Angiostatin-functionalized collagen scaffolds suppress angiogenesis but do not induce chondrogenesis by mesenchymal stromal cells in vivo.

Tissue engineering for fibrocartilage regeneration using mesenchymal stromal cells (MSC) and biomaterial scaffolds is emerging as a promising strategy, but inhibiting vascularization to prevent endochondral ossification is important to develop stable implants. The objective of this study was to investigate the effect of angiostatin on inhibition of angiogenesis and promotion of chondrogenesis by collagen scaffolds with or without MSC implanted subcutaneously in rats. One scaffold from the following groups was implanted in each animal: Collagen scaffolds only, scaffolds functionalized with angiostatin, scaffolds loaded with MSC and scaffolds functionalized with angiostatin and loaded with MSC. The various scaffolds were harvested after 2 and 8 weeks for histological analysis, Real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence quantification. Results demonstrated significantly decreased expression of inflammatory (interleukin 1 alpha and beta) and angiogenic genes (platelet and endothelial cell adhesion molecule 1) in scaffolds functionalized with angiostatin after 2 weeks in vivo. Histologically, after 8 weeks, the scaffolds with angiostatin had less inflammatory cells and more collagen matrix formation, but no fibrocartilage formation was detected. Thus, although angiostatin suppressed angiogenesis, it did not stimulate ectopic chondrogenesis in tissue engineered constructs in vivo.

Serum Biomarkers of Endothelial Dysfunction in Fabry Associated Cardiomyopathy.

Background: Fabry disease (FD) is characterized by early development of vasculopathy and endothelial dysfunction. However, it is unclear whether these findings also play a pivotal role in cardiac manifestation. As Fabry cardiomyopathy (FC) is the leading cause of death in FD, we aimed to gather a better insight in pathological mechanisms of the disease. Methods: Serum samples were obtained from 17 healthy controls, 15 FD patients with and 7 without FC. FC was defined by LV wall thickening of >12 mm in cardiac magnetic resonance imaging and serum level of proBNP, high sensitive Troponin T (hsT), and globotriaosylsphingosine (lyso-GB3) were obtained. A multiplex ELISA-Assay for 23 different angiogenesis markers was performed in pooled samples. Markers showing significant differences among groups were further analyzed in single samples using specific Elisa antibody assays. L-homoarginine (hArg), L-arginine, asymmetric (ADMA), and symmetric Dimethylarginine (SDMA) were quantified by liquid chromatography-mass spectrometry. Results: Angiostatin and matrix metalloproteinase 9 (MMP-9) were elevated in FD patients compared to controls independently of the presence of FC (angiostatin: 98 ± 25 vs. 75 ± 15 ng/mL; p = 0.001; MMP-9: 8.0 ± 3.4 vs. 5.0 ± 2.4 µg/mL; p = 0.002). SDMA concentrations were highest in patients with FC (0.90 ± 0.64 µmol/l) compared to patients without (0.57 ± 0.10 µmol/l; p = 0.027) and vs. controls (0.58 ± 0.12 µmol/l; p = 0.006) and was positively correlated with indexed LV-mass (r = 0.61; p = 0.003), hsT (r = 0.56, p = 0.008), and lyso-Gb3 (r = 0.53, p = 0.013). Accordingly, the ratio of L-homoarginine to SDMA (hArg/SDMA) was lowest in patients with FC (2.63 ± 1.78) compared to controls (4.16 ± 1.44; p = 0.005). For L-arginine, hArg and ADMA no significant differences among groups could be detected, although a trend toward higher ADMA and lower hArg levels could be observed in the FC group. Furthermore, a significant relationship between kidney and cardiac function could be revealed (p = 0.045). Conclusion: Elevated MMP-9 and angiostatin levels suggest an increased extracellular matrix turnover in FD patients. Furthermore, endothelial dysfunction may also be involved in FC, as SDMA and hArg/SDMA are altered in these patients.

