Paracrine Interactions Research Articles

Abstract 4135951: Single Nuclei RNA Sequencing Reveals Distinct Cellular Composition of Pericoronary Adipose Tissue

Introduction: Adipose tissue is a metabolically active organ with local paracrine interactions on adjacent tissues. Pericoronary adipose tissue (PcAT) is an emerging cardiovascular disease (CVD) risk factor and likely has paracrine effects on coronary vasculature that may promote coronary artery disease (CAD) development. PcAT may be functionally and clinically distinct from epicardial adipose tissue (EAT) given its proximity to the coronary artery adventitia. The cellular composition of PcAT and EAT and whether they have unique inflammatory cell signatures and functional expression at a single cell level has not been explored. Hypothesis: PcAT will have diverse cellular composition with heightened inflammatory cell content compared to EAT. Methods: Visual dissection of paired EAT not adjacent to an epicardial coronary artery and PcAT in direct apposition to an epicardial artery (left anterior descending artery) was performed from a single human heart immediately after explant (prior to heart transplantation). Nuclei were isolated using the Chromium Nuclei Isolation Kit (10x Genomics) with RNAse inhibitor. Cells were counted with a Cellometer K2 and Chromium Next GEM Single Cell libraries were prepared, followed by reverse transcription, PCR and NovaSeq 6000 platform sequencing. Raw sequence reads from single nuclei RNA-seq (snRNA-seq) were processed and after QC filtering, cells were clustered using Seurat. Clusters were annotated using singleR. Results: In PcAT and EAT, >7000 cells were successfully extracted with 73755 and 48750 mean snRNA-seq reads per cell, respectively. snRNA-seq cellular composition analysis delineated a heightened inflammatory milieu and distinct cellular composition of human PcAT vs. EAT. A UMAP (Uniform Manifold Approximation and Projection) plot displays higher inflammatory cell infiltrate with a neutrophil predominance and lesser adipocyte composition in PcAT compared with EAT ( Figure 1) . Conclusions: We have generated snRNA-seq data from human PcAT and shown distinct cellular composition compared to EAT. Given the proximity of PcAT to coronary vasculature, these results are supportive of paracrine inflammatory effects and highlight adipose tissue biology in cardiovascular disease. Understanding this biology may identify novel therapeutic targets for CVD.

CNSC-61. NEURONAL-ACTIVITY DEPENDENT MECHANISMS OF SMALL CELL LUNG CANCER PATHOGENESIS

Abstract Neural activity is increasingly recognized as a crucial regulator of cancer growth. In the brain, neuronal activity robustly influences glioma growth both through paracrine mechanisms and through electrochemical integration of malignant cells into neural circuitry via neuron-to-glioma synapses. Outside of the CNS, innervation of tumors such as prostate, breast, pancreatic, and gastrointestinal cancers by peripheral nerves similarly regulates cancer progression. However, the extent to which the nervous system regulates lung cancer progression, either in the lung or when metastatic to the brain, is less well understood. Small cell lung cancer (SCLC) is a lethal high-grade neuroendocrine tumor that exhibits a strong propensity to metastasize to the brain. Here we demonstrate that SCLC cells in the brain co-opt neuronal activity-regulated mechanisms to stimulate growth and progression. With in vivo optogenetic stimulation, we show that glutamatergic and GABAergic cortical neuronal activity each drive proliferation of SCLC through both paracrine and synaptic neuron-cancer interactions. In the brain, SCLC cells form bona fide neuron-to-SCLC synapses evident on immunoelectron microscopy for the first time for non-brain derived malignancy. Using electrophysiology and two-photon calcium imaging, we find that SCLC cells exhibit depolarizing currents with consequent calcium transients in response to neuronal activity. SCLC cell membrane depolarization is sufficient to promote the growth of intracranial tumors. We also demonstrate reciprocal effects of SCLC on neurons, manifesting as increased neuronal synaptogenesis and elevated field potentials in regions of the tumor. Finally, in the lung, vagus nerve transection markedly inhibits primary lung tumor formation and development and awards survival benefit in spontaneous genetic SCLC model, highlighting a critical role for innervation in overall SCLC growth. Taken together, these studies illustrate that neuronal activity plays a crucial role in dictating SCLC pathogenesis in both the lung and the brain.

