Co-culture Of Endothelial Cells Research Articles

Effect of M2-macrophage treated lymphatic endothelial cells on angiogenesis that promoted osteointegration

Early vascularization plays an essential role during the whole process in bone regeneration because of the function of secreting cytokines, transporting nutrients and metabolic wastes. As the preliminary basis of bone repair, angiogenesis is regulated by immune cells represented by macrophages to a great extent. However, with the discovery of the endolymphatic circulation system inside bone tissue, the role of vascularization became complicated and confusing. Herein, we developed a macrophage/ lymphatic endothelial cells (LECs)/ human umbilical vein endothelial cells (HUVECs) co-culture system to evaluate the effect of macrophage treated lymphatic endothelial cells on angiogenesis in vitro and in vivo. In this study, we collected the medium from macrophage (CM) for LECs culture. We found that CM2 could promote the expression of LECs markers and migration ability, which indicated the enhanced lymphogenesis. In addition, the medium from LECs was collected for culturing HUVECs. The CM2-treated LECs showed superior angiogenesis property including the migration capacity and expression of angiogenetic markers, which suggested the superior vascularization. Rat femoral condyle defect model was applied to confirm the hypothesis in vivo. Generally, M2-macrophage treated LECs showed prominent angiogenetic potential coupling with osteogenesis.

The calcineurin–NFATc pathway modulates the lipid mediators in BAL fluid extracellular vesicles, thereby regulating microvascular endothelial cell barrier function

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.

Osteogenic differentiation of mesenchymal stem cell spheroids: A microfluidic device and electrochemiluminescence imaging study

Human mesenchymal stem cells (MSCs) have attracted attention as a major cellular source for regenerative medicine and preclinical studies. Three-dimensional (3D) cell culture systems can be used to construct physiologically relevant MSC spheroids with enhanced tissue regeneration capacity. However, what aspect of the MSC spheroids retains the regeneration capacity is not yet fully understood. Here, we induced osteoblast differentiation of MSC spheroids and investigated their respiratory activity using electrochemiluminescence (ECL) imaging developed in our previous study. In parallel, the ability of MSC spheroids to induce angiogenesis was investigated in a microfluidic device with a co-culture of endothelial cells. We found that the osteogenic differentiation of MSCs induced changes in their metabolic activity (respiratory activity of the spheroids decreased during differentiation) and lead to the loss of their angiogenesis-inducing ability. In other words, it is suggested that maintaining the stem cell nature with in the MSC spheroids is an important factor for tissue regeneration. In this study, we demonstrated the effective use of ECL imaging and microfluidic systems for the evaluation of MSC spheroids, providing new insights into stem cell tissue engineering.

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

BACKGROUND: 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. OBJECTIVE: The aim of this study was to compare the application of AI with a manual method to morphometrically quantify in vitro angiogenesis. METHODS: 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. RESULTS: 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). CONCLUSIONS: The present AI reduces the time required for quantitative analysis of angiogenesis on large datasets, and correlates well with manual analysis.

An injectable anti-vascularization functionalized hydrogel for degenerative nucleus pulposus repair

Neovascularization and inflammatory cell invasion within the nucleus pulposus (NP) constitute pivotal pathological changes during the acceleration stage of intervertebral disc degeneration (IDD). Mesenchymal stem cells (MSCs), renowned for their remarkable capacity in intervertebral disc (IVD) regeneration, also exhibit the capability to secrete pro-angiogenic factors, expediting IDD progression under hypoxic conditions. Consequently, we developed a hydrogel comprised of methacrylated hyaluronic acid (HAMA), rat tail collagen I (COL), and MSCs, incorporating the vascular endothelial growth factor receptor (VEGFR) inhibitor cabozantinib (Cabo@HAMA-COL/MSCs hydrogel). This innovative construct aimed to facilitate NP regeneration while mitigating vascularization and inflammation. Our findings revealed that the hydrogel aptly mimicked the mechanical characteristics of NP tissue, exhibiting injectability, low cytotoxicity, and the preservation of the cellular phenotype of NP cells. Co-culturing of MSCs and human umbilical vein endothelial cells (HUVECs) promoted migration, tube formation, and sprouting of HUVECs, which will be inhibited by cabozantinib. In vivo experiments demonstrated that Cabo@HAMA-COL/MSCs hydrogel maintained disc height, protected NP, and alleviated vascularization and inflammation in a puncture-induced rat caudal IDD model. Consequently, our results substantiate that Cabo@HAMA-COL/MSCs hydrogel can prevent IDD degeneration by ameliorating the vascularization-inflammation pathological microenvironment, offering a promising therapeutic strategy for IDD.

Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

Bioengineering of vascularized porcine flaps using perfusion-recellularization

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Indirect co-culture of osteoblasts and endothelial cells in vitro based on a biomimetic 3D composite hydrogel scaffold to promote the proliferation and differentiation of osteoblasts.

The field of orthopedics has long struggled with the challenge of repairing and regenerating bone defects, which involves a complex process of osteogenesis requiring coordinated interactions among different types of cells. The crucial role of endothelial cells and osteoblasts in bone vascularization and osteogenesis underscores the importance of their intimate interaction. However, efforts to bioengineer bone tissue have been impeded by the difficulty in establishing proper angiogenesis and osteogenesis in tissue structures. This study presents a novel approach to bone tissue engineering, involving a three-dimensional composite hydrogel scaffold composed of sodium alginate microspheres encapsulated in type I collagen. Using this scaffold, a three-dimensional indirect co-culture system was established for osteoblasts and endothelial cells to evaluate the osteogenic differentiation potential of osteoblasts. Results demonstrate that the non-contact co-culture system of endothelial cells and osteoblasts constructed by the composite hydrogel scaffold loaded with microspheres holds promise for bone tissue engineering. The innovative concept of an indirect co-culture system presents exciting prospects for conducting intercellular communication studies and offers a valuable in vitro tissue platform to investigate tissue regeneration.

Abstract 2880: Radiation induces myofibroblast differentiation of pericytes

Abstract Radiation not only kills cancer cells directly through DNA damage, but also indirectly by inducing vascular changes. Endothelial cells (ECs) in small blood vessels and capillaries connect with pericytes, a type of mural cells which are important for vascular stability and structure. Effects of radiation on tumor vasculature are critical for the therapeutic efficacy, however, its impacts on pericytes remain largely unknown. In this study, we focused on the effect of radiation on pericytes and sought to elucidate the mechanism by which radiation affects pericytes. We show 20Gy for C3H10T1/2 (10T1/2) mouse embryonic mesenchymal pericyte precursor cells and 10Gy for human pericytes from placenta (hPC-PL) cells induces pericyte differentiation and activation of adhesion molecules. Radiation enhanced lymphocyte adhesion and decreased permeability in co-culture of ECs and pericytes primarily through endothelial cells, not via pericytes. Furthermore, radiation promoted transdifferentiation of pericytes into myofibroblasts. Supernatant from irradiated cells in combination with a TGFβ inhibitor showed that TGFβ signaling is involved in pericyte differentiation by radiation but not all pericyte marker genes are regulated by TGFβ. Therefore, we performed phospho-kinase array analysis and revealed Akt signaling pathway activation 10 minutes following radiation, which was verified by the phosphorylation of GSK3β and PRAS40. Along with this, generation of intracellular reactive oxygen species (ROS) occurs as early as 5 min following radiation. Inhibitors of Akt and ROS suppress the expression of myofibroblast markers increased by radiation. To investigate whether radiation induces pericyte differentiation into myofibroblast in tumors, we employed NG2DsRedBAC (NG2DsRed) transgenic mice where DsRed is expressed under the control of NG2 promoter to trace pericytes. The endogenous expression of DsRed was detected in pericytes in the tumors implanted to NG2DsRed mice. Flow cytometry analysis of tumors harvested 7 days after radiation shows radiation increased vimentin+ cells and decreased DsRed+ cells, which indicates radiation induced pericyte differentiation into myofibroblast. In conclusion, radiation promotes pericyte maturation and transdifferentiation into myofibroblast via Akt signaling. Further study using Akt inhibitor with radiation to tumor-bearing N2DsRed mice will be warranted. Citation Format: Tomoko Yamazaki, Kelley R. Jordan, Marka R. Crittenden, Michael J. Gough, Kristina H. Young. Radiation induces myofibroblast differentiation of pericytes [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 2880.

