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The combination of anti-angiogenic drugs and immune checkpoint inhibitors (ICIs) in the treatment of tumors is emerging as a way to improve ICIs-resistant tumor therapy. In addition, gut microbes (GMs) are involved in angiogenesis in the tumor microenvironment and are also associated with the antitumor function of immune checkpoint inhibitors. However, it is unclear whether gut microbes have a role in anti-tumor function in the combination of anti-angiogenic drugs and immune checkpoint inhibitors for cancer treatment. Endostatin, an angiogenesis inhibitor, has been widely used as an antiangiogenic therapy for cancer. We showed that combined therapy with an adenovirus encoding human endostatin, named Ad-E, and PD-1 blockade dramatically abrogated MC38 tumor growth. The structure of intestinal microbes in mice was changed after combination treatment. We found that the antitumor function of combination therapy was inhibited after the elimination of intestinal microbes. In mice with depleted microbiota, oral gavage of Bacteroides fragilis salvaged the antitumor effects of combination Ad-E and αPD-1 monoclonal antibody (mAb) to a certain extent. Further, Bacteroides fragilis could improve CD3 + T cells, NK cells, and IFNγ + CD8 + T cells in the tumor microenvironment to inhibit tumor growth. Besides, Bacteroides fragilis might restore antitumor function by down-regulating isobutyric acid (IBA). Our results suggested that GMs may be involved in the combination of Ad-E and αPD-1 mAb for cancer treatment, which has oncological implications for tumor growth dynamics and cancer immune surveillance., (© 2024. The Author(s).)

Angiogenesis is critical for rheumatoid arthritis (RA) progression. The effects of tofacitinib, a JAK-STAT inhibitor used for RA treatment, on angiogenesis in RA are unclear. We, therefore, evaluated the levels of angiogenic factors in two systems of a human co-culture of fibroblast (HT1080) and monocytic (U937) cell lines treated with tofacitinib and in serum samples from RA patients before and after six months of tofacitinib treatment. Tofacitinib reduced CD147 levels, matrix metalloproteinase-9 (MMP-9) activity, and angiogenic potential but increased endostatin levels and secreted proteasome 20S activity. In vitro, tofacitinib did not change CD147 mRNA but increased miR-146a-5p expression and reduced STAT3 phosphorylation. We recently showed that CD147 regulates the ability of MMP-9 and secreted proteasome 20S to cleave collagen XVIIIA into endostatin. We show here that tofacitinib-enhanced endostatin levels are mediated by CD147, as CD147-siRNA or an anti-CD147 antibody blocked proteasome 20S activity. The correlation between CD147 and different disease severity scores supported this role. Lastly, tofacitinib reduced endostatin' s degradation by inhibiting cathepsin S activity and recombinant cathepsin S reversed this in both systems. Thus, tofacitinib inhibits angiogenesis by reducing pro-angiogenic factors and enhancing the anti-angiogenic factor endostatin in a dual effect mediated partly through CD147 and partly through cathepsin S.

