Recombinant Endostatin Research Articles

Abstract A034: Thermoregulation alters adipose influence of pancreatic cancer growth

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the United States and has an abysmal 5-year overall survival of 12%. A major risk factor for PDAC is obesity- which has increased 12% between 1999-2018 in the United States. This rise correlates with the projection that PDAC is to become the second leading cause of cancer death by 2025. Clinically, reports show obese PDAC patients have a three-month decrease in overall survival when compared to lean PDAC patients. Multiple studies have implicated obese white adipose tissue (WAT) as promoting tumor progression. Yet only one study has investigated the effects of brown adipose tissue (BAT) on PDAC, which exhibited cold-mediated BAT activation steals glucose from the tumor. Aside from potential metabolic regulation, our group discovered that cold-mediated activated BAT elicited the release of anti-tumorigenic factors. We discovered this by exposing diet-induced obese mice to a subthermal environment via a thermogenic shift (TS) from thermoneutral (TN) housing. The TS BAT-conditioned media (CM) inhibited PDAC cell proliferation (EdU) and increased apoptosis (via cleaved caspase-3). Using a cytokine array, we identified endostatin as the most upregulated molecule in TS BAT-CM compared to TN BAT-CM. Incubation of PDAC cell lines with recombinant endostatin showed increased cleaved caspase-3. A shotgun-based proteomics screen uncovered novel endostatin binding partners and signaling mediators. In conclusion, we have identified a novel anti-tumorigenic role for activated BAT that further suggests its importance in relation to PDAC progression. Citation Format: Austin Eades, Bailey Bye, R. McKinnon Walsh, Michael VanSaun. Thermoregulation alters adipose influence of pancreatic cancer growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A034.

Recombinant Endostatin as a Potential Radiosensitizer in the Treatment of Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer, which is the leading cause of cancer-related deaths worldwide. Over the past decades, tumour angiogenesis has been intensely studied in the treatment of NSCLC due to its fundamental role in cancer progression. Several anti-angiogenic drugs, such as recombinant endostatin (RE), have been evaluated in several preclinical and clinical trials, with mixed and often disappointing results. However, there is currently an emerging interest in RE due to its ability to create a vascular normalization window, which could further improve treatment efficacy of the standard NSCLC treatment. This review provides an overview of preclinical and clinical studies that combined RE and radiotherapy for NSCLC treatment. Furthermore, it highlights the ongoing challenges that have to be overcome in order to maximize the benefit; as well as the potential advantage of combinations with particle therapy and immunotherapy, which are rapidly gaining momentum in the treatment landscape of NSCLC. Different angiogenic and immunosuppressive effects are observed between particle therapy and conventional X-ray radiotherapy. The combination of RE, particle therapy and immunotherapy presents a promising future therapeutic triad for NSCLC.

The role of angiogenesis in melanoma: Clinical treatments and future expectations.

The incidence of melanoma has increased rapidly over the past few decades, with mortality accounting for more than 75% of all skin cancers. The high metastatic potential of Melanoma is an essential factor in its high mortality. Vascular angiogenic system has been proved to be crucial for the metastasis of melanoma. An in-depth understanding of angiogenesis will be of great benefit to melanoma treatment and may promote the development of melanoma therapies. This review summarizes the recent advances and challenges of anti-angiogenic agents, including monoclonal antibodies, tyrosine kinase inhibitors, human recombinant Endostatin, and traditional Chinese herbal medicine. We hope to provide a better understanding of the mechanisms, clinical research progress, and future research directions of melanoma.

