The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newl... more The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newly forming blood vessels. We studied its role in vascular tube formation by a quantitative ultrastructural analysis of Egfl7-knockdown zebrafish embryos. At 24 hours postfertilization, the endothelia of dorsal aorta (DA) and posterior cardinal vein (PCV) were correctly anchored to the hypochord and endoderm, respectively, but failed to expand into the vascular area. This resulted in vessels with reduced or split lumen and open sheets of ECs. Concomitantly, the organization of hematopoietic cells-identified by the presence of previously undescribed membrane tubules-between DA and PCV, and within the vessels, was severely disturbed. Strikingly, ectopic cell junctions occurred across the obstructed vessel lumen, on the luminal EC surfaces, which in control conditions never display junctions of any kind. These data suggest that Egfl7 provides ECs with a cue for their extension into the vascular area and in establishing EC cell polarity.
Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple ... more Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple neurons and support cells. Here we show that the paired-domain protein Pax2 is necessary and sufficient for the induction of FGF8 in part by regulating the expression of Pax5&8. A network of transcription and secreted factors, including En1, Otx2, Gbx2, Grg4 and Wnt1&4, that is established independently of Pax2, further refines the expression domain and level of FGF8 at the MHB through opposing effects on Pax2 activity. Our results indicate that the expression of local organizing factors is controlled by combinatorial interaction between inductive and modulatory factors.
Map4k4 is broadly expressed across different cell types and its function is cell type and context... more Map4k4 is broadly expressed across different cell types and its function is cell type and context dependent. Although it is expressed in multiple types of immune cells, its specific function in different subsets of lymphocytes and myeloid cells have yet to be determined. Here we demonstrate that reducing MAP4K4 activity can enhance CD8 T cell functions in the context of anti-tumor and anti-viral responses. Inducible deletion of Map4k4 increases CD8 T cell activation, proliferation, cytokine production, and target cell killing in vitro, and enhances CD8 T cell-mediated suppression of tumor growth as well as antigen-specific response to viral infection in vivo. Intratumoral T cells showed evidence of enhanced activation and produced more inflammatory cytokines when stimulated ex vivo. Simultaneously reducing MAP4K4 activity and blocking PDL1 resulted in additive anti-tumor efficacy. Our data suggests that MAP4K4 could be a potential target to augment anti-tumor and immunity. Citation Format: Emel Esen, Weilan Ye, Rajiv Jesudason, Ismail Sergin. Deletion of Map4k4 enhances CD8 T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-144.
Myelodysplastic syndrome (MDS) comprises a heterogeneous group of oftenfatal hematopoietic stem c... more Myelodysplastic syndrome (MDS) comprises a heterogeneous group of oftenfatal hematopoietic stem cell disorders for which neither curative nor standard treatment exists. The complex karyotypes and multistep nature of MDS have severely restricted the identification of causative genetic mutations and thus limited insight into new and more effective therapies. Here we describe a zebrafish mutant crimsonless (crs) with a develop-mental blood defect that closely recapitulates the ineffective hematopoiesis of MDS including anemia, dysplasia, increased blood cell apoptosis, and multilineage cytopenia. By positional cloning, rescue, and morpholino knockdown experiments, we demonstrate that crs encodes a conserved mitochondrial matrix chaperone HSPA9B containing a glycine-to-glutamate substitution within the substrate-binding domain. This mutation compromises mi-tochondrial function, producing oxidative stress and apoptosis distinctly in blood cells. Thus, we identify an essential role for Hspa9b in hematopoiesis and implicate both loss of HSPA9B specifically and mitochondrial dysfunction generally in the pathogenesis of the MDS. (Blood.
As the age of the population increases in many nations, age-related degenerative diseases pose si... more As the age of the population increases in many nations, age-related degenerative diseases pose significant socioeconomic challenges. One of the key degenerative diseases that compromise quality of life is age-related macular degeneration (AMD). AMD is a multi-faceted condition that affects the central retina, which ultimately leads to blindness in millions of people worldwide. The pathophysiology and risk factors for AMD are complex, and the symptoms manifest in multiple related but distinct forms. The ability to develop effective treatments for AMD will depend on a thorough understanding of the underlying pathophysiology, risk factors, and driver molecular pathways, as well as the ability to develop useful animal models. This review provides an overview of the aforementioned aspects in AMD.
MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. I... more MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragmentbased screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.
bioRxiv (Cold Spring Harbor Laboratory), Aug 28, 2020
Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensur... more Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveal distinct requirements for Rasip1 during sprouting angiogenesis, vascular anastomosis and lumen formation. During angiogenic sprouting, Rasip1 is required for efficient cell pairing, which is essential for multicellular tube formation. High-resolution time-lapse analyses show that these cell pairing defects are caused by a destabilization of tricellular junctions suggesting that tricellular junctions may serve as a counterfort to tether sprouting endothelial cells during morphogenetic cell rearrangements. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function leads to junctional defects similar to those seen in rasip1 mutants. Analysis of radil-b single and rasip1/radil-b double mutants reveal distinct and overlapping functions of both proteins. While Rasip1 and Radil-b have similar functions during angiogenic sprouting, the junction formation during anastomosis may primarily depend on Rasip1.
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In this study we report ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In this study we report the use of pharmacodynamic (PD) biomarkers to guide the clinical development of a novel antibody targeting epidermal growth factor-like domain 7 (EGFL7). EGFL7 is a secreted protein produced by endothelial cells of nascent blood vessels in tumors and other proliferating tissues, but is expressed at low levels in healthy quiescent vessels and many non-vascular cell types (Campagnolo et al. 2005; Fitch et al. 2004; Parker et al. 2004; Soncin et al. 2003). Upon secretion, EGFL7 becomes tightly associated with the perivascular extracellular matrix (ECM), and supports endothelial cell adhesion and migration (Parker et al. 2004; Schmidt et al. 2007). EGFL7 protein also protects endothelial cells from stress-induced apoptosis (Xu et al. 2008). Anti-EGFL7 is a humanized monoclonal antibody that binds to EGFL7. In preclinical models, anti-EGFL7 when combined with anti-VEGF augments the survival benefit observed from anti-VEGF alone. Anti-EGFL7 is currently being investigated in Phase I trials (in solid tumors) in combination with bevacizumab. In addition to endothelial cells, circulating CD34Hi/CD31dim progenitor cells (CPCs) express high levels of EGFL7 transcript. This population of cells can differentiate into endothelial cells in vitro, and we found that the EGFL7 protein promotes proliferation and possibly survival of the undifferentiated CPCs in vitro. In preclinical xenograft models, administration of anti-EGFL7 resulted in delayed (day 15) reduction in CPCs, which based on the time course, is unlikely to be mediated simply by clearance of antibody-bound CPCs. Hence, evaluation of CPCs was incorporated into the dose escalation phase of anti-EGFL7 clinical development as a pharmacodynamic biomarker (PD) of drug activity. Biomarker analysis of CPCs in humans correlated with preclinical findings. For example, reduction in CPCs in response to anti-EGFL7 therapy was observed at a time point similar to that in mice. The treatment dependent decrease in CPCs may serve as a pharmacodynamic biomarker that may help guide dose selection as well as represent another potential mechanism for the anti-angiogenic properties of anti-EGFL7 Campagnolo L, Leahy A, Chitnis S, Koschnick S et al. (2005) Am J Pathol 167(1):275-84 Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H. (2004) Dev Dyn. 230(2):316-24 Parker LH, Schmidt M, Jin SW et al (2004) Nature 428(6984):754-8. Schmidt M, Paes K, De Maziere A, (2007) Development 134(16):2913-23. Soncin F, Mattot V, Lionneton F (2003) EMBO J. 22(21):5700-11 Xu D, Perez RE, Ekekezie II (2008) Am J Physiol Lung Cell Mol Physiol. 294(1):L17-23 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3284. doi:10.1158/1538-7445.AM2011-3284
Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastati... more Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression. Utilizing murine models of metastasis, longitudinal in vivo imaging of lymph transport, and function blocking antibodies against two VEGF family members, we provide evidence that distal lymphatics undergo disease course-dependent up-regulation of lymph transport coincidental with structural remodeling. Inhibition of VEGF-C activity with antibodies against VEGF-C or NRP2 prevented these disease-associated changes. Furthermore, utilizing a novel model of adjuvant treatment, we demonstrate that antagonism of VEGF-C or NRP2 decreases post SLN metastasis. These data support a potential therapeutic strategy for inhibiting distant metastatic dissemination via targeting tumor-associated lymphatic remodeling.
