Current Eye Research, Early Online, 1–11, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.921312
ORIGINAL ARTICLE
The Angiogenic Biomarker Endocan is Upregulated in Proliferative Diabetic Retinopathy and Correlates with Vascular Endothelial Growth Factor
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Ahmed M. Abu El-Asrar1, Mohd Imtiaz Nawaz1, Gert De Hertogh2, Abdullah S. Al-Kharashi1, Kathleen Van den Eynde2, Ghulam Mohammad1 and Karel Geboes2 1
Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia and 2 Laboratory of Histochemistry and Cytochemistry, University of Leuven, KU Leuven, Belgium
ABSTRACT Purpose/Aim: Endocan is a proteoglycan specifically secreted by endothelial cells, is a marker of angiogenesis and endothelial cell activation in response to proangiogenic signals. The aim of this study was to measure the levels of endocan in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and to correlate its levels with clinical disease activity and the levels of the angiogenic biomarkers vascular endothelial growth factor (VEGF), soluble vascular endothelial-cadherin (sVE-cadherin) and soluble endoglin (sEng). In addition, we investigated the expression of endocan and correlated it with the level of vascularization in PDR epiretinal membranes. Materials and methods: Vitreous samples from 44 PDR and 29 non-diabetic patients were studied by enzyme-linked immunosorbent assay. Epiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. Results: Endocan, VEGF, sVE-cadherin and sEng levels were significantly higher in PDR patients than in non-diabetic patients (p50.001; p = 0.002; p50.001; p = 0.001, respectively). Endocan levels were significantly higher in patients with active PDR than in patients with inactive PDR and non-diabetic patients (p50.001). There were significant positive correlations between endocan levels and the levels of VEGF (r = 0.574, p50.001) and sVE-cadherin (r = 0.498, p50.001). In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed endocan. There was a significant positive correlation between the number of blood vessels expressing CD34 and the number of blood vessels expressing endocan (r = 0.933, p50.001). Conclusions: Our findings suggest that upregulation of endocan expression in PDR could be a reflection of endothelial cell activation associated with angiogenesis. Keywords: Angiogenesis, endocan, endothelial cell-specific molecule-1, proliferative diabetic retinopathy, vascular endothelial-cadherin, vascular endothelial growth factor
INTRODUCTION
development, but also for progression of angiogenesis-dependent diseases such as proliferative diabetic retinopathy (PDR).1,2 Ischemia-induced pathologic growth of new blood vessels and expansion of extracellular matrix in association with the outgrowth of fibrovascular epiretinal membranes at the
Angiogenesis is defined as the process during which new vessels form by branching from the existing vascular network. Formation of new capillaries is important not only for normal growth and
Received 1 January 2014; revised 16 March 2014; accepted 27 April 2014; published online 27 May 2014 Correspondence: Ahmed M. Abu El-Asrar, Department of Ophthalmology, King Abdulaziz University Hospital, Old Airport Road, P.O. Box 245, Riyadh 11411, Saudi Arabia. Tel: +966-11-4775723. Fax: +966-11-4775724. E-mail: [email protected]; [email protected]
1
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
2
A. M. Abu El-Asrar et al.
vitreoretinal interface is the pathological hallmark in PDR and often leads to catastrophic loss of vision due to vitreous hemorrhage and/or traction retinal detachment. Angiogenesis involves a complex interplay between different cells, soluble factors and extracellular matrix components.1,2 Vascular endothelial growth factor (VEGF), an endothelial cell mitogen that also enhances vascular permeability, is thought to be the major angiogenesis factor in PDR.3 Therapeutic regulation of angiogenesis has emerged as an attractive approach for the treatment of PDR.4 To aid the progress of these strategies, a more comprehensive understanding of molecules regulating angiogenesis in PDR could be of value to identify additional therapeutic targets. As the main target of angiogenesis treatment, angiogenic endothelial cells present specific markers that potentially promise a molecular basis for vessel-targeted therapy. Among these markers, endocan, vascular endothelial (VE-cadherin) and endoglin (Eng) stand out as reliable biomarkers of angiogenesis activity. Endocan, also called endothelial cell-specific molecule-1 (ESM-1), is a novel soluble dermatan sulphate proteoglycan that is secreted by cultured endothelial cells.5,6 Endocan is involved in the regulation of cellular activities such as adhesion, migration and proliferation.7,8 Endocan has been recently described as a marker of angiogenesis9,10 and endothelial cell activation in response to proangiogenic signals in tumors.8,11 In endothelial cells, endocan is expressed at higher levels in proliferating compared with nonproliferating cells.8 Moreover, endocan interacts with growth factors such as hepatocyte growth factor/ scatter factor through its dermatan sulphate chain eliciting epithelial cell proliferation in vitro.12 Endocan is overexpressed in several tumors, preferentially in the tumor endothelium,8,11,13–18 and endocan expression has been linked to a worse prognosis and metastasis.13,15–18 Moreover, serum levels of endocan are elevated in patients with cancer and high endocan values were significantly correlated with the presence of metastasis and with limited survival suggesting that this molecule may be a useful novel biomarker associated with angiogenesis, endothelial cell activation and cancer.8,11,14,17,19 Endocan was also shown to be markedly expressed during the switch between dormant to fast-growing phenotype in experimental models of angiogenic tumors.20 Finally, human embryonic kidney 293 cells that have been genetically engineered to overexpress endocan induce tumor formation in nude mice,19 and anti-endocan antibodies inhibited tumor growth19 and tumor epithelial and vascular endothelial proliferation.15 These findings suggest that endothelial-derived endocan induces tumor growth and that antibodies to endocan may have therapeutic potential.19 In addition, endocan has been suggested to regulate inflammatory processes by inhibiting the interaction between
lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1, an important step in the firm adhesion of leukocytes to the endothelium and thus could be involved in regulating leukocyte trafficking into tissues.21 VE-cadherin is a cell adhesion molecule localized at the endothelial junction. VE-cadherin plays a key role in angiogenesis and in vascular permeability.22,23 This molecule can be shed from the cell surface and elevated serum levels of soluble VE-cadherin (sVE-cadherin) seems to be a reliable marker of angiogenic activity and/or injury.24–27 Endoglin (Eng) is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells.28,29 A soluble form of Eng (sEng) has been observed in the serum from patients with cancer and elevated levels positively correlated with tumor metastasis.30 In a previous study, we demonstrated that sVE-cadherin and sEng were upregulated in the vitreous fluid from patients with PDR suggesting that sVE-cadherin and sEng might be valuable angiogenic markers for PDR.31 The aim of this study was to investigate the expression of the novel angiogenic biomarker endocan in the vitreous fluid and epiretinal membranes from patients with PDR and to correlate its levels with clinical disease activity and the levels of other biomarkers of angiogenesis, including VEGF, sVE-cadherin and sEng. In addition, we studied the correlation between the expression of endocan and the level of vascularization as assessed with the panendothelial cell marker CD34 immunostaining in PDR epiretinal fibrovascular membranes.
MATERIALS AND METHODS Vitreous Samples and Epiretinal Membranes Specimens Undiluted vitreous fluid samples (0.3–0.6 ml) were obtained from 44 patients with PDR during pars plana vitrectomy. The indications for vitrectomy were traction retinal detachment, and/or non-clearing vitreous hemorrhage. In patients with PDR, the severity of retinal neovascular activity was graded clinically at the time of vitrectomy using previously published criteria.32 Neovascularization was considered active if there were visible perfused new vessels on the retina or optic disc present within tractional epiretinal membranes. Neovascularization was considered inactive (involuted) if only nonvascularized, white fibrotic epiretinal membranes were present. Active PDR was present in 23 patients and inactive PDR was present in 21 patients. The clinical characteristics of patients with PDR are sown in Table 1. The control group consisted of 29 patients who had undergone vitrectomy for the Current Eye Research
Expression of Endocan in PDR 3 TABLE 1. Clinical characteristics of patients with proliferative diabetic retinopathy (PDR) (44 patients). Activity of PDR
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Variables
Active (n = 23)
Age (years) 45.2 ± 7.4 Sex Male 18 (78.3%) Female 5 (21.7%) Type of diabetes IDDM 16 (69.6%) NIDDM 7 (30.4%) Duration of diabetes (years) 15.3 ± 4.7 Fasting blood sugar at presentation Controlled 3 (13.1%) Uncontrolled 20 (86.9%) Treatment for hypertension Yes 18 (78.3%) No 5 (21.7%)
Inactive (n = 21) 61.3 ± 8.4
p Value 50.001*
16 (66.7%) 5 (23.8%)
0.986
16 (76.2%) 5 (23.8%) 18 ± 6.6
0.878
4 (19.1%) 17 (80.9%) 14 (66.7%) 7 (33.3%)
0.194 0.02*
0.079
IDDM, insulin-dependent diabetes mellitus; NIDDM, noninsulin-dependent diabetes mellitus.
treatment of rhegmatogenous retinal detachment with no proliferative vitreoretinopathy. Controls were free from systemic disease and were 19 males and 10 females whose ages ranged from 22 to 59 years with a mean of 42.6 ± 15.4 years. Vitreous samples were collected undiluted by manual suction into a syringe through the aspiration line of vitrectomy, before opening the infusion line. The samples were centrifuged (500 rpm for 10 min, 4 C) and the supernatants were aliquoted and frozen at 80 C until assay. Epiretinal fibrovascular membranes were obtained from 14 patients with PDR during pars plana vitrectomy for the repair of tractional retinal detachment. Active PDR was present in five patients and inactive PDR was present in nine patients. Membranes were fixed in 10% formalin solution and embedded in paraffin. The study was conducted according to the tenets of the Declaration of Helsinki. All the patients were candidates for vitrectomy as a surgical procedure. All patients signed a preoperative informed written consent and approved the use of the excised epiretinal membranes and vitreous fluid for further analysis and clinical research. The study design and the protocol were approved by the Research Centre and Institutional Review Board of the College of Medicine, King Saud University.
