Chick Embryo Chorioallantoic Membrane Assay Research Articles

Therapeutic potency of marine collagen/pectin scaffolds - Fabrication, characterization and evaluation

Skin is an important vital organ that must be given proper care and protection from external damage and harmful microbes. If injured, it must be treated with an ideal wound dressing material with potent hemostatic and non-toxic properties. In the present study, fish collagen (FC) was extracted from the fins and tails of Black pomfret (Parastromateus niger). The isolated fish collagen was homogenized with pectin (P) and freeze dried to obtain fish collagen/pectin (FC/P) scaffolds. Scanning electron microscopic analysis showed the porous nature of scaffolds with intermittent holes. UV–Visible and Fourier infrared spectroscopic analyses demonstrated the physicochemical properties of FC/P scaffolds. Hemolytic assay performed using human blood demonstrated the percentage of hemolysis as 0.5 %. In vitro blood clotting assay carried out to determine the hemostatic behaviour displayed the formation of blood clot within 60 s in the presence of FC/P scaffolds. 95 % of cells were viable with the highest concentration of FC/P scaffold used for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Scratch wound assay demonstrated complete closure of wound in FC/P scaffold treated cells after 48 h of treatment. Chick embryo chorioallantoic membrane (CAM) assay showed the development of new blood vessels within 6 h of incubation with the FC/P scaffolds, thereby proving their angiogenic potency. These results indicate the potential use of FC/P scaffolds as effective biomaterials for tissue regenerative applications.

Chitosan-collagen biopolymer biofilm derived from cephalopod gladius; Evaluation of osteogenesis, angiogenesis and wound healing for tissue engineering application

Chitosan (Ch) and acid-soluble collagen (ASC) from Doryteuthis singhalensis gladius were isolated to test their osteogenic, angiogenic, and wound healing capabilities in male Wistar rats. The results of the study showed that the ASC yield was 18.58 %, the total protein content was 86.43 % ± 0.18 %, and the amino acid composition was as follows: glycine, 15.68 %; proline, 13.84 %. A, B, I, II, and III show FT-IR amide regional bands at 3392, 2959, 1652, 1471, and 1237 cm−1 respectively. The electrophoretic pattern of ASC validated its molecular weights of 105 kDa and 96 kDa. The 1H NMR spectra showed pure singles at 1.99 ppm, and the UV–Vis spectrum showed a particular absorbance between 238 and 220 nm. The DSC showed two endothermic peaks: one with an To value of 119.72 °C and TP of 126.28 °C, and the other with 147.42 °C and 148.47 °C. Initially, we fabricated Ch and ASC biofilms at an 8.5:1.5 ratio for tissue engineering applications. A cellular-level study demonstrated good biocompatibility and enhanced osteoblastic differentiation of collagen chitosan films (CChF). Additionally, the biofilm exhibited increased angiogenic potency, as observed in the chick embryo chorioallantoic membrane (CAM) assay. The experimental animal model demonstrated that in wound healing, the CChF treated rats (95.75 ± 2.28 %) had a greater decrease in the diameter of the wound than the control rats (22.25 ± 2.45 %), followed by the CF (collagen film) treated rats (63.25 ± 2.08 %) and ChF (chitosan film) (52.67 ± 1.58 %). Rats treated with CChF had 48.82 ± 1.25 mg/g of hydroxyproline in NFGT and 75.25 ± 1.56 mg/g of overall protein. The higher hydroxyproline levels in the CChF-treated groups corroborated these histopathological findings. These results imply that by promoting the development of scars, inflammation, and proliferation, CChF accelerates the healing process.

Rhus coriaria induces autophagic and apoptotic cell death in pancreatic cancer cells.

