Co-culture Of Human Umbilical Vein Endothelial Cells Research Articles

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.

Porous Polymeric Nanofilms for Recreating the Basement Membrane in an Endothelial Barrier-on-Chip.

Organs-on-chips (OoCs) support an organotypic human cell culture in vitro. Precise representation of basement membranes (BMs) is critical for mimicking physiological functions of tissue interfaces. Artificial membranes in polyester (PES) and polycarbonate (PC) commonly used in in vitro models and OoCs do not replicate the characteristics of the natural BMs, such as submicrometric thickness, selective permeability, and elasticity. This study introduces porous poly(d,l-lactic acid) (PDLLA) nanofilms for replicating BMs in in vitro models and demonstrates their integration into microfluidic chips. Using roll-to-roll gravure coating and polymer phase separation, we fabricated transparent ∼200 nm thick PDLLA films. These nanofilms are 60 times thinner and 27 times more elastic than PES membranes and show uniformly distributed pores of controlled diameter (0.4 to 1.6 μm), which favor cell compartmentalization and exchange of large water-soluble molecules. Human umbilical vein endothelial cells (HUVECs) on PDLLA nanofilms stretched across microchannels exhibited 97% viability, enhanced adhesion, and a higher proliferation rate compared to their performance on PES membranes and glass substrates. After 5 days of culture, HUVECs formed a functional barrier on suspended PDLLA nanofilms, confirmed by a more than 10-fold increase in transendothelial electrical resistance and blocked 150 kDa dextran diffusion. When integrated between two microfluidic channels and exposed to physiological shear stress, despite their ultrathin thickness, PDLLA nanofilms upheld their integrity and efficiently maintained separation of the channels. The successful formation of an adherent endothelium and the coculture of HUVECs and human astrocytes on either side of the suspended nanofilm validate it as an artificial BM for OoCs. Its submicrometric thickness guarantees intimate contact, a key feature to mimic the blood-brain barrier and to study paracrine signaling between the two cell types. In summary, porous PDLLA nanofilms hold the potential for improving the accuracy and physiological relevance of the OoC as in vitro models and drug discovery tools.

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.

3D-Bioprinted GelMA Scaffold with ASCs and HUVECs for Engineering Vascularized Adipose Tissue.

The purpose of tissue engineering is to reconstruct parts of injured tissues and to resolve the shortage of organ donations. However, the main concern is the limited size of engineered tissue due to insufficient oxygen and nutrition distribution in large three-dimensional (3D) tissue constructs. To provide better support for cells inside the scaffolds, the vascularization of blood vessels within the scaffold could be a solution. This study compared the effects of different culturing systems using human adipose tissue-derived stem/stromal cells (ASCs), human umbilical vein endothelial cells (HUVECs), and coculture of ASCs and HUVECs in 3D-bioprinted gelatin methacrylate (GelMA) hydrogel constructs. The in vitro results showed that the number of live cells was highest in the coculture of ASCs and HUVECs in the GelMA hydrogel after culturing for 21 days. Additionally, the tubular structure was the most abundant in the GelMA hydrogel, containing both ASCs and HUVECs. In the in vivo test, blood vessels were present in both the HUVECs and the coculture of ASCs and HUVECs hydrogels implanted in mice. However, the blood vessel density was the highest in the HUVEC and ASC coculture groups. These findings indicate that the 3D-bioprinted GelMA hydrogel coculture system could be a promising biomaterial for large tissue engineering applications.

The construction of a microenvironment with the vascular network by co-culturing fibroblasts and endothelial cells

