Human Umbilical Vein Endothelial Cells Research Articles

CLEC14A facilitates angiogenesis and alleviates inflammation in diabetic wound healing

BackgroundDelayed wound healing is a serious complication of diabetic wounds, posing a significant challenge to the treatment of patients with diabetes. Diabetic wound healing is a complex dynamic process involving angiogenesis and inflammatory responses. Currently, there are limited targeted therapies to promote diabetic wound healing. This study aimed to reveal the role of CLEC14A in the process of diabetic wound healing, with the hope of identifying new therapeutic targets to accelerate the healing of diabetic wounds. MethodsIn vivo, diabetic mice were generated by combined streptozotocin (STZ) and high-fat diet treatment. The wound healing model was established in wild-type and Clec14a−/− diabetic mice. The degree of wound healing, as well as angiogenesis and inflammation during the healing process, were evaluated through Hematoxylin and Eosin (H&E) staining, immunohistochemical staining, and immunofluorescence staining. In vitro, the angiogenic activities of Human Umbilical Vein Endothelial Cells (HUVECs) were assessed following treatment with high glucose and adenoviruses overexpressing CLEC14A, using scratch assays and tube formation assays. Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) were utilized to evaluate the levels of inflammation in HUVECs. ResultsCLEC14A expression was suppressed in diabetic wounds. Deletion of the Clec14a inhibited angiogenesis and activated inflammatory responses in vivo. High-glucose treatment led to decreased CLEC14A expression, impaired angiogenic capacity, and elevated inflammatory levels in vitro. However, adenoviral-mediated overexpression of CLEC14A reversed the response induced by high glucose. ConclusionCLEC14A accelerates diabetic wound healing by promoting angiogenesis and reducing wound inflammation.

Inhibition of lysophosphatidic acid receptor 2 attenuates neonatal chronic lung disease in mice by preserving vascular and alveolar development

AimBronchopulmonary dysplasia (BPD) is a common morbidity in extremely premature infants. Previous studies demonstrated the important role of lysophosphatidic acid (LPA) in inflammation in BPD. However, the role of LPA and its receptors in hyperoxia-induced vascular malformations in BPD remains to be elucidated. Methods and ResultsElevated plasma LPA levels were observed in mice with BPD compared to controls (792 vs. 607 ng/mL, p < 0.05). Inhibition of LPA signaling protected against hyperoxia-induced lung injury in neonatal mice, demonstrated by a 2.8-fold increase in pulmonary vascular density and a 14% reduction in alveolar enlargement. In vitro studies showed that LPA suppressed tube formation in human umbilical vein endothelial cells (HUVECs) by approximately 50%. LPA receptor 2 (LPA2) was identified as a functional LPA receptor in primary endothelial cells from the lungs of hyperoxic mice and in HUVECs under hyperoxic conditions. The LPA2 antagonist H2L5186303 enhanced the tube formation ability of HUVECs exposed to LPA, both under normoxia (4-fold) and hyperoxia (5-fold). Moreover, H2L5186303 significantly protected against hyperoxia-induced vascular malformation (2-fold) and improved alveolarization in neonatal mice (12% decrease in mean linear intercept, MLI). Early growth response 1 (EGR1) was characterized as a downstream target of LPA2, silencing EGR1 restored tube formation in HUVECs exposed to LPA and hyperoxia. ConclusionsOur in vitro and in vivo findings demonstrate that the inhibition of LPA/LPA2 signaling mitigates hyperoxia-induced pulmonary vascular malformations, suggesting the LPA/LPA2-dependent signaling pathway has therapeutic potential for extremely premature infants with BPD.

