November 2014
Volume 55, Issue 11
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Retina  |   November 2014
Mechanisms of Endothelial to Mesenchymal Transition in the Retina in Diabetes
Author Affiliations & Notes
  • Yanan Cao
    Department of Pathology, Western University, London, Ontario, Canada
    Medical Research Center, Mudanjiang Medical University, Mudanjiang, Heilongjiang, People's Republic of China
  • Biao Feng
    Department of Pathology, Western University, London, Ontario, Canada
  • Shali Chen
    Department of Pathology, Western University, London, Ontario, Canada
  • Yanhui Chu
    Medical Research Center, Mudanjiang Medical University, Mudanjiang, Heilongjiang, People's Republic of China
  • Subrata Chakrabarti
    Department of Pathology, Western University, London, Ontario, Canada
  • Correspondence: Subrata Chakrabarti, Department of Pathology, Western University, Room 4033, Dental Sciences Building, London, ON, Canada; Subrata.Chakrabarti@lhsc.on.ca. Yanhui Chu, Medical Research Center, Mudanjiang Medical University, Mudanjiang, Heilongjiang, PR China; yanhui_chu@sina.com
Investigative Ophthalmology & Visual Science November 2014, Vol.55, 7321-7331. doi:https://doi.org/10.1167/iovs.14-15167
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      Yanan Cao, Biao Feng, Shali Chen, Yanhui Chu, Subrata Chakrabarti; Mechanisms of Endothelial to Mesenchymal Transition in the Retina in Diabetes. Invest. Ophthalmol. Vis. Sci. 2014;55(11):7321-7331. https://doi.org/10.1167/iovs.14-15167.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Hyperglycemia-induced endothelial damage is a key pathogenetic factor in diabetic retinopathy. Endothelial damage may lead to phenotypic changes in the cells manifested by reduced expression of endothelial markers and increased expression of mesenchymal markers, termed endothelial to mesenchymal transition (EndMT). We investigated mechanisms of such changes in the retinal endothelial cells and in the retina of diabetic animals.

Methods.: Human retinal microvascular endothelial cells were grown in medium containing 5 mM glucose or 25 mM glucose with or without TGFβ1 peptide or TGFβ1 inhibitor or miR-200b mimic transfection. Messenger RNA levels of endothelial markers, mesenchymal markers, and specific signaling molecules of TGFβ pathway were quantified. Expression of miR-200b and histone acetylator p300 was quantified. Retinal tissues from mice with endothelial-specific overexpression of miR-200b, with or without streptozotocin-induced diabetes, were similarly examined.

Results.: Glucose caused decreased expression of mRNA and protein levels of endothelial markers and increased expression of mesenchymal markers with reduced miR-200b. A glucose-like effect was seen using TGFβ1 peptide. Such changes were mediated by miR-200b and p300. In the retinas of wild-type diabetic mice, EndMT was observed, which was prevented in miR-200b transgenic mice with diabetes.

Conclusions.: These data indicate glucose-induced EndMT in vitro and in vivo is possibly mediated through TGFβ and regulated by miR-200b and p300.

