Anti-inflammatory and Anti-thrombotic Efficacy of Targeted Ultrasound Microbubbles on LPS-induced HUVEC Cells.

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.

A Dual Receptor Targeting- and BBB Penetrating- Peptide Functionalized Polyethyleneimine Nanocomplex for Secretory Endostatin Gene Delivery to Malignant Glioma.

PURPOSE: Vascular endothelial growth factor receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1) are two prominent synergistic receptors overexpressed on new blood vessels in glioma and may be promising targets for antiglioma therapy. The aim of this study was to design a dual receptor targeting and blood-brain barrier (BBB) penetrating peptide-modified polyethyleneimine (PEI) nanocomplex that can efficiently deliver the angiogenesis-inhibiting secretory endostatin gene (pVAXI-En) to treat glioma. MATERIALS AND METHODS: We first constructed the tandem peptide TAT-AT7 by conjugating AT7 to TAT and evaluated its binding affinity to VEGFR-2 and NRP-1, vasculature-targeting ability and BBB crossing capacity. Then, TAT-AT7-modified PEI polymer (PPTA) was synthesized, and a pVAXI-En-loaded PPTA nanocomplex (PPTA/pVAXI-En) was prepared. The physicochemical properties, cytotoxicity, transfection efficiency, capacities to cross the BBB and BTB (blood-tumor barrier) and glioma-targeting properties of PPTA/pVAXI-En were investigated. Moreover, the in vivo anti-angiogenic behaviors and anti-glioma effects of PPTA/pVAXI-En were evaluated in nude mice. RESULTS: The binding affinity of TAT-AT7 to VEGFR-2 and NRP-1 was approximately 3 to 10 times greater than that of AT7 or TAT. The cellular uptake of TAT-AT7 in endothelial cells was 5-fold and 119-fold greater than that of TAT and AT7 alone, respectively. TAT-AT7 also displayed remarkable efficiency in penetrating the BBB and glioma tissue in vivo. PPTA/pVAXI-En exhibited lower cytotoxicity, stronger BBB and BTB traversing abilities, higher selective glioma targeting and better gene transfection efficiency than PEI/pVAXI-En. More importantly, PPTA/pVAXI-En significantly suppressed the tube formation and migration of endothelial cells, inhibited glioma growth, and reduced the microvasculature in orthotopic U87 glioma-bearing nude mice. CONCLUSION: Our study demonstrates that PPTA/pVAXI-En can be exploited as an efficient dual-targeting nanocomplex to cross the BBB and BTB, and hence it represents a feasible and promising nonviral gene delivery system for effective glioma therapy.

Folate-decorated, endostatin-loaded, nanoparticles for anti-proliferative chemotherapy in esophaegeal squamous cell carcinoma.

This study aims to design and synthesize Endostatin (ENT)-loaded nanoparticles using Folic acid (FA) as a driver for targeted anti-proliferative chemotherapy in Esophageal Squamous Cell Carcinoma (ESCC). An ionic gelation method was employed to formulate FA-decorated, ENT-loaded nanoparticles, which were tested in vitro on KYSE-30 cells using unbiased stereological approaches. FA-ENT nanoparticles were internalized into ESCC cells with preferential binging to the nucleus and mitochondria for necrotic and apoptotic effects. Nanoparticles showed increased proliferation inhibition of 64.71% and reduced KYSE-30 cell migration of up to 74.12% when compared to the control. Positively charged spherical nanoparticles, with selective pH responsive ENT release, were further tested in vivo employing a tumor xenograft model. Tumor mass increased up to 5505.54 mm3 in the control group while a substantial reduction occurred in the treatment group (native ENT, ENT-nano and FA-ENT-nano) down to 128.23 mm3 (97.67%). Tumor volume was reduced from 1000.2 mm3 to 567.64 mm3 (43.25%) in the native ENT group, from 324.43 mm3 to 190.25 mm3 (41.36%) in ENT-nano group (non-targeted system), and from 1374.21 mm3 to 998.67 mm3 (27.33%) in FA-ENT-nano group (targeted system) following treatment. There were no significant differences in the body weight of mice treated with the nano-formulations as opposed to the control mice. FA-decorated nanoparticles for active transport of ENT into tumor cells with an enhanced in vitro and in vivo anti-proliferative efficacy in ESCC therapy were synthesized.

Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide.

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 ß-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.

A LC-MS/MS method to monitor the concentration of HYD-PEP06, a RGD-modified Endostar mimetic peptide in rat blood.

HYD-PEP06 is a novel RGD-modified Endostar mimetic peptide with 30 amino acids that is intended to suppress the formation of neoplasm vessels. This assay was developed and validated to monitor the level of the peptide HYD-PEP06 in rat blood, using liquid chromatography tandem mass spectrometry (LC-MS/MS). HYD-PEP10, another peptide similar to the analyte, was used as an internal standard (IS). A triple quadrupole mass spectrometry in Multiple Reaction Monitoring (MRM) mode and an electrospray interface (ESI) in the positive mode were used for MS analysis. The analysis was optimized with addition of 0.3% formic acid (FA) into the mobile phase as well as with a needle washing solution to overcome the carryover effect. In addition, the carryover was reduced by optimizing the mobile phase gradient. Methanol was used as a diluent of working solutions to avoid any adsorption. Methanol:acetonitrile (1:1, v:v) containing 0.3% FA was employed to precipitate the blood samples. Unknown blood samples must be placed in ice bath immediately, and precipitating agents should be added within 30 min to ensure the stability of blood samples. The assay was established and validated. This method showed a good linear relationship for the HYD-PEP06 in the range of 10 ng·mL-1 to 2000 ng·mL-1, with R > 0.99. HYD-PEP06 was determined with accuracy values (RE%) of -5.06%-8.54%, intra- and inter-day precisions (RSD%) of 3.13%-4.87% and 4.81%-9.42%. The method was successfully in monitoring the concentration of HYD-PEP06 in rat blood.

A pre-clinical safety study of PEGylated recombinant human endostatin (M2ES) in Sprague Dawley rats.

PEGylated recombinant human endostatin (M2ES) exhibited prolonged serum half-life and enhanced antitumor activity when compared with endostatin. A pre-clinical study was performed to evaluate the safety of M2ES in rats. After intravenous (IV) infusions of M2ES at a dose level of 3, 15 and 75 mg/kg in Sprague Dawley (SD) rats, M2ES was well tolerated in animals, with no observable changes in clinical observation, body weight, food consumption, urine analysis, hematology and serum biochemical analysis. The increase of kidney weights, and slight to severe vacuolation and necrosis of proximal tubule epithelial cells in kidney were observed in 15 and 75 mg/kg M2ES groups, but this adverse-effect was reversible. In summary, the major toxicity target organ of M2ES might be kidney, and the no observed adverse effect level (NOAEL) of M2ES in rats was 3 mg/kg in this study. These pre-clinical safety data contribute to the initiation of the ongoing clinical study.

Higher Anti-angiogenesis Activity, Better Cellular Uptake and Longer Half-life of a Novel Glyco-modified Endostatin by Polysulfated Heparin.