Background: Radiation-induced pulmonary fibrosis (RIPF) is a long-term complication of thoracic radiotherapy without effective treatment available., Objective: This study aimed to establish a RIPF mouse model and explore the therapeutic effects and mechanisms of recombinant human endostatin (Endostar)., Methods: C57BL/6 mice received a 16-Gy dose of X-rays to the whole thorax with or without the administration of Endostar for 24 weeks., Results: Radiation-induced body weight loss was partially attenuated by Endostar (P<0.05). Endostar significantly reduced alveolar inflammation (P<0.05) and pulmonary fibrosis (P<0.001), as indicated by a decrease in the expression levels of collagen I and collagen IV in lung tissue (both P<0.001). Angiogenesis (as shown by CD31 immunohistochemistry) was also decreased (P<0.01). In irradiated mice, Endostar inhibited the transforming growth factor-β1 (TGF-β1)/drosophila mothers against the decapentaplegic 3 (Smad3)/extracellular regulated protein kinases (ERK) signaling pathway (all P<0.05). In vitro, Endostar treatment decreased the radiation-induced expression of TGF-β1, vascular endothelial growth factor (VEGF), p-Smad3, and p-ERK in alveolar epithelial cells and vascular endothelial cells (all P<0.05)., Conclusion: Endostar could alleviate RIPF through decreased antiangiogenic activity and inhibition of the TGF-β1/Smad3/ERK pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: This study aimed to outline the pre-clinical efficacy and safety pharmacology of PEGylated recombinant human endostatin (M2ES) according to the requirements of new drug application., Methods: The purity of M2ES was evaluated by using silver staining. Transwell migration assay was applied to detect the bioactivity of M2ES in vitro. The antitumor efficacy of M2ES was evaluated in an athymic nude mouse xenograft model of pancreatic cancer (Panc-1) and gastric cancer (MNK45). BALB/C mice were treated with different doses of M2ES (6, 12 and 24 mg/kg) intravenously, both autonomic activity and cooperative sleep were monitored before and after drug administration., Results: The apparent molecular weight of M2ES was about 50 kDa, and the purity was greater than 98%. Compared with the control group, M2ES significantly inhibits human micro-vascular endothelial cells (HMECs) migration in vitro. Notably, weekly administration of M2ES showed a significant antitumor efficacy when compared with the control group. Treatment of M2ES (24mg/kg or below) showed no obvious effect on both autonomic activity and hypnosis., Conclusion: On the basis of the pre-clinical efficacy and safety pharmacology data of M2ES, M2ES can be authorized to carry out further clinical studies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Angiogenesis is the physiological process that results in the formation of new blood vessels develop from pre-existing vasculature and plays a significant role in several physiological and pathological processes. Inhibiting angiogenesis, a crucial mechanism in the growth and metastasis of cancer, has been proposed as a potential anticancer therapy. Different studies showed the beneficial effects of angiogenesis inhibitors either in patients suffering from different cancers, alone or in combination with conventional therapies. Even though there are currently a number of efficient anti-angiogenic drugs, including monoclonal antibodies and kinase inhibitors, the associated toxicity profile and their affordability constraints are prompting researchers to search for a safe and affordable angiostatic agent for cancer treatment. Endostatin is one of the endogenous anti-angiogenic candidates that have been extensively pursued for the treatment of cancer, but even over three decades after its discovery, we have not made much advancement in employing it as an anticancer therapeutic despite of its remarkable anti-angiogenic effect with low toxicity profile. A recombinant human endostatin (rh-Es) variant for non-small cell lung cancer was approved by China in 2006 and has since been used effectively. Several other successful clinical trials related to endostatin for various malignancies are either ongoing or have already been completed with promising results. Thus, in this review, we have provided an overview of existing anti-angiogenic drugs developed for cancer therapy, with a summary of tumour angiogenesis in the context of Endostatin, and clinical status of rh-Es in cancer treatment. Furthermore, we briefly discuss the various strategies to improve endostatin features (poor pharmacokinetic properties) for developing rh-Es as a safe and effective agent for cancer treatment., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Sodium-glucose cotransporter-2 inhibitor, canagliflozin, improves myocardial perfusion to ischemic territory without accompanying changes in vascular density. We aimed to (1) characterize effects on angiogenic pathways, (2) use multiomics to identify gene expression and metabolite profiles relevant to regulation of myocardial blood flow, and (3) investigate drug effect on coronary microvascular reactivity., Methods: A nondiabetic swine model of chronic myocardial ischemia and nondiabetic rat model were used to study functional and molecular effects of canagliflozin on myocardium and in vitro microvascular reactivity., Results: Canagliflozin resulted in increased coronary microvascular vasodilation and decreased vasoconstriction (P < .05) without changes in microvascular density (P > .3). Expression of the angiogenic modulator, endostatin, increased (P = .008), along with its precursor, collagen 18 (P < .001), and factors that increase its production, including cathepsin L (P = .004). Endostatin and collagen 18 levels trended toward an inverse correlation with blood flow to ischemic territory at rest. Proangiogenic fibroblast growth factor receptor was increased (P = .03) and matrix metalloproteinase-9 was decreased (P < .001) with canagliflozin treatment. Proangiogenic vascular endothelial growth factor A (P = .13), Tie-2 (P = .10), and Ras (P = .18) were not significantly altered. Gene expression related to the cardiac renin-angiotensin system was significantly decreased., Conclusions: In chronic myocardial ischemia, canagliflozin increased absolute blood flow to the myocardium without robustly increasing vascular density or proangiogenic signaling. Canagliflozin resulted in altered coronary microvascular reactivity to favor vasodilation, likely through direct effect on vascular smooth muscle. Downregulation of cardiac renin-angiotensin system demonstrated local regulation of perfusion. VIDEO ABSTRACT., Competing Interests: Conflict of Interest Statement The authors reported no conflicts of interest. The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest., (Copyright © 2023 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest All authors of this research paper have directly participated in the planning, execution, or analysis of the study. The contents of this manuscript are not under consideration for publication elsewhere, and will not be copyrighted, submitted, or published elsewhere. There are no directly related manuscripts or abstracts, published or unpublished, by any author(s) of this paper. The authors also claim that there is no competitive financial interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Background & Aims: Liver fibrosis is an intrinsic wound-healing response to chronic injury and the major cause of liver-related morbidity and mortality worldwide. However, no effective diagnostic or therapeutic strategies are available, owing to its poorly characterized molecular etiology. We aimed to elucidate the mechanisms underlying liver fibrogenesis., Methods: We performed a quantitative proteomic analysis of clinical fibrotic liver samples to identify dysregulated proteins. Further analyses were performed on the sera of 164 patients with liver fibrosis. Two fibrosis mouse models and several biochemical experiments were used to elucidate liver fibrogenesis., Results: We identified cathepsin S (CTSS) up-regulation as a central node for extracellular matrix remodeling in the human fibrotic liver by proteomic screening. Increased serum CTSS levels efficiently predicted liver fibrosis, even at an early stage. Secreted CTSS cleaved collagen 18A1 at its C-terminus, releasing endostatin peptide, which directly bound to and activated hepatic stellate cells via integrin α5β1 signaling, whereas genetic ablation of Ctss remarkably suppressed liver fibrogenesis via endostatin reduction in vivo. Further studies identified macrophages as the main source of hepatic CTSS, and splenectomy effectively attenuated macrophage infiltration and CTSS expression in the fibrotic liver. Pharmacologic inhibition of CTSS ameliorated liver fibrosis progression in the mouse models., Conclusions: CTSS functions as a novel profibrotic factor by remodeling extracellular matrix proteins and may represent a promising target for the diagnosis and treatment of liver fibrosis., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: The molecular mechanisms by which exercise improves brain function and capillaries in the cerebral cortex are unclear. Exercise can increase the expression of nitric oxide (NO) in the brain, and endogenous NO is thought to exert beneficial effects on proangiogenic factors, antiangiogenic factors and brain function. Therefore, we hypothesized that running exercise might improve brain function and enhance angiogenesis through endogenous NO., Methods and Results: The following three groups of rats were administered intracerebroventricular (i.c.v.) injections before running exercise each day for 4 weeks: exercise+L-NAME group (i.c.v. L-NAME, an NO synthase blocker, dose: 1 μmol/μl and 5 μl/day; treadmill exercise, 20 min/day), exercise group (i.c.v. normal saline, 5 μl/day; treadmill exercise, 20 min/day), and sham group (i.c.v. normal saline, 5 μl/day; no treadmill exercise). Subsequently, the spatial learning and memory abilities were tested using a Morris water maze, and the nitric oxide synthase (NOS) activity in the cerebral cortex in each group of rats was measured using a method involving nitric acid reductase and metabolic chemistry. The parameters of the cortical capillaries were quantitatively investigated using an immunohistochemistry technique and stereological methods. The expression levels of proangiogenic factors (VEGF and FGF-2) and an antiangiogenic inhibitor (endostatin) in the cerebral cortex were tested using a Western blot analysis. Running exercise significantly improved the rats' spatial learning and memory abilities and increased NOS activity in the cortex. Running exercise also subsequently improved the expression of proangiogenic factors (VEGF and FGF-2) and the length, volume and surface area of capillaries and reduced the expression of antiangiogenic factors (endostatin) in the cortex. In contrast, the L-NAME treatment attenuated the effects of running exercise., Conclusions: Running exercise regulates proangiogenic factors, antiangiogenic factors and angiogenesis in the cerebral cortex via a partially NO-dependent mechanism, and influencing endogenous NO might potentially affect the exercise-related beneficial effects on cognitive ability and cortical capillaries., Competing Interests: Declaration of Conflict of Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The blockade of programmed cell death-1 (PD-1) and recombinant human endostatin can be used for the treatment of non-small cell lung cancer (NSCLC) and its metastasis. This study aims to explore the therapeutically potential of PD-1 blockade plus Endostar in brain metastasis of NSCLC., Methods: The mouse brain metastases model was established using Lewis lung carcinoma luciferase (LLC-Luc) and PC-9-Luc cells. Tumor metastasis in the brain and tumor burden were analyzed by using bioluminescence imaging (BLI), qRT-PCR and ELISA which were used to determine the mRNA and protein levels of biomarkers in tumor tissues. Immunohistochemical staining was used to determine the expression and location of CD31 in tumor tissues in the brain., Results: Treatment with anti-PD-1 and Endostar suppressed tumor metastasis in the brain and prolonged overall survival rate in LLC-Luc and PC-9-Luc brain metastases mouse model. In addition, treatment with anti-PD-1 and Endostar inhibited the expressions of CD31 and VEGF in tumor tissues in the brain. Furthermore, treatment with anti-PD- 1 and Endostar significantly suppressed the levels of IL1β, IFNγ, and TGFβ in the tumor tissues., Conclusion: The combination of PD-1 blockade and endostar suppressed brain metastases of NSCLC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: In our previous study, N end of the Circumsporozoite protein (CSP I-plus) modified recombinant human Endostatin (rEndostatin, endostar) (rES-CSP) was constructed, which had antiangiogenic capability and bound to hepatocellular carcinoma in vivo and in vitro. In this study, the inhibition of rES-CSP on hepatocellular carcinoma metastasis was verified in vivo and in vitro, and its possible mechanism was explored., Methods: Firstly, the impact of rES-CSP on the migration, adhesion of hepatoma cell HCCLM3 was identified by wound healing, transwell, and on metastasis of orthotopic xenograft model was identified in nude mouse. Then the expression of metastasis-associated molecules (MMP2, E-cadherin, integrinβ1) and angiogenesis-related factors (VEGFA) in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry., Results: Finally, we found that rES-CSP could inhibit the migration and invasion of HCCLM3, and decrease tumor metastasis and growth in nude mouse orthotopic xenograft models. The tumor inhibiting rates of rES-CSP and Endostar were 42.46 ± 5.39% and 11.1 ± 1.88%. The lung metastasis rates of the control, Endostar and rES-CSP were 71, 50, and 42.8%, respectively. Compared with Endostar, rES-CSP significantly down-regulated the expression of VEGFA and integrinβ1. Heparin, a competitive inhibitor of CSP I-plus, which can be bind to the highly-sulfated heparan sulfate proteoglycans (HSPGs) over-expressed in liver and hepatocellular carcinoma, alleviated the down-regulation of VEGFA and integrinβ1., Conclusions: These indicate that rES-CSP may play a role in inhibiting tumor growth and metastasis by down-regulating the angiogenic factor VEGF and the metastasis-related molecules or by interfering with HSPGs-mediated tumor metastasis., (© 2022. The Author(s).)