Background:Pancreatic cancer is a leading cause of cancer-related mortality worldwide with increasing global incidence. We previously reported the anticancer effect of Rhus coriaria ethanolic extract (RCE) in triple negative breast and colon cancer cells. Herein, we investigated the anticancer effect of RCE on human pancreatic cancer cells. Methods: Cell viability was measured using Cell Titer-Glo and staining of viable and dead cells based on differential permeability to two DNA binding dyes. Cell cycle distribution and annexin V staining was carried out in Muse cell analyzer. Protein level was determined by Western blot. Tumor growth was assessed by in ovo chick embryo chorioallantoic membrane assay. Results: We found that RCE significantly inhibited the viability and colony growth of pancreatic cancer cells (Panc-1, Mia-PaCa-2, S2-013, AsPC-1). The antiproliferative effects of RCE in pancreatic cancer cells (Panc-1 and Mia-PaCa-2) were mediated through induction of G1 cell cycle arrest, Beclin-1-independent autophagy, and apoptosis. RCE activated both the extrinsic and intrinsic pathways of apoptosis and regulated the Bax/Bcl-2 apoptotic switch. Mechanistically, we found that RCE inhibited the AKT/mTOR pathway, downstream of which, inactivation of the cell cycle regulator p70S6K and downregulation of the antiapoptotic protein survivin was observed. Additionally, we found that RCE-induced autophagy preceded apoptosis. Further, we confirmed the anticancer effect of RCE in a chick embryo xenograft model and found that RCE inhibited the growth of pancreatic cancer xenografts without affecting embryo survival. Conclusion: Collectively, our findings demonstrate that Rhus coriaria exerts potent anti-pancreatic cancer activity though cell cycle impairment, autophagy, and apoptosis, and is hence a promising source of anticancer phytochemicals.

Enhanced vascularity in gelatin scaffolds via copper-doped magnesium–calcium silicates incorporation: In-vitro and ex-ovo insights

Addressing a critical challenge in current tissue-engineering practices, this study aims to enhance vascularization in 3D porous scaffolds by incorporating bioceramics laden with pro-angiogenic ions. Specifically, freeze-dried gelatin-based scaffolds were infused with sol-gel-derived powders of Cu-doped akermanite (Ca2MgSi2O7) and bredigite (Ca7MgSi4O16) at various concentrations (10, 20, and 30 wt%). The scaffolds were initially characterized for their structural integrity, biodegradability, swelling behavior, impact on physiological pH, and cytocompatibility with human umbilical vein endothelial cells (HUVECs). The silicate incorporation effectiveness in promoting vascularity was then assessed through HUVEC attachment, capillary tube formation, and ex-ovo chick embryo chorioallantoic membrane assays. The findings revealed significant improvements in both in-vitro and ex-ovo vascularity of the gelatin scaffolds upon the addition of Cu-doped akermanite. The most effective concentrations were determined to be 10 and 20 %, which led to notable HUVEC metabolic activity, a well-spread morphology with extensive peripheral filopodia and lamellipodia at 10 % and a cobblestone phenotype indicative of in-vivo endothelium at 20 % during cell attachment, the formation of complex networks of tubular structures, and robust vascularization in chick embryo development. Moving forward, the incorporation of Cu-doped akermanite into tissue-engineering scaffolds shows great potential for addressing the limitations of vascularization, especially for critical-sized bone defects, by facilitating the controlled release of pro-angiogenic and pro-osteogenic ions.

Electrospun Gelatin Scaffolds with Incorporated Antibiotics for Skin Wound Healing.

Recent advances in regenerative medicine provide encouraging strategies to produce artificial skin substitutes. Gelatin scaffolds are successfully used as wound-dressing materials due to their superior properties, such as biocompatibility and the ability to mimic the extracellular matrix of the surrounding environment. In this study, five gelatin combination solutions were prepared and successfully electrospun using an electrospinning technique. After careful screening, the optimal concentration of the most promising combination was selected for further investigation. The obtained scaffolds were crosslinked with 25% glutaraldehyde vapor and characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. The incorporation of antibiotic agents such as ciprofloxacin hydrochloride and gentamicin sulfate into gelatin membranes improved the already existing antibacterial properties of antibiotic-free gelatin scaffolds against Pseudomonas aeruginosa and Staphylococcus aureus. Also, the outcomes from the in vivo model study revealed that skin regeneration was significantly accelerated with gelatin/ciprofloxacin scaffold treatment. Moreover, the gelatin nanofibers were found to strongly promote the neoangiogenic process in the in vivo chick embryo chorioallantoic membrane assay. Finally, the combination of gelatin's extracellular matrix and antibacterial agents in the scaffold suggests its potential for effective wound-healing treatments, emphasizing the importance of gelatin scaffolds in tissue engineering.

EPAC inhibitor suppresses angiogenesis and tumor growth of triple-negative breast cancer

AimsExchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. Main methodsWe employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. Key findingsInhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. ConclusionsOur findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.

