Recombinant Human Arresten and Canstatin Inhibit Angiogenic Behaviors of HUVECs via Inhibiting the PI3K/Akt Signaling Pathway.

Angiogenetic inhibitors are crucial in tumor therapy, and endogenous angiogenesis inhibitors have attracted considerable attention due to their effectiveness, safety, and multi-targeting ability. Arresten and canstatin, which have anti-angiogenesis effects, are the c-terminal fragments of the α1 and α2 chains of type IV collagen, respectively. In this study, human arresten and canstatin were recombinantly expressed in Escherichia coli (E. coli), and their effects on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated. Regarding the cell cycle distribution test and 5-ethynyl-2'-deoxyuridine (EdU) assays, arresten and canstatin could repress the proliferation of HUVECs at a range of concentrations. Transwell assay indicated that the migration of HUVECs was significantly decreased in the presence of arresten and canstatin, while tube formation assays suggested that the total tube length and junction number of HUVECs were significantly inhibited by these two proteins; moreover, they could also reduce the expression of vascular endothelial growth factor (VEGF) and the phosphorylation levels of PI3K and Akt, which indicated that the activation of the 3-kinase/serine/threonine-kinase (PI3K/Akt) signaling pathway was inhibited. These findings may have important implications for the soluble recombinant expression of human arresten and canstatin, and for the related therapy of cancer.

Novel COL4A1-VEGFD gene fusion in myofibroma.

Myofibroma is a benign pericytic tumour affecting young children. The presence of multicentric myofibromas defines infantile myofibromatosis (IMF), which is a life-threatening condition when associated with visceral involvement. The disease pathophysiology remains poorly characterized. In this study, we performed deep RNA sequencing on eight myofibroma samples, including two from patients with IMF. We identified five different in-frame gene fusions in six patients, including three previously described fusion transcripts, SRF-CITED1, SRF-ICA1L and MTCH2-FNBP4, and a fusion of unknown significance, FN1-TIMP1. We found a novel COL4A1-VEGFD gene fusion in two cases, one of which also carried a PDGFRB mutation. We observed a robust expression of VEGFD by immunofluorescence on the corresponding tumour sections. Finally, we showed that the COL4A1-VEGFD chimeric protein was processed to mature VEGFD growth factor by proteases, such as the FURIN proprotein convertase. In conclusion, our results unravel a new recurrent gene fusion that leads to VEGFD production under the control of the COL4A1 gene promoter in myofibroma. This fusion is highly reminiscent of the COL1A1-PDGFB oncogene associated with dermatofibrosarcoma protuberans. This work has implications for the diagnosis and, possibly, the treatment of a subset of myofibromas.

Cathepsin S degrades arresten and canstatin in infarcted area after myocardial infarction in rats.

The basement membrane surrounding cardiomyocytes is mainly composed of α1 and α2 chain of type IV collagen. Arresten and canstatin are fragments of non-collagenous C-terminal domain of α1 and α2 chain, respectively. We previously reported that the expression of canstatin was decreased in infarcted area 2 weeks after myocardial infarction in rats. In the present study, we investigated the regulatory mechanism for expression of arresten and canstatin. Myocardial infarction model rats were produced by ligating left anterior descending artery. Western blotting and immunohistochemical staining were performed to determine the protein expression and distribution. Arresten and canstatin were highly expressed in the heart. One day and three days after myocardial infarction, the expression of arresten and canstatin in infarcted area was lower than that in non-infarcted area. The expression of cathepsin S, which is known to degrade arresten and canstatin, was increased in the infarcted area. A knockdown of cathepsin S gene using small interference RNA suppressed the decline of arresten and canstatin in the infarcted area 3 days after myocardial infarction. This study for the first time revealed that arresten and canstatin are immediately degraded by cathepsin S in the infarcted area after myocardial infarction. These findings present a novel fundamental insight into the pathogenesis of myocardial infarction through the turnover of basement membrane-derived endogenous factors.

Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells.

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Nanoparticle-mediated arresten gene inhibits neointimal formation of vein grafts: an experimental research.

BACKGROUND: Recently, we established a new rat model of venous arterialization by grafting the jugular vein into carotid arteries. In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft diseases. Using this model, we studied nanoparticles that mediated the arresten gene to inhibit the neointimal formation of vein grafts. METHODS: Thirty healthy Wistar female rats were randomly divided into three groups. Rat models of grafting the jugular vein into carotid arteries were established. Before and after surgery, all rats were subjected to anticoagulant drugs; and these were subcutaneously injected through different reagents after surgery. Group A: subcutaneous injection of nanoparticles to mediate the arresten gene (0.2 mL); group B: subcutaneous injection of blank nanoparticles (0.2 mL); group C: subcutaneous injection of saline (0.2 mL). At two weeks after the operation, veins of the objective blood vessel were obtained. Pathological changes of local vascular tissues and the new intima hyperplasia of experimental vascular segments were observed. Immunohistochemistry was used to observe the expression of MMPs. RESULTS: (I) After two weeks, pathological intimal hyperplasia reactions were more obvious in groups B and C than in group A (P<0.05). The difference between groups B and C was not statistically significant (P>0.05); (II) the expression of MMP-2 could be observed in different degrees among the three groups. The expression of MMP-2 markedly increased in groups B and C compared to group A (P<0.05), but the difference between these two groups was not statistically significant (P>0.05). CONCLUSIONS: (I) Nanoparticle-mediated arresten genes can reduce intimal hyperplasia in grafts; (II) we have recently shown that this gene reduced intimal hyperplasia, and this reduction is related to the reduced expression of MMP-2. This shows that the arresten gene can inhibit the degradation of the extracellular matrix (ECM).