Gram-negative, facultatively anaerobic bacteria Salmonella Typhimurium is a candidate agent or delivery vector for cancer therapy. Effective targeted therapies in addition to radiotherapy, chemotherapy and surgery have been urgently needed as an alternative or supplement. This study expected to further improve the tumor-targeting ability of Salmonella bacteria through genetic modifications. Based on an auxotrophic Salmonella bacterial strain (D2), we constructed Salmonella mutants with altered LPS length to facilitate displaying the RGD4C targeting peptide on the outer membrane surface of Salmonella. The expression of RGD4C peptide in fusion with OmpA was identified by outer membrane protein extraction and WB detection in different mutant strains. However, flow cytometry analysis following immunofluorescence staining demonstrated that the extracellular length of Salmonella LPS did affect the surface display of RGD4C peptide. The strain D2-RGD4C that synthesized intact LPS including lipid A, core oligosaccharides and O antigen polysaccharides could hardly display RGD4C peptide, showing the same fluorescence signal intensity as the strains not expressing RGD4C peptide. Among different strains, D2 ∆rfaJ-RGD4C that synthesized truncated LPS including lipid A and partial core oligosaccharides was capable of displaying RGD4C peptide most efficiently and showed the highest ability to target HUVECs expressing αV integrin and tumor tissue with abundant neovascularization. Animal experiments also demonstrated that this tumor-targeting attenuated Salmonella strain to simultaneously deliver endostatin and TRAIL, two agents with different anti-tumor activities, could significantly inhibit tumor growth and prolong mouse survival. Thus, our studies revealed that Salmonella could be genetically engineered to improve its tumor targeting via the truncation of LPS and surface display of targeting peptides, thereby eliciting superior anti-tumor effects through targeted delivery of drug molecules., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: To investigate the synergic effect and underlying mechanism of Endostar, a recombinant human endostatin used for anti-angiogenesis, in radiotherapy for cervical cancer., Methods: The Cell Counting Kit-8 (CCK-8) assay and plate cloning experiment were first employed to analyze the proliferation of HeLa and SiHa cervical cancer cells and human umbilical vein vascular endothelial cells (HUVECs). Flow cytometry was used to detect apoptosis and cell cycle progression. A tube formation assay was used to assess angiogenesis in vitro . The expression of gamma H2A histone family member X (γ-H2AX) and activation of the vascular endothelial growth factor receptor (VEGFR) signaling pathway were detected by immunofluorescence and western blotting, respectively. In a HeLa xenograft model, tumor tissue expression of CD31 and alpha smooth muscle actin and serum expression of VEGF-A were detected by immunohistochemistry (IHC) and enzyme-linked immunosorbent assay, respectively., Results: The CCK-8 and plate cloning assays showed that Endostar and radiotherapy synergistically inhibited the growth of HUVECs but not HeLa and SiHa cells. The flow cytometric results showed that Endostar only promoted radiotherapy-induced apoptosis and G2/M phase arrest in HUVECs ( p < 0.05). Endostar combined with radiotherapy also significantly inhibited tube formation by HUVECs ( p < 0.05). Furthermore, Endostar inhibited the radiotherapy-induced expression of γH2AX ( p < 0.05) and phosphorylation of VEGFR2/PI3K/AKT/DNA-PK in HUVECs ( p < 0.05). IHC showed that Endostar enhanced the inhibitory effect of radiotherapy on the microvessel density in xenograft tumor tissues ( p < 0.05), as well as serum VEGF-A expression ( p < 0.05). The tumor volume in the combination therapy groups (1200 mm 3 ) was significantly lower than in the control group (2500 mm 3 ; p < 0.05)., Conclusions: Our findings provide experimental evidence and a theoretical basis for the application of Endostar in combination with irradiation for anti-cervical cancer treatment., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

Recombinant human endostatin (rhES) is a protein drug with poor stability and short in vivo circulation time. The present study was therefore aimed at developing sustained-release lung targeted microspheres drug delivery system and evaluating its targeting efficiency using in vivo imaging techniques with quantum dots (QDs) as the imaging material. The oil-soluble QDs were coated with amphiphilic polymers to obtain a polymer-quantum dots micelle (QDs-M) with the potential to stably disperse in water. The rhES and QDs-M were combined using covalent bonds. The rhES-QDs-M microspheres (rhES-QDs-M-MS) were prepared using electrostatic spray technology and also evaluated via in vivo imaging techniques. The pharmacodynamics was further studied in mice. The rhES-QDs-M-MS (4-8 μm) were stable in an aqueous medium with good optical properties. The in vitro studies showed that the rhES-QDs-M-MS had sustained release which was maintained for at least 15 days (cumulative release >80%) without any burst release. The rhES-QDs-M-MS had a very high safety profile and also effectively inhibited the in vitro proliferation of human umbilical vein endothelial cells by about 70%. The pharmacokinetic results showed that the rhES could still be detected at 72 h in the experimental group which meant that the rhES-QDs-M-MS had a significant sustained-release effect. The rhES-QDs-M-MS had a better lung targeting effect and higher antitumor activity compared with the rhES. The traceable rhES-QDs-M-MS served as a promising drug delivery system for the poorly stable rhES proteins and significantly increased its lung-targeted effect, sustained-release properties, and antitumor activities., (© 2022. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Ageing is characterised by a progressive loss of vascular endothelial function and integrity. Endothelial progenitor cells (EPCs) play an integral role in endothelial regeneration but are prone to age-dependent changes which may accelerate their senescence and diminish their availability and functionality. Considering these, we firstly investigated the quantity of circulating EPCs in older (73.3 ± 7.2 years) and younger (40.2 ± 14.3 years) healthy volunteers and showed sharp declines in the number of EPCs expressing stemness markers (CD34 + and/or CD133 + ) in older people. These coincided with the decreases in total anti-oxidant capacity (TAC) and concomitant increases in plasma levels of pro-inflammatory cytokine, TNF-α and anti-angiogenic factor, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional properties of outgrowth endothelial cells (OECs), the functional subtype of EPCs, showed that chronological ageing, mimicked by replicative senescence, profoundly impaired proliferation, migration, tubulogenesis, and blood-brain barrier (BBB)-forming capacity of these cells. Similar to those seen in the clinical observational studies, senescent OECs also manifested decreased TAC and increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using structurally and functionally distinct anti-oxidants, namely vitamin C or VAS2870, an NADPH oxidase inhibitor, delayed OEC senescence and restored their tubulogenic and BBB-forming capacities. In conclusion, the enhanced oxidative stress level that develops during physiological ageing may promote EPC senescence and evoke endothelial dysfunction. Effective control of oxidative stress using either compound somewhat delays both phenomena and augments EPC functionality., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of transforming growth factor beta1 (TGF-β1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling., Method: The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-β1 siRNA (gene silencing) group, ES + siTGF-β1 siRNA group, and 10 Gy radiation + ES + siTGF-β1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC 50 ) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-β1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-β1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis., Results: ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-β1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-β1 gene (P < 0.05)., Conclusion: The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-β1/Smad3/CTGF signaling pathway., (© 2022. The Author(s).)

