The role of SDF-1 and CXCR4 in ocular health and pathology is just now being elucidated given that many ocular cells are sources of SDF-1 in the eye.16
SDF-1 and CXCR4 are known to have dynamic and complementary expression patterns, with both proteins playing important roles in vascularization.17
Mice lacking CXCR4 or SDF-1 die in utero, with disruption in the development of vasculature networks in any organs.18
Typically, CXC chemokines lacking ELR motifs such as SDF-1 are antiangiogenic; interestingly, however, SDF-119
stimulates endothelial cell growth.15
Darash-Yahana et al.20
demonstrated that tumor-associated blood vessels express SDF-1. Subcutaneous xenografts of PC3 cells that overexpressed CXCR4 in mice showed significantly greater blood vessel density, functionality, and invasiveness of tumors into the surrounding tissues. Neutralizing SDF-1/CXCR4 interactions with antibodies to CXCR4 inhibited CXCR4-dependent tumor growth and vascularization.21
Vessel density and functionality, as well as metastasis to the regional lymph nodes and lungs, were significantly increased in these tumors overexpressing CXCR4.20
Similar effects of CXCR4 overexpression on tumor growth were also noted in breast cancer lines.22
Although in previous studies we focused on directly blocking SDF-1 and showed a reduction in CNV lesion size,5
in this study we used two approaches for reducing CXCR4 activation, a CXCR4 blocking antibody and a CXCR4 modulator, NefM1, that downregulates CXCR4 expression. NefM1 has been used to reduce tumor size in a rodent colon cancer model and has been shown to inhibit endothelial cell proliferation in vitro.23
In this report, we show that the inhibition of CXCR4 using a blocking antibody reduced CNV lesion size when administered as a subretinal injection but that intravitreal administration of CXCR4 antibody did not. Moreover, when NefM1 was given by way of the intraperitoneal route, it did not reduce CNV lesion size. Lima e Salva et al.4
used pharmacologic antagonists of CXCR4 (AMD 8664, TC14012, and compound 3) and demonstrated reduced CNV lesion size in the identical model system we describe here. They did not test systemic delivery but used periocular administration, which provides high ocular bioavailability, and observed CNV inhibition to a degree similar to what we observed with subretinal administration. Their results, as well as ours, suggest that the route of administration is paramount to using CXCR-4 antagonist as a therapeutic strategy for CNV.4
The concentration of NefM1 we used was previously found to be effective for the inhibition of tumor growth23
; however, it had minimal effect on CNV lesion size. Interestingly, the effect of knocking down CXCR4 expression in tumor models has not always been consistent, generating conflicting data regarding the role of SDF-1 signaling on tumor cells and associated angiogenesis,24,25
and further speaks to the complexity of the system. The benefit of targeted ocular delivery rather than systemic delivery is nevertheless clearly shown in our study of CNV.
A possible explanation for the NefM1 result is that NefM1 may not reach sufficiently high levels in the target tissue. Overall, our data suggest that not only is ocular delivery critical, local delivery (subretinal rather than intravitreal) may be imperative. Subretinal blockade may be needed because antibodies and drugs that block CXCR-4 may bind in the retina before they reach the subretinal space. The bioavailability of the agent used is also essential because small molecule antagonists such as used by Lima e Silva et al.4
may be more effective than the blocking antibodies we used. Our results support those of previous reports from Lima e Silva and confirm that the blockade of CXCR4 activation results in decreases in CNV lesion size.4
In certain cell types, NefM1 induces internalization of CXCR4; thus, NefM1may activate signaling mechanisms to cause proliferation, thus limiting its clinical usefulness as an antiangiogenic.23,26,27
Internalization of a receptor from ligand binding typically leads to receptor degradation. However, Earp et al.28
showed that binding of EGF to the receptor leads to receptor degradation, but this was counterbalanced by a 3.5-fold increase in receptor synthesis; however, this effect may be cell-type specific. Although we do not know whether this type of compensation applies to NefM1 peptide and CXCR4 specifically in our system, we may consider these possibilities to explain the disparate effect observed with this agent in ocular tissue versus tumor.
More recently, a second receptor for SDF-1, CXCR7, has been discovered.29
It is present on hematopoietic stem cells and human CD34+
EPCs (Afzal A, Grant MB, unpublished results, 2007). It is also important for signaling to more committed progenitors and mature blood cells, such as T-lymphocytes and monocytes. We also observed that after laser injury, mRNA for CXCR7 is increased, suggesting that simply blocking CXCR4 may not be sufficient to achieve optimal SDF-1 blockade but that both receptors may have to be blocked to reach optimal therapeutic reduction of CNV.
SDF-1, CXCR-4, and CXCR-7 expression are increased in CNV lesions, making this system a therapeutic target. Our in vitro studies suggest that an anti–SDF-1 approach may be used in conjunction with anti-VEGF or anti–IGF-1 strategies to improve outcomes. Our studies show that the lowest concentration of SDF-1 tested enhanced the proliferative response of endothelial cells to VEGF, and this lowest concentration of SDF-1 was actually more potent than the highest concentration in stimulating VEGF-induced proliferation. This is not entirely uncommon in that cytokines such as SDF-1 typically have a “peak” effect; thus, concentrations lower or higher than this ideal level typically result in a reduced physiological response.
The repertoire of receptor expression in EPCs and endothelial cells no doubt dictates the angiogenic outcome, with the G protein–coupled receptor CXCR4 possibly regulating the tyrosine kinase receptors IGF-1R and VEGFR. Given the potential diversity of receptors constitutively expressed by EPC and endothelial cells, such interactions may be expected to alter signal coupling.
SDF-1 does not work in isolation, but its actions are orchestrated by other growth factors such as VEGF and IGF-1. Three forms of cross-talk between G protein–coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) have been demonstrated in different cellular systems. First, RTKs can be transactivated by GPCRs.30
Second, GPCRs can be transactivated by RTKs. For example, it has been shown that IGF-1 stimulated phosphorylation of the chemokine receptor CCR5 in MCF-7 cells and that chemotaxis induced by IGF-1 was inhibited by a neutralizing antibody to the ligand CCL5. Transactivation of CCR5 by IGF-1 was. Therefore, indirect, requiring the activity of CCL5 with its receptor.31
Third, bidirectional transactivation between the two receptor systems has also been observed. For example, the TKR platelet-derived growth factor receptor is phosphorylated by sphingosine-1-phosphate (S1P), leading to the activation of downstream effectors, including Shc, and the p85 regulatory subunit of the class IA PI3K.32
PDGF has been shown to transactivate the S1P receptor, a GPCR.33
Studies by Akekawatchai et al.34
showed coprecipitation of IGF-1R, CXCR4, and the G protein subunits Giα2 and Gβ, indicating a constitutive physical association between these molecules in breast cancer cells. CXCR4/IGF-1R receptor integration may play an important role in cancer metastasis and in development because both IGF-1/IGF-1R and SDF-1/CXCR4 are essential.35,36
Taken together, our results provide new information regarding the SDF-1/CXCR4/CXCR-7 axis in CD34+ EPCs, endothelial cells, and CNV lesions. Our findings add a new dimension to the understanding of chemokine-mediated neovascularization and support a new era of potential therapeutic interventions targeting this chemokine. Our work emphasizes the requirement of local delivery for targeting CXCR-4 and supports that combination therapy with anti-VEGF or anti–IGF-1 agents and simultaneous blockade of CXCR-7 may represent the optimal therapeutic strategy for CNV.