Modeling the contribution of cardiac fibroblasts in dilated cardiomyopathy using induced pluripotent stem cells (iPSCs).

Fibrosis is implicated in nearly all forms of cardiomyopathy and significantly influences disease severity and outcomes. The primary cell responsible for fibrosis is the cardiac fibroblast, which remains understudied relative to cardiomyocytes in the context of cardiomyopathy. The development of induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) allows for the modeling of patient-specific disease characteristics and provides a scalable source of fibroblasts. iPSC-CFs are invaluable for understanding molecular pathways that affect disease progression and outcomes. This review explores various aspects of cardiomyopathy, with a focus on dilated cardiomyopathy (DCM), that can be modeled using iPSC-CFs and their application in drug discovery, given the current lack of approved therapies for cardiac fibrosis. We examine how iPSC-CFs can be utilized to study heart development, fibroblast heterogeneity, and activation, with the ultimate goal of developing better therapies for patients with cardiomyopathies. Significance Statement Here, we explore how iPSC-CFs can be used to study the fibrotic component of DCM. Most research has focused on cardiomyocytes, despite the significant contribution of fibroblasts to disease outcomes. iPSC-CFs serve as a valuable tool to elucidate molecular pathways leading to fibrosis, and paracrine interactions with cardiomyocytes, which are not fully understood. Gaining insights into these events could aid in the development of new therapies and enable the use of patient-derived iPSC-CFs for precision medicine, ultimately improving patient outcomes.

Interactions between androgen and IGF1 axes in prostate tumorigenesis.

Androgen signalling through the androgen receptor (AR)is essential for prostate tumorigenesis. However, androgen signalling pathways also interact with other growth factor-mediated signalling pathways to regulate the prostatic cell cycle, differentiation, apoptosis and proliferation in the initiation and progression of prostate cancer. Insulin-like growth factor 1 (IGF1) is one of the most prominent growth factors in prostate tumorigenesis. Clinical and experimental evidence has demonstrated that IGF1 signalling supports both androgen-dependent and androgen-independent prostate tumorigenesis, suggesting that improved understanding of the interactions between the IGF1 and androgen axes might aid the development of new therapeutic strategies. Available data have shown a dynamic role of androgen-AR signalling in the activation of IGF1-signalling pathways by augmenting transcription of the IGF1 receptor in prostatic basal epithelial cells and by increasing IGF1 secretion through the suppression of IGF-binding protein 3 expression in prostatic stromal cells. In turn, IGF1 stimulates Wnt-β-catenin signalling in prostatic basal progenitors to promote prostatic oncogenic transformation and prostate cancer development. These findings highlight the cooperative, autocrine and paracrine interactions that underlie the oncogenic effects of androgens and IGF1 and open up new opportunities for therapeutic targeting.

AMH regulates a mosaic population of AMHR2-positive cells in the ovarian surface epithelium

The function and homeostasis of the mammalian ovary depend on complex paracrine interactions between multiple cell types. Using primary mouse tissues and isolated cells, we showed in vitro that ovarian follicles secrete a factor(s) that suppresses growth of ovarian epithelial cells in culture. Most of the growth suppressive activity was accounted for by Anti-Mullerian Hormone/Mullerian Inhibitory Substance (AMH/MIS) secreted by granulosa cells of the follicles, as determined by immune depletion experiments. Additionally, conditioned medium from granulosa cells from wild type control, but not AMH knockout, suppressed epithelial cell growth. Tracing of the AMH regulated cells using AMHR2 (AMH receptor 2)-Cre:ROSA26 mutant mice indicated the presence of populations of AMHR2-positive epithelial cells on the ovarian surface and oviduct epithelia. Cells isolated from the mutant mice indicated that a subpopulation of cells marked by AMHR2-Cre:ROSA26 accounted for most cell growth and expansion in ovarian surface epithelial cells, and the AMHR2 lineage derived cells were regulated by AMH in vitro; whereas, fewer AMHR2-Cre:ROSA26 marked cells accounted for oviduct epithelial cell outgrowth. The results reveal a paracrine pathway in maintaining follicle-epithelial homeostasis in ovary and support a subpopulation of AMHR2 lineage marked epithelial cells as ovarian epithelial stem/progenitor cells with higher proliferative potential regulatable by follicle secreted AMH.