Background: This study aimed to evaluate the inhibitory effects and potential mechanisms of icotinib combined with antiangiogenic drugs on lung adenocarcinoma in vivo., Methods: A total of 72 mouse xenograft models established with human lung adenocarcinoma cells (HCC827) were randomly divided into six groups, including control, icotinib (Ic), bevacizumab (Bev), recombinant human endostatin (En), Ic + Bev and Ic + En groups. Mouse weights and tumor volumes were measured regularly. Half of the nude mice in each group were sacrificed after 16 days of drug treatment. The remaining animals were observed for another 16 days without drug supply. Immunohistochemical staining was performed to detect microvessel density in tumor heart, liver, brain specimens from the nude mice and Ki67 expression. Differential expression of vascular endothelial growth factor (VEGFA) in tumor tissue specimens was determined by ELISA and Western blot., Results: The results showed that the combined drugs inhibited tumor growth more substantially compared with single drugs, without increasing the toxic effects. The antiangiogenesis effect of the combination was better than that of single drug treatment. In addition, both types of targeted drugs and combination medication not only significantly reduced microvessel density in the tumor tissue itself, but also had a certain impact on decreasing microvessel density in the liver. The combination decreased VEGFA and Ki-67 amounts significantly more than icotinib or endostatin as a monotherapy., Conclusions: Icotinib combined with bevacizumab or rh-endostatin has a stronger inhibitory effect on tumor growth than single-target drug in vivo, with no additional side effects., (© 2021 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Background: Wet age-related macular degeneration (wAMD) is characterized by the presence of choroidal neovascularization (CNV). Although there are some clinical drugs targeting vascular endothelial growth factor (VEGF) and inhibiting CNV, two major side effects limit their application, including the excessive activity of anti-VEGF and frequent intraocular injections. To explore better treatment strategies, researchers developed a hypoxic modulator retinal pigment epithelium (RPE)- specific adeno-associated virus (AAV) vector expressing endostatin to inhibit CNV. However, the mechanism of endostatin is complex. Instead, soluble fms-like tyrosine kinase-1 (sFlt-1) can inhibit VEGF-induced angiogenesis through two simple and clear mechanisms, giving rise to sequestration of VEGF and forming an inactive heterodimer with the membrane-spanning isoforms of the VEGF receptor Flt-1 and kinase insert domain-containing receptor., Objective: In this study, we chose sFlt-1 as a safer substitute to treat wAMD by inhibiting VEGFinduced angiogenesis., Methods: The AAV2/8-Y733F-REG-RPE-sFlt-1 vector was delivered by intravitreal injection to the eyes of mice. AAV2/8-Y733F vector is a mutant of the AAV2/8 vector, and the REG-RPE promoter is a hypoxia-regulated RPE-specific promoter. Two animal models were used to evaluate the function of the vector., Results: In the cobalt chloride-induced hypoxia model, the results demonstrated that the AAV2/8- Y733F-REG-RPE-sFlt-1 vector induced the expression of the sFlt-1 gene in RPE cells through hypoxia. In the laser-induced CNV model, the results demonstrated that the AAV2/8-Y733F-REG-RPE-sFlt- 1 vector reduced laser-induced CNV., Conclusion: Hypoxia regulated, RPE-specific AAV vector-mediated sFlt-1 gene is a hypoxiaregulated antiangiogenic vector for wAMD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Endostatin (ES) can effectively inhibit neovascularization in most solid tumors and has the potential to make oxygen delivery more efficient and increase the efficacy of radiotherapy (RT). With a short half-life, ES is mainly administered systemically, which leads to low intake in tumor tissue and often toxic systemic side effects. In this study, we used hyaluronic acid-tyramine as a carrier to synthesize an ES-loaded hydrogel drug (ES/HA-Tyr) that can be injected locally. ES/HA-Tyr has a longer half-life and fewer systemic toxic side effects, and it exerts a better anti-angiogenic effect and anti-tumor effect with RT. In vitro , ES/HA-Tyr showed sustained release in the release assay and a stronger ability to inhibit the proliferation of human umbilical vascular endothelial cells (HUVECs) in the MTT assay; it exhibited a more potent effect against HUVEC invasion and a stronger anti-angiogenic effect on HUVECs in tube formation. In vivo , ES/HA-Tyr increased local drug concentration, decreased blood drug concentration, and caused less systemic toxicity. Further, ES/HA-Tyr effectively reduced tumor microvessel density, increased tumor pericyte coverage, decreased tumor hypoxia, and increased RT response. ES/HA-Tyr + RT also had increased anti-tumor and anti-angiogenic effects in Lewis lung cancer (LLC) xenograft models. In conclusion, ES/HA-Tyr showed sustained release, lower systemic toxicity, and better anti-tumor effects than ES. In addition, ES/HA-Tyr + RT enhanced anti-angiogenic effects, reduced tumor hypoxia, and increased the efficacy of RT in LLC-bearing mice.

Fibrotic diseases pose significant clinical challenges due to their broadness and complexity. Thus, a better understanding of fibrogenesis and the development of more effective treatments is imperative. Recent evidence suggests a significant antifibrotic potential of an endogenous glycoprotein, endostatin. While endostatin has been widely studied for its role as an anticancer adjuvant by inhibiting tumor angiogenesis, its possible implication in fibrosis remains largely unclear. Here, we review the role of endostatin in various cellular processes and highlight its antifibrotic activity. We hypothesize that endostatin conveys a homeostatic function in the process of fibrosis by regulating (a) TGF-β1 and its downstream signaling; (b) RhoA/ROCK pathway; (c) NF-κB signaling pathway; (d) expression of EGR-1; (e) PDGF/PDGFR pathway; (f) autophagy-related pathways; (g) pathways associated with cell proliferation and apoptosis. Finally, we propose a schematic model of the antifibrotic roles and mechanisms of endostatin; also, we outline future research directions of endostatin and aim to present a potential therapeutic approach for fibrosis.

Background/aim: The early stage of atherosclerosis (AS) demonstrates a lipid-driven inflammatory cytokine increase. In the present study, we aimed to use ultrasound-targeted microbubble delivery (UTMD) therapy with the Endostar-loaded target microbubbles (MBs) to reduce AS-related inflammatory response., Materials and Methods: Normal and lipopolysaccharide (LPS) induced human umbilical vein endothelial cells (HUVECs) were placed in a parallel-plate flow chamber. MBs were perfused through the parallel-plate flow chamber to mimic physiological blood flow. Five groups were set up: G1: Negative control (normal HUVECs); G2: LPS control (LPS induced HUVECs); G3: ICAM-1-loaded-MBs (MBi); G4: Endostar-loaded-MBs (MBe) and G5: Endostar-ICAM-1-loaded-MBs (MBei). mRNA expression of inflammatory factors and release of inflammatory cytokines were detected by RT-PCR and ELISA, respectively., Results: After treatment with MBei, the mRNA expression of cell adhesion molecule-1 (CD31) (p=0.004), endothelin-1 (ET-1) (p=0.010), von willebrand factor (vWF) (p=0.018), extracellular regulated protein kinases (ERK) (p=0.046) and nuclear factor kappa B (NF-κB) (p=0.003) were significantly reduced compared to LPS-induced HUVECs. Release of inflammatory cytokines including tissue factor (TF) (p=0.033), tissue factor pathway inhibitor (TF-PI) (p=0.019), ET-1 (p=0.014), vWF (p=0.030) and blood-coagulation factor VIIα (FVIIα) (p=0.000) were also significantly reduced compared to LPS-induced HUVECs., Conclusion: UTMD therapy can inhibit the inflammatory response by reducing atherosclerotic-related inflammatory factors, suggesting a potential treatment at the early-stage of AS., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aims: Colon cancer remains a life-threating disease with increasing morbidity and mortality worldwide despite the advancement in modern medical treatment. Therefore, novel and effective anti-colon cancers drugs are urgently needed. In this study, we investigated the anti-metastatic property EnDuo, a modified version of Endostar, and the underlying mechanisms., Methods: Colon cancer cells were treated with different concentrations of EnDuo (50 μg/mL, 100 μg/mL, 200 μg/mL), and Endostar (100 μg/mL) as positive control. Cell Counting Kit-8 assay was performed to test the effect of EnDuo on cell viability. A scratch wound assay and transwell assay were employed to evaluate the relocation and motility of malignant colon cells following treatment with EnDuo. Western blot analysis was used to determine inhibitory effects of EnDuo by detecting the phosphorylation level of AKT and ERK proteins, and the expression of MMP-2 and MMP-9 proteins., Results: Our results showed that EnDuo impedes the migration of colon cancer cells in a dose-dependent manner. At the molecular level, EnDuo induced a significant reduction in the phosphorylation of AKT and ERK proteins, and inhibited the expression of MMP-2 and MMP-9 proteins., Conclusions: Collectively, these results demonstrate that EnDuo exhibits a comparable anti-metastatic effect by suppressing the migration of colon cancer cells. Possibly, EnDuo interrupts the PI3K/AKT/ERK signaling pathway to arrest cell migration. Our study provides a novel insight to the potential clinical applications of EnDuo against colon cancers in the future., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

For over a decade, diabetic neuropathy has exhibited great emergence in diabetic patients. Though there are numerous impediments in understanding the underlying pathology it is not that enough to conclude. Initially, there was no intricate protocol for diagnosis as its symptoms mimic most of the neurodegenerative disorders and demyelinating diseases. Continuous research on this, reveals many pathological correlates which are also detectable clinically. The most important pathologic manifestation is imbalanced angiogenesis/neo-vascularization. This review is completely focused on established pathogenesis and anti-angiogenic agents which are physiological signal molecules by the origin. Those agents can also be used externally to inhibit those pathogenic pathways. Pathologically DN demonstrates the misbalanced expression of many knotty factors like VEGF, FGF2, TGFb, NF-kb, TNF-a, MMP, TIMP, and many minor factors. Their pathway towards the incidence of DN is quite interrelated. Many anti-angiogenic agents inhibit neovascularization to many extents, but out of them predominantly inhibition of angiogenic activity is shared by endostatin which is now in clinical trial phase II. It inhibits almost all angiogenic factors and it is possible because they share interrelated pathogenesis towards imbalanced angiogenesis. Endostatin is a physiological signal molecule produced by the proteolytic cleavage of collagen XVIII. It has also a broad research profile in the field of medical research and further investigation can show promising therapeutic effects for benefit of mankind.

