Endothelial Cell Co-culture Model Research Articles

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.

Modulation of SEMA4D-modified titanium surface on M2 macrophage polarization via activation of Rho/ROCK-mediated lactate release of endothelial cells: In vitro and in vivo

SEMA4D-modified titanium surfaces can indirectly regulate macrophages through endothelial cells to achieve an anti-inflammatory effect, which is beneficial for healing soft tissues around the gingival abutment. However, the mechanism of surface-induced cellular phenotypic changes in SEMA4D-modified titanium has not yet been elucidated. SEMA4D activates the RhoA signaling pathway in endothelial cells, which coordinates metabolism and cytoskeletal remodeling. This study hypothesized that endothelial cells inoculated on SEMA4D-modified titanium surfaces can direct M2 polarization of macrophages via metabolites. An indirect co-culture model of endothelial cells and macrophages was constructed in vitro, and specific inhibitors were employed. Subsequently, endothelial cell adhesion and migration, metabolic changes, Rho/ROCK1 expression, and inflammatory expression of macrophages were assessed via immunofluorescence microscopy, specific kits, qRT-PCR, and Western blotting. Moreover, an in vivo rat bilateral maxillary implant model was constructed to evaluate the soft tissue healing effect. The in vitro experiments showed that the SEMA4D group had stronger endothelial cell adhesion and migration, increased Rho/ROCK1 expression, and enhanced release of lactate. Additionally, decreased macrophage inflammatory expression was observed. In contrast, the inhibitor group partially suppressed lactate metabolism and motility, whereas increased inflammatory expression. The in vivo analyses indicated that the SEMA4D group had faster and better angiogenic and anti-inflammatory effects, especially in the early stage. In conclusion, via the Rho/ROCK1 signaling pathway, the SEMA4D-modified titanium surface promotes endothelial cell adhesion and migration and lactic acid release, then the paracrine lactic acid promotes the polarization of macrophages to M2, thus obtaining the dual effects of angiogenesis and anti-inflammation.

3D bioprinted endothelial cell-microglia coculture for diabetic retinopathy modeling

Diabetic retinopathy (DR) is a common diabetes complication leading to vision impairment or blindness due to retinal vasculature alterations. Hyperglycemia induces structural alterations, inflammation, and angiogenic factor upregulation. Current treatments targeting vascular endothelial growth factor are insufficient for approximately 20% of DR patients, necessitating alternative approaches. Microglia (MG), essential for retinal homeostasis, remains underexplored in DR. This study used digital light processing bioprinting to construct a 3D coculture model of endothelial cells (ECs) and MG under varying glucose conditions, with a hydrogel stiffness of 4.6–7.1 kPa to mimic the extracellular matrix property of retina plexiform. Our results showed that high glucose levels influenced both EC and microglial phenotypes, gene expression, and angiogenic potential. Increasing glucose from 5 mM to 25 mM reduces drug efficacy by 17% for Aflibercept in EC monoculture, and 25% and 30% for Aflibercept and Conbercept in EC-MG coculture, respectively, suggesting that diabetic condition and MG presence could interfere with drug responses. In conclusion, our findings emphasize the importance of cellular interactions and microenvironmental factors in DR therapy, aiming to identify novel strategies and improve understanding of MG’s role in disease pathogenesis.

Exosomal miR-214-3p from senescent osteoblasts accelerates endothelial cell senescence

