Migration Of HUVEC Cells Research Articles

A VEGFR targeting peptide-drug conjugate (PDC) suppresses tumor angiogenesis in a TACE model for hepatocellular carcinoma therapy

Transcatheter arterial chemoembolization (TACE) has become the preferred therapy for unresectable advanced hepatocellular carcinoma (HCC). However, the embolization of tumor-feeding arteries by TACE always leads to hypoxia-related tumor angiogenesis, which limited the therapeutic effect for HCC. In this paper, we used a VEGFR targeting peptide VEGF125 − 136 (QKRKRKKSRYKS) to conjugate with a lytic peptide (KLUKLUKKLUKLUK) to form a peptide-drug conjugate (PDC). We used cell affinity assay to detect the peptide binding ability to VEGFR highly expressed cell lines, and CCK8, cell apoptosis to confirm the cellular toxicity for different cell lines. Meanwhile, we created a VX2 tumor-bearing rabbit model to assess the in vivo anti-tumor effect of the peptide conjugate in combination with TAE. HE staining was used to verify the in vivo safety of the peptide conjugate. IHC was used to assess the anti-angiogenesis and cell toxicity of the peptide conjugate in tumor tissues. The peptide conjugate could not only target VEGFR in cell surface and inhibit VEGFR function, but also have potent anti-cancer effect. We luckily found the peptide conjugate showed potent cytotoxicity for liver cancer cell Huh7 (IC50 7.3 ± 0.74 μM) and endothelial cell HUVEC (IC50 10.7 ± 0.292 μM) and induced cell apoptosis of these two cell lines. We also found the peptide conjugate inhibited cell migration of HUVEC through wound healing assay. Besides, these peptides also showed better in vivo anti-tumor effect than traditional drug DOX through TACE in VX2 rabbit tumor model, and efficiently inhibit angiogenesis in tumor tissues with good safety. In conclusion, our work may provide an alternative option for clinical HCC therapy via TACE combination.

A high-hydrophilic Cu2O-TiO2/Ti2O3/TiO coating on Ti-5Cu alloy: Perfect antibacterial property and rapid endothelialization potential

In order to make novel antibacterial Ti-Cu alloy more suitable for cardiovascular implant application, a Cu-containing oxide coating was manufactured on Ti-Cu alloy by plasma-enhanced oxidation deposition in plasma enhanced chemical vapor deposition (PECVD) equipment to further improve the antibacterial ability and the surface bioactivity. The results of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and water contact angle indicated that a sustainably high-hydrophilic Cu2O-TiO2/Ti2O3/TiO coating with nano-morphology on Ti-5Cu was successfully constructed. The corrosion performance results showed that the coating enhanced the corrosion resistance while releasing more Cu2+, compared with Ti-5Cu. Antibacterial tests confirmed the perfect antibacterial property of the coating (R≥99.9%), superior to Ti-Cu alloy (R>90%). More delightfully, it was observed by phalloidin-FITC and DAPI staining that the coating improved the early adhesion of HUVEC cells mainly due to strong hydrophilicity and nano-morphology. It was demonstrated that the extract of the coated sample significantly promoted proliferation (RGR=112%-138% after cultivation for 1 to 3days) and migration of HUVEC cells due to the appropriate Cu2+ release concentration. Hemolysis assay and platelet adhesion results showed that the coating had excellent blood compatibility. All results suggested that the coating on Ti-Cu alloy might be a promising surface with the perfect antibacterial ability, blood compatibility and evident promoting endothelialization ability for the cardiovascular application.

Chitosan/PLGA shell nanoparticles as Tylotoin delivery platform for advanced wound healing

Wound treatment remains one of the most prevalent healthcare issues. Tylotoin is a skin repair peptide identified from salamander (Tylototriton verrucosus) and exhibits skin wound healing properties. Noticeably, the easy degradation and frequent administration limit its application in wound healing. Chitosan (CS) -PLGA-Tylotoin nanoparticles (CPT NPs) were prepared to circumvent this limitation and deliver Tylotoin for the promotion of the healing of skin wounds. Results showed that optimized CPT NPs particle size, zeta potential, encapsulation efficiency and drug loading were 297.80 ± 5.37 nm, 20.37 ± 0.83 mV, 81.00 % and 1.74 %, respectively. In vitro, CPT NPs exhibited good antibacterial properties and biocompatibility and persistently promoted the cell migration of HaCaT cells and HUVECs due to the long-term sustained release of Tylotoin within 14 days (64.81 %). In vivo, the scarless healing of skin wound promotion was evaluated in mouse back full-thickness wound models. We demonstrated that mouse back full-thickness wounds topically treated with CPT NPs once every two weeks exhibited better scarless healing than those treated with Tylotoin once daily. We envision that CPT NPs, as a Tylotoin delivery platform might, may be potentially utilized to in skin wounds healing in clinics in the future.