Dioclea violacea lectin inhibits tumorigenesis and tumor angiogenesis in vivo

Dioclea violacea seed mannose-binding lectin (DvL) has attracted considerable attention because of its interesting biological activities, including antitumor, antioxidant, and anti-inflammatory activities. This study evaluated the cytotoxic effect of DvL on tumor and normal cells using the mitochondrial activity reduction (MTT) assay, the carcinogenic and anti-carcinogenic activity by the epithelial tumor test (ETT) in Drosophila melanogaster, and the anti-angiogenic effect by the chick embryo chorioallantoic membrane (CAM) assay. Data demonstrated that DvL promoted strong selective cytotoxicity against tumor cell lines, especially A549 and S180 cells, whereas normal cell lines were weakly affected. Furthermore, DvL did not promote carcinogenesis in D. melanogaster at any concentration tested, but modulated DXR-induced carcinogenesis at the highest concentrations tested. In the CAM and immunohistochemical assays, DvL inhibited sarcoma 180-induced angiogenesis and promoted the reduction of VEGF and TGF-β levels at all concentrations tested. Therefore, our results demonstrated that DvL is a potent anticancer, anti-angiogenic, and selective cytotoxic agent for tumor cells, suggesting its potential application as a prototype molecule for the development of new drugs with chemoprotective and/or antitumor effects.

DFMG decreases angiogenesis to uphold plaque stability by inhibiting the TLR4/VEGF pathway in mice.

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 μmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140.

Haemostatic potency of sodium alginate/aloe vera/sericin composite scaffolds – preparation, characterisation, and evaluation

Fabrication of haemostatic materials with excellent antimicrobial, biocompatible and biodegradable properties remains as a major challenge in the field of medicine. Haemostatic agents play vital role in protecting patients and military individuals during emergency situations. Natural polymers serve as promising materials for fabricating haemostatic compounds due to their efficacy in promoting hemostasis and wound healing. In the present work, sodium alginate/aloe vera/sericin (SA/AV/S) scaffold has been fabricated using a simple cost-effective casting method. The prepared SA/AV/S scaffolds were characterised for their physicochemical properties such as scanning electron microscope, UV–visible spectroscopy and Fourier transform infra-red spectroscopy. SA/AV/S scaffold showed good mechanical strength, swelling behaviour and antibacterial activity. In vitro experiments using erythrocytes proved the hemocompatible and biocompatible features of SA/AV/S scaffold. In vitro blood clotting assay performed using human blood demonstrated the haemostatic and blood absorption properties of SA/AV/S scaffold. Scratch wound assay was performed to study the wound healing efficacy of prepared scaffolds. Chick embryo chorioallantoic membrane assay carried out using fertilised embryos proved the angiogenic property of SA/AV/S scaffold. Thus, SA/AV/S scaffold could serve as a potential haemostatic healthcare product due to its outstanding haemostatic, antimicrobial, hemocompatible, biocompatible and angiogenic properties.

Pedunculagin and tellimagrandin-I stimulate inflammation and angiogenesis and upregulate vascular endothelial growth factor and tumor necrosis factor-alpha in vivo

Pedunculagin (PD) and tellimagrandin-I (TL), isolated from Myrciaria cauliflora seeds and Eucaliptus microcorys leaves, respectively, have attracted great attention owing to their relevant biological activities, such as antitumor, antioxidant, and hepatoprotective activities. This study investigated the angiogenic potential of PD and TL using a chick embryo chorioallantoic membrane (CAM) assay. Using the CAM assay, our results showed that both PD and TL promoted a significant increase in the number and caliber of blood vessels, the thickness of the CAM, and the presence of fibroblasts and inflammatory cells. Moreover, an increase of tumor necrosis factor-α and vascular endothelial growth factor was observed in the CAM treated with PD and TL, indicating the induction of angiogenic factors. Thus, the remarkable profile of PD and TL in inducing angiogenesis opens up new perspectives for their potential utilization in different therapeutic approaches involving neovascularization.