A significant existing challenge for anti-angiogenic therapy is that there are no validated bioma... more A significant existing challenge for anti-angiogenic therapy is that there are no validated biomarkers to either monitor dosing, response to therapy or escape from therapy. This limitation is a more significant challenge when the therapy in question targets a novel pathway with a unique MOA. Some generalized biomarkers have been identified to follow administration of anti-angiogenic therapy, and included in this class are circulating endothelial cells or CECs. CECs are a well-established marker of on-going and active angiogenesis1. In this report we describe the utility of measuring CECs in response to targeting a novel angiogenic protein, EGFL7. EGFL7 is an extracellular matrix protein that is highly expressed in endothelial cells of angiogenic vessels, including embryonic, tumor and injured vasculature. Inhibition of EGFL7 activity by anti-EGFL7 results in a reduction of tumor microvascular density and tumor growth in genetically engineered models of cancer such as RIP-T Ags. In o...
F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bac... more F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bacterially expressed F1-20/AP-3 to bind and assemble clathrin cages. We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble clathrin as efficiently as preparations of F1-20/AP-3 from bovine brain. This establishes that the clathrin assembly activity found in F1-20/AP-3 preparations from brain extracts is indeed encoded by the cloned gene for F1-20/AP-3. It also demonstrates that post-translation modification is not required for activation of the clathrin binding or assembly function of F1-20/AP-3. Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1-20/AP-3 reveals a strikingly narrow size distribution. This may be important for the regulation of quanta1 size during neurotransmission. We also express the 33 kD NH 2 -terminus of F1-20/AP-3 in E. coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages. It has been suggested that the 33 kD NH 2 -terminus of F1-20/AP-3 constitutes a clathrin binding domain. We find that the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly. The finding that the 33 kD NH 2 -terminus of F1-20/AP-3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies. The finding that the 33 kD NH 2 -terminus of F1-20/ AP-3 fails to bind to clathrin cages is novel and potentially important. It is clear from these experiments that the 33 kD NH 2 -terminus of F1-20/AP-3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated.
Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebra... more Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. Here, we identify a Sonic hedgehog(Shh)-regulated cascade of transcription factors that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1), which project to the forebrain, but not for the induction of caudal 5-HT neurons,which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. Our findings reveal that the identity of closely rel...
The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newl... more The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newly forming blood vessels. We studied its role in vascular tube formation by a quantitative ultrastructural analysis of Egfl7-knockdown zebrafish embryos. At 24 hours postfertilization, the endothelia of dorsal aorta (DA) and posterior cardinal vein (PCV) were correctly anchored to the hypochord and endoderm, respectively, but failed to expand into the vascular area. This resulted in vessels with reduced or split lumen and open sheets of ECs. Concomitantly, the organization of hematopoietic cells-identified by the presence of previously undescribed membrane tubules-between DA and PCV, and within the vessels, was severely disturbed. Strikingly, ectopic cell junctions occurred across the obstructed vessel lumen, on the luminal EC surfaces, which in control conditions never display junctions of any kind. These data suggest that Egfl7 provides ECs with a cue for their extension into the vascular area and in establishing EC cell polarity.
Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple ... more Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple neurons and support cells. Here we show that the paired-domain protein Pax2 is necessary and sufficient for the induction of FGF8 in part by regulating the expression of Pax5&8. A network of transcription and secreted factors, including En1, Otx2, Gbx2, Grg4 and Wnt1&4, that is established independently of Pax2, further refines the expression domain and level of FGF8 at the MHB through opposing effects on Pax2 activity. Our results indicate that the expression of local organizing factors is controlled by combinatorial interaction between inductive and modulatory factors.
Map4k4 is broadly expressed across different cell types and its function is cell type and context... more Map4k4 is broadly expressed across different cell types and its function is cell type and context dependent. Although it is expressed in multiple types of immune cells, its specific function in different subsets of lymphocytes and myeloid cells have yet to be determined. Here we demonstrate that reducing MAP4K4 activity can enhance CD8 T cell functions in the context of anti-tumor and anti-viral responses. Inducible deletion of Map4k4 increases CD8 T cell activation, proliferation, cytokine production, and target cell killing in vitro, and enhances CD8 T cell-mediated suppression of tumor growth as well as antigen-specific response to viral infection in vivo. Intratumoral T cells showed evidence of enhanced activation and produced more inflammatory cytokines when stimulated ex vivo. Simultaneously reducing MAP4K4 activity and blocking PDL1 resulted in additive anti-tumor efficacy. Our data suggests that MAP4K4 could be a potential target to augment anti-tumor and immunity. Citation Format: Emel Esen, Weilan Ye, Rajiv Jesudason, Ismail Sergin. Deletion of Map4k4 enhances CD8 T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-144.
