Endothelial Human Umbilical Vein Research Articles

Multicompartment duct platform to study epithelial-endothelial crosstalk associated with lung adenocarcinoma.

Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRASG12D mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRASG12D isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular in vitro platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma.

Tumor vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Suppresses macrophage capping protein beyond STING activation

The vascular disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) induces apoptosis in vascular endothelial cells and leads to tumor hemorrhagic necrosis. While DMXAA has been proven to be a potent agonist of murine stimulator of interferon genes (mSTING), it has little effect on human-STING (hSTING). This species selectivity of DMXAA may explain its effectiveness against solid tumors in mice and its failure in clinical trials. However, DMXAA did reduce tumor volume in some patients during clinical trials. These paradoxical results have prompted us to investigate the anti-tumor mechanism of DMXAA beyond STING in the destruction of tumor vasculature in humans. In this study, we demonstrated that DMXAA binds to both human and mouse macrophage capping protein (CapG), with a KD of 5.839 μM for hCapG and a KD of 2.867 μM for mCapG, as determined by surface plasmon resonance (SPR) analysis. Homology modeling and molecular docking analysis of hCapG indicated that the critical residues involved in the hydrogen bond interaction of DMXAA with hCapG were Arg153, Thr151, and GLN141, Asn234. In addition, electrostatic pi-cation interaction occurred between DMXAA and hCapG. Further functional studies revealed that CapG protein plays a crucial role in the effects of DMXAA on human umbilical endothelial vein cell (HUEVC) angiogenesis and migration, as well as the expression of cytoskeletal proteins actin and tubulin, and the invasion of A549 lung adenocarcinoma cells. Our study has originally uncovered a novel cross-species pathway underlying the antitumor vascular disruption of DMXAA extends beyond STING activation. This finding deepens our understanding of the multifaceted actions of flavonoid VDAs in animal models and in clinical settings, and may provide insights for the precise therapy of DMXAA based on the biomarker CapG protein.

Abstract 7298: A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels

Abstract Introduction: Extra virgin olive oil is a basic component of the Mediterranean diet; it contains several molecules with antioxidant, anti-inflammatory, anti-angiogenic and pro-apoptotic properties against a variety of cancers and influencing cellular metabolism too. Olive mill waste water (OMWW), a liquid residue produced in vast amounts during olive oil extraction, represents an environmental issue but on the other side it is a rich source of phytochemicals with potential health benefits. Particularly, OMWWs are rich in different phenolic compounds, that show promise in preventing the development of several disease disorders, including cancer. We have assessed the anti-angiogenic and anti-tumor properties exerted by a nutraceutical from purified OMWW extract, in in vitro and in vivo models, and evaluated its effect in a human pilot study upon informed consent. Experimental procedures: OMWWs ability to affect cell proliferation and survival on different cancer cell lines was assessed by MTT. OMWWs interference with human umbilical endothelial vein cells (HUVECs) tube formation, migration and invasive capacities was studied by endothelial cell morphogenesis and migration assays. The inhibition of angiogenesis and tumor cell growth was investigated by the matrigel assay and tumour xenograft. To evaluate contribution of potential metabolomic biomarkers, SANIST (surface-activated chemical ionization-electrospray-NIST technology) mass spectrometric data-based platform has been employed on sera from a pilot study (Aiello et al, in preparation). Results: OMWW extract shown a potent inhibitory effect inhibiting cells proliferation of both cancer cell lines and HUVECs.OMWW inhibited several molecular targets expression, including IL8, Angiogenin, mTOR, VEGF, CXCR4, CXCL12 and CXCL8, in tumor cells and in TNF-α-stimulated HUVEC. We nalyzed sera from volunteers, after nutraceutical intervention with Oliphenoliaand we assessed several biological parameters. Endogenous vitamin D was significantly increased, as well as albumin, direct bilirubin, and calcium. We also observed glycemia and potassium decrease after Oliphenolia assumption. A previously unknow form of Vitamin D was also detected by mass spectrometry. Conclusions: OMWW extract has shown a stronger anti-angiogenic and anti-tumor properties, with potential chemo-preventive potentials. Results of human pilot study shows Oliphenolia as a promising and interesting natural dietary supplement, rich in polyphenol compounds, that can exert anti-inflammatory and antioxidant activities. Acknowledgements: This study was supported by a donation from the Fattoria La Vialla di Gianni, Antonio e Bandino Lo Franco - SAS (Arezzo, Italy). We are grateful to Professor Anna Aiello, and Professor Calogero Caruso, Palermo University for the pilot study. Citation Format: Adriana Albini, Luana Calabrone, Francesca Albini, Paola Corradino, Simone Cristoni, Douglas M. Noonan. A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7298.