Enzyme-Linked Immunosorbent Assay Kits Enzyme-linked immunosorbent assay (ELISA) kit for Human endocan (Human Endocan/ESM-1, Cat No: DIYEK-LIK-1101) was purchased from Lunginnov Sas (Lille, France). ELISA kit for human VEGF (Human vascular endothelial growth factor, Cat No: SVE00), sVE-cadherin (Human soluble VE-Cadherin, !
2014 Informa Healthcare USA, Inc.
Cat No: DCADV0) and human sEng (Human Endoglin/CD105, Cat No: DNDG00) were purchased from R&D Systems (Minneapolis, MN). The minimum detection limit of each ELISA kit for VEGF, sVE-cadherin and sEng are 9, 113 and 7 picograms/mL (pg/mL), respectively. The ELISA plate readings were done using FLUOstar OmegaMiroplate reader from BMG Labtech (Offenburg, Germany).
Measurement of Endocan, VEGF, sVE-Cadherin and sEng The quantification of human endocan, VEGF, sVE-cadherin and sEng in the vitreous fluid was determined using ELISA kits according to the manufacturer’s instruction. For each ELISA kit, the undiluted standard serves as the highest standard and calibrator diluents serves as the zero standard. Depending upon the detection range for each ELISA kit, vitreous samples were either directly used or diluted with calibrator diluents supplied with the ELISA kit. For the measurement of endocan and sEng, 100 mL of undiluted vitreous were used and added to the wells of respective ELISA plates. Whereas, for the measurement of VEGF and sVE-cadherin, 100 mL of 2- and 5-fold, respectively, diluted vitreous were used in each ELISA assay for their analysis. As instructed in the kit manual, samples were incubated into each well of ELISA plates. Antibodies against endocan, VEGF, sVE-cadherin and sEng conjugated to horseradish peroxidase were added to each well of the ELISA plate. After incubation, substrate mix solution was added for color development. The reaction was stopped by the addition of 2 N sulfuric acid and optical density was read at 450 nm in microplate reader. Each assay was performed in duplicate. Using the four-parameter fit logistic (4-PL) curve equation, the actual concentration for each sample was calculated. For the vitreous fluid that have been diluted, the correction read from the standard curve obtained using 4-PL were multiplied by the dilution factors to get the actual reading for each sample.
Immunohistochemical Staining Antigen retrieval was performed by boiling the sections in a citrate based buffer [pH 5.9–6.1] [BOND Epitope Retrieval Solution 1; Leica] for 20 min. Subsequently, the sections were incubated with the monoclonal antibodies listed in Table 2. Optimal working concentration and incubation times for the antibodies were determined earlier in pilot experiments. The sections were then incubated for 20 min with a post-primary IgG linker followed by an
4
A. M. Abu El-Asrar et al.
TABLE 2. Monoclonal antibodies used for immunohistochemical staining. Primary antibody Anti-CD34 (Clone My10) (mc) Anti-a-Smooth muscle actin (Clone 1A4) (mc) Anti-endocan (Clone 6D4) (mc)
Dilution
Incubation time (min)
Sourcea
1/50 1/200 1/200
60 60 60
BD Biosciences Dako Bio-Rad AbD SeroTec
a
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Location of manufacturers: BD Bioscience (San Jose, CA); Dako (Glostrup, Denmark); Bio-Rad AbD SeroTec (Du¨sseldorf, Germany).
alkaline phosphatase conjugated polymer. The reaction product was visualized by incubation for 15 min with the Fast Red chromogen, resulting in bright-red immunoreactive sites. The slides were then faintly counterstained with Mayer’s hematoxylin [BOND Polymer Refine Red Detection Kit; Leica]. Omission or substitution of the primary antibody with an irrelevant antibody from the same species and staining with chromogen alone were used as negative controls. Sections from patients with glioblastoma were used as positive controls for the immunohistochemical staining methods. The sections from the control patients were obtained from patients treated at the University Hospital, University of Leuven, Belgium, in full compliance with tenets of the Declaration of Helsinki. We used archived material and patients gave written consent at admission for the use of the leftover material in studies. The Ethics Committee of the University Hospital, University of Leuven approved this consent procedure.
Quantitation Immunoreactive blood vessels and cells were counted in five representative fields, using an eyepiece calibrated grid in combination with the 40 objective. These representative fields were selected based on the presence of immunoreactive blood vessels and cells. With this magnification and calibration, immunoreactive blood vessels and cells present in an area of 0.33 0.22 mm2 were counted.
Statistical Analysis Data are presented as the mean ± standard deviation. The Mann–Whitney test was used to compare means from two independent groups. The Chi-square test was used to compare proportions when analyzing data for two categorical variables. Pearson correlation coefficients were computed to investigate correlations between variables. One-way ANOVA and postANOVA pairwise comparisons of means were conducted using the Kruskal–Wallis test. For three groups, the critical Z-value for post-ANOVA pairwise mean comparisons was Z = 2.39 at a 5% level of significance. A p value less than 0.05 indicated statistical significance. SPSS version 12.0 (SPSS Inc.,
Chicago, IL) for Windows and program 3S from BioMedical Data Processing Version 2007 (BMDP 2007) Statistical Software (Cork Technology Pack, Model Farm Road, Cord, Ireland) were used for the statistical analyses.
RESULTS Angiogenesis Biomarker Levels in Vitreous Samples Endocan, sVE-cadherin and sEng were detected in all vitreous samples from patients with PDR and non-diabetic patients. VEGF was detected in 39 of 44 (88.6%) vitreous samples from patients with PDR and in 14 of 29 (48.3%) vitreous samples from nondiabetic patients, and the incidence rates of detection differed significantly (p50.001; Chi-square test). The mean levels of endocan, VEGF, sVE-cadherin and sEng in vitreous samples from PDR patients were significantly higher than that in non-diabetic patients (p50.001; p = 0.002; p50.001; p = 0.001, respectively; Mann–Whitney test; Figure 1 and Table 3).
Relationship Between Angiogenesis Biomarker Levels and Activity of PDR Patients with active PDR were significantly younger than those with inactive PDR. In addition, there were significantly more patients with uncontrolled fasting blood sugar at presentation among patients with active PDR compared with those with inactive PDR (Table 1). Comparison of the mean levels of angiogenesis biomarkers among active PDR patients, inactive PDR patients, and non-diabetic patients was conducted using the Kruskal–Wallis test and the results are shown in Table 3 and Figure 2. Mean levels differed significantly between the three groups for endocan (p50.001), VEGF (p = 0.002), sVEcadherin (p50.001) and sEng (p = 0.003). PostANOVA pairwise comparisons of means indicated that mean endocan levels were significantly higher in patients with active PDR and patients with inactive PDR than that in non-diabetic patients (Z = 6.33 and 3.06, respectively). Furthermore, mean endocan level in patients with active PDR was significantly higher than that in patients with inactive PDR (Z = 3.02). Current Eye Research
Expression of Endocan in PDR 5
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
For VEGF, the mean level in patients with active PDR was significantly higher than that in non-diabetic patients (Z = 3.59). For sVE-cadherin, the mean levels were significantly higher in patients with active PDR and patients with inactive PDR than that in non-diabetic patients (Z = 3.27 and 3.35, respectively). For sEng, the mean levels were significantly higher in patients with active PDR and patients with inactive
PDR than that in non-diabetic patients (Z = 2.42 and 3.21, respectively).
Correlations In the whole study group, there were significant positive correlations between vitreous fluid levels of
FIGURE 1. Comparisons of mean angiogenesis biomarker levels in vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RD). *The difference between the two means was statistically significant at 5% level of significance. VEGF, vascular endothelial growth factor; sVE-cadherin, soluble vascular endothelial-cadherin; sEng, soluble endoglin.
TABLE 3. Comparisons of mean angiogenesis biomarker levels in proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RD) patients and in PDR patients with or without active neovascularization. Disease group PDR RD p Value (Mann–Whitney test) Active PDR Inactive PDR RD p Value (ANOVA)
Endocan (ng/ml)
VEGF (ng/ml)
sVE-Cadherin (ng/ml)
sEng (ng/ml)
3.57 ± 1.2 0.83 ± 0.41 50.001a 4.4 ± 0.7 2.64 ± 0.9 0.83 ± 0.4 50.001a
0.97 ± 1.3 0.08 ± 0.06 0.002a 1.11 ± 1.3 0.76 ± 1.5 0.08 ± 0.06 0.002a
68.54 ± 61.3 27.66 ± 30.3 50.001a 65.7 ± 53.4 71.66 ± 70.2 27.66 ± 30.3 50.001a
3.58 ± 1.7 2.22 ± 0.8 0.001a 3.34 ± 1.6 3.83 ± 1.8 2.22 ± 0.8 0.003a
VEGF, vascular endothelial growth factor; sVE-cadherin, soluble vascular endothelial-cadherin; sEng, soluble endoglin. a Statistically significant at 5% level of significance. !
2014 Informa Healthcare USA, Inc.
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
6
A. M. Abu El-Asrar et al.
FIGURE 2. Comparisons of mean angiogenesis biomarker levels in vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) with or without active neovascularization and rhegmatogenous retinal detachment (RD). For three groups, the critical Z-value for post-ANOVA pairwise mean comparisons was Z = 2.39 at a 5% level of significance. VEGF, vascular endothelial growth factor; sVE-cadherin, soluble vascular endothelial-cadherin; sEng, soluble endoglin.
endocan and the levels of VEGF (r = 0.574, p50.001) and sVE-cadherin (r = 0.498, p50.001; Figure 3).