Myelodysplastic syndrome (MDS) comprises a heterogeneous group of oftenfatal hematopoietic stem c... more Myelodysplastic syndrome (MDS) comprises a heterogeneous group of oftenfatal hematopoietic stem cell disorders for which neither curative nor standard treatment exists. The complex karyotypes and multistep nature of MDS have severely restricted the identification of causative genetic mutations and thus limited insight into new and more effective therapies. Here we describe a zebrafish mutant crimsonless (crs) with a develop-mental blood defect that closely recapitulates the ineffective hematopoiesis of MDS including anemia, dysplasia, increased blood cell apoptosis, and multilineage cytopenia. By positional cloning, rescue, and morpholino knockdown experiments, we demonstrate that crs encodes a conserved mitochondrial matrix chaperone HSPA9B containing a glycine-to-glutamate substitution within the substrate-binding domain. This mutation compromises mi-tochondrial function, producing oxidative stress and apoptosis distinctly in blood cells. Thus, we identify an essential role for Hspa9b in hematopoiesis and implicate both loss of HSPA9B specifically and mitochondrial dysfunction generally in the pathogenesis of the MDS. (Blood.
As the age of the population increases in many nations, age-related degenerative diseases pose si... more As the age of the population increases in many nations, age-related degenerative diseases pose significant socioeconomic challenges. One of the key degenerative diseases that compromise quality of life is age-related macular degeneration (AMD). AMD is a multi-faceted condition that affects the central retina, which ultimately leads to blindness in millions of people worldwide. The pathophysiology and risk factors for AMD are complex, and the symptoms manifest in multiple related but distinct forms. The ability to develop effective treatments for AMD will depend on a thorough understanding of the underlying pathophysiology, risk factors, and driver molecular pathways, as well as the ability to develop useful animal models. This review provides an overview of the aforementioned aspects in AMD.
MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. I... more MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragmentbased screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.
bioRxiv (Cold Spring Harbor Laboratory), Aug 28, 2020
Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensur... more Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveal distinct requirements for Rasip1 during sprouting angiogenesis, vascular anastomosis and lumen formation. During angiogenic sprouting, Rasip1 is required for efficient cell pairing, which is essential for multicellular tube formation. High-resolution time-lapse analyses show that these cell pairing defects are caused by a destabilization of tricellular junctions suggesting that tricellular junctions may serve as a counterfort to tether sprouting endothelial cells during morphogenetic cell rearrangements. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function leads to junctional defects similar to those seen in rasip1 mutants. Analysis of radil-b single and rasip1/radil-b double mutants reveal distinct and overlapping functions of both proteins. While Rasip1 and Radil-b have similar functions during angiogenic sprouting, the junction formation during anastomosis may primarily depend on Rasip1.
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In this study we report ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In this study we report the use of pharmacodynamic (PD) biomarkers to guide the clinical development of a novel antibody targeting epidermal growth factor-like domain 7 (EGFL7). EGFL7 is a secreted protein produced by endothelial cells of nascent blood vessels in tumors and other proliferating tissues, but is expressed at low levels in healthy quiescent vessels and many non-vascular cell types (Campagnolo et al. 2005; Fitch et al. 2004; Parker et al. 2004; Soncin et al. 2003). Upon secretion, EGFL7 becomes tightly associated with the perivascular extracellular matrix (ECM), and supports endothelial cell adhesion and migration (Parker et al. 2004; Schmidt et al. 2007). EGFL7 protein also protects endothelial cells from stress-induced apoptosis (Xu et al. 2008). Anti-EGFL7 is a humanized monoclonal antibody that binds to EGFL7. In preclinical models, anti-EGFL7 when combined with anti-VEGF augments the survival benefit observed from anti-VEGF alone. Anti-EGFL7 is currently being investigated in Phase I trials (in solid tumors) in combination with bevacizumab. In addition to endothelial cells, circulating CD34Hi/CD31dim progenitor cells (CPCs) express high levels of EGFL7 transcript. This population of cells can differentiate into endothelial cells in vitro, and we found that the EGFL7 protein promotes proliferation and possibly survival of the undifferentiated CPCs in vitro. In preclinical xenograft models, administration of anti-EGFL7 resulted in delayed (day 15) reduction in CPCs, which based on the time course, is unlikely to be