Human Umbilical Endothelial Cells Research Articles

Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process

Background: Indoleamine 2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO) are the two principals enzymes involved in the catabolization of tryptophan (Trp) into kynurenine (Kyn). Despite their well-established role in the immune escape, their involvement in angiogenesis remains uncertain. We aimed to characterize TDO and IDO1 in human umbilical venular endothelial cells (HUVECs) and human endothelial colony-forming cells (ECFCs). Methods: qRT-PCR and immunofluorescence were used for TDO and IDO1 expression while their activity was measured using ELISA assays. Cell proliferation was examined via MTT tests and in in vitro angiogenesis by capillary morphogenesis. Results: HUVECs and ECFCs expressed TDO and IDO1. Treatment with the selective TDO inhibitor 680C91 significantly impaired HUVEC proliferation and 3D-tube formation in response to VEGF-A, while IDO1 inhibition showed no effect. VEGF-induced mTor phosphorylation and Kyn production were hindered by 680C91. ECFC morphogenesis was also inhibited by 680C91. Co-culturing HUVECs with A375 induced TDO up-regulation in both cell types, whose inhibition reduced MMP9 activity and prevented c-Myc and E2f1 upregulation. Conclusions: HUVECs and ECFCs express the key enzymes of the kynurenine pathway. Significantly, TDO emerges as a pivotal player in in vitro proliferation and capillary morphogenesis, suggesting a potential pathophysiological role in angiogenesis beyond its well-known immunomodulatory effects.

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.

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

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

Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

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

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

Ca2+ signals in human umbilical endothelial cells derived from pregnancy with fetal growth restriction associated with hypertensive disorder.

Fetal growth restriction associated with hypertensive disorders of pregnancy (FGR-HDP) is a prevalent pathology with a higher risk of perinatal morbimortality. In this condition, placental insufficiency and endothelial dysfunction serve key roles. The present prospective cohort study monitored 11 patients with an FGR-HDP and 15 with full-term normotensive pregnancies and studied post-natal intracellular calcium concentration ([Ca2+]i) signals in human umbilical vein endothelial cells (HUVECs). Small fetuses with placental insufficiency were identified using fetal biometry with Doppler velocimetry. Mean gestational age and birth weight were 31.8±4.1 weeks and 1,260±646 g for FGR-HDP and 39.2±0.8 weeks and 3,320±336 g for normal births, respectively. Abnormal umbilical artery Doppler waveforms were found in 64% of neonates with FGR-HDP. A significant percentage (86%) of FGR newborns were admitted to the neonatal intensive care unit at Gustavo Fricke hospital, Viña del Mar, Chile, with one case of death after birth. [Ca2+]i signals were measured by microfluorimetry in Fluo-3-loaded HUVECs from primary cultures. Altered [Ca2+]i signals were observed in HUVECs from FGR-HDP, where the sustained phase of ATP-induced [Ca2+]i responses was significantly reduced compared with the normotensive group. Also, the [Ca2+]i signals induced with 10 mM Ca2+ after depletion of internal Ca2+ stores were significantly higher. The present study provides a better comprehension of the role of altered cytosolic Ca2+ dynamics in endothelial dysfunction and an in vitro model to assess novel therapeutic approaches for decreasing or preventing complications in FGR-HDP.

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2 O2 -induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

An imprint-based approach to replicate nano- to microscale roughness on gelatin hydrogel scaffolds: surface characterization and effect on endothelialization

Biologization of biomaterials with endothelial cells (ECs) is an important step in vascular tissue engineering, aiming at improving hemocompatibility and diminishing the thrombo-inflammatory response of implants. Since subcellular topography in the scale of nano to micrometers can influence cellular adhesion, proliferation, and differentiation, we here investigate the effect of surface roughness on the endothelialization of gelatin hydrogel scaffolds. Considering the micron and sub-micron features of the different native tissues underlying the endothelium in the body, we carried out a biomimetic approach to replicate the surface roughness of tissues and analyzed how this impacted the adhesion and proliferation of human umbilical endothelial cells (HUVECs). Using an imprinting technique, nano and micro-roughness ranging from Sa= 402 nm to Sa= 8 μm were replicated on the surface of gelatin hydrogels. Fluorescent imaging of HUVECs on consecutive days after seeding revealed that microscale topographies negatively affect cell spreading and proliferation. By contrast, nanoscale roughnesses of Sa= 402 and Sa= 538 nm promoted endothelialization as evidenced by the formation of confluent cell monolayers with prominent VE-cadherin surface expression. Collectively, we present an affordable and flexible imprinting method to replicate surface characteristics of tissues on hydrogels and demonstrate how nanoscale roughness positively supports their endothelialization.

