-1 (ET-1)--a vasoactive, mitogenic, and pro-apoptotic peptide- levels with repercussions on logarithmic (LogMAR) visual acuity (VA). METHODS: Prospective clinical cohort study on 66 healthy patients [33 with proliferative vitreoretinopathy (PVR) and 33 with no PVR] with unilateral RD candidates for scleral buckling (SB) surgery. Using radioimmunoassay, immunoreactive ET-1 (IR-ET-1) was tested in both plasma and subretinal fluid (SRF) of these RD patients. Pearson's correlations were evaluated between preoperative RD duration and each IR-ET-1 level (plasma, SRF and the difference SRF minus plasma) and also between both variables and the LogMAR VAs (preoperative, postoperative 8 months, and the difference: postoperative 8 months minus preoperative). RESULTS: PVR was associated with higher preoperative RD duration, higher LogMAR VA values (pre- and postoperative 8 months) and higher IR-ET-1 values (plasma, SRF and the difference: SRF minus plasma) than no-PVR IR-ET-1 levels (plasma and SRF) were only correlated (r = 0.462, p = 0.007; r = 0.397, p = 0.022 respectively) with preoperative RD duration in the no-PVR group. IR-ET-1 values (plasma, SRF and the difference:SRF minus plasma) showed statistically significant correlations with pre- and with postoperative 8 months LogMAR VAs in no-PVR and with postoperative 8 months LogMAR VA and LogMAR VA difference in PVR The highest correlation between IR-ET-1 levels and LogMAR VAs was found between SRF IR-ET-1 and postoperative 8 months LogMAR VA in PVR (cases with macula-on) (r = 0.956, p < 0.0001). CONCLUSIONS: Preoperative RD duration showed statistically significant positive correlations with pre- and with postoperative 8 months LogMAR VAs in both the no-PVR and the PVR groups and with IR-ET-1 measurements (plasma and SRF: lower correlations) only in the no-PVR group. These findings support the idea of doing primary and prompt vitrectomy for RD and perhaps using coadjutant pharmacologic therapy in order to improve visual results.

Apoptosis in the trabecular meshwork of glaucomatous patients.

We established and validated an in toto method to perform TdT-mediated dUTP nick end labeling to study apoptosis in human trabecular meshwork tissue obtained during trabeculectomy in glaucoma patients. In specimens from patients with primary open-angle glaucoma and primary angle-closure glaucoma, we detected a tendency for more apoptotic cells to accumulate in patients with primary open-angle glaucoma. The utility of this method to study apoptosis in the trabecular meshwork is discussed, as well as its application as a tool in biologic samples.

Expression of nitrotyrosine and oxidative consequences in the trabecular meshwork of patients with primary open-angle glaucoma.

PURPOSE: To evaluate the possible correlation between the visual field defects in patients with primary open-angle glaucoma (POAG) and the expression and enzymatic activity of nitric oxide synthase (NOS) isoenzymes and nitrotyrosine in trabecular meshwork (TM) samples. METHODS: TM specimens were collected from 146 patients with POAG by using standard filtration surgery. Visual field defects were evaluated by perimetry. Expression of endothelial (e)NOS and inducible (i)NOS were evaluated by quantitative RT-PCR. Constitutive (Ca2+-dependent) and iNOS (Ca2+-independent) activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. In four TM specimens from POAG-affected eyes and in three human donor control eyes, 3-nitrotyrosine was localized by immunohistochemistry. The marker of lipid peroxidation malondialdehyde (MDA) was measured by the thiobarbituric acid test in samples of aqueous humor (AH) from 48 patients with either POAG or cataracts. RESULTS: The results showed an upregulation of iNOS and a downregulation of calcium-dependent NOS correlated with visual field defects. Expression and activity of iNOS increased in parallel with visual field defects. However, constitutive activity decreased as the visual field defect increased. Nitrotyrosine was observed only in the cells of the TM specimens from eyes with severe POAG. CONCLUSIONS: The increased expression and activity of iNOS in the TM of patients with POAG are proportional to the visual field defect and could lead to the increased of nitrotyrosine levels which may serve as marker of oxidative stress in the progression of cell death of the TM in POAG.