Hydrolytic instability of laser-ablatively deposited CaSi2 coatings in air and neutral water affects the behavior of bone healing-related cell types

Calcium silicide (CaSi2) instability in humid air or pH-neutral water remains unknown, although the topochemical formation of silicene from CaSi2 in various aqueous phases is a well-known process. Here we report on laser ablation of CaSi2 in the vacuum and ethanol, characterize coatings deposited on titanium substrates with electron microscopy, X-ray photoelectron, Raman, and infrared spectroscopy, and examine the instability of the deposited coatings in humid air and neutral water. We further investigate the behavior of human mesenchymal stromal cells (hMSCs), vascular cells (HUVECS, human umbilical vein endothelial cells), and macrophages (derived from THP-1 cell line) in contact with the deposited coatings submerged in cell culture medium. The observed results indicate that the CaSi2 coatings undergo topochemical conversion to silicene, which is accompanied by hydrolytic reactions leading to inorganic Ca compounds and SiO2, and that the response of all cell types in the hydrolyzed CaSi2 surface domain is negatively affected by the nature of the hydrolytic products. In contrast, cells showed a tendency for enhanced biocompatibility towards the CaSi2 particles ablated in the vacuum. The results suggest that coating approaches can significantly influence cell behavior outcomes.

Application of biogenic silver nanoparticle incorporated nanofibers in biomedical science

The applications of nanotechnology are expanding into medical science for designing new medical devices related products. These products can deliver targeted therapies to diseases such as cancer and peripheral artery treatment. Therefore, with the goal to treat peripheral artery disease, we integrated biogenic silver nanoparticles (AgNPs) into polyethylene glycol-polycaprolactone (PEG-PCL) nanofibrous (NFs). The AgNPs-NFs mats were successfully fabricated using a blended electrospinning process. AgNPs with average sizes of 35–55 nm were produced and characterized using multiple techniques such as UV–Vis spectroscopy, TEM, SEM and FTIR. Nanofibrous membranes containing 1 %, 2 %, and 3 % AgNPs by weight were developed, showing a reduction in fiber diameter from 473 nm to 179 nm due to the introduction of charged particles. The scaffold’s suitability and drug release profile were critically analyzed, revealing results consistent with optimal biomedical applications. Preliminary studies indicated that AgNPs dose-dependent inhibition of human umbilical vein endothelial cells (HUVECs) may potentially help prevent atherosclerosis. These findings underscore the potential of AgNPs integrated PEG-PCL NFs mats for advanced biomedical applications.

An open-source, battery-powered, low-cost, and dual-channel pneumatic pulse generator for microfluidic cell-stretch assays

Cells in the body are regularly subjected to mechanical forces that influence their biological fate in terms of morphology, gene expression, and differentiation. The current gold standard method to replicate these effects in vitro is to culture cells on devices with elastic substrates and to impart mechanical stretch using mechanical or pneumatic pull–push methods. Microfluidic device designs offer several advantages in this context for general uniform and controlled stretching. However, the experimental setups are bulky, not user-friendly, and often involve several components that reside outside of the tissue culture incubator. Given the wide utility of mechanical stimulation in in-vitro research, our aim was to create a turn-key research tool that bioengineers can deploy in their cell-stretch assays, without having to deal with the complexity and nuances of ad hoc experimental setups. Here, we present an open-source, battery-powered, dual-channel cyclic pneumatic pulse generator box that can reside within an incubator and is compatible with custom microfluidic cell stretch devices. Our method depends on generating pressure-vacuum pulses simply using a linear miniature pneumatic air cylinder actuated using a continuous servo motor. To the best our knowledge, this is a first example of a completely battery-powered, standalone system that doesn’t have any peripherals residing out of the incubator. We provide a detailed list of different components as well as the step-by-step assembly process. We validate its performance in a cell stretch assay using a commercially available microfluidic chip. Our results show an acute stimulation of cyclic stretching over 8 h on human umbilical vein endothelial cells (HUVECs) resulted in preferential alignment of cells perpendicular to the axis of stretch.

Catalpol regulates apoptosis and proliferation of endothelial cell via activating HIF-1α/VEGF signaling pathway.