IntroductionExtracellular matrix (ECM) synthesis and deposition in fibroblasts, and vascularization via endothelial cells are essential for successful tissue regeneration. Fibroblasts can produce both ECM, physical support for maintaining homeostasis, and bioactive molecules, such as growth factors and cytokines. Endothelial cells can secrete growth factors and form vascular networks that enable the supply of nutrients and oxygen and remove metabolic products. MethodsIn this study, we focused on combining Human Periodontal Ligament Fibroblasts (HPLF) and Human Umbilical Vein Endothelial Cells (HUVEC) for tissue regeneration in clinical applications. ResultsThe fibroblastic and angiogenic phenotypes were promoted in co-culture with HPLF and HUVEC at a ratio of 1:1 compared to HPLF or HUVEC mono-culture. The gene expression of ECM components and angiogenesis-related factors was also enhanced by HPLF/HUVEC co-culture. Despite an apparent increase in the expression of angiogenic factors, the levels of secreted growth factors decreased under co-culture conditions. These data suggest that ECM constructed by HPLF and HUVEC would act as a storage site for growth factors, which can later be released. Our results showed that cell-to-cell interactions between HPLF and HUVEC enhanced collagen synthesis and endothelial network formation, leading to the creation of highly vascularized constructs for periodontal tissue regeneration. ConclusionSuccessful periodontal tissue regeneration requires microenvironmental reconstruction and vascularization, which can be achieved using a co-culture system. In the present study, we found that fibroblastic and angiogenic phenotypes were enhanced by the co-culture of HPLF and HUVEC. The optimal culture conditions (1:1) could potentially accelerate tissue engineering, including ECM synthesis and EC tube formation, and these approaches can improve therapeutic efficacy after transplantation.

Heterogeneity of T-cells in periapical lesions and in vitro validation of the proangiogenic effect of GZMA on HUVECs.

T-cells are key immunomodulatory cells in periapical lesions. This study aimed to explore the roles of T-cells in chronic apical periodontitis (CAP) using single-cell RNA sequencing and to further investigate Granzyme A (GZMA) in angiogenesis regulation. A total of five CAP samples were collected for single-cell RNA sequencing. We performed sub-cluster and lineage-tracing analyses for T-cells. According to differential gene expression, distinct biological functions enriched in T-cells of CAP were presented by Gene Set Enrichment Analysis (GSEA) and compared with healthy gingiva (data obtained from the GEO database). CellChat was used to explore potential ligand-receptor interactions between T-cells and endothelial cells in CAP. The coculture of primary human umbilical vein endothelial cells (HUVECs) and Jurkat T-cells, as well as the addition of GZMA recombinant protein, was used to validate the predicted pair of GZMA and_Coagulation Factor II Thrombin Receptor (F2R) by RT-PCR, angiogenesis, and migration assays. A transcriptomic atlas of 44,746 individual cells was constructed from the periapical lesions of five patients with CAP by single-cell RNA-seq, and eight cell types were identified. We identified nine subsets of T cells and deciphered the cellular heterogeneity of T-cells in CAP at the functional level by sub-clustering and GSEA. Lineage-tracing revealed a distinct lineage of T-cells in CAP and predicted the transition of the T cellular state upon CAP. GSEA revealed multiple biological processes and relevant angiogenesis genes upregulated in CAP T-cells. GZMA-F2R pairs were predicted by cell-cell interactions in CAP. High expression of GZMA and F2R was observed in the coculture of HUVECs and Jurkat T-cells, and the proangiogenic capacity of the GZMA recombinant protein was emphasized by in vitro experiments. Our study provides novel insights into the heterogeneity of T-cells in periapical lesions and reveals the potential role of GZMA in T-cells in regulating angiogenesis in HUVECs.

Abstract 2654: Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein

Abstract Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2654.

Harnessing the synergy of perfusable muscle flap matrix and adipose-derived stem cells for prevascularization and macrophage polarization to reconstruct volumetric muscle loss

Muscle flaps must have a strong vascular network to support a large tissue volume and ensure successful engraftment. We developed porcine stomach musculofascial flap matrix (PDSF) comprising extracellular matrix (ECM) and intact vasculature. PDSF had a dominant vascular pedicle, microcirculatory vessels, a nerve network, well-retained 3-dimensional (3D) nanofibrous ECM structures, and no allo- or xenoantigenicity. In-depth proteomic analysis demonstrated that PDSF was composed of core matrisome proteins (e.g., collagens, glycoproteins, proteoglycans, and ECM regulators) that, as shown by Gene Ontology term enrichment analysis, are functionally related to musculofascial biological processes. Moreover, PDSF-human adipose-derived stem cell (hASC) synergy not only induced monocytes towards IL-10-producing M2 macrophage polarization through the enhancement of hASCs' paracrine effect but also promoted the proliferation and interconnection of both human skeletal muscle myoblasts (HSMMs) and human umbilical vein endothelial cells (HUVECs) in static triculture conditions. Furthermore, PDSF was successfully prevascularized through a dynamic perfusion coculture of hASCs and HUVECs, which integrated with PDSF and induced the maturation of vascular networks in vitro. In a xenotransplantation model, PDSF demonstrated myoconductive and immunomodulatory properties associated with the predominance of M2 macrophages and regulatory T cells. In a volumetric muscle loss (VML) model, prevascularized PDSF augmented neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant musculofascial tissue formation. These results indicate that hASCs' integration with PDSF enhances the cells' dual function in immunomodulation and angiogenesis. Owing in part to this PDSF-hASC synergy, our platform shows promise for vascularized muscle flap engineering for VML reconstruction.