Biocompatibility assessment of Rutin and PEG loaded novel nanoceria on human erythrocytes and human myeloid leukemia (U937) cells

Nanomaterials are increasingly being used, prompting extensive research into nanomedicine. The use of nanomaterials is constrained by the absence of comprehensive toxicity profiles, despite their impressive diagnostic and therapeutic outcomes in in vitro and in vivo models. The compatibility of the bioconjugate nanoparticles, we fabricated a novel Rutin (Rut) and polyethylene glycol (PEG) loaded cerium oxide nanoparticles (Rut-PEG-CeO2NPs). The investigation of antibacterial activity of cerium oxide nanoparticles (CeO2NPs), Rut, Rut-PEG-CeO2 NPs and antibiotic was examined against Gram-positive and Gram-negative pathogens using well-diffusion and time-kill assays. The fabricated Rut-PEG-CeO2NPs has demonstrated broad spectrum nature resulting in significant cell damage at the target site when compared to CeO2NPs and Rut. The haemolytic effect of Rut-PEG-CeO2NPs on erythrocytes from healthy donor (HDE) and diabetic donor (DDE) was evaluated using the haemolysis assay against CeO2NPs. The results exhibit that the percentage of haemolysis was more in DDE compared to that of HDE for CeO2NPs as well as Rut-PEG-CeO2NPs. The study of anticoagulation activity using plasma recalcification time method which shows the clotting time of platelet rich plasma (PRP) 280–290 s and platelet poor plasma (PPP) 245–318 s. Besides that, the anti-angiogenetic property was evaluated using chick embryo chorioallantoic membrane (CAM) assay which showed the remarkable anti-angiogenic effect of Rut-PEG-CeO2NPs on the chick embryo. Additionally, the in vitro cytotoxicity of Rut-PEG-CeO2NPs was tested on human myeloid leukemia cells (U937) against Rut and CeO2NPs. The cytotoxicity results depicted that Rut-PEG-CeO2NPs shows high percent of cell viability compared to that of CeO2NPs and Rutin. These results suggests that the fabricated Rut-PEG-CeO2NPs has novel approach towards the therapeutic applications which is potentially due to the loading of Rut on the CeO2NPs on the surface to form Rut-PEG-CeO2NPs.

#4708 RENAL ORGANOID FROM URINE-DERIVED HUMAN ADULT RENAL PROGENITOR CELLS CAN SPONTANEOUSLY GENERATE LONG RENAL TUBULES

Abstract Background and Aims Organoids are self-organizing 3D aggregations of cells that represent the structure and function of organs. Kidney organoids have the potential to advance the field of nephrology by providing a tool for the study of human kidney development and disease, by providing a tool for in vitro drug screening, and ultimately, for regenerative therapy. They can be derived from embryonic stem cells or induced pluripotent stem cells. We aimed to explore for the first time, the potentiality of human adult renal progenitor cells (ARPCs), isolated from urine of healthy subjects and patients, to generate spheroids and organoids for regenerative purpose. Method ARPCs were isolated from urine by immunolabeling. Optical microscopy, immunofluorescence experiments and cytofluorimetric analysis were used to characterize spheroids and organoids. CD133, NANOG, SSA4, OCT 3-4 and GATA-3, SOX2 stemness marker antibodies were used to study spheroids and CD13, Lotus tetragonolobus lectin, ZO-1, uromodulin, and aminopeptidase antibodies were used to investigate renal tubular markers. PKH26 was used to study cell migration from organoids to tubules. Real-time PCR was used to generate gene expression data of aquaporin profile in renal tubules. Chick embryo chorioallantoic membrane (CAM) assays were used as in vivo model for angiogenesis. Results We isolated ARPCs from urine of healthy subjects or CKD patients. Usually, organoids were generated using complicate induction protocols with several chemical factors. However, for the first time we generated renal spheroids and organoids under 3D culture conditions without any stimulation, such as chemokines or growth factors, starting from ARPCs isolated from subject urine. We showed that the spheroids express high levels of stem cell markers as CD133 and NANOG, functional and constitutional marker of ARPCs, and high levels of stem cell markers that normally are low or not at all expressed in ARPCs and are typical of embryonic stem cells: GATA-3, SSEA4, and Sox2. Moreover, the spheroids were able to induce angiogenesis when implanted in chick embryo chorioallantoic membrane. The urine-derived CD133+ organoids can spontaneously generate long renal tubules in 6–12 days, as showed by PKH26 tagging. The renal tubules were positive for the proximal tubule cell markers CD13 (aminopeptidase N), Lotus tetragonolobus lectin and ZO-1. Moreover, renal tubules expressed the aminopeptidase A (CD249) that is normally present in proximal tubules and glomeruli of the kidneys and that catalyzed the conversion of the Angiotensin II in Angiotensin III witch is important in the local regulation of blood pressure. Also uromodulin was expressed by tubules. It is normally present in epithelial cells of the thick ascending limb of Henle's loop, and after proteolytical cleavage, is secreted into urine. In addition, the renal tubules express several genes typical of renal tubules as AQP1 channel protein that facilitates the flow of water molecules into the cells of the proximal tubule and descending limb of the loop of Henle; γGlut enzyme used for the transport of amino acids across cell membranes; Na/H exchanger 1 (SLC9A1), transporter involved in the regulation of pH homeostasis, cell migration and cell volume; Na/Gluc-1 co-transporter SGLUT1, which promotes the passage of glucose across the membrane in renal tubular cells. In some cases, tubule structures were similar to that found in nephrons, including proximal convoluted tubule, Loop of Henle, Distal Convoluted tubule and collecting duct, as shown by optical microscopy. Conclusion For the first time we demonstrated that kidney organoids can be generated starting by urine-derived human adult renal progenitor cells from patients and for the first time we showed that these organoids can generate, spontaneously, without specific stimulation, long portions of renal tubules. These results open new perspectives in the field of the regenerative medicine as ARPCs can be isolated from urine of patients and used to generate spheroids for the study of human kidney development and disease, for in vitro drug screening, and ultimately, for regenerative therapy.