Tumor microenvironment governs the prognostic landscape of immunotherapy for head and neck squamous cell carcinoma: A computational model-guided analysis.

Immune checkpoint inhibition (ICI) has emerged as a critical treatment strategy for squamous cell carcinoma of the head and neck (HNSCC) that halts the immune escape of the tumor cells. Increasing evidence suggests that the onset, progression, and lack of/no response of HNSCC to ICI are emergent properties arising from the interactions within the tumor microenvironment (TME). Deciphering how the diversity of cellular and molecular interactions leads to distinct HNSCC TME subtypes subsequently governing the ICI response remains largely unexplored. We developed a cellular-molecular model of the HNSCC TME that incorporates multiple cell types, cellular states, and transitions, and molecularly mediated paracrine interactions. An exhaustive simulation of the HNSCC TME network shows that distinct mechanistic balances within the TME give rise to the five clinically observed TME subtypes such as immune/non-fibrotic, immune/fibrotic, fibrotic only and immune/fibrotic desert. We predict that the cancer-associated fibroblast, beyond a critical proliferation rate, drastically worsens the ICI response by hampering the accessibility of the CD8+ killer T cells to the tumor cells. Our analysis reveals that while an Interleukin-2 (IL-2) + ICI combination therapy may improve response in the immune desert scenario, Osteopontin (OPN) and Leukemia Inhibition Factor (LIF) knockout with ICI yields the best response in a fibro-dominated scenario. Further, we predict Interleukin-8 (IL-8), and lactate can serve as crucial biomarkers for ICI-resistant HNSCC phenotypes. Overall, we provide an integrated quantitative framework that explains a wide range of TME-mediated resistance mechanisms for HNSCC and predicts TME subtype-specific targets that can lead to an improved ICI outcome.

Development and in vitro evaluation of bioprinted plasma-infused biocarriers for mesenchymal stromal cell delivery in musculoskeletal disorder treatment

A meta-analysis revealed no advantage of surgical over non-surgical treatments, emphasizing the need for non-invasive methods, particularly for prevalent osteoarticular diseases like knee osteoarthritis. To enhance therapeutic efficacy, we developed a plasma-infused gelatin methacryloyl (GelMA) biocarrier loaded with bone marrow-derived mesenchymal stromal cells (BMSCs). These constructs were evaluated in vitro for their properties and paracrine interactions in non-inflamed and inflamed environments. GelMA infused with platelet-rich plasma (PRP) and platelet-poor plasma (FFP) were compared. Pristine GelMA was used as a control. Both PRP and FFP enhanced the proliferation and viability of BMSCs in biocarriers, promoting cell survival pathways while inhibiting necrotic and apoptotic events. Proteomic analysis showed no differences in BMSC behavior between PRP and FFP in the absence of inflammation (p = 0.550). However, both plasmas significantly modified cell behavior under inflammatory conditions (p = 0.001). PRP- and FFP-infused biocarriers activated ten key signaling pathways, including HIF-1a, neuroinflammation, and extracellular matrix turnover. PRP-specific pathways included IL-17, IL-6, and several growth factor signaling pathways. No significant differences in angiogenesis were linked to platelet dose (p = 0.079), but both PRP and FFP significantly enhanced angiogenesis compared to GelMA alone (p < 0.001 for PRP, p = 0.002 for FFP). FFP showed stronger angiogenesis than PRP under IL-1β treatment (p = 0.042). Plasma-infused biocarriers altered BMSC behavior in response to inflammatory cytokines compared to GelMA (p = 0.001). PRP specifically activated TGF-b signaling under IL-1b (Z=2.308, p-value 1.02E-35), which was not seen under TNF-a exposure. These findings suggest that PRP and FFP-infused biocarriers may offer promising improvements in regenerative therapies for inflammatory osteoarticular conditions like knee osteoarthritis.