Current research progress in targeted anti-angiogenesis therapy for osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant bone tumour with a peak in incidence during adolescence. Delayed patient presentation and diagnosis is common with approximately 15% of OS patients presenting with metastatic disease at initial diagnosis. With the introduction of neoadjuvant chemotherapy in the 1970s, disease prognosis improved from 17% to 60%‐70% 5‐year survival, but outcomes have not significantly improved since then. Novel and innovative therapeutic strategies are urgently needed as an adjunct to conventional treatment modalities to improve outcomes for OS patients. Angiogenesis is crucial for tumour growth, metastasis and invasion, and its prevention will ultimately inhibit tumour growth and metastasis. Dysregulation of angiogenesis in bone microenvironment involving osteoblasts and osteoclasts might contribute to OS development. This review summarizes existing knowledge regarding pre‐clinical and developmental research of targeted anti‐angiogenic therapy for OS with the aim of highlighting the limitations associated with this application. Targeted anti‐angiogenic therapies include monoclonal antibody to VEGF (bevacizumab), tyrosine kinase inhibitors (Sorafenib, Apatinib, Pazopanib and Regorafenib) and human recombinant endostatin (Endostar). However, considering the safety and efficacy of these targeted anti‐angiogenesis therapies in clinical trials cannot be guaranteed at this point, further research is needed to completely understand and characterize targeted anti‐angiogenesis therapy in OS.

The Construction of a Radiation-Induced Brain Injury Model and Preliminary Study on the Effect of Human Recombinant Endostatin in Treating Radiation-Induced Brain Injury.

BackgroundThe aim of this study was to construct a radiation-induced brain injury (RBI) model and assess the effects of human recombinant endostatin in the treatment of RBI.Material/MethodsIn this study, the RBI model was used. Real-time quantitative polymerase chain reaction, immunohistochemistry, hematoxylin and eosin staining were conducted to detect the mRNA and protein expression of vascular endothelial growth factor (VEGF) and assess the effects of human recombinant endostatin in the treatment of RBI.ResultsIn this study, we successfully constructed a RBI model. VEGF mRNA expression was decreased after human recombinant endostatin treatment; however, VEGF protein secretion was increased in brain endothelial cells, and the secretion of VEGF protein was decreased in glial cells and nerve cells. Body weight changes indicated that human recombinant endostatin can increase the risk of weight loss. Brain water content results showed that human recombinant endostatin might aggravate cerebral edema in the acute stage of RBI, but it can reduce the progression of cerebral edema in the early delayed stage. Survival analysis showed that human recombinant endostatin improved the survival rate only in the early stage of RBI.ConclusionsRadiation can induce vasogenic edema and is associated with the RBI occurrence and development. VEGF protein is highly relevant to the induction of edema and thrombosis in the acute phase of RBI and in the early delayed phase of RBI, including vascular repair and regeneration, thrombus ablation and other events. Human recombinant endostatin can reduce the progression of cerebral edema during the early onset of RBI.