BACKGROUND: Endostatin (ES) is a promising anti-angiogenesis protein and has been approved for the treatment of non-small cell lung cancer, but short half-life, poor stability and nonspecific delivery caused great pain to patients and produced unsatisfactory treatment effectiveness. OBJECTIVE: In this work, in order to overcome these disadvantages, ES was covalently modified by polysulfated heparin (PSH) with the expectancy of longer half-life, higher anti-angiogenesis activity and better cellular uptake. METHODS: To characterize the cellular uptake, flow cytometry and confocal laser scanning microscopy were used to study the intracellular localization of fluorescein isothiocyanate-labeled ES and PSH-ES in EAhy926 endothelial cells. Zebrafish model was used to study the anti-angiogenesis activities of ES and its derivatives in vivo. The 125I-radiolabeled ES and PSH-ES were administered to healthy BALC/c mice for the pharmacokinetics study. RESULTS: Compared with ES, better cellular uptake effects were detected in PSH-ES group. Both ES and PSH-ES showed inhibition on the intersegmental vessels formation, while PSH-ES displayed a higher one. The half-life of PSH-ES was lengthened and area under the curve (AUC) was increased. At the same time, ES and PSH-ES were both widely and rapidly distributed in the lungs, livers, kidneys and hearts with little difference. CONCLUSION: The results indicated that PSH displayed good properties as a novel glyco-modifier for protein and peptide. The results also showed that PSH-ES displayed better cellular uptake, higher antiangiogenesis activity and prolonged half-life, which would lead to better anti-tumour effects.

Antitumor activities of Liver-targeting peptide modified Recombinant human Endostatin in BALB/c-nu mice with Hepatocellular carcinoma.

In our previous study, a liver-targeting peptide CSP I-plus modified recombinant human Endostatin (rEndostatin, endostar) (rES-CSP) was constructed and showed potent antiangiogenic capability and could specifically bind to human hepatocellular carcinoma cells to make a direct inhibition in vitro. In this study, the biological activities of rES-CSP in vivo were evaluated by subcutaneous and orthotopic xenograft nude mice model of human hepatocellular carcinoma cells HepG2. We found that rES-CSP significantly decreased tumor volume to 54.9% in the nude mice with subcutaneous xenograft compared with the control. In orthotopic xenograft model, rES-CSP not only decreased tumor volume (to 39.6% compared with the control) and tumor weight, it also increased its biodistribution in the liver tissue and hepatoma tissue. Moreover, lower microvessel density (MVD) and higher apoptotic index (AI) were also observed in the tumor tissues. It had no significant side-effects on the heart, liver, spleen, lung and kidney of mice. Results indicated CSP I-plus modified Endostar may be a potential candidate for a targeting therapy on hepatocellular carcinoma.

Optimization of high-concentration endostatin formulation: Harmonization of excipients' contributions on colloidal and conformational stabilities.

Recently, increasing research efforts have been devoted into developing high-concentration protein drugs for subcutaneous injection, especially for those with short half-lives and high-dose requirement. Proteins at high concentrations normally present increased colloidal and structural instability, such as aggregation, fibrillation and gelation, which significantly challenges the high-concentration formulation development of protein drugs. Here we used endostatin, a 20kD recombinant protein, as a model drug for high-concentration formulation optimization. The colloidal and conformational stability of endostatin at high concentration of 30mg/mL were investigated in formulations containing various excipients, including saccharides (mannitol, sorbitol and sucrose), salts (ArgHCl and NaCl), and surfactants (tween 20 and 80). Protein fibrillation was characterized and semi-quantified by optical polarized light microscopy and transmission electron microscopy, and the amount of fiber formation at elevated temperature of 40°C was determined. The soluble protein aggregates were characterized by dynamic and static light scattering before and after dilution. The conformational stability were characterized by polyacrylamide gel electrophoresis, fluorescence, circular dichroism, and differential scanning calorimetry. We observed that the soluble aggregation, fibrillation and gelation, induced by conformational and colloidal instabilities of the protein solution, could be substantially optimized by using suitable stabilizers such as combinations of saccharides and surfactants; while formation of gel and soluble aggregates at high protein concentration (e.g., 30mg/mL) and elevated temperature (40°C) could be prevented by avoiding the usage of salts. It's worth emphasizing that some stabilizers, such as salts and surfactants, could show opposite contributions in conformational and colloidal stabilities of endostatin. Therefore, cautions are needed when one attempts to correlate the colloidal stability of high-concentration proteins with their conformational stability, and the colloidal and conformational protein stabilities must be harmonized by a balanced selection of various types of excipients.