Renal proximal tubule-on-a-chip in PDMS: fabrication, functionalization, and RPTEC:HUVEC co-culture evaluation

‘On-a-chip’ technology advances the development of physiologically relevant organ-mimicking architecture by integrating human cells into three-dimensional microfluidic devices. This method also establishes discrete functional units, faciliting focused research on specific organ components. In this study, we detail the development and assessment of a convoluted renal proximal tubule-on-a-chip (PT-on-a-chip). This platform involves co-culturing Renal Proximal Tubule Epithelial Cells (RPTEC) and Human Umbilical Vein Endothelial Cells (HUVEC) within a polydimethylsiloxane microfluidic device, crafted through a combination of 3D printing and molding techniques. Our PT-on-a-chip significantly reduced high glucose level, exhibited albumin uptake, and simulated tubulopathy induced by amphotericin B. Remarkably, the RPTEC:HUVEC co-culture exhibited efficient cell adhesion within 30 min on microchannels functionalized with plasma, 3-aminopropyltriethoxysilane, and type-I collagen. This approach significantly reduced the required incubation time for medium perfusion. In comparison, alternative methods such as plasma and plasma plus polyvinyl alcohol were only effective in promoting cell attachment to flat surfaces. The PT-on-a-chip holds great promise as a valuable tool for assessing the nephrotoxic potential of new drug candidates, enhancing our understanding of drug interactions with co-cultured renal cells, and reducing the need for animal experimentation, promoting the safe and ethical development of new pharmaceuticals.

Anti-Inflammatory Action of Resveratrol in the Central Nervous System in Relation to Glucose Concentration-An In Vitro Study on a Blood-Brain Barrier Model.

Unbalanced blood glucose levels may cause inflammation within the central nervous system (CNS). This effect can be reversed by the action of a natural neuroprotective compound, resveratrol (RSV). The study aimed to investigate the anti-inflammatory effect of RSV on astrocyte cytokine profiles within an in vitro model of the blood-brain barrier (BBB) under varying glucose concentrations (2.2, 5.0, and 25.0 mmol/L), corresponding to hypo-, normo-, and hyperglycemia. The model included co-cultures of astrocytes (brain compartment, BC) and endothelial cells (microvascular compartment, MC), separated by 0.4 µm wide pores. Subsequent exposure to 0.2 μM LPS in the brain compartment (BC) and 50 μM RSV in the microvascular compartment (MC) of each well was carried out. Cytokine levels (IL-1 α, IL-1 β, IL-2, IL-4, IL-6, IL-8) in the BC were assessed using a Multi-Analyte ELISArray Kit before and after the addition of LPS and RSV. Statistical analysis was performed to determine significance levels. The results demonstrated that RSV reduced the concentration of all studied cytokines in the BC, regardless of glucose levels, with the most substantial decrease observed under normoglycemic conditions. Additionally, the concentration of RSV in the BC was highest under normoglycemic conditions compared to hypo- and hyperglycemia. These findings confirm that administration of RSV in the MC exerts anti-inflammatory effects within the BC, particularly under normoglycemia-simulating conditions. Further in vivo studies, including animal and human research, are warranted to elucidate the bioavailability of RSV within the central nervous system (CNS).

Platelet rich fibrin as a bioactive matrix with proosteogenic and proangiogenic properties on human healthy primary cells in vitro

Blood concentrates like platelet rich fibrin (PRF) have been established as a potential autologous source of cells and growth factors with regenerative properties in the field of dentistry and regenerative medicine. To further analyze the effect of PRF on bone tissue regeneration, this study investigated the influence of liquid PRF matrices on human healthy primary osteoblasts (pOB) and co-cultures composed of pOB and human dermal vascular endothelial cells (HDMEC) as in vitro model for bone tissue regeneration. Special attention was paid to the PRF mediated influence on osteoblastic differentiation and angiogenesis. Based on the low-speed centrifugation concept, cells were treated indirectly with PRF prepared with a low (44 g) and high relative centrifugal force (710 g) before the PRF mediated effect on osteoblast proliferation and differentiation was assessed via gene and protein expression analyses and immunofluorescence. The results revealed a PRF-mediated positive effect on osteogenic proliferation and differentiation accompanied by increased concentration of osteogenic growth factors and upregulated expression of osteogenic differentiation factors. Furthermore, it could be shown that PRF treatment resulted in an increased formation of angiogenic structures in a bone tissue mimic co-culture of endothelial cells and osteoblasts induced by the PRF mediated increased release of proangiogenic growth factors. The effects on osteogenic proliferation, differentiation and vascularization were more evident when low RCF PRF was applied to the cells. In conclusion, PRF possess proosteogenic, potentially osteoconductive as well as proangiogenic properties, making it a beneficial tool for bone tissue regeneration.