Endothelium-specific deletion of p62 causes organ fibrosis and cardiac dysfunction

BackgroundThe autophagy adapter SQSTM1/p62 is crucial for maintaining homeostasis in various organs and cells due to its protein–protein interaction domains and involvement in diverse physiological and pathological processes. Vascular endothelium cells play a unique role in vascular biology and contribute to vascular health.MethodsUsing the Cre-loxP system, we generated mice with endothelium cell-specific knockout of p62 mediated by Tek (Tek receptor tyrosine kinase)-cre to investigate the essential role of p62 in the endothelium. In vitro, we employed protein mass spectrometry and IPA to identify differentially expressed proteins upon knockdown of p62. Immunoprecipitation assays were conducted to demonstrate the interaction between p62 and FN1 or LAMC2 in human umbilical vein endothelium cells (HUVECs). Additionally, we identified the degradation pathway of FN1 and LAMC2 using the autophagy inhibitor 3-methyladenine (3-MA) or proteasome inhibitor MG132. Finally, the results of immunoprecipitation demonstrated that the interaction between p62 and LAMC2 was abolished in the PB1 truncation group of p62, while the interaction between p62 and FN1 was abolished in the UBA truncation group of p62.ResultsOur findings revealed that p62 Endo mice exhibited heart, lung, and kidney fibrosis compared to littermate controls, accompanied by severe cardiac dysfunction. Immunoprecipitation assays provided evidence of p62 acting as an autophagy adapter in the autophagy-lysosome pathway for FN1 and LAMC2 degradation respectively through PB1 and UBA domain with these proteins rather than proteasome system.ConclusionsOur study demonstrates that defects in p62 within endothelium cells induce multi-organ fibrosis and cardiac dysfunction in mice. Our findings indicate that FN1 and LAMC2, as markers of (EndoMT), have detrimental effects on HUVECs and elucidate the autophagy-lysosome degradation mechanism of FN1 and LAMC2.

Meticulously engineered three-dimensional-printed scaffold with microarchitecture and controlled peptide release for enhanced bone regeneration.

The repair of large load-bearing bone defects requires superior mechanical strength, a feat that a single hydrogel scaffold cannot achieve. The objective is to seamlessly integrate optimal microarchitecture, mechanical robustness, vascularisation, and osteoinductive biological responses to effectively address these critical load-bearing bone defects. To confront this challenge, three-dimensional (3D) printing technology was employed to prepare a polycaprolactone (PCL)-based integrated scaffold. Within the voids of 3D printed PCL scaffold, a methacrylate gelatin (GelMA)/methacrylated silk fibroin (SFMA) composite hydrogel incorporated with parathyroid hormone (PTH) peptide-loaded mesoporous silica nanoparticles (PTH@MSNs) was embedded, evolving into a porous PTH@MSNs/GelMA/SFMA/PCL (PM@GS/PCL) scaffold. The feasibility of fabricating this functional scaffold with a customised hierarchical structure was confirmed through meticulous chemical and physical characterisation. Compression testing unveiled an impressive strength of 17.81 ± 0.83 MPa for the composite scaffold. Additionally, in vitro angiogenesis potential of PM@GS/PCL scaffold was evaluated through Transwell and tube formation assays using human umbilical vein endothelium, revealing the superior cell migration and tube network formation. The alizarin red and alkaline phosphatase staining assays using bone marrow-derived mesenchymal stem cells clearly illustrated robust osteogenic differentiation properties within this scaffold. Furthermore, the bone repair potential of the scaffold was investigated on a rat femoral defect model using micro-computed tomography and histological examination, demonstrating enhanced osteogenic and angiogenic performance. This study presents a promising strategy for fabricating a microenvironment-matched composite scaffold for bone tissue engineering, providing a potential solution for effective bone defect repair.