BackgroundOsteoporosis is a common systemic bone disease that leads to bone fragility and increases the risk of fracture. However, the pathogenesis of osteoporosis is considered to be highly complex. The exosomes can regulate the communication between cells. The specific mechanism of information transmission between osteoblasts and endothelial cells is worthy of further study.MethodsExosomes were isolated and verified from senescent osteoblasts. The source and properties of exosomes were determined by TEM, particle size analysis and western blot. We established the co-culture model of endothelial cells and senescent osteoblasts. We used qRT-PCR to identify differentially expressed miRNAs. The functional changes of vascular endothelial cells were verified by cell transfection. β-Galactosidase cell senescence assay, Hoechst cell apoptosis assay, Ki67 cell proliferation assay and Transwell migration assay were used to verify cell senescence, apoptosis, proliferation, and migration. The potential target gene of miRNA was detected by bio-informatics pathway and double luciferase report.ResultsWe discovered that senescent osteoblasts could promote the senescence and apoptosis of vascular endothelial cells and inhibit their proliferation and migration. miR-214-3p was upregulated in senescent osteoblast-derived exosomes. miR-214-3p could effectively promote senescence and apoptosis of endothelial cells and inhibit proliferation and migration ability. L1CAM is a miR-214-3p direct target gene determined by bio-informatics and double luciferase report.ConclusionsIn conclusion, senescent osteoblast-derived exosomes can accelerate endothelial cell senescence through miR-214-3p/L1CAM pathway. Our experiments reveal the role of exosomes in the skeletal microenvironment.

ROBO4 Inhibits Irradiation-Induced Microvascular Permeability, Endothelial to Mesenchymal Transition, and Improves Hematopoietic Reconstitution

Background: Hematopoietic Stem Cells Transplantation involves irradiation preconditioning which causes bone marrow endothelial cells dysfunction. While much emphasis is on the reconstitution of an adequate number of HSCs to repopulate the hematopoietic system after the death of malignant cells, EC preservation and normal functioning are indispensable to overcome the preconditioning damages. ROBO4 is an endothelial-specific receptor that is important in maintaining the vasculature. The purpose of this study is to ascertain ROBO4's role in regulating irradiation-induced damage of the endothelium and its subsequent effect on hematopoietic reconstruction. Method: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. The expression of ROBO4 in the microvascular endothelial cells was manipulated employing lentiviral mediated RNAi (shRNA) and gene overexpression technology before irradiation treatment. The permeability of endothelial cells and its mesenchymal transition (Endo-MT) was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules (CD144, Connexin 43, and Occludin, etc.), endothelial cell-specific molecules (PECAM-1, VEGFR2 and TIE2, etc.), and specific mesenchymal cell markers (Fibronectin, Vimentin, S100A4, and ACTA2, etc.). Using Transwell and wound healing migration, Matrigel tube formation, and Transwell and FITC-Dextran permeability assays, we determined the changes in endothelial functions after ROBO4 gene manipulation and irradiation. In addition, a co-culture model of endothelial cells and hematopoietic stem cells isolated from C57BL/6 mice was constructed, and the effects of irradiated endothelial cells on the proliferation, differentiation and cell cycle of hematopoietic stem cells were analyzed using flow cytometry. Results: Ionizing radiation upregulates ROBO4 expression but disrupts endothelial junctional molecules. ROBO4 deletion enhances the irradiation-induced degradation of endothelial junctions hence increasing the permeability of the endothelium and also facilitating endothelial to mesenchymal transition (Endo-MT) by activating both the canonical (Smads) and non-canonical (PI3K/AKT/mTOR) pathways. Endothelial ROBO4 overexpression substantially reduces microvascular permeability and mitigates Endo-MT following irradiation. Even though ROBO4 overexpression does not dramatically affect endothelial functions, tube formation and migration potentials of endothelial cells significantly increase upon the knockdown of ROBO4 and irradiation. We also found that EC ROBO4 silencing inhibits the proliferation of HSPC and promotes HSC quiescence. On the other hand, ROBO4 overexpression lessened the inhibitory effects of the injured endothelium on hematopoietic stem cell expansion. Lastly, we saw that ROBO4 overexpression inhibits erythrocytosis and granulocytosis after irradiation compared to the control groups suggesting that EC ROBO4 may play a regulatory role in HSC differentiation. Conclusion: ROBO4 maintains microvascular integrity after radiation preconditioning treatment by regulating permeability and Endo-MT, protects endothelial function, and reduces the inhibitory effect of injured endothelium on hematopoietic reconstitution.

