Graefes Arch Clin Exp Ophthalmol (2008) 246:1707–1713 DOI 10.1007/s00417-008-0907-3

BASIC SCIENCE

Upregulation of stromal cell–derived factor 1 (SDF-1) expression in microvasculature endothelial cells in retinal ischemia-reperfusion injury Pinghong Lai & Tao Li & Jun Yang & Chengyang Xie & Xiaobo Zhu & Hui Xie & Xiaoyan Ding & Shaofen Lin & Shibo Tang

Received: 15 February 2008 / Revised: 18 June 2008 / Accepted: 7 July 2008 / Published online: 16 August 2008 # Springer-Verlag 2008

Abstract Background Stromal cell–derived factor 1 (SDF-1) is a potent chemotactic and angiogenic factor that has been proposed to play a role in the development of neovascularization. In this study, we explored the expression of SDF-1 in a rat model of retinal ischemia-reperfusion injury and investigated the possible role of retinal microvasculature endothelium cells in generation of this chemokine. Methods Expression patterns of SDF-1 were studied in retina suffering ischemia-reperfusion insult in SpragueDawley rats by elevating the intraocular pressure to 110 mm for 60 minutes. The relative level of SDF-1 mRNA in retinas following 6, 12 and 24 hours reperfusion was determined by semi-quantitative RT-PCR. Immunohistochemical methods were used to detect specific lesions expressing SDF-1. The gene expression of SDF-1 in cultured human retinal microvasculature endothelial cells

(HRMEC) under hypoxia conditions was assessed by semi-quantitative RT-PCR. The SDF-1 protein was analyzed by immunocytochemistry and fluorescence-activated cell sorting. Results Upregulation of SDF-1 mRNA (at 6, 12, and 24 hours of reperfusion) was observed, with the expression peak occurring at 12 hours. SDF-1 positive cells appeared initially around the retinal vessels,which diffused into the inner retinal layers. Hypoxia enhanced the expression of HIF-1 and SDF-1 mRNA in HRMEC. The production of SDF-1 protein by HRMEC was increased up to 320% after 6 hours of hypoxia, as demonstrated by fluorescenceactivated cell sorting. Conclusions The results of our study indicate that endogenous SDF-1 is up-regulated in retinal microvasculature suffering ischemia insult, and that microvasculature endothelial cells are potential contributors for generation of SDF-1 in ischemic retina.

We don’t have any financial relationship with the organization that sponsored the research. We have full control of all primary data, and agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review these data.

Keywords Chemokine . Stromal-derived factor 1 . Endothelial cell . Retina . Ischemia-reperfusion injury

P. Lai : T. Li : X. Zhu : X. Ding : S. Lin : S. Tang (*) State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People’s Republic of China e-mail: [email protected] P. Lai : C. Xie : H. Xie Jiangxi Eye Center, Jiangxi Provincial People’s Hospital, Nanchang, Jiangxi Province, People’s Republic of China J. Yang Ophthalmology Department, The First People’s Hospital of Yunnan Province, 172 Jinbi Road, Kunming, Yunnan Province, People’s Republic of China

Introduction Retinal ischemia is blamed for being the major stimulator for proliferative retinopathy such as proliferative diabetic retinopathy. In events of ischemia, the release of growth factors such as vascular endothelial growth factor (VEGF) is increased at the site of injury, which function by increasing vascular permeability, promoting endothelial cell activation,proliferation, and, eventually, capillary formation [1–3]. Recent studies have suggested an important role for stromal cell–derived factor 1 (SDF-1) in the recruitment of