Molecular engineering to design a bright near-infrared red photosensitizer: cellular bioimaging and phototherapy.

Near-infrared red (NIR) fluorescence imaging guide phototherapeutic therapy (PDT) has the advantages of deep tissue penetration, real-time monitoring of drug treatment and disease, little damage to normal tissue, low cytotoxicity and almost no side effects, and thus, it is attracting increasing research attention and is expected to show promising potential for clinical tumor treatment. The photosensitizer (PS), light source and oxygen are the three basic and important factors to construct PDT technology, and highly efficient PSs are still being passionately pursued because they determine the PDT efficiency. Ideal PSs should have properties such as good biocompatibility, deep tissue penetration, and highly efficient reactive oxygen species (ROS) generation despite the hypoxic environment. Therefore, pure organic type I PSs with NIR fluorescence have been receiving increasing attention due to their deep penetration and hypoxia resistance. However, reported NIR-active type I PSs usually require complex synthetic procedures, which presents a challenge for mass production. In this research work, based on the molecular design ideas of introducing the heavy atom effect and intramolecular charge transfer, we prepared three NIR-active type I PSs (TNZ, TNZBr, and TNZCHO) using a very simple method with one or two synthetic steps. Clear characterizations of photophysical properties, ROS performance tests, and fluorescent imaging of human umbilical vein endothelial (HUVE) cells and PDT treatment of HepG2 cells were carried out. The results revealed that the heavy atom and intramolecular charge transfer (ICT) effects could obviously enhance the ROS efficiency, and both PSs produce only type I ROS without any type II ROS (1O2) generation. The good NIR fluorescence brightness and type I ROS efficiency ensure satisfactory bioimaging and PDT outcomes. This research provides the possibility of preparing NIR-active type I PSs via mass production.

Three-dimensional printing of cell-laden bioink for blood vessel tissue engineering: influence of process parameters and components on cell viability

Three-dimensional (3D) bioprinting is a potential therapeutic method for tissue engineering owing to its ability to prepare cell-laden tissue constructs. The properties of bioink are crucial to accurately control the printing structure. Meanwhile, the effect of process parameters on the precise structure is not nonsignificant. We investigated the correlation between process parameters of 3D bioprinting and the structural response of κ-carrageenan-based hydrogels to explore the controllable structure, printing resolution, and cell survival rate. Small-diameter (<6 mm) gel filaments with different structures were printed by varying the shear stress of the extrusion bioprinter to simulate the natural blood vessel structure. The cell viability of the scaffold was evaluated. The in vitro culture of human umbilical vein endothelium cells (HUVECs) on the κ-carrageenan (kc) and composite gels (carrageenan/carbon nanotube and carrageenan/sodium alginate) demonstrated that the cell attachment and proliferation on composite gels were better than those on pure kc. Our results revealed that the carrageenan-based composite bioinks offer better printability, sufficient mechanical stiffness, interconnectivity, and biocompatibility. This process can facilitate precise adjustment of the pore size, porosity, and pore distribution of the hydrogel structure by optimising the printing parameters as well as realise the precise preparation of the internal structure of the 3D hydrogel-based tissue engineering scaffold. Moreover, we obtained perfused tubular filament by 3D printing at optimal process parameters.

Exosomal lncRNA PCAT1 Promotes Tumor Circulating Cell-Mediated Colorectal Cancer Liver Metastasis by Regulating the Activity of the miR-329-3p/Netrin-1-CD146 Complex.

