Exploring the molecular role of endostatin in diabetic neuropathy.

For over a decade, diabetic neuropathy has exhibited great emergence in diabetic patients. Though there are numerous impediments in understanding the underlying pathology it is not that enough to conclude. Initially, there was no intricate protocol for diagnosis as its symptoms mimic most of the neurodegenerative disorders and demyelinating diseases. Continuous research on this, reveals many pathological correlates which are also detectable clinically. The most important pathologic manifestation is imbalanced angiogenesis/neo-vascularization. This review is completely focused on established pathogenesis and anti-angiogenic agents which are physiological signal molecules by the origin. Those agents can also be used externally to inhibit those pathogenic pathways. Pathologically DN demonstrates the misbalanced expression of many knotty factors like VEGF, FGF2, TGFb, NF-kb, TNF-a, MMP, TIMP, and many minor factors. Their pathway towards the incidence of DN is quite interrelated. Many anti-angiogenic agents inhibit neovascularization to many extents, but out of them predominantly inhibition of angiogenic activity is shared by endostatin which is now in clinical trial phase II. It inhibits almost all angiogenic factors and it is possible because they share interrelated pathogenesis towards imbalanced angiogenesis. Endostatin is a physiological signal molecule produced by the proteolytic cleavage of collagen XVIII. It has also a broad research profile in the field of medical research and further investigation can show promising therapeutic effects for benefit of mankind.

Endostatin and transglutaminase 2 are involved in fibrosis of the aging kidney.

Endostatin (EST), an antiangiogenic factor, is enriched in aging kidneys. EST is also an interactive partner of transglutaminase 2 (TG2), an enzyme that cross-links extracellular matrix proteins. Here we tested whether EST and TG2 play a role in the fibrosis of aging. In wild-type mice, aging kidneys exhibited a 2- to 4-fold increase in TG2 paralleled by increased cross-linked extracellular matrix proteins and fibrosis. Mice transgenic to express EST showed renal fibrosis at a young age. One-month delivery of EST via minipumps to young mice showed increased renal fibrosis that became more robust when superimposed on folic acid-induced nephropathy. Upregulated TG2 and impaired renal function were apparent with EST delivery combined with folic acid-induced nephropathy. Subcapsular injection of TG2 and/or EST into kidneys of young mice not only induced interstitial fibrosis, but also increased the proportion of senescent cells. Thus, kidney fibrosis in aging may represent a natural outcome of upregulated EST and TG2, but more likely it appears to be a result of cumulative stresses occurring on the background of synergistically acting geronic (aging) proteins, EST and TG2.

Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.

Inhibition of Wnt/ß-catenin signaling by a soluble collagen-derived frizzled domain interacting with Wnt3a and the receptors frizzled 1 and 8.

The Wnt/ß-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/ß-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/ß-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/ß-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced ß-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth.

Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro.

BACKGROUND: Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. METHODS: Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. RESULTS: Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. CONCLUSION: Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.

Functional characterization of neostatins, the MMP-derived, enzymatic cleavage products of type XVIII collagen.

Several anti-angiogenic factors are derived from proteolytic processing of large molecules including endostatin from type XVIII collagen and angiostatin from plasminogen. In previous studies we showed that neostatin-7, the C-terminal 28kDa endostatin-spanning proteolytic fragment, is generated from the proteolytic action of matrix metalloproteinase matrilysin (MMP)-7 on type XVIII collagen. Now, we report a second member of the neostatin family of proteins, neostatin-14. Given the small quantities of neostatin-7 and -14 generated by the breakdown of naturally occurring collagen XVIII (using MMP-7 and -14, respectively), we used two other approaches to characterize the anti-angiogenic properties of these molecules: murine recombinant neostatin in vitro, and gene therapy. We demonstrate that murine recombinant neostatin-7 inhibits calf pulmonary artery endothelial cell proliferation and that microinjection of neostatin-7 and neostatin-14 naked DNA into the corneal stroma of mice results in significant reduction of basic fibroblast growth factor-induced corneal neovascularization. These results provide supportive evidence of the possible anti-angiogenic effect of neostatins.