TC14012 enhances the anti-fibrosis effects of UC-MSCs on the liver by reducing collagen accumulation and ameliorating inflammation

BackgroundMesenchymal stem cells (MSCs) are attracting attention as a promising cell-based therapy for the treatment of liver fibrosis or cirrhosis. However, the strategies and potential mechanisms of MSCs therapy need further investigation. The CXCL12/CXCR4/CXCR7 chemokine axis is well known to regulate cell migration and is involved in the regulation of liver fibrosis. This study aims to treat MSCs with a CXCR7-specific agonist to evaluate its therapeutic effects on hepatic fibrosis and potential mechanisms.MethodsTC14012, a potent agonist of CXCR7, has been used to pretreat human umbilical cord-derived MSCs (UC-MSCs) and assess its effect on proliferation, apoptosis, migration, immunoregulation, and gene regulatory network. Then, CCl4-induced liver fibrosis mice models were used to evaluate the therapeutic effect and mechanism of TC14012-treated UC-MSCs for treating hepatic fibrosis.ResultsTC14012 increased CXCR7 expression in UC-MSCs. Notably, co-culture of liver sinusoidal endothelial cells (LSEC) with TC14012-pretreated UC-MSCs increased CXCR7 expression in LSEC. Additionally, TC14012 promoted cell migration and mediated the immunoregulation of UC-MSCs. Compared to UC-MSCs without TC14012 pretreatment, UC-MSCs treated with TC14012 ameliorated live fibrosis by restoring CXCR7 expression, reducing collagen fibril accumulation, inhibiting hepatic stellate cells activation, and attenuating the inflammatory response.ConclusionThis study suggests that TC14012 pretreatment can enhance the therapeutic effects of UC-MSCs on liver fibrosis, mainly by promoting the migration and immunoregulation of MSCs.

Abstract WMP118: Utilizing Patient-Derived Induced Pluripotent Stem Cells as a Cellular Model for Studying Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a rare cerebrovascular disease that can lead to stroke. A histopathological characteristic in MMD is the thickening of the intimal layer that causes narrowing of the vessel, suggesting dysfunctions in the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The mechanism of MMD is largely unknown, due to a lack of established MMD-specific cellular or animal models. In this study, we established patient-derived cellular co-culture models using MMD iPSC-derived ECs and VSMCs. We also generated a 3D vascular organoid model to more closely mimic the MMD vasculature and function. Methods: Peripheral blood mononuclear cells (PBMCs) from MMD patients or healthy controls were used to generate iPSCs (n=4 per group). Differentiated ECs and VSMCs were validated and their functional characteristics were investigated in single and co-cultures. For co-cultures, ECs and VSMCs were labeled with Vibrant cell labeling dyes (DiO and DiD), followed by either transwell migration assay (ECs on bottom of cell culture plate and VSMCs on top Transwell insert) or in vitro angiogenic tube formation assay. Control and MMD iPSCs were used to generate 3D cellular organoids, and their cellular characteristics were examined using immunohistochemistry with antibodies labeling ECs and VSMCs. Results: While single cultures of ECs displayed similar tube formation patterns between control and MMD, co-cultures of ECs with VSMCs showed that MMD ECs failed to stabilize in vitro angiogenic tubes and formed aggregates at 24hr, when compared to control ECs. Transwell migration assays showed MMD VSMCs migrated faster than control VSMCs. Immunostaining of organoids with EC and VSMC markers showed notable differences in cellular composition, with MMD organoid showing compromised integrity of vascular networks, when compared with controls. Conclusions: We have established 3D organoid cellular models and in vitro co-cultures using MMD patient-derived iPSCs. Our co-culture studies revealed functional impairment in MMD EC/VSMC and organoid models revealed distinct characteristics in their vascular networks, possibly contributing to MMD pathology. These results shed useful insight into the cellular and molecular mechanisms driving MMD.