Introduction
Diabetic retinopathy is the leading cause of blindness worldwide.1 Endothelial dysfunction is the predominant factor in all chronic diabetic complications including diabetic retinopathy.2 Endothelium constitutes a thin monolayer covering the inner surface of the blood vessels. Endothelial dysfunction reduces cellular capacity to maintain homeostasis, which ultimately leads to the development of vascular disease.3 Hyperglycemia-induced increased production of extracellular matrix (ECM) proteins is a characteristic feature of endothelial dysfunction in diabetes.46 Recently, similar to the process of epithelial to mesenchymal transition (EMT) in malignancies,7 endothelial phenotypic changes, that is, endothelial to mesenchymal transition (EndMT), has been demonstrated during increased ECM protein production and fibrosis in several chronic diseases.812 Such process also may be of importance in some chronic diabetic complications.8,9,11,12 Endothelial to mesenchymal transition is characterized by decreased expression of endothelial cell markers and functions together with increased expression of mesenchymal markers and functions.8,10,13 Endothelial to mesenchymal transition was first described during embryonic heart development in which mesenchymal cells of the endocardial cushion were shown to arise from endothelial cells of the endocardium.12,14 Such changes have recently been demonstrated in the glucose exposed human aortic endothelial cells and in the kidneys and heart in diabetes.9,11,12 However, it is not known whether such processes occur in the retina in diabetes, and what the regulatory mechanisms of such events are. 
Similar to EMT, TGFβ is possibly a key inducer of EndMT.15 In a Smad-dependent pathway, the TGFβR2-ALK5 complex recruits and phosphorylates Smad2 and Smad3 (R-Smad). Phosphorylated R-Smad complexes with Smad4 (Co-Smad) and translocates into the nucleus. Mechanistically, the Snail family of transcription repressors are also involved.10,13 In mouse embryonic stem cell–derived endothelial cells, TGFβ-induced EndMT was mediated through activation of Smad, MEK, PI3K, and p38 MAPK, whereas knockdown of Snail1 blocked EndMT.16,17 Snail also represses the expression of endothelial markers VE-cadherin1820 and CD31,18,19 and promotes the mesenchymal markers fibroblast-specific protein 1 (FSP1),21 fibronectin (FN),22,23 and vimentin (VIM).23 
Recently, it has been shown that upregulation of the transcription coactivator p300 is an important event in EndMT. The same study also showed that alteration of several microRNAs (miRNAs) in EndMT in nondiabetic etiology.24,25 We have previously shown that molecular events such as TFGβ-mediated increased ECM protein production26 and p300-mediated histone acetylation occur in multiple tissues including the retina in diabetes.27 We have also showed alterations of several miRNAs in the retina and in other tissues in diabetes.5,2831 
Several if not all of the aforesaid transcripts are regulated by specific miRNAs. MicroRNAs are conserved RNA sequences composed of approximately 22 nucleotides, and are involved in epigenetic regulation of eukaryotic gene expression. Aberrant expressions of specific miRNAs are associated with various the pathologic conditions.24,32,33 However, the expression levels of miRNAs and their roles in EndMT are unclear. We have previously demonstrated a role of microRNA-200b (miR-200b) in mediating early molecular changes in the retina in diabetes.28 The miR-200b also has a regulatory role on EMT in other systems.34,35 Interestingly, we have previously demonstrated that both VEGF and p300 are targets of miR-200b.28 Furthermore, Smad2 and Snail1, mediators of the TGFβ pathway, are also targets of miR-200b.36 However, the role of miR-200b in EndMT has not been elucidated. 
The purpose of this study was to examine whether EndMT is of importance in the pathogenesis of early molecular events in the retina in diabetes and to determine whether this process is mediated through TGFβ. We further examined the role of histone acetylator p300 and whether miR-200b regulates these processes. We used human retinal microvascular endothelial cells (HRMECs) and diabetic mice. To further examine the molecular interactions in vivo, we generated endothelial-specific transgenic mice overexpressing miR-200b and used them with or without chemically induced diabetes. 
Methods
Cell Culture
Human retinal microvascular endothelial cells (Olaf Pharmaceuticals, Worcester, MA, USA) were grown in endothelial basal media-2, supplemented with 10% fetal bovine serum as previously described.5,29 The cells were plated at a density of 1 × 105 cells/mL. Following 24 hours of serum-free media incubation, HRMECs were incubated with various levels of D-glucose (5[normal glucose (NG)] and 25[high glucose (HG)] mM glucose) or NG with TGFβ1 protein (5 ng/mL) (R&D System, Minneapolis, MN, USA) (NG+TGFβ1), HG with TGFβ1 inhibitor SB431542 (10 μmol/L) (HG+SB431542) or with 25 mM L-glucose (osmotic control ) for variable durations. Each experiment was performed in triplicates with three or more samples each time. 
Human retinal microvascular endothelial cells were transfected with miRIDIAN miR-200b mimic (20 nmol/L; Dharmacon, Chicago, IL, USA) using the transfection reagent Lipofectamine2000 (Invitrogen, Burlington, ON, Canada). The miRIDIAN scrambled miR control was used in parallel. The cells were recovered in full medium for 24 hours. In some experiments, after the 24-hour serum-free condition, the miR-200b mimic transfected HRMECs were treated with TGFβ1 or HG. Reagents for all experiments were obtained from Sigma (Oakville, ON, Canada) unless otherwise specified. 
Animal Studies
All procedures were conducted in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. All experiments were approved by the Western University Council on Animal Care committee. The experiments conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publ. no. 85-23, revised1996). 
To investigate the role of EndMT in diabetic animals, male C57BL/6 mice weighing 23 to 26 g were obtained from Charles River (Montreal, QC, Canada). They were randomly divided into control and diabetic groups. As previously described, diabetes was induced by three intraperitoneal injections of streptozotocin (STZ) (70 mg/kg in citrate buffer, pH 5.6) on alternate days.37 Controls were injected with same volume of citrate buffer. Diabetes was confirmed by blood glucose measurement. Diabetic animals did not receive external insulin injections. After monitoring for 2 months after onset of diabetes, retinal tissues were collected from diabetic mice and from age-matched controls and stored at −70°C for future analysis. 
Transgenic Animals