Multi-comparted microgels delivering human derived probiotics and deferoxamine for multidrug-resistant infection and healing

Infection and vascular dysfunction are two major reasons for delayed wound healing, especially in the scenario of multidrug-resistant bacterial has been a challenge for antibiotics usage. Probiotics are effective in fighting pathogens, while avoiding the destruction of skin microbiome. However, the viability of probiotics is a major obstacle in clinical application. As a non-protein proangiogenic drug, deferoxamine (DFO) has significant advantages over traditional growth factors in terms of cost, storage and transportation conditions, and stability. However, there is still a problem of short half-life that requires reliable drug delivery carriers to maximize its effect. Here, we developed multiple-chambered microgels using electrospray and microfluidics. Lactobacillus fermentum (LF) and DFO, respectively, for the management of multidrug-resistant Pseudomonas aeruginosa (MPA) and promotion of wound healing. We first tested five probiotics isolated from healthy people and found LF inhibiting the growth of MPA by lowering local pH and antimicrobial substances. The multi-chambered microgels effectively inhibited MPA by delivering probiotics, they can also protect probiotics from antibiotics. DFO promoted the angiogenesis of human umbilical venous endothelial cells. The microgel system had good biocompatibility and hemocompatibility. In a MPA infected wound, the microgel system effectively alleviated wound infection by 87.44%, improved angiogenesis by 29.32% and accelerated wound healing. This work suggests that multi-compartmented microgels loaded with probiotics and deferoxamine have great potential in wounds with multidrug-resistant bacteria.

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.

Improvement of Endothelial Cell-Polycaprolactone Interaction through Surface Modification via Aminolysis, Hydrolysis, and a Combined Approach

Polycaprolactone (PCL) is a promising material for the fabrication of alternatives to autologous grafts used in coronary bypass surgery. PCL biodegrades over time, allowing cells to infiltrate the polymeric matrix, replacing the biodegrading graft, and creating a fully functional vessel constituted of autologous tissue. However, the high hydrophobicity of PCL is associated with poor cell affinity. Surface modification of PCL can increase this cell affinity, making PCL an improved scaffold material for acellular vascular grafts. In this study, the surface of PCL films was modified by hydrolysis, aminolysis, and the combination thereof to introduce carboxyl, hydroxyl, and amino groups on the surface. Only the hydrolyzed films exhibited a significant increase in their hydrophilicity, although further testing showed that all aminolysis conditions had amino groups on the surface. Furthermore, in vitro experiments with human umbilical endothelial cells (HUVECs) were performed to assess changes in cell affinity for PCL due to the surface treatments. PCL treated with sodium hydroxide (NaOH), a hydrolysis reaction, showed a significant increase in endothelial cell adhesion after 24 hours with a significant increase in cell survival after 72 hours. Thus, NaOH treatment improves the biocompatibility and endothelialization of PCL, creating a competent candidate for artificial, acellular, biodegradable vascular grafts.

Protective role of CXCR7 activation in neonatal hyperoxia-induced systemic vascular remodeling and cardiovascular dysfunction in juvenile rats

Neonatal hyperoxia induces long-term systemic vascular stiffness and cardiovascular remodeling, but the mechanisms are unclear. Chemokine receptor 7 (CXCR7) represents a key regulator of vascular homeostasis and repair by modulating TGF-β1 signaling. This study investigated whether pharmacological CXCR7 agonism prevents neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction in juvenile rats. Newborn Sprague Dawley rat pups assigned to room air or hyperoxia (85% oxygen), received CXCR7 agonist, TC14012 or placebo for 3 weeks. These rat pups were maintained in room air until 6 weeks when aortic pulse wave velocity doppler, cardiac echocardiography, aortic and left ventricular (LV) fibrosis were assessed. Neonatal hyperoxia induced systemic vascular stiffness and cardiac dysfunction in 6-week-old rats. This was associated with decreased aortic and LV CXCR7 expression. Early treatment with TC14012, partially protected against neonatal hyperoxia-induced systemic vascular stiffness and improved LV dysfunction and fibrosis in juvenile rats by decreasing TGF-β1 expression. In vitro, hyperoxia-exposed human umbilical arterial endothelial cells and coronary artery endothelial cells had increased TGF-β1 levels. However, treatment with TC14012 significantly reduced the TGF-β1 levels. These results suggest that dysregulation of endothelial CXCR7 signaling may contribute to neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction.