A PD-L1 and VEGFR2 dual targeted peptide and its combination with irradiation for cancer immunotherapy

Although the blockade of immune checkpoint PD-1/PD-L1 has achieved great success, the lack of tumor-infiltrating immune cells and PD-L1 expression in the tumor microenvironment results in a limited response in certain tumor types. Thus, rational and optimal combination strategies were urgently needed. The combination of PD-1/PD-L1 blockade and anti-angiogenic therapy has been reported to have great potential. Here, a chimeric peptide OGS was designed by conjugating the peptides OPBP-1 (8−12) and DA7R targeting PD-L1 and VEGFR2, respectively. OGS could bind to both human and mouse PD-L1 with high affinity and block the PD-1/PD-L1 interaction, and also inhibit the migration and tube formation of HUVEC cells in wound healing and tube formation assays. To further prolong the half-life of OGS, it was modified by coupling with peptide DSP which has a high binding affinity to both human serum albumin (HSA) and mouse serum albumin (MSA) to form the peptide DSPOGS. DSPOGS could not directly affect the viability, apoptosis, and cell cycle of tumor cells in vitro, while significantly inhibiting the tumor growth in the MC38 mouse model. DSPOGS could elicit a potent anti-tumor immune response and inhibit tumor angiogenesis, with the enhancement of tumor infiltrating CD8+ T cells and the IFN-γ secreting CD8+ T cells in the spleen and tumor-draining lymph node. Further, the combination of radiotherapy with DSPOGS could dramatically improve the therapeutic efficacy. Our study could provide a promising paradigm for the combination of immune checkpoint blockade, anti-angiogenesis, and radiotherapy.

Role of GDF15/MAPK14 Axis in Chondrocyte Senescence as a Novel Senomorphic Agent in Osteoarthritis.

Osteoarthritis (OA) is most prevalent in older individuals and exerts a heavy social and economic burden. However, an effective and noninvasive approach to OA treatment is currently not available. Chondrocyte senescence has recently been proposed as a key pathogenic mechanism in the etiology of OA. Furthermore, senescent chondrocytes (SnCCs) can release various proinflammatory cytokines, proteolytic enzymes, and other substances known as the senescence-associated secretory phenotype (SASP), allowing them to connect with surrounding cells and induce senesce. Studies have shown that the pharmacological elimination of SnCCs slows the progression of OA and promotes regeneration. Growth differentiation factor 15 (GDF15), a member of the tumor growth factor (TGF) superfamily, has recently been identified as a possible aging biomarker and has been linked to a variety of clinical conditions, including coronary artery disease, diabetes, and multiple cancer types. Thus, we obtained data from a publicly available single-cell sequencing RNA database and observed that GDF15, a critical protein in cellular senescence, is highly expressed in early OA. In addition, GDF15 is implicated in the senescence and modulation of MAPK14 in OA. Tissue and synovial fluid samples obtained from OA patients showed overexpression of GDF15. Next, we treated C20A4 cell lines with interleukin (IL)-1β with or without shGDF15 then removed the conditioned medium, and cultured C20A4 and HUVEC cell lines with the aforementioned media. We observed that C20A4 cells treated with IL-1β exhibited increased GDF15 secretion and that chondrocytes cultured with media derived from IL-1β–treated C20A4 exhibited senescence. HUVEC cell migration and tube formation were enhanced after culturing with IL-1β-treated chondrocyte media; however, decreased HUVEC cell migration and tube formation were noted in HUVEC cells cultured with GDF15-loss media. We tested the potential of inhibiting GDF15 by using a GDF15 neutralizing antibody, GDF15-nAb. GDF15-nAb exerted a similar effect, resulting in the molecular silencing of GDF15 in vivo and in vitro. Our results reveal that GDF15 is a driver of SnCCs and can contribute to OA progression by inducing angiogenesis.