Objective This study explored the colorectal cancer exosome lncRNA prostate cancer associated transcript 1– (PCAT1) mediated circulating tumors and the mechanism of cell colorectal cancer liver metastasis. Methods Exosomes were extracted from the primary colorectal cancer (CRC) cell lines HCT116 and SW480 and cultured with T84 and human umbilical vein endothelial (HUVE) cells. The expression of PCAT1 and miR-329-3p was detected by real-time quantitative polymerase chain reaction (RT-qPCR), the expression of Netrin-1, CD146, and epithelial mesenchymal transition (EMT) related proteins was detected by Western blot, the proliferation activity of T84 cells was detected by cell counting kit 8 (CCK-8), and cell migration was detected by Transwell. The expression of the F-actin signal was detected by immunofluorescence after coculture of exosomes with human umbilical vein endothelial cells (HUVECs). Changes in subcutaneous tumor and liver nodule size after PCAT1 deletion were observed in a mouse model of liver metastasis from rectal cancer. Results PCAT1 expression was upregulated in primary cell lines and their exosomes. After exosomes were cocultured with colorectal cancer tumor circulating T84 cells, the expression of Netrin-1 and CD146 was upregulated, the expression of miR-329-3p was downregulated, the proliferation and migration ability of T84 cells were enhanced, and EMT occurred. After knocking down PCAT1, the above phenomenon was reversed. Similarly, after exosomes were cocultured with HUVECs, the expression of the F-actin signal increased, and after PCAT1 was knocked down, the F-actin signal also decreased. PCAT1 regulates miR-329-3p/Netrin-1 and affects the biological behavior of T84 and F-actin signal expression in HUVECs. In a mouse model of colorectal cancer liver metastasis, knocking down PCAT1 significantly reduced the nodules formed by liver metastasis in mice. Conclusions LncRNA PCAT1 derived from colorectal cancer exosomes regulates the activity of the Netrin-1-CD146 complex in circulating tumor cells (CTCs) to promote the occurrence of colorectal cancer EMT and liver metastasis and provides new molecular targets for the treatment of colorectal cancer liver metastasis.

The apoptotic effect of ozone therapy on mitochondrial activity of highly metastatic breast cancer cell line MDA-MB-231 using in vitro approaches

Ozone (O3) gas is the triatomic state of oxygen and it is used as a disinfection agent due to its strong oxidizing effect, since its discovery in the mid-nineteenth century. Ozone therapy is also an alternative therapeutic approach for some diseases like circulatory disorders, AIDS, asthma, cardiovascular diseases, and certain types of cancer by increasing the oxygen levels in the blood by external addition of ozone to the body. In this study, the therapeutic potential of ozone therapy was examined by inhibiting the growth of breast cancer cells in a dose-dependent procedure. Ozone concentrations varying from 5 to 20 ​μg/ml were applied to the MDA-MB-231, human breast adenocarcinoma and HUVEC, human umbilical vein endothelium, cell lines, and MDA cells demonstrated an increased rate of death while its migration potential decreases. RT-PCR analysis showed mRNA expression levels of pro-apoptotic genes demonstrated higher folds in MDA cells after 10 ​μg/ml treatment. In the same context, Annexin V/PI and cell cycle analysis also concluded that ozone therapy causes apoptotic cell death on breast tumor cells. The use of ozone therapy for cancer treatment requires further and extensive research. However, this research has shown that ozone therapy is a promising source for cancer treatment in a way by inhibiting the proliferation of breast tumor cells.

Conjugation of VEGFR1/R2-targeting peptide with gold nanoparticles to enhance antiangiogenic and antitumoral activity

BackgroundInhibition of tumor angiogenesis through simultaneous targeting of vascular endothelial growth factor receptor (VEGFR)-1 and -2 is highly efficacious. An antagonist peptide of VEGFA/VEGFB, referred to as VGB3, can recognize and neutralize both VEGFR1 and VEGFR2 on the endothelial and tumoral cells, thereby inhibits angiogenesis and tumor growth. However, improved efficacy and extending injection intervals is required for its clinical translation. Given that gold nanoparticles (GNPs) can enhance the efficacy of biotherapeutics, we conjugated VGB3 to GNPs to enhance its efficacy and extends the intervals between treatments without adverse effects.ResultsGNP–VGB3 bound to VEGFR1 and VEGFR2 in human umbilical vein endothelial (HUVE) and 4T1 mammary carcinoma cells. GNP–VGB3 induced cell cycle arrest, ROS overproduction and apoptosis and inhibited proliferation and migration of endothelial and tumor cells more effectively than unconjugated VGB3 or GNP. In a murine 4T1 mammary carcinoma tumor model, GNP–VGB3 more strongly than VGB3 and GNP inhibited tumor growth and metastasis, and increased animal survival without causing weight loss. The superior antitumor effects were associated with durable targeting of VEGFR1 and VEGFR2, thereby inhibiting signaling pathways of proliferation, migration, differentiation, epithelial-to-mesenchymal transition, and survival in tumor tissues. MicroCT imaging and inductively coupled plasma mass spectrometry showed that GNP–VGB3 specifically target tumors and exhibit greater accumulation within tumors than the free GNPs.ConclusionConjugation to GNPs not only improved the efficacy of VGB3 peptide but also extended the intervals between treatments without adverse effects. These results suggest that GNP–VGB3 is a promising candidate for clinical translation.Graphical

