Co-culture Of Endothelial Cells Research Articles

RNA binding protein HuD mediates the crosstalk between β cells and islet endothelial cells by the regulation of Endostatin and Serpin E1 expression

RNA binding protein HuD plays essential roles in gene expression by regulating RNA metabolism, and its dysregulation is involved in the pathogenesis of several diseases, including tumors, neurodegenerative diseases, and diabetes. Here, we explored HuD-mediated differential expression of secretory proteins in mouse insulinoma βTC6 cells using a cytokine array. Endostatin and Serpin E1 that play anti-angiogenic roles were identified as differentially expressed proteins by HuD. HuD knockdown increased the expression of α chain of collagen XVIII (Col18a1), a precursor form of endostatin, and Serpin E1 by associating with the 3′-untranslated regions (UTRs) of Col18a1 and Serpin E1 mRNAs. Reporter analysis revealed that HuD knockdown increased the translation of EGFP reporters containing 3′UTRs of Col18a1 and Serpin E1 mRNAs, which suggests the role of HuD as a translational repressor. Co-cultures of βTC6 cells and pancreatic islet endothelial MS1 cells were used to assess the crosstalk between β cells and islet endothelial cells, and the results showed that HuD downregulation in βTC6 cells inhibited the growth and migration of MS1 cells. Ectopic expression of HuD decreased Col18a1 and Serpin E1 expression, while increasing the markers of islet vascular cells in the pancreas of db/db mice. Taken together, these results suggest that HuD has the potential to regulate the crosstalk between β cells and islet endothelial cells by regulating Endostatin and Serpin E1 expression, thereby contributing to the maintenance of homeostasis in the islet microenvironment.

Effect of Co-culture of mesenchymal stem cell and glomerulus endothelial cell to promote endothelialization under optimized perfusion flow rate in whole renal ECM scaffold.

In recent era, many researches on implantable bio-artificial organs has been increased owing to large gap between donors and receivers. Comprehensive organ based researches on perfusion culture for cell injury using different flow rate have not been conducted at the cellular level. The present study investigated the co-culture of rat glomerulus endothelial cell (rGEC) and rat bone marrow mesenchymal stem cells (rBMSC) to develop micro vascularization in the kidney scaffolds culturing by bioreactor system. To obtain kidney scaffold, extracted rat kidneys were decellularized by 1% sodium dodecyl sulfate (SDS), 1% triton X-100, and distilled water. Expanded rGECs were injected through decellularized kidney scaffold artery and cultured using bioreactor system. Vascular endothelial cells adhered and proliferated on the renal ECM scaffold in the bioreactor system for 3, 7 and 14 days. Static, 1​ml/min and 2​ml/min flow rates (FR) were tested and among them, 1​ml/min flow rate was selected based on cell viability, glomerulus character, inflammation/endothelialization proteins expression level. However, the flow injury was still existed on primary cell cultured at vessel in kidney scaffold. Therefore, co-culture of rGEC​+​rBMSC found suitable to possibly solve this problem and resulted increased cell proliferation and micro-vascularization in the glomerulus, reducing inflammation and cell death which induced by flow injury. The optimized perfusion rate under rGEC​+​rBMSC co-culture conditions resulted in enhanced endocellularization to make ECM derived implantable renal scaffold and might be useful as a way of treatment of the acute renal failure.

Association of Placental Growth Factor and Angiopoietin in Human Retinal Endothelial Cell-Pericyte co-Cultures and iPSC-Derived Vascular Organoids

PurposePlacental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids.MethodsIn this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1.ResultsInhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network.ConclusionCombined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.

Sickle Cell Disease Compromises the Hematopoietic Stem Cells Bone Marrow Niche with Age

