Upregulated miR-194-5p suppresses retinal microvascular endothelial cell dysfunction and mitigates the symptoms of hypertensive retinopathy in mice by targeting SOX17 and VEGF signaling.

BACKGROUND: Hypertensive retinopathy (HR) is a retinal disease that may lead to vision loss and blindness. Sex-determining region Y (SRY)-box (SOX) family transcription factors have been reported to be involved in HR development. In this study, the role and upstream mechanism of SRY-box transcription factor 17 (SOX17) in HR pathogenesis were investigated. METHODS: SOX17 and miR-194-5p levels in Angiotensin II (Ang II)-stimulated human retinal microvascular endothelial cells (HRMECs) and retinas of mice were detected by RT-qPCR. SOX17 protein level as well as levels of tight junction proteins and vascular endothelial growth factor (VEGF) signaling-associated proteins were quantified by western blotting. Tube formation assays were performed to evaluate angiogenesis in HRMECs. The structure of mouse retinal tissues was observed by H&E staining. The interaction between miR-194-5p and SOX17 was confirmed by a luciferase reporter assay. RESULTS: SOX17 was upregulated in HRMECs treated with Ang II. SOX17 knockdown inhibited angiogenesis in Ang II-stimulated HRMECs and increased tight junction protein levels. Mechanically, SOX17 was targeted by miR-194-5p. Moreover, miR-194-5p upregulation restrained angiogenesis and increased tight junction protein levels in Ang II-treated HRMECs, and the effect was reversed by SOX17 overexpression. MiR-194-5p elevation inactivated VEGF signaling via targeting SOX17. miR-194-5p alleviated pathological symptoms of HR in Ang II-treated mice, and its expression was negatively correlated with SOX17 expression in the retinas of model mice. CONCLUSIONS: MiR-194-5p upregulation suppressed Ang II-stimulated HRMEC dysfunction and mitigates the symptoms of HR in mice by regulating the SOX17/VEGF signaling.

Preeclampsia as predisposing factor for hypertensive retinopathy: Participation by the RAAS and angiogenic factors.

Preeclampsia (PE) is a hypertensive complication of pregnancy. Its cause is still unknown and it could be a risk factor for future ophthalmic problems. Retinal vascular bed alterations have been described as a consequence of PE, suggesting a retinopathy. Factors related to angiogenesis and vascular permeability, such as vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) or components of the renin angiotensin aldosterone system (RAAS), prorrenin/renin receptor ((P)RR) and angiotensin II type I receptor (AT1R) have been located in the retina, participating in other retinopathies, but it is unknown if they could participate in PE. Our aim was to elucidate whether VEGF, PEDF, (P)RR and AT1R could be modified during PE and during hypertension induced in rats with a history of PE. We used female Wistar rats and subrrenal aortic coarctation to induce PE, and after delivery, we induced a second hit by Nω-nitro-L-arginine methyl ester (L-NAME) administration. We measured blood pressure, proteinuria and pups development. In both models, eye fundal exploration and immunoblot for VEGF, PEDF, (P)RR and AT1R were performed. We found that the development of hypertension occurred faster in previously PE rats than in normal animals. VEGF, PEDF, (P)RR and AT1R were increased in PE, but in L-NAME-induced hypertension only (P)RR and AT1R were altered. Eye fundal data indicated that PE induced a level I retinopathy, but L-NAME induced a faster and more severe retinopathy in previously PE animals compared to previously normal pregnancy rats. These results indicate that PE predisposes to development of a faster and more severe retinopathy after a second hit. They also suggest that VEGF and PEDF seem to participate only in PE retinopathy, but in both models, RAAS components seem to have a more critical participation.

Dll4 Suppresses Transcytosis for Arterial Blood-Retinal Barrier Homeostasis.

RATIONALE: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown. OBJECTIVE: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage. METHODS AND RESULTS: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage. CONCLUSIONS: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.

Chronic NOS Inhibition Affects Oxidative State and Antioxidant Response Differently in the Kidneys of Young Normotensive and Hypertensive Rats.

Deficiency of nitric oxide (NO) and oxidative stress can be a cause, a consequence, or, more often, a potentiating factor for hypertension and hypertensive renal disease. Both NO and superoxide anions are radical molecules that interact with each other, leading to oxidative damage of such organs as the kidney. In the present study, we investigated the effect of chronic-specific (neuronal NOS inhibition) and nonspecific NOS inhibition on the oxidative state and antioxidant response and associated oxidative damage of the kidney of young normotensive and hypertensive rats. Young male normotensive Wistar rats (WRs, age 4 weeks) and spontaneously hypertensive rats (SHRs, age 4 weeks) were divided into three groups for each strain by the type of administered compounds. The first group was treated with 7-nitroindazole (WR+7-NI; SHR+7-NI), the second group was treated with N(G)-nitro-L-arginine-methyl ester (WR+L-NAME; SHR+L-NAME), and the control group was treated with pure drinking water (WR; SHR) continuously for up to 6 weeks. Systolic blood pressure increased in WR+L-NAME after the first week of administration and increased slightly in SHR+L-NAME in the third week of treatment. 7-NI had no effect on blood pressure. While total NOS activity was not affected by chronic NOS inhibition in any of the WR groups, it was attenuated in SHR+7-NI and SHR+L-NAME. Nitration of proteins (3-nitrotyrosine expression) was significantly reduced in WR+7NI but not in WR+L-NAME and increased in SHR+7-NI and SHR+L-NAME. Immunoblotting analysis of SOD isoforms showed decreased SOD2 and SOD3 expressions in both WR+7-NI and WR+L-NAME followed by increased SOD activity in WR+L-NAME. Conversely, increased expression of SOD2 and SOD3 was observed in SHR+L-NAME and SHR+7-NI, respectively. SOD1 expression and total activity of SOD did not change in the SHR groups. Our results show that the antioxidant defense system plays an important role in maintaining the oxidative state during NO deficiency. While the functioning antioxidant system seeks to balance the oxidation state in the renal cortex of normotensive WRs, the impaired antioxidant activity leads to the development of oxidative damage of proteins in the kidney induced by peroxynitrite in SHRs.