Retinal detachment and proliferative vitreoretinopathy: ophthalmic artery blood velocities, intraocular pressure, and endothelin-1.

PURPOSE: To analyze if endothelin-1 (ET-1) may have an effect on ophthalmic artery (OA) blood flow velocities and intraocular pressure (IOP) in retinal detachment (RD). METHODS: Using radioimmunoassay, immunoreactive (IR) ET-1 levels were tested in both plasma and subretinal fluid (SRF) specimens from patients with RD, while only plasma specimens from normal (healthy) subjects were tested. OA Doppler sonography parameters and IOP were measured in eyes with RD, with and without proliferative vitreoretinopathy (PVR), their respective healthy fellow eyes, and normal eyes. RESULTS: RD eyes had lower OA peak systolic velocity (PSV) and end diastolic velocity (EDV), higher resistivity index (RI), lower IOP, and higher plasma IR ET-1 levels than normal eyes (P < 0.0001). Eyes with PVR had lower OA PSV and EDV, higher RI, lower IOP, higher plasma IR ET-1 levels, and higher SRF IR ET-1 than eyes without PVR (P < 0.0001). A statistically significant linear correlation was found among OA parameters, IOP, and SRF IR ET-1 measurements. CONCLUSIONS: Decreased OA blood flow velocities may explain lower IOP found in RD patients, and ET-1 levels may be responsible for both measurements.

Immunoreactive endothelin-1 in the vitreous humor and epiretinal membranes of patients with proliferative diabetic retinopathy.

PURPOSE: To investigate the potential role of endothelin-1 (ET-1), a potent vasoconstrictor with mitogenic properties, in the pathogenesis of proliferative diabetic retinopathy (PDR). METHODS: Plasma and vitreous samples were collected from normal patients (controls; n = 25), diabetic patients with PDR (n = 25), and diabetic patients with non-PDR (n = 25). The patients had to have epiretinal membranes (ERMs) or other ocular conditions that made them candidates for vitrectomy. Immunoreactive ET-1 (IR-ET-1) was assayed in plasma and vitreous samples by radioimmunoassay. IR-ET-1 was immunohistochemically localized in ERMs. Expression of endothelin receptors A (ETA) and B (ETB) was confirmed by reverse transcription-polymerase chain reaction analysis. RESULTS: IR-ET-1 levels in plasma and vitreous samples from diabetic patients were higher (P < 0.0001) than those in samples from the control group. The levels for patients with PDR were even higher (P < 0.0001) than those for patients with non-PDR. Eyes with ERMs in the PDR group had the highest vitreous IR-ET-1 levels (14.67 +/- 0.67 pg/mL). IR-ET-1 was localized in the cellular and stromal components of ERMs in diabetic and nondiabetic patients. Furthermore, the ETA and ETB receptors were expressed in both diabetic and nondiabetic ERMs. CONCLUSIONS: Diabetic patients with PDR and ERMs had the highest plasma and vitreous IR-ET-1 levels. ET-1 and its ETA and ETB receptors were present in ERMs. These data suggest that ET-1 is involved in diabetic vitreoretinal disease.

Immunoreactive ET-1 in the vitreous humor and epiretinal membranes of patients with proliferative vitreoretinopathy.