Transgenic mice, with endothelial cell specific overexpression of miR-200b, were generated at the London Regional Transgenic and Gene Targeting Facility. Briefly, pSPTg.T2FpAXK (pg52) plasmid containing a Tie2 promoter and enhancer and a SV40 PolyA signal was kindly provided by Dr Thomas Sato (Nara Institute of Science and Technology, Graduate School of Biological Sciences, Japan).38 A cDNA fragment including miR-200b was cloned into pg52. The Tie2-miR-200b transgene was then excised with SalI from the vector backbone for injection. Gel-purified Tie2- miR-200b transgene was microinjected into pronuclei of fertilized mouse eggs from C57BL/6 X CBA/J mice, which were then transferred into pseudopregnant female mice.39 Potential transgenic strains and their offspring were identified by PCR with genomic DNA prepared from tail biopsies.39 No phenotypic changes were found in the transgenic mice. Routine genotyping was performed by PCR with a forward primer (corresponding to the Tie2 promoter) (5′-CCGCCTGCTTCTGTGGTGT-3′) and a reverse primer (corresponding to the cDNA fragment including miR-200b) (5′-AGGTGTACGCTGGGTGGAAGTG-3′) to amplify a 300-bp fragment. The PCR conditions were as follows: 94°C for 10 minutes, 25 cycles at 94°C (1 minute), 55°C (1 minute) and 72°C (1 minute), and 72°C for 10 minutes. The male miR-200b transgenic mice weighing 23 to 26 g were randomly divided into control and diabetic groups. Diabetes was induced as described above and the tissues were collected. 
Messenger RNA Extraction and Quantitative PCR
Total RNA was extracted with TRIzol reagent (Invitrogen), as described.5,2831 The cDNA was synthesized with high-capacity cDNA reverse-transcription kit (Applied Biosystems, Burlington, ON, Canada). The expression level of mRNA was detected by real-time quantitative RT-PCR using the LightCycler in a reaction volume of 20 μL (Roche Diagnostics, Laval, Canada). Primers were designed using the Primer 5 software (Premier Biosoft, Palo Alto, CA, USA; Supplementary Table S1). 
MicroRNA Analysis
Retinas from mice were collected as described previously.28 MicroRNA from cells and tissues were extracted with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). The cDNA for miRNA analysis was synthesized with TaqMan microRNA Assay Reverse Transcription Primer and MultiScribe reverse transcriptase (Life Technologies, Grand Island, NY, USA). Specific primers were custom synthesized based on the mature miR-200b and real-time quantitative RT-PCR was performed with the TaqMan microRNA Assay using the LightCycler as described and the data were normalized to U6 small nuclear RNA to account for differences in reverse-transcription efficiencies and the amount of template in the reaction mixtures.5,28 
Western Blotting and ELISA
The cells and tissues were lysed with RIPA buffer. Protein concentrations were measured using the BCA protein assay kit (Fisher Scientific, Burlington, ON, Canada). Briefly, total protein (30 μg) from each sample was loaded to SDS-PAGE and transferred onto a PVDF membrane (Millipore Corp, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween-20. The membranes were incubated with primary antibodies (goat-anti-CD31 and rabbit-anti-VE-cadherin) (Santa Cruz, CA, USA), rabbit-anti-FSP1 (Abcam, Cambridge, MA, USA), mouse-anti-Smad2 (Cell Signaling, Beverly, MA, USA), followed by incubation with corresponding horseradish peroxidase conjugated anti-goat, anti-rabbit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-mouse (Invitrogen) secondary IgG antibodies using 1:1000 or 1:5000 dilutions respectively. After washing, the proteins were visualized with Amersham ECL Prime Western blotting detection reagent (GE Healthcare, Pittsburgh, PA, USA). The blots were analyzed by densitometry. Blots were stripped and re-probed with β-actin (1:1000; Abcam) as an internal control. The data were analyzed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). ELISA for FN was performed using a commercially available kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. 
Immunofluorescence
Cells were plated in eight-chamber tissue culture slides and grown to 60% to 80% confluence. Following various treatments, the cells were washed with PBS and fixed with methanol:acetone (4:1, vol:vol) and permeated with 0.5% Triton X-100 in PBS. The tissue sections were deparaffinized. After incubation with 5% normal serum, both the cells and tissue sections were incubated with one of the primary antibodies: goat-anti-CD31, rabbit-anti-VE-cadherin, goat-anti-type IV collagen α1 (COL4), goat-anti-FN and rabbit-anti-Snail1 (Santa Cruz, CA, USA), rabbit-anti-FSP1 (Abcam), or mouse-anti-Smad2 (Cell Signaling) at 1:100 dilution. Corresponding secondary antibody conjugated with Alexa Fluor 488 (donkey anti-goat IgG or Alexa Fluor 488 goat anti-rabbit IgG [Life Technologies]) was used at 1:200 dilution. Stains were detected with a fluorescent microscope (Olympus BX51; Olympus, Richmond Hill, ON, Canada) and analyzed with ImageJ software. Hoechst 33342 (1 mg/mL; Invitrogen) was used to visualize the nuclei.40 
Statistical Analysis
Data are expressed as mean ± SEM. Statistical significances were analyzed using ANOVA and the Student t-test with post hoc analyses as appropriate. Differences were considered significant at values of P ≤ 0.05. 
Results
Glucose Induces EndMT in HRMECs
Endothelial to mesenchymal transition is characterized by the phonotypic change manifested as gradual loss of endothelial markers, and acquisition of mesenchymal markers.8,10,13 To examine EndMT, we focused on multiple markers. Specifically, we used two endothelial markers, namely CD31 and VE-cadherin. These are well-established endothelial markers, widely used in clinical and in experimental studies. For mesenchymal markers we used FSP1, FN, smooth muscle protein of 22 kd (SM22), and type I collagen α1 (COL1). These are well characterized mesenchymal transcripts. All these molecules are well-established markers of EndMT and have been used by several investigators.812,1518 As retinal microvascular endothelial cells are primary targets of hyperglycemic damage in diabetes, we first examined whether such changes occur in these cells. We treated HRMECs with various levels of glucose for various durations. We found that 25-mM glucose (HG) caused maximal upregulation of ECM protein FN at 72 hours (Supplementary Fig. S1). Hence, in subsequent experiments we used such concentration and duration and compared them with cells incubated for similar duration in 5 mM glucose (NG). No alterations of FN were observed when the cells were exposed to 25 mM of L-glucose (data not shown). 
Messenger RNA expressions of endothelial markers, CD31 and VE-cadherin, were decreased (>50%) in HRMECs when the cells incubated with HG for 72 hours compared with NG (Figs. 1A, 1D). In parallel, HG caused upregulation of multiple mesenchymal markers such as FSP1, FN, smooth muscle protein of 22kd (SM22), and COL1 (Figs. 2A, 2D; Supplementary Figs. S2B, S2C), ranging from 30% to 200%. In addition, we analyzed corresponding protein expression from these transcripts. In all such instances, protein levels paralleled mRNA levels (Figs. 1, 2B, 2C, 2E, 2F). 
Figure 1
 