Human glial müller and umbilical vein endothelial cell coculture as an in vitro model to investigate retinal oxidative damage. A morphological and molecular assessment.

The aim of this study was to optimize a coculture in vitro model established between the human Müller glial cells and human umbilical vein endothelial cells, mimicking the inner blood-retinal barrier, and to explore its resistance to damage induced by oxidative stress. A spontaneously immortalized human Müller cell line MIO-M1 and human umbilical vein endothelial cells (HUVEC) were plated together at a density ratio 1:1 and maintained up to the 8th passage (p8). The MIO-M1/HUVECs p1 through p8 were treated with increasing concentrations (range 200-800μM) of H2 O2 to evaluate oxidative stress induced damage and comparing data with single cell cultures. The following features were assayed p1 through p8: doubling time maintenance, cell viability using MTS assay, ultrastructure of cell-cell contacts, immunofluorescence for Vimentin and GFAP, molecular biology (q-PCR) for GFAP and CD31 mRNA. MIO-M1/HUVECs cocultures maintained distinct cell cytotype up to p8 as shown by flow cytometry analysis, without evidence of cross activation, displaying cell-cell tight junctions mimicking those found in human retina, only acquiring a slight resistance to oxidative stress induction over the passages. This MIO-M1/HUVECs coculture represents a simple, reproducible and affordable model for in vitro studies on oxidative stress-induced retinal damages.

Creating a kidney organoid-vasculature interaction model using a novel organ-on-chip system

Kidney organoids derived from human induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study kidney development and disease. However, the lack of vascularization of these organoids often leads to insufficient oxygen and nutrient supply. Vascularization has previously been achieved by implantation into animal models, however, the vasculature arises largely from animal host tissue. Our aim is to transition from an in vivo implantation model towards an in vitro model that fulfils the advantages of vascularization whilst being fully human-cell derived. Our chip system supported culturing of kidney organoids, which presented nephron structures. We also showed that organoids cultured on chip showed increased maturation of endothelial populations based on a colocalization analysis of endothelial markers. Moreover, we observed migration and proliferation of human umbilical vein endothelial cells (HUVECs) cultured in the channels of the chip inside the organoid tissue, where these HUVECs interconnected with endogenous endothelial cells and formed structures presenting an open lumen resembling vessels. Our results establish for the first-time vascularization of kidney organoids in HUVEC co-culture conditions using a microfluidic organ-on-chip. Our model therefore provides a useful insight into kidney organoid vascularization in vitro and presents a tool for further studies of kidney development and drug testing, both for research purposes and pre-clinical applications.

Piezo1 in endothelial cells is involved in vitamin D-induced vascular calcification

The relationship between the Piezo1 channel of vascular endothelial cells and vascular calcification is unknown. In this study, after subcutaneous injection of vitamin D for 10 consecutive days, the mice showed an increase in serum calcium, aortic calcium content, vascular tension and pulse wave velocity. Piezo1channel antagonist, GsMTx4 alleviated arteriosclerosis and decreased the aortic calcium content, while Piezo1 agonist Yoda1 produced opposite effect. In addition, activation of Piezo1 by Yoda1 impaired the function of human umbilical vein endothelial cells (HUVECs), as evidenced by further decreased production of NO, reduction in expression levels of eNOS, MMP-2, PCNA and VEGFA. When co-culture of HUVECs and vascular smooth muscle cells (VSMCs), activation of Piezo1 in HUVECs enhanced expression levels of calcification-related SOX9 and Runx2 genes, increased ALP activity and calcium deposition in VSMCs. We concluded that Piezo1 in endothelial cells is involved in the pathogenesis of vascular calcification. This study provides a new experimental basis for the prevention and treatment of vascular calcification.

