Co-culture Of Endothelial Cells Research Articles

IPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

EV-Elute: A universal platform for the enrichment of functional surface marker-defined extracellular vesicle subpopulations.

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, EV functionality likely differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers. Here, we used the Design-of-Experiments (DoE) methodology to optimize a protocol, which we name 'EV-Elute', to elute intact EVs from commercially available Protein G-coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH- and incubation-time-dependent fashion. EV subpopulations showed intact integrity by electron microscopy and Proteinase K protection assays and showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells (PBMCs) and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs. Taken together, we developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined EV subpopulations. This platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV-Elute can open new opportunities to study EV functionality at the subpopulation level.

Engineering Granular Hydrogels without Interparticle Cross-Linking to Support Multicellular Organization.

Advancing three-dimensional (3D) tissue constructs is central to creating in vitro models and engineered tissues that recapitulate biology. Materials that are permissive to cellular behaviors, including proliferation, morphogenesis of multicellular structures, and motility, will support the emergence of tissue structures. Granular hydrogels in which there is no interparticle cross-linking exhibit dynamic properties that may be permissive to such cellular behaviors. However, designing granular hydrogels that lack interparticle cross-linking but support cellular self-organization remains underexplored relative to granular systems stabilized by interparticle cross-linking. In this study, we developed a polyethylene glycol-based granular hydrogel system, with average particle diameters under 40 μm. This granular hydrogel exhibited bulk stress-relaxing behaviors and compatibility with custom microdevices to sustain cell cultures without degradation. The system was studied in conjunction with cocultures of endothelial cells and fibroblasts, known for their spontaneous network formation. Cross-linking, porosity, and cell-adhesive ligands (such as RGD) were manipulated to control system properties. Toward supporting cellular activity, increased porosity was found to enhance the formation of cellular networks, whereas RGD reduced network formation in the system studied. This research highlights the potential of un-cross-linked granular systems to support morphogenetic processes, like vasculogenesis and tissue maturation, offering insights into material design for 3D cell culture systems.

Engineering Biomimetic Microvascular Capillary Networks in Hydrogel Fibrous Scaffolds via Microfluidics-Assisted Co-Axial Wet-Spinning.

The microvascular bed plays a crucial role in establishing nutrient exchange and waste removal, as well as maintaining tissue metabolic activity in the human body. However, achieving microvascularization of engineered 3D tissue constructs is still an unsolved challenge. In this work, we developed biomimetic cell-laden hydrogel microfibers recapitulating oriented microvascular capillary-like networks by using a 3D bioprinting technique combined with microfluidics-assisted coaxial wet-spinning. Highly packed and aligned bundles embedding a coculture of human bone marrow-derived mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were produced by simultaneously extruding two different bioinks. To this aim, core-shell fibers were wet-spun in a coagulation bath to collect the scaffolds later on a rotary drum. Initially, the versatility of the proposed system was assessed for the extrusion of multimaterial core-shell hydrogel fibers. Subsequently, the platform was validated for the in vitro biofabrication of samples promoting optimal cell alignment along the fiber axis. After 3 weeks of culture, such fiber configuration resulted in the development of an oriented capillary-like network within the fibrin-based core and in the endothelial-specific CD31 marker expression upon MSC/HUVEC maturation. Synergistically, the vertical arrangement of the coaxial nozzle coupled with the rotation of the fiber collector facilitated the rapid creation of tightly packed bundles characterized by a dense, oriented, and extensively branched capillary network. Notably, such findings suggest that the proposed biofabrication strategy can be used for the microvascularization of tissue-specific 3D constructs.

