Usha Nagavarapu - Academia.edu (original) (raw)
Papers by Usha Nagavarapu
Nature Biotechnology, 2001
Previously we showed the superior in vitro survival of human telomerase reverse transcriptase (hT... more Previously we showed the superior in vitro survival of human telomerase reverse transcriptase (hTERT)-transduced human endothelial cells (EC). Here we show that retroviral-mediated transduction of hTERT in human dermal microvascular EC (HDMEC) results in cell lines that form microvascular structures when subcutaneously implanted in severe combined immunodeficiency (SCID) mice. Anti-human type IV collagen basement membrane immunoreactivity and visualization of enhanced green fluorescent protein (eGFP)-labeled microvessels confirmed the human origin of these capillaries. No human vasculature was observed after implantation of HT1080 fibrosarcoma cells, 293 human embryonic kidney cells, or human skin fibroblasts. Intravascular red fluorescent microspheres injected into host circulation were found within green "telomerized" microvessels, indicating functional murine-human vessel anastamoses. Whereas primary HDMEC-derived vessel density decreased with time, telomerized HDMEC maintained durable vessels six weeks after xenografting. Modulation of implant vessel density by exposure to different angiogenic and angiostatic factors demonstrated the utility of this system for the study of human microvascular remodeling in vivo.
Journal of Neuro-Oncology, 2005
Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix ... more Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix (ECM), disruption of ECM components, and subsequent cell penetration into adjacent brain structures. Discoidin domain receptor 1 (DDR1) tyrosine kinases constitute a novel family of receptors characterized by a unique structure in the ectodomain (discoidin-I domain). These cell surface receptors bind to several collagens and facilitate cell adhesion. Little is known about DDR1 expression and function in glioblastoma multiforme. In this study we demonstrate that DDR1 is overexpressed in glioma tissues using cDNA arrays, immunohistochemistry and Western blot analysis. Functional comparison of two splice variants of DDR1 (DDR1a and DDR1b) reveal novel differences in cell based glioma models. Overexpression of either DDR1a or DDR1b caused increased cell attachment. However, glioma cells overexpressing DDR1a display enhanced invasion and migration. We also detect increased levels of matrix metalloproteinase-2 in DDR1a overexpressing cells as measured by zymography. Inhibition of MMP activity using MMP inhibitor suppressed DDR1a stimulated cell-invasion. Similarly, an antibody against DDR1 reduced DDR1a mediated invasion as well as the enhanced adhesion of DDR1a and DDR1b overexpressing cells. These results suggest that DDR1a plays a critical role in inducing tumor cell adhesion and invasion, and this invasive phenotype is caused by activation of matrix metalloproteinase-2.
Journal of Biological Chemistry, 2001
Neuronal nicotinic acetylcholine receptors (nAChRs) containing the ␣7 subunit are expressed in th... more Neuronal nicotinic acetylcholine receptors (nAChRs) containing the ␣7 subunit are expressed in the central nervous system, autonomic nervous system, retina, adrenal medulla, and PC12 cells. ␣7 nAChRs have been implicated in several important biological activities apart from synaptic transmission such as mediating neurite growth and presynaptic control of neurotransmitter release. A 178-base pair promoter was sufficient to drive high level expression of the ␣7 gene in PC12 cells. The ␣7 promoter was also cell-specific, expressing in PC12 cells but not in L6 rat muscle cells. Within our minimal rat ␣7 nAChR promoter we identified two sequences important for basal level expression. Mutation of a GC-rich sequence at ؊172 relative to the translational start site led to an increase in activity of the promoter, indicating the presence of a negative regulatory element. Upstream stimulatory factor-1 acted to regulate ␣7 expression positively by binding to an E-box at ؊116. A site directly adjacent to the upstream stimulatory factor-1 binding site was shown to bind Egr-1. Sp1 and Sp3 binding also occurred downstream from or overlapping the Egr-1 binding site in the rat ␣7 promoter. Several transcription factors interact in close proximity to control expression of the rat ␣7 nicotinic receptor gene. Neuronal nicotinic acetylcholine receptors (nAChRs) 1 mediate synaptic transmission in many parts of the vertebrate central nervous system, as well as in autonomic ganglia, retina, and adrenal medulla. Neuronal nAChRs are pentameric structures (1, 2) that function as ligand-gated ion channels and are composed of multiple ␣ and  subunits. Nine neuronal nAChR ␣ subunit genes (␣2-␣10) and three nAChR  subunit genes (2-4) have been identified (3-16). One specific subtype of neuronal nAChR is sensitive to ␣-bungarotoxin and composed of ␣7 subunits. ␣7 nAChRs possess several characteristics that set them apart from most
Oncogene, 2005
extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morph... more extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morphology and cell rounding. These results indicate that the extracellular domain may compete for unidentified ligand(s), and block the normal function of GPR56 in cell attachment. In reporter assays, overexpression of GPR56 activates the NF-jB, PAI-1 and TCF transcriptional response elements. These pathways have been implicated in cytoskeletal signaling, adhesion and tumor biology. The above results indicate that GPR56 serves as an adhesion GPCR and is involved in adhesion signaling.