Induction of Periodontal Ligament–like Cells by Coculture of Dental Pulp Cells, Dedifferentiated Cells Generated from Epithelial Cell Rests of Malassez, and Umbilical Vein Endothelial Cells

IntroductionApart from the epithelial cell rests of Malassez (ERMs), dental pulp (DP) contains the same types of mesenchymal cells as the periodontal ligament (PDL). ERMs may affect the characteristics of the mesenchymal cells in the PDL. The aim of this study was to examine whether DP cells cultured with ERMs and human umbilical vein endothelial cells (HUVECs) could transform into PDL-like cells. MethodsProgenitor-dedifferentiated into stem-like cells (Pro-DSLCs) were produced by the induction of ERMs with 5-Azacytidine and valproic acid. DP cells were cultured in mesenchymal stem cell medium for 1 week under the following conditions: DP cells alone (controls); PDL cells alone; coculture of DP cells and ERMs (DP + ERM) or Pro-DSLCs (DP + Pro-DSLC); and coculture of DP cells, HUVECs, and ERMs (DP + ERM + HUVEC) or Pro-DSLCs (DP + Pro-DSLC + HUVEC). Quantitative real-time reverse transcription polymerase chain reaction, quantitative methylation-specific polymerase chain reaction, and flow cytometry were performed. ResultsThe expression levels of PDL-related markers Msx1, Msx2, Ncam1, Postn, and S100a4 and mesenchymal stem cell–positive markers Cd29, Cd90, and Cd105 were significantly higher in the PDL cells and DP + Pro-DSLC + HUVEC cultures than in the controls (P < .05). The DNA methylation levels of Msx1 and Cd29 in the PDL cells and the DP + Pro-DSLC + HUVEC culture were significantly lower than in the controls (P < .01). We found a significant increase in the number of cells stained with MSX1 (P < .05) and CD29 (P < .01) in the DP + Pro-DSLC + HUVEC culture than in the controls. ConclusionsCoculture of DP cells with Pro-DSLCs and HUVECs induced their transformation into PDL-like cells. This method may prove to be useful for periodontal regeneration via tissue engineering.

Abstract 6313: Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia

Abstract Acute myeloid leukemia is a grievous illness. BiTE® (bispecific T cell engager) molecules redirect T cells by engaging CD3 and a tumor-associated antigen (TAA). These molecules have shown clinical efficacy but one mechanism of resistance is loss of a single TAA. We hypothesized that a BiTE® molecule targeting &amp;gt;1 TAA could reduce relapse frequency. FLT3 and CD123, were selected for a dual-targeting BiTE® (dBiTE࣪) molecule.A half-life extended (HLE) CD123-FLT3 dBiTE࣪ molecule was evaluated in vitro, in mouse xenografts, and in non-human primate (NHP) tolerability studies. The molecule had nM affinity for human and NHP FLT3, CD123, and CD3, and pM efficacy in cytotoxicity assays using human T cells or NHP PBMCs. The molecule achieved 100% killing against single-positive (sp) cells (CRISPR-generated isogenic cell lines) at potencies like those of double-positive (dp) cells (7.4 ±. 4.1 pM FLT3sp, 7.1 ±. 3.8 pM CD123sp and 3.4 ±. 1.5 pM dp, n=3). In a mouse xenograft model, the molecule induced significant activity and extended survival &amp;gt; 3 weeks (1.0, 0.1, and 0.01 mg/kg (p ≤ 0.001)). In mice with sp tumors, survival benefit could not be calculated due to high survival, demonstrating that both arms of the molecule are active. In NHP, the molecule had a half-life of 52 hours (0.3 or 3 µg/kg). FLT3 mRNA levels, a marker of FLT3-expressing cells, decreased in blood following dosing. Repeat dosing was not tolerated, and cytokine release was observed. Some cytokines were reduced while others increased. CD123 is reportedly expressed on endothelial cells (ECs), with increased expression in inflammatory conditions. An immunohistochemical survey found that CD123 is expressed on human and NHP monocyte/macrophages and ECs with limited distribution in lymphoid tissues and lamina propria of the gut. We hypothesized that cytokine release following administration may result in increased CD123 expression and in turn, further increased cytokine levels with repeat dosing. CD123 was detected on primary human umbilical vein ECs (HUVECs). In co-cultures of HUVECs and T cells, a CD123 BiTE® molecule induced expression of CD123 on HUVEC cells at concentrations that elicited redirected lysis (12.5 pM), T cell activation, and cytokine secretion. To better understand BiTE®-induced upregulation of CD123 on ECs, HUVECs were cultured with supernatants (SN) from a TDCC assay or recombinant IL-3, IL-6, TNFα, or IFNγ. Assay SN and TNFα induced &amp;gt;2-fold CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs. These data demonstrate that CD123 expression on ECs was increased upon exposure to a CD123 mono-targeting BiTE® molecule, potentially through BiTE®-induced secretion of TNFα. Additional studies are ongoing.In sum, a CD123-FLT3 HLE dBiTE࣪ molecule was active against both dp- and sp-positive target cells in vitro and in vivo. Careful selection of TAA for dBiTE࣪ molecules is necessary to increase efficacy and maintain safety. Citation Format: Rebecca Goldstein, Christine Karbowski, Anja Henn, Petra Deegen, Joachim Wahl, Katja Matthes, Christoph Dahlhoff, Brooke Rock, Sabrina Benchaar, Katie Hsu, Brandy Alexander, Matthias Friedrich, Joan Lane, Xiaoting Wang, Jonas Anlahr, Markus Muenz, Tobias Raum, Tara L. Arvedson. Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6313.

