Effect and mechanism of the long noncoding RNA MALAT1 on retinal neovascularization in retinopathy of prematurity.

The most typical pathological manifestation of retinopathy of prematurity (ROP) is Retinal neovascularization (RNV). Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to mediate angiogenesis. Our experiment aimed to research the effect and mechanism of the MALAT1 on RNV in ROP. C57 mice was used to establish oxygen-introduced retinopathy (OIR), and divided into control, hyperoxia, hyperoxia control siRNA, and hyperoxia MALAT1 siRNA groups. It was shown that MALAT1 mRNA was high expressed in the retinas of OIR mice. Further studies revealed that after intravitreal injection of MALAT1 siRNA, the degree of retinopathy was significantly reduced compared with OIR group. In addition, the protein and mRNA expression levels of CCN1, AKT and VEGF were significantly decreased. This was accompanied by a decrease in inflammatory genes including IL-1β, IL-6, and TNF-α compared with the hyperoxia control siRNA mice. The result suggested that MALAT1 may be involved in the process of RNV in ROP and MALAT1 siRNA may be a promising agent for the treatment of ROP by inhibiting RNV.

Wang Y ，Wang X ，Wang YX ，Ma Y ，Di Y ... -  《-》

The mechanism of CCN1-enhanced retinal neovascularization in oxygen-induced retinopathy through PI3K/Akt-VEGF signaling pathway.

CCN1 (also called Cyr 61) is an extracellular matrix signaling molecule that has been implicated in neovascularization through its interactions with several endothelial integrin receptors. The roles of vascular endothelial growth factor (VEGF) in angiogenesis are well described. The aim of this study was to investigate the signal transduction mechanism of CCN1-PI3K/Akt-VEGF in retinopathy of prematurity (ROP), and the effects of CCN1 knockdown on ROP. The oxygen-induced retinopathy (OIR) model was established in C57BL/6J mice exposed to a high concentration of oxygen. Retinas were obtained from the normoxia, OIR, OIR control (treated with scramble siRNA) and OIR treated (with CCN1 siRNA) groups. Retinal neovascularization (RNV) was qualitatively analyzed with ADPase staining and quantitatively analyzed by counting neovascular endothelial cell nuclei at postnatal day 17 when RNV reached a peak. mRNA level and protein expression of CCN1, p-Akt, and VEGF were measured by real-time PCR and Western blotting, and located with immunohistochemistry. CCN1 depletion resulted in less neovascularization clock hour scores in the number of preretinal neovascular cells compared with the OIR treated group (1.28±0.83 versus 4.80±0.82; and 7.12±2.50 versus 23.25±2.35, respectively, both P<0.05). Furthermore, CCN1, p-Akt and VEGF mRNA, and protein were significantly expressed in the retina of the OIR and OIR control groups. Intravitreal injection of CCN1 siRNA significantly reduced PI3K/Akt-VEGF pathway expression of the OIR mouse model (all P<0.05). CCN1 siRNA significantly enhanced the avascular area and avascular diameter of OIR model (P<0.05). CCN1 siRNA decreased the levels of IL-1β, IL-6, and TNF-α significantly compared to the OIR group (P<0.05). These results suggest that CCN1 plays an important role in RNV via the PI3K/Akt-VEGF signaling pathway. CCN1 may be a potential target for the prevention and treatment of ROP.

Di Y ，Zhang Y ，Yang H ，Wang A ，Chen X ... -  《Drug Design Development and Therapy》

CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy.

Retinal neovascularization (RNV) is a characteristic pathological finding of retinopathy of prematurity (ROP). Cysteine-rich 61 [Cyr61, also known as CCN family member 1 (CCN1)] has been reported to mediate angiogenesis. The aim of the present study was to investigate the mechanisms of CCN1/Cyr61-phosphoinositide 3-kinase (PI3K)/AKT signaling in ROP. The contribution of CCN1 to human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis under hypoxic conditions was determined using a cell counting kit‑8 (CCK-8) and Annexin V/propidium iodide (PI) staining, respectively, as well as using siRNA targeting CCN1 (CCN1 siRNA). The cells exposed to hypoxia were also treated with the PI3K/AKT inhibitor, LY294002. In addition, mouse pups with oxygen-induced retinopathy (OIR) were administered an intravitreal injection of CCN1 siRNA. RNV was assessed by magnesium-activated adenosine diphosphate-ase (ADPase) staining. RT-qPCR, western blot analysis, immunofluorescence staining and immunohistochemistry were used to detect the distribution and expression of CCN1, PI3K and AKT. Exposure to hypoxia increased the neovascularization clock hour scores (from 1.23±0.49 to 5.60±0.73, P<0.05) and the number of preretinal neovascular cells, as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (all P<0.05). The injection of CCN1 siRNA decreased the neovascularization clock hour scores and the number of preretinal neovascular cells (1.53±0.72 vs. 4.76±1.04; 12.0±2.8 vs. 31.4±2.6, respectively, both P<0.05), as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (protein, -45.3, -22.5 and -28.4%; mRNA, -43.7, -58.7 and -42.9%, respectively, all P<0.05) compared to the administration of scrambled siRNA under hypoxic conditions. Treatment with LY294002 decreased the mRNA and protein expression levels of CCN1 in the cells exposed to hypoxia (both P<0.05). The administration of CCN1 siRNA resulted in less severe neovascularization in the eyes of the the mouse pups with OIR. Thus, out data suggest that CCN1 plays an important role in RNV in ROP, and may thus be a potential target for the prevention and treatment of ROP.