Oral minocycline has been the standard of care for the treatment of non-nodular moderate to sever... more Oral minocycline has been the standard of care for the treatment of non-nodular moderate to severe inflammatory acne vulgaris due to its inhibitory effects on the acne-causing Propionibacterium acnes bacterium and its anti-inflammatory properties, Despite the availability of an oral dosage form since 1966, a commercial topical minocycline remains elusive because of the challenges in stabilizing the active pharmaceutical ingredient (API) in a liquid/semisolid while ensuring sufficient uptake into targeted lesions. Recently, an investigative topical minocycline gel (BPX-01) has been developed to address the unmet needs for localized and targeted delivery while minimizing the risks of systemic side effects. Earlier preclinical studies pertaining to transepidermal delivery of the API had depended on semi-infinite doses of the 1%, 2% and 4% formulations to elicit enough fluorescence yield. We have subsequently shown evidence of minocycline delivery of 1% and 4% BPX-01 into the pilosebace...
Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an ant... more Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify ...
SKIN The Journal of Cutaneous Medicine
Biomedical Optics Express
Lasers in Surgery and Medicine
Visualizing and Quantifying Drug Distribution in Tissue II
International Journal of Pharmaceutics: X
Biomedical optics express, 2018
Acne vulgaris is a common chronic skin disease in young adults caused by infection of the piloseb... more Acne vulgaris is a common chronic skin disease in young adults caused by infection of the pilosebaceous unit, resulting in pimples and possibly permanent scarring on the skin. Minocycline, a common antibiotic, has been widely utilized as a systemic antimicrobial treatment for acne via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline through the epidermis and into the pilosebaceous unit to achieve localized treatment with lower doses of drug. As the effectiveness of the drug is directly related to its successful delivery, there is a need to evaluate the pharmacokinetics at the cellular level within tissue. Advantageously, minocycline is naturally fluorescent and can be directly visualized using microscopy-based approaches. Due to high endogenous autofluorescence, however, imaging of weakly emitting fluorescent molecules such as minocycline in skin tissue can be challenging. Here, we demonstrate a method for the selective ...
Visualizing and Quantifying Drug Distribution in Tissue, 2017
Cancer Research, 2014
Traditional anticancer therapies are often limited in their efficacy because they often lead to d... more Traditional anticancer therapies are often limited in their efficacy because they often lead to drugresistant tumors and display toxicity to normal cells. The emergence of anti-angiogenic strategies, on the other hand, had circumvented these problems by targeting the blood supply of growing tumors. Furthermore, due to the possibility of targeting angiogenic endothelial cells, these therapies allow selective targeting of the blood supply in tumors without affecting normal blood vessels. Angiogenesis is the process of developing new blood vessels that are critical for growth of solid tumors. Hence, anti-angiogenic therapy that targets tumors indirectly, through its blood supply, becomes a rational anticancer strategy. As metastatic growth directly impacts the overall patient survival, control of angiogenesis represents one of the key goals in cancer therapy, as exemplified by targeting the VEGF pathway with bevacizumab.
