Arteriosclerosis, Thrombosis, and Vascular Biology
CRISPR/Cas9 technology-mediated genome editing has significantly improved inactivation of genes b... more CRISPR/Cas9 technology-mediated genome editing has significantly improved inactivation of genes both in vitro and in vivo in many organisms in a targeted manner. This study reports a novel CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. In this vector, the cardiac-specific, cardiac myosin light chain 2 ( cmlc2) promoter drives the Cas9 expression to silence the neuropilin1(nrp1) gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes an inimitable tool to regulate the gene knockout in both the developmental and adult stages. Hence, it widens the possibility of loss-of-function studies in zebrafish at different stages of development. In contrast to mammals, adult zebrafish hearts can regenerate after an injury. Neuropilins (nrp) are co-receptors that have been reported earlier to play crucial roles in zebrafish heart regeneration. Using this novel tool, we investigated the role of neuropilin isoforms, nrp1a and nrp1b, in ...
Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of... more Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of malignancy and an extremely unfavorable prognosis. The current efficacy of established treatments for GBM is insufficient, necessitating the prompt development of novel therapeutic approaches. The progress made in the fundamental scientific understanding of GBM is swiftly translated into more advanced stages of therapeutic studies. Despite extensive efforts to identify new therapeutic approaches, GBM exhibits a high mortality rate. The current efficacy of treatments for GBM patients is insufficient due to factors such as tumor heterogeneity, the blood–brain barrier, glioma stem cells, drug efflux pumps, and DNA damage repair mechanisms. Considering this, pharmacological cocktail therapy has demonstrated a growing efficacy in addressing these challenges. Towards this, various forms of immunotherapy, including the immune checkpoint blockade, chimeric antigen receptor T (CAR T) cell therap...
Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by meta... more Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2’s known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2.
CRISPR/Cas9 technology-mediated genome editing has significantly improved the targeted inactivati... more CRISPR/Cas9 technology-mediated genome editing has significantly improved the targeted inactivation of genesin vitroandin vivoin many organisms. In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specificcardiac myosins light chain 2 (cmlc2) promoter drives Cas9 expression to silence theneuropilin-1(nrp1) gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence, widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoformsnrp1aandnrp1bin response to cardiac injury and regeneration in adult zebrafish hearts. We observed that both the isoforms (nrp1aandnrp1b) are upregulated after the cryoinjury. Interestingly, thenrp1b-knockout significant...
Genome-wide association studies (GWAS) have discovered thousands of risk loci for common, complex... more Genome-wide association studies (GWAS) have discovered thousands of risk loci for common, complex diseases, each of which could point to genes and gene programs that influence disease. For some diseases, it has been observed that GWAS signals converge on a smaller number of biological programs, and that this convergence can help to identify causal genes1–6. However, identifying such convergence remains challenging: each GWAS locus can have many candidate genes, each gene might act in one or more possible programs, and it remains unclear which programs might influence disease risk. Here, we developed a new approach to address this challenge, by creating unbiased maps to link disease variants to genes to programs (V2G2P) in a given cell type. We applied this approach to study the role of endothelial cells in the genetics of coronary artery disease (CAD). To link variants to genes, we constructed enhancer-gene maps using the Activity-by-Contact model7,8. To link genes to programs, we a...
Hypoxia-induced endothelial cell (EC) dysfunction has been implicated as potential initiators of ... more Hypoxia-induced endothelial cell (EC) dysfunction has been implicated as potential initiators of different pathogenesis, including Alzheimer’s disease and vascular dementia. However, in-depth structural, mechanical, and molecular mechanisms leading to EC dysfunction and pathology need to be revealed. Here, we show that ECs exposed to hypoxic conditions readily enter a senescence phenotype. As expected, hypoxia upregulated the expression of vascular endothelial growth factor (VEGFs) and its receptors (VEGFRs) in the ECs. Interestingly, Knockdown of VEGFR-1 expression prior to hypoxia exposure prevented EC senescence, suggesting an important role of VEGFR-1 expression in the induction of EC senescence. Using atomic force microscopy, we showed that senescent ECs had a flattened cell morphology, decreased membrane ruffling, and increased membrane stiffness, demonstrating unique morphological and nanomechanical signatures. Furthermore, we show that hypoxia inhibited the Hippo pathway Yes...