Glucose caused downregulation of CD31 and VE-cadherin (VE-cad) in retinal endothelial cells. (A, D) Messenger RNA expression, (B, E) representative Western blot (top) with quantification, and (C, F) immunofluorescence stains showing reduced expression of these markers in 25-mM glucose (HG) compared with 5-mM glucose (NG). Such alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 1
 
Glucose caused downregulation of CD31 and VE-cadherin (VE-cad) in retinal endothelial cells. (A, D) Messenger RNA expression, (B, E) representative Western blot (top) with quantification, and (C, F) immunofluorescence stains showing reduced expression of these markers in 25-mM glucose (HG) compared with 5-mM glucose (NG). Such alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 2
 
Glucose caused upregulation of mesenchymal marker FSP1 and FN in retinal endothelial cells. (A, D) mRNA expression, (B, E) representative Western blot (top) with qualification and (C, F) immunofluorescence stain showing increased expression of these markers when the cells were incubated with 25-mM glucose (HG) compared with 5-mM glucose (NG). These alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 2
 
Glucose caused upregulation of mesenchymal marker FSP1 and FN in retinal endothelial cells. (A, D) mRNA expression, (B, E) representative Western blot (top) with qualification and (C, F) immunofluorescence stain showing increased expression of these markers when the cells were incubated with 25-mM glucose (HG) compared with 5-mM glucose (NG). These alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Transforming Growth Factor β Mediates Glucose-Induced EndMT
We then examined the role of TGFβ in mediating glucose-induced EndMT. We first established that TGFβ mRNA was upregulated following HG exposure (Fig. 3A), and reached the peak at 72 hours. To establish a cause-effect relationship, we treated the HG cells with a TGFβ1 inhibitor SB431542. Such treatment prevented glucose-induced alterations of CD31, VE-cadherin, FSP1, FN, COL1, and SM22 mRNA and protein production (Figs. 1, 2; Supplementary Fig. S2). Additionally, to examine a glucose-mimetic effect, we incubated the cells with TGFβ1 peptide. Such an approach caused a glucose-like effect reducing endothelial markers and upregulating mesenchymal proteins, characteristic of EndMT (Figs. 1, 2; Supplementary Fig. S2). These effects were seen both at the mRNA and at the protein levels and established EndMT in HRMECs and the role of TGFβ in such process. 
Figure 3
 
Glucose-induced upregulation of TGFβ1 and its downstream mediators. (A) Messenger RNA analyses showed duration-dependent upregulation of TGFβ1 in 25-mM glucose (HG) compared with 5-mM glucose (NG). High glucose also upregulated (B) Smad2 and (D) Snail1 mRNA expression and increased (C) Smad2 protein (top: Western blot, bottom: quantitative analysis) and (E) Snail1 protein (immunofluorescence stain confirming increased protein expression). High glucose further reduced levels of (F) miR-200b, and increased (G) p300 mRNA and (H) VEGF mRNA expression. Such alterations were prevented by TGFβ inhibitor SB43154. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (AD), (G), (H): mRNA and protein levels are expressed as a ratio to β-actin mRNA and normalized to NG; (F) miRNA levels are expressed as a ratio of U6 snRNA (U6) and normalized to NG; (E) Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG. Also see Supplementary Figures S2, S3.
Figure 3
 
