Secretion Of Extracellular Matrix Proteins Research Articles

Improving the mechanical and biological functions of cell sheet constructs: The interplay of human-derived periodontal ligament stem cells, endothelial cells and plasma rich in growth factors

ObjectiveThe aim of this study was to produce and characterize triple-layered cell sheet constructs with varying cell compositions combined or not with the fibrin membrane scaffold obtained by the technology of Plasma Rich in Growth Factors (mPRGF). Materials and methodsHuman primary cultures of periodontal ligament stem cells (hPDLSCs) were isolated, and their stemness nature was evaluated. Three types of triple-layered composite constructs were generated, composed solely of hPDLSCs or combined with human umbilical vein endothelial cells (HUVECs), either as a sandwiched endothelial layer or as coculture sheets of both cell phenotypes. These three triple-layered constructs were also manufactured using mPRGF as cell sheets’ support. Necrosis, glucose consumption, secretion of extracellular matrix proteins and synthesis of proangiogenic factors were determined. Histological evaluations and proteomic analyses were also performed. ResultsThe inclusion of HUVECs did not clearly improve the properties of the multilayered constructs and yet hindered their optimal conformation. The presence of mPRGF prevented the shrinkage of cell sheets, stimulated the metabolic activity and increased the matrix synthesis. At the proteome level, mPRGF conferred a dramatic advantage to the hPDLSC constructs in their ability to provide a suitable environment for tissue regeneration by inducing the expression of proteins necessary for bone morphogenesis and cellular proliferation. ConclusionshPDLSCs’ triple-layer construct onto mPRGF emerges as the optimal structure for its use in regenerative therapeutics. Clinical relevanceThese results suggest the suitability of mPRGF as a promising tool to support cell sheet formation by improving their handling and biological functions.

The regulatory role of the apelin/APJ axis in scarring: Identification of upstream and downstream mechanisms

Scarring, a prevalent issue in clinical settings, is characterized by the excessive generation of extracellular matrix within the skin tissue. Among the numerous regulatory factors implicated in fibrosis across various organs, the apelin/APJ axis has emerged as a potential regulator of fibrosis. Given the shared attribute of heightened extracellular matrix production between organ fibrosis and scarring, we hypothesize that the apelin/APJ axis also plays a regulatory role in scar development. In this study, we examined the expression of apelin and APJ in scar tissue, normal skin, and fibroblasts derived from these tissues. We investigated the impact of the hypoxic microenvironment in scars on apelin/APJ expression to identify the transcription factors influencing apelin/APJ expression. Through overexpressing or knocking down apelin/APJ expression, we observed their effects on fibroblast secretion of extracellular matrix proteins. We further validated these effects in animal experiments while exploring the underlying mechanisms. Our findings demonstrated that the apelin/APJ axis is expressed in fibroblasts from keloid, hypertrophic scar, and normal skin. The regulation of apelin/APJ expression by the hypoxic environment in scars plays a significant role in hypertrophic scar and keloid development. This regulation promotes extracellular matrix secretion through upregulation of TGF-β1 expression via the PI3K/Akt/CREB1 pathway.

Production of green-natural and “authentic” cultured meat based on proanthocyanidins-dialdehyde chitosan-collagen ternary hybrid edible scaffolds

Cultured meat has the potential to fulfill the meat demand for the growing human population, but cultured meat development will be required to simplify the production process and produce naturally cultured meat, such as no longer stripping off scaffolders and adding artificial dyes. In this study, proanthocyanidins (PC) and dialdehyde chitosan (DAC) were employed as dual crosslinkers with collagen to prepare a hybrid 3D edible scaffold for the production of high-quality cell-cultured meat. The results revealed that the scaffold was biocompatible and could offer robust mechanical support and adhesion sites for bovine myoblasts, enabling long-term cell culture. Meanwhile, the Col-PC-DAC scaffold promoted the myogenic differentiation of bovine myoblasts and extracellular matrix protein secretion, further affecting the texture of cultured meat. After cooking the cultured meat and beef, it was shown that the cultured meat had some similarities to beef in color and flavor. Importantly, our findings demonstrate that cultured meat can acquire a color remarkably similar to that of conventional beef without the need for artificial dyeing. This breakthrough not only simplifies the production process but also ensures a more natural and appealing appearance of cultured meat. In conclusion, the proanthocyanidins-dialdehyde chitosan-collagen hybrid 3D edible scaffolds provide a new option for producing cultured meat that satisfies consumer expectations.