Sickle cell disease (SCD) is characterized by chronic inflammation caused by red blood cell hemolysis, which can trigger oxidative stress and tissue injury that contribute to disease. Recent studies have shown that SCD pathophysiology reconstituted post-transplant in mouse models disrupts the architecture of bone marrow (BM) sinusoidal endothelium and causes a defect of mesenchymal stromal cells (MSCs) function. However, the impact of SCD on the ability MSCs and endothelial cells (ECs) to support of human and mouse hematopoiesis remains largely unknown. Using a mouse model of SCD, we now report that both sinusoidal (LepR+) and arteriolar MSCs (PαS) are more abundant in SCD BM, where they also display increased proliferation at 2-, 6- and 12-months of age. Additionally, 12-month-old SCD mice display a massive loss of sinusoidal ECs. Young SCD patient (n=9, age 2-18) BM also displayed increased MSC numbers relative to age-matched healthy controls (n=13). SCD MSCs isolated from 6- and 12-month old mice, maintained increased cell cycle when cultured ex vivo, showed decreased CFU-F potential, and impaired osteoblast differentiation. In ex vivo co-cultures of primary murine BM MSCs, ECs and HSCs, we found that 2-, 6-, and 12-month old SCD stroma (MSCs and ECs) failed to support non-SCD HSCs relative to controls. Phenotypic HSCs recovered from these co-cultures displayed a loss of CFU potential and transplantation activity. In tri-cultures, SCD MSCs, but not SCD ECs, drove increased HSC proliferation, apoptosis, and lymphoid-biased transplantation. In addition, MSCs isolated from BM of young SCD patients also displayed increased cell cycle, increased pro-inflammatory cytokine secretion, and decreased ability to support the expansion and CFU potential of co-cultured healthy human CD34+ cells, relative to MSCs isolated from young non-SCD donors. Bulk RNA-sequencing of 2-, 6-, and 12-month old murine SCD MSCs revealed gene signatures consistent with accelerated aging, increased proliferation, loss of extracellular matrix (ECM), impaired immune modulation, and reduced secretion of factors known to support HSCs. Genetic restoration of ECM rescued the ability of SCD MSCs to expand HSCs in ex vivo co-cultures, and support HSCs with robust CFU and transplantation potential. In sum, our data reveals a loss of HSPC supportive potential in BM MSCs in young individuals with SCD and that loss of ECM production by MSCs contributes to dysfunction in the HSC BM niche in both young and aged SCD mice.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

Enhancement of airway epithelial cell differentiation by pulmonary endothelial cell co-culture

Cross-talk between lung epithelial cells and their microenvironment has an important physiological role in development. Using an in vitro model of differentiation of human induced pluripotent stem cells (iPSCs) to air–liquid interface (ALI)-cultured lung epithelial cells, we investigated the contribution of the microenvironment to maintenance of the lung progenitor cell state. Our protocol modeled in vivo cell-to-matrix and cell-to-cell interactions. These included growth of iPSCs on inserts coated with different basement membrane proteins (collagen I, IV, fibronectin, heparan sulfate or Matrigel plus collagen IV) and co-culture with human pulmonary microvascular endothelial cells (HPMECs). Marker gene expression was measured by RT-qPCR and protein expression and localization was confirmed by immunocytochemistry. The results showed that iPSCs grown on collagen IV had the highest success rate in terms of differentiation to robust ALI-cultured lung epithelial cells, followed by fibronectin, collagen I and heparan sulfate. Coating with Matrigel mixed with collagen IV further increased the success rate to > 97 %. Co-culture of iPSCs with HPMECs enhanced the expression of key airway lineage markers (NKX2.1, KRT5, TP63, MUC5AC, MUC16, FOXJ1, CFTR and SCGB1A1) during ALI culture. Cross-talk between iPSCs and their microenvironment during cell differentiation had a significant effect on lung epithelial cell differentiation in these 3D in vitro models. Both matrix proteins and endothelial cells play critical roles in the differentiation of lung progenitor cells.

New Insight on 2D In Vitro Angiogenesis Models: All That Stretches Is Not a Tube

HighlightsTube formation on MatrigelTM and tube formation in co-culture with MSCs are two different stages of angiogenesis.uPA, uPAR, Jagged1, and Notch2 are common upregulated genes for ECs on MatrigelTM, in co-culture and in dividing/migrating cells.EndMT activated at a much greater extent in ECs in a co-culture model than in a MatrigelTM assay.Only in the MatrigelTM assay are the Notch and Hippo pathway-related genes upregulated.A Matrigel-based tube formation assay is a simple and widely accepted 2D angiogenesis model in vitro. Extracellular matrix (EM) proteins and growth factors (GFs) from MatrigelTM exclusively trigger endothelial cell (EC) tubular network (ETN) formation. Co-culture of ECs with mesenchymal stromal cells (MSCs) is another and more reliable in vitro angiogenesis assay. MSCs modulate ETN formation through intercellular interactions and as a supplier of EM and GFs. The aim of the present study was to compare the expression profile of ECs in both models. We revealed upregulation of the uPA, uPAR, Jagged1, and Notch2 genes in dividing/migrating ECs and for ECs in both experimental models at 19 h. The expression of endothelial–mesenchymal transition genes largely increased in co-cultured ECs whereas Notch and Hippo signaling pathway genes were upregulated in ECs on MatrigelTM. We showed that in the co-culture model, basement membrane (BM) deposition is limited only to cell-to-cell contacts in contrast to MatrigelTM, which represents by itself fully pre-assembled BM matrix. We suggest that ETN in a co-culture model is still in a dynamic process due to immature BM whereas ECs in the MatrigelTM assay seem to be at the final stage of ETN formation.