Glucose-induced upregulation of TGFβ1 and its downstream mediators. (A) Messenger RNA analyses showed duration-dependent upregulation of TGFβ1 in 25-mM glucose (HG) compared with 5-mM glucose (NG). High glucose also upregulated (B) Smad2 and (D) Snail1 mRNA expression and increased (C) Smad2 protein (top: Western blot, bottom: quantitative analysis) and (E) Snail1 protein (immunofluorescence stain confirming increased protein expression). High glucose further reduced levels of (F) miR-200b, and increased (G) p300 mRNA and (H) VEGF mRNA expression. Such alterations were prevented by TGFβ inhibitor SB43154. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (AD), (G), (H): mRNA and protein levels are expressed as a ratio to β-actin mRNA and normalized to NG; (F) miRNA levels are expressed as a ratio of U6 snRNA (U6) and normalized to NG; (E) Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG. Also see Supplementary Figures S2, S3.
In an attempt to further characterize glucose-induced EndMT and TGFβ signaling, we examined the downstream signaling molecules of TGFβ, Smad2, and Snail1. These molecules are thought to be key mediators of EndMT in other organs.10,16,17,25,41 The mRNA and protein expression of Smad2 and Snail1 was upregulated in the HG-treated cells and were inhibited by TGFβ1 inhibitor SB431542. As expected, TGFβ1-treated cells produced a glucose-like effect with Smad2 and Snail1 upregulation (>45% and 400% respectively) (Figs. 3B–E; Supplementary Fig. S2A). 
The miR-200b Regulates Glucose-Induced EndMT in HRMECs
We have previously shown a regulatory role of miR-200b in mediating early molecular changes in the retina in diabetes. As shown previously, along with VEGF regulation, miR-200b, through histone acetylator p300, may potentially regulate multiple transcripts.28 In addition, both Smad2 and Snail1 are targets of miR-200b.36 Hence, we further examined miR-200b expression. In keeping with our previous reports, HG caused a reduction in miR-200b (>30%) in association with upregulation of VEGF and p300 in HRMECs (Figs. 3F–H). Similar glucomimetic-effects were seen after incubation with TGFβ1. To directly assess the role of miR-200b in EndMT, we transfected the cells in HG and the cells incubated with TGFβ1 peptide with miR-200b mimic. As expected, miR-200b mimic transfection prevented glucose-induced downregulation of CD31 and VE-cadherin and upregulation of FSP1 (Figs. 4A–C). Similar protection was seen when TGFβ-treated cells were transfected. As predicted, such alterations were associated with correction of glucose-induced change of Smad2, Snail1, and p300 levels, the targets of miR-200b but not those of TGFβ1 (Figs. 4D–F, 5A, 5B). These findings established a direct role of miR-200b on glucose-induced EndMT. 
Figure 4
 
Effect of miR-200b mimic transfection on mRNA expression of EndMT and associated markers. Messenger RNA expression of (A) CD31, (B) VE-cadherin, (C) FSP1, (D) Snail1, (E) TGFβ1, and (F) p300, showing glucose- and TGFβ1-induced alterations of endothelial and mesenchymal markers are prevented by miR-200b mimic (200b) transfection but not by scrambled (S) mimic transfection. Such transfection, although having no effects on TGFβ expression, corrected Snail1 and p300 expression. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from HG.
Figure 4
 
Effect of miR-200b mimic transfection on mRNA expression of EndMT and associated markers. Messenger RNA expression of (A) CD31, (B) VE-cadherin, (C) FSP1, (D) Snail1, (E) TGFβ1, and (F) p300, showing glucose- and TGFβ1-induced alterations of endothelial and mesenchymal markers are prevented by miR-200b mimic (200b) transfection but not by scrambled (S) mimic transfection. Such transfection, although having no effects on TGFβ expression, corrected Snail1 and p300 expression. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from HG.
Figure 5
 
Effect of miR-200b mimic transfection on Smad2. (A) Messenger RNA expression of Smad2. (B) Western blot and quantitative analyses of protein expression of Smad2 showing glucose- and TGFβ-induced alterations of Smad2 is prevented by miR-200b mimic (200b) transfection by not by scrambled (S) mimic transfection. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from NG.
Figure 5
 