Immunohistochemical Analysis No staining was observed in the negative control slides (Figure 4A). All membranes showed blood vessels positive for the panendothelial cell marker CD34 (Figure 4B), with a mean number of 43.3 ± 45.4 (range: 12–125). Strong immunoreactivity for endocan was present in all membranes and was noted in the cytoplasm of stromal cells and vascular endothelial cells (Figure 4C–E). The number of immunoreactive blood vessels ranged from 5 to 70, with a mean number of 22.8 ± 21.4. The number of immunoreactive stromal cells ranged from 7 to 140, with a mean number of 55.4 ± 41.1. In serial sections, the distribution and morphology of spindle-shaped stromal cells expressing endocan were similar to
those of spindle-shaped stromal cells expressing the myofibroblast marker a-smooth muscle actin (Figure 4F). The mean numbers of blood vessels expressing CD34, were significantly higher in membranes from patients with active PDR (98.4 ± 25.6) than in membranes from patients with inactive PDR (12.7 ± 8.6; p = 0.03). In addition, the mean numbers of blood vessels expressing endocan were significantly higher in membranes from patients with active PDR (46.6 ± 16.4) than in membranes from patients with inactive PDR (9.6 ± 7.8; p = 0.03; Figure 4C and D). The difference between the mean numbers of stromal cells expressing endocan in membranes from patients with active PDR (76.0 ± 32.1) and in membranes from patients with inactive PDR (44.0 ± 42.6) was not significant (p = 0.124; Figure 5). The level of vascularization and proliferative activity in epiretinal membranes were determined Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Expression of Endocan in PDR 7
FIGURE 3. Significant positive correlations between vitreous fluid levels of endocan and levels of vascular endothelial growth factor (VEGF) (A) and soluble vascular endothelial-chaderin (sVE-cadherin).
by immunodetection of the panendothelial cell marker CD34. A significant positive correlation was detected between the numbers of blood vessels expressing CD34 and the numbers of blood vessels expressing endocan (r = 0.933, p50.001; Figure 6). On the other hand, the correlation between the numbers of blood vessels expressing CD34 and the numbers of stromal cells expressing endocan was not significant (r = 0.4, p = 0.156).
DISCUSSION In this study, the levels of endocan, VEGF, sVE-cadherin and sEng were significantly higher in the vitreous fluid from patients with PDR as compared to non-diabetic control patients. This study also described for the first time, the in situ localization of the expression of endocan in epiretinal membranes from patients with PDR. Furthermore, endocan expression was significantly higher in patients with active PDR compared with patients with inactive PDR. There were significant positive correlations between the vitreous levels of endocan and the levels of VEGF and sVE-cadherin. These findings suggest that endocan could be a new biomarker of angiogenesis in PDR. The current study is the first to demonstrate that endocan is significantly upregulated in the vitreous fluid from patients with PDR. Furthermore, the levels were significantly higher in patients with active PDR compared with patients with quiescent PDR. Another aim of this study was to determine which cell types express endocan in epiretinal membranes from patients with PDR. Using immunohistochemistry, we demonstrated that endocan protein was specifically localized in vascular endothelial cells and a-smooth muscle actin-expressing myofibroblasts. In addition, there was a significant correlation between the level of !
2014 Informa Healthcare USA, Inc.
vascularization in PDR epiretinal membranes and the number of blood vessels expressing endocan. The numbers of blood vessels expressing endocan in membranes from patients with active PDR were significantly higher than those in membranes from patients with inactive PDR. Our results are consistent with previous reports showing that endocan is overexpressed in several tumors,8,11,13–18 and that there is a strong correlation between the expression of endocan and the degree of tumor vascularity,13,15 and tumor aggressiveness.13–18 In addition, circulating levels of endocan are elevated in patients with cancer and high endocan levels were significantly correlated with the presence of metastasis and with limited survival.8,11,14,17,19 Recently, endocan has been described as a biomarker of angiogenesis and endothelial cell activation. Endocan has been described as a marker of the ‘‘tip cells’’. The tip cells are the motile endothelial cells, which mediate the sprouting of developing vessels during the process of angiogenesis. They are known to be responsive for VEGF.9,10 A function of endocan during angiogenesis has been suggested by reports of increased mRNA levels in tube-forming human capillary endothelial cells in collagen gels.33 On the basis of our findings, we propose that upregulation of endocan expression in PDR could be a reflection of endothelial cell activation associated with angiogenesis. VEGF, also called vascular permeability factor, is a major player in angiogenesis. VEGF binds with high affinity and activates two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR in humans/Flk-1 in mice). These receptors regulate physiological as well as pathological angiogenesis. From the postnatal to adult stage, VEGFR-2 is expressed mostly on vascular endothelial cells.34 VEGFR-2 has strong tyrosine kinase activity, and is the major positive signal transducer for pathological angiogenesis including cancer and diabetic retinopathy as well as
A. M. Abu El-Asrar et al.
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
8
FIGURE 4. Proliferative diabetic retinopathy (PDR) epiretinal membranes. Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining for CD34 showing blood vessels positive for CD34 (B). Immunohistochemical staining for endocan showing cytoplasmic immunoreactivity in vascular endothelial cells (arrows), stromal cells (arrowheads) in membranes from patients with active PDR (C), inactive PDR (D) and spindle-shaped cells (E). Immunohistochemical staining for a-smooth muscle actin showing immunoreactivity in spindle-shaped myofibroblasts (F) (original magnification 40).
microvascular permeability.34 Activation of VEGFR-2 stimulates endothelial cell proliferation, migration and survival.34 Our analysis showed a significant positive correlation between the vitreous levels of VEGF and endocan. Similarly, previous studies reported a strong correlation between the expression of VEGF and endocan in several tumors.11,13,33 In vitro studies demonstrated that treatment of endothelial cells with VEGF induces endocan expression at both
mRNA and protein levels and that anti-VEGF antibodies inhibit VEGF-induced endocan secretion by endothelial cells.8,11,14,35 It is suggested that the effect is mediated through VEGFR-2.8 In addition, the proangiogenic factor fibroblast growth factor-211 and the inflammatory cytokines tumor necrosis factor-a and interleukin-1b5,11,14 increased the synthesis and secretion of endocan from endothelial cells. In contrast, interferon-g downregulated the secretion of Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Expression of Endocan in PDR 9
FIGURE 5. Comparisons of mean numbers of immunoreactive blood vessels and stromal cells in fibrovascular epiretinal membranes in relation to type of proliferative diabetic retinopathy (PDR). *The difference between the two means was statistically significant at 5% level of significance.
FIGURE 6. A significant positive correlation between the numbers of blood vessels expressing CD34 and the numbers of blood vessels expressing endocan in epiretinal fibrovascular membranes.
endocan, whereas interleukin-4 has no effect.5 Recently, it was demonstrated that endocan expression was also induced by the transcriptional regulator hypoxia-inducible factor-1a.21 Several studies demonstrated that sVE-cadherin serum levels may reflect the intensity of angiogenesis in several physiological and pathological conditions.24,25,27 In vitro studies demonstrated that treatment of endothelial cells with tumor necrosis factor-a,26 VEGF,36 matrix metalloproteinase-937 and the diabetic metabolite advanced glycation end products37 resulted in shedding of the VE-cadherin extracellular domain and loss of cell–cell contact !
2014 Informa Healthcare USA, Inc.
which may lead to increased vascular permeability. In this study, we demonstrated that sVE-cadherin is significantly upregulated in the vitreous fluid from patients with PDR consistent with our previous report.31 It is well established that endothelial dysfunction is a key feature of diabetic retinopathy.37 Therefore, elevated levels of sVE-cadherin in the vitreous fluid from patients with PDR could be a reflection of endothelial cell dysfunction associated with angiogenesis and breakdown of the inner bloodretinal barrier. In addition, we demonstrated a significant positive correlation between the vitreous levels of endocan and sVE-cadherin. Our findings
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
10 A. M. Abu El-Asrar et al. suggest that similar mechanisms might be involved in shedding of the VE-cadherin extracellular domain from endothelial cells and endocan biosynthesis and secretion by endothelial cells. Consistent with our previous report,31 we demonstrated that sEng is significantly upregulated in the vitreous fluid from patients with PDR, however, there was no significant correlation between endocan levels and the levels of sEng. The potential role of endocan in the process of angiogenesis was investigated. Several studies demonstrated that endocan has no direct effect on endothelial cell proliferation or in vivo angiogenesis. Recombinant endocan did neither induce formation of endothelial sprouts in fibrin gels nor it influence VEGF-induced sprouting.35 Inhibition of endogenous endocan by a neutralizing antibody19,35 or inhibition of endocan expression by antisense oligonucleotides33 did not inhibit in vitro angiogenesis. Finally, endocan did not induce angiogenesis when used in vivo model of angiogenesis based on the chick chorioallantoic membrane.35 In vivo, endocan overexpression by nontumorigenic epithelial cells induces tumor formation, whereas overexpression by tumorigenic cells sharply increases the growth rate of resulting tumors.7,19 Therefore, the cellular target of endocan seems to be the malignant epithelial cells rather than the endothelium itself. It is suggested that endocan acts locally in a paracrine positive feedback loop to support tumor growth (i.e. angiogenic growth factors stimulate endothelial cells to secrete endocan, which in turn acts by amplifying the mitogenic effect of protumoral growth factors).11,12 Similarly, the cellular target of endocan in PDR could be other cells involved in PDR progression such as myofibroblasts.38 In this study, we demonstrated that endocan was specifically localized in a-smooth muscle actinexpressing myofibroblasts. These findings suggest that myofibroblasts synthesize and secrete endocan and that endocan acts locally to promote myofibroblast activities such as migration and proliferation7,8 and could be involved in tissue remodeling and fibrosis. A recent study suggested a possible contribution of endocan in the endothelial–mesenchymal transition process.39 Endothelial–mesenchymal transition is a complex biological process in which endothelial cells lose their specific endothelial cell markers and acquire mesenchymal or myofibroblastic phenotype. This process is now recognized to be an important mechanism in the pathogenesis of pulmonary, cardiac and kidney fibrosis.40 In conclusion, our results demonstrate that endocan is overexpressed in PDR, particularly in patients with active PDR. Endocan could therefore be a new biomarker of angiogenesis in PDR. Further investigation is needed to determine if endocan may be used not only as a biomarker of retinal angiogenesis but also as a therapeutic target in PDR.
