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Keywords:

  • diabetic retinopathy;
  • endothelial progenitor cells;
  • monocytes;
  • stromal cell-derived factor-1;
  • vasculogenesis

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Purpose:  The role of vasculogenesis, recruitment and differentiation of circulating bone-marrow-derived endothelial precursor cells into mature endothelium in proliferative diabetic retinopathy (PDR) remains undefined. We investigated the presence of bone-marrow-derived endothelial precursor cells and the expression of the chemotactic pathway SDF-1/CXCL12−CXCR4 in PDR epiretinal membranes.

Methods:  Membranes from eight patients with active PDR and nine patients with inactive PDR were studied by immunohistochemistry using antibodies against CD133, vascular endothelial growth factor receptor-2 (VEGFR-2), CD14, SDF-1 and CXCR4.

Results:  Blood vessels expressed CD133, VEGFR-2, CD14, SDF-1 and CXCR4 in 10, 10, 10, seven and seven out of 17 membranes, respectively. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing CD133 (rs = 0.646; p = 0.005), VEGFR-2 (rs = 0.704; p = 0.002), CD14 (rs = 0.564; p = 0.018) and SDF-1 (rs = 0.577; p = 0.015). Stromal cells in close association with blood vessels expressed CD133, VEGFR-2, CD14 and CXCR4 in 10, 12, 13 and 14 membranes, respectively. The number of blood vessels expressing CD133 (p = 0.013), VEGFR-2 (p = 0.005), CD14 (p = 0.008) and SDF-1 (p = 0.005), and stromal cells expressing CD133 (p = 0.003), VEGFR-2 (p = 0.013) and CD14 (p = 0.002) was significantly higher in active membranes than in inactive membranes.

Conclusion:  Bone-marrow-derived CD133+ endothelial progenitor cells and CD14+ monocytes may contribute to vasculogenesis in PDR.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ischaemia-induced retinal neovascularization in association with the outgrowth of fibrovascular epiretinal membranes at the vitreoretinal interface is the pathological hallmark feature of proliferative diabetic retinopathy (PDR). The mechanisms governing this aberrant neovascularization during PDR are still being elucidated. Until recently, it was generally accepted that in adults the formation of new blood vessels results exclusively from surrounding pre-existing vessels by sprouting (a process referred to as angiogenesis). Vasculogenesis (defined as the recruitment and in situ differentiation of vascular endothelial cells from circulating bone-marrow-derived endothelial precursor cells) was thought to occur only in the embryonic phases of vascular development. However, recent studies have shown that circulating bone-marrow-derived endothelial precursor cells home in to sites of neovascularization and differentiate into endothelial cells (Dome et al. 2008).

In peripheral blood mononuclear cells, there are several possible bone-marrow-derived endothelial precursor cells such as endothelial progenitor cells (EPCs) and CD14+ monocytes. EPCs were initially identified and isolated from peripheral blood on the basis of vascular endothelial growth factor receptor-2 (VEGFR-2) and CD34 expression by these cells. These cells have the capacity to differentiate into functional endothelial cells and were shown to be incorporated into sites of physiological and pathological neovascularization in vivo (Asahara et al. 1997; Shi et al. 1998). At present, it is widely accepted that EPCs are CD133+/CD34+/VEGFR2+ cells (Dome et al. 2008). Human CD133 (human prominin-1) is a novel 120-kDa glycosylated polypeptide that contains 5-transmembrane domains with an extracellular N-terminus and a cytoplasmic C-residue, and two large extracellular loops with eight consensus sites for N-linked glycosylation (Mizrak et al. 2008). Several studies demonstrated that CD133 is a novel marker for human haematopoietic precursor stem and EPCs (Mizrak et al. 2008). The expression of CD133 is lost once these progenitors differentiate into more mature endothelial cells (Dome et al. 2008). Quirci et al. (2001) demonstrated that CD133+ bone marrow cells, a subset of CD34+ haematopoietic progenitors, can give rise to a homogenous pure population of endothelial cells, which have a high proliferative capacity and can be activated by inflammatory cytokines. In addition, Harraz et al. (2001) reported that peripheral blood CD14+ monocytes also have the potential to differentiate into the cell lineage with endothelial cell markers in vitro and to incorporate into the endothelium of blood vessels in mouse ischaemic limbs.