Conjugation of gold nanoparticles and recombinant human endostatin modulates vascular normalization via interruption of anterior gradient 2-mediated angiogenesis.

Several studies have revealed the potential of normalizing tumor vessels in anti-angiogenic treatment. Recombinant human endostatin is an anti-angiogenic agent which has been applied in clinical tumor treatment. Our previous research indicated that gold nanoparticles could be a nanoparticle carrier for recombinant human endostatin delivery. The recombinant human endostatin-gold nanoparticle conjugates normalized vessels, which improved chemotherapy. However, the mechanism of recombinant human endostatin-gold nanoparticle-induced vascular normalization has not been explored. Anterior gradient 2 has been reported to be over-expressed in many malignant tumors and involved in tumor angiogenesis. To date, the precise efficacy of recombinant human endostatin-gold nanoparticles on anterior gradient 2-mediated angiogenesis or anterior gradient 2-related signaling cohort remained unknown. In this study, we aimed to explore whether recombinant human endostatin-gold nanoparticles could normalize vessels in metastatic colorectal cancer xenografts, and we further elucidated whether recombinant human endostatin-gold nanoparticles could interrupt anterior gradient 2-induced angiogenesis. In vivo, it was indicated that recombinant human endostatin-gold nanoparticles increased pericyte expression while inhibit vascular endothelial growth factor receptor 2 and anterior gradient 2 expression in metastatic colorectal cancer xenografts. In vitro, we uncovered that recombinant human endostatin-gold nanoparticles reduced cell migration and tube formation induced by anterior gradient 2 in human umbilical vein endothelial cells. Treatment with recombinant human endostatin-gold nanoparticles attenuated anterior gradient 2-mediated activation of MMP2, cMyc, VE-cadherin, phosphorylation of p38, and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human umbilical vein endothelial cells. Our findings demonstrated recombinant human endostatin-gold nanoparticles might normalize vessels by interfering anterior gradient 2-mediated angiogenesis in metastatic colorectal cancer.

Controlled release of an endostatin peptide using chitosan nanoparticles.

Whereas several anticancer peptides are in different stages of clinical development, their administration is limited by the fast elimination from the systemic circulation. Peptide loading on nano-carriers can pave the way for their future application. We have recently indicated that a disulfide loop rather than a Zn-binding loop improves the anti-angiogenic and antitumor activities of the N-terminal fragment of endostatin. In this study, chitosan nanoparticles (CS NPs) are used for the controlled release of the engineered peptide. Loading of the peptide into CS NPs using the ionic gelation method was confirmed by FTIR and resulted in final particle size, poly-dispersity index and surface charge of 186.5 ± 24.0 nm, 0.26 ± 0.02 and 20.1 ± 0.4 mV respectively. The SEM morphological analysis revealed spherical particles with an average size of 80 ± 5 nm. Peptide loading studies revealed that CS NPs are able to adsorb the peptide as ~70%. The release measurements indicated an initial burst release by 49% after 2 hr and complete release after 80 hr. According to in vitro studies, the loaded peptide was much more toxic for endothelial cells than different cancer cell lines. These results underscore the promise of CS NPs as therapeutics nanosystems and open a perspective for improving the clinical applications of peptide drugs.

Preparation of endostatin-loaded chitosan nanoparticles and evaluation of the antitumor effect of such nanoparticles on the Lewis lung cancer model.