Antileukemia Activity and Mechanism of Platinum(II)-Based Metal Complexes.

Transition metal complexes have continued to constitute an appealing class of medicinal compounds since the exceptional discovery of cisplatin in the late 1960s. Pt(II)-based complexes are endowed with a broad range of biological properties, which are mainly exerted by targeting DNA. In this study, we report a significant biological investigation into and computation analyses of four Pt(II)-complexes, namely, LDP-1-4, synthesized and characterized according to previously reported procedures. Molecular-modelling studies highlighted that the top two LDP compounds (i.e., LDP-1 and LDP-4) might bind to both matched and mismatched base pair sites of the oligonucleotide 5'-(dCGGAAATTACCG)2-3', supporting their anticancer potential. These two complexes displayed noteworthy cytotoxicity in vitro (sub-micromolar-micromolar range) against two leukaemia cell lines, i.e., CCRF-CEM and its multi-drug-resistant counterpart CEM/ADR5000, and remarkable anti-angiogenic properties (in the sub-micromolar range) evaluated in an in vivo model, i.e., a chick embryo chorioallantoic membrane (CAM) assay.

LncRNA SPRY4-IT1 facilitates cell proliferation and angiogenesis of glioma via the miR-101-3p/EZH2/VEGFA signaling axis.

SPRY4-IT1 (SPRY4 intronic transcript 1) is a long non-coding RNA (lncRNA) that has been identified as a novel oncogene in various cancers, including glioma. However, its function and underlying mechanism in glioma remain largely unclear. Here, we investigated the role of SPRY4-IT1 in the development of glioma and its underlying mechanism. Bioinformatics analysis and RT-qPCR assay were used to examine the expression of SPRY4-IT1 in glioma tissues. The CCK-8, EdU, and Xenograft tumor assays wereperformed to assess the proliferation effect of glioma cells. The tube forming assay and Chick Embryo Chorioallantoic Membrane (CAM) assay were conducted to detect the angiogenesis effect of HUVECs. RNA-sequencing, western blotting, RT-qPCR, ELISA, and IHC assays were employed to verify the regulatory mechanism of the SPRY4-IT1/ miR-101-3p/EZH2/VEGFA axis. Analysis of the TCGA dataset and data from our own cohort demonstrated that SPRY4-IT1 was overexpressed in patients with glioma, and high SPRY4-IT1 expression correlated with poor prognosis. In vitro and in vivo experiments showed that SPRY4-IT1 promoted the proliferation of glioma cells. RNA sequencing and Gene Ontology (GO) enrichment analysis indicated significant enrichment of angiogenesis. HUVEC tube forming assay and CAM assay confirmed that SPRY4-IT1 could induce angiogenesis of glioma cells in vitro and in vivo. Mechanistically, SPRY4-IT1 upregulated EZH2 expression by sponging miR-101-3p to induce VEGFA expression in glioma cells. Moreover, SPRY4-IT1 activated the VEGFR2/AKT/ERK1/2 pathway in HUVECs mediated by glioma cells. Rescue experiments further confirmed that SPRY4-IT1 promoted glioma cell proliferation and angiogenesis via the miR-101-3p/EZH2/VEGFA signaling axis. Our findings provide compelling evidence showing that SPRY4-IT1 upregulated EZH2 to induce VEGFA by sponging miR-101-3p, thereby achieving cell proliferation and angiogenesis in glioma. Therefore, targeting SPRY4-IT1/miR-101-3p/EZH2/VEGFA axis may improve the outcomes of patients with glioma.

