Human Umbilical Cord Vein Endothelial Cells Research Articles

3D bioprinting microgels to construct implantable vascular tissue

Engineered implantable functional thick tissues require hierarchical vasculatures within cell-laden hydrogel that can mechanically withstand the shear stress from perfusion and facilitate angiogenesis for nutrient transfer. Yet current extrusion-based 3D printing strategies are unable to recapitulate hierarchical networks, highlighting the need for bioinks with tunable properties. Here, we introduce an approach whereby crosslinkable microgels enhance mechanical stability and induce spontaneous microvascular networks comprised of human umbilical cord vein endothelial cells (HUVECs) in a soft gelatin methacryoyl (GelMA)-based bioink. Furthermore, we successfully implanted the 3D printed multi-branched tissue, being connected from the rat carotid artery to the jugular vein direct surgical anastomosis. The work represents a significant step toward in the field of large vascularized tissue fabrication and may have implications for the treatment of organ failure in the future.

Epigenetic Reprogramming via Synergistic Hypomethylation and Hypoxia Enhances the Therapeutic Efficacy of Mesenchymal Stem Cell Extracellular Vesicles for Bone Repair.

Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time-dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration.

Vascular and pulmonary effects of ibuprofen on neonatal lung development

BackgroundIbuprofen is a nonsteroidal anti-inflammatory drug that is commonly used to stimulate closure of a patent ductus arteriosus (PDA) in very premature infants and may lead to aberrant neonatal lung development and bronchopulmonary dysplasia (BPD).MethodsWe investigated the effect of ibuprofen on angiogenesis in human umbilical cord vein endothelial cells (HUVECs) and the therapeutic potential of daily treatment with 50 mg/kg of ibuprofen injected subcutaneously in neonatal Wistar rat pups with severe hyperoxia-induced experimental BPD. Parameters investigated included growth, survival, lung histopathology and mRNA expression.ResultsIbuprofen inhibited angiogenesis in HUVECs, as shown by reduced tube formation, migration and cell proliferation via inhibition of the cell cycle S-phase and promotion of apoptosis. Treatment of newborn rat pups with ibuprofen reduced pulmonary vessel density in the developing lung, but also attenuated experimental BPD by reducing lung inflammation, alveolar enlargement, alveolar septum thickness and small arteriolar wall thickening.ConclusionsIn conclusion, ibuprofen has dual effects on lung development: adverse effects on angiogenesis and beneficial effects on alveolarization and inflammation. Therefore, extrapolation of the beneficial effects of ibuprofen to premature infants with BPD should be done with extreme caution.

The isolation of VCAM-1+ endothelial cell-derived extracellular vesicles using microfluidics

Background: Vascular cell adhesion molecule-1 (VCAM-1+) endothelial cell-derived extracellular vesicles (EC-EVs) are augmented in cardiovascular disease, where they can signal the deployment of immune cells from the splenic reserve. Endothelial cells in culture activated with pro-inflammatory tumor necrosis factor-α (TNF-a) also release VCAM-1+ EC-EVs. However, isolating VCAM-1+ EC-EVs from conditioned cell culture media for subsequent in-depth analysis remains challenging. Aim: We utilized the extracellular vesicles (EV) microfluidics herringbone chip (EVHB-Chip), coated with anti-VCAM-1 antibodies, for selective capture of VCAM-1+ cells and EC-EVs. Methods and Results: Engineered EA.hy926 endothelial cells overexpressing VCAM-1 (P < 0.001 versus control) showed increased binding to the VCAM-1- EVHB-Chip versus an IgG device. TNF-α-stimulated human umbilical cord vein endothelial cells (HUVECs) exhibited elevated VCAM-1 protein levels (P < 0.001) and preferential binding to the VCAM-1- EVHB-Chip versus the IgG device. HUVECs stimulated with TNF-α showed differential gene expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.001) and VCAM-1 (P < 0.001) by digital droplet PCR versus control cells. HUVEC-derived EC-EVs were positive for CD9, CD63, HSP70, and ALIX and had a modal size of 83.5 nm. Control and TNF-α-stimulated HUVEC-derived EC-EV cultures were captured on the VCAM-1- EVHB-Chip, demonstrating selective capture. VCAM-1+ EC-EV were significantly enriched for ICAM-1 (P < 0.001) mRNA transcripts. Conclusion: This study presents a novel approach using the EVHB-Chip, coated with anti-VCAM-1 antibodies and digital droplet PCR for the study of VCAM-1+ EC-EVs. Isolation of VCAM-1+ EC-EV from heterogeneous sources such as conditioned cell culture media holds promise for subsequent detailed characterization, and may facilitate the study of VCAM-1+ EC-EVs in cardiovascular and metabolic diseases, for disease monitoring and therapeutic insights.