Kinetin inhibits hepatic stellate cell activation and induces apoptosis via interactions with the TGF-β1/Smad signaling pathway

Hepatic fibrosis is the pathological repair response of the liver to chronic injury; hepatic stellate cell (HSC) activation is the central link in the pathogenesis of hepatic fibrosis. Previously, we showed that kinetin, a plant cytokinin hormone, has a protective effect on CCl4-induced liver injury in mice. However, the role of kinetin in liver fibrosis remains unclear. We aimed to study these protective effects and to determine the mechanisms by which kinetin mediates HSC activation and apoptosis. For this purpose, the human HSC line LX-2 was treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 24 h to stimulate activation. We found that treatment with kinetin at the sub-cytotoxic dose of 40 μg/ml for 48 h reduced the expression of the HSC activation marker α-SMA and inhibited the secretion of extracellular matrix proteins. In addition, kinetin was found to inhibit the proliferation and migration of LX-2 cells. We found that kinetin induced apoptosis in LX-2 cells by increasing the level of cleaved-caspase 3 and the Bax-to-Bcl-2 ratio. Interestingly, these effect were not observed in quiescent HSCs, suggesting that they are activation-dependent. Further study showed that kinetin attenuates activation and promotes apoptosis of LX-2 cells in vitro in part by suppressing the TGF-β1/Smad signaling pathway.

Stromal DDR2 Promotes Ovarian Cancer Metastasis through Regulation of Metabolism and Secretion of Extracellular Matrix Proteins.

DDR2 is highly expressed by stromal cells in ovarian cancer that can mediate metastasis and is a potential therapeutic target in ovarian cancer.

P53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Dually fibronectin/CD44-mediated nanoparticles targeted disrupt the Golgi apparatus and inhibit the hedgehog signaling in activated hepatic stellate cells to alleviate liver fibrosis

Liver fibrosis is featured by activation of hepatic stellate cells (HSCs) and excessive accumulation of extracellular matrix (ECM). The Golgi apparatus in HSCs plays a vital role in synthesis and secretion of ECM proteins, while its targeted disruption in activated HSCs could be considered as a promising approach for liver fibrosis treatment. Here, we developed a multitask nanoparticle CREKA–CS–RA (CCR) to specifically target the Golgi apparatus of activated HSCs, based on CREKA (a specific ligand of fibronectin) and chondroitin sulfate (CS, a major ligand of CD44), in which retinoic acid (a Golgi apparatus-disturbing agent) chemically conjugated and vismodegib (a hedgehog inhibitor) encapsulated. Our results showed that CCR nanoparticles specifically targeted activated HSCs and preferentially accumulated in the Golgi apparatus. Systemic administration of CCR nanoparticles exhibited significantly accumulation in CCl4-induced fibrotic liver, which was attributed to specific recognition with fibronectin and CD44 on activated HSCs. CCR nanoparticles loaded with vismodegib not only disrupted Golgi apparatus structure and function but also inhibited the hedgehog signaling pathway, thus markedly suppressing HSC activation and ECM secretion in vitro and in vivo. Moreover, vismodegib-loaded CCR nanoparticles effectively inhibited the fibrogenic phenotype in CCl4-induced liver fibrosis mice without causing obvious toxicity. Collectively, these findings indicate that this multifunctional nanoparticle system can effectively deliver therapeutic agents to the Golgi apparatus of activated HSCs, thus has potential treatment of liver fibrosis with minimal side effects.

Poly-l-lactide-co-ε-caprolactone (PLCL) and poly-l-lactic acid (PLLA)/gelatin electrospun subacromial spacer improves extracellular matrix (ECM) deposition for the potential treatment of irreparable rotator cuff tears

Biodegradable subacromial spacer implantation has become practicable for the treatment of irreparable rotator cuff tears (IRCT). However, the relative high degradation rate and inferior tissue regeneration properties of current subacromial spacer may lead to failure regards to long-term survival. It is reported that satisfactory clinical results lie in the surrounding extracellular matrix (ECM) deposition after implantation. This study aims to develop a biological subacromial spacer that would enhance tissue regeneration properties and results in better ECM deposition. Physicochemical properties were characterized on both poly-l-lactide-co-ε-caprolactone (PLCL) dip-coating spacer (monolayer spacer, MS) and PLCL dip-coating + Poly-l-Lactic Acid (PLLA)/Gelatin electrospun spacer (Bilayer Spacer, BS). Cytocompatibility, angiogenesis, and collagen inducibility were evaluated with tendon fibroblasts and endothelial cells. Ultrasonography and histomorphology were used to analyze biodegradability and surrounding ECM deposition after the implantation in vivo. BS was successfully fabricated with the dip-coating and electrospinning technique, based on the human humeral head data. In vitro studies demonstrated that BS showed a greater cytocompatibility, and increased secretion of ECM proteins comparing to MS. In vivo studies indicated that BS promoted ECM deposition and angiogenesis in the surrounding tissue. Our research highlights that BS exhibits better ECM deposition and reveals a potential candidate for the treatment of IRCT in future.