Proceedings of the National Academy of Sciences, 2002
The ␣4 laminin subunit is a component of the basement membrane of blood vessels where it codistri... more The ␣4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins ␣v3, ␣31, and ␣61. An antibody against the G domain (residues 919-1207; G 919-1207) of the ␣4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G 919-1207 to support endothelial cell adhesion. In particular, endothelial cell adhesion to G 919-1207 is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins ␣v3 and 1 and a combination of antibodies against ␣3 and ␣6 integrin subunits inhibit endothelial cell attachment to G 919-1207. Moreover, both ␣v3 and ␣31 integrin bind with high affinity to G 919-1207. Together, our studies demonstrate that the G domain of laminin ␣4 chain is a specific, high affinity ligand for the ␣v3 and ␣31 integrin heterodimers and that these integrins, together with ␣61, function cooperatively to mediate endothelial cell-␣4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the ␣v3 integrin in angiogenesis. matrix ͉ matrix receptor ͉ blood vessels L aminins, heterotrimeric molecules composed of ␣, , and ␥ Abbreviations: TrHBMEC, immortalized human bone marrow endothelial cells; HUVEC, human umbilical vascular endothelial cells.
Microcirculation, 2004
Microvasculature plays an important role in a variety of physiological and pathological processes... more Microvasculature plays an important role in a variety of physiological and pathological processes. The authors have previously shown that primary cultures of human microvascular endothelial cells consist of 2 distinct populations of blood vascular and lymphatic endothelial cells. These subtypes are ephemeral and lose purity through passaging. To generate reproducible in vitro and in vivo experiments stable blood and lymphatic endothelial cell lines are an essential prerequisite. In this study they have used human telomerase gene-immortalized nontransformed human microvascular endothelial cell cloned pure cultures of blood and lymphatic endothelial cell subpopulations. Flow cytometry, immunofluorescence, Northern and Western blotting, microarray gene analysis, as well as basic functional assays were used to characterize these clones. Immortalized blood and lymphatic subpopulations are stable and functionally specialized cell lineages that expressed pan-endothelial and cell-type-specific markers. They are excellent candidates for long-term culture studies on microvascular-related diseases.
Nature Biotechnology, 2001
Previously we showed the superior in vitro survival of human telomerase reverse transcriptase (hT... more Previously we showed the superior in vitro survival of human telomerase reverse transcriptase (hTERT)-transduced human endothelial cells (EC). Here we show that retroviral-mediated transduction of hTERT in human dermal microvascular EC (HDMEC) results in cell lines that form microvascular structures when subcutaneously implanted in severe combined immunodeficiency (SCID) mice. Anti-human type IV collagen basement membrane immunoreactivity and visualization of enhanced green fluorescent protein (eGFP)-labeled microvessels confirmed the human origin of these capillaries. No human vasculature was observed after implantation of HT1080 fibrosarcoma cells, 293 human embryonic kidney cells, or human skin fibroblasts. Intravascular red fluorescent microspheres injected into host circulation were found within green "telomerized" microvessels, indicating functional murine-human vessel anastamoses. Whereas primary HDMEC-derived vessel density decreased with time, telomerized HDMEC maintained durable vessels six weeks after xenografting. Modulation of implant vessel density by exposure to different angiogenic and angiostatic factors demonstrated the utility of this system for the study of human microvascular remodeling in vivo.
Journal of Neuro-Oncology, 2005
Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix ... more Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix (ECM), disruption of ECM components, and subsequent cell penetration into adjacent brain structures. Discoidin domain receptor 1 (DDR1) tyrosine kinases constitute a novel family of receptors characterized by a unique structure in the ectodomain (discoidin-I domain). These cell surface receptors bind to several collagens and facilitate cell adhesion. Little is known about DDR1 expression and function in glioblastoma multiforme. In this study we demonstrate that DDR1 is overexpressed in glioma tissues using cDNA arrays, immunohistochemistry and Western blot analysis. Functional comparison of two splice variants of DDR1 (DDR1a and DDR1b) reveal novel differences in cell based glioma models. Overexpression of either DDR1a or DDR1b caused increased cell attachment. However, glioma cells overexpressing DDR1a display enhanced invasion and migration. We also detect increased levels of matrix metalloproteinase-2 in DDR1a overexpressing cells as measured by zymography. Inhibition of MMP activity using MMP inhibitor suppressed DDR1a stimulated cell-invasion. Similarly, an antibody against DDR1 reduced DDR1a mediated invasion as well as the enhanced adhesion of DDR1a and DDR1b overexpressing cells. These results suggest that DDR1a plays a critical role in inducing tumor cell adhesion and invasion, and this invasive phenotype is caused by activation of matrix metalloproteinase-2.