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endothelial progenitor cells (EPCs) to home to sites of ischemic injury and facilitate repair. SDF-1 has been shown to be upregulated in many damaged tissues as part of the injury response. In a model of hypoxia-induced retinal neovascularization, SDF-1 levels are elevated in the vitreous humor, and administration of an anti–SDF-1 antibody prevents recruitment of endothelial precursors in the eye [4–7]. Although these observations strongly suggest a role for SDF-1 in ischemic retinopathy, the underlying mechanisms by which SDF-1 is involved in retinal injury response, and its possible cellular source in an ischemia retinal context remain to be elucidated [8]. Previously, Hill et al. demonstrated that SDF-1 is expressed perivascularly in the injured region in an adult mouse model of stroke [9]. Recently, they showed that SDF-1 cellular expression was largely localized to reactive astrocytes around small blood vessels in neonatal hypoxic– ischemic injury [10]. However, whether the vessel endothelium cells, a component of small vessels, are contributors to the production of SDF-1 remains unknown. Considering ischemia is the primary stimulator to retinal angiogenesis diseases, here we used a retinal ischemia-reperfusion model to investigate the in vivo expression of SDF-1; meanwhile, we used cultured human retinal microvasculature endothelial cells (HRMEC) under hypoxic conditions to investigate the in vitro expression of this chemokine [11]. In addition, the expression of hypoxia inducible factor 1 (HIF-1), a transcription factor mediating cellular responses to hypoxia, was characterized in HRMEC to explore the possible mechanism underlying SDF-1 expression regulation. Overall, the purpose of the present study was to characterize the expression pattern of SDF-1 in ischemia-reperfusion retina and to test the hypothesis that microvasculature endothelial cells contribute to the generation of SDF-1.

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reservoir containing saline. Retinal ischemia was induced by elevating the intraocular pressure to 110 mm, and was confirmed by ophthalmoscopic examination by noting the blanching of retinal arteries and loss of the red reflex. One hour later, the cannulating needle was removed to allow for reperfusion of the retinal vasculature. Animals were killed at 6 hours, 12 hours and 24 hours respectively, following reperfusion with a pentobarbital overdose. The eyes were enucleated. Six untreated normal Sprague-Dawley rats were used as control. Endothelial cells culture and hypoxia treatment HRMEC cells were obtained from a healthy donor and were grown on fibronectin-coated dishes in endothelial cells basal medium (EBM) containing 2% fetal bovine serum and growth medium supplement with penicillin 100 UL/ml and streptomycin 100 ug/ml [12–14]. Before normoxic or hypoxic exposure, cells were washed with phosphate-buffered saline, and fresh medium was added. Exposure to normoxia was performed under standard culture conditions in a humidified incubator maintained at 5% CO2, 95% room air at 37°C. For hypoxic exposure, cells were placed in a controlled environment chamber flushed with 1% O2, 5% CO2, and 94% N2 for 6 hours [15]. Media were changed twice a week. The viability of cells after incubation was assessed using trypan blue dye. Cells incubated under standard normoxic conditions (95% air, 5% CO2) from the same batch and passage were used as controls. All experiments were performed on cells approaching confluence. Only passages 3–5 were used. Cells were identified as endothelial cells on the basis of their cobblestone morphology and immunoreactivity with anti-factor VIII antibody. Immunohistochemical and immunocytochemistry of SDF-1

Materials and methods Experimental retina ischemia and reperfusion injury A total of 24 Sprague-Dawley rats, within the weight range between 200 and 250 g, were included in this study. All animals were treated in accordance with the ARVO statement for the care and use of laboratory animals and were approved by the Animal Care and Use Committee of Sun Yat-sen University. Transient retinal ischemia was induced as previously described [11]. Briefly, rats were anesthetized with ketamine–xylazine (10 mg/kg and 4 mg/kg respectively) intramuscularly, The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride (Santen, Japan). The anterior chamber of the eye was cannulated with a 25-gauge needle connected to an elevated

Eyes from untreated rats (n=6) and eyes from rats treated with re-perfusion after 6, 12 and 24 hours (n = 6), respectively, were obtained and fixed in 4% paraformaldehde in 0.1 M phosphate-buffered saline (PBS; pH 7.4) at 4°C overnight. Eyes were then processed, embedded in paraffin and sectioned. Immunohistochemistry studies were performed following the avidin-biotin complex (ABC) method using a rabbit anti-rat SDF-1 antobody (1:100; Santa Cruz, USA). The immunochemistry staining of the adhesive HRMEC cells was carried out directly on the culture plates according to the manufacturer’s instructions. Briefly, cells were fixed for 3 minutes at room temperature with 4% paraformaldehyde, washed three times in 0.1M PBS and then incubated in 5% normal goat serum with 0.1%Tixton×100 for 20 minutes at room temperature. After washing with PBS, the cultures were