ACKNOWLEDGEMENTS The authors thank Mr. Dustan Kanagave, MSc for statistical assistance, Mr. Wilfried Versin for technical assistance and Ms. Connie B. Unisa-Marfil for secretarial work.
DECLARATION OF INTEREST All the authors do not have any conflict of interests with any trademark mentioned in the manuscript. The authors alone are responsible for the content and writing of the paper. This work was supported by Dr. Nasser Al-Rasheed Research Chair in Ophthalmology (Abu El-Asrar AM).
REFERENCES 1. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–660. 2. Conway EM, Collen D, Carmeliet P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2011;49: 507–521. 3. Spranger J, Pfeiffer AP. New concepts in pathogenesis and treatment of diabetic retinopathy. Exp Clin Endocrinol Diabetes 2001;109:S438–S450. 4. Salam A, Mathew R, Sivaprasad S. Treatment of proliferative diabetic retinopathy with anti-VEGF agents. Acta Ophthalmol 2011;89:405–411. 5. Lassalle P, Molet S, Janin A, Heyden JV, Tavernier J, Fiers W, et al. ESM-1 is a novel human endothelial cellspecific molecule expressed in lung and regulated by cytokines. J Biol Chem 1996;271:20458–20464. 6. Ho M, Yang E, Matcuk G, Deng D, Sampas N, Tsalenko A, et al. Identification of endothelial cell genes by combined database mining and microarray analysis. Physiol Genomics 2003;13:249–262. 7. Sarrazin S, Adam E, Lyon M, Depontieu F, Motte V, Landolfi C, et al. Endocan or endothelial cell specific molecule-1 (ESM-1): a potential novel endothelial cell marker and a new target for cancer therapy. Biochim Biophys Acta 2006;1765:25–37. 8. Abid MR, Yi X, Yano K, Shih SC, Aird WC. Vascular endocan is preferentially expressed in tumor endothelium. Microvasc Res 2006;72:136–145. 9. Strasser GA, Kaminker JS, Tessier-Lavigne M. Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 2010;115:5102–5110. 10. del Toro R, Prahst C, Mathivet T, Siegfried G, Kaminker JS, Larrivee B, et al. Identification and functional analysis of endothelial tip cell-enriched genes. Blood 2010;116: 4025–4033. 11. Grigoriu BD, Depontieu F, Scherpereel A, Gourcerol D, Devos P, Ouata T, et al. Endocan expression and relationship with survival in human non-small cell lung cancer. Clin Cancer Res 2006;12:4575–4582. 12. Be´chard D, Gentina T, Delehedde M, Scherpereel A, Lyon M, Aumercier M, et al. Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity. J Biol Chem 2001;276:48341–48349. Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Expression of Endocan in PDR 11 13. Chen LY, Liu X, Wang SL, Qin CY. Over-expression of the Endocan gene in endothelial cells from hepatocellular carcinoma is associated with angiogenesis and tumour invasion. J Int Med Res 2010;38:498–510. 14. Leroy X, Aubert S, Zini L, Franquet H, Kervoaze G, Villers A, et al. Vascular endocan (ESM-1) is markedly overexpressed in clear cell renal cell carcinoma. Histopathology 2010;56:180–187. 15. Liu N, Zhang LH, Du H, Hu Y, Zhang GG, Wang XH, et al. Overexpression of endothelial cell specific molecule-1 (ESM-1) in gastric cancer. Ann Surg Oncol 2010;17: 2628–2639. 16. Kang YH, Ji NY, Lee CI, Lee HG, Kim JW, Yeom YI, et al. ESM-1 silencing decreased cell survival, migration, and invasion and modulated cell cycle progression in hepatocellular carcinoma. Amino Acids 2011;40:1003–1013. 17. Cornelius A, Cortet-Rudelli C, Assaker R, Kerdraon O, Gevaert MH, Pre´vot V, et al. Endothelial expression of endocan is strongly associated with tumor progression in pituitary adenoma. Brain Pathol 2012; 22:757–764. 18. Kim JH, Park MY, Kim CN, Kim KH, Kang HB, Kim KD, et al. Expression of endothelial cell-specific molecule-1 regulated by hypoxia inducible factor-1a in human colon carcinoma: impact of ESM-1 on prognosis and its correlation with clinicopathological features. Oncol Rep 2012;28: 1701–1708. 19. Scherpereel A, Gentina T, Grigoriu B, Se´ne´chal S, Janin A, Tsicopoulos A, et al. Overexpression of endocan induces tumor formation. Cancer Res 2003;63:6084–6089. 20. Almog N, Ma L, Raychowdhury R, Schwager C, Erber R, Short S, et al. Transcriptional switch of dormant tumors to fast-growing angiogenic phenotype. Cancer Res 2009;69: 836–844. 21. Be´chard D, Scherpereel A, Hammad H, Gentina T, Tsicopoulos A, Aumercier M, et al. Human endothelialcell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1. J Immunol 2001;167:3099–3106. 22. Vestweber D. VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arterioscler Thromb Vasc Biol 2008;28:223–232. 23. Harris ES, Nelson WJ. VE-cadherin: at the front, center, and sides of endothelial cell organization and function. Curr Opin Cell Biol 2010;22:651–658. 24. Wrobel T, Mazur G, Wolowiec D, Jazwiec B, Sowinska E, Kuliczkowski K. sVE-cadherin and sCD146 serum levels in patients with multiple myeloma. Clin Lab Haem 2006;28: 36–39. 25. Rabascio C, Muratori E, Mancuso P, Calleri A, Raia V, Foutz T, et al. Assessing tumor angiogenesis: increased circulating VE-cadherin RNA in patients with cancer indicates viability of circulating endothelial cells. Cancer Res 2004;64:4373–4377. 26. Sidibe¯ A, Mannic T, Arboleas M, Subileau M, GulinoDebrac D, Bouillet L, et al. Soluble VE-cadherin in
!
2014 Informa Healthcare USA, Inc.
27.
28. 29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
rheumatoid arthritis patients correlates with disease activity: evidence for tumor necrosis factor a-induced VE-cadherin cleavage. Arthritis Rheum 2012;64:77–87. Shih SC, Robinson GS, Perruzzi CA, Calvo A, Desai K, Green JE, et al. Molecular profiling of angiogenesis markers. Am J Pathol 2002;161:35–41. ten Dijke P, Goumans MJ, Pardali E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 2008;11:79–89. Duff SE, Li C, Garland JM, Kumar S. CD105 is important for angiogenesis: evidence and potential applications. FASEB J 2003;17:984–992. Li C, Guo B, Wilson PB, Stewart A, Byrne G, Bundred N, et al. Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer. Int J Cancer 2000;89:122–126. Abu El-Asrar AM, Nawaz MI, Kangave D, Abouammoh M, Mohammad G. High-mobility group box-1 and endothelial cell angiogenic markers in the vitreous from patients with proliferative diabetic retinopathy. Mediators Inflamm 2012;2012:697489. Aiello LP, Avery RL, Arrig PG, Keyt BA, Jampel HD, Shah ST, et al. Vascular endothelial growth factor in ocular fluid in patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994;331:1480–1487. Aitkenhead M, Wang SJ, Nakatsu MN, Mestas J, Heard C, Hughes CC. Identification of endothelial cell genes expressed in an in vitro model of angiogenesis: induction of ESM-1, (beta)ig-h3, and NrCAM. Microvasc Res 2002;63: 159–171. Shibuya M. Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol 2006;39:469–478. Rennel E, Mellberg S, Dimberg A, Petersson L, Botling J, Ameur A, et al. Endocan is a VEGF-A and P13K regulated gene with increased expression in human renal cancer. Exp Cell Res 2007;313:1285–1294. Villasante A, Pacheco A, Ruiz A, Pellicer A, Garcia-Velasco JA. Vascular endothelial cadherin regulates vascular permeability: implicatins for ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 2007;92:314–321. Navaratna D, McGuire PG, Menicucci G, Das A. Proteolytic degradation of VE-cadherin alters the bloodretinal barrier in diabetes. Diabetes 2007;56:2380–2387. Abu El-Asrar AM, Struyf S, Kangave D, Geboes K, Van Damme J. Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy. Eur Cytokine Netw 2006;17:155–165. Carillo LM, Arciniegas E, Rojas H, Ramirez R. Immunolocalization of endocan during the endothelialmesenchymal transition process. Eur J Histochem 2011; 55:e13. Piera-Velazquez S, Li Z, Jimenez SA. Role of endothelialmesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders. Am J Pathol 2011;179:1074–1080.