Effect of microvascular pericytes of cochlear stria vascularis on endothelial cell permeability in C57BL/6J mice

Objective: To study the changes in the permeability of the blood labyrinth barrier of the aging cochlea in mice, and to establish a non-contact co-culture model of endothelial cells (EC) and pericytes (PC) to furtherly investigate the cochlear stria vascularis microvascular pericytes impact on the permeability of endothelial cells. Methods: C57BL/6J mice were divided into two groups, three months old as young group, 12 months old as senile group. Cell experiment was divided into four groups, EC group, EC+PC co-culture group, D-gal+EC group and D-gal+EC+PC co-culture group. Auditory brainstem response (auditory brain response, ABR) was used to detect the auditory function of the two groups of mice. Evans blue staining was applied to detect the permeability of the cochlear blood labyrinth barrier of the two groups of mice. Transmission electron microscopy was used to observe the ultrastructure of blood labyrinth barrier endothelial cells, pericytes and tight junctions in the two groups of mice. Immunohistochemistry was used to detect the expression levels of tight junction proteins in the stria vascularis of the cochlea of the two groups of mice. Transwell chamber was used to detect the permeability of endothelial cells. Western blot and immunofluorescence technology were used to detect the expression level of tight junction protein on endothelial cells. SPSS 20.0 software was used to analyze the data. Results: Compared with the young group, the ABR threshold of the aging group was significantly increased, the latency of wave I was prolonged (t=10.25, P<0.01;t=5.61, P<0.05), the permeability of the cochlear blood labyrinth barrier was increased and the expression of tight junction protein on the vascular stria was decreased (P<0.05). The cochlear ultrastructure showed that the cochlear vascular stria microvascular lumen was deformed, the basement membrane thickened and the tight junction gap between endothelium enlarged. The positive rate of ECs and PCs in primary culture was more than 95%. The cells induced by 15 g/L D-gal were determined to be senescent cells. Compared with EC group, the expression of tight junction protein in endothelial cells of D-gal+EC group decreased（t=7.42，P<0.01；t=13.19，P<0.05）and the permeability increased (t=11.17, P<0.01). In the co-culture group, the expression of tight junction protein between endothelial cells in EC+PC co-culture group and D-gal+EC+PC co-culture group increased and the permeability decreased. Conclusions: In aging mice, the permeability of cochlear blood labyrinth barrier will increase and the level of tight junction protein will decrease; in aging state, cochlear vascular stria microvascular pericytes may affect endothelial cell permeability by regulating the expression of tight junction protein.

Development of alveolar-capillary-exchange (ACE) chip and its application for assessment of PM2.5-induced toxicity

Although standard two-dimensional (2D) cell culture is an effective tool for cell studies, monolayer cultivation can yield imperfect or misleading information about numerous biological functions. In this study, we developed an alveolar-capillary exchange (ACE) chip aiming to simulate the cellular microenvironment at the alveolar-capillary interface. The ACE chip was designed with two chambers for culturing alveolar epithelial cells and vascular endothelial cells separately, which are separated by a microporous polycarbonate film that allows for the exchange of soluble biomolecules. Using this model, we further tested the toxic effects of fine particulate matter (PM2.5), a form of airborne pollutant known to induce adverse effects on human respiratory system. These effects are largely associated with the ability of PM2.5 to penetrate the alveoli, where it negatively affects the pulmonary function. Our results indicate that alveolar epithelial cells cultured in the ACE chip in solo and coculture with vascular endothelial cells underwent oxidative injury-induced apoptosis mediated via the PEAK-eIF2α signaling pathway of endoplasmic reticulum stress. The use of ACE chip in an alveolar epithelial cell-vascular endothelial cell coculture model revealed cellular vulnerability to PM2.5. Therefore, this chip provides a feasible surrogate approach in vitro for investigating and simulating the cellular microenvironment responses associated with ACE in vivo.

Long non-coding RNA Rian promotes the expression of tight junction proteins in endothelial cells by regulating perivascular-resident macrophage-like melanocytes and PEDF secretion.