Di Y ，Zhang Y ，Nie Q ，Chen X ... -  《-》

Mini-peptide RPL41 attenuated retinal neovascularization by inducing degradation of ATF4 in oxygen-induced retinopathy mice.

Endoplasmic reticulum (ER) stress signaling is activated in retinal degeneration disease. Activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response (UPR), is a key element that maintains cell survival and proliferation in hypoxic conditions. Our previous studies showed that a small ribosomal protein L41 (RPL41) inhibits ATF4 by inducing its phosphorylation and degradation. In the present study, the effects of mini-peptide RPL41 on retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) mice was investigated. We induced OIR in C57BL/6 mice and obtained retinas from normoxia, OIR, OIR control (treated with PBS), and OIR treated (treated with RPL41) mice. Our results showed that ER stress signaling was activated and ATF4 was overexpressed in the retinas of OIR mice. After intravitreal injection of RPL41, the size of RNV and vaso-obliteration, and the number of preretinal neovascular cell nuclei in the retinas of OIR mice were significantly decreased. Western blot analysis and quantitative real-time polymerase chain reaction (qPCR) showed ATF4 and VEGF expression decreased after intravitreal injection of RPL41. Furthermore, the expression levels of inflammatory genes including TNF-α, IL-1β, and IL-6 were significantly decreased compared with the OIR control mice. In conclusion, RPL41 prevented pathologic neovascularization and exerted anti-inflammatory effects by degrading the important ER stress factor ATF4, thus, RPL41 could be a promising therapeutic agent for the treatment of neovascular eye diseases, especially retinopathy of prematurity (ROP).

Geng W ，Qin F ，Ren J ，Xiao S ，Wang A ... -  《-》

Differences between rat strains in models of retinopathy of prematurity.

Genetic factors appear to modulate the incidence and severity of retinopathy of prematurity (ROP). Different rat strains may be analogous to genetic differences across human ethnic groups. We investigated the incidence and severity of neovascularization (NV) in Brown Norway (BN) and Sprague Dawley (SD) rats in oxygen-induced retinopathy (OIR) and acidosis-induced retinopathy (AIR) models for ROP. We also studied whether there was a difference in retinal vascular endothelial growth factor (VEGF) mRNA levels in OIR animals. Newborn SD and BN rats (110 in both groups) were raised in standardized litters of ten (four OIR, twelve AIR, six non-gavaged room air controls). Beginning on day 1 of life, OIR litters were exposed to seven cycles of hyperoxia (80% O2, 20.5 h) and hypoxia (10% O2, 0.5 h) with a gradual return to 80% O2 over 3 h. This was followed by room air recovery for five days. OIR and OIR control rats were sacrificed on day 13 of life. AIR rats were gavaged twice daily with NH4Cl (10 mmol/kg) from day 2 to day 7 of life, or 15 mmol/kg twice daily on day 2 and then 10 mmol/kg twice daily from day 3 to day 7. AIR and AIR control rats were sacrificed on day 10 or day 13. Retinas from left eyes were dissected, ADPase stained and flatmounted. Presence and severity of NV (clock hours, 0 to 12) was scored by a masked observer. Right retinas from OIR and room air controls were processed for VEGF mRNA analysis. In OIR rats, the incidence of NV was higher in BN than SD rats (100% compared to 5%, p<0.0001). NV was more severe in BN rats (1 to 10 clock hours, median 7 clock hours compared to 0 to 1 clock hours, p=0.0001). In contrast, the incidence of NV in AIR rats was similar in BN and SD rats (4% compared to 0%, p=1.0) in the 10 mmol/kg study, and 18% compared to 0%, (p= 0.15) in the 15 mmol/kg study. Increased levels of retinal VEGF mRNA were found in BN OIR animals when compared to BN room air controls (1.4 fold increase) whereas retinal VEGF mRNA levels were similar between SD OIR and SD room air control animals. BN rats differ from SD rats in incidence and severity of NV in OIR. The findings in AIR were limited by the low incidence of NV and intolerance to higher multiple doses of NH4Cl. In OIR, the higher severity of NV was associated with higher retinal VEGF mRNA in BN OIR rats. Studies are warranted to investigate the genetic differences between Brown Norway and Sprague Dawley rats in models of ROP. These genetic studies may yield further clues into the pathogenesis of clinical ROP.

Floyd BN ，Leske DA ，Wren SM ，Mookadam M ，Fautsch MP ，Holmes JM ... -  《MOLECULAR VISION》