ORIGINAL ARTICLE
The Angiogenic Biomarker Endocan is Upregulated in Proliferative Diabetic Retinopathy and Correlates with Vascular Endothelial Growth Factor
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Ahmed M. Abu El-Asrar1, Mohd Imtiaz Nawaz1, Gert De Hertogh2, Abdullah S. Al-Kharashi1, Kathleen Van den Eynde2, Ghulam Mohammad1 and Karel Geboes2 1
Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia and 2 Laboratory of Histochemistry and Cytochemistry, University of Leuven, KU Leuven, Belgium
ABSTRACT Purpose/Aim: Endocan is a proteoglycan specifically secreted by endothelial cells, is a marker of angiogenesis and endothelial cell activation in response to proangiogenic signals. The aim of this study was to measure the levels of endocan in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and to correlate its levels with clinical disease activity and the levels of the angiogenic biomarkers vascular endothelial growth factor (VEGF), soluble vascular endothelial-cadherin (sVE-cadherin) and soluble endoglin (sEng). In addition, we investigated the expression of endocan and correlated it with the level of vascularization in PDR epiretinal membranes. Materials and methods: Vitreous samples from 44 PDR and 29 non-diabetic patients were studied by enzyme-linked immunosorbent assay. Epiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. Results: Endocan, VEGF, sVE-cadherin and sEng levels were significantly higher in PDR patients than in non-diabetic patients (p50.001; p = 0.002; p50.001; p = 0.001, respectively). Endocan levels were significantly higher in patients with active PDR than in patients with inactive PDR and non-diabetic patients (p50.001). There were significant positive correlations between endocan levels and the levels of VEGF (r = 0.574, p50.001) and sVE-cadherin (r = 0.498, p50.001). In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed endocan. There was a significant positive correlation between the number of blood vessels expressing CD34 and the number of blood vessels expressing endocan (r = 0.933, p50.001). Conclusions: Our findings suggest that upregulation of endocan expression in PDR could be a reflection of endothelial cell activation associated with angiogenesis. Keywords: Angiogenesis, endocan, endothelial cell-specific molecule-1, proliferative diabetic retinopathy, vascular endothelial-cadherin, vascular endothelial growth factor
INTRODUCTION
development, but also for progression of angiogenesis-dependent diseases such as proliferative diabetic retinopathy (PDR).1,2 Ischemia-induced pathologic growth of new blood vessels and expansion of extracellular matrix in association with the outgrowth of fibrovascular epiretinal membranes at the
Angiogenesis is defined as the process during which new vessels form by branching from the existing vascular network. Formation of new capillaries is important not only for normal growth and
Received 1 January 2014; revised 16 March 2014; accepted 27 April 2014; published online 27 May 2014 Correspondence: Ahmed M. Abu El-Asrar, Department of Ophthalmology, King Abdulaziz University Hospital, Old Airport Road, P.O. Box 245, Riyadh 11411, Saudi Arabia. Tel: +966-11-4775723. Fax: +966-11-4775724. E-mail: [email protected]; [email protected]
1
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
2
A. M. Abu El-Asrar et al.
vitreoretinal interface is the pathological hallmark in PDR and often leads to catastrophic loss of vision due to vitreous hemorrhage and/or traction retinal detachment. Angiogenesis involves a complex interplay between different cells, soluble factors and extracellular matrix components.1,2 Vascular endothelial growth factor (VEGF), an endothelial cell mitogen that also enhances vascular permeability, is thought to be the major angiogenesis factor in PDR.3 Therapeutic regulation of angiogenesis has emerged as an attractive approach for the treatment of PDR.4 To aid the progress of these strategies, a more comprehensive understanding of molecules regulating angiogenesis in PDR could be of value to identify additional therapeutic targets. As the main target of angiogenesis treatment, angiogenic endothelial cells present specific markers that potentially promise a molecular basis for vessel-targeted therapy. Among these markers, endocan, vascular endothelial (VE-cadherin) and endoglin (Eng) stand out as reliable biomarkers of angiogenesis activity. Endocan, also called endothelial cell-specific molecule-1 (ESM-1), is a novel soluble dermatan sulphate proteoglycan that is secreted by cultured endothelial cells.5,6 Endocan is involved in the regulation of cellular activities such as adhesion, migration and proliferation.7,8 Endocan has been recently described as a marker of angiogenesis9,10 and endothelial cell activation in response to proangiogenic signals in tumors.8,11 In endothelial cells, endocan is expressed at higher levels in proliferating compared with nonproliferating cells.8 Moreover, endocan interacts with growth factors such as hepatocyte growth factor/ scatter factor through its dermatan sulphate chain eliciting epithelial cell proliferation in vitro.12 Endocan is overexpressed in several tumors, preferentially in the tumor endothelium,8,11,13–18 and endocan expression has been linked to a worse prognosis and metastasis.13,15–18 Moreover, serum levels of endocan are elevated in patients with cancer and high endocan values were significantly correlated with the presence of metastasis and with limited survival suggesting that this molecule may be a useful novel biomarker associated with angiogenesis, endothelial cell activation and cancer.8,11,14,17,19 Endocan was also shown to be markedly expressed during the switch between dormant to fast-growing phenotype in experimental models of angiogenic tumors.20 Finally, human embryonic kidney 293 cells that have been genetically engineered to overexpress endocan induce tumor formation in nude mice,19 and anti-endocan antibodies inhibited tumor growth19 and tumor epithelial and vascular endothelial proliferation.15 These findings suggest that endothelial-derived endocan induces tumor growth and that antibodies to endocan may have therapeutic potential.19 In addition, endocan has been suggested to regulate inflammatory processes by inhibiting the interaction between
lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1, an important step in the firm adhesion of leukocytes to the endothelium and thus could be involved in regulating leukocyte trafficking into tissues.21 VE-cadherin is a cell adhesion molecule localized at the endothelial junction. VE-cadherin plays a key role in angiogenesis and in vascular permeability.22,23 This molecule can be shed from the cell surface and elevated serum levels of soluble VE-cadherin (sVE-cadherin) seems to be a reliable marker of angiogenic activity and/or injury.24–27 Endoglin (Eng) is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells.28,29 A soluble form of Eng (sEng) has been observed in the serum from patients with cancer and elevated levels positively correlated with tumor metastasis.30 In a previous study, we demonstrated that sVE-cadherin and sEng were upregulated in the vitreous fluid from patients with PDR suggesting that sVE-cadherin and sEng might be valuable angiogenic markers for PDR.31 The aim of this study was to investigate the expression of the novel angiogenic biomarker endocan in the vitreous fluid and epiretinal membranes from patients with PDR and to correlate its levels with clinical disease activity and the levels of other biomarkers of angiogenesis, including VEGF, sVE-cadherin and sEng. In addition, we studied the correlation between the expression of endocan and the level of vascularization as assessed with the panendothelial cell marker CD34 immunostaining in PDR epiretinal fibrovascular membranes.
MATERIALS AND METHODS Vitreous Samples and Epiretinal Membranes Specimens Undiluted vitreous fluid samples (0.3–0.6 ml) were obtained from 44 patients with PDR during pars plana vitrectomy. The indications for vitrectomy were traction retinal detachment, and/or non-clearing vitreous hemorrhage. In patients with PDR, the severity of retinal neovascular activity was graded clinically at the time of vitrectomy using previously published criteria.32 Neovascularization was considered active if there were visible perfused new vessels on the retina or optic disc present within tractional epiretinal membranes. Neovascularization was considered inactive (involuted) if only nonvascularized, white fibrotic epiretinal membranes were present. Active PDR was present in 23 patients and inactive PDR was present in 21 patients. The clinical characteristics of patients with PDR are sown in Table 1. The control group consisted of 29 patients who had undergone vitrectomy for the Current Eye Research
Expression of Endocan in PDR 3 TABLE 1. Clinical characteristics of patients with proliferative diabetic retinopathy (PDR) (44 patients). Activity of PDR
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Variables
Active (n = 23)
Age (years) 45.2 ± 7.4 Sex Male 18 (78.3%) Female 5 (21.7%) Type of diabetes IDDM 16 (69.6%) NIDDM 7 (30.4%) Duration of diabetes (years) 15.3 ± 4.7 Fasting blood sugar at presentation Controlled 3 (13.1%) Uncontrolled 20 (86.9%) Treatment for hypertension Yes 18 (78.3%) No 5 (21.7%)
Inactive (n = 21) 61.3 ± 8.4
p Value 50.001*
16 (66.7%) 5 (23.8%)
0.986
16 (76.2%) 5 (23.8%) 18 ± 6.6
0.878
4 (19.1%) 17 (80.9%) 14 (66.7%) 7 (33.3%)
0.194 0.02*
0.079
IDDM, insulin-dependent diabetes mellitus; NIDDM, noninsulin-dependent diabetes mellitus.
treatment of rhegmatogenous retinal detachment with no proliferative vitreoretinopathy. Controls were free from systemic disease and were 19 males and 10 females whose ages ranged from 22 to 59 years with a mean of 42.6 ± 15.4 years. Vitreous samples were collected undiluted by manual suction into a syringe through the aspiration line of vitrectomy, before opening the infusion line. The samples were centrifuged (500 rpm for 10 min, 4 C) and the supernatants were aliquoted and frozen at 80 C until assay. Epiretinal fibrovascular membranes were obtained from 14 patients with PDR during pars plana vitrectomy for the repair of tractional retinal detachment. Active PDR was present in five patients and inactive PDR was present in nine patients. Membranes were fixed in 10% formalin solution and embedded in paraffin. The study was conducted according to the tenets of the Declaration of Helsinki. All the patients were candidates for vitrectomy as a surgical procedure. All patients signed a preoperative informed written consent and approved the use of the excised epiretinal membranes and vitreous fluid for further analysis and clinical research. The study design and the protocol were approved by the Research Centre and Institutional Review Board of the College of Medicine, King Saud University.