Perivascular-resident macrophage-like melanocytes (PVM/Ms) can upregulate the expression of tight junction-related proteins in endothelial cells (ECs) by secreting pigment epithelial-derived factor (PEDF), and thereby regulate the permeability of the intrastrial fluid-blood barrier critical for maintaining inner ear homeostasis. This study aimed to investigate the effects of long non-coding RNA (lncRNA) Rian on cell growth of PVM/Ms and PVM/Ms regulation of intrastrial fluid-blood barrier integrity mediated by PEDF. Rian was downregulated in the aged cochlea from 12-month-old C57BL/6 mice. Rian overexpression inhibited cell apoptosis and promoted cell viability of hypoxia-injured PVM/Ms as well as increased the concentration and expression of PEDF secreted by PVM/Ms. In contrast, Rian silencing exerted the opposite effects. Furthermore, in a cell co-culture model of ECs and PVM/Ms, Rian overexpression in PVM/Ms increased the expression of the junction-associated proteins in co-cultured ECs, and this effect was abrogated by blockade of PEDF by anti-PEDF in PVM/Ms. Further mechanistical investigation revealed that Rian promoted STAT3 nuclear translocation and activation by binding to FUS, and thereby promoted the secretion of PEDF. Collectively, Rian attenuates PVM/Ms injury and strengthens the ability of PVM/Ms to maintain the integrity of the endothelial barrier by promoting PEDF expression.

Abstract P793: Extracellular Vesicles Enhance Blood-Brain Barrier Integrity by Nf-κB Dependent Regulation of Abcb1 in Stroke Mice

Extracellular vesicles (EVs) derived from neural progenitor cells (NPCs) enhance post-stroke neurological recovery, albeit the underlying mechanisms remain elusive. Since previous research described an enhanced post-stroke integrity of the blood-brain barrier (BBB) upon systemic transplantation of NPCs, we wondered if NPC-derived EVs affect BBB stability and which cellular mechanisms might be involved in the process. Using an in vitro co-culture model of brain endothelial cells (ECs) and astrocytes, cells were treated with EVs or PBS and exposed to oxygen-glucose-deprivation (OGD) injury. The readout parameters focused on the expression of ABCB1, an ATP-binding cassette (ABC) transporter expressed on ECs contributing to BBB integrity. Further in vitro analysis examined the pro-inflammatory NF-κB pathway, the permeability and the transcellular electrical resistance (TER) of cultured ECs. In vitro data was finally confirmed using a rodent stroke model. Cultured ECs displayed increased ABCB1 levels when exposed to OGD, which was reversed by treatment with EVs. The latter was due to an EV-induced inhibition of the NF-κB pathway. Using a BBB co-culture model of ECs and astrocytes exposed to OGD, EVs stabilized the BBB and ABCB1 levels without affecting TER. Likewise, EVs yielded reduced Evans blue extravasation, decreased ABCB1 expression as well as an inhibition of the NF-κB pathway and downstream matrix metalloprotease-9 activity in stroke mice. The EV-induced inhibition of the NF-κB pathway resulted in a post-stroke modulation of immune responses. Our findings suggest that EVs enhance post-stroke BBB integrity via ABCB1 and MMP-9 regulation, attenuating inflammatory cell recruitment by inhibition of the NF-κB pathway.

Effects of salvianolate lyophilized injection combined with Xueshuantong injection in regulation of BBB function in a co-culture model of endothelial cells and pericytes

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. The salvianolate lyophilized injection (SLI) and Xueshuantong Injection (XST) are two standardized Chinese medicine injections which have been widely used in the treatment of cerebrovascular diseases. Salvianolic acid B (Sal B) and Notoginsenoside R1 (NR1) is respectively one of the active constituents of SLI and XST, which have certain effects on stroke. In this study, we established a co-culture of endothelial cells and pericytes for oxygen-glucose deprivation/reperfusion (OGD/R) injury model to study the effects of SLI and Sal B or XST and NR1 alone, or with their combinations (1S1X) in regulation of BBB function. The results showed that compared with the OGD/R group, treatment with SLI, XST and SalB and NR1 can significantly increase the TEER, reduce the permeability of Na-Flu, enhance the expression of tight junctions (TJs) between cells, and stabilize the basement membrane (BM) composition. In addition, the combination of 1S1X is superior to the XST or SLI alone in enhancing the TJs between cells and stabilizing the BM. And the active components SalB and NR1 can play a strong role in these two aspects, even with the whole effects. Furthermore, the study showed that XST, Sal B and NR1 increases in Ang-1and Tie2, while decrease in Ang-2 and VEGF protein expressions. Overall, these findings suggest that SLI combined with XST (1X1S) has protective effects on co-culture of endothelial cells and pericytes after OGD/R. Moreover, its protective effect might be associated with increase of TJs and BMs through activation of Ang/Tie-2 system signaling pathway.