mediated simply by clearance of antibody-bound CPCs. Hence, evaluation of CPCs was incorporated into the dose escalation phase of anti-EGFL7 clinical development as a pharmacodynamic biomarker (PD) of drug activity. Biomarker analysis of CPCs in humans correlated with preclinical findings. For example, reduction in CPCs in response to anti-EGFL7 therapy was observed at a time point similar to that in mice. The treatment dependent decrease in CPCs may serve as a pharmacodynamic biomarker that may help guide dose selection as well as represent another potential mechanism for the anti-angiogenic properties of anti-EGFL7 Campagnolo L, Leahy A, Chitnis S, Koschnick S et al. (2005) Am J Pathol 167(1):275-84 Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H. (2004) Dev Dyn. 230(2):316-24 Parker LH, Schmidt M, Jin SW et al (2004) Nature 428(6984):754-8. Schmidt M, Paes K, De Maziere A, (2007) Development 134(16):2913-23. Soncin F, Mattot V, Lionneton F (2003) EMBO J. 22(21):5700-11 Xu D, Perez RE, Ekekezie II (2008) Am J Physiol Lung Cell Mol Physiol. 294(1):L17-23 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3284. doi:10.1158/1538-7445.AM2011-3284
Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastati... more Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression. Utilizing murine models of metastasis, longitudinal in vivo imaging of lymph transport, and function blocking antibodies against two VEGF family members, we provide evidence that distal lymphatics undergo disease course-dependent up-regulation of lymph transport coincidental with structural remodeling. Inhibition of VEGF-C activity with antibodies against VEGF-C or NRP2 prevented these disease-associated changes. Furthermore, utilizing a novel model of adjuvant treatment, we demonstrate that antagonism of VEGF-C or NRP2 decreases post SLN metastasis. These data support a potential therapeutic strategy for inhibiting distant metastatic dissemination via targeting tumor-associated lymphatic remodeling.
A significant existing challenge for anti-angiogenic therapy is that there are no validated bioma... more A significant existing challenge for anti-angiogenic therapy is that there are no validated biomarkers to either monitor dosing, response to therapy or escape from therapy. This limitation is a more significant challenge when the therapy in question targets a novel pathway with a unique MOA. Some generalized biomarkers have been identified to follow administration of anti-angiogenic therapy, and included in this class are circulating endothelial cells or CECs. CECs are a well-established marker of on-going and active angiogenesis1. In this report we describe the utility of measuring CECs in response to targeting a novel angiogenic protein, EGFL7. EGFL7 is an extracellular matrix protein that is highly expressed in endothelial cells of angiogenic vessels, including embryonic, tumor and injured vasculature. Inhibition of EGFL7 activity by anti-EGFL7 results in a reduction of tumor microvascular density and tumor growth in genetically engineered models of cancer such as RIP-T Ags. In o...
F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bac... more F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bacterially expressed F1-20/AP-3 to bind and assemble clathrin cages. We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble clathrin as efficiently as preparations of F1-20/AP-3 from bovine brain. This establishes that the clathrin assembly activity found in F1-20/AP-3 preparations from brain extracts is indeed encoded by the cloned gene for F1-20/AP-3. It also demonstrates that post-translation modification is not required for activation of the clathrin binding or assembly function of F1-20/AP-3. Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1-20/AP-3 reveals a strikingly narrow size distribution. This may be important for the regulation of quanta1 size during neurotransmission. We also express the 33 kD NH 2 -terminus of F1-20/AP-3 in E. coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages. It has been suggested that the 33 kD NH 2 -terminus of F1-20/AP-3 constitutes a clathrin binding domain. We find that the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly. The finding that the 33 kD NH 2 -terminus of F1-20/AP-3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies. The finding that the 33 kD NH 2 -terminus of F1-20/ AP-3 fails to bind to clathrin cages is novel and potentially important. It is clear from these experiments that the 33 kD NH 2 -terminus of F1-20/AP-3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated.
Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebra... more Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. Here, we identify a Sonic hedgehog(Shh)-regulated cascade of transcription factors that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1), which project to the forebrain, but not for the induction of caudal 5-HT neurons,which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. Our findings reveal that the identity of closely rel...
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