Effect of miR-200b mimic transfection on Smad2. (A) Messenger RNA expression of Smad2. (B) Western blot and quantitative analyses of protein expression of Smad2 showing glucose- and TGFβ-induced alterations of Smad2 is prevented by miR-200b mimic (200b) transfection by not by scrambled (S) mimic transfection. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from NG.
Retinal EndMT in Diabetic miR-200b Transgene Mice
To further translate our findings and to establish that miR-200b–mediated EndMT indeed occurred in diabetic animals, we examined transgenic mice with endothelial-specific overexpression of miR-200b. We first confirmed that endothelial cells, isolated from these mice, show miR-200b overexpression compared with wild-type mice (Supplementary Fig. S3).47 We further examined retinal tissues from the littermate control and miR-200b transgenic mice with or without STZ-induced diabetes. In keeping with our hypothesis, mRNA expression of endothelial markers CD31 and VE-cadherin were reduced in wild-type mice with diabetes in association with increased mRNA expression of mesenchymal markers FSP1 and VIM confirming EndMT in animals with diabetes (Figs. 6A–D). Such changes were associated with increased expression of EndMT inducer, TGFβ, and that of two miR-200b target genes Smad2 and Snail1, which are mediators of TGFβ signaling (Figs. 6E–G) and p300 (Fig. 6H). In addition, immunofluorescence staining of the retinal tissue showed reduced CD31 and increased FSP1 stain in the retinal capillary endothelial cells in diabetes (Supplementary Fig. S4). All such abnormalities were prevented in the miR-200b transgenic mice with diabetes, further establishing the pathogenetic mechanism involving miR-200b in retinal EndMT (Fig. 6). 
Figure 6
 
Analysis of mRNA expression of EndMT and associated markers in the B6 mice retina; (A) CD31, (B) VE-cadherin (VE-cad), (C) FSP1, (D) VIM, (E) TGFβ1, (F) Smad2, (G) Snail1, and (H) p300, showing alteration of these markers in the retina of diabetic mice (D) compared with age- and sex-matched controls (C). These alterations were prevented in transgenic mice with endothelial-specific overexpression of miR-200b (200bTG). Messenger RNA levels are expressed as a ratio to β-actin and normalized to B6C. *Significantly different from C; n = 5–7/group.
Figure 6
 
Analysis of mRNA expression of EndMT and associated markers in the B6 mice retina; (A) CD31, (B) VE-cadherin (VE-cad), (C) FSP1, (D) VIM, (E) TGFβ1, (F) Smad2, (G) Snail1, and (H) p300, showing alteration of these markers in the retina of diabetic mice (D) compared with age- and sex-matched controls (C). These alterations were prevented in transgenic mice with endothelial-specific overexpression of miR-200b (200bTG). Messenger RNA levels are expressed as a ratio to β-actin and normalized to B6C. *Significantly different from C; n = 5–7/group.
Discussion
In these experiments, we demonstrated glucose-induced EndMT in the retinal endothelial cells. We further showed similar changes in the retinal tissues of animals with short-term diabetes. At various levels of complexity, we demonstrated in vivo, that TGFβ plays a key role in this process and that in diabetes; such changes are regulated by miR-200b and histone acetylator p300. Using transgenic mice with endothelial-specific miR-200b overexpression we further confirmed the presence of such molecular abnormalities in the retinal tissues of diabetic animals. 
Endothelial cells are known to be a main target of glucose-induced retinal tissue damage in diabetes.2,4,42 Hyperglycemia-induced metabolic alteration leads to a series of cellular changes.4 These alteration lead to changes in the cellular transcriptional machinery causing altered synthesis of macromolecules.57 Through epigenetic mechanisms, regulation of protein production may happen at the level of nucleus by acetylation, methylation, and so forth, or at the posttranscription level through miRNAs.24,32,33 Although we and several others have previously demonstrated some aspects of EndMT (e.g., increased ECM protein production in the retina of animals with diabetes), the phenomena of EndMT has not been previously established in this context.5,9,11,12 In this study we have shown glucose-induced reduced expression of endothelial cell markers such as CD31, VE-cadherin and increased expression of multiple mesenchymal markers (e.g., FSP1, FN, COL1, VIM), confirming this phenotypic change. We have further shown that histone acetylator p300 and miR-200b regulate such process. In addition, as miR-200b regulates production of p300, as shown in this and in our previous report, a large number of molecules causing EndMT may further be produced through p300-dependant histone acetylation.28,35,36 
In the heart, where EndMT was first demonstrated, it has been shown that this transition of endothelial cells is key to the formation of cardiac valves during development.14 Additionally, such process may act as a key switch in cardiac fibrosis in various diseases and provide potential treatment target.9,10 Transforming growth factor β signaling pathway through Smad and Snail converge in the cell nucleus to change the transcriptional machinery.36 It has been demonstrated that oxidative stress may be a key initiating factor in such process in other tissues.43,44 We also used serum-free condition in our assays to avoid effects of TGFβ or other growth factors in the serum. It should be noted that overt fibrosis, as in diabetic nephropathy or cardiomyopathy, is not observed in the retina in diabetes. However, increased production of ECM proteins and endothelial dysfunctions are well-established features of retinal affection in diabetes.5,6,26,27 The data generated from the current study indicate molecular and phenotypic changes linking these processes. Results of the current study further indicate involvement of specific transcriptional machinery in retinal EndMT in diabetes. We have previously demonstrated the role of miR-200b in augmented VEGF production in diabetes.28 Here we have shown that the same miR also modulates EndMT through modulation of signaling molecules in the TGFβ pathway. The miR-200b also regulates production of transcription coactivator p300.28 The p300 alteration has been shown in cardiac EndMT.24 In addition, p300 has been thought to be a master regulator of transcriptional machinery.8,9,26,45 Conceptually, through P300, miR-200b may regulate multiple molecules. As shown in in vitro studies and in the experiments using endothelial-specific miR-200b transgenic mice, we have demonstrated such phenomena. We have also demonstrated earlier that other members on the miR200 cluster may not be involved in such process.28 In addition, this study has further supported the important role played by endothelial cells in the pathogenesis of molecular changes in diabetic retinopathy.28,40,46 This study further opens up new targets to block EndMT. Based on the results of this study, either miR-200b or p300 may act as potential targets to prevent these changes. The miR-200b, being a regulator of p300 as shown in this study and in other studies, may actually lend itself as a target of RNA-based therapeutics in the context of early molecular changes in the retina in diabetes. Such an approach may be beneficial to block changes like EndMT, as shown in this study, or augmented production of VEGF as demonstrated by us previously.28 However, whether such an approach may indeed be beneficial in established diabetic retinopathy needs to be further validated by carefully conducted long-term studies. A diagrammatic outline of the mechanisms demonstrated in this study has been provided in Figure 7
Figure 7
 
