Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities.

The aim of the present study is to identify an effective and efficient expression system for purification of recombinant antiangiogenic protein tumstatin. The sequence encoding carboxy-terminal non-collagenous domain of α3 chain Type IV collagen, α3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLT-A to express it in bacteria, mammalian and Sf-9 insect cells respectively. Expression and purification profiles of tumstatin were evaluated by coomassie staining and immunoblotting, and the efficiency was determined based on the yields of soluble protein.

Role of Chemokines and Chemokine Receptors in Prostate Cancer Development and Progression.

Prostate cancer (PC) is the second leading cause of cancer deaths in men in America and Western Europe. Epidemiological studies suggest that prostate cancer incidences increased in last few years in Asian. The causes or consequences of increasing trend of prostate cancer incidence are not completely known.

Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing.

Background: Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds.

Inhibitory effects of arresten on bFGF-induced proliferation, migration, and matrix metalloproteinase-2 activation in mouse retinal endothelial cells.

Purpose: The potential role of arresten (alpha1(IV)NC1) as an endogenous angiogenesis inhibitor in the prevention of bFGF mediated retinal angiogenesis and regulation of matrix metalloproteinase-2 activation has not been explored.

Methods: Mouse retinal endothelial cells (MREC) were cultured on type IV collagen and treated with basic fibroblast growth factor (bFGF) alone or in the presence of arresten at concentrations ranging from 1 to 10 microg/ml. The proliferation of MRECs were evaluated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, and bFGF stimulated endothelial cell migration was assessed using Boyden chamber.

FAK and p38-MAP kinase-dependent activation of apoptosis and caspase-3 in retinal endothelial cells by alpha1(IV)NC1.

Purpose: To determine the impact of the antiangiogenic factor alpha1(IV)NC1 on vascular endothelial growth factor-mediated proangiogenic activity in mouse retinal endothelial cells (MRECs).

Methods: Primary culture of MRECs was established as previously described and was used to determine the effects of alpha1(IV)NC1 on the proangiogenic activity of VEGF. Cell proliferation was evaluated using [(3)H]-thymidine incorporation and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric assays.

Characterization of the anti-angiogenic properties of arresten, an alpha1beta1 integrin-dependent collagen-derived tumor suppressor.

Physiological and pathological turnover of basement membranes liberates biologically active cryptic molecules. Several collagen-derived fragments possess anti-angiogenic activity. Arresten is the 26-kDa non-collagenous domain of type IV collagen alpha1 chain.

Inhibition of tumor angiogenesis by tumstatin: insights into signaling mechanisms and implications in cancer regression.

Growing tumors develop additional new blood vessels to meet the demand for adequate nutrients and oxygen, a process called angiogenesis. Cancer is a highly complex disease promoted by excess angiogenesis; interfering with this process poses for an attractive approach for controlling tumor growth. This hypothesis led to the identification of endogenous angiogenesis inhibitors generated from type IV collagen, a major component of vascular basement membrane (VBM).

Molecular Cloning and Functional Characterization of Mouse α3(IV)NC1.

Non-collagenous α3 chain of type IV collagen or α3(IV)NC1, a 28 kDa C-terminal domain of collagen type IV is a specific inhibitor of endothelial cell translation and angiogenesis. In the present study we have cloned and expressed mouse α3(IV)NC1 in baculovirus system. The recombinant protein was expressed in soluble form and tested for several of its biological functions.

Regulation of COX-2 mediated signaling by alpha3 type IV noncollagenous domain in tumor angiogenesis.

Human alpha3 chain, a noncollagenous domain of type IV collagen [alpha3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of alpha3(IV)NC1 to alphaVbeta3 and alpha3beta1 integrins. alpha3(IV)NC1 binds alphaVbeta3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways.

Signaling mechanisms of endogenous angiogenesis inhibitors derived from type IV collagen.

Vascular basement membrane (VBM) derived molecules are regulators of certain biological activities such as cell growth, differentiation and angiogenesis. Angiogenesis is regulated by a systematic controlled balance between VBM derived antiangiogenic factors and proangiogenic growth factors. In the normal physiological state, equilibrium is maintained between the antiangiogenic and proangiogenic factors.

Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype.

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated.

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells.

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed.

Human alpha1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by alpha1beta1 integrin.

Human noncollagenous domain 1 of the alpha1 chain of type IV collagen [alpha1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human alpha1(IV)NC1 binds to alpha1beta1 integrin, competes with type IV collagen binding to alpha1beta1 integrin, and inhibits migration, proliferation, and tube formation by ECs.

Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors.

Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth.

Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression.

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors.

Physiological levels of tumstatin, a fragment of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alphaV beta3 integrin.

We demonstrate a physiological role for tumstatin, a cleavage fragment of the alpha3 chain of type IV collagen (Col IValpha3), which is present in the circulation. Mice with a genetic deletion of Col IValpha3 show accelerated tumor growth associated with enhanced pathological angiogenesis, while angiogenesis associated with development and tissue repair are unaffected. Supplementing Col IValpha3-deficient mice with recombinant tumstatin to a normal physiological concentration abolishes the increased rate of tumor growth.

Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins.

Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as alpha 3 chain of type IV collagen and alpha1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors.

Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria.

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria.

Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors.

Background & Aims: In liver fibrosis, alterations within the space of Disse microenvironment occur and facilitate further progression of chronic liver disease. The normal basement membrane-like matrix present within the space of Disse converts to a matrix rich in fibril-forming collagens during fibrosis.

Methods: To further understand the pathogenesis of liver fibrosis, we modified an in vitro Boyden chamber system to partially mimic in vivo conditions of hepatic stellate cells (HSCs) during health and disease.

NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele.

Mutations in NPHS2, encoding podocin, have been identified in childhood onset focal and segmental glomerulosclerosis (FSGS). The role of NPHS2 in adult disease is less well defined. We studied 30 families with FSGS and apparent autosomal recessive inheritance and 91 individuals with primary FSGS.

Determinants of vascular permeability in the kidney glomerulus.

The human kidneys filter 70 liters of blood plasma every day. The hallmark of almost all kidney diseases, whether acquired or genetic, is the leakage of plasma proteins into the urine because of alterations in the glomerular filtration unit of the kidney. In this regard, the human mutations in nephrin, podocin, alpha-actinin-4, COL4A3, and COL4A5 genes expressed in the glomeruli have been implicated to cause alterations in glomerular filtration apparatus.

Tumstatin, an endothelial cell-specific inhibitor of protein synthesis.

Tumstatin is a 28-kilodalton fragment of type IV collagen that displays both anti-angiogenic and proapoptotic activity. Here we show that tumstatin functions as an endothelial cell-specific inhibitor of protein synthesis. Through a requisite interaction with alphaVbeta3 integrin, tumstatin inhibits activation of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3-kinase), protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR), and it prevents the dissociation of eukaryotic initiation factor 4E protein (eIF4E) from 4E-binding protein 1.