Stromal cell-derived factor-1 (SDF-1/CXCL12) is a member of the CXC chemokine family that was originally isolated from murine bone marrow stromal cells and characterized as a pre-B-cell growth-stimulating factor. CXCR4, a 7-transmembrane-spanning G protein-coupled receptor, is one of the two receptors for SDF-1 (Murphy et al. 2000; Burns et al. 2006). Recent studies have shown that SDF-1/ CXCR4 interaction plays an important role in EPC migration differentiation, proliferation and survival (Yamaguchi et al. 2003; De Falco et al. 2004; Walter et al. 2005; Aghi et al. 2006; Reddy et al. 2008; Stellos et al. 2008).

The role of vasculogenesis in the pathogenesis of PDR remains undefined. Therefore, the aim of the present study was to determine whether circulating bone-marrow-derived endothelial precursor cells contribute to neovascularization in the epiretinal membranes from patients with PDR and whether the components of the SDF-1/CXCR4 chemotactic pathway are expressed in these membranes. The level of vascularization and proliferative activity in epiretinal membranes was determined by immunodetection of the panendothelial marker CD34 and the proliferating cell marker Ki-67.

Materials and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Epiretinal membrane specimens

Epiretinal fibrovascular membranes were obtained from 17 patients with PDR during pars-plana vitrectomy. Membranes were fixed in 10% formalin solution and embedded in paraffin. The clinical ocular findings were graded at the time of vitrectomy for the presence or absence of patent new vessels on the retina or optic disc. Patients with active PDR were graded as such on the basis of visible patent new vessels on the retina or optic disc or their absence (inactive PDR). Active PDR was present in eight patients and inactive PDR was present in nine. The study was conducted according to the tenets of the Declaration of Helsinki, and informed consent was obtained from all patients. The study was approved by the Research Centre at the College of Medicine, King Saud University.

Immunohistochemical staining

Endogenous peroxidase was abolished with 2% hydrogen peroxide in methanol for 20 min, and non-specific background staining was blocked by incubating the sections for 5 min in normal swine serum. For Ki-67, CD133, VEGFR-2 and SDF-1 detection, antigen retrieval was performed by boiling the sections in 10 mm Tris–ethylene-diaminetetraacetic acid (EDTA) buffer (pH 9) for 30 min. For CD34 and CXCR4 detection, antigen retrieval was performed by boiling the sections in 10 mm citrate buffer (pH 6) for 30 min. Subsequently, the sections were incubated with the monoclonal and polyclonal antibodies listed in Table 1. Optimal working concentration and incubation time for the antibodies were determined in earlier pilot experiments. The sections were then incubated for 30 min with goat anti-rabbit or anti-mouse immunoglobulins conjugated to peroxidase-labelled dextran polymer (EnVision+; Dako, Carpinteria, California, USA). The reaction product was visualized by incubation for 10 min in 0.05 m acetate buffer at pH 4.9, containing 0.05% 3-amino-9-ethylcarbazole (Sigma-Aldrich, Bornem, Belgium) and 0.01% hydrogen peroxide, resulting in bright-red immunoreactive sites. The slides were then faintly counterstained with Harris haematoxylin.

Table 1.   Monoclonal and polyclonal antibodies used in this study.
Primary antibodyDilutionIncubation time Source*
  1. mc, monoclonal; pc, polyclonal; VEGF, vascular endothelial growth factor.

  2. * Location of manufacturers: BD Biosciences, San Jose, California, USA; BioGenex, San Ramon, California, USA; Abcam, Cambridge, UK; Nova Castra, A. Menarini Diagnostics, Zaventem, Belgium; R&D Systems, Abingdon, UK.

Anti-CD34 (clone My 10) (mc)1/5060 minBD Biosciences
Anti-Ki-67 (clone MIB-1) (mc)1/10060 minBioGenex
Anti-CD133 (catalogue no. ab19898) (pc)1/100OvernightAbcam
Anti-VEGF receptor-2 (catalogue no. ab2349) (pc)1/5060 minAbcam
Anti-CD14 (clone 7) (mc)1/2060 minNova Castra
Anti-CXCR4 (clone 44716) (mc)1/10060 minR&D Systems
Anti-SDF-1/CXCL12 (clone 79018) (mc)1/5060 minR&D Systems

To identify the phenotype of cells expressing VEGFR-2 and CXCR4, sequential double immunohistochemistry was performed as described previously (Abu El-Asrar et al. 2008).