Jatrorrhizine Improves Endothelial Function in Diabetes and Obesity through Suppression of Endoplasmic Reticulum Stress.

Jatrorrhizine (JAT) is one of the major bioactive protoberberine alkaloids found in rhizoma coptidis, which has hypoglycemic and hypolipidemic potential. This study aimed to evaluate the vasoprotective effects of JAT in diabetes and obesity and the underlying mechanism involved. Mouse aortas, carotid arteries and human umbilical cord vein endothelial cells (HUVECs) were treated with risk factors (high glucose or tunicamycin) with and without JAT ex vivo and in vitro. Furthermore, aortas were obtained from mice with chronic treatment: (1) control; (2) diet-induced obese (DIO) mice fed a high-fat diet (45% kcal% fat) for 15 weeks; and (3) DIO mice orally administered JAT at 50 mg/kg/day for the last 5 weeks. High glucose or endoplasmic reticulum (ER) stress inducer tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations (EDRs) in mouse aortas, induced oxidative stress in carotid arteries and HUVECs, downregulated phosphorylations of Akt at Ser473 and eNOS at Ser1177 and enhanced ER stress in mouse aortas and HUVECs, and these impairments were reversed by cotreatment with JAT. JAT increased NO release in high-glucose-treated mouse aortas and HUVECs. In addition, chronic JAT treatment restored endothelial function with EDRs comparable to the control, increased Akt/eNOS phosphorylation, and attenuated ER stress and oxidative stress in aortas from DIO mice. Blood pressure, glucose sensitivity, fatty liver and its morphological change, as well as plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and plasma lipid profile, were also normalized by JAT treatment. Collectively, our data may be the first to reveal the vasoprotective effect of JAT that ameliorates endothelial dysfunction in diabetes and obesity through enhancement of the Akt/eNOS pathway and NO bioavailability, as well as suppression of ER stress and oxidative stress.

3D engineered neural co-culture model and neurovascular effects of marine fungi-derived citreohybridonol

Marine-based biomolecules are emerging metabolites that have gained attention for developing novel biomaterials, drugs, and pharmaceutical in vitro platforms. Here, we developed a 3D engineered neural co-culture model via a 3D prototyped sliding frame-platform for multi-step UV lithography and investigated the neurovascular potential of citreohybridonol in neuroblastoma treatment. Citreohybridonol was isolated from a sponge-derived fungus Penicillium atrovenetum. The model was characterized by Fourier-transform infrared spectroscopy and scanning electron microscopy analysis. Human umbilical cord vein endothelial cells (HUVECs) and neuroblastoma (SH-SY5Y) cell lines were encapsulated in gelatin methacrylate (GelMA) with and without citreohybridonol. The effect of citreohybridonol on the proliferation capacity of cells was assessed via cell viability and immunostaining assays. GelMA and 3D culture characterization indicated that the cells were successfully encapsulated as axenic and mixed with/without citreohybridonol. The cytotoxic test confirmed that the 3D microenvironment was non-toxic for cultural experiments, and it showed the inhibitory effects of citreohybridonol on SH-SY5Y cells and induced the proliferation of HUVECs. Finally, immunohistochemical staining demonstrated that citreohybridonol suppressed SH-SY5Y cells and induced vascularization of HUVECs in mixed 3D cell culture.