Deciphering influence of donor age on adipose-derived stem cells: in vitro paracrine function and angiogenic potential

Background: As fat grafting is commonly used as a filler, Adipose-derived stem/stromal cells (ASC) have been reported to be key player in retention rate. Paracrine and differentiation potential of those cells confer them strong pro-angiogenic capacities. However, a full characterization of the influence of aging on ASC has not been reported yet. Here we’ve investigated the effect of age on paracrine function, stemness and angiogenic potential of ASC. Methods: ASC were extracted from young and old adult donors. We assessed stromal vascular fraction cell populations repartition, ASC stemness potential, capability to differentiate into mesenchymal lineages as well as their secretome. Angiogenic potential was assessed using a sprouting assay, an indirect co-culture of ASC and dermal microvascular endothelial cells (EC). Total vascular sprout length was measured, and co-culture soluble factors were quantified. Pro-angiogenic factors alone or in combination as well as ASC-conditioned medium (CM) were added to EC to assess sprouting induction. Results: Decrease of endothelial cells yield and percentage is observed in cells extracted from adipose tissue of older patients, whereas ASC percentage increased with age. Clonogenic potential of ASC is stable with age. ASC can differentiate into adipocytes, chondrocytes and osteoblasts, and aging does not alter this potential. Among the 25 analytes quantified, high levels of pro-angiogenic factors were found, but none is significantly modulated with age. ASC induce a significantly longer vascular sprouts compared to fibroblasts, and no difference was found between young and old ASC donors on that parameter. Higher concentrations of FGF-2, G-CSF, HGF and IL-8, and lower concentrations of VEGF-C were quantified in EC/ASC co-cultures compared to EC/fibroblasts co-cultures. EC/ASC from young donors secrete higher levels of VEGF-A compared to old ones. Neither soluble factor nor CM without cells are able to induce organized sprouts, highlighting the requirement of cell communication for sprouting. CM produced by ASC supporting development of long vascular sprouts promote sprouting in co-cultures that establish shorter sprouts. Conclusion: Our results show cells from young and old donors exhibit no difference in all assessed parameters, suggesting all patients could be included in clinical applications. We emphasized the leading role of ASC in angiogenesis, without impairment with age, where secretome is a key but not sufficient actor.

Expression of soluble guanylate cyclase (sGC) and its ability to form a functional heterodimer are crucial for reviving the NO-sGC signaling in PAH

In order to determine the underpinnings of a dysfunctional NO-sGC signal pathway which occurs in pulmonary arterial hypertension (PAH), we investigated pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients. We found low expression of sGC, a poor sGCα1β1 heterodimer and this correlated with low expression of its facilitator chaperon, hsp90. Treating PASMCs overnight (16 h) with low micromolar doses of a slow release NO donor DETANONOate, reinstated the sGCα1β1 heterodimer and restored its NO-heme dependent activity. Transwell co-culture of HEK cells stably expressing eNOS with PAH PASMCs also restored the sGC heterodimer and its heme-dependent activity with sGC stimulator, BAY 41–2272. To determine whether the dysfunctionality in the NO-sGC pathway stems from a dysfunctional eNOS producing negligible NO, we did transwell co-cultures of pulmonary arterial endothelial cells (PAECs) with PASMCs. Our results indicated that PAECs from both control and PAH samples when activated for eNOS restored both sGC heterodimer and its heme-dependent sGC activity in the corresponding PASMCs, suggesting that PAECs from PAH can also generate NO. In line with these results expression of eNOS, its support chaperon hsp90, its specific kinase Akt, p-Akt or post-translational modifications (PTMs) like OGlcNAc or phospho-tyrosine were unchanged in PAH relative to controls. Additionally there was uniform expression of Hbα/β and Mb in PASMCs or PAECs in PAH or controls and these globins can effectively scavenge the eNOS generated NO, as there was evidence of strong eNOS-Hb/Mb interactions. Our studies suggest that factors such as globin NO scavenging along with vascular remodeling in PAH can cause hampered vasodilation which in the face of poor NO levels as occurs in PAH are additional impediments for effective vasodilation. However importantly our studies suggests that future therapies can use low doses of NO along with sGC stimulators as a potential drug for PAH subjects.

Using Polycaprolactone Nanofibers for the Proof-of-Concept Construction of the Alveolar-Capillary Interface.

The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface invitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.

Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite.