Molecular incompatibility between pig CD200 and human CD200 receptor in in vitro xenogeneic immune responses.

Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages againstporcine endothelial cells. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species. We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells and compared their suppressive activity against U937-derived human macrophage-like cells (hMCs) and primary macrophages. In xenogeneic coculture of porcine endothelial cellsand human macrophage-like cells or macrophages, hCD200-porcine endothelial cells suppressed phagocytosis and cytotoxicity of human macrophages to a greater extent than pCD200-porcine endothelial cells. Secretion of tumor necrosis factor-α, interleukin-1β, and monocyte chemoattractant protein-1 from human macrophages and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. The above data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in porcine endothelial cellscould be mediated by overcoming the molecular incompatibility across the species barrier rather than by simple overexpression effects of CD200.

Development of an in vitro microfluidic model to study the role of microenvironmental cells in oral cancer metastasis.

Metastasis occurs when cancer cells leave the primary tumour and travel to a secondary site to form a new lesion. The tumour microenvironment (TME) is recognised to greatly influence this process, with for instance the vascular system enabling the dissemination of the cells into other tissues. However, understanding the exact role of these microenvironmental cells during metastasis has proven challenging. Indeed, in vitro models often appear too simplistic, and the study of the interactions between different cell types in a 3D space is limited. On the other hand, even though in vivo models incorporate the TME, observing cells in real-time to understand their exact role is difficult. Horizontal compartmentalised microfluidic models are a promising new platform for metastasis studies. These devices, composed of adjacent microchannels, can incorporate multiple cell types within a 3D space. Furthermore, the transparency and thickness of these models also enables high quality real-time imaging to be performed. This paper demonstrates how these devices can be successfully used for oral squamous cell carcinoma (OSCC) metastasis studies, focusing on the role of the vascular system in this process. Conditions for co-culture of OSCC cells and endothelial cells have been determined and staining protocols optimised. Furthermore, several imaging analysis techniques for these models are described, enabling precise segmentation of the different cell types on the images as well as accurate assessment of their phenotype. These methods can be applied to any study aiming to understand the role of microenvironmental cell types in cancer metastatic dissemination, and overcome several challenges encountered with current in vitro and in vivo models. Hence, this new in vitro model capable of recapitulating important aspects of the cellular complexity of human metastatic dissemination can ultimately contribute to replacing animal studies in this field.

DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Co-culture of STRO1 + human gingival mesenchymal stem cells and human umbilical vein endothelial cells in 3D spheroids: enhanced in vitro osteogenic and angiogenic capacities.

Stem cell spheroid is a promising graft substitute for bone tissue engineering. Spheroids obtained by 3D culture of STRO1+ Gingival Mesenchymal Stem Cells (sGMSCs) (sGMSC spheroids, GS) seldom express angiogenic factors, limiting their angiogenic differentiation in vivo. This study introduced a novel stem cell spheroid with osteogenic and angiogenic potential through 3D co-culture of sGMSCs and Human Umbilical Vein Endothelial Cells (HUVECs) (sGMSC/HUVEC spheroids, GHS). GHS with varying seeding ratios of sGMSCs to HUVECs (GHR) were developed. Cell fusion within the GHS system was observed via immunofluorescence. Calcein-AM/PI staining and chemiluminescence assay indicated cellular viability within the GHS. Furthermore, osteogenic and angiogenic markers, including ALP, OCN, RUNX2, CD31, and VEGFA, were quantified and compared with the control group comprising solely of sGMSCs (GS). Incorporating HUVECs into GHS extended cell viability and stability, initiated the expression of angiogenic factors CD31 and VEGFA, and upregulated the expression of osteogenic factors ALP, OCN, and RUNX2, especially when GHS with a GHR of 1:1. Taken together, GHS, derived from the 3D co-culture of sGMSCs and HUVECs, enhanced osteogenic and angiogenic capacities in vitro, extending the application of cell therapy in bone tissue engineering.

Cenicriviroc prevents dysregulation of astrocyte/endothelial cross talk induced by ischemia and HIV-1 via inhibiting the NLRP3 inflammasome and pyroptosis.