Journal of Biological Chemistry, 2001
Neuronal nicotinic acetylcholine receptors (nAChRs) containing the ␣7 subunit are expressed in th... more Neuronal nicotinic acetylcholine receptors (nAChRs) containing the ␣7 subunit are expressed in the central nervous system, autonomic nervous system, retina, adrenal medulla, and PC12 cells. ␣7 nAChRs have been implicated in several important biological activities apart from synaptic transmission such as mediating neurite growth and presynaptic control of neurotransmitter release. A 178-base pair promoter was sufficient to drive high level expression of the ␣7 gene in PC12 cells. The ␣7 promoter was also cell-specific, expressing in PC12 cells but not in L6 rat muscle cells. Within our minimal rat ␣7 nAChR promoter we identified two sequences important for basal level expression. Mutation of a GC-rich sequence at ؊172 relative to the translational start site led to an increase in activity of the promoter, indicating the presence of a negative regulatory element. Upstream stimulatory factor-1 acted to regulate ␣7 expression positively by binding to an E-box at ؊116. A site directly adjacent to the upstream stimulatory factor-1 binding site was shown to bind Egr-1. Sp1 and Sp3 binding also occurred downstream from or overlapping the Egr-1 binding site in the rat ␣7 promoter. Several transcription factors interact in close proximity to control expression of the rat ␣7 nicotinic receptor gene. Neuronal nicotinic acetylcholine receptors (nAChRs) 1 mediate synaptic transmission in many parts of the vertebrate central nervous system, as well as in autonomic ganglia, retina, and adrenal medulla. Neuronal nAChRs are pentameric structures (1, 2) that function as ligand-gated ion channels and are composed of multiple ␣ and  subunits. Nine neuronal nAChR ␣ subunit genes (␣2-␣10) and three nAChR  subunit genes (2-4) have been identified (3-16). One specific subtype of neuronal nAChR is sensitive to ␣-bungarotoxin and composed of ␣7 subunits. ␣7 nAChRs possess several characteristics that set them apart from most
Oncogene, 2005
extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morph... more extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morphology and cell rounding. These results indicate that the extracellular domain may compete for unidentified ligand(s), and block the normal function of GPR56 in cell attachment. In reporter assays, overexpression of GPR56 activates the NF-jB, PAI-1 and TCF transcriptional response elements. These pathways have been implicated in cytoskeletal signaling, adhesion and tumor biology. The above results indicate that GPR56 serves as an adhesion GPCR and is involved in adhesion signaling.
Oral minocycline has been the standard of care for the treatment of non-nodular moderate to sever... more Oral minocycline has been the standard of care for the treatment of non-nodular moderate to severe inflammatory acne vulgaris due to its inhibitory effects on the acne-causing Propionibacterium acnes bacterium and its anti-inflammatory properties, Despite the availability of an oral dosage form since 1966, a commercial topical minocycline remains elusive because of the challenges in stabilizing the active pharmaceutical ingredient (API) in a liquid/semisolid while ensuring sufficient uptake into targeted lesions. Recently, an investigative topical minocycline gel (BPX-01) has been developed to address the unmet needs for localized and targeted delivery while minimizing the risks of systemic side effects. Earlier preclinical studies pertaining to transepidermal delivery of the API had depended on semi-infinite doses of the 1%, 2% and 4% formulations to elicit enough fluorescence yield. We have subsequently shown evidence of minocycline delivery of 1% and 4% BPX-01 into the pilosebace...
Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an ant... more Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify ...