Anti-angiogenic collagen fragment arresten is increased from 16 weeks' gestation in pre-eclamptic plasma.

INTRODUCTION: Arresten and canstatin are endogenous anti-angiogenic factors derived from type IV collagen α-chains COL4A1 and COL4A2 respectively. While their functions are explored in cancer studies, little is known about their role in pregnancy. Pre-eclampsia (PE) is a common, serious hypertensive disorder of pregnancy that is characterised by systemic endothelial dysfunction. COL4A1 and COL4A2 are maternal PE susceptibility genes that have increased mRNA expression in PE decidua. Our study aim was to determine the levels of arresten and canstatin in plasma and decidua from PE and gestational age matched normotensive patients. METHODS: Plasma was collected from normotensive (n = 44) and PE (n = 39) women during the second and third trimesters of pregnancy. Third trimester decidua was collected at delivery from normotensive and PE women (n = 4 each). Levels of arresten and canstatin were determined by Western immunoblotting. RESULTS: Arresten levels were significantly increased in second and third trimester PE plasma, and in third trimester PE decidua (p < 0.05). Third trimester PE plasma arresten levels also significantly correlated with the need for MgSO4 treatment, where a 1.7 fold increase was observed in patients requiring MgSO4 treatment (p < 0.05). No significant change in canstatin levels was observed between normotensive and PE patients. DISCUSSION: This is the first study to report significant increases in the levels of collagen fragment arresten in PE plasma and decidua. Given its significant increase before the onset of clinical disease and associations with clinical severity in the third trimester, arresten may be a useful biomarker for predicting PE and monitoring its severity.

Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells / 华中科技大学学报（医学）（英德文版）

The eukaryotic expression of human arresten geneand its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells,while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-αactin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40.154, P＜0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

Prokaryotic Expression and Biological Activity Analysis of Human Ar-resten Gene / 华中科技大学学报（医学）（英德文版）

To express recombinant arresten in Escherichia coli (E. Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E. Coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0. 992 1 ku) amounted to 29 % of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.

Expression in E.coli JM109 and pharmacology on anti-angiogenesis of endogenetic angiogenesis inhibitor Arresten gene / 中国药理学通报

Aim To construct prokaryotic expression vector for endogenetic angiogenesis inhibitor Arresten gene and to express recombinant.Pharmacology experiments found that the expression product had the function of restrain blood vessel growing on the chorioallantoic membrane(CAM).Methods The total RNA was extracted from the placenta organize of the normal puerpera,and Arresten gene was amplified by RT-PCR to construct recombinant vector pBV220-Arr.Then,it was transformed into E.coli JM109 for yielding recombinant human Arresten protein.The method to detect inhibiting angiogenesis activity was CAM study.Results The plasmid pBV220-Arr.could be expressed in E.coli JM109 from two hours to eight hours,and the expression yield reached the highest in two hours.The Arresten protein could restrain blood vessel growth of CAM evidently than that of angiostatin.Conclusion The recombinant plasmid pBV220-Arr was construted successfully and the target protein Arresten can express in E.coli JM109,the protein of Arresten had the evident effect of inhibite the activity of angiogenesis.

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells / 中国病理生理杂志

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction(RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8(CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 ?m in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group(P

Construction of prokaryotic expression vector of human angiogenesis inhibitor arresten and its expression in E.coli / 中国病理生理杂志

AIM: To construct prokaryotic expression vector of human angiogenesis inhibitor arresten gene and express recombinant arresten in Escherichia coli. METHODS: Human arresten gene was amplified from recombinant plasmid pGEM-Arr with polymerase chain reaction (PCR), and then cloned into prokaryotic expression vector pRSET by means of recombinant gene technology. The recombinant plasmid pRSET-Arr was transformed into E.coli BL21(DE3), and recombinant arresten was expressed in the bacteria under induction of IPTG. The expressed products were detected by SDS-PAGE analysis. RESULTS: Restriction analysis indicated that the arresten gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant arresten was successfully expressed in Escherichia coli; its molecular weight was about 26 kD and its amount was approximately 30% of total bacterial proteins.CONCLUSION: The successful construction of prokaryotic expression vector containing human arresten gene and the effective expression of recombinant arresten in Escherichia coli laid the foundation for further study on its biological functions.