Antioxidant Properties of Cerium Oxide Nanoparticles Prevent Retinal Neovascular Alterations In Vitro and In Vivo.

In this study, we investigated whether cerium oxide nanoparticles (CeO2-NPs), a promising antioxidant nanomaterial, may contrast retinal vascular alterations induced by oxidative damage in vitro and in vivo. For the in vivo experiments, the light damage (LD) animal model of Age-Related Macular Degeneration (AMD) was used and the CeO2-NPs were intravitreally injected. CeO2-NPs significantly decreased vascular endothelial growth factor (VEGF) protein levels, reduced neovascularization in the deep retinal plexus, and inhibited choroidal sprouting into the photoreceptor layer. The in vitro experiments were performed on human retinal pigment epithelial (ARPE-19) cells challenged with H2O2; we demonstrated that CeO2-NPs reverted H2O2-induced oxidative stress-dependent effects on this cell model. We further investigated the RPE–endothelial cells interaction under oxidative stress conditions in the presence or absence of CeO2-NPs through two experimental paradigms: (i) treatment of human umbilical vein endothelial cells (HUVECs) with conditioned media from ARPE-19 cells, and (ii) coculture of ARPE-19 and HUVECs. In both experimental conditions, CeO2-NPs were able to revert the detrimental effect of H2O2 on angiogenesis in vitro by realigning the level of tubule formation to that of the control. Altogether, our results indicate, for the first time, that CeO2-NPs can counteract retinal neovascularization and may be a new therapeutic strategy for the treatment of wet AMD.

Concentrated Growth Factors Promote hBMSCs Osteogenic Differentiation in a Co-Culture System With HUVECs.

Osteogenesis is a complex physiologic process that occurs during bone regeneration. This process requires several growth factors that act on bone marrow-derived mesenchymal stem cells (BMSCs). Concentrated growth factor (CGF) is a new-generation platelet-rich derivative that is an appealing autologous material for application in tissue repair and bone regenerative medicine because it contains a variety of fibrin and growth factors. In this study, the effects of CGF on the proliferation and osteogenic differentiation of hBMSCs and human umbilical vein endothelial cells (HUVECs) were explored with in vitro cell co-culture experiments. HBMSCs and HUVECs were directly co-cultured at the ratio of 1:2 under different concentrations (0, 2, 5, 10, 20%) of CGF for 7 days. Alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction were used to detect the effects of CGF on the expression of osteogenic genes (ALP, osteocalcin [OCN], type I collagen [COL-1], Runt-related transcription factor 2 [RUNX2]) and connexin 43 (CX43). RNA sequencing was used to explore potential regulated genes and signaling pathways that affect the osteogenesis of co-cultured hBMSCs exposed to CGF. The results showed higher expressions of ALP, COL-1, RUNX2, OCN, and CX43 in the direct co-culture group containing 10% CGF compared to the direct co-culture group without CGF and the indirect co-culture group. In summary, 10% CGF can significantly promote osteogenesis in hBMSCs directly co-cultured with HUVECs. Intercellular communication between the direct co-culture of hBMSCs and HUVECs through CX43 may be one of the main regulatory mechanisms.

Chemokine (C-C motif) ligand 2-enhanced adipogenesis and angiogenesis of human adipose-derived stem cell and human umbilical vein endothelial cell co-culture system in adipose tissue engineering.