The purpose of this study was to prepare ES-loaded chitosan nanoparticles (ES-NPs) and evaluate the antitumor effect of these particles on the Lewis lung cancer model. ES-NPs were prepared by a simple ionic cross-linking method. The characterization of the ES-NPs, including size distribution, zeta potential, loading efficiency and encapsulation efficiency (EE), was performed. An in vitro release test was also used to determine the release behavior of the ES-NPs. Cell viability and cell migration were assayed to detect the in vitro antiangiogenic effect of ES-NPs. In order to clarify the antitumor effect of ES-NPs in vivo, the Lewis lung cancer model was used. ES-NPs were successfully synthesized and shown to have a suitable size distribution and high EE. The nanoparticles were spherical and homogeneous in shape and exhibited an ideal releasing profile in vitro. Moreover, ES-NPs significantly inhibited the proliferation and migration of human umbilical vascular endothelial cells (HUVECs). The in vivo antiangiogenic activity was evaluated by ELISA and immunohistochemistry analyses, which revealed that ES-NPs had a stronger antiangiogenic effect for reinforced anticancer activity. Indeed, even the treatment cycle in which ES-NPs were injected every seven days, showed stronger antitumor effect than the free ES injected for 14 consecutive days. Our study confirmed that the CS nanoparticle is a feasible carrier for endostatin to be used in the treatment of lung cancer.

Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.

The antiangiogenic and antitumor activities of the N-terminal fragment of endostatin augmented by Ile/Arg substitution: The overall structure implicated the biological activity.

The antiangiogenic and antitumor activities of the 27-amino acid fragment corresponding to the N-terminal domain of endostatin were shown to be dependent on a Zn-binding loop in the N-terminus. To investigate whether the regions outside of the N-terminal loop play a role in the peptide function, the structure and function of a variant containing Ile26Arg mutation (ES-R) were compared with those of the native peptide (ES-Zn). Structural analysis using far-UV CD, intrinsic fluorescence and molecular dynamics simulation provided information regarding the overall changes upon the mutation. In addition, the docking simulations predicted a higher affinity of ES-R to integrins αvß3 and α5ß1 than ES-Zn and a profound reorganization of the binding residues throughout the sequence. In Human Umbilical Vein Endothelial Cells (HUVECs), ES-R inhibited the tube formation and activated caspase-3 more strongly than do ES-Zn. Based on in vivo studies, the growth of breast tumor and expression of CD31, Bcl-2 and nonfunctional p53 were inhibited more effectively by ES-R than by ES-Zn. We conclude that the C-terminal region is involved in the peptide function through some global structural effects.

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

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

Infra Red Dye and Endostar Loaded Poly Lactic Acid Nano Particles as a Novel Theranostic Nanomedicine for Breast Cancer.

Endostar, a novel recombinant human endostatin, has been proven to inhibit tumor angiogenesis and is utilized as an anticancer drug. While free drugs can display limited efficacy, nanoscaled anticancer drugs have been fabricated and proven to possess superior therapeutic effects. Poly(lactic acid) (PLA) is a FDA-approved biomaterial displaying excellent biocompatibility and low toxicity. In this study, Endostar-loaded PLA nanoparticles (EPNPs) were first prepared, and a near-infrared (NIR) dye, IRDye 800CW, was conjugated to the surface for detecting nanoparticle biodistribution through fluorescence molecular imaging (FMI) using an orthotopic breast tumor mouse model. The antitumor efficacy of EPNPs was examined using bioluminescence imaging (BLI) and immunohistology. To further improve the antitumor effects, we combined EPNPs with zoledronic acid monohydrate (ZA), which is known to decrease the tumor-associated macrophages (TAM) and inhibit tumor progression. We found that EPNPs decreased human umbilical vein endothelial cell (HUVEC) viability by inhibiting tumor growth gene expression more significantly than free Endostar in vitro. In vivo, EPNPs displayed better tumor growth inhibitory effects compared with free Endostar, and the combination of EPNPs with ZA exhibited more significant antitumor effects. As confirmed by CD31 and CD11b immunohistochemistry, the combination of EPNPs and ZA showed synergistic effects in reducing tumor angiogenesis and TAM accumulation in tumor regions. Taken together, this study presents a novel and effective form of nanoscaled Endostar for the treatment of breast cancer that displays synergistic antitumor effects in combination with ZA.