Understanding the niche interactions between blood and bone through the in vitro co-culture of osteo-competent cells and endothelial cells is a key factor in unraveling therapeutic potentials in bone regeneration. This can be additionally supported by employing numerical simulation techniques to assess local physical factors, such as oxygen concentration, and mechanical stimuli, such as shear stress, that can mediate cellular communication. In this study, we developed a Mesenchymal Stem Cell line (MSC) and a Human Umbilical Vein Endothelial Cell line (HUVEC), which were co-cultured under flow conditions in a three-dimensional, porous, natural pullulan/dextran scaffold that was supplemented with hydroxyapatite crystals that allowed for the spontaneous formation of spheroids. After 2 weeks, their viability was higher under the dynamic conditions (>94%) than the static conditions (<75%), with dead cells central in the spheroids. Mineralization and collagen IV production increased under the dynamic conditions, correlating with osteogenesis and vasculogenesis. The endothelial cells clustered at the spheroidal core by day 7. Proliferation doubled in the dynamic conditions, especially at the scaffold peripheries. Lattice Boltzmann simulations showed negligible wall shear stress in the hydrogel pores but highlighted highly oxygenated zones coinciding with cell proliferation. A strong oxygen gradient likely influenced endothelial migration and cell distribution. Hypoxia was minimal, explaining high viability and spheroid maturation in the dynamic conditions.

APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROflUIDICS

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

Zinc and gallium doped borate bioactive glasses influence in-vitro angiogenesis: New evidence in cell co-culture studies

Borate bioactive glasses doped with 1 mol% ZnO or Ga2O3 were produced by melt-quenching. The cytocompatibility of fibroblast and endothelial cells in response to glass extracts was assessed. The angiogenic potential was examined through fibroblast-derived vascular endothelial growth factor secretion and tubular formation analysis in an indirect co-culture of fibroblast and endothelial cells. The results confirm that Zn and Ga ion-releasing bioactive glasses are attractive biomaterials with angiogenic properties.

Bruton's tyrosine kinase inhibition suppresses pathological retinal angiogenesis.

Pathological retinal angiogenesis is a typical manifestation of vision-threatening ocular diseases. Many patients exhibit poor response or resistance to anti-vascular endothelial growth factor (VEGF) agents. Bruton's tyrosine kinase (BTK) controls the proliferation and function of immune cells. Therefore, we examined the anti-inflammatory and anti-angiogenic effects of BTK inhibition on retinal angiogenesis. Retinal neovascularisation and vascular leakage in oxygen-induced retinopathy in C57/BL6J mice were assessed by whole-mount retinal immunofluorescence. PLX5622 was used to deplete microglia and Rag1-knockout mice were used to test the contribution of lymphocytes to the effects of BTK inhibition. The cytokines, activation markers, inflammatory and immune-regulatory activities of retinal microglia/macrophages were detected using qRT-PCR and immunofluorescence. NLRP3 was detected by western blotting, and the effects of BTK inhibition on the co-culture of microglia and human retinal microvascular endothelial cells (HRMECs) were examined. BTK inhibition suppressed pathological angiogenesis and vascular leakage, and significantly reduced retinal inflammation, which involved microglia/macrophages but not lymphocytes. BTK inhibition increased anti-inflammatory factors and reduced pro-inflammatory cytokines that resulted from NLRP3 inflammasome activation. BTK inhibition suppressed the inflammatory activity of microglia/macrophages, and acted synergistically with anti-VEGF without retinal toxicity. Moreover, the supernatant of microglia incubated with BTK-inhibitor reduced the proliferation, tube formation and sprouting of HRMECs. BTK inhibition suppressed retinal neovascularisation and vascular leakage by modulating the inflammatory activity of microglia and macrophages. Our study suggests BTK inhibition as a novel and promising approach for alleviating pathological retinal angiogenesis.

Modelling the maternal-fetal interface: An invitro approach to investigate nutrient and drug transport across the human placenta.

The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological invitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our invitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.

Empowering High-Throughput High-Content Analysis of Microphysiological Models: Open-Source Software for Automated Image Analysis of Microvessel Formation and Cell Invasion.