Immune and Non-Immune Inflammatory Cells Involved in Autoimmune Fibrosis: New Discoveries.

Fibrosis is an important health problem and its pathogenetic activation is still largely unknown. It can develop either spontaneously or, more frequently, as a consequence of various underlying diseases, such as chronic inflammatory autoimmune diseases. Fibrotic tissue is always characterized by mononuclear immune cells infiltration. The cytokine profile of these cells shows clear proinflammatory and profibrotic characteristics. Furthermore, the production of inflammatory mediators by non-immune cells, in response to several stimuli, can be involved in the fibrotic process. It is now established that defects in the abilities of non-immune cells to mediate immune regulation may be involved in the pathogenicity of a series of inflammatory diseases. The convergence of several, not yet well identified, factors results in the aberrant activation of non-immune cells, such as epithelial cells, endothelial cells, and fibroblasts, that, by producing pro-inflammatory molecules, exacerbate the inflammatory condition leading to the excessive and chaotic secretion of extracellular matrix proteins. However, the precise cellular mechanisms involved in this process have not yet been fully elucidated. In this review, we explore the latest discoveries on the mechanisms that initiate and perpetuate the vicious circle of abnormal communications between immune and non-immune cells, responsible for fibrotic evolution of inflammatory autoimmune diseases.

Passing the post: roles of posttranslational modifications in the form and function of extracellular matrix.

The extracellular matrix (ECM) is central to the physiology of animal tissues, through its multifaceted roles in tissue structure, mechanical properties, and cell interactions, and by its cell-signaling activities that regulate cell phenotype and behavior. The secretion of ECM proteins typically involves multiple transport and processing steps within the endoplasmic reticulum and the subsequent compartments of the secretory pathway. Many ECM proteins are substituted with various posttranslational modifications (PTMs) and there is increasing evidence of how PTM additions are required for ECM protein secretion or functionality within the extracellular milieu. The targeting of PTM-addition steps may thus offer opportunities to manipulate ECM quality or quantity, in vitro or in vivo. This review discusses selected examples of PTMs of ECM proteins for which the PTM has known importance for anterograde trafficking and secretion of the core protein, and/or loss-of-function of the respectively modifying enzyme leads to alterations of ECM structure or function with pathophysiological consequences in humans. Members of the protein disulfide isomerase (PDI) family have central roles in disulfide bond formation and isomerization within the endoplasmic reticulum, and are discussed in relation to emerging knowledge of the roles of certain PDIs in ECM production in the pathophysiological context of breast cancer. Cumulative data suggest the possible applicability of inhibition of PDIA3 activity to modulate ECM composition and functionality within the tumor microenvironment.

Taurocholic acid promotes hepatic stellate cell activation via S1PR2/p38 MAPK/YAP signaling under cholestatic conditions.

Disrupted bile acid regulation and accumulation in the liver can contribute to progressive liver damage and fibrosis. However, the effects of bile acids on the activation of hepatic stellate cells (HSCs) remain unclear. This study investigated the effects of bile acids on HSC activation during liver fibrosis, and examined the underlying mechanisms. The immortalized HSCs, LX-2 and JS-1cells were used for the in vitro study. in vitro, the adeno-associated viruses adeno-associated virus-sh-S1PR2 and JTE-013 were used to pharmacologically inhibit the activity of S1PR2 in a murine model of fibrosis induced by a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Histological and biochemical analyses were performed to study the involvement of S1PR2 in the regulation of fibrogenic factors as well as the activation properties of HSCs. S1PR2 was the predominant S1PR expressed in HSCs and was upregulated during taurocholic acid (TCA) stimulation and in cholestatic liver fibrosis mice. TCA-induced HSC proliferation, migration and contraction and extracellular matrix protein secretion were inhibited by JTE-013 and a specific shRNA targeting S1PR2 in LX-2 and JS-1 cells. Meanwhile, treatment with JTE-013 or S1PR2 deficiency significantly attenuated liver histopathological injury, collagen accumulation, and the expression of fibrogenesis-associated genes in mice fed a DDC diet. Furthermore, TCAmediated activation of HSCs through S1PR2 was closely related to the yes-associated protein (YAP) signaling pathway via p38 mitogen-activated protein kinase (p38 MAPK). TCA-induced activation of the S1PR2/p38 MAPK/YAP signaling pathways plays a vital role in regulating HSC activation, which might be therapeutically relevant for targeting cholestatic liver fibrosis.

