Akulapalli Sudhakar

Physiological levels of tumstatin, a fragment of collagen IV α3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via αVβ3 integrin

We demonstrate a physiological role for tumstatin, a cleavage fragment of the α3 chain of type IV collagen (Col IVα3), which is present in the circulation. Mice with a genetic deletion of Col IVα3 show accelerated tumor growth associated with enhanced pathological angiogenesis, while angiogenesis associated with development and tissue repair are unaffected. Supplementing Col IVα3-deficient mice with recombinant tumstatin to a normal physiological concentration abolishes the increased rate of tumor growth. The suppressive effects of tumstatin require αVβ3 integrin expressed on pathological, but not on physiological, angiogenic blood vessels. Mice deficient in matrix metalloproteinase-9, which cleaves tumstatin efficiently from Col IVα3, have decreased circulating tumstatin and accelerated growth of tumor. These results indicate that MMP-generated fragments of basement membrane collagen can have endogenous function as integrin-mediated suppressors of pathologic angiogenesis and tumor growth.

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. We screened family members for NPHS2 mutations. NPHS2 mutations appeared to be responsible for disease in nine of these families. In six families, the affected individuals were compound heterozygotes for a nonconservative R229Q amino acid substitution. This R229Q variant has an allele frequency of 3.6% in a control population. In these families, R229Q was the only mutation identified on one of the two disease-associated NPHS2 alleles. We used in vitro-translated podocin and purified nephrin to investigate the effect of R229Q on their interaction and found decreased nephrin binding to the R229Q podocin. These data suggest that this common polymorphism contributes to the development of FSGS. Chromosomes bearing the R229Q mutation share a common haplotype defining an approximately 0.2-Mb region. R229Q appears to enhance susceptibility to FSGS in association with a second mutant NPHS2 allele. Identification of R229Q mutations may be of clinical importance, as NPHS2-associated disease appears to define a subgroup of FSGS patients unresponsive to corticosteroids.

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. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen α3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.

Characterization of the anti-angiogenic properties of arresten, an α1β1 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. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models, but its anti-angiogenic mechanism is not completely known. Here we show that arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family; Bcl-2 and Bcl-xL. Although the pro-apoptotic effect of arresten is endothelial cell specific in vitro, in mouse tumors arresten induced apoptosis both in endothelial and tumor cells. The tumor cell apoptosis is likely an indirect effect due to the inhibition of blood vessel growth into the tumor. The active site of arresten was localized by deletion mutagenesis within the C-terminal half of the molecule. We have previously shown that arresten binds to alpha1beta1 integrin on human umbilical vein endothelial cells. However, the microvascular endothelial cells (MLECs) are more important in the context of tumor vasculature. We show here that arresten binds also to the microvascular endothelial cells via alpha1beta1 integrin. Furthermore, it has no effect on Matrigel neovascularization or the viability of integrin alpha1 null MLECs. Tumors implanted on integrin alpha1 deficient mice show no integrin alpha1 expression in the host-derived vascular endothelium, and thus arresten does not inhibit the tumor growth. Collectively, this data sheds more light into the anti-angiogenic mechanism of arresten.

Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase β and DNA-ligase

Synthetic deoxy‐oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase β. High levels of DNA polymerase β supplementation resulted in gap‐filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32‐mer). However, at lower concentrations of DNA polymerase β, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5′‐PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA‐ligase in addition to recombinant DNA polymerase β. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase β and DNA‐ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.

From Normal Cells to Malignancy: Distinct Role of Pro-inflammatory Factors and Cellular Redox Mechanisms

The genesis of many solid cancers is a complex, multistep process that includes cellular neoplastic transformation, resistance to apoptosis, loss of control of cell cycle, angiogenesis and acquisition of invasive properties. Among a number of factors, the simultaneous existence of chronic inflammatory mechanism and downregulation of antioxidant defense mechanism of cells are emerging major causes of neoplastic transformation and the progression of many solid cancers. The Longer the inflammation persists, higher the risk of developing many age related malignancies such as organs of colon, stomach and prostate etc. Concurrent occurrence of these processes that may affects DNA mutations in cells by excessive generation of free radicals, Reactive Oxygen Species (ROS) and other active intermediates that exceed the limit of the cells ability to neutralize it. Chronic inflammatory conditions may activate a variety of pro-tumorigenic activities, such as stimulation of proliferative pathway, chemotactic activity, increased invasive potential of cells and angiogenesis. However, many of the antioxidant or cellular redox molecules play a crucial role in maintaining cellular homeostasis and response to oxidative damage but down-regulation of some of the cellular molecules of this process may leads to malignant transformation. We provide an overview of the possible mechanism(s) of interaction of pro-inflammatory factors in down-regulating the antioxidant and cellular redox mechanism(s) making cells susceptible to carcinogenesis. The elucidation of specific effects and interactions of these factors may provide the opportunity for the identification of new target molecules at early stage of human malignancies.

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. Our results indicate that, baculovirus expression system was efficient for scalable yields of soluble protein that could be purified in its biologically active form. This baculovirus expressed tumstatin was used to evaluate its anti-angiogenic and anti-tumarogenic functions such as inhibition of endothelial cell proliferation, cell viability, migration, tube formation, cap dependent protein translation and the associated signaling mechanism including in-vivo tumor study. Our evaluated approaches using a modified baculovirus expression system shows high expression and high yield of biologically active tumstatin, as compared to two expression systems, indicating baculovirus expression system to be an ideal method for bulk production of soluble tumstatin that needed for pre-clinical and clinical trails.