Discovery and Development of Tumor Angiogenesis Assays.

The angiogenesis process was described in its basic concepts in the works of the Scottish surgeon John Hunter and terminologically assessed in the early twentieth century. An aberrant angiogenesis is a prerequisite for cancer cells in solid tumors to grow and metastasize. The sprouting of new blood vessels is one of the major characteristics of cancer and represents a gateway for tumor cells to enter both the blood and lymphatic circulation systems. In vivo, ex vivo, and in vitro models of angiogenesis have provided essential tools for cancer research and antiangiogenic drug screening. Several in vivo studies have been performed to investigate the various steps of tumor angiogenesis and in vitro experiments contributed to dissecting the molecular bases of this phenomenon. Moreover, coculture of cancer and endothelial cells in 2D and 3D matrices have contributed to improve the recapitulation of the complex process of tumor angiogenesis, including the peculiar conditions of tumor microenvironment.

E. coli strain-dependent lipid alterations in cocultures with endothelial cells and neutrophils modeling sepsis.

Dysregulated lipid metabolism is common in infection and inflammation and is a part of the complex milieu underlying the pathophysiological sequelae of disease. Sepsis is a major cause of mortality and morbidity in the world and is characterized by an exaggerated host response to an infection. Metabolic changes, including alterations in lipid metabolism, likely are important in sepsis pathophysiology. Here, we designed an in vitro cell culture model using endothelial cells, E. coli, and neutrophils to mimic sepsis in a simplified cell model. Lipid alterations were studied in the presence of the pathogenic E. coli strain CFT073 and non-pathogenic E. coli strain JM109. We employed untargeted lipidomics to first identify lipid changes and then targeted lipidomics to confirm changes. Both unique and shared lipid signatures were identified in cocultures with these E. coli strains. In the absence of neutrophils, the CFT073 strain elicited alterations in lysophosphatidylcholine and diglyceride molecular species during coculture while both strains led to increases in phosphatidylglycerols. Lipid alterations in these cocultures changed with the addition of neutrophils. In the presence of neutrophils with E. coli and endothelial cells, triglyceride increases were a unique response to the CFT073 strain while phosphatidylglycerol and diglyceride increases occurred in response to both strains. Phosphatidylethanolamine also increased in neutrophils, E. coli and endothelial cells cocultures, and this response was greater in the presence of the CFT073 strain. We further evaluated changes in phosphatidylethanolamine in a rat model of sepsis, which showed multiple plasma phosphatidylethanolamine molecular species were elevated shortly after the induction of sepsis. Collectively, these findings demonstrate unique lipid responses by co-cultures of E. coli with endothelial cells which are dependent on the E. coli strain as well as the presence of neutrophils. Furthermore, increases in phosphatidylethanolamine levels in CFT073 urosepsis E. coli, endothelial cell, neutrophil cocultures were similarly observed in the plasma of septic rats.

Spatial patterning of PCL µ-scaffolds directs 3D vascularized bio-constructs morphogenesis in vitro

Modular tissue engineering (mTE) strategies aim to build three-dimensional tissue analogues in vitro by the sapient combination of cells, micro-scaffolds (μ-scaffs) and bioreactors. The translation of these newly engineered tissues into current clinical approaches is, among other things, dependent on implant-to-host microvasculature integration, a critical issue for cells and tissue survival in vivo. In this work we reported, for the first time, a computer-aided modular approach suitable to build fully vascularized hybrid (biological/synthetic) constructs (bio-constructs) with micro-metric size scale control of blood vessels growth and orientation. The approach consists of four main steps, starting with the fabrication of polycaprolactone μ-scaffs by fluidic emulsion technique, which exhibit biomimetic porosity features. In the second step, layers of μ-scaffs following two different patterns, namely ordered and disordered, were obtained by a soft lithography-based process. Then, the as obtained μ-scaff patterns were used as template for human dermal fibroblasts and human umbilical vein endothelial cells co-culture, aiming to promote and guide the biosynthesis of collagenous extracellular matrix and the growth of new blood vessels within the mono-layered bio-constructs. Finally, bi-layered bio-constructs were built by the alignment, stacking and fusion of two vascularized mono-layered samples featuring ordered patterns. Our results demonstrated that, if compared to the disordered pattern, the ordered one provided better control over bio-constructs shape and vasculature architecture, while minor effect was observed with respect to cell colonization and new tissue growth. Furthermore, by assembling two mono-layered bio-constructs it was possible to build 1 mm thick fully vascularized viable bio-constructs and to study tissue morphogenesis during 1 week of in vitro culture. In conclusion, our results highlighted the synergic role of μ-scaff architectural features and spatial patterning on cells colonization and biosynthesis, and pave the way for the possibility to create in silico designed vasculatures within modularly engineered bio-constructs.