Burn injuries, especially severe ones, causes microcirculation disorders in local wounds and distant tissues, leading to ischemia and hypoxia of body tissues and organs. The key to prevent and treat complications and improve prognosis after burns is to improve the state of ischemia and hypoxia of tissue and restore the blood supply of organs. Catalpol is an iridoid glycoside compound isolated from Rehmannia radix, which has been widely reported to have various of functions, including antioxidative stress, anti-inflammation, anti-apoptosis, and neuroprotection. However, the pharmacologic action and underlying mechanism of Catalpol in angiogenesis after burn injury remains unclear. The study investigated whether Catalpol regulates apoptosis and proliferation following vascular injury induced by burns using an in vitro model of oxygen-glucose deprivation (OGD) with a human umbilical vein endothelial (HUVE) cell line. The results showed that treatment with Catalpol reduces the level of apoptosis and promotes proliferation of endothelial cell. Mechanistically, Catalpol increases the expression of vascular endothelial growth factor (VEGF) by activating Hypoxia-inducible factor-1α (Hif-1α), resulting in increased expression of related downstream effector molecules. The current study suggested that Catalpol is a promising compound for endothelial protection in burns. It may be an efficient Hif-1α activator for endothelial cell deprived of oxygen and glucose.

Increased caveolin 1 by human antigen R exacerbates Porphyromonas gingivali-induced atherosclerosis by modulating oxidative stress and inflammatory responses

Objective: Many different types of infectious oral diseases have been identified clinically, including chronic periodontitis. Porphyromonas gingivalis is the main pathogen causing chronic periodontitis, which is closely related to atherosclerosis (AS) and can promote the expression levels of caveolin 1 (Cav-1) and induced ribonucleic acid (RNA)-binding protein human antigen R (HuR). However, the roles of Cav-1 and its relationship with HuR in P. gingivalis-mediated AS progression remain largely unknown. Here, we aimed to detect the role and molecular mechanisms of Cav-1 in P. gingivalis-mediated AS. Material and Methods: To investigate the role of Cav-1 in P. gingivalis-mediated AS, we infected human umbilical vein endothelial cells (HUVECs) with P. gingivalis at a multiplicity of infection of 100:1 for 6, 12, and 24 h to simulate P. gingivalis-induced AS models in vitro and then transfected them with Cav-1 small interfering RNA to silence Cav-1. Combining molecular biology experimental techniques such as cell counting kit-8 assay, enzyme-linked immunosorbent assay, immunofluorescence staining, flow cytometry, Western blotting, and Oil Red O staining, and apolipoprotein E-deficient AS model mice, the impacts of Cav-1 on cell viability, inflammation, oxidative stress, apoptosis, Cav-1 and intercellular cell adhesion molecule-1 (ICAM-1) levels, and atherosclerotic plaque formation were investigated. Then, the relationship between Cav-1 and HuR was investigated through biotin pull-down and RNA immunoprecipitation assays, reverse transcription quantitative polymerase chain reaction, and Western blot. Results: P. gingivalis can induce Cav-1 expression in a time- and dose-dependent manner (P &lt; 0.05). This effect can inhibit the proliferation of HUVECs (P &lt; 0.05). Cav-1 interference repressed inflammatory response, reactive oxygen species (ROS) and ICAM-1 levels, and apoptosis in the HUVECs (P &lt; 0.05). Cav-1 messenger RNA was stabilized by HuR, which can bind to the 3’ untranslated region of Cav-1. Increase in HuR level reversed the effects of Cav-1 silencing on ROS and ICAM-1 levels and apoptosis in the HUVECs (P &lt; 0.05). In addition, the levels of inflammatory response, oxidative stress, and atherosclerotic plaque formation induced by P. gingivalis in the mouse model were significantly reduced after Cav-1 expression was inhibited (P &lt; 0.05). Conclusion: HuR-activated Cav-1 may promote atherosclerotic plaque formation by modulating inflammatory response and oxidative stress, leading to AS.

Research on the TSPAN6 regulating the secretion of ADSCs-Exos through syntenin-1 and promoting wound healing.

