Endothelial Cell Spheroids Research Articles

Secretome from iPSC-derived MSCs exerts proangiogenic and immunosuppressive effects to alleviate radiation-induced vascular endothelial cell damage

BackgroundRadiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass, irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications, neurodegeneration, and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity, homeostasis, and repair. Here, we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines.MethodsWe generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome, their modulation, and the molecular pathways they elicit.ResultsIncreasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ≤ 5 Gy) were treated with iMSC CM, endothelial cell viability, adherence, spheroid compactness, and proangiogenic sprout formation were significantly ameliorated, and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently, iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore, iMSC CM suppressed IR-induced NFκB activation, TNF-α release, and ROS production in THP1 cells. Additionally, iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3, iMSCs, or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2, IL6, IL8/CXCL8, ANG (Angiogenin), GROα/CXCL1, and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors, the PI3K/AKT/mTOR modulator PRAS40 and β-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism, translation, mitochondrial respiration, DNA damage repair, and neurodevelopment.ConclusionsThe iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.

CD133-containing microvesicles promote colorectal cancer progression by inducing tumor angiogenesis

Angiogenesis is an indispensable mechanism in cancer progression, as cancer cells need to establish blood vessels to supply oxygen and nutrients. Extracellular vesicles (EVs) derived from cancer cells act as messengers in the tumor microenvironment and induce resistance to anti-angiogenic cancer treatment. EVs can be classified into two categories: exosomes and microvesicles (MVs). Although exosomes are involved in angiogenesis, the role of MVs in angiogenesis and cancer progression remains unclear. CD133 plays a key role in MV formation and oncoprotein trafficking. In this study, we investigated the role of CD133-containing MVs derived from colorectal cancer (CRC) in angiogenesis and cancer progression. CRC-derived MVs were incorporated into endothelial cells and increased the mesh area and tube length of endothelial cells. CD133-containing MVs also stimulate vessel sprouting in endothelial cell spheroids and mouse thoracic aortas. However, MVs derived from CD133-knockdown CRC cells exerted a limited effect on tube formation and vessel sprouting. CD133-containing MVs induced angiogenesis through p38 activation and angiogenesis induced by CD133-containing MVs was insensitive to the anti-vascular endothelial growth factor antibody bevacizumab. Survival analysis revealed that high expression level of CD133 correlated with poor prognosis in patients with metastatic CRC. These findings suggest that CD133-containing MVs act as key regulators of angiogenesis and are related to the prognosis of CRC patients.

Notch signaling pathway induces expression of type IV collagen in angiogenesis.

Mural cell adhesion is important for the localization of basement membrane components during angiogenesis, and cell-cell interactions are thought to be critical for basement membrane formation. Type IV collagen, a component of the basement membrane, and non-triple helical type IV collagen α1 chain (NTH α1(IV)) co-localize in the basement membrane of neovascular vessels. However, it remains unclear how type IV collagen and NTH α1(IV) are produced around the basement membrane. In the present study, we developed a de novo angiogenesis model using human umbilical vein endothelial cell spheroids and TIG-1 fibroblast cells and demonstrated that NTH α1(IV), probably with α1(IV) chain before forming triple helix molecule, was localized in the fibroblasts in contact with vascular endothelial cells. This localization was disrupted by DAPT, a Notch signaling inhibitor. DAPT treatment also reduced type IV collagen and NTH α1(IV) secretion in TIG-1 fibroblasts, along with diminished COL4A1 and COL4A2 gene expression. Downregulation of Notch3 in TIG-1 fibroblasts decreased the secretion of type IV collagen and NTH α1(IV). Taken together, these findings suggest that heterogeneous and homogeneous intercellular Notch signaling via Notch3 induces type IV collagen and NTH α1(IV) expression in fibroblasts and contributes to basement membrane formation in neovascular vessels.