Injectable Therapeutic Organoids Using Sacrificial Hydrogels

SummaryOrganoids are becoming widespread in drug-screening technologies but have been used sparingly for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility in size and cellular organization. We introduce a method of using hydrogels as sacrificial scaffolds, which allow cells to form self-organized clusters followed by gentle release, resulting in highly reproducible multicellular structures on a large scale. We demonstrated this strategy for endothelial cells and mesenchymal stem cells to self-organize into blood-vessel units, which were injected into mice, and rapidly formed perfusing vasculature. Moreover, in a mouse model of peripheral artery disease, intramuscular injections of blood-vessel units resulted in rapid restoration of vascular perfusion within seven days. As cell therapy transforms into a new class of therapeutic modality, this simple method—by making use of the dynamic nature of hydrogels—could offer high yields of self-organized multicellular aggregates with reproducible sizes and cellular architectures.

Photobiomodulation (PBM) promotes angiogenesis in-vitro and in chick embryo chorioallantoic membrane model

The application of light in various therapeutic settings known as Photobiomodulation (PBM) is well established. Indications are the improvement of wound healing and tissue regeneration, scarring, and perfusion as well as pain therapy. Tissue perfusion is mandatory for successful wound healing. Nevertheless, there is a lack of mechanistic studies. We investigate the potential effect of PBM from light emitting diodes (LED) at 635 nm, 80 mW/cm2, 24 J/cm2 on angiogenesis in a two-part study: 1.) Investigation of the effect of PBM on the proliferation of endothelial cells and on vasculogenesis in a co-culture model of endothelial cells and stem cells. 2.) Investigation of the influence of PBM at chick egg chorioallantoic membrane (CAM) assays with fresh human skin xenografts. In both study phases, we observed a stimulating effect of PBM at 635 nm; in part 1: for proliferation of HUVEC (human umbilical vein endothelial cells) (25833 ± 12859 versus 63002 ± 35760 cells/well, p < 0.05, for cellular network formation (2.1 ± 2.1 versus 4.6 ± 3.5, p < 0.05) and for less cell compactness p = 0.01; in part 2: for the increase of number of vessel junctions per ROI (region of interest) (15.9 ± 2.6 versus 20.8 ± 5.4, p < 0.05). Our results suggest significant promotion of angiogenesis by PBM at 635 nm in vitro and in vivo.

Differential effects of sulforaphane in regulation of angiogenesis in a co-culture model of endothelial cells and pericytes.

Aberrant neovascularization supports nutrients and the oxygen microenvironment in tumour growth, invasion and metastasis. Recapitulation of functional microvascular structures invitro could provide a platform for the study of vascular conditions. Sulforaphane (SFN), an isothiocyanate, has been reported to possess chemopreventive properties. In the present study, the effects of SFN on cell proliferation and tubular formation have been investigated using endothelial cells (ECs) and pericytes in coculture. SFN showed a dose-dependent inhibition on the growth of ECs and pericytes with IC50 values 46.7 and 32.4µM, respectively. SFN (5-20µM) inhibited tube formation in a 3D coculture although a lower dose (1.25µM) promoted 30% more endothelial tube formation than control. Moreover, SFN affected intercellular communication between ECs and pericytes via inhibition of angiogenic factor such as vascular endothelial growth factor (VEGF) expression in pericytes. However, the expression of its receptor (VEGFR-2) was found significantly increased in ECs. These effects were associated with downregulation of prolyl hydroxylase domain-containing protein 1 and 2 (PHD1/2) and activation of hypoxia-inducible factor-1 (HIF) pathway by SFN. Furthermore, thioredoxin reductase-1 was also upregulated by SFN treatment, suggesting that anti-oxidant and redox regulation are involved in angiogenesis. Taken together, the results of the present study suggest that SFN differentially regulates endothelial cells and pericytes disrupting their interplay through the VEGF-VEGFR signalling pathway. Anti-angiogenesis property of SFN indicates that it has potential role as an anticancer agent.