Tanshinone IIA improves cardiac function via regulating miR-499-5p dependent angiogenesis in myocardial ischemic mice

Background/aimsMyocardial ischemia-reperfusion injury leads to aggravated cardiac remodeling and heart failure. After myocardial infarction (MI), angiogenesis plays a vital role in the repair and regeneration of tissue. The purpose of the current study was to determine the effect of Tanshinone IIA (Tan IIA) on angiogenesis and elucidate its related mechanism. MethodsThe C57BL/6 mice MI model was established to evaluate the therapeutic effect of Tan IIA in vivo. MicroRNA (miRNA) microarray and bioinformatics analysis were performed to determine the differential expressions of miRNAs after Tan IIA administration. Cell proliferation, migration, and angiogenesis capacity were detected by EdU, Transwell, and Tube formation assay in vitro, respectively. The relationship between miR-499-5p (miR-499) and paired phosphate and tension homolog deleted on chromosome ten (PTEN) was confirmed by using a Dual-luciferase reporter assay. ResultsOur results showed that Tan IIA administration improved cardiac function after MI by activating angiogenesis. Further miRNA microarray and bioinformatics analysis revealed that miR-499 was significantly down-regulated, while PTEN was remarkably upregulated after Tan IIA administration post-MI. In addition, we found that miR-499 knock-down effectively promotes cell proliferation, migration, and tube formation ability of HUVECs. Dual-luciferase reporter assay demonstrated that PTEN contains a direct binding site for miR-499-5p. ConclusionTan IIA improves cardiac function post-MI by inducing angiogenesis. In terms of the mechanism, Tan IIA promotes therapeutic angiogenesis by regulating miR-499-5p/PTEN signaling pathway.

Tumor-Derived Exosome FGD5-AS1 Promotes Angiogenesis, Vascular Permeability, and Metastasis in Thyroid Cancer by Targeting the miR-6838-5p/VAV2 Axis.

Exosomes are small vesicles with a diameter of 30~150 nm secreted by cells, which are rich in mRNA, microRNA, and long noncoding RNA (lncRNA). The biological functions of most exosomal lncRNAs are not well understood. Studies have shown that tumor exosome FGD5-AS1 plays an important role in the proliferation, migration, and invasion of tumor cells. In this study, SW1736 and KAT18 TC cells with high expression of FGD5-AS1 were screened. Exosomes with high expression of FGD5-AS1 were collected. The collected exosomes were then added to HUVEC cells. After incubation for 24 h, the effects on the proliferation and migration of HUVEC cells and vascular permeability were detected. The results showed that TC cells SW1736 and KAT18 could secrete a large number of exosomes, which could be taken up by HUVEC cells. Overexpression of FGD5-AS1 enhanced proliferation, migration, angiogenesis, and permeability of HUVEC. This effect is achieved through activation of the miR-6838-5p/VAV2 axis. These results suggest that FGD5-AS1 in tumor-derived exoskeleton promotes angiogenesis, vascular permeability, and metastasis by regulating the endothelial miR-6838-5p/VAV2 axis and ultimately promotes the occurrence and development of TC.

Improvement of polydopamine-loaded salidroside on osseointegration of titanium implants

BackgroundMicroarc oxidation (MAO) on the surface of medical pure titanium can improve its histocompatibility, and loading drugs on the surface can resist excessive intimal hyperplasia.MethodsIn this study, salidroside (SAL) was loaded on the surface of porous titanium (Ti) with polydopamine (PDA) carrier. The effects of SAL on the osteogenesis and angiogenesis of Ti implants were studied by phalloidin staining, alizarin red staining, ALP staining, wound-healing assay, cell transwell assay, matrigel tube formation, and osteogenic and angiogenic genes and proteins expression detected by PCR and western blot in vitro. The bone defect model experiments in rats was established in vivo including X-ray, micro CT, hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Goldner's trichrome analysis, Safranin O-fast green staining and determination of contents of TNF-α and IL-6 in serum.ResultsEDS and EDS mapping showed that SAL could be loaded on the surface of the MAO coating by PDA. A drug release experiment showed that SAL loaded on the Ti coating could release slowly and stably without sudden release risk. In vitro cell experiments showed that the SAL coating could promote the proliferation, morphology, calcification and alkaline phosphate activity of MC3T3-E1 cells. At the same time, it promoted the migration and tube formation of HUVEC cells. The SAL coating promoted osteogenesis and angiogenesis by promoting the expression of genes and proteins related to. In vivo experiments, HE and IHC showed that SAL significantly promoted the expression of COL-1 and CD31. Goldner's trichrome and Safranin O-fast green staining showed that SAL coating could increase the new bone tissue around the implantation site. The SAL coating had anti-inflammatory activity by reducing the levels of TNF-α and IL-6 in vivo.ConclusionTherefore, SAL could improve osteogenesis and angiogenesis in conjunction with the Ti-PDA coating.

VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types.