Enzyme-Linked Immunosorbent Assay Kits Enzyme-linked immunosorbent assay (ELISA) kit for Human endocan (Human Endocan/ESM-1, Cat No: DIYEK-LIK-1101) was purchased from Lunginnov Sas (Lille, France). ELISA kit for human VEGF (Human vascular endothelial growth factor, Cat No: SVE00), sVE-cadherin (Human soluble VE-Cadherin, !
2014 Informa Healthcare USA, Inc.
Cat No: DCADV0) and human sEng (Human Endoglin/CD105, Cat No: DNDG00) were purchased from R&D Systems (Minneapolis, MN). The minimum detection limit of each ELISA kit for VEGF, sVE-cadherin and sEng are 9, 113 and 7 picograms/mL (pg/mL), respectively. The ELISA plate readings were done using FLUOstar OmegaMiroplate reader from BMG Labtech (Offenburg, Germany).
Measurement of Endocan, VEGF, sVE-Cadherin and sEng The quantification of human endocan, VEGF, sVE-cadherin and sEng in the vitreous fluid was determined using ELISA kits according to the manufacturer’s instruction. For each ELISA kit, the undiluted standard serves as the highest standard and calibrator diluents serves as the zero standard. Depending upon the detection range for each ELISA kit, vitreous samples were either directly used or diluted with calibrator diluents supplied with the ELISA kit. For the measurement of endocan and sEng, 100 mL of undiluted vitreous were used and added to the wells of respective ELISA plates. Whereas, for the measurement of VEGF and sVE-cadherin, 100 mL of 2- and 5-fold, respectively, diluted vitreous were used in each ELISA assay for their analysis. As instructed in the kit manual, samples were incubated into each well of ELISA plates. Antibodies against endocan, VEGF, sVE-cadherin and sEng conjugated to horseradish peroxidase were added to each well of the ELISA plate. After incubation, substrate mix solution was added for color development. The reaction was stopped by the addition of 2 N sulfuric acid and optical density was read at 450 nm in microplate reader. Each assay was performed in duplicate. Using the four-parameter fit logistic (4-PL) curve equation, the actual concentration for each sample was calculated. For the vitreous fluid that have been diluted, the correction read from the standard curve obtained using 4-PL were multiplied by the dilution factors to get the actual reading for each sample.
Immunohistochemical Staining Antigen retrieval was performed by boiling the sections in a citrate based buffer [pH 5.9–6.1] [BOND Epitope Retrieval Solution 1; Leica] for 20 min. Subsequently, the sections were incubated with the monoclonal antibodies listed in Table 2. Optimal working concentration and incubation times for the antibodies were determined earlier in pilot experiments. The sections were then incubated for 20 min with a post-primary IgG linker followed by an
4
A. M. Abu El-Asrar et al.
TABLE 2. Monoclonal antibodies used for immunohistochemical staining. Primary antibody Anti-CD34 (Clone My10) (mc) Anti-a-Smooth muscle actin (Clone 1A4) (mc) Anti-endocan (Clone 6D4) (mc)
Dilution
Incubation time (min)
Sourcea
1/50 1/200 1/200
60 60 60
BD Biosciences Dako Bio-Rad AbD SeroTec
a
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Location of manufacturers: BD Bioscience (San Jose, CA); Dako (Glostrup, Denmark); Bio-Rad AbD SeroTec (Du¨sseldorf, Germany).
alkaline phosphatase conjugated polymer. The reaction product was visualized by incubation for 15 min with the Fast Red chromogen, resulting in bright-red immunoreactive sites. The slides were then faintly counterstained with Mayer’s hematoxylin [BOND Polymer Refine Red Detection Kit; Leica]. Omission or substitution of the primary antibody with an irrelevant antibody from the same species and staining with chromogen alone were used as negative controls. Sections from patients with glioblastoma were used as positive controls for the immunohistochemical staining methods. The sections from the control patients were obtained from patients treated at the University Hospital, University of Leuven, Belgium, in full compliance with tenets of the Declaration of Helsinki. We used archived material and patients gave written consent at admission for the use of the leftover material in studies. The Ethics Committee of the University Hospital, University of Leuven approved this consent procedure.
Quantitation Immunoreactive blood vessels and cells were counted in five representative fields, using an eyepiece calibrated grid in combination with the 40 objective. These representative fields were selected based on the presence of immunoreactive blood vessels and cells. With this magnification and calibration, immunoreactive blood vessels and cells present in an area of 0.33 0.22 mm2 were counted.
Statistical Analysis Data are presented as the mean ± standard deviation. The Mann–Whitney test was used to compare means from two independent groups. The Chi-square test was used to compare proportions when analyzing data for two categorical variables. Pearson correlation coefficients were computed to investigate correlations between variables. One-way ANOVA and postANOVA pairwise comparisons of means were conducted using the Kruskal–Wallis test. For three groups, the critical Z-value for post-ANOVA pairwise mean comparisons was Z = 2.39 at a 5% level of significance. A p value less than 0.05 indicated statistical significance. SPSS version 12.0 (SPSS Inc.,
Chicago, IL) for Windows and program 3S from BioMedical Data Processing Version 2007 (BMDP 2007) Statistical Software (Cork Technology Pack, Model Farm Road, Cord, Ireland) were used for the statistical analyses.
RESULTS Angiogenesis Biomarker Levels in Vitreous Samples Endocan, sVE-cadherin and sEng were detected in all vitreous samples from patients with PDR and non-diabetic patients. VEGF was detected in 39 of 44 (88.6%) vitreous samples from patients with PDR and in 14 of 29 (48.3%) vitreous samples from nondiabetic patients, and the incidence rates of detection differed significantly (p50.001; Chi-square test). The mean levels of endocan, VEGF, sVE-cadherin and sEng in vitreous samples from PDR patients were significantly higher than that in non-diabetic patients (p50.001; p = 0.002; p50.001; p = 0.001, respectively; Mann–Whitney test; Figure 1 and Table 3).
Relationship Between Angiogenesis Biomarker Levels and Activity of PDR Patients with active PDR were significantly younger than those with inactive PDR. In addition, there were significantly more patients with uncontrolled fasting blood sugar at presentation among patients with active PDR compared with those with inactive PDR (Table 1). Comparison of the mean levels of angiogenesis biomarkers among active PDR patients, inactive PDR patients, and non-diabetic patients was conducted using the Kruskal–Wallis test and the results are shown in Table 3 and Figure 2. Mean levels differed significantly between the three groups for endocan (p50.001), VEGF (p = 0.002), sVEcadherin (p50.001) and sEng (p = 0.003). PostANOVA pairwise comparisons of means indicated that mean endocan levels were significantly higher in patients with active PDR and patients with inactive PDR than that in non-diabetic patients (Z = 6.33 and 3.06, respectively). Furthermore, mean endocan level in patients with active PDR was significantly higher than that in patients with inactive PDR (Z = 3.02). Current Eye Research
Expression of Endocan in PDR 5
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
For VEGF, the mean level in patients with active PDR was significantly higher than that in non-diabetic patients (Z = 3.59). For sVE-cadherin, the mean levels were significantly higher in patients with active PDR and patients with inactive PDR than that in non-diabetic patients (Z = 3.27 and 3.35, respectively). For sEng, the mean levels were significantly higher in patients with active PDR and patients with inactive
PDR than that in non-diabetic patients (Z = 2.42 and 3.21, respectively).
Correlations In the whole study group, there were significant positive correlations between vitreous fluid levels of
FIGURE 1. Comparisons of mean angiogenesis biomarker levels in vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RD). *The difference between the two means was statistically significant at 5% level of significance. VEGF, vascular endothelial growth factor; sVE-cadherin, soluble vascular endothelial-cadherin; sEng, soluble endoglin.
TABLE 3. Comparisons of mean angiogenesis biomarker levels in proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RD) patients and in PDR patients with or without active neovascularization. Disease group PDR RD p Value (Mann–Whitney test) Active PDR Inactive PDR RD p Value (ANOVA)
Endocan (ng/ml)
VEGF (ng/ml)
sVE-Cadherin (ng/ml)
sEng (ng/ml)
3.57 ± 1.2 0.83 ± 0.41 50.001a 4.4 ± 0.7 2.64 ± 0.9 0.83 ± 0.4 50.001a
0.97 ± 1.3 0.08 ± 0.06 0.002a 1.11 ± 1.3 0.76 ± 1.5 0.08 ± 0.06 0.002a
68.54 ± 61.3 27.66 ± 30.3 50.001a 65.7 ± 53.4 71.66 ± 70.2 27.66 ± 30.3 50.001a
3.58 ± 1.7 2.22 ± 0.8 0.001a 3.34 ± 1.6 3.83 ± 1.8 2.22 ± 0.8 0.003a
VEGF, vascular endothelial growth factor; sVE-cadherin, soluble vascular endothelial-cadherin; sEng, soluble endoglin. a Statistically significant at 5% level of significance. !
2014 Informa Healthcare USA, Inc.
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
6
A. M. Abu El-Asrar et al.
FIGURE 2. Comparisons of mean angiogenesis biomarker levels in vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) with or without active neovascularization and rhegmatogenous retinal detachment (RD). For three groups, the critical Z-value for post-ANOVA pairwise mean comparisons was Z = 2.39 at a 5% level of significance. VEGF, vascular endothelial growth factor; sVE-cadherin, soluble vascular endothelial-cadherin; sEng, soluble endoglin.
endocan and the levels of VEGF (r = 0.574, p50.001) and sVE-cadherin (r = 0.498, p50.001; Figure 3).