S1P receptor-1 signaling reduces arterial thrombosis by upregulation of endothelial thrombomodulin expression

Abstract Background Sphingosine-1-phosphate (S1P) is a bioactive lipid known for its cardioprotective effects. The extent to which S1P also has effects on arterial thrombosis has not yet been fully elucidated. Another important factor in arterial thrombosis is thrombomodulin (TM), an important endothelial antithrombotic factor. To what extent S1P, TM, and, in addition, platelet adhesion as an initial part influences arterial thrombosis is unclear and the aim of this work. Methods The influence of S1P on human umbilical cord venous endothelial cells (HUVECs) was investigated in cell culture. To investigate effects of S1P and TM on platelet adhesion, flow chamber experiments were performed. In addition, murine arterial thrombus formation was analyzed by intravital microscopy. Results S1P increased TM expression on HUVECs. This effect was abolished by S1P receptor1 (S1PR1) inhibition with W146 (S1P: 1.081 ± 0.091 vs. S1P + W146: 0.951 ± 0.073, normalized to control, p&amp;lt;0.0001). Sphingosine kinase-2 deficient mice (SphK1-/-) lack the kinase primarily producing sphingosine. Accordingly, these animals have less S1P. Here, TM expression was significantly lower in the aorta compared to wild-type mice (WT: 207.2 ± 45.8 pg/g aorta vs. SphK1-/- 124.5 ± 40.2 pg/g aorta, p&amp;lt;0.0001). S1P treatment decreased platelet adhesion from whole blood to endothelial cells in the flow chamber (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p&amp;lt;0.0001). W146 reversed this effect by S1PR1 inhibition (S1P: 80.91 ± 22.85%, vs. S1P + W146: 118.60 ± 33.65%, p=0.0067). Platelet adhesion to endothelial cells was assessed in the presence of a TM-neutralizing antibody. The TM antibody abolished the anti-adhesive effects of S1P (S1P: 54.79 ± 29.59% vs. S1P+AB: 105.54 ± 35.43%, p=0.0174). TM inhibited intravital murine arterial thrombus formation (time to first occlusion: con: 9.38 ± 1.02 min vs. TM: 60 ± 0 min, p=0.0286). Furthermore, the duration of the first occlusion was significantly shorter and the embolic rate as a marker for thrombus stability was higher. Conclusion We demonstrated that S1P inhibits arterial thrombosis via regulation of endothelial TM. In particular, platelet adhesion to endothelial cells is reduced. This effect is mediated via S1PR1. The extent to which S1PR1 represents a potential antithrombotic target must be clarified in future studies.

Controlled release of therapeutic antibody using hydrolytically degradable microgels.

Monoclonal antibodies have gained significant interest as potential therapeutics for treating various diseases. However, these therapies are not always effective due to poor treatment compliance associated with multiple administrations and drug resistance. Thus, there is a growing interest in developing advanced monoclonal antibody delivery systems that can customize pharmacokinetics to enhance therapeutic outcomes. This work aimed to engineer hydrolytic 4-arm PEG maleimide (PEG-4MAL) microgels for the controlled delivery of therapeutic antibodies, specifically anti-angiogenic bevacizumab, to overcome the limitations of current monoclonal antibody therapies. Through a PEGylation reaction with a thiol-terminated PEG linker, the antibody was covalently conjugated to the macromer backbone before microgel synthesis. The PEGylation reaction was simple, effective, and did not affect antibody bioactivity. Antibody release kinetics was tuned by changing the concentration of the hydrolytic linker (0-2 mM) and/or PEG-4MAL:protein molar ratio (1000:1, 2000:1, and 5000:1) in the macromer precursor solution during microgel fabrication. The bioactivity of the released antibody was assessed on human umbilical endothelial vascular cells (HUVEC), demonstrating that extracts from hydrolytic microgels reduced cell proliferation over time. Collectively, this study demonstrates the development of highly tunable delivery platform based on degradable PEG-4MAL microgels that can be adapted for therapeutic antibody-controlled release.

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.

Effects of Acetyl-L-Carnitine on Oxidative Stress in Amyotrophic Lateral Sclerosis Patients: Evaluation on Plasma Markers and Members of the Neurovascular Unit.

Oxidative stress, the alteration of mitochondrial function, and the neurovascular unit (NVU), play a role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis. We aimed to demonstrate the changes in the plasma redox system and nitric oxide (NO) in 32 new ALS-diagnosed patients in treatment with Acetyl-L-Carnitine (ALCAR) compared to healthy controls. We also evaluated the effects of plasma on human umbilical cord-derived endothelial vascular cells (HUVEC) and astrocytes. The analyses were performed at the baseline (T0), after three months (T1), and after six months (T2). In ALS patients at T0/T1, the plasma markers of lipid peroxidation, thiobarbituric acid reactive substances (TBARS) and 4-hydroxy nonenal (4-HNE) were higher, whereas the antioxidants, glutathione (GSH) and the glutathione peroxidase (GPx) activity were lower than in healthy controls. At T2, plasma TBARS and 4-HNE decreased, whereas plasma GSH and the GPx activity increased in ALS patients. As regards NO, the plasma levels were firmly lower at T0-T2 than those of healthy controls. Cell viability, and mitochondrial membrane potential in HUVEC/astrocytes treated with the plasma of ALS patients at T0-T2 were reduced, while the oxidant release increased. Those results, which confirmed the fundamental role of oxidative stress, mitochondrial function, and of the NVU in ALS pathogenesis, can have a double meaning, acting as disease markers at baseline and potential markers of drug effects in clinical practice and during clinical trials.