Overview of the angiogenic effect of probiotics (Lactobacillus acidophilus and Lactobacillus rhamnosus) at human umbilical vein endothelial cells

Introduction: Angiogenesis (neovascularization), which means new vessel construction, is normal and physiologically, wound healing, embryogenesis, a necessary menstrual cycle it's a mechanism. When taken in appropriate amounts together with or separately with nutrients, mucosal and by regulating systemic immunity, ensuring nutritional and microbial balance in the intestines living nonpatogenic microorganisms that positively affect the health of the host it is called "probiotics". Lactic acid bacteria, the most probiotic microorganisms it constitutes its important group. Where probiotics have an effect on angiogenesis, and it is thought to help heal wounds through the road. With this research indicated that roles of Lactobacillus acidophilus and Lactobacillus rhamnosus on angiogenesis if present to demonstrate in vitro methods and the gene expression responsible for the formation of these effects it is intended to reveal.
 Material and Method: This study is an experimental study conducted in vitro human umbilical cord vein endothelial cell (HUVEC) MTT test in cell culture with (3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) evaluation of viability and proliferation wound healing model, tube formation method and gene expression with real rime-polymer chain reaction (RT-PCR) methods of appointment were used.
 Results: HUVEC cells L. acidophilus 10⁹ CFU\ml after extract application statistical of mRNA expression of VEGF and FGF genes by control group 24 per hour it was found to increase significantly. L. rhamnosus 10 6 CFU\ml and 109 CFU\ml after application of extracts VEGF gene mRNA by control group 24 per hour its expression was found to be statistically significantly increased. Also L. rhamnosus extracts cell proliferation and migration of in vitro wound model it was found to increase statistically significantly.
 Conclusion: In this study, in vitro L. acidophilus 10⁹ CFU\ml extract and 10⁶ CFU\ml and 10⁹ CFU\ml extract of L. rhamnosus, VEGF gene mRNA revealed to be effective on angiogenesis in HUVEC cells by increasing expression it is.

Berberine Suppresses Leukocyte Adherence by Downregulating CX3CL1 Expression and Shedding and ADAM10 in Lipopolysaccharide-Stimulated Vascular Endothelial Cells.

Berberine exerts therapeutic effects in inflammation-associated diseases. In a lipopolysaccharide (LPS)-induced endotoxemic acute lung injury (ALI) rat model, berberine alleviated lung injury through different anti-inflammatory mechanisms; however, treatment effects on CX3CL1 expression and shedding remain to be examined. As these processes play important roles in promoting the binding of leukocytes to the endothelium, the CX3CL1/CX3CR1 axis and its related pathways may serve as potential targets for the clinical treatment of ALI. The anti-inflammatory effects of berberine were investigated in LPS-stimulated rats, human umbilical cord vein endothelial cells (HUVECs), and THP-1 monocytic cells. Cx3cl1 expression in rat pulmonary tissues was examined using immunohistochemistry. CX3CL1, CX3CR1, RELA, STAT3, and ADAM10 levels were examined using Western blotting. CX3CL1 and ADAM10 mRNA levels were examined using quantitative real-time polymerase chain reaction. Soluble fractalkine levels in LPS-stimulated rats and HUVECs were examined using the enzyme-linked immunosorbent assay. Berberine significantly mitigated the LPS-induced upregulation of fractalkine and soluble fractalkine in rats and cultured HUVECs. Berberine mitigated the LPS-induced activation of the NF-κB and STAT3 signaling pathways. In THP-1 cells, berberine mitigated the LPS-induced upregulation of CX3CR1. Furthermore, the membrane expression of ADAM10 in LPS-stimulated HUVECs was suppressed by the berberine treatment. Berberine dose-dependently inhibited the LPS-induced activation of the CX3CL1/CX3CR1 axis and fractalkine shedding through ADAM10. These findings reveal a novel molecular mechanism underlying the inhibitory effect of berberine on monocyte adherence to the endothelium during inflammation.

Robotic Intracellular Electrochemical Sensing for Adherent Cells.