The primary aim of this study was to develop an open-source Python-based software for the automated analysis of dynamic cell behaviors in microphysiological models using non-confocal microscopy. This research seeks to address the existing gap in accessible tools for high-throughput analysis of endothelial tube formation and cell invasion in vitro, facilitating the rapid assessment of drug sensitivity. Our approach involved annotating over 1000 2mm Z-stacks of cancer and endothelial cell co-culture model and training machine learning models to automatically calculate cell coverage, cancer invasion depth, and microvessel dynamics. Specifically, cell coverage area was computed using focus stacking and Gaussian mixture models to generate thresholded Z-projections. Cancer invasion depth was determined using a ResNet-50 binary classification model, identifying which Z-planes contained invaded cells and measuring the total invasion depth. Lastly, microvessel dynamics were assessed through a U-Net Xception-style segmentation model for vessel prediction, the DisPerSE algorithm to extract an embedded graph, then graph analysis to quantify microvessel length and connectivity. To further validate our software, we reanalyzed an image set from a high-throughput drug screen involving a chemotherapy agent on a 3D cervical and endothelial co-culture model. Lastly, we applied this software to two naive image datasets from coculture lumen and microvascular fragment models. The software accurately measured cell coverage, cancer invasion, and microvessel length, yielding drug sensitivity IC50 values with a 95% confidence level compared to manual calculations. This approach significantly reduced the image processing time from weeks down to h. Furthermore, the software was able to calculate cell coverage, microvessel length, and invasion depth from two additional microphysiological models that were imaged with confocal microscopy, highlighting the versatility of the software. Our free and open source software offers an automated solution for quantifying 3D cell behavior in microphysiological models assessed using non-confocal microscopy, providing the broader Cellular and Molecular Bioengineering community with an alternative to standard confocal microscopy paired with proprietary software.This software can be found in our GitHub repository: https://github.com/fogg-lab/tissue-model-analysis-tools. The online version contains supplementary material available at 10.1007/s12195-024-00821-2.

APP-CD74 axis mediates endothelial cell-macrophage communication to promote kidney injury and fibrosis.

Kidney injuries often carry a grim prognosis, marked by fibrosis development, renal function loss, and macrophage involvement. Despite extensive research on macrophage polarization and its effects on other cells, like fibroblasts, limited attention has been paid to the influence of non-immune cells on macrophages. This study aims to address this gap by shedding light on the intricate dynamics and diversity of macrophages during renal injury and repair. During the initial research phase, the complexity of intercellular communication in the context of kidney injury was revealed using a publicly available single-cell RNA sequencing library of the unilateral ureteral obstruction (UUO) model. Subsequently, we confirmed our findings using an independent dataset from a renal ischemia-reperfusion injury (IRI) model. We treated two different types of endothelial cells with TGF-β and co-cultured their supernatants with macrophages, establishing an endothelial cell and macrophage co-culture system. We also established a UUO and an IRI mouse model. Western blot analysis, flow cytometry, immunohistochemistry and immunofluorescence staining were used to validate our results at multiple levels. Our analysis revealed significant changes in the heterogeneity of macrophage subsets during both injury processes. Amyloid β precursor protein (APP)-CD74 axis mediated endothelial-macrophage intercellular communication plays a dominant role. In the in vitro co-culture system, TGF-β triggers endothelial APP expression, which subsequently enhances CD74 expression in macrophages. Flow cytometry corroborated these findings. Additionally, APP and CD74 expression were significantly increased in the UUO and IRI mouse models. Immunofluorescence techniques demonstrated the co-localization of F4/80 and CD74 in vivo. Our study unravels a compelling molecular mechanism, elucidating how endothelium-mediated regulation shapes macrophage function during renal repair. The identified APP-CD74 signaling axis emerges as a promising target for optimizing renal recovery post-injury and preventing the progression of chronic kidney disease.

The effect of CGRP and SP and the cell signaling dialogue between sensory neurons and endothelial cells

Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca2+ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca2+ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway.

Application of an artificial intelligence for quantitative analysis of endothelial capillary beds in vitro.

The use of endothelial cell cultures has become fundamental to study angiogenesis. Recent advances in artificial intelligences (AI) offer opportunities to develop automated assessment methods in medical research, analyzing larger datasets. The aim of this study was to compare the application of AI with a manual method to morphometrically quantify in vitro angiogenesis. Co-cultures of human microvascular endothelial cells and fibroblasts were incubated mimicking endothelial capillary-beds. An AI-software was trained for segmentation of endothelial capillaries on anti-CD31-labeled light microscope crops. Number of capillaries and branches and average capillary diameter were measured by the AI and manually on 115 crops. The crops were analyzed faster by the AI than manually (3 minutes vs 1 hour per crop). Using the AI, systematically more capillaries (mean 48/mm2 vs 27/mm2) and branches (mean 23/mm2 vs 11/mm2) were counted than manually. Both methods had a strong linear relationship in counting capillaries and branches (r-capillaries = 0.88, r-branches = 0.89). No correlation was found for measurements of the diameter (r-diameter = 0.15). The present AI reduces the time required for quantitative analysis of angiogenesis on large datasets, and correlates well with manual analysis.