Bioprinting 3D muscle tissue supplemented with endothelial-spheroids for neuromuscular junction model

The neuromuscular junction (NMJ) plays a critical role in muscle contraction, and its dysfunction can result in various neuromuscular disorders. In vitro models for studying NMJ are essential for understanding their functions and pathology. However, the engineering of muscle tissue presents challenges for the organization of myofiber-like oriented muscle bundles as well as the induction of vessel formation and innervation. To address these challenges, we fabricated a hybrid muscle construct comprising uniaxially aligned muscle struts and endothelial cell spheroids using a combination of in situ electric field-assisted bioprinting (E-printing) and microdroplet-based spheroid-forming bioprinting (MDS-printing) techniques. This resulted in self-aggregation of human umbilical vein endothelial cells (HUVECs) into spheroids without attachment to the structures. We tested various fabrication parameters, such as electric field and cross-linking conditions, for E-printing and the deposited cell density of MDS-printing, to stabilize the alignment of the human muscle progenitor cells (hMPCs) and HUVEC spheroids, respectively. The stimulated hMPCs efficiently formed fully aligned myofibers, and the incorporation of HUVEC spheroids induced highly upregulated crosstalk between different cell types compared to a simple E-printed hMPC/HUVEC mixture-loaded construct. This improved myogenesis and vessel formation in vitro. In addition, when co-cultured with a motor neuron-like cell (NSC-34) spheroid separated by a channel, we observed considerably improved neuromuscular junction formation compared to those formed with the normally mixed cell-bearing structures. Our findings suggest that this hybrid muscle construct has the potential to enhance muscle tissue engineering by improving biological activities through the incorporation of HUVEC-spheroids and facilitating neuromuscular junction formation through co-culture with NSC-34 spheroids.

Tunable Synthetic Hydrogels to Study Angiogenic Sprouting.

Angiogenic sprouting, the formation of new blood vessels from pre-existing vasculature, is tightly regulated by the properties of the surrounding tissue microenvironment. Although the extracellular matrix has been shown to be a major regulator of this process, it is not clear how individual biochemical and mechanical properties influence endothelial cell sprouting. This information gap is largely due to the lack of suitable in vitro models that recapitulate angiogenic sprouting in a 3D environment with independent control over matrix properties. Here, we present protocols for the preparation of endothelial cell spheroid-laden synthetic, dextran-based hydrogels, which serve as a highly tunable 3D scaffold. The adjustment of the hydrogels' adhesiveness, stiffness, and degradability is demonstrated in detail. Finally, we describe assays to elucidate how individual matrix properties regulate angiogenic sprouting, including their analysis by immunofluorescence staining and imaging. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of methacrylated dextran (DexMA) Basic Protocol 2: Generation of endothelial cell spheroids in microwells Basic Protocol 3: Endothelial cell sprouting in hydrogels of tunable stiffness Basic Protocol 4: Endothelial cell sprouting in hydrogels of tunable adhesiveness Basic Protocol 5: Endothelial cell sprouting in hydrogels of tunable degradability Basic Protocol 6: Imaging of endothelial cell spheroid-laden hydrogels Support Protocol 1: Preparation of pro-angiogenic cocktail for endothelial cell sprouting.

Development of in vitro and in vivo tools to evaluate the antiangiogenic potential of melatonin to neutralize the angiogenic effects of VEGF and breast cancer cells: CAM assay and 3D endothelial cell spheroids

Melatonin is a molecule with different antitumor actions in breast cancer and has been described as an inhibitor of vascular endothelial growth factor (VEGF). Despite the recognition of the key role exerted by VEGF in tumor angiogenesis, limitations arise when developing models to test new antiangiogenic molecules. Thus, the aim of this study was to develop rapid, economic, high capacity and easy handling angiogenesis assays to test the antiangiogenic effects of melatonin and demonstrate its most effective dose to neutralize and interfere with the angiogenic sprouting effect induced by VEGF and MCF-7. To perform this, 3D endothelial cell (HUVEC) spheroids and a chicken embryo chorioallantoic membrane (CAM) assay were used. The results showed that VEGF and MCF-7 were able to stimulate the sprouting of the new vessels in 3D endothelial spheroids and the CAM assay, and that melatonin had an inhibitory effect on angiogenesis. Specifically, as the 1 mM pharmacological dose was the only effective dose able to inhibit the formation of ramifications around the alginate in the CAM assay model, this inhibition was shown to occur in a dose-dependent manner. Taken together, these techniques represent novel tools for the development of antiangiogenic molecules such as melatonin, with possible implications for the therapy of breast cancer.