Nanopipette-based observation of intracellular biochemical processes is an important approach to revealing the intrinsic characteristics and heterogeneity of cells for better investigation of disease progression or early disease diagnosis. However, the manual operation needs a skilled operator and faces problems such as low throughput and poor reproducibility. This paper proposes an automated nanopipette-based microoperation system for cell detection, three-dimensional nonovershoot positioning of the nanopipette tip in proximity to the cell of interest, cell approaching and proximity detection between nanopipette tip and cell surface, and cell penetration and detection of the intracellular reactive oxygen species (ROS). A robust focus algorithm based on the number of cell contours was proposed for adherent cells, which have sharp peaks while retaining unimodality. The automated detection of adherent cells was evaluated on human umbilical cord vein endothelial cells (HUVEC) and NIH/3T3 cells, which provided an average of 95.65% true-positive rate (TPR) and 7.59% false-positive rate (FPR) for in-plane cell detection. The three-dimensional nonovershoot tip positioning of the nanopipette was achieved by template matching and evaluated under the interference of cells. Ion current feedback was employed for the proximity detection between the nanopipette tip and cell surface. Finally, cell penetration and electrochemical detection of ROS were demonstrated on human breast cancer cells and zebrafish embryo cells. This work provides a systematic approach for automated intracellular sensing for adherent cells, laying a solid foundation for high-throughput detection, diagnosis, and classification of different forms of biochemical reactions within single cells.

Brain‐targeted intranasal formulation of Centella asiatica: A potential strategy for treating Alzheimer’s disease

AbstractBackgroundA major drawback associated with the conventional AD therapy is the inability of drugs to cross Blood Brain Barrier (BBB). Although, many drugs show potential to dissolve amyloid protein in vitro, only few mimics this action in vivo due to hindrance by BBB. One such drug is Centella asiatica containing asiaticoside that is proven to dissolves amyloid protein in vitro but lacks in vivo effects.MethodThe study was designed to develop a nasal formulation of an herbal drug (Centella asiatica) using brain targeted polymers to bypass the BBB to enhance the penetration of Centella in the brain.The extract of C. asiatica was first encapsulated into β‐ cyclodextrin by acid‐base precipitation method. These particles were nanonised and encapsulated into thiolated eudragit (TE) by ionic gelation method. The nanoparticles were characterized by FTIR, XRD, DLS, SEM, AFM and Ex ‐ vivo permeation studies. MTT assay was performed using Human Umbilical cord Vein Endothelial Cells (HUVECs) mimicking Blood‐Brain‐Barrier (BBB) to establish the safety and permeability of NPs. Further, the targeting efficacy was confirmed by molecular docking studies against receptors Sphingosine‐1‐Phosphate receptor 1 and Low‐density lipoprotein Receptor‐related Protein associated with BBB.ResultFTIR and XRD studies confirmed encapsulation of Centella into beta cyclodextrin and TE. Nanoparticle characterization confirmed the size and stability of the particles with zeta potential of −10.43 mV and Pdi of 0.260. NPs showed no signs of nasal ciliotoxicity and had good permeation of 88.44 ± 2.65 % (Mean ± SD, n=3) at 8h across nasal mucosa. MTT assay showed that the nanocomposite had lesser toxicity and better permeability compared to free drug (IC50 of Centella – 269.1 μg/mL and IC50 of nanoformulation – 310.625 μg/mL). The affinity of polymer to the receptors associated to BBB was proved by docking studies proves the ability of polymer‐based NPs to concentrate in brain post nasal administration.ConclusionThe study successfully showed that the NPs containing Centella had better permeability across BBB that could be beneficial in treating AD when administered intranasally.

EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/\u03b2-catenin signaling

BackgroundOsteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs), playing a key role in the interaction between osteogenesis and angiogenesis.MethodsWe evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis.ResultsEGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/β-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6.ConclusionEGFL6 enhanced the osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis. Thus, increasing EGFL6 secretion appeared to underpin the therapeutic benefit by promoting angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.

MicroRNA-128 Confers Anti-Endothelial Adhesion and Anti-Migration Properties to Counteract Highly Metastatic Cervical Cancer Cells' Migration in a Parallel-Plate Flow Chamber.

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells’ adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells’ adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells’ adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.

4-hexylresorcinol-induced protein expression changes in human umbilical cord vein endothelial cells as determined by immunoprecipitation high-performance liquid chromatography.