Enhancing bone regeneration: Unleashing the potential of magnetic nanoparticles in a microtissue model.

Bone tissue engineering addresses the limitations of autologous resources and the risk of allograft disease transmission in bone diseases. In this regard, engineered three-dimensional (3D) models emerge as biomimetic alternatives to natural tissues, replicating intracellular communication. Moreover, the unique properties of super-paramagnetic iron oxide nanoparticles (SPIONs) were shown to promote bone regeneration via enhanced osteogenesis and angiogenesis in bone models. This study aimed to investigate the effects of SPION on both osteogenesis and angiogenesis and characterized a co-culture of Human umbilical vein endothelial cells (HUVEC) and MG-63 cells as a model of bone microtissue. HUVECs: MG-63s with a ratio of 4:1 demonstrated the best results among other cell ratios, and 50 μg/mL of SPION was the optimum concentration for maximum survival, cell migration and mineralization. In addition, the data from gene expression illustrated that the expression of osteogenesis-related genes, including osteopontin, osteocalcin, alkaline phosphatase, and collagen-I, as well as the expression of the angiogenesis-related marker, CD-31, and the tube formation, is significantly elevated when the 50 μg/mL concentration of SPION is applied to the microtissue samples. SPION application in a designed 3D bone microtissue model involving a co-culture of osteoblast and endothelial cells resulted in increased expression of specific markers related to angiogenesis and osteogenesis. This includes the design of a novel biomimetic model to boost blood compatibility and biocompatibility of primary materials while promoting osteogenic activity in microtissue bone models. Moreover, this can improve interaction with surrounding tissues and broaden the knowledge to promote superior-performance implants, preventing device failure.

Curcumin Reduces Hypoxia/Reperfusion Injury of Cardiomyocytes byStimulating Vascular Endothelial Cells to Secrete FGF2.

Endothelial cells (ECs) can provide cell protection for cardiomyocytes (CMs) under hypoxia-reoxygenation (HR) conditions by secreting derived factors. This study aimed to explore the role of curcumin (CUR) in ECs for protecting CMs from HR injury. A co-culture system for ECs and CMs was set up, and subjected to HR. The transcription, expression, and secretion of FGF2 were detected by RT-qPCR, western blot, and ELISA, respectively. siRNAs specifically targeting FGF2 were transfected into ECs. FGF2 receptor- specific inhibitors (AZD4547) were used to treat CMs. The co-culture with ECs did not affect the proliferation of CMs, while CUR and ECs co-culture had a synergistic effect on promoting the proliferation of CMs in HR. Furthermore, the co-culture with ECs did not affect the apoptosis and autophagy of CMs in HR. However, the co-culture of ECs after CUR treatment inhibited the apoptosis and autophagy of CMs in HR. CUR treatment significantly enhanced FGF2 mRNA, protein, and secretion levels of ECs in HR. In addition, CUR treatment increased FGF2 levels in the CMs medium in the ECs and CMs co-culture system. The reduction of FGF2 levels in the medium and the inhibition of FGF2 receptors significantly inhibited the proliferation of CMs and significantly promoted the apoptosis and autophagy of CMs in HR. Focusing on the protective effects of CUR and ECs on cardiomyocytes is of great significance for the treatment of clinical myocardial HR injury.

P02-39 Doxorubicin induced change in filtration permeability in podocytes and glomerular microvascular endothelial cells in co-culture at physiological oxygen tensions

P02-39 Doxorubicin induced change in filtration permeability in podocytes and glomerular microvascular endothelial cells in co-culture at physiological oxygen tensions

Pericyte-to-Endothelial Cell Communication via Tunneling Nanotubes Is Disrupted by a Diol of Docosahexaenoic Acid.

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte-endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte-endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.