Increased Expression of TGF-β1 by 4-hexylresorcinol Is Mediated by Endoplasmic Reticulum and Mitochondrial Stress in Human Umbilical Endothelial Vein Cells

In our previous study, 4-hexylresorcinol (4HR) increased the expression level of vascular endothelial growth factor in human umbilical vein endothelial cells (HUVECs) via the transforming growth factor-β1 (TGF-β1)-mediated pathway. Endoplasmic reticulum (ER) and mitochondrial stress is a positive regulator of cellular differentiation. As TGF-β1 is a master regulator for cellular differentiation, 4HR treatment may increase TGF-β1 expression via ER stress. In this study, HUVECs were treated using 4HR (1–100 μM) for 24 h. The 4HR treatment increased ER stress-associated markers and mitochondrial stress. Increased TGF-β1 expression by 4HR administration was alleviated by tauroursodeoxycholate (ER stress inhibitor) treatment. Combining these activities with the elevated acetylation level of histone 3 (H3) by 4HR treatment, TGF-β1 expression was increased in HUVECs. Overall, 4HR increased TGF-β1 expression through upregulation of the stress response of ER as well as H3 acetylation in HUVECs.

High-Resolution Novel Indirect Bioprinting of Low-Viscosity Cell-Laden Hydrogels via Model-Support Bioink Interaction.

Bioprinting of unmodified soft extracellular matrix into complex 3D structures has remained challenging to fabricate. Herein, we established a novel process for the printing of low-viscosity hydrogel by using a unique support technique to retain the structural integrity of the support structure. We demonstrated that this process of printing could be used for different types of hydrogel, ranging from fast crosslinking gelatin methacrylate to slow crosslinking collagen type I. In addition, we evaluated the biocompatibility of the process by observing the effects of the cytotoxicity of L929 and the functionality of the human umbilical vein endothelium primary cells after printing. The results show that the bioprinted construct provided excellent biocompatibility as well as supported cell growth and differentiation. Thus, this is a novel technique that can be potentially used to enhance the resolution of the extrusion-based bioprinter.

Long-Term Protective Effects of Single-Dose Cardioplegic Solutions in Cell Culture Models

Despite the popularity of single-dose cardioplegic techniques, the time window and targeted population for successful reperfusion remain unclear. We tested currently available techniques based on cell viability and integrity to demonstrate long-term cardioprotection and clarify whether these solutions were performed on neonatal/adult endothelium and myocardium by examining different cell lines. Cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test proliferation assay and membrane integrity with the lactic dehydrogenase (LDH) cytotoxicity test were documented in a cell culture/microscopy setting on adult (human umbilical vein endothelium [HUVEC]), neonatal (H9C2-cardiomyocytes), and myofibroblast (L929) cell lines. Apoptotic cell activity and necrosis were evaluated by acridine orange/propidium iodide (AO/PI) staining. Twenty-four hours after seeding, cells were incubated in control (Dulbecco’s modified Eagle), St. Thomas and blood cardioplegia (4:1), histidine–tryptophan–ketoglutarate (HTK), and del Nido solutions at 32°C followed by an additional 6, 24, and 48 hours in standard conditions (37°C, 5% CO2). Experiments were repeated eight times. In MTT cell viability analysis, HTK protection was significantly better than the control medium in L929 cell lines at 48th hours follow-up and acted markedly better on the HUVEC cell line at 24th and 48th hours. del Nido and HTK provided significantly better protection on H9C2 (at 24th and 48th hours). Apoptotic and necrotic cell scoring as a result of AO/PI staining was found consistent with MTT results. The LDH test demonstrated that the level of cell disruption was significantly higher for St. Thomas and blood cardioplegia in H9c2 cells. Experimental studies on cardioplegia aimed at assessing myocardial protection use time-consuming and often expensive approaches that are unrealistic in clinical practice. We have focused on identifying the most effective cell types and the direct consequences of different cardioplegia solutions to document long-term effects that we believe are the most underestimated ones in the cardioplegia literature.