4-Hexylresorcinol (4HR) is used as a food preservative and an ingredient of toothpaste and cosmetics. The present study was performed using 233 antisera to determine the changes in protein expression induced by 4HR in human umbilical cord vein endothelial cells (HUVECs), and evaluated the 4HR-induced effects in comparison with previous results (Kim et al., 2019). Similar to RAW 264.7 cells, 4HR-treated HUVECs showed decreases in the expression of the proliferation-related proteins, cMyc/MAX/MAD network proteins, p53/RB and Wnt/β-catenin signaling, and they showed inactivation of DNA transcription and protein translation compared to the untreated controls. 4HR upregulated growth factors (TGF-β1, β2, β3, SMAD2/3, SMAD4, HGF-α, Met, IGF-1) and RAS signaling proteins (RAF-B, p38, p-p38, p-ERK-1, and Rab-1), and induced stronger expression of the cellular protection-, survival-, and differentiation-related proteins in HUVECs than in RAW 264.7 cells. 4HR suppressed NFkB signaling in a manner that suggests potential anti-inflammatory and wound healing effects by reducing M1 macrophage polarization and increasing M2 macrophage polarization in both cells. 4HR-treated HUVECs tended to increase the ER stress mediators by upregulating eIF2AK3, ATF4, ATF6, lysozyme, and LC3 and downregulating eIF2α and GADD153 (CHOP), resulting in PARP-1/AIF-mediated apoptosis. These results indicate that 4HR has similar effects on the protein expression of HUVECs and RAW 264.7 cells, but their protein expression levels differ according to cell types. The 4HR-treated cells showed global protein expression characteristic of anticancer and wound healing effects, which could be alleviated simultaneously by other proteins exerting opposite functions. These results suggest that although 4HR has similar effects on the global protein expression of HUVECs and RAW 264.7 cells, the 4HR-induced molecular interferences in those cells are complex enough to produce variable protein expression, leading different cell functions. Moreover, HUVECs have stronger wound healing potential to overcome the impact induced by 4HR than RAW 264.7 cells.

Effect of anthocyanin-rich sour cherry extract on the level of IL-8 in LPS-induced endothelial cell

The anthocyanin content of the Hungarian sour cherry is remarkable. Nutraceutical and pharmaceutical effects of the anthocyanins and their role in disease prevention have been studied extensively. Endothelial cells are involved in the pathogenesis of several inflammatory diseases. The objective of this work was to investigate pure sour cherry extract on human umbilical cord vein endothelial cells (HUVECs) as an inflammatory model. HUVECs were treated with 100 ng/mL lipopolysaccharide (LPS) and 50 mg/mL sour cherry extract or M199 medium as control. The optimal concentration range of the sour cherry extract was investigated and selected based on MTT assay measuring the conversion of the tetrazolium salt to formazan by mitochondrial dehydrogenases. The level of interleukine-8 (IL-8), a pro-inflammatory cytokine, was measured in Luminex MagPlex assay. LPS treatment significantly increased the secretion of IL-8. The pure sour cherry extract was able to attenuate this increment indicating the potent anti-inflammatory effect of pure sour cherry extract. Our results emphasize that pure sour cherry extract could reduce the LPS-induced inflammatory response thereby may improve endothelial dysfunction.

Effect of Anthocyanin-Rich Extract of Sour Cherry for Hyperglycemia-Induced Inflammatory Response and Impaired Endothelium-Dependent Vasodilation.