Steppogenin suppresses tumor growth and sprouting angiogenesis through inhibition of HIF-1α in tumors and DLL4 activity in the endothelium

BackgroundHypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, angiogenesis mediated by HIF-1α is involved in the growth and metastasis of tumor cells. During the angiogenic process, differentiated tip endothelial cells (ECs) characterized by high expression of DLL4 promote angiogenic germination through filopodia. Inhibitors of HIF-1α or DLL4 have been widely studied PurposeWe tried to find inhibitors targeting both HIF-1α and DLL4 in tumor which have not yet been developed. Study designIn this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1α and DLL4 under hypoxic conditions. MethodsAfter examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1α and DLL4 transcriptional activity using a dual-luciferase reporter assay. Western blot analysis, immunofluoresecnt assay and real-time qPCR were performed to identify expression of proteins, such as HIF-1α and DLL4, as well as HIF-1α target genes under hypoxic conditions. In vitro angiogenesis assay and in vivo allograft tumor experiment were performed to investigate inhibition of tumor growth through anti-angiogenic activity. ResultsAmong these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1α under hypoxic conditions in HEK293T cells and vascular endothelial growth factor (VEGF)-induced DLL4 expression in vascular ECs in a dose-dependent manner. In tumor cells and retinal pigment epithelial cells, steppogenin significantly suppressed HIF-1α protein levels under hypoxic conditions as well as VEGF-induced DLL4 expression in ECs. Furthermore, steppogenin suppressed hypoxia-induced vascular EC proliferation and migration as well as VEGF-induced sprouting of EC spheroids. ConclusionThese results suggest that the natural compound steppogenin could potentially be used to treat angiogenic diseases, such as those involving solid tumors, because of its dual inhibition of HIF-1α and DLL4.

Hybrid cell constructs consisting of bioprinted cell-spheroids.

Bioprinted cell constructs have been investigated for regeneration of various tissues. However, poor cell-cell interactions have limited their utility. Although cell-spheroids offer an alternative for efficient cell-cell interactions, they complicate bioprinting. Here, we introduce a new cell-printing process, fabricating cell-spheroids and cell-loaded constructs together without preparation of cell-spheroids in advance. Cells in mineral oil droplets self-assembled to form cell-spheroids due to the oil-aqueous interaction, exhibiting similar biological functions to the conventionally prepared cell-spheroids. By controlling printing parameters, spheroid diameter and location could be manipulated. To demonstrate the feasibility of this process, we fabricated hybrid cell constructs, consisting of endothelial cell-spheroids and stem cells loaded decellularized extracellular matrix/β-tricalcium phosphate struts for regenerating vascularized bone. The hybrid cell constructs exhibited strong angiogenic/osteogenic activities as a result of increased secretion of signaling molecules and synergistic crosstalk between the cells.

Electrochemical microwell sensor with Fe-N co-doped carbon catalyst to monitor nitric oxide release from endothelial cell spheroids.

Endothelial cells have been widely used for vascular biology studies; recent progress in tissue engineering have offered three-dimensional (3D) culture systems for vascular endothelial cells which can be considered as physiologically relevant models. To facilitate the studies, we developed an electrochemical device to detect nitric oxide (NO), a key molecule in the vasculature, for the evaluation of 3D cultured endothelial cells. Using an NO-sensitive catalyst composed of Fe-N co-doped reduced graphene oxide, the real-time monitoring of NO release from the endothelial cell spheroids was demonstrated.

TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload.