Abstract P3064: Immunoengineered Tantalum Carbide Mxene Quantum Dots For Prevention Of Transplant Vasculopathy

Transplant vasculopathy is an aggressive form of atherosclerosis that manifests uniquely in transplanted hearts, lungs, and kidneys. Recently, immunoengineered transition metal carbides, or MXenes, have demonstrated unique immunomodulatory properties and this can potentially be leveraged to prevent transplant vasculopathy. Here, we present the synthesis, characterization and application of novel zero-dimensional tantalum carbide (Ta 4 C 3 T x ) MXene quantum dots (MQDs) for immunomodulation. A facile hydrofluoric acid-free etching protocol was developed to synthesize zero-dimensional Ta 4 C 3 T x MQDs from Ta 4 AlC 3 MAX phase. The resultant MQDs are 3 to 7 nm in diameter and are surface modified with carboxyl, hydroxyl, and amine functional groups for biological interactions. Immunomodulation was assessed in vitro through co-cultures of human umbilical vein endothelial cells (ECs) and peripheral blood mononuclear cells. We found that MQDs interact with activated human ECs in vitro to reduce activation and pro-inflammatory Th1 polarization of allogeneic CD4 + lymphocytes. Mechanistically, we showed using quantitative PCR that treatment with MQDs significantly increased endothelial surface expression of the T-cell co-inhibitory molecule PD-L1 and decreased the co-stimulatory molecule CD86. Furthermore, exploratory bulk RNA-sequencing of co-cultured lymphocytes showed decreases in the immunologic signatures of naïve T-cells and increases in the immunologic signatures of CD25 + regulatory T-cells, suggesting activation of this suppressive T-cell population. Finally, when applied in an in vivo rat model of transplant vasculopathy, treatment with MQDs reduced lymphocyte infiltration and preserved medial smooth muscle cell integrity within transplanted aortic segments. These findings suggest that these novel MQDs have potential as an effective treatment to ameliorate transplant vasculopathy.

Understanding 3D culture in smooth muscle cell biology and the modelling of atherosclerosis

Background and Aims : Even though the advantages of 3D culture in mimicking cellular environment and drug discovery is widely reported in cancer biology, there are limited studies on 3D culture in vascular biology and atherosclerosis modelling. Our study aims to investigate the significance of 3D spheroid culture on cellular physiology and in atherosclerosis modelling using vascular smooth muscle cells(VSMC). We also explore 3D vessel modelling via co-culturing of endothelial cells(EC) and VSMCs.

Promotion of angiogenesis in vitro by Astragalus polysaccharide via activation of TLR4 signaling pathway.