PURPOSE: Endothelin one (ET-1) is a vasomodulator peptide that plays a role on ocular blood flow, glial proliferation, and collagen matrix contraction by retinal pigmented epithelial (RPE) cells. Both glial and RPE cells have been involved in the formation of epiretinal membranes (ERMs). This investigation was conducted to determine whether ET-1 may be associated with ERMs, either idiopathic (IERMs) or from proliferative vitreoretinopathy (PVR). METHODS: Plasma and vitreous samples were collected from patients classified by the presence of PVR membranes, retinal detachment (RD), and other ocular conditions, such as IERMs, that made the patients candidates for vitrectomy. Immunoreactive endothelin one (IR-ET-1) was tested in plasma and vitreous by radioimmunoassay. Immunoreactive-ET-1 was localized in IERMs and PVR membranes immunohistochemically. Expression of endothelin receptors A (ETA) and B (ETB) was confirmed by reverse transcription-polymerase chain reaction. RESULTS: IR-ET-1 levels in plasma and vitreous were higher in patients with PVR and in patients with RD than in those of the control group. Eyes with IERMs also showed higher IR-ET-1 levels than the control group cases. IR-ET-1 levels in eyes with PVR were higher than those in eyes with IERMs. IR-ET-1 levels in eyes with RD were also higher than those of eyes with IERMs. Immunoreactive ET-1 was localized in the cellular and stromal components of both IERMs and PVR membranes. Furthermore, ETA and ETB receptors were expressed in both IERMs and PVR membranes. CONCLUSIONS: IR-ET-1 in human vitreous is elevated in PVR, RD, and IERMs. ET-1 and its receptors ETA and ETB are present in epiretinal tissue of both idiopathic and PVR membranes. These data suggest an involvement of ET-1 in retinal disease.

Downregulation of the atrial natriuretic peptide/natriuretic peptide receptor-C system in the early stages of diabetic retinopathy in the rat.

PURPOSE: Atrial natriuretic peptide (ANP) is a known vascular antipermeability and antiangiogenic factor, but its possible alteration during the early stages of diabetic retinopathy has not yet been explored. The present study sought to investigate the expression of ANP and its receptors using a model of streptozotocin (STZ) induced diabetes in the rat. METHODS: Diabetes was induced in male Wistar rats by an intraperitoneal injection of STZ. Age matched animals served as control. One and 3 months after the onset of diabetes, the expression of ANP mRNA and that of its receptors (NPRA, NPRB, NPRC) and the immunoreactive ANP was quantified in retinal tissue by quantitative real time reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay, respectively. The locations of ANP and glial fibrillary acidic protein (GFAP) in normal and diabetic retinas were also established by immunohistochemistry. RESULTS: No alteration in the gene expression of the retinal natriuretic peptide system was noted after 1 month of diabetes. However, 3 months after the onset of diabetes, significantly diminished ANP and NPRC mRNA levels were detected in the retina of diabetic rats compared to controls, while NPRA, NPRB mRNA levels remained unchanged. At this time point, retinal ANP concentrations were significantly diminished in the diabetic rats compared to control rats. However, at 1 month retinal ANP concentrations in diabetic retina were similar to control rats. Diabetes caused the downregulation of ANP protein expression in the layers of the retina at 3 months after the induction of diabetes. ANP immunoreactivity was detected in the cell bodies of the astrocytes and in their processes enveloping vessels. CONCLUSIONS: The downregulation of ANP and NPRC in retinas of diabetic rats suggests a role for this peptide in experimental diabetic retinopathy. Further studies should address the possible involvement of the ANP/NPRC system in the pathophysiology of diabetic retinopathy.

Atrial natriuretic peptide in the vitreous humor and epiretinal membranes of patients with proliferative diabetic retinopathy.

PURPOSE: Atrial natriuretic peptide (ANP) has been recently described as an endogenous inhibitor of the synthesis and angiogenic action of vascular endothelial growth factor (VEGF). Given VEGF's key role in promoting neovascularization in proliferative diabetic retinopathy (PDR), this study was designed to evaluate the possibility that ANP could be involved in the neovascular and fibrotic complications of PDR. METHODS: We determined ANP by radioimmunoassay in plasma and vitreous humor samples collected from diabetic patients with and without PDR and from non-diabetic subjects. ANP was also immunohistochemically localized in the epiretinal membranes of patients with PDR. RESULTS: Vitreous ANP concentrations were significantly higher in patients with active PDR compared to patients with quiescent PDR, diabetes without PDR or controls <0.05. Significant differences were also observed between vitreous ANP levels in diabetic patients without PDR and control subjects. There was no significant correlation between serum and vitreous ANP levels in any of the patient groups. ANP was detected in the fibrovascular epiretinal tissue of patients with PDR. CONCLUSIONS: Diabetic patients with active neovascularization have significantly higher levels of ANP in the vitreous humor than those without active PDR. Diabetic patients without PDR were also found to have significantly higher vitreous ANP levels than non-diabetic patients. Since plasma and vitreous ANP concentrations were found to be unrelated, we suggest intraocular ANP synthesis and/or an increase in the release of ANP into the vitreous, as opposed to diffusion from the blood, as the main factors contributing to the high vitreous ANP levels observed in diabetic patients. In the fibrovascular epiretinal tissue of these patients, ANP was found to be localized in vascular, glial, fibroblast-like and retinal pigment epithelium cells. Our findings suggest a role for ANP in PDR.