The movement of cell-bound membrane vesicles (CBMVs) on migrating cells is poorly understood. We hypothesized that the movement of CBMVs on migrating cells is different from that on non-migrating cells and can be interfered by external stimuli. To test it, single-vesicle tracking was performed to analyze motion type, speed, displacement, and direction of CBMVs on migrating cells treated with different reagents (Ang-1, TNF-α, LPS, VEGFα, endostatin, Cytochalasin D, and nocodazole) among which the former four promoted cell migration whereas the others inhibited cell migration. We found that cell migration changed CBMVs from non-directed to directed motion and that most CBMVs on untreated migrating cells moved along the migration axis. Interestingly, the migration-promoting reagents played positive roles in CBMV movement (improving directed motion, speed and/or maximal displacement, upregulating the amount of vesicles moving in migration direction) whereas the migration-inhibiting reagents played negative roles (impairing/abolishing directed motion, speed and/or maximal displacement, downregulating the vesicles moving forward or causing an even distribution of motion direction). The cytoskeleton (particularly microtubules) probably played vital roles in CBMV movement on migrating cells and mediated the effects of stimuli on vesicle movement. The data may provide important information for understanding the properties, behaviors, and functions of CBMVs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Background: Immunotherapy has been shown to be effective as a first-line treatment option for non-small cell lung cancer (NSCLC) patients. Unfortunately, it has failed to acquire an anticipant anti-tumour effect for relatively lower clinical benefit rates. It is therefore important to identify novel strategies for improving immunotherapy. Endostar is a novel recombinant human endostatin that exerts its anti-angiogenic effects via vascular endothelial growth factor (VEGF)-related signalling pathways. Anti-programmed death receptor 1 (PD-1) antibody is an immune checkpoint inhibitor that was developed to stimulate the immune system. In this study, the synergy of PD-1 blockade and endostar was assessed in a lung carcinoma mouse model., Methods: Lewis lung carcinoma (LLC)-bearing mice were randomly assigned into three groups: controls, anti-PD-1 and anti-PD-1+endostar. The levels of cytokines such as interleukin (IL)-17, transforming growth factor-β1 (TGF-β1) and interferon-γ (IFN-γ) were measured with enzyme-linked immune sorbent assay (ELISA). The expression of VEGF, CD34 and CD31 was assessed with immunohistochemistry (IHC). The proportion of mature dendritic cells (mDC) and myeloid-derived suppressor cells (MDSC) was analysed with flow cytometry. The major proteins in PI3K/AKT/mTOR and autophagy were quantified with Western blot., Results: Anti-PD-1 combined with endostar dramatically suppressed tumour growth in LLC mouse models. This synergistic effect resulted in decreased pro-inflammatory cytokine IL-17 and immunosuppressive factor TGF-β1 levels, increased IFN-γ secretion, reduced myeloid-derived suppressor cell (MDSC) accumulation, and reversed CD8 + T cell suppression. The expression of VEGF, CD34 and CD31 was significantly down-regulated, while tumour cell apoptosis and PI3K/AKT/mTOR-mediated autophagy was up-regulated., Conclusion: The combination of anti-PD-1 and endostar has a remarkably synergic effect on LLC tumour growth by means of improving the tumour microenvironment and activating autophagy., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019. Published by Elsevier Masson SAS.)

Endostar (ES) inhibits metastasis in some tumors, but its role in ovarian cancer invasion has not been elucidated. In this study, the effects of ES on ovarian cancer cells were further analyzed, to excavate an effective strategy for treating ovarian cancer. Ovarian cancer cell lines (SKOV3 and HO-8910PM) were treated with different concentrations of ES. Cell activity and half-maximal inhibitory concentration (IC50) detected by MTT were used for subsequent experiments. The migration and invasion abilities of treated cells were detected by wound healing and Transwell assays. The expressions of epithelial-mesenchymal transition (EMT)-related proteins in treated cells were determined by western blot analysis. Moreover, in vitro angiogenesis, the expressions of related proteins in treated cells and STAT3, and PD-L1 expressions were determined. We found that with the increase of ES concentrations, the cell activity showed a decreasing trend, and that the compositive IC50 of SKOV3 and HO-8910PM was 50 μg/ml. Moreover, ES observably inhibited migration, invasion, and EMT of ovarian cancer cell lines. In angiogenesis experiments, the angiogenesis ability and the expressions of related proteins in ovarian cancer cell lines were downregulated after ES treatment. Furthermore, ES reduced the expression of PD-L1 and suppressed the phosphorylation of STAT3 in ovarian cancer cell lines. ES blocked the metastasis, invasion, and angiogenesis of ovarian cancer cells by suppressing the activation of PD-L1 and STAT3, which might be considered as the potential mechanism of ES in the treatment of ovarian cancer.

Wnt/β-catenin signaling pathway modulates miscellaneous biological events in cells including gene expression, cell growth, apoptosis, metabolism and transition. The aim of this study was to investigate the effect of endostatin on Wnt signaling pathway of stem-like cells in bladder cancer in tumor microenvironment. The qRT-PCR assay and western blot were conducted to evaluate related factors expressions of Wnt signaling pathway in both bladder cancer 5637 cells and stem cells. Loss of function assays were carried out to detect the influence of endostatin on the proliferation, migration, cell proliferation and apoptosis of bladder cancer cells. We demonstrated that endostatin triggered the degradation of β-catenin, a key mediator of Wnt signaling. The activation of the endostatin blocked β-catenin function and inhibited cell growth and migration of bladder cancer. In order to verify that the Wnt/β-catenin signaling pathway was inhibited by endostain in 5637 bladder cancer cells and stem cells, the Wnt/β-catenin signaling pathway-associated molecules, including DKK1, LRP5, TCF4, β-catenin, cyclin D1, and c-Myc, were evaluated in 5637 bladder cancer cells and stem cells. The western blotting results showed that expressions of these molecules were remarkably increased in the 5637 bladder cancer cells and stem cells compared to the control group. In summary, our study demonstrated that endostatin inhibited angiogenesis. The downregulation of the Wnt/β-catenin pathway may be engaged in the suppression of angiogenesis by endostatin in bladder cancer cells and cancer stem cells.

In this study, we used endostatin (ES)-induced apoptosis of endothelial cells to study the role of Hexokinase2 (HK2) in the control of angiogenesis in melanoma. Real-time polymerase chain reaction and Western blot analysis were performed to explore the effect of HK2, lactate, and ES on the levels of caspase-9/3, ATP, and p38/MAPK activation. ES increased the levels of caspase-9/3 while decreasing the level of ATP, whereas ES + HK2 and lactate both restored the normal levels of caspase-9/3 and ATP. In addition, cells transfected with HK2 short hairpin RNA1 (HK2shRNA1) and HK2shRNA2 showed an evident decrease in the levels of caspase-9/3 along with an obvious increase in the level of ATP. Knockdown of HK2 also increased O 2 consumption while decreasing the extracellular level of lactate and the phosphorylation of p38-mitogen-activated protein kinase (MAPK). On the other hand, the lactate treatment elevated the phosphorylation of p38-MAPK under time- and concentration-dependent manner. In the study, we clarified the role of HK2 in the control of apoptosis of ECs, which plays an important role in the angiogenesis of melanoma by promoting aerobic glycolysis and activating the p38-MAPK signaling., (© 2019 Wiley Periodicals, Inc.)

Purpose: To explore the clinical efficacy of recombinant human endostatin combined with apatinib mesylate in patients with middle and advanced non-small cell lung cancer (NSCLC)., Methods: A total of 64 patients with middle and advanced NSCLC were randomly divided into the control group (n=32) and observation group (n=32). The patients in control group received paclitaxel monotherapy, while those in the observation group were treated with recombinant human endostatin combined with apatinib mesylate. The short-term efficacy, the lung function and levels of immunoglobulin and T lymphocyte subsets before and after treatment and the adverse drug reactions of patients were compared between the two groups. All patients were followed up for 5 years, and the survival rate in the two groups was observed., Results: The short-term efficacy and lung function in observation group were better than those in control group (p<0.05). Compared with those in the control group, the levels of immunoglobulin G (IgG), IgA, IgM, cluster of differentiation 3+ (CD3+), CD4+ and CD4+/CD8+ were increased, while the CD8+ level was lowered in the observation group (p<0.05). The rate of adverse drug reactions in the observation group was lower than that in the control group (p<0.05). The 5-year survival rate was significantly higher in the observation group than that in the control group (p<0.05)., Conclusion: Recombinant human endostatin combined with apatinib mesylate achieves a better therapeutic effect in the treatment of middle and advanced NSCLC, with improved immune resistance of patients and less side effects. Therefore, it is worthy of popularization and application in clinical practice.