Background: Cardiac hypertrophy (CH) occurs with an increase in myocardium mass as an adaptive compensation to increased stress. Prolonged CH causes decompensated heart failure (HF). Enhanced angiogenesis by vascular endothelial growth factor (VEGF) is observed in hypertrophied hearts; impaired angiogenesis by angiotensin II (AngII) is observed in failing hearts. Angiogenesis is executed by vascular endothelial cells (ECs). Abnormal Ca2+ homeostasis is a hallmark feature of hypertrophied and failing hearts. Ca2+-activated chloride channel transmembrane protein 16A (TMEM16A) is expressed in cardiomyocytes and ECs but its role in heart under stress remains unknown. Methods: Pressure-overload-induced CH and HF mouse models were established. Echocardiography was performed to evaluate cardiac parameters. Quantitative real-time PCR, traditional and simple western assays were used to quantify molecular expression. Whole-cell patch-clamp experiments were used to detect TMEM16A current (ITMEM16A) and action potential duration (APD) of cardiomyocytes. VEGF and AngII were used separately in ECs culture to simulate enhanced or impaired angiogenesis, respectively. TMEM16A low-expressed and over-expressed ECs were obtained by siRNA or lentivirus transfection. Wound healing, tube formation and ECs spheroids sprouting assays were performed to assess migration and angiogenesis. Results: Neither TMEM16A molecular expression levels nor whole-cell ITMEM16A density varied significantly during the development of CH and HF. ITMEM16A comprises transient outward current, but doesn’t account for APD prolongation in hypertrophied or failing cardiomyocytes. In cultured ECs, TMEM16A knockdown inhibited migration and angiogenesis, TMEM16A overexpression showed opposite result. Promotion of migration and angiogenesis by VEGF was decreased in TMEM16A low-expressed ECs but was increased in TMEM16A over-expressed ECs. Inhibition of migration and angiogenesis by AngII was enhanced in TMEM16A low-expressed ECs but was attenuated in TMEM16A over-expressed ECs. Conclusion: TMEM16A contributes insignificantly in myocardium remodeling during pressure-overload. TMEM16A is a positive regulator of migration and angiogenesis under normal condition or simulated stress. TMEM16A may become a new target for upregulation of angiogenesis in ischemic disorders like ischemic heart disease.

Bone tissue engineering supported by bioprinted cell constructs with endothelial cell spheroids.

In bone tissue engineering, efficient formation of vascularized bone tissue is a challenging issue. Here, we introduce a new strategy for effectively using multiple cells laden in a hybrid structure, such as endothelial cell (EC) spheroids and homogeneously distributed human adipose stem cells (hASCs) for bone regeneration.Methods: To fabricate the EC spheroids, cell-mixed mineral oil was used, and microscale droplets of the cell mixture were interlayered between the bioprinted hASC-laden struts. In vitro cellular responses of spheroid-laden multiple-cell constructs have been evaluated by comparing with the cell constructs bioprinted with the mixture of hASCs and ECs. In addition, mastoid obliterated rat model has been used to observe in vivo bone formation of those cell constructs.Results: The spheroid-laden multiple-cell constructs induced outstanding angiogenesis and osteogenic activities compared to a conventionally bioprinted multiple-cell construct. The enhanced biological results were clearly due to the EC spheroids, which triggered highly cooperative crosstalk between ECs and stem cells. The co-culture of the hASC constructs with the EC spheroids exhibited enhanced osteogenic- and angiogenic-related gene expression in vitro. In addition, in a rat obliterated mastoid model, considerably greater new bone formation and more competent development of new blood vessels were observed compared to those achieved with the normally bioprinted multiple cell-loaded structure.Conclusion: In vitro and in vivo results demonstrated that the bioprinted spheroid-laden multiple-cell construct is a potential candidate for use in bone tissue engineering.

ENOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Natural Presentation of Glycosaminoglycans in Synthetic Matrices for 3D Angiogenesis Models.

