Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.

BACKGROUND: A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. OBJECTIVE: To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. METHODS: Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. RESULTS: Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. CONCLUSIONS: DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.

Effect of thoracic hyperthermic perfusion with recombinant human endostatin plus nedaplatin in treating pleural effusion in patients with advanced non-small cell lung cancer.

PURPOSE: To explore the efficacy and safety of thoracic hyperthermia perfusion with recombinant human endostatin plus nedaplatin in the treatment of pleural effusion in patients with advanced non-small cell lung cancer (NSCLC). METHODS: A retrospective analysis was conducted on the clinical data of 122 advanced NSCLC patients with pleural effusion, and among them, 61 received thoracic hyperthermic perfusion with recombinant human endostatin (ES) plus nedaplatin (Endostatin group), while the other 61 underwent thoracic hyperthermic perfusion with cisplatin alone (Cisplatin group). The short-term efficacy, changes in the pleural effusion and serum immunological indicators before and after treatment, quality of life, and incidence of adverse reactions were compared between the two groups of patients. Finally, the progression of pleural effusion in patients were followed up and recorded. RESULTS: After treatment, the overall response rate of patients in Endostatin group was considerably higher than that in Cisplatin group (p=0.030). At 2 weeks after treatment, the level of alanine transferase (ALT) rose notably, while that of carcinoembryonic antigen (CEA) declined dramatically in both groups of patients, and the patients in Endostatin group had markedly lower levels of ALT and CEA than those in Cisplatin group (p=0.007, p=0.003). After treatment, the Karnofsky Performance status (KPS) score of patients was prominently raised in the two groups, and Endostatin group exhibited considerably higher KPS scores than Cisplatin group (p=0.045). The incidence rates of nausea and vomiting as well as diarrhea in Endostatin group were prominently lower than those in Cisplatin group (p=0.039, p=0.048). According to the follow-up results, the median time to the progression of pleural effusion in Endostatin group was markedly longer than that in Cisplatin group (p=0.008). CONCLUSIONS: Compared with the thoracic hyperthermic perfusion with cisplatin alone, the thoracic hyperthermic perfusion with recombinant human endostatin plus nedaplatin showed dramatically potential efficacy, decrease of the incidence rate of adverse reactions in the digestive system, improvement of quality of life of patients, and prolongation of progression of pleural effusion.

Study on glyco-modification of endostatin-derived synthetic peptide endostatin2 (ES2) by soluble chitooligosaccharide.

Soluble O-(2-hydroxyl)propyl-3-trimethyl ammonium chitooligosaccharide chloride (HTCOSC) was covalently conjugated to the 11-amino-acid peptide derived from amino terminus of endostatin (endostatin2, ES2, IVRRADRAAVP) to overcome its poor stability, low cell affinity and instable activity. Nuclear magnetic resonance spectroscopy and mass spectrometry was used to study the structure and molecular weight information. The anti-angiogenic activities were evaluated using cell counting Kit-8 assay, flow cytometry assay, wounding migration assay, transwell migration assay, chicken chorioallantoic membrane (CAM) assay and zebra fish angiogenesis assay. In contrast with ES2, the novel carbohydrate-polymer HTCOSC-ES2 displayed improved heat stability, higher cell affinity, better inhibition on endothelial cell proliferation, tube formation, 2-dimensional and 3-dimensional migration in vitro. According to the evaluation in CAM and zebra fish, HTCOSC-ES2 also displayed better anti-angiogenic activity than ES2 in vivo. These results indicate that HTCOSC has good properties as potential candidate for protein/peptide modifier and HTCOSC-ES2 has good potential in angiogenesis related diseases treatment.

Recombinant human endostatin endostar suppresses angiogenesis and lymphangiogenesis of malignant pleural effusion in mice.