Human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) co-cultured in vitro are widely used in adipose tissue engineering but exhibit various limitations. Chemokine (C-C motif) ligand 2 (CCL2) has been proved essential during adipogenesis and angiogenesis in vivo. We examined whether adipogenesis and angiogenesis could also be directly promoted by CCL2 in vitro. Cells were cultured with 0, 10, 50, and 100ng/ml CCL2. The effects of CCL2 on adipogenesis of hADSCs, and lipid accumulation in the positive control group (hADSCs), blank control group (hADSCs+HUVECs), and experimental group (hADSCs+HUVECs+CCL2) in the hADSC and HUVEC direct co-culture system were evaluated by Oil Red O staining. Angiogenesis in the presence of CCL2 was evaluated by Matrigel tube formation assay. Angiogenic- and adipogenic-associated gene and protein expression in the co-culture system were measured by Quantitative Real-time Polymerase Chain Reaction and western blotting, respectively. All concentrations of CCL2 promoted hADSC adipogenic differentiation and HUVEC tube formation (P<0.05). Following direct co-culture, the experimental group accumulated more lipid droplets than the positive control (P<0.0001), whereas the latter showed better adipogenesis than the blank control group. 50ng/ml CCL2 exhibited stronger adipogenic and angiogenic potential than other concentrations. After 72h of direct co-culture, the mRNA expression of adipogenic differentiation (peroxisome proliferators-activated receptorsγ, CCAAT/enhancer binding protein-α, Leptin, and lipoprotein lipase) and angiogenic genes (vascular endothelial growth factor-A, vascular endothelial growth factor receptor 2, matrix metalloprotein (MMP) 9, and 14) in the experimental group was much higher than in the control (P<0.05). The addition of 50ng/ml CCL2 in the system resulted in elevated phosphorylated Protein kinase B/AKT expression. In summary, CCL2 directly promoted adipogenesis of hADSCs and angiogenesis of HUVECs under both mono-culture and co-culture condition in vitro possibly by enhancing AKT phosphorylation. An optimal concentration of 50ng/ml CCL2 could improve the adipogenesis and angiogenesis of hADSC and HUVEC co-culture system.

Co-culture of BMSCs and HUVECs with simvastatin-loaded gelatin nanosphere/chitosan coating on Mg alloy for osteogenic differentiation and vasculogenesis

Mg alloys are increasingly being investigated as a versatile and economical alternative for developing bone repair implants because of their high mechanical strength, wide availability, adjustable structure and properties. In this study, magnesium alloy WE43 is coated on both sides with gelatin nanosphere/chitosan (GNs/CTS), a coating enhanced by incorporating simvastatin (SIM). SIM-loaded GNs/CTS coated magnesium alloy can promote the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). BMSCs and human umbilical vein endothelial cells (HUVECs) are co-cultured through transwell systems. The release of SIM from the coating is found to increase the secretion of chemokine and angiogenic factors from BMSCs, which promote the migration and tube formation of HUVECs, respectively. Bone morphogenetic protein secreted by HUVECs is seen to increase by the release of SIM from the coating, promoting the osteogenic differentiation of BMSCs. The secretion of chemokines from HUVECs promote the migration of BMSCs. The coated magnesium alloy substrate loaded with SIM is found to regulate the osteogenic differentiation of BMSCs. The study of the paracrine interaction between BMSCs and HUVECs proves that the applied coating promotes both osteogenic differentiation and vascularization, thus demonstrating a new approach for the design of bone repair materials based on magnesium alloys.

Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells.