Glycosaminoglycans (GAGs) are long, linear polysaccharides that occur in the extracellular matrix of higher organisms and are either covalently attached to protein cores, as proteoglycans or in free form. Dependent on their chemical composition and structure, GAGs orchestrate a wide range of essential functions in tissue homeostasis. Accordingly, GAG-based biomaterials play a major role in tissue engineering. Current biomaterials exploit crosslinks between chemically modified GAG chains. Due to modifications along the GAG chains, they are limited in their GAG-protein interactions and accessibility to dissect the biochemical and biophysical properties that govern GAG functions. Herein, a natural presentation of GAGs is achieved by a terminal immobilization of GAGs to a polyethylene glycol (PEG) hydrogel. A physicochemical characterization showed that different end-thiolated GAGs can be incorporated within physiological concentration ranges, while the mechanical properties of the hydrogel are exclusively tunable by the PEG polymer concentration. The functional utility of this approach was illustrated in a 3D cell culture application. Immobilization of end-thiolated hyaluronan enhanced the formation of capillary-like sprouts originating from embedded endothelial cell spheroids. Taken together, the presented PEG/GAG hydrogels create a native microenvironment with fine-tunable mechanobiochemical properties and are an effective tool for studying and employing the bioactivity of GAGs.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

Matrix stiffness modulates tip cell formation through the p-PXN-Rac1-YAP signaling axis

Endothelial tip cell outgrowth of blood-vessel sprouts marks the initiation of angiogenesis which is critical in physiological and pathophysiological procedures. However, how mechanical characteristics of extracellular matrix (ECM) modulates tip cell formation has been largely neglected. In this study, we found enhanced CD31 expression in the stiffening outer layer of hepatocellular carcinoma than in surrounding soft tissues. Stiffened matrix promoted sprouting from endothelial cell (EC) spheroids and upregulated expressions of tip cell-enriched genes in vitro. Moreover, tip cells showed increased cellular stiffness, more actin cytoskeleton organization and enhanced YAP nuclear transfer than stalk and phalanx ECs. We further uncovered that substrate stiffness regulates FAK and Paxillin phosphorylation in focal adhesion of ECs promoting Rac1 transition from inactive to active state. YAP is subsequently activated and translocated into nucleus, leading to increased tip cell specification. p-Paxillin can also loosen the intercellular connection which also facilitates tip cell specification. Collectively our present study shows that matrix stiffness modulates tip cell formation through p-PXN-Rac1-YAP signaling axis, shedding light on the role of mechanotransduction in tip cell formation. This is of special significance in biomaterial design and treatment of some pathological situations.

Effects of Tunneling Nanotubes on the Mitochondrial Regulation of the Amount and Subcellular Localization of Β-Catenin During Osteogenesis of MSC/HUVEC Spheroids

Tunneling nanotubular expressways (TNTs), which allow direct cell-to-cell transfer of intracellular organelles, have been widely identified in various cell types. However, the precise functions of TNTs in intercellular communication and their practical application in tissue regeneration is still uncertain. Mesenchymal stem cells (MSCs) are commonly employed as seed cells in tissue engineering. The differentiation of MSCs requires sufficient energy, which can be regulated by the fusion and fission of mitochondria. The phenomenon of mitochondrial shuttle between cells has been observed, and has led to the hypothesis that applying TNTs to deliver mitochondria into MSCs might be a promising approach to stimulate osteogenic differentiation. In proliferating endothelial cells (ECs), cellular dynamics including the fusion and fission of mitochondria is increased, and thus ECs are considered as ideal candidates for mitochondria donors. In order to exploit the application of TNT-mediated mitochondria transfer, we employed mesenchymal stem cell/ human umbilical vein endothelial cell (MSC/HUVEC) spheroids as a research model, and investigated the transfer among them, as well as the underlying mechanism. Fluorescence staining showed that directional transfer of mitochondria between MSC-HUVEC pairs, especially from HUVEC to MSC. Through TNT-mediated mitochondrial transfer, osteogenesis markers were up-regulated, accompanied by an increased amount of β-catenin in MSCs. Moreover, the improved generation of pre-vascular network has also been observed in the spheroids, as a result of β-catenin translocation to the periphery of HUVECs. However, all of these effects are abolished by the destruction of TNTs. Collectively, our results indicate that the TNT strategy can be applied widely to various aspects of biological research, such as but not limited to tissue regeneration and targeted drug delivery.