Bulk RNA-seq and scRNA-seq analysis reveal an activation of immune response and compromise of secretory function in major salivary glands of obese mice

Obesity affects the function of multiple organs/tissues including the exocrine organ salivary glands. However, the effects of obesity on transcriptomes and cell compositions in the salivary glands have yet been studied by bulk RNA-sequencing and single-cell RNA-sequencing. Besides, the cell types in the sublingual gland, one of the three major salivary glands, have yet been characterized by the approach of single-cell RNA-sequencing. In this report, we find that the histological structure of the three major salivary glands are not obviously affected in the obese mice. Bulk RNA-sequencing analysis shows that the most prominent changes observed in the three major salivary glands of the obese mice are the mobilization of transcriptomes related to the immune response and down-regulation of genes related to the secretory function of the salivary glands. Based on single-cell RNA-sequencing analysis, we identify and annotate 17 cell clusters in the sublingual gland for the first time, and find that obesity alters the relative compositions of immune cells and secretory cells in the major glands of obese mice. Integrative analysis of the bulk RNA-sequencing and single-cell RNA-sequencing data confirms the activation of immune response genes and compromise of secretory function in the three major salivary glands of obese mice. Consequently, the secretion of extracellular matrix proteins is significantly reduced in the three major salivary glands of obese mice. These results provide new molecular insights into understanding the effect of obesity on salivary glands.

Paxlovid accelerates cartilage degeneration and senescence through activating endoplasmic reticulum stress and interfering redox homeostasis

BackgroundThe COVID-19 pandemic has become a huge threat to human health, infecting millions of people worldwide and causing enormous economic losses. Many novel small molecule drugs have been developed to treat patients with COVID-19, including Paxlovid, which block the synthesis of virus-related proteins and replication of viral RNA, respectively. Despite satisfactory clinical trial results, attention is now being paid to the long-term side effects of these antiviral drugs on the musculoskeletal system. To date, no study has reported the possible side effects, such as osteoarthritis, of Paxlovid. This study explored the effects of antiviral drug, Paxlovid, on chondrocyte proliferation and differentiation.MethodsIn this study, both in vitro and in vivo studies were performed to determine the effect of Paxlovid on chondrocyte degeneration and senescence. Furthermore, we explored the possible mechanism behind Paxlovid-induced acceleration of cartilage degeneration using transcriptome sequencing and related inhibitors were adopted to verify the downstream pathways behind such phenomenon.ResultsPaxlovid significantly inhibited chondrocyte extracellular matrix protein secretion. Additionally, Paxlovid significantly induced endoplasmic reticulum stress, oxidative stress, and downstream ferroptosis, thus accelerating the senescence and degeneration of chondrocytes. In vivo experiments showed that intraperitoneal injection of Paxlovid for 1 week exacerbated cartilage abrasion and accelerated the development of osteoarthritis in a mouse model.ConclusionsPaxlovid accelerated cartilage degeneration and osteoarthritis development, potentially by inducing endoplasmic reticulum stress and oxidative stress. Long-term follow-up is needed with special attention to the occurrence and development of osteoarthritis in patients treated with Paxlovid.

Tridimensional cell culture of dermal fibroblasts promotes exosome-mediated secretion of extracellular matrix proteins

Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts. We report that tridimensional (3D) cell culture of dermal fibroblasts promotes secretion of exosomes carrying a large quantity of proteins involved in the formation, organisation and remodelling of the ECM. In our 3D model, gene expression was highly modulated and linked to ECM, cellular migration and proliferation, as well as inflammatory response. Mass spectrometry analysis of exosomal proteins, isolated from 3D cultured fibroblast-conditioned media, revealed ECM protein enrichment, of which many were associated with the matrisome. We also show that the cytokine interleukin 6 (IL-6) is predicted to be central to the signalling pathways related to ECM formation and contributing to cell migration and proliferation. Overall, our data suggest that dermal fibroblast-derived EVs participate in many steps of the establishment of dermis’s ECM.