Lower limb ischemia caused by diabetic foot (DF) is one of the most serious complications of diabetes. The therapeutic role of VEGF in DF is well documented. However, the mechanism for action of VEGF is still not clear. The present study aimed to explore the effects of VEGF-mediated skeletal muscle fiber type switch in angiogenesis and the treatment of DF. C57BL/6 mice housed in cages equipped with a voluntary running wheel were used to access VEGF protein level and citrate synthase activity (by ELISA) as well as muscle fiber type changes (by immunofluorescence) in the gastrocnemius muscle. C57BL/6 mice were fed on a high-fat diet for 6 weeks and then injected with streptozocin to induce diabetic lower limb ischemia model. Control adenovirus (Ad-GFP) or Ad-VEGF-GFP were then injected into the left gastrocnemius of the ischemic diabetic mice. Blood flow perfusion was examined by laser Doppler imaging at 1, 3, 7 and 14 days after adenovirus transduction. On day 14, all mice were anesthetized and sacrificed. VEGF expression levels, citrate synthase activity and muscle fiber type changes in the gastrocnemius muscle were assayed by ELISA and immunofluorescence analysis of myosin heavy chain IIa (MHCIIa) expression, respectively. Transwell assays were performed to determine whether VEGF-treated C2C12 myotubes played a role on tubule formation and migration of HUVECs. It was found that VEGF levels and citrate synthase activity were upregulated after voluntary exercise, along with the increased frequency of oxidized muscle fibers. Notably, adenovirus-mediated VEGF overexpression in the muscle also increased the frequency of oxidized (MHCIIa-positive) muscle fibers, enhanced citrate synthase activity and ameliorated lower limb ischemia in diabetic mice. VEGF treatment enhanced the phosphorylation of PI3K, Akt and AMPK (assayed by western blotting), as well as glucose consumption and metabolism (assayed by western blotting and glucose uptake assay), in the C2C12 myotubes. Interestingly, VEGF-treated C2C12 myotubes promoted the migration and tubule formation of HUVEC cells. The present findings suggest that skeletal muscle fiber conversion might be a potential approach for VEGF-mediated angiogenesis and disease treatment, which may provide new options for the prevention and treatment of DF.

Development of quercetin-loaded PVCL–PVA–PEG micelles and application in inhibiting tumor angiogenesis through the PI3K/Akt/VEGF pathway

Quercetin (Que) exhibits excellent biological activity; however, its clinical development is hindered owing to the poor water solubility. In this study, Que. was loaded on polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PVCL–PVA–PEG, Soluplus) micelles through a thin-film hydration process, and their tumor angiogenesis inhibition ability was investigated. The particle size of Soluplus-Que micelles was 55.3 ± 1.8 nm, and the micelles stayed stability within 9 months. Soluplus-Que micelles can enhance the cell uptake of Que. and transport the micelles to intracellular lysosomes and mitochondria. The MTT assay results revealed that Soluplus-Que micelles enhanced the cytotoxicity of Que. on HUVEC cells. Furthermore, Soluplus-Que micelles inhibited migration and invasion of HUVEC cells, as well as inhibited the neovascularization of chick embryo allantoic membrane (CAM). The in vivo study revealed that Soluplus-Que micelles significantly inhibit the growth of H22 solid tumors, with low toxic side effects. Soluplus-Que inhibited the expression of CD31 (a marker of angiogenesis) and the PI3K/Akt/VEGF pathway in tumor tissues, indicating its potential to hold back tumor growth via the inhibition of angiogenesis. Our findings indicated that as a delivery system, Soluplus micelles demonstrate potential for the delivery of poorly soluble drugs for tumor treatment.

RhoJ facilitates angiogenesis in glioblastoma via JNK/VEGFR2 mediated activation of PAK and ERK signaling pathways.

Glioblastoma (GBM) is a highly vascularized malignant tumor that depends on new blood vessel formation. Small molecules targeting the angiogenic process may be an effective anti-GBM therapeutic strategy. We previously demonstrated that RhoJ promoted the progression and invasion of GBM. RhoJ has also been shown to be expressed in endothelial cells and plays an important role in regulating endothelial cell migration and tumor angiogenesis. Therefore, we aimed to evaluate the role and mechanism of actions of RhoJ in GBM angiogenesis. We analyzed the expression of RhoJ in different grade gliomas and investigated its role in GBM angiogenesis in vivo and in vitro. Furtherly, RNA sequencing (RNA-seq), Western blotting and immunofluorescence were performed to identify the molecular mechanism of RhoJ in regulating endothelial cell behavior and GBM angiogenesis. Here, we found that silencing RhoJ resulted in inhibition of HUVEC cell migration and blood vessel formation. Overexpression of RhoJ promoted the expression of CD31, EpCAM and moesin, suggesting RhoJ facilitated angiogenesis and the malignant progression of GBM. RNA-seq data showed that VEGF/TNF signaling pathway positively regulated RhoJ. The expression levels of RhoJ was upregulated with the stimulation of VEGF, and reduced by the treatment of JNK inhibitor SP600125. It was also found that the activity of PAK-BRAF-ERK was down-regulated upon RhoJ and JNK knockdown. In conclusion, these results suggested that RhoJ plays an essential role in regulating GBM angiogenesis through the JNK/VEGFR2-PAK-ERK signaling pathway and there might exist a VEGF-JNK/ERK-VEGF circuitry. Thus, RhoJ may be a candidate therapeutic target for anti-angiogenesis treatment in GBM.