Abstract A34: Hypoxic cancer-associated fibroblasts secrete regulators of angiogenesis: Novel potential players revealed by MS

Abstract Hypoxia correlates with increased risk of metastasis and poor prognosis in breast cancer patients. Cancer-associated fibroblasts (CAFs) aid cancer spread by inducing the formation of an aberrant tumor stroma through secretion of growth factors, cytokines, ECM proteins, and ECM modifiers; this altered stroma can foster a pro-angiogenic environment in tumors. However, the role of CAFs under hypoxic conditions is still controversial. Here we aim to understand how hypoxia modulates the repertoire of intracellular and secreted proteins of mammary CAFs and how CAFs affect the angiogenic response of endothelial cells (ECs) in the hypoxic environment. To tackle this question, we combined a 3D co-culture model of ECs and CAFs which enabled to evaluate the impact of CAF-secreted factors on EC sprouting with SILAC-based MS secretomics approach. Using the 3D angiogenesis assay we found that ECs co-cultured with CAFs under hypoxia or exposed to the conditioned medium (CM) of hypoxic CAFs sprouted significantly more than the normoxic counterpart. The increased sprouting was the result of both autocrine and paracrine signals, although the pro-angiogenic capacity of CAFs was more pronounced than the one of the ECs. Furthermore, we found that the CM of hypoxic CAFs was able to significantly enhance EC sprouting even upon VEGFA blockage with bevacizumab, thus indicating the presence of other hypoxia-induced pro-angiogenic factors. These findings prompted us to profile the proteome and secretome of hypoxic mammary CAFs in culture. The proteome analysis revealed regulation of metabolic proteins, particularly up-regulation of glycolysis-related proteins and down-regulation of proteins involved in mitochondrial metabolism, mirroring the known metabolic switch induced by hypoxia. The secretomics analysis unveiled increased levels of known HIF1α targets, including VEGFA and STC1, and, interestingly, additionally identified a subset of proteins which have not been previously related to hypoxia nor functionally characterized. Integrated bioinformatic analysis revealed that the chromosomic region flanking these genes contains hypoxia responsive elements (HRE), suggesting that their transcription is driven by HIFs. We are currently investigating the pro-angiogenic capacity of these newly identified secreted proteins. Our findings points out that there are hypoxia-induced players still uncharacterized, whose investigation may lead to a better understanding of the CAFs response to hypoxia and how these cells affect tumor progression. Citation Format: Fernanda Grande Kugeratski, Juan Ramon Hernandez, Gabriela Kalna, Sara Zanivan. Hypoxic cancer-associated fibroblasts secrete regulators of angiogenesis: Novel potential players revealed by MS. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A34.

Abstract 3382: A physiological model of the tumor microenvironment for screening drug delivery systems