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incubated with rabbit anti-human HIF-1 and SDF-1, respectively, at 4°C overnight. After washing, cultures were incubated with bio-labeled goat-rabbit IgG antibody at 37°C for 30 minutes. After washing, cells were instructed with strept actividin-biotin complex (BOSTER, China) for 20 minutes and then with 3-amino-9-ethylcarbazole (MAIXIN, China). Photographs were taken using a Nikon SLR camera. SDF-1 and HIF-1 mRNA isolation and reverse transcription polymerase chain reaction (semi-quantitative RT-PCR) To characterize the regulation of SDF-1 expression in ischemia-reperfused retina, total RNA was isolated from retinas 6, 12 and 24 hours after ischemia reperfusion injury, or from non-treated eyes (n= 6). For cultured HRMEC cells, RNA was isolated from approximately 106 cells using an RNAgents RNA Isolation Kit (Promega). RNA was subjected to a semi-quantitative RT-PCR analysis. The primers were, for SDF-1: 5′-tgc atc agt gac ggt aag cca-3′ and 3′-ttg tcc agg tac tct tgg atc-5′;for HIF1: 5′-aca agt cac cac agg aca gta cag-3′ and 3′-cca gtg act ctg gac ttg atc ta-5′;for β-actin:5′-ctc gaa ccc taa ggc caa3′,3′-tca cgc cac gat ttc cct c-5′.. The PCR cycling conditions were 10 min at 94°C, followed by 35 cycles at 94°C for 30 s, and 60°C for 30 s, 72°C for 20 s, with a final extension step at 72°C for 5 min. PCR products were analyzed by agarose/ethidium bromide gel electrophoresis. Densitometry was performed on all gels, and each signal was normalized to the corresponding β-actin signal using an NHI imaging system. Each PCR reaction was repeated three times.

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Results Temporal expression pattern of SDF-1 mRNA in retina following Ischemia-reperfusion injury Induction of SDF-1 mRNA in ischemia-reperfusion retinas was confirmed by RT-PCR. SDF-1 mRNA was expressed at a low basal level in control retina. At 6 hours after reperfusion, SDF-1 mRNA was up-regulated (by 2-fold), peaked at 12 hours (by 4-fold), and then declined at 24 hours (Fig. 1). Localization of SDF-1 protein in the retina We analyzed the distribution of SDF-1 protein in the retina after ischemia-reperfusion injury by immunohistochemical staining. SDF-1 expression was barely detectable in the uninjured retina, and was only weakly detectable in the inner sclera (data not shown). In contrast, 6 hours after ischemia-reperfusion insult, prominent staining was seen in the inner retina, in a vascular and perivascular distribution. At 12 hours, SDF-1 protein diffused in the inner retinal layer. By 24 hours, faint staining for SDF-1 were observed in the whole retina, particularly along the outer nuclear layer. (Figure 2).

Fluorescence-activated cell sorting (FACS) analysis FACS analysis was performed on cultured cells of P2–4 in both normoxia and hypoxia systems. Cells were harvested, washed with PBS, and then fixed with 0.5% paraformaldehde for 1 hour at 4°C, 20 ug of propidium iodide and 10 ug of NaN3 in PBS were added. The monoclonal antibodies used in the study were PE-conjugated anti-SDF1 MoAb and PE-conjugated anti-HIF-1 MoAb (Sigma, USA).Isotype-identical antibodies were used as controls. After 10 min incubation at room temperature, samples were analyzed on a FACS flow cytometer (Beckman-Coulter, USA). Data were analyzed, and the percentages of SDF-1 and HIF-1 positive cells were calculated. Statistical analysis Statistical comparisons between experimental groups in all the above assays were evaluated by Student’s t-test; a P value≤0.05 was considered significant.

Fig. 1 SDF-1 mRNA expression in ischemia-reperfusion retina. a Bar graph of semiquantitative results of the mean densitometry from four rats. b PCR amplification followed by gel electrophoresis and ethidium bromide staining of the samples. The relative level of mRNA expression was quantified, and corrected for the levels of βactin mRNA expression. The values in each column represent the mean ± SD (n=3)