Omission or substitution of the primary antibody with an irrelevant antibody of the same species and staining with chromogen alone were used as negative controls. Sections from patients with colorectal carcinoma and breast cancer were used as positive controls.

Quantitation

Blood vessels and cells were counted in five representative fields, using an eyepiece calibrated grid in combination with the 40× objective. With this magnification and calibration, the blood vessels and cells present in an area of 0.33 × 0.22 mm were counted. Data were expressed as mean values ± standard deviation (SD) and analysed by the Mann–Whitney test. Spearman’s rank correlation coefficients were computed to investigate the linear relationship between the variables investigated. A p-value < 0.05 indicated statistical significance. BMDP 2007 statistical package was used for the statistical analysis. (BMDP Statistical Software, Inc., Los Angeles, CA, USA).

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Immunohistochemical analysis

There was no staining in the negative control slides (Fig. 1A). Fourteen (82.3%) membranes showed blood vessels positive for the panendothelial marker CD34, with a mean number of 18.9 ± 19.8 (range 0–66). In addition, stromal cells expressing CD34 were noted in close association with blood vessels (Fig. 1B). Nuclear immunoreactivity for the proliferating cell marker Ki-67 (Fig. 1C) was present in seven (41.2%) membranes, with a mean number of 15.5 ± 27.8 (range 0–102). Immunoreactivity for CD133 was present in 10 (58.8%) specimens. CD133+ small blood vessels were noted (Fig. 1D) [mean 8.1 ± 10.8 (range 0–35)]. Stromal cells expressing CD133 (Fig. 1E) [mean 17.7 ± 19.8 (range 0–65)] and CD133+ intravascular cells (Fig. 1F) were also noted.

image

Figure 1.  Negative control slide that was treated identically with an irrelevant antibody showing no labelling (A) (original magnification ×40). Immunohistochemical staining for CD34 showing blood vessels and stromal cells (arrows) positive for CD34 (B) (original magnification ×40). Immunohistochemical staining for Ki-67 showing nuclear immunoreactivity in proliferating cells (C). Immunohistochemical staining for CD133 showing CD133+ blood vessels (D), CD133+ stromal cells (E) and CD133+ intravascular cells (F) (original magnification ×100).

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Immunoreactivity for VEGFR-2 was present in 12 (70.6%) membranes. Expression of VEGFR-2 by vascular endothelial cells (Fig. 2A) was noted in 10 (58.8%) specimens [mean 11.5 ± 3.3 (range 0–39)]. Stromal cells expressing VEGFR-2 (Fig. 2B) were present in close association with blood vessels in 12 (70.6%) specimens [mean 34.3 ± 38.8 (range 0–125)]. Immunoreactivity for VEGFR-2 was observed in the nucleus and in the cytoplasm. This pattern of expression is consistent with previous studies that demonstrated rapid internalization of VEGFR-2 in the nucleus induced by VEGF stimulation or in vitro wounding of endothelial cell monolayers (Santos et al. 2007).

image

Figure 2.  Immunohistochemical staining for vascular endothelial growth factor receptor-2 (VEGFR-2) showing VEGFR-2+ endothelial cells (A) and VEGFR-2+ stromal cells (B). Immunohistochemical staining for CD14 showing CD14+ blood vessels (C), CD14+ stromal cells (D) and CD14+ intravascular cells (E). Immunohistochemical staining for stromal cell-derived factor-1 (SDF-1) showing SDF-1-expressing blood vessels (F) (original magnification ×100).

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Immunoreactivity for CD14 was noted in 13 (76.5%) membranes. CD14+ blood vessels (Fig. 2C) were detected in 10 (58.8%) membranes [mean 5.5 ± 6.7 (range 0–20)]. Stromal cells expressing CD14 (Fig. 2D) were present in close association with blood vessels in 13 (76.5%) membranes [mean 15.1 ± 15.3 (range 0–55)]. CD14+ intravascular cells were also noted (Fig. 2E).