The Effects of Antiplatelet Agents on Endocytosis

Almost every platelet-derived protein originates from either megakaryocytes (MKs), or the endocytosis of factors within blood circulation. These endocytosed factors can be locally released upon platelet activation to regulate hemostasis, or to promote the growth and neovascularization of solid tumors. Although platelet endocytosis has long been recognized, our mechanistic understanding remains largely confined to receptor-mediated endocytosis of fibrinogen via integrin αIIbβ3. Whether antiplatelet therapy and/or different disease states influence platelet endocytosis remain understudied areas of investigation. In this project, we examined how various antiplatelet agents affect the endocytosis of vascular endothelial growth factor (VEGF) and endostatin, which are broad pro-angiogenic and anti-angiogenic regulators, respectively. For our initial experiments, human platelets in platelet-rich plasma were exposed to either recombinant VEGF or endostatin for 1 hour, then washed to remove residual/unbound protein. Platelets were then lysed and changes in VEGF or endostatin concentration determined by ELISA. Using this approach, we demonstrated that platelets endocytose VEGF and endostatin in a concentration-dependent manner. We next established that following platelet activation, lysate concentrations of endocytosed VEGF and endostatin decreased and supernatant concentrations increased. This finding implies that endocytosed VEGF and endostatin are packaged into platelet alpha granules and are released following their activation, which we aim to confirm by measuring co-localization with endosomal and alpha granule markers. We have also developed two assays to track endocytosis into platelets, one where proteins are conjugated to pH-sensitive dyes that increase their fluorescence upon endosomal compartmentalization, and another utilizing fluorophore-targeting antibodies to quench eternal fluorescence of labeled factors. We next assessed if antiplatelet agents modulated endocytosis into platelets. We found that pre-treating platelets with aspirin, vorapaxar or ticagrelor resulted in dose-dependent inhibition of VEGF and fibrinogen but not endostatin endocytosis. This finding suggests that antiplatelet agents selectively inhibit the uptake of pro-angiogenic regulators, a statement we aim to support by measuring the endocytosis of additional angiogenic proteins. Our observations also affirm previous studies, claiming that fibrinogen and VEGF are differentially packaged into a subset of alpha granules distinct from endostatin. Experiments are ongoing to measure platelet levels of VEGF and endostatin in human subjects before and after aspirin intervention, to assess if our findings hold physiological relevance. The fact that several antiplatelet agents impaired VEGF endocytosis suggests a common inhibitory mechanism linked to suppressing basal levels of platelet activation. We tested this hypothesis by either simulating platelets with low-dose adenosine diphosphate or by chelating intracellular calcium to prevent basal activation. Neither of these processes influenced VEGF or endostatin endocytosis, suggesting that the process is not associated with basal platelet activation. We are currently investigating if antiplatelet agents alter the activity of small GTPases Arf6 and Dynamin-2, which have been shown to regulate receptor-mediated endocytosis of fibrinogen by platelets. Future experiments will aim to elucidate the mechanism by which antiplatelet agents inhibit protein uptake. It has previously been shown that platelets endocytose tumor-derived factors and RNA that may promote disease progression or act as biomarkers for liquid biopsies. We are currently studying if the daily administration of aspirin prevents the endocytosis of tumor-derived factors in a mouse model of tumor generation. In summary, our data show platelets endocytose and release key angiogenic regulators, a process which can be blocked through pre-treatment with various antiplatelet agents. Given the important role of platelets to tumor neovascularization, preventing the endocytosis of pro-angiogenic mediators could represent a novel mechanism that contributes to the inhibitory effects of antiplatelet agents on tumor growth and metastasis. Disclosures Flaumenhaft: Relay Therapeutics: Consultancy; PlateletDiagnostics: Consultancy, Other: Founder. Italiano:Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

Well‐controlled pleural effusion indicated pseudoprogression after immunotherapy in lung cancer: A case report

Squamous cancer (SqCC) of the lung has a poor prognosis. With the advent of immunotherapy, prognosis has tended to improve; however, pseudoprogression poses a challenge to the management of immunotherapy. Herein, we discuss the case of a 47‐year‐old heavy smoker with advanced SqCC. The patient had recurrent disease after initial successful control of the tumor by concurrent radiochemotherapy, together with ample pleural effusion. Pleural effusion was well controlled with systematic nivolumab and intra‐thoracic recombinant endostatin; however with simultaneous deterioration of performance and tumor progression. Nivolumab was maintained with the addition of nab‐paclitaxel. The combination soon led to a partial response and rapid improvement of the patient's performance. During treatment of this case, we advocated the early control of pleural effusion as an indicator for pseudoprogression. Our experience might be helpful to identify pseudoprogression for the clinical management of immunotherapy.

重组人血管内皮抑制素联合顺铂治疗非小细胞肺癌恶性胸腔积液的疗效及安全性的Meta分析

重组人血管内皮抑制素联合顺铂治疗非小细胞肺癌恶性胸腔积液的疗效及安全性的Meta分析

411O - Humanized recombinant endostatin combined with vinorelbine plus cisplatin followed by maintenance therapy with humanized recombinant endostatin in advanced non-small cell lung cancer: A multicentre, double-blind, randomised phase 3 trial

411O - Humanized recombinant endostatin combined with vinorelbine plus cisplatin followed by maintenance therapy with humanized recombinant endostatin in advanced non-small cell lung cancer: A multicentre, double-blind, randomised phase 3 trial

Continuous Intravenous Infusion Recombinant Human Endostatin Combined with Concurrent Chemoradiation Therapy in Unresectable Stage III Non–small Lung Cancer—Results of a Multicenter Phase 2 Study (NCT01733589)