During the implantation of functional tissue-engineered constructs for treating bone defects, a functional vascular network is critical for the survival of the construct. One strategy to achieve rapid angiogenesis for this application is the co-culture of outgrowth endothelial cells (OECs) and primary human osteoblasts (POBs) within a scaffold prior to implantation. In the present study, we aim to investigate whether Astragalus polysaccharide (APS) promotes angiogenesis or vascularization via the TLR4 signaling pathway in a co-culture of OECs and POBs. The co-cultures were treated with various concentrations of APS for 24 h and, subsequently, another 7 days, followed by CD31 staining and analysis of micro-vessel-formation areas using software. Additionally, APS (0.4mg/ml for 24 h) was added to monocultures of OECs or POBs for evaluating proliferation, apoptosis, angiogenesis, osteogenesis, TLR4 signaling pathway, and inflammatory cytokine release. We found that APS promoted angiogenesis in the co-culture at the optimal concentration of 0.4mg/ml. TLR4 activation by APS up-regulated the expression level of TLR4/MyD88 and enhanced angiogenesis and osteogenesis in monocultures of OECs and POBs. The levels of E-selectin adhesion molecules, three cytokines (IL-6, TNF-α, and IFN-γ), and VEGF and PDGF-BB, which can induce angiogenesis, increased significantly (p < .05) following APS treatment. Therefore, APS appears to promote angiogenesis and ossification in the co-culture system via the TLR4 signaling pathway. PRACTICAL APPLICATIONS: This study demonstrates that APS may promote angiogenesis and osteocyte proliferation in OEC and POB co-culture systems through the MyD88-dependent TLR4 signaling pathway. APS might represent a potential therapeutic strategy in tissue-engineered bone implantation for the treatment of large bone defects; additionally, it has the advantage of safety, as it exhibits low or no side effects. In the future, it is expected to be used in vitro for the construction of tissue-engineered bone and in vivo after implantation in patients with bone defects for promoting rapid vascularization and ossification of tissue-engineered bone and early fusion with the recipient's bone. In addition, as a food additive, Astragalus membranaceus can be used as a tonic material in patients recovering from a fracture for promoting blood-vessel formation at the fracture site and fracture recovery. Combining traditional Chinese medicine with tissue engineering can provide further strategies for promoting the development of regenerative medicine.

Redesigning of 3-Dimensional Vascular-Muscle Structure Using ADSCs/HUVECs Co-Culture and VEGF on Engineered Skeletal Muscle ECM.

Objective The main objective of this study is to determine the myogenic effects of skeletal muscle extracellular matrix, vascular endothelial growth factor and human umbilical vein endothelial cells on adipose-derived stem cells to achieve a 3-dimensional engineered vascular-muscle structure. Materials and MethodsThe present experimental research was designed based on two main groups, i.e. monoculture of adipose tissue-derived stem cells (ADSCs) and co-culture of ADSCs and human umbilical vein endothelial cells (HUVECs) in a ratio of 1:1. Skeletal muscle tissue was isolated, decellularized and its surface was electrospun using polycaprolactone/gelatin parallel nanofibers and then matrix topography was evaluated through H&E, trichrome staining and SEM. The expression of MyHC2 gene and tropomyosin protein were examined through real-time reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence, respectively. Finally, the morphology of mesenchymal and endothelial cells and their relationship with each other and with the engineered scaffold were examined by scanning electron microscopy (SEM).Results According to H&E and Masson’s Trichrome staining, muscle tissue was completely decellularized. SEM showed parallel Polycaprolactone (PCL)/gelatin nanofibers with an average diameter of about 300 nm. The immunofluorescence proved that tropomyosin was positive in the ADSCs monoculture and the ADSCs/HUVECs co- culture in horse serum (HS) and HS/VEGF groups. There was a significant difference in the expression of the MyHC2 gene between the ADSCs and ADSCs/HUVECs culture groups (P<0.05) and between the 2D and 3D models in HS/ VEGF differentiation groups (P<001). Moreover, a significant increase existed between the HS/VEGF group and other groups in terms of endothelial cells growth and proliferation as well as their relationship with differentiated myoblasts (P<0.05). ConclusionCo-culture of ADSCs/HUVECs on the engineered cell-free muscle scaffold and the dual effects of VEGF can lead to formation of a favorable engineered vascular-muscular tissue. These engineered structures can be used as an acceptable tool for tissue implantation in muscle injuries and regeneration, especially in challenging injuries such as volumetric muscle loss, which also require vascular repair.

Dose-dependent dominance: How cell densities design stromal cell functions during soft tissue healing.

Regular soft tissue healing relies on the well-organized interaction of different stromal cell types with endothelial cells. However, spatiotemporal conditions might provoke high densities of one special stromal cell type, potentially leading to impaired healing. Detailed knowledge of the functions of rivaling stromal cell types aiming for tissue contraction and stabilization as well as vascular support is mandatory. By the application of an in vitro approach comprising the evaluation of cell proliferation, cell morphology, myofibroblastoid differentiation, and cytokine release, we verified a density-dependent modulation of these functions among juvenile and adult fibroblasts, pericytes, and adipose-derived stem cells during their interaction with microvascular endothelial cells in cocultures.Results indicate that juvenile fibroblasts rather support angiogenesis via paracrine regulation at the early stage of healing, a role potentially compromised in adult fibroblasts. In contrast, pericytes showed a more versatile character aiming at angiogenesis, vessel stabilization, and tissue contraction. Such a universal character was even more pronounced among adipose-derived stem cells. The explicit knowledge of the characteristic functions of stromal cell types is a prerequisite for the development of new analytical and therapeutic approaches for impaired soft tissue healing. The present study delivers new considerations concerning the roles of rivaling stromal cell types within a granulation tissue, pointing to extraordinary properties of pericytes and adipose-derived stem cells.