Characterization and diabetic wound healing benefits of protein-polysaccharide complexes isolated from an animal ethno-medicine Periplaneta americana L.

Periplaneta americana L. (PA), a type of animal medicine, has been widely used for wound healing in clinical settings. In order to further investigate the bioactive wound healing substances in PA, crude PA protein-polysaccharide complexes were further purified by cellulose DE-52 and Sephadex G100 chromatography in succession. Among these isolated fractions, two fractions eluted by 0.3 M and 0.5 M NaCl with the higher yield, respectively named PaPPc2 and PaPPc3 respectively, were chosen for the wound healing experiments. Mediated by HPGPC, amino acid and monosaccharide composition analysis, circular dichroism spectrum, glycosylation type, FT-IR, and 1H NMR analysis, the characterization of PaPPc2 and PaPPc3 was implemented. And then, the benefits of PaPPcs to promote cell proliferation, migration, and tube formation of HUVECs were determined in vitro, indicated these fractions would facilitate angiogenesis. Finally, as proof of concept, PaPPc2 and PaPPc3 were employed to accelerate the acute wounds of diabetic mice, involving in increase blood vessels and the amounts of angiogenesis-related cytokines (α-SMA, VEGF, and CD31). In short, this study provides an experimental basis to demonstrate the protein-polysaccharide complexes of Periplaneta americana L. as its wound healing bioactive substances.

Metformin enhances the osteogenesis and angiogenesis of human umbilical cord mesenchymal stem cells for tissue regeneration engineering.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a potential clinical material in regenerative medicine applications. Metformin has shown safety and effectiveness as a clinical drug. However, the effect of metformin as a treatment on hUC-MSCs is unclear. Our research aimed to explore the effects of metformin on the osteogenesis, adipogenesis and angiogenesis of hUC-MSCs, and attempted to explain the molecular fluctuations of metformin through the mapping of protein profiles. Proliferation assay, osteogenic and adipogenic differentiation induction, cell cycle, flow cytometry, quantitative proteomics techniques and bioinformatics analysis were used to detect the influences of metformin treatment on hUC-MSCs. Our results demonstrated that low concentrations of metformin promoted the proliferation of hUC-MSCs, but high concentrations of metformin inhibited it. Metformin exhibited promotion of osteogenesis but inhibition of adipogenesis. Metformin treated hUC-MSCs up-regulated the expression of osteogenic marker ALP, OCN and RUNX2, but down-regulated the expression of adipogenic markers PPARγ and LPL. Proteomics analysis found that up-regulation of differentially expressed proteins in metformin treatment group involved the biological process of cell migration in Gene Ontology analysis. Metformin enhanced cell migration of HUVEC in a co-culture system, and hUC-MSCs treated with metformin exhibited stronger angiogenesis in vitro and in vivo compared to the hUC-MSCs group. The results of RT-qPCR revealed that the SCF and VEGFR2 were raised in metformin treatment. This study can promote the application of hUC-MSCs treated with metformin to tissue engineering for vascular reconstruction and angiogenesis.

Bioinformatics and Network Pharmacology Identify the Therapeutic Role and Potential Mechanism of Melatonin in AD and Rosacea.

Rosacea is significantly associated with dementia, particularly Alzheimer’s disease (AD). However, the common underlying molecular mechanism connecting these two diseases remains limited. This study aimed to reveal the common molecular regulatory networks and identify the potential therapeutic drugs for rosacea and AD. There were 747 overlapped DEGs (ol-DEGs) that were detected in AD and rosacea, enriched in inflammation-, metabolism-, and apoptosis-related pathways. Using the TF regulatory network analysis, 37 common TFs and target genes were identified as hub genes. They were used to predict the therapeutic drugs for rosacea and AD using the DGIdb/CMap database. Among the 113 predicted drugs, melatonin (MLT) was co-associated with both RORA and IFN-γ in AD and rosacea. Subsequently, network pharmacology analysis identified 19 pharmacological targets of MLT and demonstrated that MLT could help in treating AD/rosacea partly by modulating inflammatory and vascular signaling pathways. Finally, we verified the therapeutic role and mechanism of MLT on rosacea in vivo and in vitro. We found that MLT treatment significantly improved rosacea-like skin lesion by reducing keratinocyte-mediated inflammatory cytokine secretion and repressing the migration of HUVEC cells. In conclusion, this study contributes to common pathologies shared by rosacea and AD and identified MLT as an effective treatment strategy for rosacea and AD via regulating inflammation and angiogenesis.