Investigation of biodegradability and cellular activity of PCL/PLA and PCL/PLLA electrospun webs for tissue engineering applications.

Biodegradability and cellular activity are key performance indicators that should be prioritized for tissue engineering applications. Biopolymer selection, determination of necessary structural properties, and their synergistic interactions play an active role in obtaining the expected biodegradability and biological activity from scaffolds. In this study, it is aimed to produce electrospun webs with improved biocompatibility by blending polycaprolactone (PCL) with polylactic acid (PLA) and poly-l-lactide (PLLA), and examine the effect of biopolymer selection and blend ratio on the biodegradability and cellular activity of surfaces. In this context, fibrous webs are produced from PCL/PLA and PCL/PLLA blends with a weight ratio of 80/20 and 50/50 and pure polymers of PCL, PLA, and PLLA by electrospinning method and subjected to morphological and biological analyses. The biodegradation tests are carried out hydrolytically while the cell viability and cell proliferation analyses are performed with adult human primary dermal fibroblasts and human umbilical endothelial cells (HUVECs). The results show that the fiber diameters of the fabricated webs ranged from 0.747 to 1.685 μm. At the end of the 5th month, it is observed that the biodegradation rates of the webs blended 50% with PLA and PLLA, in comparison to PCL ones, increase from 3.7% to 13.33% and 7.69%, respectively. On the other hand, cell culture results highlight that the addition of 20% PLA and PLLA improves the cellular activity of both cell types, but increased PLA or PLLA ratio in PCL webs has a negative effect as it makes the structure stiff and brittle.

DZ2002 alleviates corneal angiogenesis and inflammation in rodent models of dry eye disease via regulating STAT3-PI3K-Akt-NF-κB pathway.

Dry eye disease (DED) is a prevalent ocular disorder with a multifactorial etiology. The pre-angiogenic and pre-inflammatory milieu of the ocular surface plays a critical role in its pathogenesis. DZ2002 is a reversible type III S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitor, which has shown excellent anti-inflammatory and immunosuppressive activities in vivo and in vitro. In this study, we evaluated the therapeutic potential of DZ2002 in rodent models of DED. SCOP-induced dry eye models were established in female rats and mice, while BAC-induced dry eye model was established in female rats. DZ2002 was administered as eye drops (0.25%, 1%) four times daily (20 μL per eye) for 7 or 14 consecutive days. We showed that topical application of DZ2002 concentration-dependently reduced corneal neovascularization and corneal opacity, as well as alleviated conjunctival irritation in both DED models. Furthermore, we observed that DZ2002 treatment decreased the expression of genes associated with angiogenesis and the levels of inflammation in the cornea and conjunctiva. Moreover, DZ2002 treatment in the BAC-induced DED model abolished the activation of the STAT3-PI3K-Akt-NF-κB pathways in corneal tissues. We also found that DZ2002 significantly inhibited the proliferation, migration, and tube formation of human umbilical endothelial cells (HUVECs) while downregulating the activation of the STAT3-PI3K-Akt-NF-κB pathway. These results suggest that DZ2002 exerts a therapeutic effect on corneal angiogenesis in DED, potentially by preventing the upregulation of the STAT3-PI3K-Akt-NF-κB pathways. Collectively, DZ2002 is a promising candidate for ophthalmic therapy, particularly in treating DED.

Hypoxia Affects Autophagy in Human Umbilical Vein Endothelial Cells via the IRE1 Unfolded Protein Response.

The purpose of this study was to investigate the role of the unfolded protein response, specifically the inositol-requiring enzyme 1 (IRE1) signaling pathway, in hypoxia-induced autophagy in human umbilical venous endothelial cells (HUVECs). The expression of IRE1 and autophagy relative protein in HUVECs with hypoxia was explored by Western blotting, qRT-PCR and confocal microscopy. Further, we evaluated the biological effects of HUVECs by tube formation assay and wound healing assay in vitro. Finally, we examined the function of IRE1 in local blood vessels through animal models. Hypoxia activated the IRE1 signaling pathway and induced autophagy in a time-dependent manner in HUVECs and further influenced the biological effects of HUVECs. Intraperitoneal injection of IRE1 inhibitors inhibited local vascular autophagy levels and lipid accumulation in model animals. Hypoxia can induce autophagy and activate the IRE1 signaling pathway in HUVECs and the IRE1 signaling pathway is involved in autophagy in hypoxic conditions.