1,25-dihydroxyvitamin-D3 distinctly impacts the paracrine and cell-to-cell contact interactions between hPDL-MSCs and CD4+ T lymphocytes.

Human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) possess a strong ability to modulate the immune response, executed via cytokine-boosted paracrine and direct cell-to-cell contact mechanisms. This reciprocal interaction between immune cells and hPDL-MSCs is influenced by 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). In this study, the participation of different immunomodulatory mechanisms on the hPDL-MSCs-based effects of 1,25(OH)2D3 on CD4+ T lymphocytes will be elucidated using different co-culture models with various cytokine milieus. hPDL-MSCs and CD4+ T lymphocytes were co-cultured indirectly and directly with inserts (paracrine interaction only) or directly without inserts (paracrine and direct cell-to-cell contact interaction). They were stimulated with TNF-α or IL-1β in the absence/presence of 1,25(OH)2D3. After five days of co-cultivation, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the gene expression of soluble and membrane-bound immunomediators was determined in hPDL-MSCs. In the indirect and direct co-culture model with inserts, 1,25(OH)2D3 decreased CD4+ T lymphocyte proliferation and viability. The direct co-culture model without inserts caused the opposite effect. 1,25(OH)2D3 mainly decreased the CD4+ T lymphocyte-associated secretion of cytokines via hPDL-MSCs. The degree of these inhibitions varied between the different co-culture setups. 1,25(OH)2D3 predominantly decreased the expression of the soluble and membrane-bound immunomediators in hPDL-MSCs to a different extent, depending on the co-culture models. The degree of all these effects depended on the absence and presence of exogenous TNF-α and IL-1β. These data assume that 1,25(OH)2D3 differently affects CD4+ T lymphocytes via the paracrine and direct cell-to-cell contact mechanisms of hPDL-MSCs, showing anti- or pro-inflammatory effects depending on the co-culture model type. The local cytokine microenvironment seems to be involved in fine-tuning these effects. Future studies should consider this double-edged observation by executing different co-culture models in parallel.

SIRT1 activation by Ligustrazine ameliorates migraine via the paracrine interaction of microglia and neurons

BackgroundNeuroinflammation with associated oxidative stress aggravates the pathogenesis and progression of migraine. Ligustrazine (LGZ) is a key component from traditional edible-medicinal herb Ligusticum chuanxiong Hort., and has the effects of anti-platelet aggregation, expanding small arteries, improving microcirculation and promoting blood circulation and removing blood stasis in clinic. Hypothesis/PurposeThis study aims to investigate the pharmacological effect and mechanism of LGZ in migraine. Study Design/MethodsA mouse model of migraine was induced by nitroglycerin (NTG), and LPS/IFN-γ stimulated microglial cell model was conducted to investigate neuroinflammation, the paracrine interactions between microglia and neurons were determined by the co-culture system, and the effect of LGZ on stability of SIRT1 protein was measured by cellular thermal shift assay (CETSA). Whilst, the SIRT1 inhibitor EX527 was used alone or co-treatment with LGZ in vitro or in vivo. ResultsLGZ significantly attenuated migraine-like behaviors in NTG-induced mice, and ameliorated neuroinflammation and related oxidative damage in brain tissue, but co-treatment with SIRT1 inhibitor EX527 abolished the protective effects of LGZ. Mechanistically, LGZ mitigated neuroinflammation by upregulating SIRT1 expression and subsequently inhibiting the activation of NF-κB pathway in microglia. CETSA indicated that LGZ significantly maintained the stability of SIRT1 protein in microglia. While, in the co-culture system, culture medium from LPS/IFN-γ-treated microglia exacerbated neuronal damage and oxidative stress, which was suppressed by treating LPS/IFN-γ-induced microglia with LGZ, this effect might be related to the activation of Nrf2 signals in neurons. Notably, SIRT1 inhibitor EX527 abrogated the effects of LGZ both in vitro and in vivo. ConclusionConsequently, SIRT1 might be an important pharmacological target of LGZ, which attenuates migraine associated neuroinflammation and oxidative stress by interfering the crosstalk between microglia and neurons, thereby relieving migraine.