Immunoreactivity for the chemokine SDF-1 was present on vascular endothelial cells (Fig. 2F) in seven (41.2%) membranes [mean 3.8 ± 6.4 (range 0–17)]. Immunoreactivity for the chemokine receptor CXCR4 was present in 14 (82.3%) specimens. Small blood vessels positive for CXCR4 (Fig. 3A) were present in seven (41.2%) membranes [mean 2.7 ± 5.0 (range 0–20)]. Stromal cells expressing CXCR4 (Fig. 3B) were noted in 14 (82.3%) membranes [mean 24.4 ± 26.9 (range 0–95)]. The CXCR4+ cells were closely associated with the new vessels within the membranes. Double immunohistochemistry indicated that VEGFR-2+ CD34+ cells (Fig. 3C) and CXCR4+ CD34+ cells (Fig. 3D) were present in the vascular endothelium of small blood vessels and in the stroma closely associated with new vessels.

image

Figure 3.  Immunohistochemical staining for CXCR4 showing CXCR4+ blood vessels (A) and CXCR4+ stromal cells in close association with blood vessels (B). Double immunohistochemistry for vascular endothelial growth factor receptor-2 (VEGFR-2) (red) and CD34 (blue) showing cells co-expressing VEGFR-2 and CD34 in the vascular endothelium (arrows) and in close association with blood vessels (arrowheads) (C). Double immunohistochemistry for CXCR4 (red) and CD34 (blue) showing cells co-expressing CXCR4 and CD34 in the vascular endothelium (arrows) and in close association with blood vessels (arrowheads) (D) (original magnification ×100).

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Correlations and relationship with PDR activity

The mean number of blood vessels expressing CD34 (p = 0.001), CD133 (p = 0.013), VEGFR-2 (p = 0.005), CD14 (P = 0.008) and SDF-1 (p = 0.005), and stromal cells expressing Ki-67 (p = 0.005), CD133 (p = 0.003), VEGFR-2 (p = 0.013) and CD14 (p = 0.002) were significantly higher in membranes from patients with active PDR than in membranes from patients with inactive PDR (Table 2). Table 3 shows Spearman’s rank correlation coefficients between variables studied.

Table 2.   Mean numbers in relation to type of proliferative diabetic retinopathy (PDR).
VariableActive PDR (n = 8) (mean ± SD)Inactive PDR (n = 9) (mean ± SD)P-value (Mann–Whitney test)
  1. SD, standard deviation; VEGFR-2, vascular endothelial growth factor receptor-2; SDF-1, stromal cell-derived factor-1.

  2. * Statistically significant at 5% level of significance.

Blood vessels expressing CD3433.9 ± 19.25.7 ± 6.40.001*
Cells expressing Ki-6732.5 ± 33.80.44 ± 1.30.005*
Blood vessels expressing CD13314.0 ± 11.92.8 ± 6.60.013*
Cells expressing CD13332.5 ± 19.04.4 ± 7.00.003*
Blood vessels expressing VEGFR-220.8 ± 13.43.3 ± 5.90.005*
Cells expressing VEGFR-260.3 ± 42.511.2 ± 12.40.013*
Blood vessels expressing CD1410.1 ± 7.01.4 ± 2.70.008*
Cells expressing CD1426.3 ± 14.65.1 ± 6.60.002*
Blood vessels expressing SDF-17.9 ± 7.50.11 ± 0.330.005*
Blood vessels expressing CXCR44.1 ± 6.81.3 ± 2.20.387
Cells expressing CXCR436.6 ± 32.413.4 ± 15.60.123
Table 3.   Spearman’s rank correlation coefficients.
VariableBlood vessels expressing CD34Blood vessels expressing CD133Cells expressing CD133Blood vessels expressing VEGFR-2Cells expressing VEGFR-2Blood vessels expressing CD14Cells expressing CD14Blood vessels expressing SDF-1Blood vessels expressing CXCR4Cells expressing CXCR4
  1. VEGFR-2, vascular endothelial growth factor receptor-2; SDF-1, stromal cell-derived factor-1.

  2. * Statistically significant at 5% level of significance. Where the row and column meet is the correlation coefficient and P-value for the two variables.