Exosomes (Exos) from adipose-derived stem cells (ADSCs) have a high inclusion content and low immunogenicity, which helps to control inflammation and accelerate the healing of wounds. Unfortunately, the yield of exosomes is poor, which raises the expense and lengthens the treatment period in addition to impairing exosomes' therapeutic impact. Thus, one of the key problems that needs to be resolved in the current exosome study is increasing the exosome yield. Tetraspanin-6 (TSPAN6) overexpression and knockdown models of ADSCs were constructed to determine the number of exosomes secreted by each group of cells as well as the number of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) within the cells. Subsequently, the binding region of the interaction between TSPAN6 and syntenin-1 was identified using the yeast two-hybrid assay, and the interaction itself was identified by immunoprecipitation. Finally, cellular and animal studies were conducted to investigate the role of each class of exosomes. When compared to the control group, the number of intracellular MVBs and ILVs was significantly larger, and the number of ADSCsTSPAN6+-Exos was more than three times higher. However, TSPAN6's ability to stimulate exosome secretion was reduced as a result of syntenin-1 knockdown. Additional yeast two-hybrid assay demonstrated that the critical structures for their interaction were the N-terminal, Postsynaptic density protein 95/Discs large protein/Zonula occludens 1 (PDZ1), and PDZ2 domains of syntenin-1, and the C-terminal of TSPAN6. In animal trials, the wound healing rate was best in the ADSCsTSPAN6+-Exos group, while cellular experiments demonstrated that ADSCsTSPAN6+-Exos better enhanced the proliferation and migration of human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs). TSPAN6 stimulates exosome secretion and formation, as well as the creation of MVBs and ILVs in ADSCs. Syntenin-1 is essential for TSPAN6's stimulation of ADSCs-Exos secretion. Furthermore, ADSCsTSPAN6+-Exos has a greater ability to support wound healing, angiogenesis, and the proliferation and migration of a variety of cells.

Large Scale Ultrafast Manufacturing of Wireless Soft Bioelectronics Enabled by Autonomous Robot Arm Printing Assisted by a Computer Vision-Enabled Guidance System for Personalized Wound Healing.

A Customized wound patch for Advanced tissue Regeneration with Electric field (CARE), featuring an autonomous robot arm printing system guided by a computer vision-enabled guidance system for fast image recognition is introduced. CARE addresses the growing demand for flexible, stretchable, and wireless adhesive bioelectronics tailored for electrotherapy, which is suitable for rapid adaptation to individual patients and practical implementation in a comfortable design. The visual guidance system integrating a 6-axis robot arm enables scans from multiple angles to provide a 3D map of complex and curved wounds. The size of electrodes and the geometries of power-receiving coil are essential components of the CARE and are determined by a MATLAB simulation, ensuring efficient wireless power transfer. Three heterogeneous inks possessing different rheological behaviors can be extruded and printed sequentially on the flexible substrates, supporting fast manufacturing of large customized bioelectronic patches. CARE can stimulate wounds up to 10 mm in depth with an electric field strength of 88.8 mVmm-1. In vitro studies reveal the ability to accelerate cell migration by a factor of 1.6 and 1.9 for human dermal fibroblasts and human umbilical vein endothelial cells, respectively. This study highlights the potential of CARE as a clinical wound therapy method to accelerate healing.

NSUN2 methylates IRF4 to affect the capacity of macrophages attached to titanium implant on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs in vitro.

We aimed to investigate the effect and underlying mechanism of titanium (Ti) implant on the polarization of macrophages and subsequent effects on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs) affected by macrophages. Firstly, the regulatory effect of Ti implant on macrophage polarization was investigated. Levels of M1 polarization markers and M2 polarization markers in macrophages were evaluated by immunofluorescence staining method and qPCR analysis. The osteogenic differentiation capacity of PDLSCs cultured with supernatants of macrophages of each group was evaluated by the Alizarin Red S (ARS) staining method and qPCR. Angiogenesis related genes were also evaluated in HUVECs cultured in supernatants of macrophages. To explore whether RNA m5C modification can modulate the effects of Ti implant on macrophage regulation of osteogenic differentiation and angiogenesis, we analyzed the main genes related to m5C in macrophages using RNA m5C dot blotting and qPCR methods. The interaction between NSUN2 and IRF4 was verified by m5C-RIP, RIP, and double-luciferase gene report experiments. Macrophages were activated as M1 macrophages under the interference of LPS, and macrophages attached to Ti implants were more easily activated as M2 macrophages under the action of LPS. Macrophages activated by Ti implant enhanced osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. NSUN2 level was up-regulated in macrophages treated with LPS and was down-regulated by Ti implant. Over-expression of NSUN2 attenuated the effect of Ti implant on M1 polarization promotion of macrophages and enhanced the M2 polarization promotion of macrophages. Up-regulation of NSUN2 weakened the effects of Ti implant on promotion the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. The KEGG analysis suggested that IRF4 was enriched in several inflammatory signaling pathways. Moreover, NSUN2 methylates IRF4 to affect the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. Taken together, macrophages of M1 type can be stimulated by Ti implants in vitro, promoting osteogenic differentiation of PDLSCs and angiogenesis in HUVECs through NSUN2-mediated methylation of IRF4.

Apoptosis-induced exosomes from human exfoliated deciduous teeth enhance angiogenesis in human umbilical vein endothelial cells.

Exosomes derived from the stem cells of human exfoliated deciduous teeth (SHED) hold promise for tissue regeneration. Apoptotic cells release a variety of extracellular vesicles that affect intercellular communication. This study aimed to investigate the angiogenic effects of SHED-derived exosomes modified via apoptosis induction on human umbilical vein endothelial cells (HUVECs). Apoptosis was induced in SHED via serum starvation for 3 weeks and confirmed by the upregulation of the apoptotic genes, caspase 3 and 9, and via annexin V staining. The apoptotic SHED-derived exosomes were isolated, characterized, and subjected to proteomic analysis. In vitro experiments were performed to assess the effects of apoptotic SHED exosomes on the proliferation, migration, and tube formation of HUVECs. The apoptosis-induced SHED showed increased cell viability and decreased numbers of dead cells compared with those of conventional cultures while retaining their identity as mesenchymal stem cells positive for CD44, CD73, and CD90. The apoptotic SHED-derived exosomes exhibited characteristic features, such as standard size, cup-shaped morphology, and positive staining, for exosomal markers CD9, CD63, and CD81. Proteins associated with apoptosis, programmed cell death, and cellular senescence were downregulated in the apoptotic SHED exosomes, whereas those associated with extracellular matrix organization were upregulated, indicating positive angiogenesis. HUVECs treated with apoptotic SHED exosomes exhibited significantly enhanced proliferation and migration compared with those treated with normal SHED exosomes. The mesh-like structures in the apoptotic SHED exosomes exhibited significantly increased signs of angiogenesis. The findings of this study provide new insights into the potential use of apoptotic SHED-derived exosomes in regenerative medicine.

Divergent Proteomic Profiles and Uptake Mechanisms of Exosomes Derived from Human Dental Pulp Stem Cells, Endothelial Cells, and Fibroblasts.

Effective intercellular communication is crucial for tissue repair and regeneration, with exosomes playing a key role in mediating these processes by transferring proteins, lipids, and nucleic acids between cells. This study explored the mechanisms underlying the uptake of exosomes derived from human dental pulp stem cells (hDPSCs), human umbilical vein endothelial cells (HUVECs), and human fibroblasts (HFBs). Our findings revealed that hDPSCs exhibited the greatest capacity for exosome uptake across all three cell types. Moreover, exosomes originating from hDPSCs were also taken up in the highest amounts by all three cell types. Proteomic analysis uncovered significant differences in protein expression among exosomes from these different cell types, particularly in proteins related to endocytosis. Clathrin-dependent endocytosis emerged as the primary pathway for exosome uptake in hDPSCs and HUVECs, while HFBs appeared to use a different mechanism. Additionally, proteins such as fibronectin and tetraspanins were found to be highly expressed in hDPSC-derived exosomes, suggesting their potential involvement in exosome-cell interactions. This study offers new insights into exosome uptake mechanisms and highlights the potential of exosomes in advancing tissue engineering and regenerative medicine.

Abstract 4145829: Fatty Acid Binding Protein 4 as Potential Indicator for Gestational Diabetes Mellitus-Induced Fetal Endothelial Dysfunction

Introduction/Background: Gestational diabetes mellitus (GDM) is the most common complication during pregnancy. GDM is leading to an elevated risk for the development of cardiovascular diseases both in the mother and the child in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential role as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. Research Question/Hypothesis: Does FABP4 affect the function of human endothelial cells from patients with GDM? Goals/Aim: Our aim is a better understanding of the role of FABP4 in fetal endothelial dysfunction of patients with GDM and the development of potential therapeutic strategies. Methods/Approach: Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords obtained from normoglycemic and GDM pregnancies. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression pattern. HUVECs were subjected to functional analyses assessing proliferation, migration, NO and insulin signaling. Finally, human endothelial cells were exposed to high laminar flow (30 dyn/cm 2 ) or simvastatin (10 nM) treatment to investigate their impact on FABP4 expression and endothelial function. Results/Data: Bulk RNA sequencing data analysis revealed FABP4 as a gene overexpressed in GDM HUVECs compared to normoglycemic controls. Gene set enrichment analysis showed in addition an enrichment of genes involved in angiogenesis and Notch signaling pathways in human endothelial cells from GDM patients. GDM HUVECs had a significantly higher wound healing capacity compared to normoglycemic controls. The PI3K/AKT/mTOR pathway was enriched in GDM HUVECs. GDM HUVECs displayed impaired activation of AKT and eNOS (expression and phosphorylation) after stimulation with insulin, suggesting a possible insulin resistance. Finally, subjecting HUVECs to high laminar shear stress or simvastatin treatment caused a reduction of FABP4 mRNA expression and an increase of eNOS expression, indicating potential therapeutic strategies to improve endothelial function. Conclusions: We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. High laminar shear stress and medications activating eNOS signaling could protect the endothelium against the negative impact of FABP4 in GDM.

Abstract 4141461: Zonal Coating Application System for Left Atrial Appendage Occlusion Devices: A Novel Approach to Reduce Peri-Device Leakage and Device Related Thrombus.

Introduction: Left atrial appendage occlusion (LAAO) is an effective method of stroke prevention in patients with atrial fibrillation (AF). However, peri-device leakage (PDL) and device related thrombus (DRT) are common limitations of LAAO. Notably, PDL is associated with an increased risk of thromboembolic events. In order to minimize DRT, the Watchman FLX Pro is coated with PVDF-HFP. We aim to minimize both DRT and PDL with our novel zonal coating application system (ZCAS). ZCAS integrates a two-part thromboresistant and endothelialization promoting coating onto LAAO devices (Figure 1). In order to determine the optimal coating for the endothelialization promoting zone of ZCAS, we validated coating candidates in an in vitro model of LAAO device endothelialization (Figure 2). Hypothesis: We hypothesized that polyethylene terephthalate (PET) mesh coated with collagen type IV, fibronectin + VEGF, or poly-L-lysine would improve endothelial cell viability and proliferation. Methods: Human umbilical vein endothelial cells (HUVEC) were cultured in DMEM/F12 supplemented with ATCC’s Endothelial Cell Growth Kit and antibiotics. For the cell viability and proliferation assay, HUVEC cells were cultured in DMEM/F12 supplemented with 2% FBS, 10 mM L-glutamine, and antibiotics. A 96 well plate with PET inserts was coated with three coating candidates. Each coating was applied in three concentrations ([1x], [10x], and [100x]) with triplicates for each concentration. The coatings included collagen type IV ([1x]: 1 ug/cm2), fibronectin ([1x]: 1 ug/cm2) + VEGF ([1x]: 10 ng/cm2), and poly-L-lysine ([1x]: 4 ug/cm2). An MTS assay was performed at 48 hours after plating to assess cell viability and proliferation. Results: MTS absorbance relative to the uncoated control (MTS absorbance coated well/MTS absorbance uncoated well) for collagen type IV at [1x], [10x], and [100x] was 0.81, 1.29, and 0.84 respectively (Table 1). Fibronectin + VEGF at [1x], [10x], and [100x] was 1.31, 1.62, and 0.96. Finally, poly-L-lysine at [1x], [10x], and [100x] was 0.69, 0.90, and 0.91 respectively. Conclusion(s): Among the coating candidates, fibronectin (1 ug/cm2) + VEGF (100 ng/cm2) had the greatest increase in cell viability and proliferation compared to the uncoated PET control. The significance of this finding lies in the potential for ZCAS to improve the endothelial seal between the LAAO device and LAA orifice, which may significantly reduce PDL post-implantation.

TMIC-41. RBBP4/P300 COMPLEX CONTROLS GENES MODULATING ANGIOGENESIS AND IMMUNOSUPPRESSION IN GLIOBLASTOMA MICROENVIRONMENT

Abstract The RBBP4/p300 complex was recently shown to control genes critical for DNA damage repair and may play a role in glioblastoma (GBM) therapy responsiveness, especially to temozolomide (TMZ). However, the role of this complex in the modulation of the GBM tumor microenvironment (GBM-TME) remains elusive. Because pro-tumorigenic GBM-TME is primarily due to angiogenesis and immune tolerance, we evaluated whether the RBBP4/p300 complex may control these critical processes. To that end, we used shRNA to silence each RBBP4/p300 complex member in GBM43 cells and RNAseq to identify angiogenesis and immune genes regulated by this complex. Our data demonstrated that silencing RBBP4 or p300 suppressed the expression of HIF1α and several other hypoxia response genes, including VEGFA and DDIT4, suggesting that RBBP4/p300 complex may regulate angiogenesis. Consistent with a role in regulating angiogenesis, culture medium conditioned by GBM43 cells expressing the control non-targeting shRNA (shNT) significantly induced angiogenic tube formation by human umbilical vein endothelial cells (HUVEC). In contrast, this effect was blocked by the medium conditioned by the shRBBP4 or shp300 expressing cells. Both shRBBP4 and shp300 expressing cells failed to activate HIF1α accumulation and other signaling in response to hypoxia. Additionally, the control shNT-conditioned medium inhibited IFN-γ production while the medium conditioned by the shRBBP4 or shp300 expressing cells supported IFN-ɣ production from CD8+ T cells. Intriguingly, silencing RBBP4 or p300 also suppressed the expression of CCL2 and PD-L1 (CD274). CCL2 protein is a chemokine that promotes TME infiltration by myeloid-derived suppressor cells (MDSCs), while PD-L1 interacts with PD1 to suppress cytotoxic CD8+ T cell activation. Although this is an ongoing investigation, we conclude that RBBP4/p300 complex mediates the expression of hypoxia response genes, including HIF1α, and immunosuppressive genes, including PD-L1, and this activity may enhance angiogenic and immunosuppressed GBM-TME, making this complex a potential target for GBM therapy.

Phenolic Metabolites Protocatechuic Acid and Vanillic Acid Improve Nitric Oxide Bioavailability via the Akt-eNOS Pathway in Response to TNF-α Induced Oxidative Stress and Inflammation in Endothelial Cells

Background/Objectives: Reduced nitric oxide (NO) bioavailability secondary to excess-superoxide-driven oxidative stress is central to endothelial dysfunction. Previous studies suggest that phenolic metabolites may improve NO bioavailability, yet limited research is available in response to an inflammatory mediator. Therefore, we assessed the effects of cyanidin-3-glucoside (C3G) and its phenolic metabolites protocatechuic acid (PCA) and vanillic acid (VA) on NO bioavailability in a TNF-α induced inflammatory environment. Methods: Primary human umbilical vein endothelial cells (HUVECs) were supplemented with either C3G, PCA, or VA at 1 μM for 24 h before being stimulated with TNF-α 20 ng/mL for an additional 24 h. Measurements included cell viability, apoptosis, reactive oxygen species (ROS), nitrite concentrations, and endothelial nitric oxide synthase (eNOS) and Akt at the mRNA and protein level. Results: Phenolic metabolites did not increase the eNOS expression or nitrite levels in the unstimulated environment; rather, the metabolites mediated NO bioavailability in response to TNF-α induced oxidative stress, with increased viability, eNOS mRNA, phosphorylation, and nitrite levels. Conclusions: Phenolic metabolites, in the presence of TNF-α, can improve NO bioavailability at physiologically relevant concentrations via the Akt-eNOS pathway. This demonstrates that the induction of inflammation is a prerequisite for phenolic metabolites to promote protective properties in endothelial cells by activating the Akt-eNOS pathway.

Evaluating the Biodegradability and Cellular Compatibility of Mg-Zn-Nd Alloy for Vascular Stent Application.

The study is designed to evaluate the corrosion behavior, biocompatibility, and cytotoxicity of a novel magnesium alloy, Mg-2Zn-0.5Nd (ZN20), for potential use as biodegradable scaffolding in cerebrovascular stents. Magnesium alloy (AZ31) and ZN20 are co-cultured with Human Umbilical Vein Endothelial Cells (HUVEC)and human neuroblastoma cell (SH-SY5Y), respectively. The corrosion of AZ31 and ZN20 in different time periods is detected by electron microscope, the effects of AZ31 and ZN20 on the expression level of inflammatory factors are detected by ELISA, the PH value of cells in each group is detected, and the cell proliferation is detected by cck-8. Cell-related apoptosis protein, the expression of Platelet endothelial cell adhesion molecule-1 (CD31)and VE-cad is detected, and the pathological analysis of rat vascular tissue is carried out by HE experiment. In contrast to the AZ31 group, ZN20 exhibits mild, uniform corrosion and does not significantly deter HUVEC proliferation or increase inflammatory markers and In vivo testing reveals better endothelization with ZN20, as demonstrated by higher expression of endothelial markers CD31, and intact endothelial structure group. Western blotting shows favorable expression levels of apoptotic and anti-apoptotic markers in the ZN20 group. ZN20 alloy demonstrates enhanced corrosion resistance, favorable endothelial compatibility, and reduced cytotoxicity, endorsing its safe application in vascular stent use.

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ10, alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ10 or CoQ10 with trolox. The combination of CoQ10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ10 and trolox reduced protein expression of VEGFA. CoQ10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

Functionally distinct anticoagulant mechanisms of endothelial cells

Antithrombin and tissue factor pathway inhibitor (TFPI) provide different anticoagulant mechanisms. Having established a potent anticoagulant role of cultured human umbilical vein endothelial cells in vessel-on-a-chip microfluidic models, we now investigated how these cells modulated thrombin generation under stasis through antithrombin and TFPI pathways. We observed that endothelial monolayers in 96 well-plates strongly delayed and suppressed the thrombin generation process induced by tissue factor, regardless of the presence of whole blood, platelet-rich plasma or platelet-free plasma. Intervention studies indicated that the blocking of heparin-like proteoglycans with polybrene or protamine sulphate, similarly as the absence of antithrombin in plasma, reverted the endothelial anticoagulant activity. Heparinase treatment of the cells also reduced the anticoagulant potential. Furthermore, the presence of andexanet-alpha (inactivated factor Xa) and an anti-TFPI antibody were able to revert the endothelial effects. Jointly, these data point to additive anticoagulant mechanisms of endothelial cells through surface-expressed heparin-like proteoglycans and TFPI, both contributing to thrombin inhibition.

Dual Gold Nanostructures-Based Stretchable Electrochemiluminescence Sensors for Hydrogen Peroxide Monitoring in Endothelial Mechanotransduction.

Hydrogen peroxide (H2O2) release during blood flow is commonly provoked by the cyclic stretch and dynamic shear stress of endothelial cells and is of vital significance for maintaining vascular function. Flexible and stretchable electrochemical sensors show great capability in retrieving mechanical stimulation-induced H2O2 variation; however, cell secretions, especially electroactive constituents' interferences, remain a big concern for sensing accuracy. Herein, we developed a stretchable electrochemiluminescence (ECL) sensor by synthesizing L012-reduced gold nanospheres and decorating them onto a polydimethylsiloxane film-supported gold nanotubes substrate (Au NTs/PDMS) to form dual gold nanostructure-modified meshwork interface. Given the specific reaction between L012 and H2O2, the as-prepared Au-L012/Au NTs/PDMS exhibited outstanding selectivity toward H2O2 quantification. Through culturing human umbilical vein endothelial cells (HUVECs), real-time monitoring of transient H2O2 release from mechanically sensitive HUVECs in stretching states was realized. This work successfully incorporated the ECL sensing model into in situ cellular sensing, therefore expanding the application mode of the ECL approach for health care and biomedical investigation.