Biomedical subjects

Usha Nagavarapu

Publications and source records attributed to Usha Nagavarapu.

6 recordsLinked to original sources

PubMed2006

Discoidin domain receptor-1a (DDR1a) promotes glioma cell invasion and adhesion in association with matrix metalloproteinase-2.

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.

Blotting, Western↗

PubMed2005

GPR56 is a GPCR that is overexpressed in gliomas and functions in tumor cell adhesion.

GPR56 (also known as TM7XN1) is a newly discovered orphan G-protein-coupled receptor (GPCR) of the secretin family that has a role in the development of neural progenitor cells and has been linked to developmental malformations of the human brain. GPR56 diverges from other secretin-like family members in that it has an extremely large N-terminal extracellular region (381 amino acids) and contains a novel feature among this new subclass, consisting of four cysteine residues that define a GPCR proteolytic site (GPS motif) located just before the first transmembrane spanning domain. The rest of the amino-terminal domain contains a large number of possible N- and O-linked glycosylation sites similar to mucin-like proteins. These features suggest a role in cell-cell, or cell-matrix interactions. Here, we demonstrate upregulation of GPR56 in glioblastoma multiforme tumors using functional genomics. Immunohistochemistry studies confirmed the expression of GPR56 protein in a majority of glioblastoma/astrocytoma tumor samples with undetectable levels of expression in normal adult brain tissue. Immunofluorescence analysis of human glioma cells using anti-GPR56 antibodies demonstrate that GPR56 is expressed on the leading edge of membrane filopodia and colocalizes with alpha-actinin. Purified recombinant GPR56 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-kappaB, 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.

Cell Adhesion↗

PubMed2002

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo.

The alpha4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins alphavbeta3, alpha3beta1, and alpha6beta1. An antibody against the G domain (residues 919-1207; G(919-1207)) of the alpha4 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 alphavbeta3 and beta1 and a combination of antibodies against alpha3 and alpha6 integrin subunits inhibit endothelial cell attachment to G(919-1207). Moreover, both alphavbeta3 and alpha3beta1 integrin bind with high affinity to G(919-1207). Together, our studies demonstrate that the G domain of laminin alpha4 chain is a specific, high affinity ligand for the alphavbeta3 and alpha3beta1 integrin heterodimers and that these integrins, together with alpha6beta1, function cooperatively to mediate endothelial cell-alpha4 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 alphavbeta3 integrin in angiogenesis.

Antibodies, Monoclonal↗

PubMed2002

Membrane type 1 matrix metalloproteinase regulates cellular invasiveness and survival in cutaneous epidermal cells.

Membrane type 1 matrix metalloproteinase is a member of the membrane-anchored matrix metalloproteinase family and is involved in tissue remodeling events ranging from tumor invasion and angiogenesis to growth and development. We sought to clarify the role of membrane type 1 matrix metalloproteinase in cutaneous epidermal cells using anti-sense cDNA expression in human keratinocytes. Modulation of membrane type 1 matrix metalloproteinase transcript and protein levels was achieved via retroviral expression of a 5' 1.4 kb anti-sense membrane type 1 matrix metalloproteinase construct and a 3.4 kb full-length sense membrane type 1 matrix metalloproteinase construct in primary and immortalized keratinocytes and SCC-25 cells. Maximal reductions were observed 48-72 h after transduction with 1.4 kb anti-sense membrane type 1 matrix metalloproteinase construct that correlated with significant decreased pro-matrix metalloproteinase-2 activation. Functionally, we found decreased cell migration, reduced cellular proliferation, and increased apoptotic nuclear fragmentation after 1.4 kb anti-sense membrane type 1 matrix metalloproteinase construct expression. Our findings suggest a role for membrane type 1 matrix metalloproteinase in human cutaneous epidermal cell invasion and survival mechanisms in vivo.

Antisense Elements (Genetics)↗

PubMed2002

Aging and survival of cutaneous microvasculature.

The growth and turnover of blood vessels in the skin is fundamental in normal development, wound repair, hair follicle cycling, tumor cell metastasis, and in many different states of cutaneous pathology. Whereas many investigations are focused on mechanisms of angiogenesis in the skin, the influence of cellular aging and replicative senescence (i.e., the inability, after a critical number of population doublings, to replicate) on microvascular remodeling events has received relatively less attention. In this article, we review the clinical and pathologic relationships associated with cutaneous vascular aging and update current knowledge of endothelial cell survival characteristics. A hypothesis is presented in which endothelial cell aging and survival are linked to molecular mechanisms controlling cell proliferation, quiescence, apoptosis, and cellular senescence. We review recent results demonstrating how activation of telomerase in human dermal microvascular endothelial cells affects their durability both in vitro and in vivo and conclude by linking these studies with current concepts involving endothelial cell precursors, control of postnatal somatic cell telomerase activity, and murine model systems.

Aging↗

PubMedDate not supplied

Telomerase-immortalized lymphatic and blood vessel endothelial cells are functionally stable and retain their lineage specificity.

OBJECTIVE: 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. METHODS: 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. RESULTS AND CONCLUSIONS: 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.

Biomarkers↗