Background: Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. They have recently emerged as important regulators of angiogenesis. To identify novel lncRNAs that may be involved in the regulation of angiogenesis, we detected the mRNA of 84 lncRNAs in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia for 24h. One of these, rhabdomyosarcoma 2-associated transcript (RMST), is significantly upregulated by hypoxia. Little is known about the presence and roles of RMST in EC function.Objective: The main objective of the study was to investigate the regulation of RMST in ECs and to determine its role in EC survival, proliferation, migration, and differentiation.Methods: Using qPCR, basal mRNA levels of 10 RMST isoforms in HUVECs were measured. Levels were then measured in response to 24h of hypoxia, 7days of differentiation in a co-culture assay, and exposure to four different angiogenesis factors. Functional roles of RMST in EC survival, migration, and differentiation were quantified by using a loss-of-function approach (transfection with single-stranded antisense LNA GapmeRs). EC survival was measured using cell counts and crystal violet assays. Cell migration and differentiation were measured using scratch wound healing and Matrigel® differentiation assays, respectively.Results: Five RMST isoforms (RMST-202, -203, -204, -206, and -207) were detected in HUVECs and human microvascular endothelial cells (HMEC-1s). Other types of vascular cells, including human aortic valve interstitial cells and human aortic smooth muscle cells, did not display this expression profile. RMST was significantly upregulated in response to 24h of hypoxia and in response to 7days of HUVEC co-culture with human lung fibroblasts. RMST was significantly downregulated by angiopoietin-2 (Ang-2), but not by VEGF, FGF-2, or angiopoietin-1 (Ang-1). Selective knockdown of RMST demonstrated that it promotes EC survival in response to serum deprivation. It is also required for VEGF- and Ang-1-induced EC survival and migration, but not for differentiation.Conclusion: We conclude that RMST is expressed in human ECs and that this expression is upregulated in response to hypoxia and during differentiation into capillary-like structures. We also conclude that RMST plays important roles in EC survival and migration.

Spheroid Coculture of Human Gingiva-Derived Progenitor Cells With Endothelial Cells in Modified Platelet Lysate Hydrogels.

Cell coculture strategies can promote angiogenesis within tissue engineering constructs. This study aimed to test the angiogenic potential of human umbilical vein endothelial cells (HUVEC) cocultured with gingiva-derived progenitor cells (GPC) as spheroids in a xeno-free environment. Human platelet lysate (HPL) was used as a cell culture supplement and as a hydrogel matrix (HPLG) for spheroid encapsulation. HUVEC and HUVEC + GPC (1:1 or 5:1) spheroids were encapsulated in various HPLG formulations. Angiogenesis was assessed via in vitro sprouting and in vivo chick chorioallantoic membrane (CAM) assays. HUVEC revealed characteristic in vitro sprouting in HPL/HPLG and this was significantly enhanced in cocultures with GPC (p < 0.05). A trend for greater sprouting was observed in 5:1 vs 1:1 HUVEC + GPC spheroids and in certain HPLG formulations (p > 0.05). Both HUVEC and HUVEC + GPC spheroids in HPLG revealed abundant and comparable neoangiogenesis in the CAM assay (p > 0.05). Spheroid coculture of HUVEC + GPC in HPLG represents a promising strategy to promote angiogenesis.

Repair of Osteoporotic Bone Defects Using Adipose-Derived Stromal Cells and Umbilical Vein Endothelial Cells Seeded in Chitosan/Nanohydroxyapatite-P24 Nanocomposite Scaffolds

Background. The cell regeneration and blood supply of bone defect lesions are restricted under osteoporotic pathological conditions, which make the healing of bone defect of osteoporosis still a great challenge. The current therapeutic strategies that mainly inhibit bone resorption are not always satisfactory for osteoporotic bone defects, which make the development of new therapies an urgent need. Methods. Previously, we prepared chitosan/nanohydroxyapatite (CS/nHA) biomimetic nanocomposite scaffolds for controlled delivery of bone morphogenetic protein 2-derived peptide (P24). In this study, we determined the effect of coculturing adipose-derived stromal cells (ADSCs) and human umbilical vein endothelial cells (HUVECs) with the CS-P24/nHA nanocomposite scaffolds on osteoporotic bone defect healing. In vitro mixed coculture models were employed to assess the direct effects of coculture. Results. ADSCs cocultured with HUVECs showed significantly greater osteogenic differentiation and mineralization compared with ADSCs or HUVECs alone. The CS-P24/nHA scaffold cocultured with ADSCs and HUVECs was more effective in inducing osteoporotic bone repair, as demonstrated by micro-computed tomography and histology of critical-sized calvariae defects in ovariectomized rats. Calvariae defects treated with the CS-P24/nHA nanocomposite scaffold plus ADSC/HUVEC coculture had a greater area of repair and better reconstitution of osseous structures compared with defects treated with the scaffold plus ADSCs or the scaffold plus HUVECs after 4 and 8 weeks. Conclusion. Taken together, coculture of ADSCs and HUVECs with the CS-P24/nHA nanocomposite scaffold is an effective combination to repair osteoporotic bone defects.