Immunohistochemical Analysis No staining was observed in the negative control slides (Figure 4A). All membranes showed blood vessels positive for the panendothelial cell marker CD34 (Figure 4B), with a mean number of 43.3 ± 45.4 (range: 12–125). Strong immunoreactivity for endocan was present in all membranes and was noted in the cytoplasm of stromal cells and vascular endothelial cells (Figure 4C–E). The number of immunoreactive blood vessels ranged from 5 to 70, with a mean number of 22.8 ± 21.4. The number of immunoreactive stromal cells ranged from 7 to 140, with a mean number of 55.4 ± 41.1. In serial sections, the distribution and morphology of spindle-shaped stromal cells expressing endocan were similar to
those of spindle-shaped stromal cells expressing the myofibroblast marker a-smooth muscle actin (Figure 4F). The mean numbers of blood vessels expressing CD34, were significantly higher in membranes from patients with active PDR (98.4 ± 25.6) than in membranes from patients with inactive PDR (12.7 ± 8.6; p = 0.03). In addition, the mean numbers of blood vessels expressing endocan were significantly higher in membranes from patients with active PDR (46.6 ± 16.4) than in membranes from patients with inactive PDR (9.6 ± 7.8; p = 0.03; Figure 4C and D). The difference between the mean numbers of stromal cells expressing endocan in membranes from patients with active PDR (76.0 ± 32.1) and in membranes from patients with inactive PDR (44.0 ± 42.6) was not significant (p = 0.124; Figure 5). The level of vascularization and proliferative activity in epiretinal membranes were determined Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Expression of Endocan in PDR 7
FIGURE 3. Significant positive correlations between vitreous fluid levels of endocan and levels of vascular endothelial growth factor (VEGF) (A) and soluble vascular endothelial-chaderin (sVE-cadherin).
by immunodetection of the panendothelial cell marker CD34. A significant positive correlation was detected between the numbers of blood vessels expressing CD34 and the numbers of blood vessels expressing endocan (r = 0.933, p50.001; Figure 6). On the other hand, the correlation between the numbers of blood vessels expressing CD34 and the numbers of stromal cells expressing endocan was not significant (r = 0.4, p = 0.156).
DISCUSSION In this study, the levels of endocan, VEGF, sVE-cadherin and sEng were significantly higher in the vitreous fluid from patients with PDR as compared to non-diabetic control patients. This study also described for the first time, the in situ localization of the expression of endocan in epiretinal membranes from patients with PDR. Furthermore, endocan expression was significantly higher in patients with active PDR compared with patients with inactive PDR. There were significant positive correlations between the vitreous levels of endocan and the levels of VEGF and sVE-cadherin. These findings suggest that endocan could be a new biomarker of angiogenesis in PDR. The current study is the first to demonstrate that endocan is significantly upregulated in the vitreous fluid from patients with PDR. Furthermore, the levels were significantly higher in patients with active PDR compared with patients with quiescent PDR. Another aim of this study was to determine which cell types express endocan in epiretinal membranes from patients with PDR. Using immunohistochemistry, we demonstrated that endocan protein was specifically localized in vascular endothelial cells and a-smooth muscle actin-expressing myofibroblasts. In addition, there was a significant correlation between the level of !
2014 Informa Healthcare USA, Inc.
vascularization in PDR epiretinal membranes and the number of blood vessels expressing endocan. The numbers of blood vessels expressing endocan in membranes from patients with active PDR were significantly higher than those in membranes from patients with inactive PDR. Our results are consistent with previous reports showing that endocan is overexpressed in several tumors,8,11,13–18 and that there is a strong correlation between the expression of endocan and the degree of tumor vascularity,13,15 and tumor aggressiveness.13–18 In addition, circulating levels of endocan are elevated in patients with cancer and high endocan levels were significantly correlated with the presence of metastasis and with limited survival.8,11,14,17,19 Recently, endocan has been described as a biomarker of angiogenesis and endothelial cell activation. Endocan has been described as a marker of the ‘‘tip cells’’. The tip cells are the motile endothelial cells, which mediate the sprouting of developing vessels during the process of angiogenesis. They are known to be responsive for VEGF.9,10 A function of endocan during angiogenesis has been suggested by reports of increased mRNA levels in tube-forming human capillary endothelial cells in collagen gels.33 On the basis of our findings, we propose that upregulation of endocan expression in PDR could be a reflection of endothelial cell activation associated with angiogenesis. VEGF, also called vascular permeability factor, is a major player in angiogenesis. VEGF binds with high affinity and activates two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR in humans/Flk-1 in mice). These receptors regulate physiological as well as pathological angiogenesis. From the postnatal to adult stage, VEGFR-2 is expressed mostly on vascular endothelial cells.34 VEGFR-2 has strong tyrosine kinase activity, and is the major positive signal transducer for pathological angiogenesis including cancer and diabetic retinopathy as well as
A. M. Abu El-Asrar et al.
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
8
FIGURE 4. Proliferative diabetic retinopathy (PDR) epiretinal membranes. Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining for CD34 showing blood vessels positive for CD34 (B). Immunohistochemical staining for endocan showing cytoplasmic immunoreactivity in vascular endothelial cells (arrows), stromal cells (arrowheads) in membranes from patients with active PDR (C), inactive PDR (D) and spindle-shaped cells (E). Immunohistochemical staining for a-smooth muscle actin showing immunoreactivity in spindle-shaped myofibroblasts (F) (original magnification 40).
microvascular permeability.34 Activation of VEGFR-2 stimulates endothelial cell proliferation, migration and survival.34 Our analysis showed a significant positive correlation between the vitreous levels of VEGF and endocan. Similarly, previous studies reported a strong correlation between the expression of VEGF and endocan in several tumors.11,13,33 In vitro studies demonstrated that treatment of endothelial cells with VEGF induces endocan expression at both
mRNA and protein levels and that anti-VEGF antibodies inhibit VEGF-induced endocan secretion by endothelial cells.8,11,14,35 It is suggested that the effect is mediated through VEGFR-2.8 In addition, the proangiogenic factor fibroblast growth factor-211 and the inflammatory cytokines tumor necrosis factor-a and interleukin-1b5,11,14 increased the synthesis and secretion of endocan from endothelial cells. In contrast, interferon-g downregulated the secretion of Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Expression of Endocan in PDR 9
FIGURE 5. Comparisons of mean numbers of immunoreactive blood vessels and stromal cells in fibrovascular epiretinal membranes in relation to type of proliferative diabetic retinopathy (PDR). *The difference between the two means was statistically significant at 5% level of significance.
FIGURE 6. A significant positive correlation between the numbers of blood vessels expressing CD34 and the numbers of blood vessels expressing endocan in epiretinal fibrovascular membranes.
endocan, whereas interleukin-4 has no effect.5 Recently, it was demonstrated that endocan expression was also induced by the transcriptional regulator hypoxia-inducible factor-1a.21 Several studies demonstrated that sVE-cadherin serum levels may reflect the intensity of angiogenesis in several physiological and pathological conditions.24,25,27 In vitro studies demonstrated that treatment of endothelial cells with tumor necrosis factor-a,26 VEGF,36 matrix metalloproteinase-937 and the diabetic metabolite advanced glycation end products37 resulted in shedding of the VE-cadherin extracellular domain and loss of cell–cell contact !
2014 Informa Healthcare USA, Inc.
which may lead to increased vascular permeability. In this study, we demonstrated that sVE-cadherin is significantly upregulated in the vitreous fluid from patients with PDR consistent with our previous report.31 It is well established that endothelial dysfunction is a key feature of diabetic retinopathy.37 Therefore, elevated levels of sVE-cadherin in the vitreous fluid from patients with PDR could be a reflection of endothelial cell dysfunction associated with angiogenesis and breakdown of the inner bloodretinal barrier. In addition, we demonstrated a significant positive correlation between the vitreous levels of endocan and sVE-cadherin. Our findings
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
10 A. M. Abu El-Asrar et al. suggest that similar mechanisms might be involved in shedding of the VE-cadherin extracellular domain from endothelial cells and endocan biosynthesis and secretion by endothelial cells. Consistent with our previous report,31 we demonstrated that sEng is significantly upregulated in the vitreous fluid from patients with PDR, however, there was no significant correlation between endocan levels and the levels of sEng. The potential role of endocan in the process of angiogenesis was investigated. Several studies demonstrated that endocan has no direct effect on endothelial cell proliferation or in vivo angiogenesis. Recombinant endocan did neither induce formation of endothelial sprouts in fibrin gels nor it influence VEGF-induced sprouting.35 Inhibition of endogenous endocan by a neutralizing antibody19,35 or inhibition of endocan expression by antisense oligonucleotides33 did not inhibit in vitro angiogenesis. Finally, endocan did not induce angiogenesis when used in vivo model of angiogenesis based on the chick chorioallantoic membrane.35 In vivo, endocan overexpression by nontumorigenic epithelial cells induces tumor formation, whereas overexpression by tumorigenic cells sharply increases the growth rate of resulting tumors.7,19 Therefore, the cellular target of endocan seems to be the malignant epithelial cells rather than the endothelium itself. It is suggested that endocan acts locally in a paracrine positive feedback loop to support tumor growth (i.e. angiogenic growth factors stimulate endothelial cells to secrete endocan, which in turn acts by amplifying the mitogenic effect of protumoral growth factors).11,12 Similarly, the cellular target of endocan in PDR could be other cells involved in PDR progression such as myofibroblasts.38 In this study, we demonstrated that endocan was specifically localized in a-smooth muscle actinexpressing myofibroblasts. These findings suggest that myofibroblasts synthesize and secrete endocan and that endocan acts locally to promote myofibroblast activities such as migration and proliferation7,8 and could be involved in tissue remodeling and fibrosis. A recent study suggested a possible contribution of endocan in the endothelial–mesenchymal transition process.39 Endothelial–mesenchymal transition is a complex biological process in which endothelial cells lose their specific endothelial cell markers and acquire mesenchymal or myofibroblastic phenotype. This process is now recognized to be an important mechanism in the pathogenesis of pulmonary, cardiac and kidney fibrosis.40 In conclusion, our results demonstrate that endocan is overexpressed in PDR, particularly in patients with active PDR. Endocan could therefore be a new biomarker of angiogenesis in PDR. Further investigation is needed to determine if endocan may be used not only as a biomarker of retinal angiogenesis but also as a therapeutic target in PDR.