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi.

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.

Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells

Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3−/− endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development.

Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow.

BackgroundThe microanatomical steps of malignant pleural mesothelioma (MPM) vascularization and the resistance mechanisms to anti-angiogenic drugs in MPM are unclear.MethodsWe investigated the vascularization of intrapleurally implanted human P31 and SPC111 MPM cells. We also assessed MPM cell’s motility, invasion and interaction with endothelial cells in vitro.ResultsP31 cells exhibited significantly higher two-dimensional (2D) motility and three-dimensional (3D) invasion than SPC111 cells in vitro. In co-cultures of MPM and endothelial cells, P31 spheroids permitted endothelial sprouting (ES) with minimal spatial distortion, whereas SPC111 spheroids repealed endothelial sprouts. Both MPM lines induced the early onset of submesothelial microvascular plexuses covering large pleural areas including regions distant from tumor colonies. The development of these microvascular networks occurred due to both intussusceptive angiogenesis (IA) and ES and was accelerated by vascular endothelial growth factor A (VEGF-A)-overexpression. Notably, SPC111 colonies showed different behavior to P31 cells. P31 nodules incorporated tumor-induced capillary plexuses from the earliest stages of tumor formation. P31 cells deposited a collagenous matrix of human origin which provided “space” for further intratumoral angiogenesis. In contrast, SPC111 colonies pushed the capillary plexuses away and thus remained avascular for weeks. The key event in SPC111 vascularization was the development of a desmoplastic matrix of mouse origin. Continuously invaded by SPC111 cells, this matrix transformed into intratumoral connective tissue trunks, providing a route for ES from the diaphragm.ConclusionsHere, we report two distinct growth patterns of orthotopically implanted human MPM xenografts. In the invasive pattern, MPM cells invade and thus co-opt peritumoral capillary plexuses. In the pushing/desmoplastic pattern, MPM cells induce a desmoplastic response within the underlying tissue which allows the ingrowth of a nutritive vasculature from the pleura.

Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone

To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group ( n=6) and porous β-TCP scaffold as the control group ( n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel ( P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group ( P<0.05), except for area fraction at 4 weeks after implantation ( P>0.05). The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.

Abstract 195: Transcriptional Regulation Of Transcriptional Regulation Of Triggering Receptor Expressed On Myeloid Cells-1 By Low Shear Stress In A Three-dimensional Co-culture Of Coronary Artery Smooth Muscle Cells, Endothelial Cells, And Monocytes

Objectives: To identify transcriptional factors involved in the expression of Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) in response to low shear stress in an in-vitro model of coronary artery atherosclerosis. Methods: Human coronary artery smooth muscle cells were mixed with neutralized type I collagen solution and plated on glass coverslips. The human coronary artery endothelial cells (HCAECs) were then plated on top of the collagen gels followed by plating monocytes (THP-1 cells) on top of the HCAECs. After 48 h, the co-culture was treated with or without low shear stress (LSS, 5±3 dyne/cm 2 ) using the Flexcell Streamer for 30 min. Protein and mRNA of TREM-1 expression was assessed by immunofluorescence staining and real time RT-PCR. The expression of transcriptional factors involved in the expression of TREM-1 was assessed by ChIP assay. Results: LSS significantly stimulated TREM-1 expression as evidenced by IF staining and mRNA expression (fold increase vs control: 43.2 ± 9.7, Fig 1A and 1B ). ChIP assay demonstrated that, under control condition (no flow), NF-κB (p65), PU.1 and activation transcription factor 2 (ATF2) bound to TREM-1 promoter region 2, but not to region 1 ( Fig 2A ). In response to LSS for 30 min, however, binding of the NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 2 was significantly enhanced; furthermore, LSS treatment induced binding of NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 1 ( Fig 2B) . Conclusions: Transcriptional factors NF-κB (p65), PU.1 and ATF2 are critically involved in the TREM-1 expression in response to LSS stimulation in an in-vitro model of human coronary artery atherosclerosis.