Simple Summary Pancreatic cancer is among the most lethal cancers. The expression of PLEXIND1, a ... more Simple Summary Pancreatic cancer is among the most lethal cancers. The expression of PLEXIND1, a receptor, is upregulated in many cancers (including pancreatic cancer). Traditionally, PLEXIND1 is known to be involved in neuron development and mediate semaphorin signaling. However, its role and signaling in cancer is not fully understood. In our study, we present a new mechanism through which PLEXIND1 mediates its roles in cancer. For the first time, we demonstrate that it can function as a transforming growth factor beta coreceptor and modulate SMAD3 signaling. Around 90% of pancreatic cancer patients have mutant KRAS. Our work suggests that PLEXIND1 functions differently in pancreatic cancer cell lines, and the difference correlates with KRAS mutational status. Additionally, we demonstrate a novel peptide based therapeutic approach to target PLEXIND1 in cancer cells. Our work is valuable to both neuroscience and cancer fields, as it demonstrates an association between two previousl...
Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-... more Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-2 to control vasculogenesis and angiogenesis. Dysregulation of these physiological processes contributes to the pathologies of heart disease, stroke, and cancer. Protein kinase D (PKD) plays a crucial role in the regulation of angiogenesis by modulating endothelial cell proliferation and migration. In human umbilical vein endothelial cells (HUVEC) and human blood outgrowth endothelial cells (BOEC), knockdown of PKD-1 or PKD-2 downregulates VEGFR-2 and significantly inhibits VEGF-induced endothelial cell proliferation and migration. We sought to determine the molecular mechanism through which PKD modulates VEGFR-2 expression. Based on bioinformatics data, activating enhancer binding protein 2 (AP2) binding sites exist within the VEGFR-2 promoter. Thus, we hypothesized PKD may downregulate VEGFR-2 through AP2-mediated transcriptional repression of the VEGFR-2 promoter. Indeed, AP2β binds t...
Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is assoc... more Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 21, 2018
Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to ... more Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aβ peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aβ1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aβ oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aβ1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aβ1-42 monomers, and 5 µM Aβ1-42 fibrils, respectively. Brain ECs treated with Aβ1-42 oligomers showed increased senescence-associated β-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aβ1-42 monomer or Aβ1-42 fibrils did not induce senes...
Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in p... more Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis (MS), however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ) -induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1 dependent manner. Moreover, endothelial specific NRP1 knockout mice, VECadherin-Cre-ERT2/NRP1(flox/flox) mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry, and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neur...
Arteriosclerosis, Thrombosis, and Vascular Biology
CRISPR/Cas9 technology-mediated genome editing has significantly improved inactivation of genes b... more CRISPR/Cas9 technology-mediated genome editing has significantly improved inactivation of genes both in vitro and in vivo in many organisms in a targeted manner. This study reports a novel CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. In this vector, the cardiac-specific, cardiac myosin light chain 2 ( cmlc2) promoter drives the Cas9 expression to silence the neuropilin1(nrp1) gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes an inimitable tool to regulate the gene knockout in both the developmental and adult stages. Hence, it widens the possibility of loss-of-function studies in zebrafish at different stages of development. In contrast to mammals, adult zebrafish hearts can regenerate after an injury. Neuropilins (nrp) are co-receptors that have been reported earlier to play crucial roles in zebrafish heart regeneration. Using this novel tool, we investigated the role of neuropilin isoforms, nrp1a and nrp1b, in ...
Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of... more Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of malignancy and an extremely unfavorable prognosis. The current efficacy of established treatments for GBM is insufficient, necessitating the prompt development of novel therapeutic approaches. The progress made in the fundamental scientific understanding of GBM is swiftly translated into more advanced stages of therapeutic studies. Despite extensive efforts to identify new therapeutic approaches, GBM exhibits a high mortality rate. The current efficacy of treatments for GBM patients is insufficient due to factors such as tumor heterogeneity, the blood–brain barrier, glioma stem cells, drug efflux pumps, and DNA damage repair mechanisms. Considering this, pharmacological cocktail therapy has demonstrated a growing efficacy in addressing these challenges. Towards this, various forms of immunotherapy, including the immune checkpoint blockade, chimeric antigen receptor T (CAR T) cell therap...
Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by meta... more Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2’s known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2.
CRISPR/Cas9 technology-mediated genome editing has significantly improved the targeted inactivati... more CRISPR/Cas9 technology-mediated genome editing has significantly improved the targeted inactivation of genesin vitroandin vivoin many organisms. In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specificcardiac myosins light chain 2 (cmlc2) promoter drives Cas9 expression to silence theneuropilin-1(nrp1) gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence, widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoformsnrp1aandnrp1bin response to cardiac injury and regeneration in adult zebrafish hearts. We observed that both the isoforms (nrp1aandnrp1b) are upregulated after the cryoinjury. Interestingly, thenrp1b-knockout significant...