A diagrammatic outline of the mechanisms demonstrated in this study.
Figure 7
 
A diagrammatic outline of the mechanisms demonstrated in this study.
We also recognize that several other miRNAs may also be altered in EndMT.5,8,9,29,31 Such findings are expected, as one miRNA may regulate multiple targets and one gene may be regulated by multiple miRNAs. It is, however, of interest to examine whether similar changes or additional miRNA changes occur in the context of diabetic retinopathy. In the similar context, we have previously shown that miR-146a regulates increased ECM protein FN in the retina in diabetes.5 Whether miR-146a is also involved in EndMT requires further investigations. 
In summary, we have shown that in the retinal endothelial cells, HG causes phenotypic changes leading to increased production of mesenchymal markers and reduced production of endothelial markers. We have further shown that this process involves TGFβ and is also regulated by miR-200b and histone acetylator p300. We have further confirmed such findings in a novel genetically engineered mouse model with endothelium-specific overexpression of miR-200b. The data obtained from this study also suggest potential new therapeutic targets to prevent such early changes in the retina in diabetes through specific miRNA or histone acetylation. 
Acknowledgments
Supported by grants from the Canadian Diabetes Association (OG-3-13-4173-SC), the Heart and Stroke Foundation of Canada (T7366), and the National Natural Science Foundation of China (Grant 81200305). 
Disclosure: Y. Cao, None; B. Feng, None; S. Chen, None; Y. Chu, None; S. Chakrabarti, None 
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Figure 1
 
Glucose caused downregulation of CD31 and VE-cadherin (VE-cad) in retinal endothelial cells. (A, D) Messenger RNA expression, (B, E) representative Western blot (top) with quantification, and (C, F) immunofluorescence stains showing reduced expression of these markers in 25-mM glucose (HG) compared with 5-mM glucose (NG). Such alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 1
 
Glucose caused downregulation of CD31 and VE-cadherin (VE-cad) in retinal endothelial cells. (A, D) Messenger RNA expression, (B, E) representative Western blot (top) with quantification, and (C, F) immunofluorescence stains showing reduced expression of these markers in 25-mM glucose (HG) compared with 5-mM glucose (NG). Such alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 2
 
Glucose caused upregulation of mesenchymal marker FSP1 and FN in retinal endothelial cells. (A, D) mRNA expression, (B, E) representative Western blot (top) with qualification and (C, F) immunofluorescence stain showing increased expression of these markers when the cells were incubated with 25-mM glucose (HG) compared with 5-mM glucose (NG). These alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 2
 
Glucose caused upregulation of mesenchymal marker FSP1 and FN in retinal endothelial cells. (A, D) mRNA expression, (B, E) representative Western blot (top) with qualification and (C, F) immunofluorescence stain showing increased expression of these markers when the cells were incubated with 25-mM glucose (HG) compared with 5-mM glucose (NG). These alterations were prevented by TGFβ inhibitor SB431542. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (A, B, D, E) mRNA and protein levels are expressed as a ratio to β-actin and normalized to NG. Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG.
Figure 3
 