Blood vessels expressing CD133
 rs0.646         
 P0.005*         
Cells expressing CD133
 rs0.7100.916        
 P0.001*< 0.001*        
Blood vessels expressing VEGFR-2
 rs0.7040.6180.561       
 P0.002*0.008*0.019*       
Cells expressing VEGFR-2
 rs0.5350.7830.7000.714      
 P0.027*< 0.001*0.002*0.001*      
Blood vessels expressing CD14
 rs0.5640.4580.6040.3990.485     
 P0.018*0.0640.010*0.1120.048*     
Cells expressing CD14
 rs0.6760.7670.8170.6320.7450.860    
 P0.003*< 0.001*< 0.001*0.006*0.001*< 0.001*    
Blood vessels expressing SDF-1
 rs0.5770.5850.5650.6560.6990.2080.476   
 P0.015*0.014*0.018*0.004*0.002*0.4230.054   
Blood vessels expressing CXCR4
 rs−0.0330.0090.161−0.095−0.0770.3140.273−0.147  
 P0.9000.9740.5370.7170.7690.2200.2890.573  
Cells expressing CXCR4
 rs0.1230.3650.504−0.0050.3220.5430.5360.0700.557 
 P0.6380.1500.039*0.9850.2080.024*0.027*0.7890.020* 
Cells expressing Ki-67
 rs0.5570.6980.7090.5920.7620.3600.5870.662−0.0230.186
 P0.020*0.002*0.001*0.012*< 0.001*0.1560.013*0.004*0.9300.476

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the present study, we tested the hypothesis that circulating bone-marrow-derived endothelial precursor cells contribute to neovascularization in PDR epiretinal membranes. Using immunohistochemical techniques, we demonstrated for the first time the presence of cells expressing the novel haematopoietic precursor stem and EPC marker CD133 (Mizrak et al. 2008). In addition, cells expressing the monocyte marker CD14 were detected. CD133+ and CD14+ cells were located in the vascular endothelium of small vessels, in the stroma and in intravascular spaces. The numbers of blood vessels and stromal cells expressing CD133 and CD14 in membranes from patients with active PDR were significantly higher than those in membranes from patients with inactive PDR. In addition, there were significant correlations between the number of blood vessels expressing the panendothelial marker CD34 and cells expressing the proliferating cell marker Ki-67 and the number of blood vessels and cells expressing CD133 and CD14. Taken together, our findings suggest that CD133+ EPCs and CD14+ monocytes may contribute to vasculogenesis in PDR epiretinal membranes.

Recent studies demonstrated that the recruitment of bone-marrow-derived EPCs plays an essential role in tumour vasculogenesis (Hilbe et al. 2004; Igreja et al. 2007). In addition, direct differentiation of bone-marrow-derived stem/progenitor cells into endothelial cells lining blood vessel lumens is known to be important in tissue revascularization after ischaemic events (Rafii & Lyden 2003). In the present study, we observed the presence of CD133+ small vascular structures in PDR membranes. This observation is consistent with previous studies that demonstrated the presence of CD133+ EPCs in the endothelial tubes of human tumour capillaries (Hilbe et al. 2004; Igreja et al. 2007). Moreover, the presence of CD133+ EPCs correlated with tumour size and with increased angiogenesis (Igreja et al. 2007). Murayama et al. (2002) investigated the quantitative contribution of bone-marrow-derived EPCs to newly formed vascular structures in an in vivo model. They demonstrated that EPCs make a significant contribution to angiogenic growth-factor-induced neovascularization, which may account for up to 26% of all endothelial cells. Previous phenotypic analyses revealed that most CD34+ VEGFR-2+ cells expressed the haematopoietic stem-cell marker CD133 (Peichev et al. 2000). In agreement with these studies, we found significant correlations between the number of blood vessels and stromal cells expressing CD133 and the number of blood vessels and stromal cells expressing VEGFR-2. In addition to EPCs, several studies have demonstrated that in the presence of angiogenic growth factors, human peripheral blood CD14+ monocytes develop an endothelial phenotype with expression of specific endothelial lineage markers and form cord- and tubular-like structures in vitro (Harraz et al. 2001; Kim et al. 2005). In vivo studies have shown that circulating CD14+ monocytic progenitors improved healing, accelerated blood flow restoration and induced vascular growth in ischaemic limbs of diabetic mice (Awad et al. 2006).