Long-Term Protective Effects of Single-Dose Cardioplegic Solutions in Cell Culture Models.

Despite the popularity of single-dose cardioplegic techniques, the time window and targeted population for successful reperfusion remain unclear. We tested currently available techniques based on cell viability and integrity to demonstrate long-term cardioprotection and clarify whether these solutions were performed on neonatal/adult endothelium and myocardium by examining different cell lines. Cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test proliferation assay and membrane integrity with the lactic dehydrogenase (LDH) cytotoxicity test were documented in a cell culture/microscopy setting on adult (human umbilical vein endothelium [HUVEC]), neonatal (H9C2-cardiomyocytes), and myofibroblast (L929) cell lines. Apoptotic cell activity and necrosis were evaluated by acridine orange/propidium iodide (AO/PI) staining. Twenty-four hours after seeding, cells were incubated in control (Dulbecco's modified Eagle), St. Thomas and blood cardioplegia (4:1), histidine-tryptophan-ketoglutarate (HTK), and del Nido solutions at 32°C followed by an additional 6, 24, and 48 hours in standard conditions (37°C, 5% CO2). Experiments were repeated eight times. In MTT cell viability analysis, HTK protection was significantly better than the control medium in L929 cell lines at 48th hours follow-up and acted markedly better on the HUVEC cell line at 24th and 48th hours. del Nido and HTK provided significantly better protection on H9C2 (at 24th and 48th hours). Apoptotic and necrotic cell scoring as a result of AO/PI staining was found consistent with MTT results. The LDH test demonstrated that the level of cell disruption was significantly higher for St. Thomas and blood cardioplegia in H9c2 cells. Experimental studies on cardioplegia aimed at assessing myocardial protection use time-consuming and often expensive approaches that are unrealistic in clinical practice. We have focused on identifying the most effective cell types and the direct consequences of different cardioplegia solutions to document long-term effects that we believe are the most underestimated ones in the cardioplegia literature.

C-C Motif Chemokine 8 promotes angiogenesis in vascular endothelial cells.

Angiogenesis is an important progress associated with several pathological situations. Several chemokines have been reported to act as regulators of angiogenesis. The current study aimed to find whether C-C Motif Chemokine 8 is involved in angiogenesis regulation. To verify whether C-C Motif Chemokine 8 is related to angiogenesis in plaques, carotid plaques were collected from patients with severe carotid stenosis and analysed using CD31 immunohistochemistry and real-time PCR. To further clarify the relation between C-C Motif Chemokine 8 and angiogenesis, human umbilical vein endothelium cells and human dermal microvascular endothelial cells were treated with C-C Motif Chemokine 8 in the presence or absence of C-C motif chemokine receptor 2-Ab and extracellular regulated MAP kinase 1/2 inhibition (FR180204). Proliferation and migration of human umbilical vein endothelium cells and human dermal microvascular endothelial cells were examined with Cell Counting Kit-8 and Transwell chamber assay, respectively. In vitro angiogenesis stimulated by C-C Motif Chemokine 8 was examined using tube formation assay. Ex vivo and in vivo angiogenesis were assessed by mice aortic ring assay and Matrigel plug assay, respectively. C-C motif chemokine receptors of human umbilical vein endothelium cells were examined with real-time PCR, and C-C motif chemokine receptor 1, C-C motif chemokine receptor 2, extracellular regulated MAP kinase 1/2 and phosphorylation-extracellular regulated MAP kinase 1/2 were examined with western blotting assay. C-C Motif Chemokine 8 was increased in carotid plaques with severe angiogenesis in both RNA and protein level. C-C Motif Chemokine 8 (5 ng/ml) weakly increased human umbilical vein endothelium cell proliferation, but not on human dermal microvascular endothelial cells. Migration and tube formation could be induced by C-C Motif Chemokine 8 in both human umbilical vein endothelium cells and human dermal microvascular endothelial cells. In mice aortic ring assay and Matrigel plug assay, C-C Motif Chemokine 8 could promote angiogenesis compared to vehicle groups. Phosphorylation of extracellular regulated MAP kinase 1/2 was increased with C-C Motif Chemokine 8 stimulation. The migration and tube formation promoted by C-C Motif Chemokine 8 could be largely blocked by C-C motif chemokine receptor 2-Ab or extracellular regulated MAP kinase 1/2 inhibition (FR180204). C-C Motif Chemokine 8 could promote both in vitro and in vivo angiogenesis. C-C motif chemokine receptor 2 played an important role in the activation of C-C Motif Chemokine 8 and extracellular regulated MAP kinase 1/2 signalling pathway was involved in this mechanism.