Glucose-induced upregulation of TGFβ1 and its downstream mediators. (A) Messenger RNA analyses showed duration-dependent upregulation of TGFβ1 in 25-mM glucose (HG) compared with 5-mM glucose (NG). High glucose also upregulated (B) Smad2 and (D) Snail1 mRNA expression and increased (C) Smad2 protein (top: Western blot, bottom: quantitative analysis) and (E) Snail1 protein (immunofluorescence stain confirming increased protein expression). High glucose further reduced levels of (F) miR-200b, and increased (G) p300 mRNA and (H) VEGF mRNA expression. Such alterations were prevented by TGFβ inhibitor SB43154. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (AD), (G), (H): mRNA and protein levels are expressed as a ratio to β-actin mRNA and normalized to NG; (F) miRNA levels are expressed as a ratio of U6 snRNA (U6) and normalized to NG; (E) Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG. Also see Supplementary Figures S2, S3.
Figure 3
 
Glucose-induced upregulation of TGFβ1 and its downstream mediators. (A) Messenger RNA analyses showed duration-dependent upregulation of TGFβ1 in 25-mM glucose (HG) compared with 5-mM glucose (NG). High glucose also upregulated (B) Smad2 and (D) Snail1 mRNA expression and increased (C) Smad2 protein (top: Western blot, bottom: quantitative analysis) and (E) Snail1 protein (immunofluorescence stain confirming increased protein expression). High glucose further reduced levels of (F) miR-200b, and increased (G) p300 mRNA and (H) VEGF mRNA expression. Such alterations were prevented by TGFβ inhibitor SB43154. Incubation of cells with 5 ng/mL TGFβ1 produced a glucose-like effect. (AD), (G), (H): mRNA and protein levels are expressed as a ratio to β-actin mRNA and normalized to NG; (F) miRNA levels are expressed as a ratio of U6 snRNA (U6) and normalized to NG; (E) Nuclear, Hoechst 33342 stain. *Significantly different from NG; #Significantly different from HG. Also see Supplementary Figures S2, S3.
Figure 4
 
Effect of miR-200b mimic transfection on mRNA expression of EndMT and associated markers. Messenger RNA expression of (A) CD31, (B) VE-cadherin, (C) FSP1, (D) Snail1, (E) TGFβ1, and (F) p300, showing glucose- and TGFβ1-induced alterations of endothelial and mesenchymal markers are prevented by miR-200b mimic (200b) transfection but not by scrambled (S) mimic transfection. Such transfection, although having no effects on TGFβ expression, corrected Snail1 and p300 expression. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from HG.
Figure 4
 
Effect of miR-200b mimic transfection on mRNA expression of EndMT and associated markers. Messenger RNA expression of (A) CD31, (B) VE-cadherin, (C) FSP1, (D) Snail1, (E) TGFβ1, and (F) p300, showing glucose- and TGFβ1-induced alterations of endothelial and mesenchymal markers are prevented by miR-200b mimic (200b) transfection but not by scrambled (S) mimic transfection. Such transfection, although having no effects on TGFβ expression, corrected Snail1 and p300 expression. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from HG.
Figure 5
 
Effect of miR-200b mimic transfection on Smad2. (A) Messenger RNA expression of Smad2. (B) Western blot and quantitative analyses of protein expression of Smad2 showing glucose- and TGFβ-induced alterations of Smad2 is prevented by miR-200b mimic (200b) transfection by not by scrambled (S) mimic transfection. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from NG.
Figure 5
 
Effect of miR-200b mimic transfection on Smad2. (A) Messenger RNA expression of Smad2. (B) Western blot and quantitative analyses of protein expression of Smad2 showing glucose- and TGFβ-induced alterations of Smad2 is prevented by miR-200b mimic (200b) transfection by not by scrambled (S) mimic transfection. Messenger RNA expressed as a ratio to β-actin mRNA and normalized to NG. *Significantly different from NG; #Significantly different from NG.
Figure 6
 
Analysis of mRNA expression of EndMT and associated markers in the B6 mice retina; (A) CD31, (B) VE-cadherin (VE-cad), (C) FSP1, (D) VIM, (E) TGFβ1, (F) Smad2, (G) Snail1, and (H) p300, showing alteration of these markers in the retina of diabetic mice (D) compared with age- and sex-matched controls (C). These alterations were prevented in transgenic mice with endothelial-specific overexpression of miR-200b (200bTG). Messenger RNA levels are expressed as a ratio to β-actin and normalized to B6C. *Significantly different from C; n = 5–7/group.
Figure 6
 
Analysis of mRNA expression of EndMT and associated markers in the B6 mice retina; (A) CD31, (B) VE-cadherin (VE-cad), (C) FSP1, (D) VIM, (E) TGFβ1, (F) Smad2, (G) Snail1, and (H) p300, showing alteration of these markers in the retina of diabetic mice (D) compared with age- and sex-matched controls (C). These alterations were prevented in transgenic mice with endothelial-specific overexpression of miR-200b (200bTG). Messenger RNA levels are expressed as a ratio to β-actin and normalized to B6C. *Significantly different from C; n = 5–7/group.
Figure 7
 
A diagrammatic outline of the mechanisms demonstrated in this study.
Figure 7
 
A diagrammatic outline of the mechanisms demonstrated in this study.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
Supplementary Table S1
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