Enteroendocrine cells regulate intestinal homeostasis and epithelial function

Enteroendocrine cells (EECs) are well-known for their systemic hormonal effects, especially in the regulation of appetite and glycemia. Much less is known about how the products made by EECs regulate their local environment within the intestine. Here, we focus on paracrine interactions between EECs and other intestinal cells as they regulate three essential aspects of intestinal homeostasis and physiology: 1) intestinal stem cell function and proliferation; 2) nutrient absorption; and 3) mucosal barrier function. We also discuss the ability of EECs to express multiple hormones, describe in vitro and in vivo models to study EECs, and consider how EECs are altered in GI disease.

Bilayer 3D co‐culture platform inducing the differentiation of normal fibroblasts into cancer‐associated fibroblast like cells: New in vitro source to obtain cancer‐associated fibroblasts

AbstractThis study presents a novel in vitro bilayer 3D co‐culture platform designed to obtain cancer‐associated fibroblasts (CAFs)‐like cells. The platform consists of a bilayer hydrogel structure with a collagen/polyethylene glycol (PEG) hydrogel for fibroblasts as the upper layer and an alginate hydrogel for tumor cells as the lower layer. The platform enabled paracrine interactions between fibroblasts and cancer cells, which allowed for selective retrieval of activated fibroblasts through collagenase treatment for further study. Fibroblasts remained viable throughout the culture periods and showed enhanced proliferation when co‐cultured with cancer cells. Morphological changes in the co‐cultured fibroblasts resembling CAFs were observed, especially in the 3D microenvironment. The mRNA expression levels of CAF‐related markers were significantly upregulated in 3D, but not in 2D co‐culture. Proteomic analysis identified upregulated proteins associated with CAFs, further confirming the transformation of normal fibroblasts into CAF within the proposed 3D co‐culture platform. Moreover, co‐culture with CAF induced radio‐ and chemoresistance in pancreatic cancer cells (PANC‐1). Survival rate of cancer cells post‐irradiation and gemcitabine resistance increased significantly in the co‐culture setting, highlighting the role of CAFs in promoting cancer cell survival and therapeutic resistance. These findings would contribute to understanding molecular and phenotypic changes associated with CAF activation and provide insights into potential therapeutic strategies targeting the tumor microenvironment.

Modulation of Breast Cancer Cell Apoptosis and Macrophage Polarization by Mistletoe Lectin in 2D and 3D Models.

Korean mistletoe (Viscum album L. var. coloratum) is renowned for its medicinal properties, including anti-cancer and immunoadjuvant effects. This study aimed to elucidate the mechanisms by which Korean mistletoe lectin (V. album L. var. coloratum agglutinin; VCA) modulates breast cancer cell apoptosis and macrophage polarization. The specific objectives were to (1) investigate the direct effects of VCA on MCF-7 breast cancer cells and THP-1-derived M1/M2 macrophages; (2) analyze the impact of VCA on the paracrine interactions between these cell types; and (3) compare the efficacy of VCA in 2D vs. 3D co-culture models to bridge the gap between in vitro and in vivo studies. We employed both 2D and 3D models, co-culturing human M1/M2 macrophages with human MCF-7 breast cancer cells in a Transwell system. Our research demonstrated that M1 and M2 macrophages significantly influenced the immune and apoptotic responses of breast cancer cells when exposed to VCA. M1 macrophages exhibited cytotoxic characteristics and enhanced VCA-induced apoptosis in both 2D and 3D co-culture models. Conversely, M2 macrophages initially displayed a protective effect by reducing apoptosis in breast cancer cells, but this protective effect was reversed upon exposure to VCA. Furthermore, our findings illustrate VCA's ability to modulate M1 and M2 polarization in breast cancer cells. Finally, the use of magnetic 3D cell cultures suggests their potential to yield results comparable to conventional 2D cultures, bridging the gap between in vitro and in vivo studies.