BACKGROUND: Malignant pleural effusion (MPE) is a common complication of lung cancer. One widely used treatment for MPE is Endostar, a recombined humanized endostatin based treatment. However, the mechanism of this treatment is still unclear. The aim of this study was to investigate the effects of Endostar in mice with MPE. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cell line expressing enhanced green fluorescent protein (EGFP) was injected into pleural cavity to establish MPE mice model. Mice were randomly divided into four groups. High dose of Endostar (30 mg/kg), low dose of Endostar (8 mg/kg), normal saline, or Bevacizumab (5 mg/kg) was respectively injected into pleural cavity three times with 3-day interval in each group. Transverse computed tomography (CT) was performed to observe pleural fluid formation 14 days after LLC cells injection. Mice were anesthetized and sacrificed 3 days after final administration. The volume of pleural effusion n was measured using 1 ml syringe. Micro blood vessel density (MVD), Lymphatic micro vessel density (LMVD), the expression level of vascular endothelial growth factor A (VEGF-A) and VEGF-C were observed by immunohistochemistry (IHC) staining. RESULTS: The volume of pleural effusion as well as the number of pleural tumor foci, MVD and the expression of VEGF-A were significantly reduced in high dose of Endostar treat group. More importantly, LMVD and the expression of VEGF-C were markedly lower in treat group than those in the other three control groups. CONCLUSION: Our work demonstrated that Endostar played an efficient anti-cancer role in MPE through its suppressive effect on angiogenesis and lymphangiogenesis, which provided a certain theoretical basis for the effectiveness of Endostar on the MPE treatment.

Single and combined effects of alphavbeta3- and alpha5beta1-integrins on capillary tube formation in a human fibrinous matrix.

The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-alpha and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the alphavbeta3-integrin inhibiting mAb LM609 or the alpha5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when alphavbeta3- and alpha5beta1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. alphavbeta5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. alphav-specific cRGD only inhibited angiogenesis when alpha5beta1-integrin was simultaneously blocked. Endostatin mimicked the effect of alpha5beta1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of alphavbeta3- and alpha5beta1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.

The HUVEC/Matrigel assay: an in vivo assay of human angiogenesis suitable for drug validation.

The future ability to manipulate the growth of new blood vessels (angiogenesis) holds great promise for treating ischemic disease and cancer. Several models of human in vivo angiogenesis have been described, but they seem to depend on transgenic support and have not been validated in a therapeutic context. Here, we describe an in vivo assay that mimics human angiogenesis in which native human umbilical vein-derived endothelial cells are suspended in a liquid laminin/collagen gel (Matrigel), injected into immunodeficient mice, and develop into mature, functional vessels that vascularize the Matrigel plug in the course of 30 d. Moreover, we demonstrate how to target this process therapeutically by sustained delivery of the angiogenesis inhibitor endostatin from subcutaneously implanted microosmotic pumps.

Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors.

Tumor escape from immunity, as well as the failure of several anti-cancer vaccination and cellular immunotherapy approaches, is suggested to be due to the angiogenesis-mediated suppression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions. We hypothesized that inhibition of angiogenesis would overcome this escape from immunity. We investigated this in vivo by means of intravital microscopy and ex vivo by immunohistochemistry in two mouse tumor models. Angiogenesis inhibitors anginex, endostatin, and angiostatin, and the chemotherapeutic agent paclitaxel were found to significantly stimulate leukocyte-vessel wall interactions by circumvention of EC anergy in vivo, i.e., by the up-regulation of endothelial adhesion molecules in tumor vessels. This was confirmed by in vitro studies of cultured EC at the protein and mRNA levels. The new angiostatic designer peptide anginex was most potent at overcoming EC anergy; the enhanced leukocyte-vessel interactions led to an increase in the numbers of tumor infiltrating leukocytes. While anginex inhibited tumor growth and microvessel density significantly, the amount of infiltrated leukocytes (CD45), as well as the number of CD8+ cytotoxic T lymphocytes, was enhanced markedly. The current results suggest that immunotherapy strategies can be improved by combination with anti-angiogenesis.

Antiangiogenic strategies in neuroblastoma.

Promising new antiangiogenic strategies are emerging for the treatment of cancer and the inhibition of angiogenesis could represent a powerful adjunct to traditional therapy of malignant tumors. Over the last ten years several reports have been published concerning the relationship between tumor progression and angiogenesis in neuroblastoma in experimental models in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumor growth on active angiogenesis. Here, we present an overview of recent advances in antiangiogenesis in neuroblastoma and describe the most important active substances, preclinical and clinical data, as well as future perspectives.