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Hypoxia-induction of HIF-1 and SDF-1 expression in HRMEC cells Cells cultured under hypoxia conditions showed no morphologic changes by light microscopy after exposures over 24 hours. The majority cells excluded trypan blue dye (>98%), and could subsequently be passaged normally. Sub-confluent HRMEC cultures were exposed to hypoxia for 6 hours. HIF-1, SDF-1 mRNA was analyzed by RTPCR. Our results showed low basal HIF-1 and SDF-1 mRNAs in normoxic cells. However, hypoxia markedly enhanced the expression of both SDF-1 and HIF-1 mRNAs in HRMEC cells (Fig. 3). The expression of HIF-1 and SDF-1 was studied by immunohistochemistry. Under normoxic conditions, HIF-1 expression was primarily detected in the nuclei of endothelial cells, while SDF-1 was expressed in a cytoplasmic pattern. Under hypoxic conditions, HIF-1and SDF-1 were consistently augmented (Fig. 4). Augmentation of HIF-1 and SDF-1 by hypoxia was also confirmed by FACS assays which revealed that the ratio of HIF-1 positive cells in normoxia culture was 21.6± 0.6%, whereas in hypoxia-induced cultures it was 92.1± 0.8% (n=3, P<0.001) (Fig. 5). Similarly, a proportion (37%) of endothelial cells (HRMECs) expressed SDF-1 during normoxia culture (Fig. 3, middle). Together, these data confirm that hypoxia enhanced the expression of HIF1 and SDF-1 in HRMEC cells.

Discussion In this study, we characterized the time course and localization of the SDF-1 expression in retina suffering ischemia-reperfusion in rat. We found that SDF-1 appeared initially in ischemic tissue in a vascular and perivascular distribution, and then diffused into other inner retinal layers. Similarly, upregulation of SDF-1 gene and protein

Fig. 2 Histological distribution of SDF-1 in ischemia-reperfusion retina. Rats suffering retinal ischemia induced by elevating the intraocular pressure were enucleated at 6 hours, 12 hours and 24 hours following reperfusion and stained by anti-SDF-1 antibody. a Six hours after ischemia-reperfusion insult, prominent staining was seen around small blood vessels in the inner retina. b At 12 hours, SDF-1 diffused in the inner retinal layer. Bar=150 mm. c By 24 hours, faint staining for SDF-1 in the whole retina, particularly along the outer nuclear layer

Fig. 3 Hypoxia induction of SDF-1, HIF-1 gene expression in cultured HRMEC cells. HRMEC cells were cultured under normoxic or hypoxic conditions for up to 24 hours. The electrophoresis pattern of PCR products for SDF-1, HIF-1 in cultured HRMEC cells. Lane 1, Normoxia; Lane 2, Hypoxia. Lane 3, DNA mark

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Fig. 4 Hypoxia induction of SDF-1, HIF-1 protein expression in cultured HRMEC cells. HRMEC cells were cultured under normoxic or hypoxic conditions for 6 hours. HIF-1, SDF-1 protein was detected by immunocytomistry. Lowstaining of SDF-1 and HIF-1 in HRMEC under normoxia conditions. Strong SDF-1 staining in HRMEC suffering hypoxia. Red, SDF-1+ cells. Blue, HIF-1+ cells. Bar=100 mm

expression was observed in cultured HRMEC cells exposed to hypoxia, suggesting that microvasculature endothelial cells contribute to the source of SDF-1. These results are consistent with the hypothesis that endothelium cells are involved in the generation of SDF-1 in ischemia-induced retinopathy. Furthermore, we demonstrated that the pattern of induction of SDF-1 in HRMEC cells under hypoxia was similar to that seen in HIF-1, suggesting HIF-1 may be a mediator for SDF-1 expression in hypoxia environment. Among the range of inflammation chemokines expressed in the ischemia microenvironment, SDF-1 has emerged as a major attractant of reparative cells. In addition to targeting hematopoietic cells, SDF-1 is also known to play a critical role in adult injury repair, including neurogenesis, neuroblast migration, and neuronal organization as well as vasculogenesis [16–18]. Therefore, it is important to determine the spatial expression of SDF-1 relative to injury. SDF-1 has been reported in the literature to be expressed by bone marrow endothelium cells, fibroblasts, osteoblasts and platelets [5, 17–19]. It is, however, also secreted by stromal and endothelial cells of organs such as heart, skeletal muscle, liver, brain, and kidney [20–23]. Some studies have shown that SDF-1 expression was localized in endothelial cells, whereas others showed that SDF-1 mainly colocalized with pre-endothelial cells of fibroblastic or smooth muscle nature. More recently, SDF-1 was shown to localize in glia cells near the surface of the retina [24,25]. In a study, the principal localization of SDF-1 expression