Several studies have demonstrated that EPCs express CXCR4 and that SDF-1 induces a concentration-dependent migration of EPCs. This migration was CXCR4-dependent, as confirmed by the total inhibition by a CXCR4-specific peptide antagonist (Yamaguchi et al. 2003; Walter et al. 2005). SDF-1 is upregulated in ischaemic tissue, establishing an SDF-1 gradient favouring recruitment of EPCs from peripheral blood to sites of ischaemia, thereby contributing to accelerated neovascularization (Yamaguchi et al. 2003; De Falco et al. 2004). In addition, SDF-1 promoted the chemotaxis of bone-marrow-derived CD34+ stem cells and their differentiation into EPCs in ischaemic tissue and tumours (De Falco et al. 2004; Reddy et al. 2008; Stellos et al. 2008). Moreover, SDF-1 attenuated EPC apoptosis (Yamaguchi et al. 2003). Recently, Reddy et al. (2008) demonstrated that upregulation of SDF-1 in the tumour results in the formation of enlarged, lumen-bearing, functional blood vessels, implying that this chemokine may influence vascular remodelling via a direct action on endothelial cells. Furthermore, they showed that SDF-1-mediated vasculogenesis may represent an alternative pathway that could be utilized by tumours to sustain growth and neovasculature expansion after anti-VEGF therapy. Similarly, Aghi et al. (2006) demonstrated that SDF-1 secretion is both necessary and sufficient to induce vasculogenesis in intracranial gliomas. VEGF alone failed to cause vasculogenesis in this model but enhanced the vasculogenesis of SDF-1-secreting tumours independent of the ability of VEGF to stimulate SDF-1 secretion. CXCR4 blockade profoundly inhibited VEGF- and SDF-1-induced migration of EPCs and impaired incorporation of EPCs into sites of ischaemia-induced neovascularization (Walter et al. 2005; Aghi et al. 2006). The finding that VEGF-mediated migration of EPCs was also influenced by CXCR4 antibodies points toward a more general involvement of CXCR4 and its downstream signalling in the homing mechanisms of EPCs.

Another aim of the present study was to determine whether the SDF-1/CXCR4 chemokine axis is expressed in PDR epiretinal membranes. We demonstrated the presence of CXCR4+ CD34+ cells in the vascular endothelium and in the stroma and that SDF-1 protein was specifically localized in vascular endothelial cells. Stromal CXCR4+ CD34+ cells were closely associated with the new vessels within the epiretinal membranes. SDF-1 expression by endothelial cells might provide an important role in triggering CXCR4+ CD34+ cell arrest and migration into preretinal membranes. It might also function to localize and retain CXCR4+ CD34+ cells adjacent to blood vessels to form new vessels. Our findings thereby confirm the contribution of the SDF-1/CXCR4 signalling pathway to the trafficking of circulating EPCs to PDR membranes.

Our study supports previous studies showing that SDF-1/CXCR4 pathway is implicated in diabetic retinopathy. Butler et al. (2005) reported increased SDF-1 levels in vitreous from patients with PDR. In a murine model of retinal ischaemia, upregulation of SDF-1 and CXCR4 was detected in ischaemic retinas. A substantial amount of the increase in CXCR4 was caused by influx of CXCR4-expressing bone-marrow-derived cells. Pharmacological blockade of CXCR4 suppressed ischaemia- and VEGF-induced retinal neovascularization (Lima e Silva et al. 2007). In a murine model of proliferative retinopathy, Butler et al. (2005) demonstrated that intravitreal injection of blocking antibodies to SDF-1 prevented retinal neovascularization, even in the presence of VEGF.

In conclusion, we have shown that bone-marrow-derived CD133+ EPCs and CD14+ monocytes can be found in PDR epiretinal membranes and we hypothesize that these cells contribute to vasculogenesis in PDR epiretinal membranes. Thus, novel strategies aimed at treating PDR might target the participation of bone-marrow-derived endothelial precursors and the modulation of their activity. Therefore, inhibition of the SDF-1/CXCR4 signalling pathway might provide a new therapeutic approach to treating PDR.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Mr Dustan Kangave for statistical assistance, Ms Lieve Ophalvens, Ms Christel Van den Broeck and Mr Johan Van Evan for technical assistance, and Ms Connie B. Unisa-Marfil for secretarial work.

This work was supported by the College of Medicine Research Centre, King Saud University, Medical Research Chair Funded by Dr Nasser Al-Rasheed and the European Union 6FP EC Contract INNOCHEM. S.S. is a senior research assistant from the Fund for Scientific Research of Flanders (FWO-Vlaanderen).

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References