Daily crowding stress has limited, yet detectable effects on skin and head kidney gene expression in surgically tagged atlantic salmon (Salmo salar)

To ensure welfare-friendly and effective internal tagging, the tagging process should not cause a long-term burden on individuals given that tagged fish serve as representatives for the entire population in telemetry applications. To some extent, stress is inevitable within regular aquaculture practices, and thus, the consequences of long-term stress should be described in terms of their effects on internal tagging. In fish, stressors activate the Hypothalamus-Pituitary-Interrenal (HPI) and Brain-Sympathetic-Chromaffin Cell (BSC) axes, leading to neuroimmunoendocrine communication and paracrine interactions among stress hormones. The interrelation between wound healing and stress is complex, owing to their shared components, pathways, and energy demands. This study assessed 14 genes (mmp9, mmp13, il-2, il-4, il-8a, il-10, il-12, il-17d, il-1b, tnfa, ifng, leg-3, igm, and crh) in the skin (1.5 cm from the wound) and head kidney over eight weeks. These genes, associated with cell signaling in immunity, wound healing, and stress, have previously been identified as influenced and regulated by these processes. Half of a group of Atlantic salmon (n = 90) with surgically implanted dummy smart-tags were exposed to daily crowding stress. The goal was to investigate how this gene panel responds to a wound alone and then to the combined effects of wounding and daily crowding stress. Our observations indicate that chronic stress impacts inflammation and impedes wound healing, as seen through the expression of matrix metalloproteinases genes in the skin but not in the head kidney. This difference is likely due to the ongoing internal wound repair, in contrast to the externally healed wound incision. Cytokine expression, when significant in the skin, was mainly downregulated in both treatments compared to control values, particularly in the study's first half. Conversely, the head kidney showed initial cytokine downregulation followed by upregulation. Across all weeks observed and combining both tissues, the significantly expressed gene differences were 12 % between the Wound and Stress+ groups, 28 % between Wound and Control, and 25 % between Stress+ and Control. Despite significant fluctuations in cytokines, sustained variations across multiple weeks are only evident in a few select genes. Furthermore, Stress+ individuals demonstrated the most cytokine correlations within the head kidney, which may suggest that chronic stress affects cytokine expression. This investigation unveils that the presence of stress and prolonged activation of the HPI axis in an eight weeklong study has limited yet detectable effects on the selected gene expression within immunity, wound healing, and stress, with notable tissue-specific differences.

Unraveling the Dynamics of Estrogen and Progesterone Signaling in the Endometrium: An Overview.

The endometrium is crucial for the perpetuation of human species. It is a complex and dynamic tissue lining the inner wall of the uterus, regulated throughout a woman's life based on estrogen and progesterone fluctuations. During each menstrual cycle, this multicellular tissue undergoes cyclical changes, including regeneration, differentiation in order to allow egg implantation and embryo development, or shedding of the functional layer in the absence of pregnancy. The biology of the endometrium relies on paracrine interactions between epithelial and stromal cells involving complex signaling pathways that are modulated by the variations of estrogen and progesterone levels across the menstrual cycle. Understanding the complexity of estrogen and progesterone receptor signaling will help elucidate the mechanisms underlying normal reproductive physiology and provide fundamental knowledge contributing to a better understanding of the consequences of hormonal imbalances on gynecological conditions and tumorigenesis. In this narrative review, we delve into the physiology of the endometrium, encompassing the complex signaling pathways of estrogen and progesterone.