Efficacy of recombinant human endostatin combined with TP regimen on patients with advanced EGFR wild type squamous cell lung carcinoma / 国际肿瘤学杂志

Objective To evaluate the efficacy of recombinant human endostatin combined with TP(paclitaxel + cisplatin) in the treatment of advanced epidermal growth factor receptor (EGFR) squamous cell lung carcinoma.Methods From January 2012 to February 2015,100 patients with squamous cell lung carcinoma in Chongqing Three Gorges Central Hospital for medical treatment were selected as the subjects.According to different treatment methods,they were divided into single group (n =50) and combined group (n =50).The single group used the TP protocol and the combined group was treated with recombinant human endostatin on the basis of the TP protocol.The therapeutic effect was evaluated according to the Response Evaluation Criteria In Solid Tumors 1.1 (RECIST 1.1).The short-term effects,long-term effects,adverse reactions,hospitalization time and expenses were also analyzed.Results The objective effective rate in the combined group [52.0％ (26/50)] was significantly higher than that in the single group [16.0％ (8/50)],with a significant difference (x2 =14.429,P =0.007).The disease control rate in the combined group was higher than that in the single group (80.0％ vs.52.0％),with a significant difference (x2 =8.734,P =0.009).Compared with the patients in the single group,the median progression free survival (8.4 months vs.6.3 months)and overall survival (17.7 months vs.11.5 months) in the combined group were prolonged obviously,with significant differences (x2 =5.390,P =0.025;x2 =3.993,P =0.035).The incidence rates of adverse reactions in single group were basically the same compared with the combined group,and the difference was not statistically significant (all P ＞0.05).Compared with the single group,the hospitalization time in combined group was longer [(23.5 ± 2.8) weeks vs.(18.2 ± 3.5) weeks],and the expenses in combined group was higher [(112 453.9 ± 994.9) yuan vs.(87 821.4 ± 943.2) yuan],with significant differences (t =8.361,P ＜0.001;t =127.051,P ＜0.001).But the satisfaction of patients in combined group was significantly higher than that in single group (88.0％ vs.64.0％,x2 =4.210,P =0.017).Conclusion Recombinant human endostatin combined with TP regimen is effective in the treatment of advanced EGFR wild-type squamous cell lung carcinoma,and has a low incidence of adverse reactions.It is suitable for clinical application.

Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors Reduce Reactive Gliosis and Improve Angiostatin Levels in Retina of Diabetic Rats.

Diabetic retinopathy (DR) is a multifactorial disease characterized by reactive gliosis and disbalance of angiogenesis regulators, contributing to endothelial dysfunction and microvascular complications. This study was organized to elucidate whether poly(ADP-ribose) polymerase-1 (PARP-1) inhibition could attenuate diabetes-induced damage to macroglia and correct angiogenic disbalance in diabetic rat retina. After 8 weeks of streptozotocin (STZ)-induced diabetes, Wistar male rats were treated with PARP-1 inhibitors, nicotinamide (NAm) or 3-aminobenzamide (3-AB) (100 and 30 mg/kg/daily i.p., respectively), for 14 days. After the 10-weeks experiment period, retinas were undergone an immunohistochemical staining for glial fibrillary acidic protein (GFAP), while western blots were performed to evaluate effects of PAPR-1 inhibitors on the levels of PARP-1, poly(ADP-ribosyl)ated proteins (PARs), GFAP, and angiostatin isoforms. Diabetes induced significant up-regulation and activation of retinal PARP-1, reactive gliosis development, and GFAP overexpression compared to non-diabetic control. Moreover, extensive fragmentation of both PARP-1 and GFAP (hallmarks of apoptosis and macroglia reactivation, respectively) in diabetic retina was also observed. Levels of angiostatin isoforms were dramatically decreased in diabetic retina, sustaining aberrant pro-angiogenic condition. Both NAm and 3-AB markedly attenuated damage to macroglia, evidenced by down-regulation of PARP-1, PARs and total GFAP compared to diabetic non-treated group. PARP-1-inhibitory therapy prevented formation of PARP-1 and GFAP cleavage-derived products. In retinas of anti-PARP-treated diabetic animals, partial restoration of angiostatin's levels was shown. Therefore, PARP-1 inhibitors counteract diabetes-induced injuries and manifest retinoprotective effects, including attenuation of reactive gliosis and improvement of angiogenic status, thus, such agents could be considered as promising candidates for DR management.