RETRACTED ARTICLE: M2 macrophage-derived extracellular vesicles promote gastric cancer progression via a microRNA-130b-3p/MLL3/GRHL2 signaling cascade

BackgroundTransfer of noncoding microRNAs (miRNAs) by extracellular vesicles (EVs) promotes the development of chemoresistance in many tumor types. Additionally, restoration or depletion of several miRNAs has been observed in multiple cancer types including gastric cancer (GC). In this present study, we aimed to investigate the mechanism of miR-130b-3p in M2 macrophage-derived EVs in the development of GC through regulation of mixed lineage leukemia 3 (MLL3) and grainyhead-like 2 (GRHL2).MethodsExpression of miR-130b-3p and GRHL2 was quantified in 63 pairs of cancerous and noncancerous gastric tissues. The predicted binding between miR-130b-3p and MLL3, together with the enrichment of MLL3, H3K4me1, and H3K27ac in gene enhancer region, was verified by luciferase activity assay and chromatin immunoprecipitation. Effects of miR-130b-3p on GC cell proliferation, apoptosis, migration and invasion, as well as tube formation of human umbilical endothelial vein cells (HUEVCs) were further determined by gain- and loss-of function assays in vitro.ResultsmiR-130b-3p was upregulated in GC tissues, and miR-130b-3p promoted survival, metastasis and angiogenesis of GC cells as well as enhanced tumor formation and angiogenesis in GC in vivo. Additionally, miR-130b-3p delivered in M2 macrophage-derived EVs promoted survival, migration, invasion, and angiogenesis of GC cells. Notably, MLL3 inhibited GC cell proliferation, migration, invasion, and vessel-like tube formation of HUEVCs by increasing GRHL2. Furthermore, downregulation of miR-130b-3p in M2 macrophage-derived EVs or upregulation of GRHL2 inhibited tumor formation and angiogenesis in GC.ConclusionThis study highlights that EVs loaded with the specific miRNA cargo miR-130b-3p mediate communication between M2 macrophages and cancer cells in the tumor microenvironment through the modulation of MLL3 and GRHL2 in GC.

Novel fibrin-fibronectin matrix accelerates mice skin wound healing

Plasma fibrinogen (F1) and fibronectin (pFN) polymerize to form a fibrin clot that is both a hemostatic and provisional matrix for wound healing. About 90% of plasma F1 has a homodimeric pair of γ chains (γγF1), and 10% has a heterodimeric pair of γ and more acidic γ′ chains (γγ′F1). We have synthesized a novel fibrin matrix exclusively from a 1:1 (molar ratio) complex of γγ′F1 and pFN in the presence of highly active thrombin and recombinant Factor XIII (rFXIIIa). In this matrix, the fibrin nanofibers were decorated with pFN nanoclusters (termed γγ′F1:pFN fibrin). In contrast, fibrin made from 1:1 mixture of γγF1 and pFN formed a sporadic distribution of “pFN droplets” (termed γγF1+pFN fibrin). The γγ′F1:pFN fibrin enhanced the adhesion of primary human umbilical vein endothelium cells (HUVECs) relative to the γγF1+FN fibrin. Three dimensional (3D) culturing showed that the γγ′F1:pFN complex fibrin matrix enhanced the proliferation of both HUVECs and primary human fibroblasts. HUVECs in the 3D γγ′F1:pFN fibrin exhibited a starkly enhanced vascular morphogenesis while an apoptotic growth profile was observed in the γγF1+pFN fibrin. Relative to γγF1+pFN fibrin, mouse dermal wounds that were sealed by γγ′F1:pFN fibrin exhibited accelerated and enhanced healing. This study suggests that a 3D pFN presentation on a fibrin matrix promotes wound healing.