Fig. 5 Flow cytometric analysis of surface HIF-1 /SDF-1 expression in HRMEC cells cultured in normoxia or hypoxia condition. Cells were stained with either specific mouse anti-human-HIF-1/SDF-1 antibodies or isotype-matched control antibody. Red: normoxia; Black: hypoxia

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following injury was astrocytes in small blood vessels [10]. However, whether the vessel endothelium cells, a component of small vessels, contribute to the expression of SDF-1 is uncertain. In the present study, we clearly confirmed that vessel endothelium cells in ischemia microenvironment also contributed to SDF-1 expression. This notion is supported by three lines of evidences. Firstly, SDF-1 mRNA was found more than 2-fold in the injured tissue in comparison to the normal tissue (Fig. 1). Secondly, SDF1 was abundant in the inner retina suffering ischemiareperfusion insult (Fig. 2), suggesting that retinal small vessels represent the sources of upregulation of SDF-1. Thirdly, hypoxia increases the low basal SDF-1 mRNA and protein levels in normoxic cells (Figs. 3, 4), providing direct evidence that endothelial cells play a role in generation of this potent chemokine. These findings are consistent with a previous study. Using a colocalization of CD31 and SDF-1 immunostaining, Ceradini et al. demonstrate that endothelial cells are a source of SDF-1 expression in ischemic tissue in a model of soft-tissue ischemia [25]. In our study, the spatial location and temporal expression pattern of SDF-1 in I/H retinal is similar to that seen in mouse neonatal brain having undergone I/H injury [10]. More importantly, the expression of SDF-1 by vessel endothelium cells resembles a temporal pattern similar to that seen in the ischemia-reperfusion retina, with low basal SDF-1 mRNA and protein levels in normoxic cells that increased with hypoxia (Figs. 1, 2). This up-regulation was evident at 6 hours, and continued to increase at 12 hours. These observations were parallel with the expression pattern of in vivo SDF-1 in retinas suffering ischemiareperfusion insult. The physiological mechanisms underlying the localized expression of SDF-1 in injured tissue are not completely understood. Many factors generated during tissue injury could potentially regulate SDF-1 expression [25].However, the primary stimulator may involve the transcription factor HIF-1, which is activated by hypoxia [25, 26]. Karshovska et al. demonstrated a direct contribution of HIF-1 to SDF1–mediated neointima formation after vascular injury [27]. Ceradini et al. found that SDF-1 expression was directly proportional to reduced local tissue oxygen tensions [25, 26]. In our study, the time course of upregulation of HIF-1 mRNA was similar to that of SDF-1 in HRMEC cells. Through FACS analysis, we clearly demonstrated that hypoxic induction of SDF-1 production was accompanied by the hypoxic induction of HIF-1 production in HRMEC cells (Fig. 5). Therefore, the hypoxia-HIF-1-SDF-1 functional axis may represent a critical pathway in ischemia microenvironment. The current findings in our study may have important implications, because the cell type expressing SDF-1 and its

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localization is highly related to its function [10, 28]. Previously, SDF-1 cellular expression was in large part localized to astrocytes around small blood vessels [10]. Our study demonstrates that hypoxia augments the expression and production of SDF-1 in HRMEC cells. Though HRMEC cells may not be the major source of SDF-1 seen in the ischemia microenvironment, endothelial cell–derived SDF-1 may play a unique role in pathologic blood vessel formation. It has been suggested that SDF-1, especially the surface-bound form, activates the adhesion molecules of hematopoietic progenitors, thereby enhancing adhesion of the cells to endothelial cells [16, 20]. It is possible that HEMEC-derived SDF-1 enhances the transendothelial migration of hematopoietic progenitor cells [29]. Given that SDF-1 and its receptor (CXCR4) are expressed by endothelial cells and are induced by HIF-1, endogenous SDF-1–CXCR4 may provide critical signals directing vascular remodeling and neovascularization [30]. Further studies on the implications of modulation of SDF-1 production by HRMEC are indicted. In summary, out findings suggest that microvasculature endothelial cells may represent a critical source of SDF-1 in retina suffering ischemia-reperfusion injury. Acknowledgments This study was supported by the 5010 Project of Sun Yat-sen University in China (Grant No. 2006-45) and the Clinical Science Project of Ministry of Health in China (Grant No. 2007-353) and a scientific-technological program of Jiangxi Health department (200732020).

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