Blood-brain barrier (BBB) breakdown is one of the pathophysiological characteristics of ischemic stroke, which may contribute to the progression of brain tissue damage and subsequent neurological impairment. Human immunodeficiency virus (HIV)-infected individuals are at greater risk for ischemic stroke due to diminished immune function and HIV-associated vasculopathy. Studies have shown that astrocytes are involved in maintaining BBB integrity and facilitating HIV-1 infection in the brain. The present study investigated whether targeting astrocyte-endothelial cell signaling with cenicriviroc (CVC), a dual chemokine receptor (CCR)2 and CCR5 antagonist, may protect against dysregulation of cross talk between these cells after oxygen-glucose deprivation/reoxygenation (OGD/R) combined with HIV-1 infection. Permeability assay with 10 kDa fluorescein isothiocyanate (FITC)-dextran demonstrated that CVC alleviated endothelial barrier disruption in noncontact coculture of human brain microvascular endothelial cells (HBMECs) with HIV-1-infected human astrocytes, and reversed downregulation of tight junction protein claudin-5 induced by OGD/R- and HIV-1. Moreover, CVC attenuated OGD/R- and HIV-1-triggered upregulation of the NOD-like receptor protein-3 (NLRP3) inflammasome and IL-1β secretion. Treatment with CVC also suppressed astrocyte pyroptosis by attenuating cleaved caspase-1 levels and the formation of cleaved N-terminal GSDMD (N-GSDMD). Secretome profiling revealed that CVC ameliorated secretion levels of chemokine CC chemokine ligand 17 (CCL17), adhesion molecule intercellular adhesion molecule-1 (ICAM-1), and T cell activation modulator T cell immunoglobulin and mucin domain 3 (TIM-3) by astrocytes synergistically induced by OGD/R and HIV-1. Overall, these results suggest that CVC contributes to restoring astrocyte-endothelial cross interactions in an astrocyte-dependent manner via protection against NLRP3 activation and pyroptosis.NEW & NOTEWORTHY The present study reveals the role of astrocytic NOD-like receptor protein-3 (NLRP3) inflammasome in dysfunctional astrocyte-endothelial cross interactions triggered in response to oxygen/glucose deprivation injury associated with human immunodeficiency virus type 1 (HIV-1) infection. Our results suggest that blocking NLRP3 inflammasome activation and pyroptosis-mediated inflammation with cenicriviroc (CVC) may constitute a potentially effective therapeutic strategy for blood-brain barrier (BBB) protection during HIV-1-associated ischemic stroke.

Ang1/Tie2/VE-Cadherin Signaling Regulates DPSCs in Vascular Maturation

Adding dental pulp stem cells (DPSCs) to vascular endothelial cell–formed vessel-like structures can increase the longevity of these vessel networks. DPSCs display pericyte-like cell functions and closely assemble endothelial cells (ECs). However, the mechanisms of DPSC-derived pericyte-like cells in stabilizing the vessel networks are not fully understood. In this study, we investigated the functions of E-DPSCs, which were DPSCs isolated from the direct coculture of human umbilical vein endothelial cells (HUVECs) and DPSCs, and T-DPSCs, which were DPSCs treated by transforming growth factor beta 1 (TGF-β1), in stabilizing blood vessels in vitro and in vivo. A 3-dimensional coculture spheroid sprouting assay was conducted to compare the functions of E-DPSCs and T-DPSCs in vitro. Dental pulp angiogenesis in the severe combined immunodeficiency (SCID) mouse model was used to explore the roles of E-DPSCs and T-DPSCs in vascularization in vivo. The results demonstrated that both E-DPSCs and T-DPSCs possess smooth muscle cell–like cell properties, exhibiting higher expression of the mural cell–specific markers and the suppression of HUVEC sprouting. E-DPSCs and T-DPSCs inhibited HUVEC sprouting by activating TEK tyrosine kinase (Tie2) signaling, upregulating vascular endothelial (VE)–cadherin, and downregulating vascular endothelial growth factor receptor 2 (VEGFR2). In vivo study revealed more perfused and total blood vessels in the HUVEC + E-DPSC group, HUVEC + T-DPSC group, angiopoietin 1 (Ang1) pretreated group, and vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor pretreated group, compared to HUVEC + DPSC group. In conclusion, these data indicated that E-DPSCs and T-DPSCs could stabilize the newly formed blood vessels and accelerate their perfusion. The critical regulating pathways are Ang1/Tie2/VE-cadherin and VEGF/VEGFR2 signaling.