Synthesis of AuNPs using plant polyphenols and their potential treatment for age-related macular degeneration

The primary idea behind this study is to synthesize Saudi Origanum vulgare L. extract mediated AuNPs and to investigate their efficacy in significantly treating AMD. The biocompatible AuNPs were tested for their radical scavenging efficiency in vitro. Also, The effects of AuNPs in suppressing the angiogenic protein expression and inflammatory cytokine levels were analyzed in ARPE-19 (human retinal pigment epithelium-19) as well as and human umbilical vein endothelium cell lines in vitro. From the ORAC findings, it was evident that the bio synthesized AuNPs have presented momentous anti-oxidative activity in reducing the apoptosis in H2O2 treated ARPE-19 cell lines. In addition, the biocompatible AuNPs exhibited significant potential in suppressing the LPS stimulated inflammatory reaction in ARPE-19 cell lines while demonstrating inhibitory expression of growth factor in ARPE-19 as well as in umbilical vein endothelium cell lines. The experimental findings in this study have suggested that the synthesized AuNPs are biocompatible with significant potential in defending against angiogenesis, apoptosis, and generation of pro-inflammatory cytokines in AMD disease.

Epigallocatechin-3-gallate inhibits tumor angiogenesis: involvement of endoglin/Smad1 signaling in human umbilical vein endothelium cells

Strategies targeting endoglin are currently being investigated in clinical trials as an anti-angiogenic therapy. The redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis was verified. Increased endoglin signaling after an anti-VEGF treatment was observed in patients. Treatment with an endoglin-neutralizing antibody increased VEGF signaling in endothelial cells. Therefore, strategies targeting both the endoglin and VEGF pathways were applied to determine whether the anti-angiogenic effects were increased in vitro. Five possible hits for endoglin were identified from 2000 compounds in the Traditional Chinese Medicine Database using Discovery Studio 4.5 Epigallocatechin-3-gallate (EGCG) attenuates angiogenesis by downregulating VEGF; however, researchers have not determined whether its anti-angiogenic effects are mediated by endoglin/Smad1 signaling. A major contribution of this study is that EGCG significantly inhibited the upregulation of endoglin in semaxanib-treated human umbilical vein endothelial cell. Thus, a combination treatment with EGCG and a VEGF tyrosine kinase inhibitor would be appropriate to reverse drug resistance. EGCG alone significantly decreased endoglin/pSmad1 levels in HUVECs. In the angiogenesis assay, the migration, invasion, and tube formation of HUVECs were markedly suppressed by higher concentrations of EGCG. A combination treatment with EGCG and semaxanib further produced increased anti-angiogenic effects. The main contribution of the study indicated that EGCG significantly decreased the semaxanib-induced overexpression of endoglin. Therefore, a combination treatment including EGCG will probably solve the drug resistance to anti-VEGF treatments.

TNF-alpha inhibits pregnancy-adapted Ca2+ signaling in uterine artery endothelial cells

Enhancement of vasodilation of uterine arteries during pregnancy occurs through increased connexin (Cx)43 gap junction (GJ) communication supporting more frequent and sustained Ca2+ ‘bursts’. Such adaptation is lacking in subjects with preeclampsia (PE). Here we show TNF-alpha, commonly increased in PE subjects, inhibits Cx43 function and Ca2+ bursts in pregnancy-derived ovine uterine artery endothelial cells (P-UAEC) via Src and MEK/ERK phosphorylation of Cx43, and this can be reversed by PP2 or U0126. Of relevance to humans: (1) the nutraceutical Src antagonist t10, c12 CLA also recovers Ca2+ bursting in P-UAEC. (2) TNF-alpha can reduce and PP2 rescue Ca2+ bursting and NO output in human umbilical vein endothelium (HUV Endo) preparations. (3) Treatment of HUV Endo from PE subjects with PP2 alone can rescue bursting and NO output. We conclude TNF-alpha acts via Src more than MEK/ERK to inhibit GJ Cx43 function in PE subjects, and CLA may offer a potential therapy.