Vascular Interstitial Cells in Retinal Arteriolar Annuli Are Altered During Hypertension.

Purpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, ß-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. Methods: Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Results: The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed ß-actin, nestin, and CD44, but they did not express CD31 and α-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-ß). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. Conclusions: A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease.

Ischemic Retinopathies: Oxidative Stress and Inflammation.

Ischemic retinopathies (IRs), such as retinopathy of prematurity (ROP), diabetic retinopathy (DR), and (in many cases) age-related macular degeneration (AMD), are ocular disorders characterized by an initial phase of microvascular changes that results in ischemia, followed by a second phase of abnormal neovascularization that may culminate into retinal detachment and blindness. IRs are complex retinal conditions in which several factors play a key role during the development of the different pathological stages of the disease. Increasing evidence reveals that oxidative stress and inflammatory processes are important contributors to the pathogenesis of IRs. Despite the beneficial effects of the photocoagulation and anti-VEGF therapy during neovascularization phase, the need to identify novel targets to prevent initial phases of these ocular pathologies is still needed. In this review, we provide an update on the involvement of oxidative stress and inflammation in the progression of IRs and address some therapeutic interventions by using antioxidants and anti-inflammatory agents.

The association between endothelial dysfunction and hypertensive retinopathy in essential hypertension.

BACKGROUND: Endothelial dysfunction, which is characterized by an imbalance between relaxing and contracting factors, procoagulant and anticoagulant substances, and between pro-inflammatory mediators, may play a particularly significant role in the pathogenesis of atherosclerosis. Numerous experimental and clinical reports suggest that a high von Willebrand factor (vWF) level reflects endothelial damage or dysfunction. Hypertensive retinopathy (HR) is a condition characterized by a spectrum of retinal vascular signs in people with elevated blood pressure. The pathophysiological mechanism of HR is not completely understood. Elevated blood pressure alone does not fully account for the extent of retinopathy. Endothelial dysfunction and mechanisms known to be involved in vascular lesions may be involved in the pathophysiological mechanism of HR. Therefore, this study was designed to answer the following questions: (i) Do vWf levels change in HR? and (ii) Is there any relation between degree of HR and vWf levels? MATERIAL AND METHODS: This study included 80 hypertensive patients with HR. Group 1 comprised 40 patients with grade I HR, and group 2 comprised 40 patients with grade II HR. We selected 40 healthy subjects for the control group. RESULTS: Level of vWf in group 2 was significantly higher than in group 1 (p=0.017) and the control group (p<0.001), and it was also higher in group 1 than in the control group (p<0.005). Also, vWf showed positive correlation with degree of HR in the hypertensive group (r=0.284, p=0.009). CONCLUSIONS: Our study suggests that endothelial dysfunction, which is a mechanism known to be involved in vascular lesions, may promote the development of HR.

Ghrelin in ocular pathophysiology: from the anterior to the posterior segment.

Ghrelin is a 28 amino acid acylated peptide produced in several organs that binds the growth hormone secretagogues receptor type 1a (GHSR-1a). It acts over a wide range of systems, e.g. the endocrine, cardiovascular, musculoskeletal and immune systems and the eye. The aim of this work is to review the physiologic and pathologic implications of the ghrelin-GHSR-1a in the eye. A systematic revision of studies published between 2000 and 2013 in English, Spanish or Portuguese in MEDLINE, EMBASE and Scopus was performed. Search words used included: ghrelin, GHSR-1a, ocular production, iris muscular kinetics, ciliary body, glaucoma, retinopathy and uvea. The production of ghrelin by the ocular tissue has been detected both in the anterior and posterior segments, as well as the presence of GHSR-1a. This peptide promotes the relaxation of the iris sphincter and dilator muscles, being this effect independent from GHSR-1a and dependent on prostaglandins release in the first case and dependent on GHSR-1a in the second. Regarding ocular pathology, ghrelin levels in the aqueous humor appear to be decreased in individuals with glaucoma. Moreover, ghrelin has been shown to decrease the intraocular pressure in animal models of ocular hypertension through GHSR-1a. In the posterior segment, the ghrelin-GHSR-1a system interferes with the development of oxygen-induced retinopathy, being protective in the vaso-obliterative phase and deleterious in the vaso-proliferative stage of the disease. Thus, the ghrelin-GHSR-1a system presents as a possible local regulatory mechanism in the eye, with pathophysiological implications, constituting a target for future clinical and therapeutic research and interventions.