Natriuretic peptide system in the human retina.

PURPOSE: The natriuretic peptide (NP) family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Natriuretic peptides are known to inhibit vascular cell growth and regulate vessel tone. There is also much evidence to suggest they modulate vascular permeability and angiogenesis, as well as regulating aqueous humor production in the eye. All these data indicate that the natriuretic peptide system might be involved in the development of diabetic retinopathy and glaucoma. Given the expression pattern of natriuretic peptides (NPs) and their receptors, natriuretic peptide receptor A (NPRA), natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC) in the human retina has not yet been established, the present study was designed to determine ANP, BNP and CNP gene expression and localize the mature peptides in this tissue. The expression pattern of the genes encoding the different NP receptor subtypes was also examined. METHODS: Eyes (n=10) from human donors with no history of eye disease were fixed and processed for routine paraffin embedding. The cellular location of the NPs was established by immunohistochemistry. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of NP and NP receptor genes in neural retinas obtained from the contralateral eyes. RESULTS: Immunohistochemistry revealed the presence of NPs in the neural retina and retinal pigment epithelium. Positive NP immunostaining was observed within the astrocytes and in their processes enveloping vessels. In the anterior portion of the optic nerve, NPs were intensely labeled in neural bundles. We were able to detect NP gene expression in the human retina. The levels of NP receptor-encoding transcripts detected indicated no significant differential expression of genes coding for the different receptor subtypes. CONCLUSIONS: Our finding that NP receptor transcripts are expressed along with ANP, BNP, and CNP mRNA in the human retina provides evidence for a local system in this tissue. The expression of NPs in neural retinal, glial, and vascular elements of the normal adult retina suggests a role for these peptides in maintaining both the neural and vascular integrity of the mature retina.

Localization of endothelin-1 mRNA expression and immunoreactivity in the anterior segment of human eye: expression of ETA and ETB receptors.

PURPOSE: Endothelin-1 (ET-1), a potent vasoactive peptide, is an important regulator of intraocular pressure. Actually, there is evidence of a role for ET-1 in the pathogenesis of glaucoma. However, the expression pattern of ET-1 and its receptors, ETA and ETB, in the anterior segment of human eye are not known. In the current study, we have examined the expression and distribution of ET-1 as well as the expression profile of ETA and ETB genes in the iris, ciliary muscle, and ciliary processes of human eyes. METHODS: Six normal human eyes with no history of eye diseases were fixed, embedded in paraffin and sectioned. Cellular localization of ET-1 was identified by in situ hybridization and immunohistochemistry. Iris, ciliary processes, and ciliary muscles were dissected from six normal human eyes and quantitative real time RT-PCR was used to quantify the expression of ETA and ETB. RESULTS: In situ hybridization revealed the presence of ET-1 transcripts in the iris, nonpigmented epithelial ciliary cells, and ciliary muscle. Immunohistochemical studies showed that ET-1-like immunoreactivity appeared in the same regions where ET-1 mRNA was expressed as well as in trabecular cells, inner and outer endothelial cells lining Schlemm's canal, corneal epithelial, and limbus cells. Quantitative real time RT-PCR demonstrated that the expression of ETA and ETB receptors is greatest in the iris, followed by ciliary muscle and ciliary processes. CONCLUSIONS: ET-1 and its receptors ETA and ETB are constitutively expressed in the anterior segment of human eye. These results indicate that ET-1 may play a physiological role in the regulation of intraocular pressure through its ETA and ETB receptors in human eye. In addition, ET-1 present in corneal epithelium and limbus may function in regulating cell proliferation and/or differentiation.