[Role of angiostatins in diabetic complications].

Angiogenesis is a process through which new blood vessels form from pre-existing vessels. Angiogenesis is regulated by a number of factors of peptide nature. Disbalance of angiogenic system appears to be the major causative factor contributing vascular abnormalities in diabetes mellitus, resulting in various complications. Angiostatins, which are kringle-containing fragments of plasminogen/plasmin, are known to be powerful physiological inhibitors of neovascularization. In the present review, current literature data on peculiarities of production of angiostatins and their functioning at diabetes mellitus are summarized and analyzed for the first time. Also, role of angiostatins in the pathogenesis of typical diabetic complications, including retinopathies, nephropathies and cardiovascular diseases, is discussed. Data presented in this review may be useful for elaboration of novel effective approaches for diagnostics and therapy of vascular abnormalities in diabetes mellitus.

Inhibition of tumor growth by intramuscular administration of the canstatin gene delivered by electroporation / 中国医师杂志

Objective To construct human canstatin gene eukaryotic expression vector and investigate the therapeutic effect of intramuscular canstatin gene delivered by electroporation on tumor growth.Methods Canstatin cDNA was amplified from total RNA extracted from fresh fetal liver by reversing transcription polymerase chain reaction (RT-PCR).The canstatin cDNA fragment was in serted into pEGFP-N1 eukaryotic expression vector.The recombination plasmid was delivered to the quadriceps of the mice with Lewis lung carcinomas by electroporation intramuscular.Fluorescence intension measured by fluorescence microscope,reverse-PCR assay,and immunohistochemistry assay were performed to detect the expression of canstatin gene in the muscle and in circulation.The tumor weight and volume were used to detect the biological effects of canstatin gene delivery.Results Recombinant eukaryotic expression vector of recombinant human canstatin was successfully constructed.The canstatin mRNA was significantly increased in the skeletal muscle and intramuscular delivery of canatatin gene by electroporation acquired the expression of enhanced green fluorescent protein (EGFP)/canstatin protein in the circulation and significantly inhibited tumor growth.The percent of the inhibition of tumor weight was 57.7 ％.Conclusions Electroporation mediated gene transfer efficiency in skeletal muscle was compared to simple plasmid injection and lasted for a long time.It was an efficient and safe,convenient and economic,gene transfer methods and might have certain clinical application value.Electroporation mediated canstatin gene transfer in skeletal muscle had obvious inhibitory effect on Lewis lung cancer in mice subcutaneous xenograft tumor growth.

Study on relationship between extravascular lung water,TNF-α,E-selectin,vasostatin-2 and the prognosis of patients with ARDS / 中国基层医药

Objective To analyze the correlations between acute respiratory distress syndrome ( ARDS) and extravascular lung water(EVLW),tumor necrosis factor alpha (TNF-α),E-selectin,vasostatin-2.To discuss the value of EVLW, TNF-α, E-selectin and vasostatin-2 in evaluating the severity of ARDS and prognosis of this disease . Methods There are thirty-six patients with ARDS were selected .According to the level of EVLWI shown by pulse indicator continuous cardiac output ( PiCCO) monitor,they were divided into the two groups:EVLWI≥14mL/kg group (n=18) and EVLWI0.05).EVLWI had positive correlation with PVPI,TNF-αand E-selectin (r=0.605,0.649, 0.549,all P0.05).Conclusion There were significant correla-tions between EVLWI and TNF-α,E-selectin in patients with ARDS.The levels of EVLWI,TNF-α,E-selectin were higher,and the mortality rate was higher,the the stay time in ICU was longer.The levels of EVLWI,TNF-αand E-selectin can provide a good reference value to the judgement of the severity of disease and prognosis in patients with ARDS.The level of vasostatin-2 had no correlation with EVLWI .