Purpose: Local treatments for isolated synchronous or metachronous liver metastases in colorectal cancer (CRC) have been shown to improve overall survival (OS). The aim of this study was to investigate the factors affecting OS in CRC patients with isolated liver metastasis in whom the primary tumor and corresponding liver metastasis were treated with curative intent using local ablative or surgical methods., Methods: A total 47 surgical operated CRC patients presenting with an initial or subsequent isolated liver metastasis, who were treated with local surgical or ablative treatment for liver metastasis with curative intent, were enrolled in this study between 2007 and 2017. The possible factors affecting OS were analyzed., Results: Of the 47 patients, 35 (74.5%) were male. The median age was 61 (25 - 80) years. Thirty-four (72.3%) patients underwent liver metastasectomy, while 13 (27.7%) patients were treated with non-surgical local ablative therapies (NSLAT) for liver metastasis. Median OS (mOS) could not be reached in patients who underwent metastasectomy at the time of diagnosis compared to 55 months in those undergoing metastasectomy following a chemotherapy period (p = 0.03). Patients treated with NSLAT had a mOS of 60 months compared to ''not reached'' in those who underwent liver metastasectomy (p = 0.45). mOS was higher in patients with pT4 stage vs. with

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Aim: Though Endostar (ES) could inhibit tumor growth by inhibiting tumor angiogenesis, other possible mechanisms have been less reported. This study aims to investigate the role of ES in the treatment of lung cancer from the perspective of macrophage-mediated epithelial mesenchymal transformation (EMT)., Methods: THP1 cells were induced to polarized macrophages (MΦ). A549 and H1795 cells were separately treated with MΦ conditioned medium, ES (12.5 μg/ml) and HGF (5 ng/ml) for 24 h at 37 °C. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of CCL17, CD163, hepatocyte growth factor (HGF), Epidermal Growth Factor (EGF), transforming growth factor (TGF)-β1 and interleukin (IL)-6. Western blot was carried out to detect the p-MET, MET and EMT-related proteins (E-cadherin, N-cadherin, Snail and vimentin). Fibroblast-like A549 and H1975 cells were observed by a microscope. Cell invasion and migration were observed and analyzed by transwell and scratch assays., Results: The expression levels of CCL17 and CD163 were significant higher in MΦ. ES significantly inhibited the expression of HGF in MΦ. Moreover, ES could restore the abnormal expressions of EMT-related proteins and inhibit MΦ-induced and HGF-induced fibroblast-like lung cancer cells. Furthermore, ES suppressed the MΦ-induced and HGF-induced migration and invasion of lung cancer cells. ES was also found to down-regulate HGF-Met signaling in HGF-treated lung cancer cells., Conclusion: ES suppresses lung cancer progression by down-regulating HGF-Met signaling, revealing the possible mechanism of ES in the process of treating lung cancer patients., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Obstructive sleep apnea (OSA) is associated with cancer incidence and mortality. The underlying mechanism is unclear. This study aims to evaluate the influence of intermittent hypoxia (IH), a novel hallmark of OSA, on tumor and to access the anti-tumor effect of endostatin on a mouse model with OSA., Methods: The C57BL/6 J mice were randomly classified into four groups: control (normoxia) (CTL), control plus endostatin (CTL + ED), IH, and IH plus endostatin (IH + ED). Mice in IH and IH + ED groups were subjected to IH 8 h per day in 5 weeks. Lewis lung cancer cells were injected into the flank of each mouse after 1 week of IH exposure. Endostatin was also intraperitoneally injected after tumor volume reached about 200 mm 3 . The maximum standard uptake values (SUVmax) were detected by micro-positron emission tomography-computed tomography (micro-PET-CT) imaging prior and post-endostatin administration. Microvessel density (MVD) and vascular endothelial growth factor (VEGF) were determined for evaluating the anti-tumor effect of endostatin among the normoxia and IH conditions., Results: Mice had higher SUVmax in the IH group than the CTL group (p < 0.01). When compared with mice in the CTL group, those in the IH group had significantly greater MVD values (p < 0.001). The SUVmax can be attenuated by endostatin both in the CTL (p < 0.01) and IH conditions (p < 0.001). When compared with CTL group, mice in the IH group had increased MVD values (p < 0.001) and VEGF expression both at mRNA (p < 0.05) and protein levels (p < 0.001 in western blotting results). Treatment with endostatin attenuated serum and tissue VEGF levels, lowering the MVD values. As compared to normoxia condition, the endostatin-therapeutic effects were more significant under the IH condition (p < 0.05 in western blotting results)., Conclusions: Micro-PET-CT imaging is a promising non-invasive technique to evaluate the tumor metabolic characteristics under IH condition in vivo. The anti-tumor effect of endostatin under IH condition is superior to that of the normoxia condition.

Purpose: Ultrasound-targeted microbubble destruction (UTMD) has been reported to be a meritorious technique for drug targeting delivery. In this study, we aimed to evaluate the synergistic antiangiogenic effect of UTMD combined with Endostar on triple-negative breast carcinoma tumors., Materials and Methods: The lipid-shelled microbubbles (MBs) conjugated with Endostar were constructed using a biotin-avidin bridging chemistry method, and the morphological characteristics and drug-conjugating content were determined. MBs were administered intravenously to nude mice bearing MDA-MB-231 breast carcinoma xenografts and ultrasound exposure followed. The tumor microcirculation was observed by contrast-enhanced ultrasonography (CEUS) and the Endostar biodistribution was detected by enzyme-linked immunosorbent assay. Twenty-four breast carcinoma-bearing nude mice were divided into four groups. After treatment, every 3 days for 15 days the in vivo antitumor effects were assessed by calculating the tumor growth inhibition rate (TGIR). The tumor microcirculation was observed by CEUS, the tumor microvessel density (MVD) was calculated by immunohistochemistry under a microscope, and the vascular endothelial growth factor (VEGF) gene expression was detected by real-time quantitative polymerase chain reaction., Results: The prepared Endostar-conjugated MBs were round and well-dispersed with a mean size of 2.8 ± 0.7 µm and a drug conjugating content of 800.72 ± 70.53 µg/10 8  MBs. UTMD blocked the tumor microcirculation, and improved Endostar release in the targeted tumor tissue with a drug content of 1.12 ± 0.43 µg/gram protein, which was about three times higher than that in Endostar group or Endostar conjugated MBs group. Endostar-conjugated MBs combined with UTMD treatment achieved the optimal antitumor effects in vivo with a TGIR of 46.29%, and apparent antiangiogenic effects with minimal tumor blood perfusion, MVD and VEGF gene expression level., Conclusion: UTMD can improve Endostar delivery in the targeting tumor tissue and mediate synergistic antiangiogenetic and antitumor effects, which may be a potential therapeutic strategy for refractory breast cancer.

Endostar, a potent endogenous antiangiogenic factor, is wildly used in clinics. However, it was easily degraded by enzymes and rapidly cleared by the kidneys. To overcome these shortcomings, PEGylated recombinant human endostatin was developed. In this study, the purity of M 2 ES was evaluated by silver stain and reversed-phase high-performance liquid chromatography. Ultraviolet spectrum was used to examine the structural of M 2 ES and endostar. The bioactivity and antitumor efficacy of M 2 ES were evaluated using an in vitro endothelial cell migration model and athymic nude mouse xenograft model of a heterogeneous lung adenocarcinoma, respectively. A preclinical study was performed to evaluate the acute toxicity and safety pharmacology in rhesus monkeys. The purity of M 2 ES was more than 98%; PEG modification has no effect on endostatin structure. Compared with the control group, M 2 ES dramatically retards endothelial cell migration and tumor growth. After intravenous (IV) infusions of M 2 ES at a dose level of three and 75 mg/kg in rhesus monkeys, there was no observable serious adverse event in both acute toxicity and safety pharmacology study. On the basis of the quality and bioactivity study data of M 2 ES and the absence of serious side effect in rhesus monkeys, M 2 ES was authorized to initiate a phase I clinical trial., (© 2018 Wiley Periodicals, Inc.)