Abstract Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. Tumor microvasculature has many unique features including unusual transport characteristics, high interstitial pressure, and enhanced permeability and retention (EPR) effect. Current in vitro models of tumor drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. The objective of this study is to develop and characterize a synthetic 3D solid tumor - endothelium model in a novel microfluidic platform that mimics the tumor microenvironment observed in vivo. The novel synthetic tumor model consists of a vascular network of tumor derived endothelial cells forming a complete lumen in communication with 3D solid tumors. Primary human breast tumor associated endothelial cells (HBTAECs) were co-cultured under physiological fluid flow conditions with 3D tumor cells from metastastic (MDA-MB-231) or non-metastatic (MCF-7) origins to study the effect of metastatic potential on the integrity of the adjacent endothelial cell lining. Extravasation of fluorescently labeled liposomes across the endothelium to the tumors was measured following HBTAECs treatment with normal media, tumor conditioned media (TCM), or TNF-α. Tight junction formation was characterized using ZO-1 immunostaining. MDA-MB-231 cells quickly invaded into the vascular network from their primary culture location whereas MCF-7 cells rarely grew beyond the boundary of tumor origin. Permeability of liposomes across the endothelium was significantly higher with TCM treatment compared to both control and TNF-α treated cells (P &amp;lt; 0.01, one-way ANOVA). ZO-1 staining indicated strong tight junction formation when HBTAECs were treated with normal media as compared to TCM or TNF-α. We have successfully established an in vitro 3D tumor - endothelial cell co-culture model in a novel microfluidic platform that closely mimics the tumor microenvironment in vivo. This system reproduces the tumor permeability and retention (EPR) effect on a chip. This realistic in vitro model can have great potential in applications such as cell-cell/cell-drug carrier interaction studies, drug delivery carrier screening, and drug efficacy testing. Citation Format: Yuan Tang, Fariborz Soroush, Sudhir Deosarkar, Bin Wang, Balabhaskar Prabhakarpandian, Mohammad Kiani. A physiological model of the tumor microenvironment for screening drug delivery systems. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3382.

Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte&amp;ndash;endothelial cell coculture model

Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte–endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease.

A novel coculture model of HUVECs and HUASMCs by hyaluronic acid micropattern on titanium surface

Orientation smooth muscle cell environment plays a positive role in the development of a functional, adherent endothelium. Therefore, building an orientation coculture model of endothelial cells (ECs) and smooth muscle cells (SMCs) on biomaterials surface may provide more help for understanding the interaction between the two cells in vitro. In the present study, a "SMCs-ColIV-ECs" coculture model was built on the hyaluronic acid (HA) patterned titanium (Ti) surface, and compared with the previous "SMCs-HAa-ECs" model on endothelial cell number, morphology index, nitric oxide (NO), and prostacyclin2 (PGI2) release, anticoagulation property, human umbilical artery smooth muscle cells (HUASMCs) inhibition property and retention under fluid flow shear stress. The result indicated that "SMCs-ColIV-ECs" model could enhance the number, spreading area, and major/minor index of human umbilical vein endothelial cells (HUVECs), which contributed to the retention of HUVECs on the surface. Greater major/minor index may produce more NO and PGI2 release, contributing to the anticoagulation property and HUASMCs inhibition property. In summary, this novel "SMCs-ColIV-ECs" coculture model improved the previous "SMCs-HAa-ECs" model, and may provide more inspiration for the human vascular intima building on the biomaterials in vitro.

Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

BackgroundThe inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear.MethodsHuman dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls.ResultsCell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC.ConclusionThe increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds.

Hypothermia Prevents Inflammation but Leads to Cellular Stress in a Co-Culture Model of Endothelial Cells and Macrophages: Possible Role of Vitamin C Application During Transplantation