NETosis in the pathogenesis of acute lung injury following cutaneous chemical burns.

Despite the high morbidity and mortality among patients with extensive cutaneous burns in the intensive care unit due to the development of acute respiratory distress syndrome, effective therapeutics remain to be determined. This is primarily because the mechanisms leading to acute lung injury (ALI) in these patients remain unknown. We test the hypothesis that cutaneous chemical burns promote lung injury due to systemic activation of neutrophils, in particular, toxicity mediated by the deployment of neutrophil extracellular traps (NETs). We also demonstrate the potential benefit of a peptidyl arginine deiminase 4 (PAD4) inhibitor to prevent NETosis and to preserve microvascular endothelial barrier function, thus reducing the severity of ALI in mice. Our data demonstrated that phenylarsine oxide (PAO) treatment of neutrophils caused increased intracellular Ca2+-associated PAD4 activity. A dermal chemical burn by lewisite or PAO resulted in PAD4 activation, NETosis, and ALI. NETs disrupted the barrier function of endothelial cells in human lung microvascular endothelial cell spheroids. Citrullinated histone 3 alone caused ALI in mice. Pharmacologic or genetic abrogation of PAD4 inhibited lung injury following cutaneous chemical burns. Cutaneous burns by lewisite and PAO caused ALI by PAD4-mediated NETosis. PAD4 inhibitors may have potential as countermeasures to suppress detrimental lung injury after chemical burns.

BNO 1095, a Standardized Dry Extract from the Fruits of Vitex agnus-castus, Impairs Angiogenesis-related Endothelial Cell Functions In Vitro.

BNO 1095, a standardized dry extract from the fruits of Vitex agnus-castus, represents an approved herbal medicinal product for the treatment of premenstrual syndrome. Angiogenesis, the formation of new blood vessels from pre-existing capillaries, plays a major role in physiological situations, such as wound healing or tissue growth in female reproductive organs, but it is also of great importance in pathophysiological conditions such as chronic inflammatory diseases or cancer. Angiogenesis is a highly regulated multi-step process consisting of distinct key events that can be influenced pharmacologically. Few studies suggested anti-angiogenic actions of V.agnus-castus fruit extracts in in vivo and ex vivo models. Here, we provide for the first time profound in vitro data on BNO 1095-derived anti-angiogenic effects focusing on distinct angiogenesis-related endothelial cell functions that are inevitable for the process of new blood vessel formation. We found that V.agnus-castus extract significantly attenuated undirected and chemotactic migration of primary human endothelial cells. Moreover, the extract efficiently inhibited endothelial cell proliferation and reduced the formation of tube-like structures on Matrigel. Of note, the treatment of endothelial cell spheroids almost blocked endothelial sprouting in a 3D collagen gel. Our data present new and detailed insights into the anti-angiogenic actions of BNO 1095 and, therefore, suggest a novel scope of potential therapeutic applications of the extract for which these anti-angiogenic properties are required.

Photo-crosslinked gelatin methacrylate hydrogels with mesenchymal stem cell and endothelial cell spheroids as soft tissue substitutes

Photo-crosslinked gelatin methacrylate hydrogels with mesenchymal stem cell and endothelial cell spheroids as soft tissue substitutes

An Approach to Study Melanoma Invasion and Crosstalk with Lymphatic Endothelial Cell Spheroids in 3D Using Immunofluorescence.

Three-dimensional (3D) cell culture has allowed a deeper understanding of complex pathological and physiological processes, overcoming some of the limitations of 2D cell culture on plastic and avoiding the costs and ethical issues related to experiments involving animals. Here we describe a protocol to embed single melanoma cells alone or together with primary human lymphatic endothelial cells in a 3D cross-linked matrix, to investigate the invasion and molecular crosstalk between these two cell types, respectively. After fixation and staining with antibodies and fluorescent conjugates, phenotypic changes in both cell types can be specifically analyzed by confocal microscopy.