Endostatin (ES) has attracted considerable attention for the treatment of anti-angiogenesis-related disorders. An 11-amino-acid peptide (ES2, IVRRADRAAVP) from the amino terminal of ES is of interest because it is the main active fragment of ES. However, both ES and ES2 have a poor stability and a short half-life, and other disadvantages need to be further resolved. Thus, we conjugated ES2 to glycol-split heparin derivatives (GSHPs) to yield the polymer-peptide conjugate, GSHP-ES2. This study showed that GSHP-ES2 exhibited increased stability, a wider pH activity range, better inhibition of endothelial cell proliferation, migration and tube formation in vitro, better anti-angiogenic activity and a longer half-life in vivo compared with ES2. These results also indicate that GSHP-ES2 has good potential for the treatment of angiogenesis-related diseases, either alone or in combination with other chemicals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: To testify the hypothesis that endostatin exerts antifibrotic effects in hepatic stellate cells (HSCs) by modulating RhoA (ras homolog gene family, member A)/ROCK 1 (Rho-associated protein kinase 1) signal pathways., Materials and Methods: HSCs-T6 of passages 3-5 were cultured in DMEM and serum starved for 48 hours. HSCs were grouped as follows: control group, TGF-β1 (transforming growth factor β1) group, endostatin+TGF-β1 group, PDGF-BB (platelet-derived growth factor-BB) group, and endostatin+PDGF-BB group. In the PDGF-BB group, HSCs were treated with PDGF-BB (200 ng/mL) for 72 hours; in the TGF-β1 group, they were treated with TGF-β1 (10 ng/mL) for 72 hours. In the Endostatin+TGF-β1 group or Endostatin+PDGF-BB group, HSCs were treated with TGF-β1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours after pretreatment with endostatin (5 µg/mL) for 1 hour. In the control group, HSCs were only treated with serum-free DMEM for 72 hours. Collagen I was analyzed with ELISA. F-actin was detected with immunofluorescent staining. The mRNAs and proteins of α-smooth muscle actin, RhoA, and ROCK1 were analyzed by using real-time PCR and Western blot, respectively., Results: TGF-β1 and PDGF-BB promote the proliferation of HSCs significantly at 48 and 72 hours. Endostatin inhibits the proliferation effect induced by TGF-β1 or PDGF-BB significantly ( P <0.01). The expression of collagen I and F-actin was significantly upregulated in both TGF-β1 and PDGF-BB groups than in the control group ( P <0.01). Both the collagen I and F-actin expression were downregulated significantly in the endostatin-treated groups ( P <0.05). Endostatin significantly inhibited the upregulated expression of α-smooth muscle actin, RhoA, and ROCK1 induced by TGF-β1 or PDGF-BB ( P <0.01)., Conclusion: These results suggested that endostatin inhibited TGF-β1- or PDGF-BB-induced fibrosis in HSCs by modulating RhoA/ROCK signal pathways., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: Recombined humanized endostatin (Rh-endostatin) exhibits a potent anti-cancer effect involving multiple molecular targets and signaling pathways. HMGB1 is a highly conserved DNA-binding protein involved in cancer development. The therapeutic effect of Rh-endostatin on HMGB1 has not been reported, thus we investigate the effect in non-small cell lung cancer (NSCLC) cells., Methods: Quantitative real-time PCR and Western blot were used to analyze the messenger RNA and protein expression of HMGB1 in A549 cancer cells, while enzyme-linked immunosorbent assay was used to detect the release of HMGB1. Western blot was performed to evaluate HMGB1 expression in SK-MES-1 and H661 NSCLC cells., Results: Rh-endostatin inhibited the proliferation of A549 cancer cells and distinctly downregulated the expression and release of HMGB1 in dose and time dependent manners. Rh-endostatin-induced HMGB1 downregulation was confirmed in different types of NSCLC cells., Conclusion: These results demonstrate the general phenomenon that Rh-endostatin can induce HMGB1 suppression in a variety of NSCLC cells. Rh-endostatin may suppress HMGB1 expression and release in A549 cancer cells, thus inhibiting cell proliferation., (© 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Objective: To investigate the effect of endostatin on the proliferation, invasion and epithelial-mesenchymal transition (EMT) of human basal cell carcinoma (BCC) cells (A431)., Patients and Methods: CCK-8 assay and transwell chamber assay were performed to detect cell proliferation and invasion abilities, respectively. Western blot was performed for the detection of the expressions of EMT-related proteins levels. The therapeutic effect of endostatin on tumor formation was tested using a mouse xenograft model., Results: After endostatin treatment, transwell assay showed that the number of invasive cells in the observation group and control group were (38.25±8.13) and (98.25±9.14), respectively; the relative expression level of E-cadherin protein in the observation group was (0.34±0.03), which was significantly higher than that in the control group (0.14±0.01); the relative expression levels of N-cadherin protein in the observation group was (0.18±0.05), which was significantly lower than that in the control group (0.43±0.03), (all p<0.05)., Conclusions: The expression levels of Vimentin and Fibronectin proteins were significantly lower, while the expression levels of α-smooth muscle Actin (α-SMA) were significantly higher in the observation group than those in the control group. Treatment with endostatin significantly inhibited tumor growth in the mouse xenograft model. Therefore, endostatin can inhibit the proliferation, invasion and EMT in BCC.

In adult rodents, neuroblasts originating from the subventricular zone migrate tangentially through the rostral migratory stream (RMS) toward the olfactory bulb where they differentiate into interneurons. Neuroblasts in the RMS migrate in chains for a long distance along specifically arranged blood vessels which promote their migration. Although blood vessels in the neurogenic region of the forebrain are present early in development, their rearrangement into this specific pattern takes place during the first postnatal weeks. Here we examined the relevance of this rearrangement to the migration-guiding "scaffold" for the neurogenic processes in the RMS such as cell migration and proliferation. To disturb the reorganization of blood vessels, endostatin - an inhibitor of angiogenesis, was administered systemically to rat pups during the first postnatal week. Ten days or three months later, the arrangement of blood vessels, migration and proliferation of cells in the RMS were assessed. As we expected, the inhibition of angiogenesis disrupted rearrangement of blood vessels in the RMS. The rearrangement's failure resulted in a strong disruption of the mode and direction of neuroblast migration. Chain migration failed and neuroblasts migrated out of the RMS. The inhibition caused a slight increase in the number of proliferating cells in the RMS. The consequences were more obvious ten days after the inhibition of angiogenesis, although they persisted partly into adulthood. Altogether, here we show that the process of rearrangement of blood vessels in the RMS during the early postal period is crucial to ensure the regular course of postnatal neurogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Recombinant human endostatin (rh-endostatin) plus standard chemotherapy in advanced non-small cell lung cancer (NSCLC) patients has shown improved efficacy; however, it is unclear whether it is effective and safe when added to pemetrexed/cisplatin and used as maintenance therapy., Methods: We retrospectively evaluated the data of untreated NSCLC patients administered rh-endostatin plus pemetrexed/cisplatin or pemetrexed/cisplatin. The primary endpoint was progression-free survival (PFS)., Results: Fifty-six and 39 patients received rh-endostatin plus pemetrexed/cisplatin and pemetrexed/cisplatin, and 34 and 29 underwent maintenance treatment, respectively. The median PFS was 10 months (95% confidence interval [CI] 5.85-14.15) in the rh-endostatin and 8.2 months (4.04-12.36) in the chemotherapy group, but the difference was not statistically significant (P = 0.13). In patients administered maintenance treatment, rh-endostatin plus pemetrexed was associated with prolonged PFS compared to single-agent pemetrexed when PFS was calculated from first dosing (13.7 [9.41-17.99] vs. 8.2 [4.16-12.24]; P = 0.032); however, PFS did not differ between the groups (hazard ratio 0.618; 95% CI 0.368-1.038; P = 0.069) after adjusting for clinical factors. No difference was observed in the objective response rate between the groups (48.2% vs. 38.5%; P = 0.346), with the exception of men (62.1% vs. 33.3%; P = 0.032) or in the incidence of drug-related or grade 3-4 adverse events., Conclusion: In previously untreated, advanced-stage NSCLC patients, first-line treatment with pemetrexed/cisplatin plus rh-endostatin did not prolong PFS or overall survival when compared to pemetrexed/cisplatin, but a trend of improved PFS was observed in patients administered maintenance rh-endostatin plus pemetrexed., (© 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