Design and development of in-vitro co-culture device for studying cellular crosstalk in varied tissue microenvironment

Despite of being in different microenvironment, breast cancer cells influence the bone cells and persuade cancer metastasis from breast to bone. Multiple co-culture approaches have been explored to study paracrine signaling between these cells and to study the progression of cancer. However, lack of native tissue microenvironment remains a major bottleneck in existing co-culture technologies. Therefore, in the present study, a tumorigenic and an osteogenic microenvironment have been sutured together to create a multi-cellular environment and has been appraised to study cancer progression in bone tissue. The PCL-polystyrene and PCL-collagen fibrous scaffolds were characterized for tumorigenic and osteogenic potential induction on MDA-MB-231 and MC3T3-E1 cells respectively. Diffusion ability of crystal violet, glucose, and bovine serum albumin across the membrane were used to access the potential paracrine interaction facilitated by device. While in co-cultured condition, MDA-MB-231 cells showed EMT phenotype along with secretion of TNFα and PTHrP which lower down the expression of osteogenic markers including alkaline phosphatase, RUNX2, Osteocalcin and Osteoprotegerin. The cancer progression in bone microenvironment demonstrated the role and necessity of creating multiple tissue microenvironment and its contribution in studying multicellular disease progression and therapeutics.

Reciprocal Interactions of Human Monocytes and Cancer Cells in Co-Cultures In Vitro.

The tumor microenvironment (TME) includes immune and stromal cells and noncellular extracellular matrix (ECM) components. Tumor-associated macrophages (TAMs) are the most important immune cells in TME and are crucial for carcinomas' progression. The purpose was to analyze direct and indirect interactions in co-culture of tumor cells with monocytes/macrophages and, additionally, to indicate which interactions are more important for cancer development. Cytokines, reactive oxygen species, nitric oxide level, tumor cell cycle and changes in tumor cell morphology after human tumor cells (Hep-2 and RK33 cell lines) with human monocyte/macrophage (THP-1 cell line) interactions were tested. Morphology and cytoskeleton organization of tumor cells did not change after co-culture with macrophages. In co-culture of tumor cells with human monocyte, changes in the percentage of tumor cells in cell cycle phases was observed. No significant changes in reactive oxygen species (ROS) were found in the co-culture as compared to the tumor cell mono-culture. Monocytes produced about three times higher ROS than tumor cells. In co-cultures, a lower nitric oxide (NOx) level was found as compared to the sum of the production by both mono-cultures. Co-culture conditions limited the production of cytokines (IL-4, IL-10 and IL-13) as compared to the sum of their level in mono-cultures. In conclusion, macrophages influence tumor cell growth and functions. Mutual (direct and paracrine) interactions between tumor cells and macrophages changed cytokine production and tumor cell cycle profile. The data obtained may allow us to initially indicate which kind of interactions may have a greater impact on cancer development processes.

1759-P: Delta Cell Potentiates Secretion of Insulin and Glucagon in a Novel Model of Islet Paracrine Interactions

1759-P: Delta Cell Potentiates Secretion of Insulin and Glucagon in a Novel Model of Islet Paracrine Interactions