Comparisons of biophysical properties and bioactivities of mono-PEGylated endostatin and an endostatin analog.

BACKGROUND: Endostatin (ES) is a well-established potent endogenous antiangiogenic factor. An ES variant, called zinc-binding protein-ES (ZBP-ES), is clinically available; however, its use is limited by rapid renal clearance and short residence time. PEGylation has been exploited to overcome these shortcomings, and mono-PEGylated ES (called M2ES) as well as mono-PEGylated ZBP-ES (MZBP-ES) are developed in our study. This study aimed to compare the biophysical properties and biological effects of M2ES and MZBP-ES to evaluate their druggability. METHODS: Circular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M2ES and MZBP-ES. Their resistance to trypsin digestion and guanidinium chloride (GdmCl)-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence, respectively. The biological effects of M2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays, and the uptake of M2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence. RESULTS: Structural analyses revealed that M2ES has a more compact tertiary structure than MZBP-ES. Moreover, M2ES was more resistant to trypsin digestion and GdmCl-induced unfolding compared with MZBP-ES. In addition, although M2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells, M2ES displayed an increased ability to enter cells compared with MZBP-ES, possibly caused by the enhanced interaction with nucleolin. CONCLUSIONS: M2ES has a more compact tertiary structure, is more stable for trypsin digestion and GdmCl-induced unfolding, exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES, indicating that M2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.

Selective Binding of Endostatin Peptide 4 to Recombinant VEGF Receptor 3 In Vitro.

We previously reported that neostatin, a proteolytic fragment of collagen XVIII that includes endostatin, inhibits basic fibroblast growth factor-induced corneal angiogenesis and lymphangiogenesis. In experiments to determine which fragments in neostatin are responsible for binding to VEGF receptors (VEGFRs), we previously showed that a 28- mer sequence at the C-terminal of endostatin, known as endostatin peptide 9, preferentially binds VEGFR3-Fc over VEGFR1-Fc and VEGFR2-Fc. In the present study, we show that a different endostatin fragment, endostatin peptide 4 (26 mers long), also selectively binds VEGFR3-Fc and not VEGFR1-Fc or VEGFR2-Fc. From surface plasmon resonance data, the KD and Chi² (RU²) values for endostatin peptide 4 binding to VEGFR3-Fc are 5.72 × 10⁻8 M and 0.354, respectively. In conclusion, endostatin peptides 4 and 9 may be responsible for endostatin binding to VEGFR3-Fc, and this improved understanding of endostatin peptide binding to VEGFR3-Fc may support the development of therapeutics targeting lymphangiogenic processes.

Possible role of endostatin in the antiangiogenic therapy of diabetic retinopathy.

Diabetic retinopathy is one of various complications of diabetes mellitus, which is one of the most prevalent chronic disorders in the modern world. Diabetic retinopathy is one of the secondary complications encountered by the patients suffering from chronic diabetes mellitus. Two major characterizing features of diabetic retinopathy are - macular edema and angiogenesis. It has been noted in the past few years that by controlling or completely inhibiting the factors contributing to the progression of events leading to angiogenesis, there is a noticeable amount of progress seen in the prevention and cure of the animal models of diabetic retinopathy. Endostatin is one such antiangiogenic agent being studied at present. It is a carbon terminal protein fragment obtained after cleavage from the carbon terminus of collagen XVIII. It is one of the most potent inhibitors of angiogenesis known at present and is currently undergoing clinical trials. Although the exact mechanism of action of endostatin is not completely known, various factors which are altered/influenced by the action of endostatin are being studied. These include the downregulation and activation/inactivation of various factors which have been proven to have some role in the progression of angiogenesis. Endostatin could be well exploited as a durable agent in the antiangiogenic therapy, once the clinical trials show positive results.

Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative, anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights.

Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and ß structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to αVß3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth.