Umbilical Cord Endothelial Cells Research Articles

Abstract LB387: Microphysiological modeling of gastric cancer vasculature using iPSC-derived endothelial cells

Abstract Generated induced pluripotent stem cells (iPSCs) from the blood of patients with advanced gastric cancer, differentiating them into endothelial cells and fibroblasts. Utilizing microfluidic 3D cell culture, we intricately reconstructed the observed abnormal blood vessels in gastric cancer patients in a three-dimensional format, capturing real-time neovasculature. Applying deep learning techniques, we virtually labeled the growing blood vessels with fluorescence, unveiling thin and tortuous morphological characteristics specific to patient iPSCs, distinct from those derived from human umbilical cord endothelial cells. The development of a tumor model, vascularized with cancer cells from the same donor, establishes a versatile testbed for evaluating personalized cancer vascular models. Through comprehensive morphological and functional assessments, this platform facilitates screening for patient-specific combination therapies and holds promise as a model for immune response testing. Citation Format: Jihoon Ko, Noo Li Jeon, Young Sun Oh. Microphysiological modeling of gastric cancer vasculature using iPSC-derived endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB387.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

LAPONITE® nano-silicates potentiate the angiogenic effects of FG-4592 and osteogenic effects of BMP-2.

LAPONITE®-based drug delivery systems offer many advantages due to the unique ionic and physical properties of LAPONITE®. The high ionicity and large surface area of LAPONITE® nanoparticles enable the intercalation and dissolution of biomolecules. In this study, we explored the potential of LAPONITE® as a carrier for FG-4592 to support angiogenesis and as a carrier for bone morphogenic protein-2 (BMP-2) to support osteogenesis. Interestingly, we found that LAPONITE® promoted the FG-4592 induced upregulation of vascular endothelial growth factor (VEGF) gene expression of human umbilical cord endothelial cells (HUVECs). Additionally, we observed that LAPONITE® could provide a sustained release of BMP-2 and significantly potentiate the osteogenic effects of BMP-2 on adipose derived mesenchymal stem cells (AMSCs). Overall, current findings on the LAPONITE®-drug/protein model system provide a unique way to potentiate the angiogenic activities of FG-4592 on HUVECs and osteogenic effects of BMP-2 on AMSCs for tissue engineering application. Future studies will be directed towards gaining a deeper understanding of these effects on a co-culture system of HUVECs and AMSCs.

Toxic effects of bisphenol S on mice heart and human umbilical cord endothelial cells

Bisphenol S (BPS) exerts toxic effects on hippocampal HT22 cells, endocrine secretion, and reproductive capacity. However, whether BPS exerts toxic effects on the heart requires further investigation. Therefore, we investigated the effects of BPS on mouse heart tissues and predicted possible underlying molecular mechanisms of action. Our study showed that BPS induced apoptosis, increased oxidative stress response. Using electron microscopy, we found that BPS disrupted sarcomere arrangement in myocardial cells and caused reduction in the number of plasmalemmal vesicles in endothelial cells in the mouse heart tissues. Also, BPS increased expression levels of P-NF-κB in mouse heart tissues. Furthermore, we found that BPS induced reactive oxygen species (ROS) generation, NF-κB activation, promoted apoptosis, elevated expression of BAX and Caspase 3, and decreased expression of Bcl-2 in H9c2 cells and HUVECs. However, after the addition of n-acetylcysteine or pyrrolidinedithiocarbamate, ROS generation, NF-κB activation, apoptosis, and expression of BAX and Caspase 3 were reduced, whereas expression of Bcl-2 was elevated. Our results demonstrated that BPS induced apoptosis of myocardial and endothelial cells through oxidative stress by activation of NF-κB signaling pathway.

The Paracrine Effect of Hyaluronic Acid-Treated Endothelial Cells Promotes BMP-2-Mediated Osteogenesis.

The combination of hyaluronic acid (HA) and BMP-2 has been reported to promote bone regeneration. However, the interaction of endothelial cells and bone marrow mesenchymal stem cells (BMSCs) during HA + BMP-2 treatment is not fully understood. This study aimed to analyze the direct effect of HA, as well as the paracrine effect of HA-treated endothelial cells, on the BMP-2-mediated osteogenic differentiation of BMSCs. The angiogenic differentiation potential of HA at different molecular weights and different concentrations was tested. The direct effect of HA, as well as the indirect effect of HA-treated human umbilical cord endothelial cells (HUVECs, i.e., conditioned medium (CM)-based co-culture) on the BMP-2-mediated osteogenic differentiation of BMSCs was analyzed using alkaline phosphatase (ALP) staining and activity, alizarin red S (ARS) staining, and RT-qPCR of osteogenic markers. Angiogenic differentiation markers were also analyzed in HUVECs after treatment with HA + BMP-2. The bone regeneration potential of BMP-2 and HA + BMP-2 was analyzed in a rat ectopic model. We found that 1600 kDa HA at 300 µg/mL promoted tube formation by HUVECs in vitro and upregulated the mRNA expression of the angiogenic markers CD31, VEGF, and bFGF. HA inhibited, but conditioned medium from HA-treated HUVECs promoted, the BMP-2-mediated osteogenic differentiation of BMSCs, as indicated by the results of ALP staining and activity, ARS staining, and the mRNA expression of the osteogenic markers RUNX-2, ALP, COLI, and OPN. HA + BMP-2 (50 ng/mL) upregulated the expression of the angiogenesis-related genes VEGF and bFGF in HUVECs and bone regeneration in vivo compared to BMP-2 treatment. In conclusion, the paracrine effect of hyaluronic acid-treated endothelial cells promotes BMP-2-mediated osteogenesis, suggesting the application potential of HA + BMP-2 in bone tissue engineering.

Exploring the expression and potential function of follicle stimulating hormone receptor in extragonadal cells related to abdominal aortic aneurysm.

Follicle stimulating hormone (FSH) is identified to play a role in postmenopausal disease and hypothesized to affect abdominal aortic aneurysm (AAA) onset/progression in postmenopausal women. We aimed to detect FSHR gene expression in AAA tissue and cell types involved in AAA formation. FSH stimulation of human umbilical cord endothelial cells (HUVECs), smooth muscle cells (HUCs) and PMA-differentiated macrophages to assess gene expression of FSHR and various markers. Human macrophages activated with various stimuli were assessed for FSHR gene expression. AAA dataset, AAA tissue samples and AAA-derived smooth muscle cells (SMC) obtained from elderly female donors were assessed for FSHR gene expression. AAA-SMCs were stimulated with FSH to assess its effect on gene expression. Lastly, oxidized low-density-lipoprotein (ox-LDL) uptake and abundance of cell surface protein markers were assessed by flow cytometry after FSH stimulation of human monocytes. FSH stimulation showed similar levels of gene expression in HUVECs and HUCs. Only ACTA2 was downregulated in HUCs. In PMA-differentiated macrophages, gene expression of inflammation markers was unchanged after FSH stimulation. FSHR gene expression was found to be low in the AAA datasets. Female AAA-SMCs show occasional FSHR gene expression at a very low level, yet stimulation with FSH did not affect gene expression of SMC- or inflammation markers. FSH stimulation did not impact ox-LDL uptake or alter cell surface protein expression in monocytes. While FSHR gene expression was detected in human testis tissue, it was below quantification level in all other investigated cell types, even upon activation of macrophages with various stimuli. Despite previous reports, we did not detect FSHR gene expression in various extragonadal cell types, except in occasional female AAA-SMCs. No clear effect on cell activation was observed upon FSH stimulation in any cell type. Our data suggest that a direct effect of FSH in AAA-related extragonadal cells is unlikely to influence AAA.

Controlled drug delivery from chitosan-coated heparin-loaded nanopores anodically grown on nitinol shape-memory alloy

Nitinol (NiTi shape-memory alloy) is an interesting candidate in various medical applications like dental, orthopedic, and cardiovascular devices, owing to its unique mechanical behaviors and proper biocompatibility. The aim of this work is the local controlled delivery of a cardiovascular drug, heparin, loaded onto nitinol treated by electrochemical anodizing and chitosan coating. In this regard, the structure, wettability, drug release kinetics, and cell cytocompatibility of the specimens were analyzed in vitro. The two-stage anodizing process successfully developed a regular nanoporous layer of Ni-Ti-O on nitinol, which considerably decreased the sessile water contact angle and induced hydrophilicity. The application of the chitosan coatings controlled the release of heparin mainly by a diffusional mechanism, where the drug release mechanisms were evaluated by the Higuchi, first-order, zero-order, and Korsmeyer-Pepass models. Human umbilical cord endothelial cells (HUVECs) viability assay also showed the non-cytotoxicity of the samples, so that the best performance was found for the chitosan-coated samples. It is concluded that the designed drug delivery systems are promising for cardiovascular, particularly stent applications.

Abnormal expression of miRNA-122 in cerebral infarction and related mechanism of regulating vascular endothelial cell proliferation and apoptosis by targeting CCNG1

ObjectiveTo analyze the value of serum miRNA-122 expression in the diagnosis, severity, and prognosis of Acute Cerebral Infarction (ACI) and the correlation mechanism of serum miRNA-122 on the proliferation and apoptosis of vascular endothelial cells in ACI. MethodA total of 60 patients with ACI who were admitted to the emergency department of the Taizhou People's Hospital from January 1, 2019, to December 30, 2019, and 30 healthy controls during the same period were selected. General clinical data of all patients at admission were collected. Including age, sex, medical history, and inflammatory factors (C-Reactive Protein [CRP], Interleukin-6 [IL-6], Procalcitonin [PCT], Neutrophil Gelatinase-Associated Lipid carrier protein [NGAL]). The National Institutes of Health Stroke Scale (NIHSS) score at admission and short-term prognosis (the Modified Rankin Score [mRS]) score at 3 months after onset were recorded. The expression level of miRNA-122 in the serum of patients with ACI and normal controls was detected by reverse-transcription quantitative Real-Time Polymerase Chain Reaction (RT-QPCR), and the correlation between the expression level of miRNA-122 in the serum of patients with ACI and the level of inflammatory factors, NIHSS and mRS scores were analyzed. The expression levels of miRNA-122 in the serum of patients with ACI, normal people, and Human Umbilical cord Endothelial Cells (HUVECs) cultured in a blank control group were detected by RT-QPCR and statistically analyzed. MTT and flow cytometry was used to compare the proliferation and apoptosis of vascular endothelial cells in the miRNA-122 mimics and inhibitors transfection groups and the corresponding negative control group. The mRNA and protein levels of apoptosis-related factors Bax, Bcl-2, Caspase-3, and angiogenesis-related proteins Hes1, Notch1, Vascular Endothelial Growth Factors (VEGF), and CCNG1 were detected by RT-QPCR and Western blot. Bioinformatics methods predicted CCNG1 to be the target of miRNA-122, and the direct targeting relationship between CCNG1 and miRNA-122 was verified by a dual-luciferase reporting assay. ResultSerum miRNA-122 expression in patients with ACI was significantly higher than that in healthy controls, with an area under the receiver operating characteristic curve of 0.929, 95% Confidence Interval of 0.875‒0.983, and an optimal cut-off value of 1.397. The expression levels of CRP, IL-6, and NGAL in patients with ACI were higher than those in healthy control groups, p < 0.05; miRNA-122 was positively correlated with CPR, IL-6, NIHSS score, and mRS score. At 48h and 72h, the proliferation rate of HUVECs cells in the miRNA-122 mimics group decreased and the apoptosis rate increased. Cell proliferation rate increased, and apoptosis rate decreased significantly in the groups transfected with miRNA-122 inhibitors. The mRNA and protein levels of pro-apoptotic factors Bax and caspase-3 were significantly increased in the miRNA-122 mimics transfection group, while those of anti-apoptotic factor Bcl-2 were significantly decreased compared to those of the control group. The expression of Bax and Caspase-3 decreased, and the expression of anti-apoptotic factor Bcl-2 increased in the transfected miRNA-122 inhibitors group. mRNA expression levels of Hes1, Notch1, VEGF, and CCNG1 in the miRNA-122 mimic transfected group were significantly decreased, while mRNA expression levels in the miRNA-122 inhibitors transfected group were significantly increased. Bioinformatics showed that there was a miRNA-122 binding site in the 3′UTR region of CCNG1, and dual luciferase assay confirmed that CCNG1 was the target of miRNA-122. ConclusionSerum miRNA-122 increased significantly after ACI, which may be a diagnostic marker of ACI. miRNA-122 may be involved in the pathological process of ACI and is related to the degree of neurological impairment and short-term prognosis in patients with ACI. miRNA-122 may play a regulatory role in ACI by inhibiting cell proliferation, increasing apoptosis, and inhibiting vascular endothelial cell regeneration through the CCNG1 channel.

The relationship between nuclear factor-erythrocyte-related factor 2 and antioxidant enzymes in the placenta of patients with gestational diabetes and the metabolism of umbilical cord endothelial cells.

The paper aimed to explore the relationship between nuclear factor erythroid 2 related factor 2 (Nrf2), antioxidant enzymes and the metabolism of umbilical cord endothelial cells in the placenta of patients with gestational diabetes (GDM). A total of 200 pregnant women who underwent an obstetric examination at the Municipal Maternity and Child Health Hospital from March 2018 to December 2019 were selected, with an average age of 30.91±3.24. According to the plasma glucose level and oral glucose tolerance test (OGTT), pregnant women were divided into a control group and an observation group. Blood samples were collected from these pregnant women, serum was removed, put into a centrifuge tube and stored in the refrigerator of the laboratory at - 80 °C. Placental tissue was collected for biochemical analysis. GSH level was detected by absorbance kit, and serum MDA content was detected by spectrophotometry. The expression levels of Heme oxygenase-1(HO-1), Nrf2, and NQO1 protein in placental tissue were analyzed by gel electrophoresis. Western blot analysis was used to detect the protein expression levels of Bach1 and Keap1 in endothelial cells. PCR real-time analysis was used to detect the expression of GSH and NQO1 mRNA. Results showed that the SOD and GSH levels in the serum of the observation group were lower than those of the control group (P<0.05). The protein expression levels of HO-1, Nrf2 and NQO1 in the observation group were higher than those in the control group (P<0.05). The GSH level of the HNE+ observation group was lower than that of the HNE+ control group before stimulation (P<0.05). 1.5 hours after the stimulation, the GSH levels of the two groups of cells were decreased. After 6 hours, the GSH levels of the two groups began to increase. The GSH level of HUVEC in the HNE+ observation group was lower than that of the HNE+ control group after 48 hours. The expression level of Bach1 protein in the HNE+ observation group was lower than that in the HNE+ control group (P<0.05). The expression level of Keap1 protein in the HNE+ observation group and HNE+ control group did not change (P>0.05). The expression levels of GSH and NQO1 mRNA in the HNE+Nrf2 silence group were lower than that in the Nrf2 silence group (P<0.05). The expression levels of GSH and NQO1 mRNA in the HNE+Nrf2 overexpression observation group were higher than those of the HNE+Nrf2 silence group (P<0.05). The apoptosis of trophoblast in the placenta of GDM patients was significantly decreased. The continuous lack of redox signals in fetal endothelial cells in patients with gestational diabetes can destroy the defense ability of cells in the uterus against oxidative stress. Nrf2 antioxidant defense pathway can provide therapeutic targets for reducing oxidative stress associated with diabetes and aging.

Healthberry 865® and a Subset of Its Single Anthocyanins Attenuate Oxidative Stress in Human Endothelial In Vitro Models.

Oxidative stress and inflammation play a pivotal role in the development of cardiovascular diseases, an ever-growing worldwide problem. As a non-pharmacological approach, diet, especially a flavonoid-rich diet, showed promising results in the reduction of cardiovascular diseases and alleviation of their symptoms. In this study, in vitro systems based on human microvascular endothelial cells (hmvEC) and human umbilical cord endothelial cells (HUVEC) were established to determine the effect of Healthberry 865® (HB) and ten of its relating single anthocyanins on oxidative stress. Furthermore, five metabolites were used in order to examine the effect of anthocyanin’s most common breakdown molecules. The results showed an effect of HB in both models after 24 h, as well as most of its single anthocyanins. Cyanidin-rutinoside, peonidin-galactoside, and petunidin-glucoside had a model-specific effect. For the metabolites, phloroglucinaldeyhde (PGA) showed an effect in both models, while vanillic acid (VA) only had an effect in HUVEC. When combined, a combination of several anthocyanins did not have a cumulative effect, except for combining glucosides in hmvEC. The combination of PGA and VA even revealed an inhibitive behavior. Overall, the study demonstrates the antioxidative effect of HB and several of its single anthocyanins and metabolites, which are partially model specific, and coincides with animal studies.

In vitro modeling of liver fibrosis in 3D microtissues using scalable micropatterning system.

Liver fibrosis is the late consequence of chronic liver inflammation which could eventually lead to cirrhosis, and liver failure. Among various etiological factors, activated hepatic stellate cells (aHSCs) are the major players in liver fibrosis. To date, various in vitro liver fibrosis models have been introduced to address biological and medical questions. Availability of traditional in vitro models could not fully recapitulate complicated pathology of liver fibrosis. The purpose of this study was to develop a simple and robust model to investigate the role of aHSCs on the progression of epithelial to mesenchymal transition (EMT) in hepatocytes during liver fibrogenesis. Therefore, we applied a micropatterning approach to generate 3D co-culture microtissues consisted of HepaRG and human umbilical cord endothelial cells (HUVEC) which co-cultured with inactivated LX-2 cells or activated LX-2 cells, respectively, as normal or fibrotic liver models in vitro. The result indicated that the activated LX-2 cells could induce EMT in HepaRG cells through activation of TGF-β/SMAD signaling pathway. Besides, in the fibrotic microtissue, physiologic function of HepaRG cells attenuated compared to the control group, e.g., metabolic activity and albumin secretion. Moreover, our results showed that after treatment with Galunisertib, the fibrogenic properties decreased, in the term of gene and protein expression. In conclusion, it is proposed that aHSCs could lead to EMT in hepatocytes during liver fibrogenesis. Furthermore, the scalable micropatterning approach could provide enough required liver microtissues to prosper our understanding of the mechanisms involved in the progression of liver fibrosis as well as high throughput (HT) drug screening.

Loss of CXC-Chemokine Receptor 1 Expression in Chorioamnionitis Is Associated with Adverse Perinatal Outcomes.

Background: Chorioamnionitis complicates about 1–5% of deliveries at term and causes about one-third of stillbirths. CXC-chemokine receptor 1 (CXCR1) binds IL-8 with high affinity and regulates neutrophil recruitment. We aimed to determine the immunoexpression of CXCR1 in placentas with chorioamnionitis, and its association with adverse perinatal outcomes. Methods: A total of 101 cases of chorioamnionitis and 32 cases of non-chorioamnionitis were recruited over a period of 2 years. CXCR1 immunohistochemistry was performed, and its immunoexpression in placentas was evaluated. The adverse perinatal outcomes included intrauterine death, poor APGAR score, early neonatal death, and respiratory complications. Results: Seventeen cases (17/101, 16.8%) with chorioamnionitis presented as preterm deliveries. Lung complications were more common in mothers who were >35 years (p = 0.003) and with a higher stage in the foetal inflammatory response (p = 0.03). Notably, 24 cases (23.8%) of histological chorioamnionitis were not detected clinically. Interestingly, the loss of CXCR1 immunoexpression in the umbilical cord endothelial cells (UCECs) was significantly associated with foetal death (p = 0.009). Conclusion: The loss of CXCR1 expression in UCECs was significantly associated with an increased risk of adverse perinatal outcomes and could be used as a biomarker to predict adverse perinatal outcomes in chorioamnionitis. Further study is warranted to study the pathophysiology involved in the failure of CXCR1 expression in these cells.

An Easy-to-Fabricate Microfluidic Shallow Trench Induced Three-Dimensional Cell Culturing and Imaging (STICI3D) Platform.

Compared to the established monolayer approach of two-dimensional cell cultures, three-dimensional (3D) cultures more closely resemble in vivo models; that is, the cells interact and form clusters mimicking their organization in native tissue. Therefore, the cellular microenvironment of these 3D cultures proves to be more clinically relevant. In this study, we present a novel easy-to-fabricate microfluidic shallow trench induced 3D cell culturing and imaging (STICI3D) platform, suitable for rapid fabrication as well as mass manufacturing. Our design consists of a shallow trench, within which various hydrogels can be formed in situ via capillary action, between and fully in contact with two side channels that allow cell seeding and media replenishment, as well as forming concentration gradients of various molecules. Compared to a micropillar-based burst valve design, which requires sophisticated microfabrication facilities, our capillary-based STICI3D can be fabricated using molds prepared with simple adhesive tapes and razors alone. The simple design supports the easy applicability of mass-production methods such as hot embossing and injection molding as well. To optimize the STICI3D design, we investigated the effect of individual design parameters such as corner radii, trench height, and surface wettability under various inlet pressures on the confinement of a hydrogel solution within the shallow trench using Computational Fluid Dynamics simulations supported with experimental validation. We identified ideal design values that improved the robustness of hydrogel confinement and reduced the effect of end-user dependent factors such as hydrogel solution loading pressure. Finally, we demonstrated cultures of human mesenchymal stem cells and human umbilical cord endothelial cells in the STICI3D to show that it supports 3D cell cultures and enables precise control of cellular microenvironment and real-time microscopic imaging. The easy-to-fabricate and highly adaptable nature of the STICI3D platform makes it suitable for researchers interested in fabricating custom polydimethylsiloxane devices as well as those who are in need of ready-to-use plastic platforms. As such, STICI3Ds can be used in imaging cell–cell interactions, angiogenesis, semiquantitative analysis of drug response in cells, and measurement of transport through cell sheet barriers.

Tumor-Derived Extracellular Vesicles Induce CCL18 Production by Mast Cells: A Possible Link to Angiogenesis.

Mast cells (MCs) function as a component of the tumor microenvironment (TME) and have both pro- and anti-tumorigenic roles depending on the tumor type and its developmental stage. Several reports indicate the involvement of MCs in angiogenesis in the TME by releasing angiogenic mediators. Tumor cells and other cells in the TME may interact by releasing extracellular vesicles (EVs) that affect the cells in the region. We have previously shown that tumor-derived microvesicles (TMVs) from non-small-cell lung cancer (NSCLC) cells interact with human MCs and activate them to release several cytokines and chemokines. In the present study, we characterized the MC expression of other mediators after exposure to TMVs derived from NSCLC. Whole-genome expression profiling disclosed the production of several chemokines, including CC chemokine ligand 18 (CCL18). This chemokine is expressed in various types of cancer, and was found to be associated with extensive angiogenesis, both in vitro and in vivo. We now show that CCL18 secreted from MCs activated by NSCLC-TMVs increased the migration of human umbilical cord endothelial cells (HUVECs), tube formation and endothelial- to-mesenchymal transition (EndMT), thus promoting angiogenesis. Our findings support the conclusion that TMVs have the potential to influence MC activity and may affect angiogenesis in the TME.

Static Adhesion Assay for Human Peripheral Blood Mononuclear Cells.

Blood endothelial cells (ECs) constitute the primary physical barrier to be crossed by circulating leukocytes, once attracted to a site of ongoing inflammation/infection. Upon a pro-inflammatory stimulus, such as tumor necrosis factor (TNF), ECs upregulate adhesion molecule expression to favor the adhesion and, subsequently, the transendothelial migration of the attracted lymphocytes. To address the ability of a cell to transmigrate through a monolayer of ECs, the classical transmigration assay is usually performed (Muller and Luscinskas, 2008). In the present protocol, adapted from Safuan et al. (2012), we describe an in vitro assay for assessing the functionality of the second step of the transendothelial migration, i.e., the firm adhesion of peripheral blood mononuclear cells (PBMCs) to ECs, under static conditions. By pre-incubating primary human umbilical cord ECs (HUVECs) with either innate lymphoid cell progenitors (ILCPs) or TNF, we were able to upregulate adhesion molecules on the EC surface. Then, by adding total PBMCs, we were able to both quantitatively and qualitatively analyze the cellular subtype and number of PBMCs that adhered to the pre-treated ECs. The important advantage of this technique is the possibility to perform functional studies on ECs biology since, differently from transwell-based strategies, it allows a good recovery of ECs at the end of the assay. Overall, this assay enables to interrogate how/if different stimulations/cell types can influence EC ability to retain PBMCs in vitro, under static conditions. Graphic abstract: The workflow of the Static Adhesion Assay.

Samchulkunbi-Tang Alleviates Vascular Endothelial Disorder and Renal Dysfunction in Nitric Oxide-Deficient Hypertensive Rats.

Samchulkunbi-tang (SCT, Shen Zhu Jian pi tang in Chinese) is said to have been first recorded by Zheng Zhi Zhun Sheng during the Ming Dynasty in China. Records of SCT in Korea are known to have been cited in Donguibogam (Dong Yi Bao Jian in Chinese), Uibang Hwaltu (Yi Fang Huo Tao in Chinese), and Bang Yak Hapyeon (Fang Yao He Bian in China). Although SCT is widely used in treating chronic gastritis and gastric ulcers, the beneficial effect on renal vascular function is unknown. Hypertension is a risk factor for cardiovascular disease and endothelial dysfunction in humans and experimental animal models of arterial hypertension. In addition, kidney dysfunction is characterized by hypertension diseases. This study was conducted to evaluate the effect of SCT on the vascular function in vitro (human umbilical cord endothelial cells, HUVECs) and in vivo (NG‐nitro‐L‐arginine methyl ester, L-NAME-induced hypertensive rats). The phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) is closely related to nitric oxide (NO) production in HUVECs, and SCT in this study significantly increased these. For three weeks, hypertensive rat models were induced by L-NAME administration (40 mg/kg/day) with portable water. It was followed by oral administration with 100 and 200 mg/kg/day for two weeks to confirm the effectiveness of SCT. As a result, systolic blood pressure decreased in the SCT-treated groups, compared with that in the L-NAME-induced hypertensive group. SCT treatment restored vasorelaxation by stimulating acetylcholine and cGMP production in the thoracic aorta. In addition, SCT treatment decreased intima-media thickness, attenuated the reduction of eNOS expression, and increased endothelin-1 expression. It also increased p-Akt and p-eNOS expression in hypertensive rat aorta. Furthermore, regarding renal function parameters, SCT ameliorated urine osmolality, urine albumin level, serum creatinine, and blood urea nitrogen levels. These results demonstrate that the oriental medicine SCT exerts potent vascular and renal protective effects on nitric oxide-deficient hypertensive rats and HUVECs

An NIR-Triggered Au Nanocage Used for Photo-Thermo Therapy of Chronic Wound in Diabetic Rats Through Bacterial Membrane Destruction and Skin Cell Mitochondrial Protection.

Bacterial infection and its severe oxidative stress reaction will cause damage to skin cell mitochondria, resulting in long-lasting wound healing and great pain to patients. Thus, delayed wound healing in diabetic patients with Staphylococcus aureus infection is a principal challenge worldwide. Therefore, novel biomaterials with multifunction of bacterial membrane destruction and skin cell mitochondrial protection are urgently needed to be developed to address this challenge. In this work, novel gold cage (AuNCs) modified with epigallocatechin gallate (EGCG) were prepared to treat delayed diabetic wounds. The results showed that Au-EGCG had a high and stable photothermal conversion efficiency under near-infrared irradiation, and the scavenging rate of Au-EGCG for S. aureus could reach 95%. The production of large amounts of reactive oxygen species (ROS) leads to the disruption of bacterial membranes, inducing bacterial lysis and apoptosis. Meanwhile, Au-EGCG fused into hydrogel (Au-EGCG@H) promoted the migration and proliferation of human umbilical cord endothelial cells, reduced cellular mitochondrial damage and oxidative stress in the presence of infection, and significantly increased the basic fibroblast growth factor expression and vascular endothelial growth factor. In addition, animal studies showed that wound closure was 97.2% after 12 days of treatment, and the healing of chronic diabetic wounds was significantly accelerated. Au-EGCG nanoplatforms were successfully prepared to promote cell migration and angiogenesis in diabetic rats while removing S. aureus, reducing oxidative stress in cells, and restoring impaired mitochondrial function. Au-EGCG provides an effective, biocompatible, and multifunctional therapeutic strategy for chronic diabetic wounds.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells

PurposeLimbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. MethodsHuman corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. ResultsLMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. ConclusionThese findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

Flow-through isolation of human first trimester umbilical cord endothelial cells

Human umbilical vein and artery endothelial cells (HUVEC; HUAEC), placental endothelial cells (fpAEC), and endothelial colony-forming cells (ECFC) from cord blood are a widely used model for researching placental vascular development, fetal and placental endothelial function, and the effect of adverse conditions in pregnancy thereon. However, placental vascular development and angiogenesis start in the first weeks of gestation, and adverse conditions in pregnancy may also affect endothelial function before term, suggesting that endothelial cells from early pregnancy may respond differently. Thus, we established a novel, gentle flow-through method to isolate pure human umbilical endothelial cells from first trimester (FTUEC). FTUEC were characterized and their phenotype was compared to the umbilical endothelium in situ as well as to other fetal endothelial cell models from term of gestation, i.e. HUVEC, fpAEC, ECFC. FTUEC possess a CD34-positive, juvenile endothelial phenotype, and can be expanded and passaged. We regard FTUEC as a valuable tool to study developmental processes as well as the effect of adverse insults in pregnancy in vitro.

Endothelial cells from umbilical cord of women affected by gestational diabetes: A suitable in vitro model to study mechanisms of early vascular senescence in diabetes.

Human umbilical cord endothelial cells (HUVECs) obtained from women affected by gestational diabetes (GD-HUVECs) display durable pro-atherogenic modifications and might be considered a valid in vitro model for studying chronic hyperglycemia effects on early endothelial senescence. Here, we demonstrated that GD- compared to C-HUVECs (controls) exhibited oxidative stress, altered both mitochondrial membrane potential and antioxidant response, significant increase of senescent cells characterized by a reduced NAD-dependent deacetylase sirtuin-1 (SIRT1) activity together with an increase in cyclin-dependent kinase inhibitor-2A (P16), cyclin-dependent kinase inhibitor-1 (P21), and tumor protein p53 (P53) acetylation. This was associated with the p300 activation, and its silencing significantly reduced the GD-HUVECs increased protein levels of P300 and Ac-P53 thus indicating a persistent endothelial senescence via SIRT1/P300/P53/P21 pathway. Overall, our data suggest that GD-HUVECs can represent an "endothelial hyperglycemic memory" model to investigate in vitro the early endothelium senescence in cells chronically exposed to hyperglycemia in vivo.