Approaches to efficient production of recombinant angiogenesis inhibitor rhVEGI‐192 and characterization of its structure and antiangiogenic function

Abstract

Methods to prepare pure, bioactive recombinant human vascular endothelial growth inhibitor (rhVEGI), a potent inhibitor of angiogenesis potentially applicable in antiangiogenic cancer therapy, are in urgent demand for preclinical investigation as well as future clinical trials of the protein. Here, we report expression and purification of rhVEGI-192, a recombinant VEGI isoform, comparatively using host strains BL21 (DE3) pLysS and Origami B (DE3) with IPTG-induction and autoinduction techniques. Our study identified that a combined use of Origami B (DE3) strain and autoinduction expression system gave rise to a high yield of purified rhVEGI-192 at 105.38 mg/L culture by immobilized-metal affinity chromatography on Ni-NTA column. The antiangiogenic activity was effectively restored after the insoluble fractions being dissolved in 8M urea and subsequently subjected to a gradient-dialysis refolding process. Functional tests demonstrated that the purified rhVEGI-192 potently inhibited endothelial growth, induced endothelial apoptosis and suppressed neovascularization in chicken chorioallantoic membrane, indicating that the developed method allows preparation of rhVEGI-192 with high yield, solubility, and bioactivity. Most importantly, our study also demonstrates that VEGI-192 is capable of forming polymeric structure, which is possibly required for its antiangiogenic activity.

Suppression of renal cell carcinoma growth in vivo by forced expression of vascular endothelial growth inhibitor

“…Further investigation has shown that the density of the endothelial cells exhibited an 88% decrease within 1 week of treatment with recombinant human VEGI and a further decrease within 3 weeks in Lewis lung cancer murine tumor models. Parr et al reported that patients with breast tumors expressing reduced levels of VEGI had a higher local recurrence, shorter survival time and an overall poorer prognosis than those patients expressing high levels of VEGI (39). Chen et al also reported that purified rhVEGI-192 potently inhibited endothelial growth, induced endothelial apoptosis and suppressed neovascularization in chicken chorioallantoic membrane and demonstrated that VEGI-192 is capable of forming polymeric structure, which is possibly required for its anti-angiogenic activity (40).…”

DANCR Promotes Metastasis and Proliferation in Bladder Cancer Cells by Enhancing IL-11-STAT3 Signaling and CCND1 Expression

“…Western blotting was performed as previously described. 64,65 Primary antibodies specific to MMP9, CCND1, STAT3, phospho (p)-STAT3(Tyr705), JAK2, p-JAK2(Tyr1007), GAPDH (1:1,000, Cell Signaling Technology, Danvers, MA, USA), PLAU, Ki-67 (1:1,000, Abcam,), and LRPPRC (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. The blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology), and they were visualized using enhanced chemiluminescence.…”

Increased expression of TNF ligand-related molecule 1A and death receptor 3 in bladder tissues of patients with painful bladder syndrome/interstitial cystitis

“…With regard to the evidence that TNF-related apoptosis-inducing ligand and TNFSF14 are also important in PBS/IC (6,8), it may be suggested that TL1A and DR3 elevation is one of the factors contributing to the inflammation and apoptosis in PBS/IC. Secondly, TL1A has been shown to have an anti-angiogenic function (25,26). Upregulation of TL1A and DR3 may inhibit angiogenesis which then causes bladder ischemia and destroys the blood-urine barrier.…”