Background: During cardiac transplantation patients are often cooled to a temperature of 14-18°C for organ protection during ischemia. Reperfusion injury and prolonged inflammation are common complications following organ transplantation. We hypothesized that hypothermia and rewarming time have an important influence on tissue and organ function after heart transplantation. The aim of our studies was therefore to investigate the effects of deep hypothermia and rewarming on the inflammatory response of human endothelial cells and macrophages. Methods: We established an in vitro co-culture model consisting of human umbilical vein endothelial cells (HUVEC) and macrophages (THP-1 cells). The co-culture cells were exposed to dynamic temperature changes: deep hypothermia (20°C for 20 min), slow rewarming (30 min up to 37°C) and normothermia (24h at 37°C). To imitate inflammatory reaction, endothelial cells and macrophages were stimulated with 500 U/mL TNF-a throughout the cooling and rewarming period. Vitamin C (50 uM) was administrated at the beginning of the experiment and remained on the cells during the whole experiment. We investigated cell morphology (fluorescence microscopy), cell viability (MTT assay, LDH assay), cell differentiation (FACS analysis), cytokine secretion (ELISA), intracellular reactive oxygen species concentration (ROS), Adenosin- Triphsophat (ATP) content as well as pErk and IkappaB-a protein expression (Western Blot Analysis). Results: Hypothermia decreased the inflammatory reaction of the co-culture cells as proven in significant diminished extracellular IL-8, IL-6 and MCP-1 content after hypothermia and rewarming. On the other hand hypothermia led to a significant increase of extracellular ROS concentration after 24 hours which is in line with a significantly decreased intracellular ATP content in the hypothermia treated cells compared to normothermic control cells. Moreover, we could demonstrate that hypothermia and rewarming diminished the IkappaB-a protein levels, led to a significant up-regulation of pErk during the rewarming period. Vitamin C treatment during the whole experiment inhibited this hypothermia induced ROS production and restored the intracellular ATP content despites the hypothermic treatment of the cells. Conclusion: In a co-culture model consisting of endothelial cells and macrophages, hypothermia led to diminished inflammation. However, there is evidence for hypothermia induced cellular stress with diminished intracellular ATP concentration. Treating the co-culture cells with Vitamin C abolished this adverse effect of hypothermic treatment. We could demonstrate that Vitamin C acts as an antioxydizing defence. Hereby further understanding of the effects of hypothermia on the regulation of intracellular signalling and the counter-regulatory mechanisms that limit this process will improve our ability to predict the effects of hypothermia on inflammation and reperfusion injury after heart transplantation.

OS041. Apolipoprotein A-I protects normal integration of the trophoblast into endothelial cellular networks in an in vitro model of preeclampsia

Failure of the trophoblast to appropriately invade uterine spiral arteries is thought to be an initiating event in preeclampsia, a disorder associated with endothelial dysfunction. A dyslipidemia characterised by low plasma levels of high density lipoproteins (HDL) and elevated triglycerides has also been described in preeclampsia. The pro-inflammatory cytokine TNF-α inhibits trophoblast invasion of uterine endothelial cells. Previous work using an in vitro JEG-3 cell/Uterine endothelial cell co-culture model investigated the effect of apoliopoprotein A-I, the main apolipoprotein component of HDL, on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. These effects are now investigated using the human invasive trophoblast cell line HTR-8/SVneo. This study asks if apoA-I, which has established anti-inflammatory properties, can protect against the deleterious effect of TNF-α on trophoblast-endothelial cell interactions. The in vitro trophoblast-uterine endothelial cell co-culture model was used to investigate the effect of apoA-I on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. Uterine endothelial cells were pre-incubated with lipid free apoA-I (final apoA-I concentration 1 mg/mL) for 16h prior to seeding on matrigel coated plates. Tubules formed within 4h. Fluorescence-labelled HTR-8/SVneo trophoblast cells were then co-cultured with the endothelial cells±TNF-α (final concentration of 0.2ng/mL). Bright field and fluorescent images were captured after 24h. The effect of TNF-α on HTR-8/SVneo cell invasion was quantified with Image J software. Integration of HTR-8/SVneo trophoblast cells into uterine endothelial tubular networks was also imaged using live cell imaging techniques (Zeiss Axiovert). TNF-α inhibited HTR-8/SVneo (trophoblast) cell integration into endothelial tubular structures by 24.1±3.7% p<0.001. This effect was reversed when the endothelial cells were pre-incubated for 16h with lipid free apoA-I (p<0.001 compared to non-incubated cells). Live cell images of the co-culture clearly demonstrate a disruption to the normal integration of trophoblast into endothelial tubular structures in the presence of TNF-α. The protective effect conferred by pre-incubation of endothelial cells with apoA-I against TNF-α is also clearly visible. Apolipoprotein A-I has been shown to enhance trophoblast-endothelial cell integration in the presence of a pro-inflammatory stimulus. A healthy lipid profile may affect pregnancy outcomes by priming endothelial cells in preparation for trophoblast invasion.