Concurrent radiotherapy is the standard treatment of unresectable stage III non-small lung cancer (NSCLC) but with unsatisfied results. Tumor angiogenesis is one of the most important targets for novel agents’ exploration. Recombinant human endostatin, an angiogenesis blocker, has been indicated to normalize the tumor vasculature to make it more efficient for oxygen delivery, thereby provide a window of opportunity for enhanced sensitivity to radiation treatment. To evaluate the efficacy and safety of the addition of continuous intravenous infusion of recombinant human endostatin to concurrent chemoradiotherapy in inoperable stage III NSCLC, we designed this phase 2 study. This trial was approved by the institutional ethics committees of the four participating institutions. The study was registered on the website of ClinicalTrials.gov (NCT01733589).The eligible patients should meet the following criteria before treatment: untreated histologically or cytologically diagnosed NSCLC, inoperable stage IIIA or IIIB according to AJCC seventh staging system, performance score of 0 to 1, blood coagulation is normal. Patients with preexisting bleeding diatheses were excluded. All of the enrolled patients received thoracic radiation with dose of 60-66 Gy, continuous intravenous infusion of recombinant human endostain (7.5 mg/m2/24 h, 120 h, d-6-d-2, d8-d12, d22-d26, d36-d40, from the beginning of radiotherapy) and two cycles of etoposide (50 mg/m2, d1-d5, d29-d33) plus cisplatin (50 mg/m2, d1, d8, d29, d36). The primary endpoint was progression free survival (PFS) and the secondary endpoints were response, overall survival (OS), local regional free survival (LRFS) and toxicities. Calculating with 20% bilateral Z test (alpha = 0.05, beta = 0.2), 72 patients were needed. From November 2012 to June 2015, seventy-three patients were enrolled and 67 patients were assessable. Fifty-six patients were male and 11 patients were female. The median age was 59 years old. The majority (65.7%) of the patients were diagnosed as squamous cell carcinoma. Severe adverse events were observed in 2 patients (1 hemoptysis and 1 pneumonia). The most common adverse events was leucopenia (95.5%, Grade 3-4:44.8%). The complete response and partial response rate were 11.9% and 64.2%, respectively. The median follow-up time was 37.1 months (20.1-52.1 months). The median time of OS, PFS, and LRFS was 34.7 months, 13.3 months and 27.1 months respectively. The 1-, 2-, and 3-year PFS were 50.7%, 34.8%, and 28.2%, OS were 82.1%, 59.9%, and 47.7%, and LRFS were 72.7%, 54.7%, and 49.9%, respectively. The addition of continuous intravenous human recombinant endostatin to concurrent chemoradiotherapy is effective and tolerable in unresectable stage III NSCLC. Further randomized phase III study is warranted.

Continuous intravenous infusion of recombinant human endostatin combined with concurrent etoposide plus cisplatin and radiotherapy for treatment of unresectable stage III non-small-cell lung cancer (HELPER): a phase 2, multicentre study