Diabetes mellitus (DM)-related morbidity and mortality are steadily rising worldwide, affecting about half a billion people worldwide. A significant proportion of diabetic cases are in the elderly, which is concerning given the increasing aging population. Proper nutrition is an important component in the effective management of diabetes in the elderly. A plethora of active substances of plant origin exhibit potency to target the pathogenesis of diabetes mellitus. The nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. In this study, the effect of Hungarian sour cherry, which is rich in anthocyanins, on hyperglycemia-induced endothelial dysfunction was tested using human umbilical cord vein endothelial cells (HUVECs). HUVECs were maintained under both normoglycemic (5 mM) and hyperglycemic (30 mM) conditions with or without two concentrations (1.50 ng/µL) of anthocyanin-rich sour cherry extract. Hyperglycemia-induced oxidative stress and inflammatory response and damaged vasorelaxation processes were investigated by evaluating the level of reactive oxygen species (ROS) and gene expression of four proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1α (IL-1α), as well as the gene expression of nitric oxide synthase (NOS) endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1). It was found that hyperglycemia-induced oxidative stress was significantly suppressed by anthocyanin-rich sour cherry extract in a concentration-dependent manner. The gene expression of the tested proinflammatory cytokines increased under hyperglycemic conditions but was significantly reduced by both 1 and 50 ng/µL anthocyanin-rich sour cherry extract. Further, although increased ET-1 and ECE-1 expression due to hyperglycemia was reduced by anthocyanin-rich sour cherry extract, NOS expression was increased by the extract. Collectively, these data suggest that anthocyanin-rich sour cherry extract could alleviate hyperglycemia-induced endothelial dysfunction due to its antioxidant, anti-inflammatory, and vasorelaxant effects.

Biochemical Research of the Effects of Essential Oil Obtained from the Fruit of Myrtus communis L. on Cell Damage Associated with Lipopolysaccharide-Induced Endotoxemia in a Human Umbilical Cord Vein Endothelial Cells.

The aim of this study to investigate the potential effects of essential oils and compounds obtained from MC fruit on sepsis induced endothelial cell damage in human umbilical cord vein endothelial cells (HUVECs) at molecular and cellular levels on in vitro sepsis model. A sepsis model was induced by the application of LPS. The HUVEC treatment groups were as follows: control, LPS, MC, MC plus LPS, 1.8 cineole, 1.8 cineole plus LPS, α-pinene, α-pinene plus LPS, α-terpineol, and α-terpineol plus LPS. Following the treatments, cell proliferation was analyzed using the xCELLigence® system. The mRNA expression of various cytokines [tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and IL-6] and endothelial nitric oxide (eNOS) were determined by quantitative polymerase chain reaction (qPCR) analysis. The 1.8 cineole and α-pinene treatments at specific doses showed toxic effects on α-terpineine, although it did not result in a change in the cellular index as compared with that of the control group. The application of LPS to HUVECs led to a significant decrease in the cellular index, depending on the treatment time. It did not correct the decreased cell index of MC plus LPS and α-terpineol plus LPS groups as compared with that of the LPS-only group. The 1.8 cineole plus LPS treatment and α-pinene plus LPS treatment significantly increased the cell index as compared with that of the LPS-only treatment, and the cell index in these groups was closer to that of the control. According to the results of the qPCR analysis, neither the MC-only treatment nor the α-terpineol-only treatment significantly reduced cellular damage caused by LPS-induced increases in TNF-α, IL-1β, IL-6, and eNOS mRNA expression. However, both the 1.8 cineole treatment and α-pinene treatments significantly decreased TNF-α, IL-1β, IL-6, and eNOS mRNA expression induced by LPS. Volatile oil obtained from MC fruit and the MC compound α-terpineol had no effect on the decreased cell index and increased cytokine response due to LPS-induced endothelial cell damage. However, 1.8 cineole and α-pinene, other major components of MC fruit, ameliorated LPS-induced damage in HUVECs at cellular and biomolecular levels (TNF-α, IL-1β, IL-6, and eNOS).

Cobalt-containing bioactive glass mimics vascular endothelial growth factor A and hypoxia inducible factor 1 function.