Development of in vitro and in vivo tools to evaluate the antiangiogenic potential of melatonin to neutralize the angiogenic effects of VEGF and breast cancer cells: CAM assay and 3D endothelial cell spheroids

Melatonin is a molecule with different antitumor actions in breast cancer and has been described as an inhibitor of vascular endothelial growth factor (VEGF). Despite the recognition of the key role exerted by VEGF in tumor angiogenesis, limitations arise when developing models to test new antiangiogenic molecules. Thus, the aim of this study was to develop rapid, economic, high capacity and easy handling angiogenesis assays to test the antiangiogenic effects of melatonin and demonstrate its most effective dose to neutralize and interfere with the angiogenic sprouting effect induced by VEGF and MCF-7. To perform this, 3D endothelial cell (HUVEC) spheroids and a chicken embryo chorioallantoic membrane (CAM) assay were used. The results showed that VEGF and MCF-7 were able to stimulate the sprouting of the new vessels in 3D endothelial spheroids and the CAM assay, and that melatonin had an inhibitory effect on angiogenesis. Specifically, as the 1 mM pharmacological dose was the only effective dose able to inhibit the formation of ramifications around the alginate in the CAM assay model, this inhibition was shown to occur in a dose-dependent manner. Taken together, these techniques represent novel tools for the development of antiangiogenic molecules such as melatonin, with possible implications for the therapy of breast cancer.

Enhanced angiogenic potential of adipose-derived stem cell sheets by integration with cell spheroids of the same source

BackgroundAdipose-derived stem cell (ASC) has been considered as a desirable source for cell therapy. In contrast to combining scaffold materials with cells, ASCs can be fabricated into scaffold-free three-dimensional (3D) constructs to promote regeneration at tissue level. However, previous reports have found decreased expression of vascular endothelial growth factor (VEGF) in ASC sheets. In this study, we aimed to integrate ASC spheroids into ASC sheets to enhance the angiogenic capability of cell sheets.MethodsASCs were seeded in agarose microwells to generate uniform cell spheroids with adjustable size, while extracellular matrix deposition could be stimulated by ascorbic acid 2-phosphate to form ASC sheets. RNA sequencing was performed to identify the transcriptomic profiles of ASC spheroids and sheets relative to monolayer ASCs. By transferring ASC spheroids onto ASC sheets, the spheroid sheet composites could be successfully fabricated after a short-term co-culture, and their angiogenic potential was evaluated in vitro and in ovo.ResultsRNA sequencing analysis revealed that upregulation of angiogenesis-related genes was found only in ASC spheroids. The stimulating effect of spheroid formation on ASCs toward endothelial lineage was demonstrated by enhanced CD31 expression, which maintained after ASC spheroids were seeded on cell sheets. Relative to ASC sheets, enhanced expression of VEGF and hepatocyte growth factor was also noted in ASC spheroid sheets, and conditioned medium of ASC spheroid sheets significantly enhanced tube formation of endothelial cells in vitro. Moreover, chick embryo chorioallantoic membrane assay showed a significantly higher capillary density with more branch points after applying ASC spheroid sheets, and immunohistochemistry also revealed a significantly higher ratio of CD31-positive area.ConclusionIn the spheroid sheet construct, ASC spheroids can augment the pro-angiogenesis capability of ASC sheets without the use of exogenous biomaterial or genetic manipulation. The strategy of this composite system holds promise as an advance in 3D culture technique of ASCs for future application in angiogenesis and regeneration therapies.