BackgroundConcurrent chemoradiotherapy is the standard treatment for unresectable stage III non-small lung cancer, with frequently unsatisfactory results. We aimed to evaluate the efficacy and safety of the addition of continuous intravenous infusion of recombinant human endostatin (Endostar) to concurrent chemoradiotherapy. MethodsWe did this prospective, phase 2 study (HELPER) in patients with inoperable stage III non-small-cell lung cancer (defined by the American Joint Committee on Cancer 7th edn staging system) with an E performance score of 0–2; diagnoses were made on the basis of pathology or cytology results. All enrolled patients received thoracic radiation at doses of 60–66 Gy and continuous intravenous infusions of Endostar (7·5 mg/m2 per 24h, 120 h continuously, 120 h per cycle; from day −5 to day −1, days 10–13, days 24–28, and days 38–42). Additionally, patients received two cycles of etoposide (50 mg/m2; days 1–5 and days 29–33) plus cisplatin (50 mg/m2; day 1, 8, 29, and 36). The primary endpoint was progression-free survival and the secondary endpoints were objective response, overall survival, local recurrence-free survival, and toxicities. FindingsFrom November, 2012, to June, 2015, 73 patients were enrolled and 67 patients were evaluable. 56 patients were male and 11 patients were female. The median age was 59 years (range 31–69). Most patients (44, 66%) had squamous cell carcinoma. 27 patients (40%) had stage IIIa disease, and 40 (60%) had stage IIIb disease. Severe adverse events were observed in two patients (one had massive haemoptysis and one had pneumonia). The most common adverse event was leucopenia (96%; 45% were grade 3–4). The complete response was 12% and partial response was 64%. The median follow-up time was 37·1 months (range 20·1–52·1). The median overall survival was 34·7 months (21·9–47·4), progression-free survival was 13·3 months (5·5–21·0), and local recurrence-free survival was 27·1 months. Progression-free survival was 51% at 1 year, 35% at 2 years, and 28% at 3 years, overall survival was 82% at 1 year, 60% at 2 years, and 48% at 3 years, and local recurrence-free survival was 73% at 1 year, 55% at 2 years, and 50% at 3 years. InterpretationFor patients with unresectable stage III non-small-cell lung cancer, continuous intravenous human recombinant endostatin in combination with concurrent chemoradiotherapy is an effective treatment with tolerable toxicities. A phase 3 study is warranted. FundingNone.

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es) is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs) is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli) expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A) of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

Smooth Muscle Cell Proangiogenic Phenotype Induced by Cyclopentenyl Cytosine Promotes Endothelial Cell Proliferation and Migration

Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) are in close contact with blood vessels. SMC phenotypes can be altered during pathological vascular remodeling. However, how SMC phenotypes affect EC properties remains largely unknown. In this study, we found that PDGF-BB-induced synthetic SMCs suppressed EC proliferation and migration while exhibiting increased expression of anti-angiogenic factors, such as endostatin, and decreased pro-angiogenic factors, including CXC motif ligand 1 (CXCL1). Cyclopentenyl cytosine (CPEC), a CTP synthase inhibitor that has been reported previously to inhibit SMC proliferation and injury-induced neointima formation, induced SMC redifferentiation. Interestingly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration because of an increase in CXCL1 along with decreased endostatin production in SMCs. Addition of recombinant endostatin protein or blockade of CXCL1 with a neutralizing antibody suppressed the EC proliferation and migration induced by CPEC-conditioned SMC medium. Mechanistically, CPEC functions as a cytosine derivate to stimulate adenosine receptors A1 and A2a, which further activate downstream cAMP and Akt signaling, leading to the phosphorylation of cAMP response element binding protein and, consequently, SMC redifferentiation. These data provided proof of a novel concept that synthetic SMC exhibits an anti-angiogenic SMC phenotype, whereas contractile SMC shows a pro-angiogenic phenotype. CPEC appears to be a potent stimulator for switching the anti-angiogenic SMC phenotype to the pro-angiogenic phenotype, which may be essential for CPEC to accelerate re-endothelialization for vascular repair during injury-induced vascular wall remodeling.

Human Recombinant Endostatin Combined with Cisplatin Based Doublets in Treating Patients with Advanced NSCLC and Evaluation by CT Perfusion Imaging.

To study the effectiveness of human recombinant endostatin injection (Endostar®) combined with cisplatin doublets in treating advanced non-small cell lung cancer (NSCLC), and to evaluate outcome by CT perfusion imaging. From April 2011 to September 2014, 76 patients with advanced NSCLC who were treated with platinum-based doublets were divided into group A (36 patients) and group B (40 patients). Endostar® 15 mg/day was administered 4 days before chemotherapy and combined with chemotherapy from day 5 in group A, and combined with chemotherapy from the first day in Group B. Endostar® in the two groups was injected intravenously for 14 days. Treatment effectiveness in the two groups differed with statistical significance (p<0.05). Effectiveness evaluated by CT perfusion imaging, BF, BV, MTT and PS also demonstrated significant differences (all p<0.05). Adverse reactions in the two groups did not significantly vary (p>0.05). The response rate with Endostar® administered 4 days before chemotherapy and combined with chemotherapy from day 5 in group A was better than Endostar® combined with chemotherapy from the first day, and CT perfusion imaging could be a reasonable method for evaluation of patient outcomes.