Anti-angiogenesis effect of adeno-associated virus-mediated recombinant angiostatin combined with celastrol on intracranial C6 glioma in rats / 肿瘤

Objective: To examine the effects of therapeutic alliance of adeno-associated virus-mediated recombinant angiostatin (AAV-AS) combined with celastrol on tumor growth, microvessel density and apoptosis of intracranial glioma in rats, and to give a prospective of this therapeutic alliance. Methods: A rat intracranial C6 glioma model was established, and then the rats (n=40) were randomly assigned into four groups after 7 days, which were saline control group, AAV-AS group, celastrol group and therapeutic alliance group. The tumor growth was examined by magnetic resonance imaging (MRI) every 7 days, and the volume of tumor was calculated. The rats were killed after 22 days, and the expression of AS protein, the microvessel density and the apoptosis of tumor cells were detected. Results: The expression of AS protein was detectable in AAV-AS group and the therapeutic alliance group, which confirmed the achievement of genetic transduction. The tumor volume, microvessel density and apoptosis index were significantly different in the therapeutic alliance group compared with the other three groups (P<0.05). The therapeutic alliance of AAV-AS combined with celastrol could inhibit the tumor growth, reduce the microvessel density of tumor, and enhance the apoptosis. Conclusion: Gene therapy of AAV-AS combined with celastrol can inhibit glioma growth by inhibiting tumor vasculogenesis. This therapeutic alliance exerts a synergic effect and compensates the shortage of single drug. Copyright© 2011 by TUMOR.

Clinical study of recombinant human endostatin with GP regimen in the treatment of advanced non-small-cell lung cancer / 肿瘤研究与临床

Objective To evaluate the efficacy and security of combined recombinant human endostatin with GP chemotherapy for the treatment of advanced non-small-cell lung cancer (NSCLC).Methods Non- randomized concurrent control was used.32 patients were treated by recombinant human endostatin combined with chemotherapy as test group,40 patients of control group only received chemotherapy.The response rate (RR),the clinical benefit rate (CBR) and the time to progression (TTP) were observed.Results The total RR in two groups were 40.6 ％ and 20.0 ％ (x2 =3.66,P =0.07).The total CBR were 68.8 ％ and 42.5 ％ (x2 =4.93,P =0.034).The total time to progression were 5.2 months and 3.9 months (P =0.042).Incidence of adverse reactions of experimental group and control group was no significant difference.Conclusion Combined recombinant human endostatin and chemotherapy can improve the curative effect (RR,CBR and TTP) of advanced NSCLC.

Effects of macrophage metalloelastase on angiostatin generation and growth of tumor vessels in murine colon carcinoma / 中华消化杂志

Objective To determine the pathway of macrophage metalloelastase (MME)generate active angiostatin by decomposing plasminogen and its effect on inhibiting growth of tumor and microvessel density (MVD) in vivo in mouse models. Methods The recombined plasmid pEGFPC1-MME was constructed. Thirty mice were subcutaneously inoculated with CT-26 cells that were stably transfected with pEGFP-C1-MME (MME-transfected group), 30 with CT-26 cells transfected with empty vector pEGFP-C1 (vector-transfected group) and 30 with CT-26 cells (non-transfected group). Radioiodination and radioisotope tracer were used to explore the pathway of angiostatin generation in vivo. Results SDS-PAGE electrophoresis analysis revealed that, in the PAGE gel contained the protein with molecular weights of 35 000 and 38 000, radioactivity in MME-transfected group was significantly higher than vector-transfected and non-transfected groups (P = 0. 00).Western blotting analysis demonstrated two bands containing 35 000 and 38 000 fragments in three groups. Quantification of the protein signals by image analysis revealed that the levels of 35 000 and 38 000 fragments were obviously increased in MME-transfected group (9.32±1.52 and 5.61±2.24,respectively) than those in vector-transfected (2.47 ± 0.23 and 0. 67 ± 0. 12, respectively) and nontransfected (1.21±0. 69 and 0. 86 ± 0.44, respectively) groups (P= 0.00). The average value of MVD and fluorescent express of vascular endothelial growth factor (VEGF) were lower in MMEtransfected group when compared with those in vector-transfected and non-transfected groups (P =0.00). The average tumor size in MME-transfected group was small in comparison with vectortransfected and non-transfected groups (P= 0.00). Conclusions MME is demonstrated to be one of matrix metalloproteinase that closely related with angiostatin production and has inhibitory effect on tumor growth in tumor-bearing mice.

Comparative study on inhibitory effects of angiostatin gene and endostatin gene on growth of hepatoma / 肿瘤

Objective: To make a comparative study on therapeutic action of angiostatin gene and endostatin gene on hepatoma. Methods: The xenografted hepatoma models were established in rats. The tumor-bearing rats were randomly divided into four groups. Each group were locally injected 200 μL of endostation gene, angiostatin gene, endostation gene plus angiostatin gene, and 0.9% NaCl solution, respectively. The microvessel density (MVD), tumor apoptosis rate, the number of pulmonary metastasis, survival time, and tumor growth rate were recorded. Results: The tumor growth was inhibited in all the therapeutic groups. The inhibitory action was most significant in the double gene therapy group. There was significant difference in the number of pulmonary metastasis, MVD, apoptosis index (AI) between the therapeutic groups and control group (P 0.05). The survival time of rats was the longest in the double gene therapy group and the shortest in the control group. Conclusion: Endostatin and angiostatin inhibit the tumor growth and metastasis by repressing tumor angiogenesis and migration. Both statins have synergistic action in inhibiting tumor growth.

Coordinate repression of angiostatin and Fas gene on human colon carcinoma / 中国医师杂志

Objective To investigate the coordinate repression of angiostatin(AS)and Fas gene on human colon carcinoma LOVO.Methods Plasmid pcDNA3-AS and pcDNA3-Fas were constructed,and AS,Fas,AS and Fas gene were transfected to human colon carcinoma LOVO cells by liposome method.The expressions of target protein were detected by Western blot.The effects of transfection of AS and Fas gene on the growth of human colon carcinoma cells were detected by MTr methods.AS,Fas,AS and Fas gene were transfected to the human colon carcinoma subcutaneously implanted in nude mice by direct injection into the tumor.The tumor sizes were detected after 14 days of the first gene transfection.Results The effect of gene transfeetion in LOVO cell after twelve hours,24 h,48 h and 72 h were observed and compared with control group,co-transfection of Fas and AS gene group and Fas group significantly inhibited the growth of human colon carcinoma LOVO line cells(P＜0.01).Fourteen days after plasmid transfection,the tumor volume in group Fas and group co-transfection were signifieandy smaller than that of control,AS and Fas group(P＜0.05).The tumor volume in AS group and Fas group were significantly smaller than that of control(P＜0.05).Conclusion Angiostatin and Fas gene have coordinate repression effect on human colon carcinoma in vivo.

The Role of Angiostatin, Vascular Endothelial Growth Factor, Matrix Metalloproteinase 9 and 12 in the Angiogenesis of Hepatocellular Carcinoma / 대한간학회지

BACKGROUND/AIMS: Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. Hepatocellular carcinoma (HCC) has become a common malignant tumor worldwide. It is characterized by a high vascularity. METHODS: We studied the immunohistochemical expression of angiostatin, vascular endothelial cell growth factor (VEGF), matrix metalloproteinase (MMP)-9 and MMP-12, and the relationship between these results and the microvessel density (MVD) in 48 HCC specimens. To determine whether HCC cells express angiostatin per se, we examined the expression of angiostatin, MMP-9 and MMP-12 by Western blotting in four HCC cell lines. RESULTS: Expression of angiostatin and MMP-12 (but not MMP-9) were strongly correlated with decreased MVD in HCCs (P=0.006, P=0.038, respectively). VEGF positive tumors showed a significantly higher MVD than VEGF negative tumors (P=0.01). We divided the 48 cases into the following four groups: group A, angiostatin (+), MMP-9 or -12 (+), and VEGF (-); group B, angiostatin (-) and VEGF (-); group C, angiostatin (+), MMP-9 or -12 (+), and VEGF (+); group D, angiostatin (-) and VEGF (+). There was a significant correlation with MVD among these groups (P<0.001). Angiostatin was detected by Western blotting in 2 out of 4 HCC cell lines and was associated with plasminogen and MMP expression. CONCLUSIONS: These results indicate that angiogenesis in HCC is a complex process involving multiple factors including angiostatin, VEGF, and MMP. Our results suggest that angiostatin is generated by MMP-mediated proteolysis of plasminogen in HCC cells.