Preparation and characterization of electrospun nanofibers-based facial mask containing hyaluronic acid as a moisturizing component and huangshui polysaccharide as an antioxidant component

A novel nanofiber material incorporating hyaluronic acid (HA), crude huangshui polysaccharide (cHSP) and polyvinyl alcohol (PVA) were prepared to obtain dry facial masks (PVA/HA/cHSP) via electrospinning. The facial mask was characterized by scanning electron microscopy (SEM), water absorption and retention evaluation, in vitro release study, DPPH and hydroxyl free radical scavenging measurements, red blood cell hemolysis and chick embryo chorioallantoic membrane assay. The results suggested that the fiber mask was densely homogeneous, uniform and well distributed with a diameter <260 nm. Meanwhile, the water absorption rate was >300 %, DPPH and hydroxyl free radical half-inhibitory concentrations of cHSP aqueous solution scavenging were 0.2781 and 1.029 mg/mL, respectively. The PVA/HA/cHSP could retain the antioxidant capacity of cHSP, possessing simultaneous excellent moisture retention and oxidation resistance. Importantly, the mucous membrane irritation experiment indicated that it was mild and safe.

Human Lung Tissue Implanted on the Chick Chorioallantoic Membrane as a Novel In Vivo Model of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first invivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.

Blends based on amino acid functionalized poly (ethylene-alt-maleic anhydride) polyelectrolytes and PEO for nanofiber elaboration: Biocompatible and angiogenic polyelectrolytes

A wide variety of polymers have been electrospun to obtain nanofibers. However, obtaining nanofibers from polyelectrolytes is less frequent due to the charges of these polymers, which hinder the electrospinning process. Poly (ethylene-alt-maleic anhydride) (PEMA) was modified with a series of amino acids (Aa). The functionalization of PEMA with Aa (PEMA-Aa) was demonstrated by FT-IR, 1H NMR, and 13C NMR. Blends of PEMA-Aa and poly (ethylene oxide), PEO, with different ratios were prepared. Nanofibers were obtained by electrospinning using blends of 10–20% w/v of PEMA-Aa and 10% w/v of PEO. The conductivity of blends decreased, and the surface tension increased as the quantity of PEO in the blends was increased. TGA showed intermediate thermal properties compared with the blend components. Nanofibers were obtained for all PEMA-Aa/PEO blends, with diameters between 170 and 350 nm. Continuous fibers without morphological defects were obtained at concentrations of 20% w/v and 10% w/v of PEMA-Aa and PEO. Wharton’s Jelly Mesenchymal Stem Cells viability, chicken embryo chorioallantoic membrane (CAM) assay and embryo viability measurements were realized for PEMA-Aa. Cytotoxicity test showed both composition and concentration-dependent behavior for PEMA-Aa, with higher WJ-MSC viability at 0.1 mg/mL at 24 h. CAM assay showed the formation of a high number of blood vessels and chicken embryo viability was close to 100% in the presence of polyelectrolytes. This, study demonstrates that electrospun nanofibers obtained from PEMA-Aa/PEO modified polyelectrolyte blends can be considered as a promising material for biomedical applications.

LncRNA CRART16/miR-122-5p/FOS axis promotes angiogenesis of gastric cancer by upregulating VEGFD expression.

Background: We previously identified a novel lncRNA, CRART16, that could induce cetuximab resistance in colorectal cancer cells. This study explored the relationship of CRART16 expression to gastric cancer progression and the molecular mechanisms involved.Methods: We evaluated CRART16 expression in gastric cancer tissues and adjacent normal tissues from the TCGA database and our hospital. Besides, we assessed its relationship with the overall survival (OS) of patients with gastric cancer. The effects of CRART16 on gastric cancer angiogenesis were determined by endothelial tube formation assay, spheroid sprouting assay, HUVEC invasion assay, and chick embryo chorioallantoic membrane (CAM) assay. The involvement of the lncRNA CRART16/miR-122-5p/FOS axis was analyzed by western blotting and dual-luciferase reporter assay. The functions of CRART16 were confirmed in xenograft mouse models.Results: We found that CRART16 was substantially overexpressed in gastric cancer tissues compared with normal tissues, based on the TCGA database and our clinical samples. High expression of CRART16 correlated with more advanced tumor stages and poor prognosis. Overexpression of CRART16 in gastric cancer cells promoted proliferation, colony formation, angiogenesis, and bevacizumab resistance in vitro, and it promoted tumor growth and angiogenesis in vivo, and vice versa. CRART16 was found to downregulate miR-122-5p by acting as a sponge, upregulating the target oncogene FOS. Afterward, the increased FOS expression led to the upregulation of VEGFD.Conclusion: Our findings demonstrate that CRART16 promotes angiogenesis in vitro and in vivo, and CRART16 is a prognostic marker and therapeutic target in gastric cancer.