Bioactive glasses (BGs) have shown great potential for tissue regeneration and their composition flexibility allows the incorporation of different ions with physiological activities and therapeutic properties in the glass network. Among the many ions that could be incorporated, cobalt (Co) is a significant one, as it mimics hypoxia, triggering the formation of new blood vessels by the vascular endothelial growth factor A (VEGFA), due to the stabilizing effect on the hypoxia inducible factor 1 subunit alpha (HIF1A), an activator of angiogenesis-related genes, and is therefore of great interest for tissue engineering applications. However, despite its promising properties, the effects of glasses incorporated with Co on angiogenesis, through human umbilical cord vein endothelial cells (HUVECs) studies, need to be further investigated. Therefore, this work aimed to evaluate the biocompatibility and angiogenic potential of a new sol-gel BG, derived from the SiO2 -CaO-P2 O5 -CoO system. The structural evaluation showed the predominance of an amorphous glass structure, and the homogeneous presence of cobalt in the samples was confirmed. in vitro experiments showed that Co-containing glasses did not affect the viability of HUVECs, stimulated the formation of tubes and the gene expression of HIF1A and VEGFA. in vivo experiments showed that Co-containing glasses stimulated VEGFA and HIF1A expression in blood vessels and cell nuclei, respectively, in the deep dermis layer of the dorsal region of rats, featuring considerable local stimulation of the angiogenesis process due to Co-release. Co-containing glasses showed therapeutic effect, and Co incorporation is a promising strategy for obtaining materials with superior angiogenesis properties for tissue engineering applications.

Interactome of miRNAs and transcriptome of human umbilical cord endothelial cells exposed to short-term simulated microgravity

Adaptation of humans in low gravity conditions is a matter of utmost importance when efforts are on to a gigantic leap in human space expeditions for tourism and formation of space colonies. In this connection, cardiovascular adaptation in low gravity is a critical component of human space exploration. Deep high-throughput sequencing approach allowed us to analyze the miRNA and mRNA expression profiles in human umbilical cord vein endothelial cells (HUVEC), cultured under gravity (G), and stimulated microgravity (MG) achieved with a clinostat. The present study identified totally 1870 miRNAs differentially expressed in HUVEC under MG condition when compared to the cells subjected to unitary G conditions. The functional association of identified miRNAs targeting specific mRNAs revealed that miRNAs, hsa-mir-496, hsa-mir-151a, hsa-miR-296-3p, hsa-mir-148a, hsa-miR-365b-5p, hsa-miR-3687, hsa-mir-454, hsa-miR-155-5p, and hsa-miR-145-5p differentially regulated the genes involved in cell adhesion, angiogenesis, cell cycle, JAK-STAT signaling, MAPK signaling, nitric oxide signaling, VEGF signaling, and wound healing pathways. Further, the q-PCR based experimental studies of upregulated and downregulated miRNA and mRNAs demonstrate that the above reported miRNAs influence the cell proliferation and vascular functions of the HUVEC in MG conditions effectively. Consensus on the interactome results indicates restricted fluctuations in the transcriptome of the HUVEC exposed to short-term MG that could lead to higher levels of endothelial functions like angiogenesis and vascular patterning.

Fabrication of Stiffness Gradients of GelMA Hydrogels Using a 3D Printed Micromixer.

Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost-efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin-methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co-culture consisting of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.

Anti-angiogenic effects of testis-specific gene antigen 10 on primary endothelial cells.

Growing evidence indicates the antitumor and antiangiogenesis activities of testis-specific gene antigen 10 (TSGA10). However, the underlying mechanisms and precise role of TSGA10 in angiogenesis are still elusive. In this study, we isolated human umbilical cord vein endothelial cells (HUVECs) and stably transfected with pcDNA3.1 carrying TSGA10 coding sequence. We demonstrated that TSGA10 over-expression significantly decreases HUVEC tubulogenesis and interconnected capillary network formation. HUVECs over-expressing TSGA10 exhibited a significant decrease in migration and proliferation rates. TSGA10 over-expression markedly decreased expression of angiogenesis-related genes, including VEGF-A, VEGFR-2, Ang-1, Ang-2, and Tie-2. Our ELISA results showed the decrease in VEGF-A mRNA expression level is associated with a significant decrease in its protein secretion. Additionally, over-expressing TSGA10 decreased expression levels of marker genes of cell migration (MMP-2, MMP-9, and SDF-1a) and proliferation (PCNA and Ki-67. Furthermore, ERK-1 and AKT phosphorylation significantly reduced in HUVECs over-expressing TSGA10. Our findings suggest a potent anti-angiogenesis activity of TSGA10 in HUVECs through down-regulation of ERK and AKT signalling pathways, and may provide therapeutic benefits for the management of different pathological angiogenesis.