Current Research Projects
- CMG2 proteomics using IP-MS and proximity proteomics to identify protein-protein interactions that control endothelial cell chemotaxis
- Identification of tear film biomarkers for neovascular eye disease
Currently Funded or Submitted Grant Applications
Functional Requirement of CMG2 for Endothelial Cell Chemotaxis and Resulting Angiogenesis
Corneal neovascularization greatly increases the risk for corneal graft rejection, and thus contributes to severe vision loss, risk of endophthalmitis, and life-threatening meningitis. It afflicts up to 1.4 million new patients annually and together with other pathological angiogenesis is the leading cause of blindness in the United States. Angiogenesis also contributes to diseases that range from cancer to arthritis. A wide range of protein growth factors (e.g. VEGF, bFGF, PDGF) stimulate angiogenesis, but only VEGF is currently targeted for antiangiogenic therapy in the eye. CMG2 is an integrin-like transmembrane protein with an extracellular domain that binds ECM proteins and an intracellular tail without homology to domains of known function. We find that targeting CMG2 via the protein inhibitor PASSSR, CMG2-binding small molecules, or CMG2 knockout profoundly inhibits corneal neovascularization, but the mechanism underlying this effect has been unknown.
Angiogenesis requires endothelial cells to migrate towards growth factors. This migration has both a movement component (motility = chemokinesis) and a directional component (chemotaxis); however, these differences can only be readily observed using a microfluidic migration assay that visualizes individual cells, not with traditional (wound scratch or transwell) assays. Using a microfluidic assay, we recently discovered that CMG2 targeting completely disrupts chemotaxis, an effect observed with multiple growth factors (bFGF, VEGF, PDGF) and all targeting methods tried thus far (CRISPR knockout, PASSSR, blocking peptide). Thus, we hypothesize that CMG2 is a key intermediary in a pathway required for growth-factor induced chemotaxis and efficient angiogenesis. We will test this hypothesis by: 1) identifying intracellular interactors required for CMG2-mediated chemotaxis; 2) identifying extracellular interactions required for CMG2-mediated chemotaxis in response to growth factors, and 3) evaluating the contribution of RhoA to CMG2-directed chemotaxis.
Our working model of CMG2 signaling is based on preliminary data from our lab and others that indicates that CMG2 localizes near RhoA and several Rho pathway members. Thus, CMG2 is positioned to directly regulate the cell polarity required for directional migration (chemotaxis). Indeed, we observe that inhibiting RhoA phenocopies CMG2 inhibition. Finally, we can bypass CMG2 signaling by activating RhoA via the S1P receptor, so that chemotaxis is no longer sensitive to CMG2 targeting. Thus, RhoA is downstream of CMG2.
Successful completion of proposed work will identify the mechanism underlying the strong antiangiogenic effects observed upon CMG2 targeting in vivo and accelerate exploitation of this potential target for broad-spectrum antiangiogenic therapy. In addition, this work will enable the development of pharmacodynamic assays to rapidly evaluate drug leads. Finally, key aspects of this proposal are designed to produce possible therapeutic leads. Drugs arising from these studies could supplement anti-VEGF therapies to treat blindness caused by ocular neovascularization as well as many other angiogenesis-dependent diseases.
Novel Peptide Inhibitors of CMG2 for Treatment of Pathologic Angiogenesis
Corneal neovascularization dramatically increases the risk of corneal graft rejection, contributes to graft failure that results in severe visual acuity loss. Neovascularization afflicts up to 1.4 million new patients annually and, together with other angiogenesis-related ocular pathologies, is the leading cause of blindness in the United States. Angiogenesis also contributes to diseases that range from cancer to arthritis. A wide range of protein growth factors (e.g., VEGF, bFGF, PDGF) stimulate angiogenesis, but only VEGF is currently targeted for anti-angiogenic therapy in the eye. However, VEGF targeting can be accompanied by serious ocular and systemic side effects including disruption of wound healing, is complicated by breakthrough of alternate angiogenic pathways, not uniformly effective for all angiogenic disorders, and is not approved as a treatment for corneal neovascularization. Hence, there remains a great need for alternate anti-angiogenic targets and therapies. CMG2 is an integrin-like transmembrane protein with an extracellular domain that binds ECM proteins. We have found that targeting CMG2 via the protein inhibitor PASSSR, CMG2-binding small molecules, or a CMG2 knockout has profound anti-angiogenic effects that are mediated through CMG2’s ability to control directional cell mobility. Angiogenesis requires migration of endothelial cells towards growth factors as a necessary first step; this migration has both a movement component (motility = chemokinesis) and a directional component (chemotaxis) that can only be readily separated using a microfluidic migration assay to visualize individual cells. Using this microfluidic assay, we have recently discovered that CMG2 targeting completely disrupts chemotaxis, an effect observed with multiple growth factors (bFGF, VEGF, PDGF) and all targeting methods tried thus far (CRISPR knockout, PASSSR, blocking peptide). Based on these data, our central hypothesis is that CMG2 binding of endogenous ligand(s) is required for chemotaxis and resulting angiogenesis and that inhibition of this interaction by competing ligands will inhibit pathological angiogenesis, including corneal neovascularization. Consistent with this hypothesis, we have demonstrated that targeting CMG2 with peptides derived from ECM protein sequence(s) abolishes endothelial cell chemotaxis. We propose improvement and testing of peptide CMG2 antagonists from the primary sequences of Collagen IV (Col4) and VI (Col6). Col6 is widely expressed in eye tissue and is among the ECM molecules first encountered by neovascular tip cells as they pilot new vessels, while Col4 is found in the microvascular basement membrane, where it can serve as a ligand for neovascular stalk cells. CMG2-binding peptides derived from Col6 and Col4 will compete with physiological ligands, resulting in the inhibition of chemotaxis and resulting angiogenesis. Given the high structural homology of CMG2 to integrins and the significant number of known effective peptide integrin antagonists, we expect that these CMG2-binding peptides can be developed into effective therapies for corneal neovascularization and other angiogenesis-related diseases.