Abstract 10791: Regeneration-Associated Cells Derived Exosomes Beneficial Than Mesenchymal Stromal Cells Derived Exosome in the Context of Myocardial Ischemia-Injury

Introduction: Vasculogenic conditioning of pro-inflammatory cells such as macrophages type 1, T cells, and primitive EPCs shifted their phenotype to pro-regenerative cells such as vasculogenic EPCs, M2 macrophages, and reg T cells, also known as regeneration-associated cells (RACs). Here, we investigated therapeutic efficacy of RAC-derived extracellular vesicles (RACev) in comparison with MSC-derived EVs (MSCev) in the context of myocardial ischemia-reperfusion injury (IRI). Methods: RACev and MSCev were isolated via ultracentrifugation. EVs quantity and quality were characterized by nanotracking analysis and CD9, CD63, Alix expression. EVs miR was sequenced and regenerative responsible miRs were validated using TargetScan and miRBase. Allogeneic immune response to the RACev and MSCev were evaluated. The function of RACev and MSCev were evaluated using HUVECs proliferation/cell-cycle assays in vitro, and repetitive (30min, d1, and d3 after IRI) systemic infusion with either RACev or MSCev in a myocardial IRI model. Results: Nanotracking analysis showed significant differences in quantity RACev vs. MSC (P= 0.03). In vitro , RACev markedly enhanced cell viability, proliferation, and migration of HUVECs in a dose-dependent manner compared to MSCev. Systemic injection of RACs (5x10 5 ) derived EVs improved cardiac functions such as ejection fraction (P= 0.005) than MSCev treated group. In histology, RACev transplanted group showed less interstitial fibrosis and enhanced capillary densities (P =0.05) compared to MSCev. These beneficial effects are coupled with significant expression of angiogenesis, anti-inflammation, cardiomyogenesis, and anti-fibrosis-related miRs in RACev but not in MSCev. In vivo bioluminescence and labeled EVs histology tracking analyses depicted preferential accumulation of RACev into the IRI myocardium (P=0.01) than MSCev. Immune phenotyping analysis confirmed similar immunomodulatory effects of MSCev and RACev. Conclusion: Repetitive systemic transplantation of RACev is superior to MSCev in terms of cardiac function enhancements via crucial angiogenesis, anti-fibrosis, anti-inflammation, cardiomyogenesis, and anti-apoptosis-related miRs delivery to the ischemic tissue.

Debio-0932, a second generation oral Hsp90 inhibitor, induces apoptosis in MCF-7 and MDA-MB-231 cell lines

Heat shock protein 90 (Hsp90) is a key chaperone that is abnormally expressed in cancer cells, and therefore, designing novel compounds to inhibit chaperone activities of the Hsp90 is a promising therapeutic approach for cancer drug discovery. Debio-0932 is a second-generation Hsp90 inhibitor that exhibited promising anticancer activity against a wide variety of cancer types with a strong binding affinity for Hsp90 and high oral bioavailability. Anticancer activities of the Debio-0932 were tested in MCF-7 and MDA-MB-231 cell lines. Molecular docking results indicated that Debio-0932 was selectively bound to the ATP binding pocket of the Hsp90 with an estimated free energy of binding - 7.24 kcal/mol. Antiproliferative activity of Debio-0932 was determined by XTT assay and Debio-0932 exhibited a cytotoxic effect on MCF-7 and MDA-MB-231 cells in a time and dose-depended manner. Apoptosis inducer role of Debio-0932 was evaluated in MCF-7 and MDA-MB-231 cells with fluorometric apoptosis/necrosis detection kit. Treatment with Debio-0932 stimulated apoptosis in both breast cancer cell lines. mRNA and protein expression levels of Bax, Bcl-2 and Casp-9 were determined in MCF-7 and MDA-MB-231 cells by RT-PCR and Western blotting respectively. Debio-0932 stimulated the down-regulation of anti-apoptotic protein Bcl-2 and the up-regulation of apoptotic protein Bax and cleavage of Casp-9 in cancer cells. Moreover, the anti-invasive potential of Debio-0932 was evaluated in endothelial cells (HUVEC) by wound-healing assay. Debio-0932 decreased the migration of HUVEC cells as compared to the control group. These results indicate that Debio-0932 is a promising compound to treat triple-negative breast cancer and hormone receptor-positive breast cancer, and their metastases.