SKIN The Journal of Cutaneous Medicine
Biomedical Optics Express
Lasers in Surgery and Medicine
Visualizing and Quantifying Drug Distribution in Tissue II
International Journal of Pharmaceutics: X
Biomedical optics express, 2018
Acne vulgaris is a common chronic skin disease in young adults caused by infection of the piloseb... more Acne vulgaris is a common chronic skin disease in young adults caused by infection of the pilosebaceous unit, resulting in pimples and possibly permanent scarring on the skin. Minocycline, a common antibiotic, has been widely utilized as a systemic antimicrobial treatment for acne via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline through the epidermis and into the pilosebaceous unit to achieve localized treatment with lower doses of drug. As the effectiveness of the drug is directly related to its successful delivery, there is a need to evaluate the pharmacokinetics at the cellular level within tissue. Advantageously, minocycline is naturally fluorescent and can be directly visualized using microscopy-based approaches. Due to high endogenous autofluorescence, however, imaging of weakly emitting fluorescent molecules such as minocycline in skin tissue can be challenging. Here, we demonstrate a method for the selective ...
Visualizing and Quantifying Drug Distribution in Tissue, 2017
Cancer Research, 2014
Traditional anticancer therapies are often limited in their efficacy because they often lead to d... more Traditional anticancer therapies are often limited in their efficacy because they often lead to drugresistant tumors and display toxicity to normal cells. The emergence of anti-angiogenic strategies, on the other hand, had circumvented these problems by targeting the blood supply of growing tumors. Furthermore, due to the possibility of targeting angiogenic endothelial cells, these therapies allow selective targeting of the blood supply in tumors without affecting normal blood vessels. Angiogenesis is the process of developing new blood vessels that are critical for growth of solid tumors. Hence, anti-angiogenic therapy that targets tumors indirectly, through its blood supply, becomes a rational anticancer strategy. As metastatic growth directly impacts the overall patient survival, control of angiogenesis represents one of the key goals in cancer therapy, as exemplified by targeting the VEGF pathway with bevacizumab.
Proceedings of the National Academy of Sciences, 2002
The ␣4 laminin subunit is a component of the basement membrane of blood vessels where it codistri... more The ␣4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins ␣v3, ␣31, and ␣61. An antibody against the G domain (residues 919-1207; G 919-1207) of the ␣4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G 919-1207 to support endothelial cell adhesion. In particular, endothelial cell adhesion to G 919-1207 is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins ␣v3 and 1 and a combination of antibodies against ␣3 and ␣6 integrin subunits inhibit endothelial cell attachment to G 919-1207. Moreover, both ␣v3 and ␣31 integrin bind with high affinity to G 919-1207. Together, our studies demonstrate that the G domain of laminin ␣4 chain is a specific, high affinity ligand for the ␣v3 and ␣31 integrin heterodimers and that these integrins, together with ␣61, function cooperatively to mediate endothelial cell-␣4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the ␣v3 integrin in angiogenesis. matrix ͉ matrix receptor ͉ blood vessels L aminins, heterotrimeric molecules composed of ␣, , and ␥ Abbreviations: TrHBMEC, immortalized human bone marrow endothelial cells; HUVEC, human umbilical vascular endothelial cells.
Microcirculation, 2004
Microvasculature plays an important role in a variety of physiological and pathological processes... more Microvasculature plays an important role in a variety of physiological and pathological processes. The authors have previously shown that primary cultures of human microvascular endothelial cells consist of 2 distinct populations of blood vascular and lymphatic endothelial cells. These subtypes are ephemeral and lose purity through passaging. To generate reproducible in vitro and in vivo experiments stable blood and lymphatic endothelial cell lines are an essential prerequisite. In this study they have used human telomerase gene-immortalized nontransformed human microvascular endothelial cell cloned pure cultures of blood and lymphatic endothelial cell subpopulations. Flow cytometry, immunofluorescence, Northern and Western blotting, microarray gene analysis, as well as basic functional assays were used to characterize these clones. Immortalized blood and lymphatic subpopulations are stable and functionally specialized cell lineages that expressed pan-endothelial and cell-type-specific markers. They are excellent candidates for long-term culture studies on microvascular-related diseases.