Clinical observation of recombinant human vascular endostatin combined with pemetrexed and cisplatin in the treatment of advanced lung adenocarcinoma

Objective To study the clinical effects and side effects of recombinant human vascular endostatin combined with pemetrexed and cisplatin in the treatment of advanced lung adenocarcinoma. Methods Forty-seven patients with advanced lung adenocarcinoma were recruited from Jan 2011 to Jan 2013, and they were randomly divided into experimental group and control group. The experimental group (n = 24) was added with recombinant human vascular endostatin based on pemetrexed and cisplatin,whereas the control group(n = 23) was administered with pemetrexed and cisplatin only. The objective response rate (ORR), disease control rate (DCR), progressive disease (PD) rate, progression-free survival (PFS), overall survival (OS) and the side effects of 2 groups were evaluated. Results In the experimental group, ORR, DCR, PD rate, PFS and OS were 41.7 % (10/24), 79.2 % (19/24), 20.8 % (5/24), 8.0 months and 12.5 months respectively, while those of control group were 34.7 % (8/23), 47.8 % (11/23), 52.2 % (12/23), 6.2 months and 10.0 months. DCR, PD rate and PFS of experimental group had significant differences compared with control group (P 0.05). The side effects of 2 groups were mainly hematologic toxicities, digestive reactions and fatigue, and the incidence rates were not significantly different between 2 groups (P> 0.05). Conclusions Recombinant human vascular endostatin combined with pemetrexed and cisplatin in treatment of patients with advanced lung adenocarcinoma improves the DCR, decreases the PD rate, prolongs the PFS. There is an increasing trend in the OS of experimental group, and with tolerable side effects. Key words: Lung neoplasms; Recombinant human vascular endostatin; Antineoplastic combined chemotherapy protocols

Human recombinant endostatin Endostar attenuates hepatic sinusoidal endothelial cell capillarization in CCl4‑induced fibrosis in mice.

The aim of the present study was to detect the effect of the recombinant human endostatin Endostar on hepatic sinusoidal capillarization in CCl4‑induced murine models of liver fibrosis. The liver fibrosis model was induced in BALB/c mice using intraperitoneal injection of CCl4 for 6 weeks. Animals were divided into the following six treatment groups: Group 1, normal animals; group 2, CCl4‑induced liver fibrosis; group 3, CCl4+Endostar 20 mg/kg/day for 6 weeks; group 4, CCl4+Endostar 10 mg/kg/day for 6 weeks; group 5, CCl4+Endostar 20 mg/kg/day for 4 weeks; and group 6, CCl4+Endostar 10 mg/kg/day for 4 weeks. The average number of fenestrae per hepatic sinusoid was determined using transmission electron microscopy. Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 1 and 2 expression was detected by western blot analysis. There were significant differences in the number of fenestrae per sinusoid between the normal control and untreated model fibrotic mice (P<0.01), and between the untreated model and Endostar‑treated mice (P<0.05). Endostar treatment was associated with reduced levels of VEGFR1 and VEGFR2 in liver tissues (P<0.01), as well as with decreased hepatic sinusoidal endothelial cell capillarization in CCl4‑induced mouse models of liver fibrosis, and this effect may involve the VEGF pathway. However, further studies are required to confirm its involvement in other causes of liver fibrosis.

Endostatin induces proliferation of oral carcinoma cells but its effect on invasion is modified by the tumor microenvironment

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen–fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment.

Inhibitory effect of polysulfated heparin endostatin on alkali burn induced corneal neovascularization in rabbits.