CXCL1 stimulates decidual angiogenesis via the VEGF-A pathway during the first trimester of pregnancy.

Angiogenesis is critical to establishing a successful pregnancy. Thechemokine (C-X-C motif) ligand 1(CXCL1) is a smallcytokinebelonging to the CXCchemokine family that is an important chemokine involved in the processes of angiogenesis andarteriogenesis; however, little is known about its role in decidual angiogenesis. Effects of CXCL1 on cell proliferation and migration (propidium iodide staining and wound healing assays) of HUVEC cells were determined. The angiogenesis roles of CXCL1 in HUVEC-HTR8/SVneo co-culture system were detected by the tube formation assay. Signal transduction pathways in HUVEC cells in response to CXCL1 were determined by in-cell western analyses. In vivo, mice were injected with (1) PBS (Group A) or (2) CXCL1-neutralizing antibody (Group B) or (3) CXCL1-neutralizing antibody plus recombinant VEGF-A protein (Group C) from E1 to E5 and sacrificed at E6.5 of pregnancy. The decidual angiogenesis in mice was examined by immunohistochemistry of cluster designation 34 (CD34), and the expression levels of vascular endothelial growth factor-A (VEGF-A) in the decidual cells and vascular endothelial growth factor receptor 2 (VEGFR2) in decidual vascular endothelial cells were also tested. Exogenous recombinant human CXCL1 supported endothelial cell proliferation and migration, and this effect was blocked by CXCL1-neutralizing antibody or CXCR2 inhibitor SB265610. The tube formation of HUVEC-HTR8/SVneo co-culture system was significantly stimulated by CXCL1, but this effect was markedly abrogated once they were pretreated with CXCL1-neutralizing antibody or CXCR2 inhibitor SB265610. In addition, the level of vascular endothelial growth factor A (VEGF-A) expression in HUVEC cells was increased by CXCL1, and this level was suppressed by CXCL1-neutralizing antibody or CXCR2 inhibitor SB265610. In vivo, compared with Group A (n = 3), decidual angiogenesis was significantly reduced in Group B by CD34 immunostaining. But compared with Group B, decidual angiogenesis was significantly increased in Group C. In addition, the expression of VEGF-A and VEGFR2 was significantly increased after neutralizing of CXCL1 in Group B. In conclusions, CXCL1 may play essential roles in decidual angiogenesis during the first trimester, and this function may be mediated in part via altering VEGF-A expression.

The mechanism of lncRNA‐CRNDE in regulating tumour‐associated macrophage M2 polarization and promoting tumour angiogenesis

M2 macrophages can promote liver cancer metastasis by promoting tumour angiogenesis; however, the mechanism underlying macrophage polarization has not been completely revealed. In this study, we mainly explored the mechanism underlying long non‐coding RNA‐CRNDE (lncRNA‐CRNDE) in regulating M2 macrophage polarization and promoting liver cancer angiogenesis. The expression of CRNDE was up‐regulated or down‐regulated in THP‐1 cells (CRNDE‐/‐‐THP‐1 cells and pcDNA3.1‐CRNDE‐THP‐1). THP‐1 cells were co‐cultured with liver cancer cell line H22, and M2 polarization was induced in THP‐1 by IL‐4/13 to simulate tumour‐induced macrophage polarization. As a result, after CRNDE overexpression, THP‐1 cell viability was up‐regulated, the expression of M2 membrane marker CD163 was up‐regulated, and the proportion of F4/80 + CD163+ cells was also up‐regulated. ELISA assay showed that the expression of M2 markers (including TGF‐β1 and IL‐10) and chemokines (including CCl22 and CCL22) was up‐regulated, and the expression of key signals (including STAT6, JAK‐1, p‐AKT1, and Arg‐1) was also up‐regulated, which were significantly different compared with the control group (Con). In addition, the intervention effect of CRNDE on THP‐1 was consistent between co‐culture with H22 cells and IL‐4/13 induction assay. The induced M2 THP‐1 cells were co‐cultured with HUVEC. As a result, THP‐1 cells with CRNDE overexpression can promote the migration and angiogenesis of HUVEC cells in vitro and simultaneously up‐regulate the expression of Notch1, Dll4 and VEGFR2, indicating that THP‐1 M2 polarization induced by CRNDE could further promote angiogenesis. The H22 cell tumour‐bearing mouse model was constructed, followed by injection of CRNDE anti‐oligosense nucleotides and overexpression plasmids to interfere CRNDE expression in tumour‐bearing tissues. Consequently, down‐regulation of CRNDE could down‐regulate tumour volume, simultaneously down‐regulate the expression of CD163 and CD31 in tissues, decrease the expression of key proteins (including JAK‐1, STAT‐6, p‐STAT6 and p‐AKT1), and down‐regulate the expression of key angiogenesis‐related proteins (including VEGF, Notch1, Dll4 and VEGFR2). In this study, we found that CENDE could indirectly regulate tumour angiogenesis by promoting M2 polarization of macrophages, which is also one of the mechanisms of microenvironmental immune regulation in liver cancer.

