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Angiogenesis

Current Funded Research Projects (newest to oldest)

  • CMG2 as a target for safe and effective treatment of endometriosis-associate pain (in collaboration with the Dr. Michael Rogers Lab at Harvard Medical School/Boston Children's Hospital through the NIH; R01HD110922; 2022-2026)
  • Functional requirement of CMG2 for endothelial cell chemotaxis and resulting angiogenesis (in collaboration with the Dr. Michael Rogers Lab at Harvard Medical School/Boston Children's Hospital through the NIH; R01EY033354; 2022-2027)
  • Identification of tear film biomarkers for neovascular eye disease (BYU-CPMS CHIRP Award; 2022)

Currently Funded or Submitted Grant Applications

CMG2 as a target for safe and effective treatment of endometriosis-associate pain

Endometriosis-associated pain is an important driver of opioid use in women. Endometriosis is an estrogen-sensitive, angiogenesis-dependent disease that affects ~10% of women of childbearing age and is found in half of the women with chronic pelvic pain. Endometriosis increases the likelihood of chronic opioid use, opioid dependence/abuse, and opioid overdose. Endometriosis is currently treated with NSAIDs, hormonal therapy targeting estrogen production, and surgery, but these options are not durably effective for ~30% of patients. Thus, new targets are needed. CMG2 is an integrin-like extracellular matrix receptor that we have shown regulates angiogenesis. It is also overexpressed in endometriosis tissue vs. normal endometrium. In mouse models, published work and our preliminary data show that treatment with CMG2 antagonists that we discovered (PGG, PGM) decreases lesion incidence, growth, and endometriosis-associated pain. These observations suggest that CMG2 may be a useful target for treating endometriosis-associated pain. However, key gaps in our understanding of CMG2 biology would slow any development program with CMG2 as a target. The first key gap is that the cell type whose targeting resulted in decreased pain upon CMG2 antagonist treatment is unclear. Second, the molecular mechanism underlying CMG2 signaling in endometriosis is not known. Finally, both the maximum efficacy and, importantly, safety of CMG2 targeting are not known. We propose to fill these gaps by using cell-type-specific CMG2 knockout mice to identify cell types where CMG2 expression supports endometriosis lesion growth and pain. Because CMG2 lacks any catalytic domains, we hypothesize that CMG2 signals via differential protein interaction. Then, the expression of CMG2 in appropriate cells in human lesions will be confirmed. Next, we will use proximity proteomics to identify downstream molecules interacting differently with CMG2 ±inhibitor. Candidate mediators of CMG2 signaling will be confirmed using CRISPR knockout and cell-based assays of CMG2 function. Finally, we will evaluate the safety and efficacy of CMG2 targeting in a mouse model using a well-characterized, high-specificity CMG2 inhibitor. Throughout ex vivo and in vivo assays, RNAseq and scRNAseq will be used to identify transcriptional signatures of CMG2 blockade, both to validate in vitro assays and to generate potential pharmacodynamic markers of CMG2 inhibitors. Completing the proposed work will validate CMG2 as a target for treating endometriosis-associated pain. It will also provide key insights into endometriosis pathophysiology that will enable the generation of novel therapeutics and may identify additional targets for treating the disease. The development of such drugs will improve the lives of many women and decrease the use of opioids to treat this disease, thereby improving many lives.

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 toward 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 indicate 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 the proposed work will identify the mechanism underlying the strong antiangiogenic effects observed upon CMG2 targeting in vivo and accelerate the 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 and many other angiogenesis-dependent diseases.

Identification of tear film biomarkers for neovascular eye disease

Tear film holds great potential as a disease screening medium as it can be sampled rapidly and non-invasive. Recently, protein biomarkers have been identified in human tear film for various pathologies, including glaucoma, dry eye disease, multiple sclerosis, Parkinson’s disease, and cancer (2, 3). However, there remains an unmet need for biomarkers in many disease states. Specifically, there are currently no human tear film biomarkers for neovascular eye diseases.
Ocular neovascular diseases such as diabetic retinopathy (DR), age-related macular degeneration, and neovascular glaucoma are blinding pathologies that are increasing in prevalence throughout the world (4, 5) and DR is the leading cause of blindness and vision impairment in working age adults (6). Vision loss from neovascular diseases can often be prevented if diagnosed promptly, but if left undiagnosed, they can lead to total blindness in a matter of hours to days (7). Thus, early detection and diagnosis is critical to preserve vision.
Neovascularization for most eye diseases is diagnosed upon clinical observation. Common pathologies such as cataracts and corneal opacifications inhibit a physician's ability to detect and diagnose retinal neovascularization. Left undetected, neovascularization puts the patient at extremely high risk for rapid and severe vision loss secondary to neovascular-related retinal detachments, vitreal hemorrhages, and retinal edema. Therefore, early detection of ocular neovascularization using a tear film screen for neovascular biomarkers could aid in prompt diagnoses with or without clinical observation. Such a screen would help prevent blindness for millions of people with neovascular diseases throughout the world.
However, before clinical screenings for neovascular disease can become a reality, biomarkers must be identified and validated. Until recently, relatively few tear film proteins had been identified due limitations in mass spectrometry (MS) resolution and primitive liquid chromatography-mass spectrometry (LC-MS) protocols for evaluating tear samples. For example, a study performed by Csosz et al. in 2012 to identify biomarkers for diabetic retinopathy identified 53 total proteins and 6 potential biomarkers (8). Further evaluation of the proposed biomarkers revealed that none were sensitive or specific enough to be a true biomarkers (9). [RR1] However, the recent use of newer, high-resolution Orbitrap mass spectrometers and improved LC-MS techniques have expanded the view of the tear proteome from 17 human tear film protein identifications in 2005 to more than 1,500 today (10). As the view of the tear proteome increases, so does the ability to identify protein biomarkers. We hypothesize that previously undiscovered protein biomarkers exist for ocular neovascular diseases in human tear film.

Novel Peptide Inhibitors of CMG2 for Treatment of Pathologic Angiogenesis

Corneal neovascularization dramatically increases the risk of corneal graft rejection and contributes to graft failure, resulting 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 the 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 improving and testing 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. At the same time, 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.