To investigate anti-angiogenic effects of polysulfated heparin endostatin (PSH-ES) on alkali burn induced corneal neovascularization (NV) in rabbits. An alkali burn was made on rabbit corneas to induce corneal NV in the right eye of 24 rabbits. One day after burn creation, a 0.2 mL subconjunctival injection of 50 µg/mL PSH-ES, 50 µg/mL recombinant endostatin (ES), or normal saline was administered every other day for a total of 14d (7 injections). Histology and immunohistochemisty were used to examine corneas. Corneal NV growth was evaluated as microvessel quantity and corneal vascular endothelial growth factor (VEGF) expression was measured by immunohistochemical assay. Subconjunctival injection of ES and PSH-ES resulted in significant corneal NV suppression, but PSH-ES had a more powerful anti-angiogenic effect than ES. Mean VEGF concentration in PSH-ES treated corneas was significantly lower than in ES treated and saline treated corneas. Histological examination showed that corneas treated with either PSH-ES or ES had significantly fewer microvessels than eyes treated with saline. Additionally corneas treated with PSH-ES had significantly fewer microvessels than corneas treated with ES. Both PSH-ES and recombinant ES effectively inhibit corneal NV induced by alkali burn. However, PSH-ES is a more powerful anti-angiogenic agent than ES. This research has the potential to provide a new treatment option for preventing and treating corneal NV.

Endostatin stimulates proliferation and migration of adult rat cardiac fibroblasts through PI3K/Akt pathway

Endostatin, a non-collagenous fragment of type XVIII collagen, has anti-angiogenic roles. Although the expression level of endostatin increases in some experimental models of cardiac diseases, its effects on cardiac remodeling have not been clarified. In this study, we investigated the effect of endostatin on proliferation, migration and collagen synthesis of cardiac fibroblasts, which are activated during the cardiac remodeling following myocardial infarction. Cardiac fibroblasts were isolated from adult male Wistar rats and treated with recombinant endostatin for 20min to 24h. Cell counting assay was used to determine a cell proliferation. Boyden chamber assay was performed to determine a cell migration. Wound-healing assay was performed for detecting a wound-induced migration. Expression of collagen type I and phosphorylation of Akt, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were detected by Western blotting. Endostatin (100–3000ng/ml) stimulated cell proliferation, migration and wound-induced migration but not collagen type I expression. Endostatin stimulated phosphorylation of Akt (Ser473) but not ERK, JNK and p38 MAPK. LY294002 (10μM), a PI3K/Akt pathway inhibitor, significantly inhibited the endostatin-induced proliferation and migration. N-acetyl-l-cysteine (NAC) (5mM), an antioxidant, significantly inhibited the endostatin-induced Akt phosphorylation. This study for the first time demonstrated that endostatin stimulates cell proliferation, migration and wound-induced migration of adult rat cardiac fibroblasts at least partly through the reactive oxygen species-dependent activation of Akt. Our findings suggest a novel function of endostatin except for an anti-angiogenic activity during the wound-healing process following myocardial infarction.

Suppression of choroidal neovascularization by Endostar in rats.

Choroidal neovascularization (CNV) is common in various retinal and choroidal diseases, and may result in severe and irreversible loss of vision. Our previous studies suggested that Endostar, a novel recombinant endostatin, is able to inhibit the proliferation and migration of choroid‑retinal endothelial cells. To further evaluate the effect of Endostar on the formation of CNV in vivo, a rat model of laser‑induced CNV was constructed and Endostar or phosphate‑buffered saline treatment was administered intravitreally every other day. Using fluorescein angiography (FA), reduced CNV incidence and leakage grade was observed in the Endostar group. In addition, CNV area and maximal thickness were prominently reduced in the Endostar group measured by choroid flat mounts and sections. Furthermore, vascular endothelial growth factor (VEGF), hypoxia‑inducible factor 1α and chemokine C‑X‑C motif ligand 1 were markedly reduced in the Endostar group as determined by quantitative polymerase chain reaction and downregulation of VEGF was also verified by western blot analysis at the protein level. This study demonstrates that Endostar suppressed CNV in a rat model, which may be largely mediated by the downregulation of VEGF and other angiogenic molecules.