MiR-18b suppresses high-glucose-induced proliferation in HRECs by targeting IGF-1/IGF1R signaling pathways
Introduction
Diabetic retinopathy (DR) is the most common micro-vascular complication of diabetes and one of the main reasons of blindness in adults in developed countries (Fong et al., 2004, Moss et al., 1998). Retinal changes in diabetes are thought to be initiated by sustained hyperglycemia, which has been recognized as a major risk factor responsible for the pathogenesis of diabetic complications and the development of micro-vascular diseases (Aronson, 2008, Das Evcimen and King, 2007). Retinal microvascular endothelial cell (REC) proliferation and neovascularization at the back of the eyes are hallmarks of proliferative diabetic retinopathy (PDR) (Fong et al., 2004, Moss et al., 1998), and dysfunction of the RECs contributes to the pathogenesis of micro-vascular complications in diabetes. Generally, numerous growth factors such as vascular endothelial growth factor (VEGF) are reported to increase in patients with PDR (Selim et al., 2010), in the retinas of diabetic rats (Jin et al., 2010, Yu et al., 2010) and in high-glucose-induced endothelial cells (Li et al., 2010, Sun et al., 2010). Thus, VEGF is considered to be a key modulator of the vascular alterations in DR pathology. Moreover, VEGF promotes the proliferation of RECs. The excessive secretion of retinal VEGF stimulates neovascularization or angiogenesis in the eyes of diabetic patients, whereas the specific inhibition of VEGF prevents these complications in animal models (Qaum et al., 2001). There are already several reports that show the expression of VEGF in the retinas of STZ-induced diabetic rats, and a number of preclinical and clinical studies have shown a very strong correlation between increased VEGF expression and the development of PDR in response to hyperglycemia or hypoxia (Aiello et al., 1994). However, even under tight glycemic control, the number of DR patients continues to grow, and the therapeutic options are very limited (Ioacara et al., 2009). Therefore, it is necessary to understand the molecular mechanisms underlying the altered expression of VEGF during the pathogenesis of DR and to develop effective measures for treating and preventing RECs proliferation and neovascularization.
MicroRNAs (miRNAs) are a well-recognized group of small (21–25 nucleotides), single stranded, noncoding RNA molecules that bind to the 3′ untranslated region (3′ UTR) of specific target transcript, regulating mRNA activity by inducing their degradation or suppressing their translation (Bartel, 2009, He and Hannon, 2004). Previous studies have shown that miRNAs are involved and play critical roles in a variety of biological processes in the endothelial cells, including their proliferation (Suarez et al., 2007), migration (Sabatel et al., 2011), and survival (Zhang et al., 2009). Recently, miRNAs are shown to be potential mediators of glucose responses (Patella and Rainaldi, 2012). Our previous work using miRNA expression profiling of RECs in normal and diabetic rats indicates that several miRNAs are significantly down-regulated in the RECs of diabetic rats compared with controls, suggesting that miRNAs might be important in the regulation of the pathogenesis of DR (Wu et al., 2012, Kovacs et al., 2011). However, the biological roles of miRNAs in endothelial dysfunction and DR induced by hyperglycemia are still largely unknown and require further investigation.
Insulin-like growth factor-1 (IGF-1) has mitogenic actions in several cells and tissues, including retina (Spoerri et al., 2003). It is reported that IGF-1 possesses a key role in retinal development and pathological states, such as diabetic retinopathy (Hellstrom et al., 2003). In addition, elevated vitreal and serum levels of IGF-1 have been reported to contribute to PDR in patients and also, are known to stimulate endothelial cell proliferation in vitro and in vivo (Payne et al., 2011). Moreover, IGF-1 can act directly as a mitogenic factor on endothelial cells and indirectly induce neovascularization by regulating the expression of potential factors including VEGF (Ruberte et al., 2004). Abundant evidences suggest that the IGF family is a multicomponent network of molecules in regulating pathophysiological growth processes in DR. For example, when RECs exposed to high glucose, IGF-1 binding to insulin growth factor-1 receptor (IGF-1R) activates the phosphatidylinositol 3 kinase (PI3K) or mitogen-activated protein kinase (MAPK) pathway, and then promotes the proliferation (DeBosch et al., 2002, Panjala and Steinle, 2011). IGF-1 participates in the pathogenesis of DR by activating the PI3K/Akt signaling pathway and inducing retinal VEGF expression (Poulaki et al., 2004). Though there is strong experimental and clinical evidence indicating that IGF-1 is associated with the development of DR, the regulated mechanism of IGF-1 in pathogenesis of DR is not well understood and remains controversial.
Based on our previous work of miRNA differential expression in retinal tissues from normal and STZ-induced diabetic rats (Wu et al., 2012), we identified that microRNA-18b (miR-18b) was significantly decreased in DR. To clarify whether miR-18b played a role in DR induced by hyperglycemia, we assessed the effects of gaining and/or losing miR-18b in primary HRECs that cultured in both normal and high glucose mediums. We found that the decrease of miR-18b in high-glucose-induced HRECs promoted cell proliferation and VEGF production. Notably, we demonstrated that miR-18b performed its function by targeting IGF-1, consequently reducing activation of the pathway downstream of IGF-1/IGF1R, suggesting a potential new therapeutic target in the treatment of DR.
Section snippets
HRECs culture and identification
HRECs were respectively isolated from the eyes of 4 donors (Table 1) who had traffic accidents and were treated at either Changhai hospital or Renji hospital as previously described (Chen et al., 2013). HRECs isolated methods were carried out in accordance with the approved guidelines. This study was approved by the Ethics Committee of the Changhai Hospital of the Second Military Medical University and Renji hospital. Retinas from four different donors were respectively harvested, cut into
High glucose promotes the proliferation of HRECs
Primary HRECs were isolated from adult humans as described in Section 2 of this paper. HRECs were respectively identified using morphological and immunofluorescence analyses followed with staining by endothelial markers. Primary HRECs from all four donors exhibited a cobblestone-like morphology and could be passaged without any observable morphological changes (Fig. 1A). Furthermore, cultured HRECs were positive for CD31, von Willebrand factor (vWF) and VE-cadherin, all of which were biological
Discussion
DR is a major complication of both type I and type II diabetes during the later phase and is present in almost every patient who has been diabetic for over 15 years (Ioacara et al., 2009). DR can be divided into two stages, non-proliferative and proliferative, according to its development. Abnormal proliferation of RECs is a characteristic of early proliferative DR. HG is considered to be essential for the development of DR. In this study, cultured HRECs under different high-glucose conditions
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was funded by the National Natural Science Foundation of China (30801268, 81371044 to Jinhui Wu), the Natural Science Foundation of Shanghai, China (13ZR1409600 to Lin Liu), the Project in Committee of Science of Shanghai (124119a9500 to Lin Liu), and the Project of National Health and Family Planning Commission of the PRC (PW2013D-1 to Lin Liu).
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