The structure and function of a 27-a.a. fragment of the N-terminal sequence of human endostatin (ES-Zn) were compared to those of the mutant peptide (ES-SSZn) obtained by adding Cys-Pro-Ala to the endostatin N-terminus and substituting Asn16 for Cys ensuring formation of a disulfide bond. Structural comparison of ES-Zn and ES-SSZn by far-UV circular dichroism (CD), intrinsic fluorescence, and molecular dynamics simulation methods revealed significant structural perturbations in ES-SSZn, such as elimination of the β-sheet conformer, modification of the N-terminal loop structure, and reorganization of dynamic properties of the entire peptide backbone. ES-SSZn was approximately 2 and 3 times less efficient than ES-Zn and the full-length human endostatin, respectively, in the induction of caspase-3-dependent apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro (p < 0.05). In contrast, treatment of metastatic 4T1 breast tumors in mice with ES-Zn and ES-SSZn (5 mg/kg body weight daily) for 14 days resulted in similar regression of tumor size, comparable downregulation of angiogenesis (CD31 and CD34) and cell proliferation (Ki67), and therefore, the same extent of apoptosis induction (TUNEL, p53, and Bcl-2) for both peptides (as compared to the untreated controls). Western blot analysis of HUVEC and 4T1 tumor lysates revealed the same levels of suppression of key signaling mediators Akt and ERK1/2 by ES-Zn and ES-SSZn. Contrary to the earlier studies, our results showed that the function of the 1-27 endostatin fragment is independent of its overall structure. Stabilization of the N-terminal loop structure by the disulfide bond incorporation causes relief from structural deviations.

The poor prognosis in non-small-cell lung cancer has driven the development of novel targeted therapies. Vascular endothelial growth factor is the most potent force in mediating tumor angiogenesis, and many angiogenesis inhibitors have been developed for oncology treatment. We performed a study to characterize the efficacy, safety and tumor suppression of three lung cancer related anti-angiogenic drugs (bevacizumab, endostar and apatinib) using transgenic zebrafish embryo and human lung cancer xenotransplantation model. All three drugs demonstrated remarkable angiogenesis and tumor inhibition effect in the zebrafish model, within the nonlethal dose range. Endostar and bevacizumab showed competitive anti-tumor efficacy. The anti-tumor performance of apatinib was hamstrung by its elevated toxicity at 35 °C. The addition of pemetrexed to anti-angiogenesis therapy had no obvious additional benefit in tumors.

Valve stiffening is a hallmark of aortic valve stenosis caused by excess extracellular matrix accumulation by myofibroblasts. We aimed to elucidate whether matrix stiffness regulates endothelial-to-mesenchymal transition (EndMT) of adult valvular endothelial cells (VECs) to myofibroblasts as a mechanism to further promote valve fibrosis. In addition, we specifically examined the role of the Wnt/β-catenin signaling pathway in the development of myofibroblasts during EndMT, as Wnt/β-catenin signaling has been implicated in EndMT during heart development, is reactivated in valve disease, and is required for mechanically-regulated myofibrogenesis of valve interstitial cells. Clonally derived porcine VECs were cultured on soft (5 kPa) or stiff (50 kPa) silicone Sylgard 527 substrates and treated with transforming growth factor (TGF)-β1 to induce EndMT. Immunofluorescent staining revealed that TGF-β1 preferentially promoted EndMT in VECs on stiffer substrates, evidenced by a decrease in the endothelial marker VE-cadherin and an increase in the myofibroblast marker α-smooth muscle actin (α-SMA). These changes were accompanied by β-catenin nuclear localization both in vitro and in vivo, assessed by immunostaining. Degradation of β-catenin with endostatin reduced VEC myofibroblast transition, as indicated by decreased α-SMA fiber expression. We conclude that TGF-β1-induced EndMT in aortic VECs is dependent on matrix stiffness and Wnt/β-catenin signaling promotes myofibrogenesis during EndMT.

Pathological angiogenesis can be a significant barrier to effective cancer therapy. Recent evidence suggests that Endostar may induce vascular normalization, thereby improving tumor perfusion and systemic chemotherapy. However, the molecular mechanism by which Endostar makes chemotherapy more effective remains to be fully elucidated. In this study, established 4T1 breast tumor-bearing animals treated with Endostar were evaluated at serial time points for treatment-associated changes in vascular architecture. As a result, Endostar induced a morphologically and functionally normalized vascular network. Combined Endostar and doxorubicin exhibited significant antitumor (34% of control size) and antimetastatic effects (29% of control metastatic nodules) in vivo. Finally, a two-dimensional gel electrophoresis and MALDIQ-TOF MS/MS-based proteomics approach was used to identify differentially expressed proteins involved in vascular normalization during Endostar administration. SRCIN1 was detected as one of the most significantly increased proteins. SRCIN1 is a novel Src-binding protein that regulates Src activation through C-terminal Src kinase, and attenuated Src activation during Endostar treatment was further confirmed by immunoblotting. Collectively, these data provided a molecular basis for vascular normalization, which were associated with the observed synergistic effect in vivo.

Myofibroblasts contribute to the healing of infarcted areas after myocardial infarction through proliferation, migration, and production of extracellular matrix (ECM). Expression of endostatin, a cleaved fragment of type XVIII collagen, increases in the heart tissue of an experimental myocardial infarction model. In the present study, we examined the effect of endostatin on the function of myofibroblasts derived from an infarcted area. The myocardial infarction model was created by ligating the left anterior descending artery in rats. Two weeks after the operation, α-smooth muscle actin (α-SMA)-positive myofibroblasts were isolated from the infarcted area. Endostatin significantly increased the proliferation and migration of myofibroblasts in vitro. On the other hand, endostatin had no effect on the production of type I collagen, a major ECM protein produced by myofibroblasts. Endostatin activated Akt and extracellular signal-regulated kinase (ERK), and the pharmacological inhibition of these signaling pathways suppressed the endostatin-induced proliferation and migration. A knockdown of the COL18A1 gene in the myocardial infarction model rats using small interference RNA (siRNA) worsened the cardiac function concomitant with wall thinning and decreased the α-SMA-positive myofibroblasts and scar formation compared with that of control siRNA-injected rats. In summary, we demonstrated for the first time that endostatin might be an important factor in the healing process after myocardial infarction through the activation of myofibroblasts., Competing Interests: The authors declare no conflict of interest.

Aim: To evaluate the role of endostatin (ES) gene therapy on myeloid-derived suppressor cells (MDSC) in a metastatic model of renal cell carcinoma (RCC)., Materials and Methods: Balb/C mice bearing orthotopic Renca tumors were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. At the end of in vivo experiment, plasma and tissue lung samples were collected. Plasma ES and granulocyte colony stimulating factor (G-CSF) levels were measured by ELISA and Milliplex, respectively. Quantification of CD11b + Gr-1 + cells and their subsets was performed by flow cytometry. Reactive oxygen species (ROS) production was measured in CD11b + Gr-1 + MDSC using the DCFDA marker by flow cytometry., Results: Metastatic RCC (mRCC) induced expansions of CD11b + Gr-1 + MDSC and promoted accumulation of these cells and their subtypes in lymphoid organ and metastases. ES treatment promoted low G-CSF plasmatic levels which were produced by the tumor microenvironment, reflecting the reduced metastatic accumulation of CD11b + Gr-1 + MDSC in the lungs. However, the therapy was selective for granulocytic cells, thus reducing the production of ROS., Conclusion: These findings confirm the expansion of MDSC during metastatic progression of RCC and indicate the important role of ES in reducing MDSC and possible use of ES therapy in combined anticancer treatment.