ACKNOWLEDGEMENTS The authors thank Mr. Dustan Kanagave, MSc for statistical assistance, Mr. Wilfried Versin for technical assistance and Ms. Connie B. Unisa-Marfil for secretarial work.
DECLARATION OF INTEREST All the authors do not have any conflict of interests with any trademark mentioned in the manuscript. The authors alone are responsible for the content and writing of the paper. This work was supported by Dr. Nasser Al-Rasheed Research Chair in Ophthalmology (Abu El-Asrar AM).
REFERENCES 1. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–660. 2. Conway EM, Collen D, Carmeliet P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2011;49: 507–521. 3. Spranger J, Pfeiffer AP. New concepts in pathogenesis and treatment of diabetic retinopathy. Exp Clin Endocrinol Diabetes 2001;109:S438–S450. 4. Salam A, Mathew R, Sivaprasad S. Treatment of proliferative diabetic retinopathy with anti-VEGF agents. Acta Ophthalmol 2011;89:405–411. 5. Lassalle P, Molet S, Janin A, Heyden JV, Tavernier J, Fiers W, et al. ESM-1 is a novel human endothelial cellspecific molecule expressed in lung and regulated by cytokines. J Biol Chem 1996;271:20458–20464. 6. Ho M, Yang E, Matcuk G, Deng D, Sampas N, Tsalenko A, et al. Identification of endothelial cell genes by combined database mining and microarray analysis. Physiol Genomics 2003;13:249–262. 7. Sarrazin S, Adam E, Lyon M, Depontieu F, Motte V, Landolfi C, et al. Endocan or endothelial cell specific molecule-1 (ESM-1): a potential novel endothelial cell marker and a new target for cancer therapy. Biochim Biophys Acta 2006;1765:25–37. 8. Abid MR, Yi X, Yano K, Shih SC, Aird WC. Vascular endocan is preferentially expressed in tumor endothelium. Microvasc Res 2006;72:136–145. 9. Strasser GA, Kaminker JS, Tessier-Lavigne M. Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 2010;115:5102–5110. 10. del Toro R, Prahst C, Mathivet T, Siegfried G, Kaminker JS, Larrivee B, et al. Identification and functional analysis of endothelial tip cell-enriched genes. Blood 2010;116: 4025–4033. 11. Grigoriu BD, Depontieu F, Scherpereel A, Gourcerol D, Devos P, Ouata T, et al. Endocan expression and relationship with survival in human non-small cell lung cancer. Clin Cancer Res 2006;12:4575–4582. 12. Be´chard D, Gentina T, Delehedde M, Scherpereel A, Lyon M, Aumercier M, et al. Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity. J Biol Chem 2001;276:48341–48349. Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Mcgill University on 02/26/15. For personal use only.
Expression of Endocan in PDR 11 13. Chen LY, Liu X, Wang SL, Qin CY. Over-expression of the Endocan gene in endothelial cells from hepatocellular carcinoma is associated with angiogenesis and tumour invasion. J Int Med Res 2010;38:498–510. 14. Leroy X, Aubert S, Zini L, Franquet H, Kervoaze G, Villers A, et al. Vascular endocan (ESM-1) is markedly overexpressed in clear cell renal cell carcinoma. Histopathology 2010;56:180–187. 15. Liu N, Zhang LH, Du H, Hu Y, Zhang GG, Wang XH, et al. Overexpression of endothelial cell specific molecule-1 (ESM-1) in gastric cancer. Ann Surg Oncol 2010;17: 2628–2639. 16. Kang YH, Ji NY, Lee CI, Lee HG, Kim JW, Yeom YI, et al. ESM-1 silencing decreased cell survival, migration, and invasion and modulated cell cycle progression in hepatocellular carcinoma. Amino Acids 2011;40:1003–1013. 17. Cornelius A, Cortet-Rudelli C, Assaker R, Kerdraon O, Gevaert MH, Pre´vot V, et al. Endothelial expression of endocan is strongly associated with tumor progression in pituitary adenoma. Brain Pathol 2012; 22:757–764. 18. Kim JH, Park MY, Kim CN, Kim KH, Kang HB, Kim KD, et al. Expression of endothelial cell-specific molecule-1 regulated by hypoxia inducible factor-1a in human colon carcinoma: impact of ESM-1 on prognosis and its correlation with clinicopathological features. Oncol Rep 2012;28: 1701–1708. 19. Scherpereel A, Gentina T, Grigoriu B, Se´ne´chal S, Janin A, Tsicopoulos A, et al. Overexpression of endocan induces tumor formation. Cancer Res 2003;63:6084–6089. 20. Almog N, Ma L, Raychowdhury R, Schwager C, Erber R, Short S, et al. Transcriptional switch of dormant tumors to fast-growing angiogenic phenotype. Cancer Res 2009;69: 836–844. 21. Be´chard D, Scherpereel A, Hammad H, Gentina T, Tsicopoulos A, Aumercier M, et al. Human endothelialcell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1. J Immunol 2001;167:3099–3106. 22. Vestweber D. VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arterioscler Thromb Vasc Biol 2008;28:223–232. 23. Harris ES, Nelson WJ. VE-cadherin: at the front, center, and sides of endothelial cell organization and function. Curr Opin Cell Biol 2010;22:651–658. 24. Wrobel T, Mazur G, Wolowiec D, Jazwiec B, Sowinska E, Kuliczkowski K. sVE-cadherin and sCD146 serum levels in patients with multiple myeloma. Clin Lab Haem 2006;28: 36–39. 25. Rabascio C, Muratori E, Mancuso P, Calleri A, Raia V, Foutz T, et al. Assessing tumor angiogenesis: increased circulating VE-cadherin RNA in patients with cancer indicates viability of circulating endothelial cells. Cancer Res 2004;64:4373–4377. 26. Sidibe¯ A, Mannic T, Arboleas M, Subileau M, GulinoDebrac D, Bouillet L, et al. Soluble VE-cadherin in
!
2014 Informa Healthcare USA, Inc.
27.
28. 29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
rheumatoid arthritis patients correlates with disease activity: evidence for tumor necrosis factor a-induced VE-cadherin cleavage. Arthritis Rheum 2012;64:77–87. Shih SC, Robinson GS, Perruzzi CA, Calvo A, Desai K, Green JE, et al. Molecular profiling of angiogenesis markers. Am J Pathol 2002;161:35–41. ten Dijke P, Goumans MJ, Pardali E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 2008;11:79–89. Duff SE, Li C, Garland JM, Kumar S. CD105 is important for angiogenesis: evidence and potential applications. FASEB J 2003;17:984–992. Li C, Guo B, Wilson PB, Stewart A, Byrne G, Bundred N, et al. Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer. Int J Cancer 2000;89:122–126. Abu El-Asrar AM, Nawaz MI, Kangave D, Abouammoh M, Mohammad G. High-mobility group box-1 and endothelial cell angiogenic markers in the vitreous from patients with proliferative diabetic retinopathy. Mediators Inflamm 2012;2012:697489. Aiello LP, Avery RL, Arrig PG, Keyt BA, Jampel HD, Shah ST, et al. Vascular endothelial growth factor in ocular fluid in patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994;331:1480–1487. Aitkenhead M, Wang SJ, Nakatsu MN, Mestas J, Heard C, Hughes CC. Identification of endothelial cell genes expressed in an in vitro model of angiogenesis: induction of ESM-1, (beta)ig-h3, and NrCAM. Microvasc Res 2002;63: 159–171. Shibuya M. Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol 2006;39:469–478. Rennel E, Mellberg S, Dimberg A, Petersson L, Botling J, Ameur A, et al. Endocan is a VEGF-A and P13K regulated gene with increased expression in human renal cancer. Exp Cell Res 2007;313:1285–1294. Villasante A, Pacheco A, Ruiz A, Pellicer A, Garcia-Velasco JA. Vascular endothelial cadherin regulates vascular permeability: implicatins for ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 2007;92:314–321. Navaratna D, McGuire PG, Menicucci G, Das A. Proteolytic degradation of VE-cadherin alters the bloodretinal barrier in diabetes. Diabetes 2007;56:2380–2387. Abu El-Asrar AM, Struyf S, Kangave D, Geboes K, Van Damme J. Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy. Eur Cytokine Netw 2006;17:155–165. Carillo LM, Arciniegas E, Rojas H, Ramirez R. Immunolocalization of endocan during the endothelialmesenchymal transition process. Eur J Histochem 2011; 55:e13. Piera-Velazquez S, Li Z, Jimenez SA. Role of endothelialmesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders. Am J Pathol 2011;179:1074–1080.