A novel bioengineered fragment peptide of Vasostatin-1 exerts smooth muscle pharmacological activities and anti-angiogenic effects via blocking VEGFR signalling pathway

Chromogranin A (CgA) is a hydrophilic glycoprotein released by post-ganglionic sympathetic neurons. CgA consists of a single peptide chain containing numerous paired basic residues, which are typical cleavage sites in prohormones to generate bioactive peptides. It is recognized as a diagnostic and prognostic serum marker for neuroendocrine tumours. Vasostatin-1 is one of the most conserved regions of CgA and has diverse inhibitory biological activities. In this study, a novel peptide fragment that contains three typical functional structures of Vasostatin-1 was synthesized. This unique bioengineered Vasostatin-1 Derived Peptide (named V1DP) includes a highly conserved domain between vertebrate species in its N-terminal region, comprising a disulphide bridge formed by two cysteine residues at amino acid positions 17 and 38, respectively. Besides, V1DP contains two significant tripeptide recognition sequences: the amino acid triplets, RGD and KGD. Our data demonstrated that V1DP could induce a dose-dependent relaxation of rat arterial smooth muscle and also increase the contraction activity of rat uterus smooth muscle. More importantly, we found that V1DP inhibits cancer cell proliferation, modulate the HUVEC cell migration, and exhibit anti-angiogenesis effect both in vitro and in vivo. We further investigated the actual mechanism of V1DP, and our results confirmed that V1DP involves inhibiting the vascular endothelial growth factor receptor (VEGFR) signalling. We docked V1DP to the apo structures of VEGFR2 and examined the stability of the peptide in the protein pockets. Our simulation and free energy calculations results indicated that V1DP can bind to the catalytic domain and regulatory domain pockets, depending on whether the conformational state of the protein is JM-in or JM-out. Taken together, our data suggested that V1DP plays a role as the regulator of endothelial cell function and smooth muscle pharmacological homeostasis. V1DP is a water-soluble and biologically stable peptide and could further develop as an anti-angiogenic drug for cancer treatment.

Acteoside as a potential therapeutic option for primary hepatocellular carcinoma: a preclinical study

BackgroundHepatocellular carcinoma (HCC) is a common malignant tumor with characteristics of poor prognosis, high morbidity and mortality worldwide. In particular, only a few systemic treatment options are available for advanced HCC patients, and include sorafenib and the recently described atezolizumab plus bevacizumab regimen as possible first-line treatments. We here propose acteoside, a phenylethanoid glycoside widely distributed in many medicinal plants as a potential candidate against advanced HCC.MethodsCell proliferation, colony formation and migration were analyzed in the three human HCC cell lines BEL7404, HLF and JHH-7. Angiogenesis assay was performed using HUVESs. The BEL7404 or JHH-7 xenograft nude mice model was established to analyze the possible antitumor effects of acteoside. qRT-PCR and western blotting were used to reveal the potential antitumor mechanisms of acteoside.ResultsActeoside inhibited cell proliferation, colony formation and migration in all the three human HCC cell lines BEL7404, HLF and JHH-7. The prohibition of angiogenesis by acteoside was revealed by the inhibition of tube formation and cell migration of HUVECs. The combination of acteoside and sorafenib produced stronger inhibition of cell colony formation and migration of the HCC cells as well as of angiogenesis of HUVECs. The in vivo antitumor efficacy of acteoside was further demonstrated in BEL7404 or JHH-7 xenograft nude mice model, with an enhancement when combined with sorafenib in inhibiting the growth of JHH-7 xenograft. Further treatment of JHH-7 cells with acteoside revealed an increase in the level of tumor suppressor protein p53 as well as a decrease of kallikrein-related peptidase (KLK1, 2, 4, 9 and 10) gene level with no significant changes of the rest of KLK1–15 genes.ConclusionsActeoside exerts an antitumor effect possibly through its up-regulation of p53 levels as well as inhibition of KLK expression and angiogenesis. Acteoside could be useful as an adjunct in the treatment of advanced HCC in the clinic.