Angiogenesis inhibition by angiostatin, endostatin and TNP-470 prevents cyclophosphamide induced cystitis.

Angiogenesis, the induction of vessel growth is involved in numerous physiological and pathological processes. While the anti-tumor effect of angiogenesis inhibitors has been extensively investigated in malignant tumors, there is very little information on the effect of angiogenesis inhibitors on inflammation induced angiogenesis. In this report, we utilized a murine model of acute chemically induced cystitis to investigate the ability of three different angiogenesis inhibitors, angiostatin, endostatin and TNP-470, to inhibit the angiogenesis stimulated by this injury. We demonstrate herein, that prophylactic application of the angiogenesis inhibitors led to a significant reduction of each of the inflammatory parameters that were measured. We conclude that anti-angiogenic therapy with angiostatin, endostatin and TNP-470 inhibits inflammation associated angiogenesis induced in this model. We also propose that anti-angiogenic agents may serve as a valuable addition to a standard cyclophosphamid chemotherapy regimen to help reduce the chemotherapy-related side effects while potentially adding an anti-tumor effect.

Antiangiogenesis therapy for endometriosis.

It is known that angiogenesis is of pivotal importance for the development of endometriosis. However, in the treatment of endometriosis patients, prevention of endometriosis lesion development only will not be sufficient as a therapy. Treatment options, aimed at interfering with established lesions, have to be developed. In this study we evaluated whether inhibition of angiogenesis by angiostatic therapy is also effective in antagonizing the sustentation of endometriosis. We evaluated the effect of the angiostatic compounds antihuman vascular endothelial growth factor, TNP-470, endostatin, and anginex on the growth of established endometriosis lesions in the nude mouse model. We show that human endometrium in the proliferative endometrium is highly angiogenic and that vascular endothelial growth factor-A is the most important angiogenesis promotory factor. The angiostatic compounds significantly decreased microvessel densities and the number of established endometriosis lesions. In the remaining lesions, the number of pericyte-protected vessels is not different in control and treated mice; however, the number of unprotected vessels was significantly reduced in the groups treated with the angiostatic agents. Our data demonstrate that inhibitors of angiogenesis effectively interfere with the maintenance and growth of endometriosis by inhibiting angiogenesis. This suggests that the use of angiostatic agents may be promising as a therapy for endometriosis.

Disparate effects of endostatin on tumor vascular perfusion and hypoxia in two murine mammary carcinomas.

PURPOSE: Recent results in the literature have demonstrated that the antiangiogenic agent endostatin can enhance antitumor effects when administered before or during radiotherapy. To better understand the underlying pathophysiologic basis for this radiosensitization, the current study investigated whether short-term endostatin administration is linked to alterations in tumor vascular perfusion and oxygen delivery. METHODS AND MATERIALS: Three daily doses of recombinant endostatin (20 mg/kg) were administered to two murine mammary carcinomas, the highly vascularized MCa-35 and the less vascularized MCa-4. Image analysis techniques were used to quantify (1) total and perfused vascular spacing, and (2) changes in tumor hypoxia as a function of distance from the nearest blood vessel. RESULTS: In MCa-35 tumors, endostatin had no effect on vessel spacing, tumor hypoxia, or tumor growth. In MCa-4 tumors, total and perfused vessel spacings were also unchanged, but tumor growth was inhibited, and tumor hypoxia significantly decreased. These tumors demonstrated an increased vascular functionality suggestive of an increase in the number of intermittently perfused vessels, without corresponding alterations in tumor oxygen consumption rate. CONCLUSIONS: Poorly vascularized, hypoxic mammary carcinomas were much more responsive to short-term endostatin treatment than well-vascularized, more homogeneously oxygenated tumors. Oxygen levels in the responsive tumors were transiently improved after treatment, which could have substantial implications with respect to the therapeutic effectiveness of combining antiangiogenic agents with conventional therapies.