Cardiomyocyte-stromal cell interplay modulates the pathogenesis of arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Transnational Research Projects on Cardiovascular Diseases Background Arrhythmogenic Cardiomyopathy (ACM) is an inherited heart disorder characterized by a high incidence of sudden death at young age. Mutations linked to ACM occur primarily in desmosomal genes (e.g. PKP2) [1]. Different cell types individually contribute to set the ACM phenotype with either functional abnormalities (cardiomyocytes; CM) [2]; or fibro-fatty substitution (cardiac mesenchymal stromal cells; cMSC) [3]. The relative contribution of the different cell type derangements to determine disease evolution and electrical instability is still unclear. Purpose We hypothesize that the use of a multicellular cardiac system, modelling the functional interaction between CM and cMSC, will allow the understanding of the relative contribution of the different cell types to the ACM phenotypes. Methods Primary cMSC (carrier of the PKP2 mutation c.2013delC) have been collected from a right ventricle biopsy sample; control cMSC obtained from a biobank. iPSC reprogrammed from the same ACM patient and the isogenic line in which the PKP2 mutation was corrected [4] were used. Cardiac cocultures (cc) were assembled by combining 85% iPSC-CM and 15% primary cMSC (as in Figure 1A), either mutated or control, in different combination (as in Figure 1B). Fibro-fatty accumulation have been evaluated by immunofluorescence analysis. Movies of contracting clusters have been collected and analysed by the MUSCLEMOTION tool [5]. Cell-cell communication was studied in silico by InterCellar analysis [6] on the single culture transcriptomes. The assessment of the paracrine interactions was performed on the cc secretome by Olink technology. Results Immunofluorescence analysis highlighted that ACM cc (cc 4) accumulate more lipids and collagen than healthy control (HC) ones (cc 1). Since intermediate fibro-fatty levels were observed in ‘mixed cc’ (cc 2 and 3), we conclude that ACM iPSC-CM are able to influence fibro-adipo-commitment of cMSC. Contraction studies showed a high propensity for cc monolayers which include ACM iPSC-CM (cc3 and cc4) to display arrhythmic events. An increase in the percentage of arrhythmic events was triggered when the ACM cMSC were replacing HC cMSC, indicating that ACM cMSC can affect iPSC-CM rhythm. The most abundant ligand-receptor couplets regarded cell-cell adhesion, cell-matrix coupling, cell differentiation and inflammation. Differential abundance between the four cc was observed mainly for secreted factors involved in cardiac fibrosis, inflammation, adipogenesis / lipid metabolism and heart failure. In particular, DLK-1, secreted only by HC CM, is validated as novel inhibitor of ACM fibro-adipose differentiation. Conclusion Overall, our novel simple multicellular ACM cell model can recapitulate different phenotypes of ACM and contributed to understand the relative contribution of the different cell types to ACM features (e.g. fibro-adipose replacement, contractile defects and proarrhythmic events).

DNA methyltransferase isoforms regulate endothelial cell exosome proteome composition

Extrinsic and intrinsic pathological stimuli in vascular disorders induce DNA methylation based epigenetic reprogramming in endothelial cells, which leads to perturbed gene expression and subsequently results in endothelial dysfunction (ED). ED is also characterized by release of exosomes with altered proteome leading to paracrine interactions in vasculature and subsequently contributing to manifestation, progression and severity of vascular complications. However, epigenetic regulation of exosome proteome is not known. Hence, our present study aimed to understand influence of DNA methylation on exosome proteome composition and their influence on endothelial cell (EC) function. DNMT isoforms (DNMT1, DNMT3A, and DNMT3B) were overexpressed using lentivirus in ECs. Exosomes were isolated and characterized from ECs overexpressing DNMT isoforms and C57BL/6 mice plasma treated with 5-aza-2′-deoxycytidine. 3D spheroid assay was performed to understand the influence of exosomes derived from cells overexpressing DNMTs on EC functions. Further, the exosomes were subjected to TMT labelled proteomics analysis followed by validation. 3D spheroid assay showed increase in the pro-angiogenic activity in response to exosomes derived from DNMT overexpressing cells which was impeded by inclusion of 5-aza-2′-deoxycytidine. Our results showed that exosome proteome and PTMs were significantly modulated and were associated with dysregulation of vascular homeostasis, metabolism, inflammation and endothelial cell functions. In vitro and in vivo validation showed elevated DNMT1 and TGF-β1 exosome proteins due to DNMT1 and DNMT3A overexpression, but not DNMT3B which was mitigated by 5-aza-2′-deoxycytidine indicating epigenetic regulation. Further, exosomes induced ED as evidenced by reduced expression of phospho-eNOSser1177. Our study unveils epigenetically regulated exosome proteins, aiding management of vascular complications.

Myocardial B cells have specific gene expression and predicted interactions in dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.

Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with arrhythmogenic right ventricular cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. We identified 1,100 B cells, including naive B cells and plasma cells. Cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interactions; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.