Genome-wide association studies (GWAS) have discovered thousands of risk loci for common, complex... more Genome-wide association studies (GWAS) have discovered thousands of risk loci for common, complex diseases, each of which could point to genes and gene programs that influence disease. For some diseases, it has been observed that GWAS signals converge on a smaller number of biological programs, and that this convergence can help to identify causal genes1–6. However, identifying such convergence remains challenging: each GWAS locus can have many candidate genes, each gene might act in one or more possible programs, and it remains unclear which programs might influence disease risk. Here, we developed a new approach to address this challenge, by creating unbiased maps to link disease variants to genes to programs (V2G2P) in a given cell type. We applied this approach to study the role of endothelial cells in the genetics of coronary artery disease (CAD). To link variants to genes, we constructed enhancer-gene maps using the Activity-by-Contact model7,8. To link genes to programs, we a...
Hypoxia-induced endothelial cell (EC) dysfunction has been implicated as potential initiators of ... more Hypoxia-induced endothelial cell (EC) dysfunction has been implicated as potential initiators of different pathogenesis, including Alzheimer’s disease and vascular dementia. However, in-depth structural, mechanical, and molecular mechanisms leading to EC dysfunction and pathology need to be revealed. Here, we show that ECs exposed to hypoxic conditions readily enter a senescence phenotype. As expected, hypoxia upregulated the expression of vascular endothelial growth factor (VEGFs) and its receptors (VEGFRs) in the ECs. Interestingly, Knockdown of VEGFR-1 expression prior to hypoxia exposure prevented EC senescence, suggesting an important role of VEGFR-1 expression in the induction of EC senescence. Using atomic force microscopy, we showed that senescent ECs had a flattened cell morphology, decreased membrane ruffling, and increased membrane stiffness, demonstrating unique morphological and nanomechanical signatures. Furthermore, we show that hypoxia inhibited the Hippo pathway Yes...
Simple Summary Pancreatic cancer is among the most lethal cancers. The expression of PLEXIND1, a ... more Simple Summary Pancreatic cancer is among the most lethal cancers. The expression of PLEXIND1, a receptor, is upregulated in many cancers (including pancreatic cancer). Traditionally, PLEXIND1 is known to be involved in neuron development and mediate semaphorin signaling. However, its role and signaling in cancer is not fully understood. In our study, we present a new mechanism through which PLEXIND1 mediates its roles in cancer. For the first time, we demonstrate that it can function as a transforming growth factor beta coreceptor and modulate SMAD3 signaling. Around 90% of pancreatic cancer patients have mutant KRAS. Our work suggests that PLEXIND1 functions differently in pancreatic cancer cell lines, and the difference correlates with KRAS mutational status. Additionally, we demonstrate a novel peptide based therapeutic approach to target PLEXIND1 in cancer cells. Our work is valuable to both neuroscience and cancer fields, as it demonstrates an association between two previousl...
Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-... more Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGFR-2 to control vasculogenesis and angiogenesis. Dysregulation of these physiological processes contributes to the pathologies of heart disease, stroke, and cancer. Protein kinase D (PKD) plays a crucial role in the regulation of angiogenesis by modulating endothelial cell proliferation and migration. In human umbilical vein endothelial cells (HUVEC) and human blood outgrowth endothelial cells (BOEC), knockdown of PKD-1 or PKD-2 downregulates VEGFR-2 and significantly inhibits VEGF-induced endothelial cell proliferation and migration. We sought to determine the molecular mechanism through which PKD modulates VEGFR-2 expression. Based on bioinformatics data, activating enhancer binding protein 2 (AP2) binding sites exist within the VEGFR-2 promoter. Thus, we hypothesized PKD may downregulate VEGFR-2 through AP2-mediated transcriptional repression of the VEGFR-2 promoter. Indeed, AP2β binds t...
Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is assoc... more Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 21, 2018
Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to ... more Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aβ peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aβ1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aβ oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aβ1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aβ1-42 monomers, and 5 µM Aβ1-42 fibrils, respectively. Brain ECs treated with Aβ1-42 oligomers showed increased senescence-associated β-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aβ1-42 monomer or Aβ1-42 fibrils did not induce senes...
Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in p... more Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis (MS), however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ) -induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1 dependent manner. Moreover, endothelial specific NRP1 knockout mice, VECadherin-Cre-ERT2/NRP1(flox/flox) mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry, and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neur...
Uploads
Papers by Ramcharan Angom