A Biocompatible Self-Powered Piezoelectric Poly(vinyl alcohol)-Based Hydrogel for Diabetic Wound Repair.

Acute and chronic wounds, caused by trauma, tumors, diabetic foot ulcers, etc., are usually difficult to heal, while applying exogenous electrical stimulation to enhance the endogenous electric field in the wound has been proven to significantly accelerate wound healing. However, traditional electrical stimulation devices require an additional external power supply, making them poor in portability and comfort. In this work, a self-powered piezoelectric poly(vinyl alcohol) (PVA)/polyvinylidene fluoride (PVDF) composite hydrogel is constructed by establishing a distinctive preparation process of freezing/thawing-solvent replacement-annealing-swelling. The hydrogen bonding in the hydrogel is remarkably enhanced by the annealing-swelling process, which is stronger between PVA/PVDF molecules than that between PVA molecules, promoting transformation of the α-phase into the electroactive β-phase PVDF and facilitating formation of a much more crystalline structure with high cross-linking density. Hence, an obvious piezoelectric response with high piezoelectric coefficient and electrical signal output with superior stability and sensitivity and excellent mechanical strength and stretchability was achieved for hydrogels. PVA/PVDF composite hydrogels with good cytocompatibility significantly promote proliferation, migration, and secretion of extracellular matrix proteins and growth factors of fibroblasts, possibly through activating the AKT and ERK1/2 signaling pathways. In a wound model of diabetic rats, piezoelectric hydrogels could not only rapidly attract wound exudate and maintain the wet environment of the wound bed but also convert the mechanical energy generated by rats' physical activities into electrical energy, so as to provide local piezoelectric stimulation to the wound bed evenly and symmetrically in real time. Such an effect significantly promotes re-epithelialization and collagen deposition and increases angiogenesis and secretion of growth factors in wound tissue. Besides, it regulates the macrophage phenotype from the M1 subtype (pro-inflammatory subtype) to the M2 subtype (anti-inflammatory subtype) and reduces the expression levels of inflammatory factors, thus accelerating wound healing. The development of such a novel piezoelectric hydrogel provides new therapeutic strategies for chronic wound healing.

Nano-encapsulation of melatonin into polydiacetylene-phospholipid assembly for sustained-release and enhanced bone formation in zebrafish

The low bioavailability of melatonin limits its effective antiosteoporosis activity. In the present study, the polymer-supported liposome is used to encapsulate the melatonin in order to enhance its bone formation activity. More specifically, the melatonin drug has enwrapped into a polymer/lipid hybrid nanovesicle (Lip-mel) by the self-assembly of 10,12-pentacosadiynoic acid (PCDA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) through thin-film hydration method. The liposome-encapsulated melatonin was characterised well by UV–Vis absorption spectroscopy, fluorescence microscopy, energy-dispersive X-ray analysis (EDX), and dynamic light scattering (DLS) measurements. The conjugated yne-ene chain of polymeric backbone of polydiacetylene (PDA) facilitates the formation of stable nanoaggregate from which only 50% of the encapsulated melatonin was leached out even after 72 h under physiological conditions. The Lip-mel shows alkaline phosphatase activity (ALP) and calcium deposition in mouse mesenchymal stem cells more efficient than non-encapsulated melatonin. Furthermore, the Lip-mel elevated the expression of key transcription factors (Runx2, type1 col mRNAs) and secretion of extracellular matrix proteins that are related to osteoblast differentiation. Interestingly, the bone formation in zebrafish model was also enhanced after exposure of Lip-mel compared to melatonin. Thus, the nano formulation of melatonin could be developed as efficient antiosteoporosis drug with better pharmacokinetic properties than that of free form of melatonin.

NHE1 is Involved in the Cytoskeletal Remodeling and ECM Deposition of Lung Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a progressive lung disease characterized by the deregulation of the wound healing process leaving an accumulation of fibroblasts and scarring tissue. Fibroblasts, under normal conditions, support growth and health of connective tissues and maintain the extracellular matrix (ECM). However, as the disease progresses, increases in profibrotic agonists lead to activation of fibroblasts causing stiffening of the cells, increasing in secretion of ECM proteins in the bronchial. The chronic nature of the disease leads fibroblast differentiating into myofibroblast cells making the tissue less compliant. The affected patient’s alveolar architecture becomes damaged which then leads to lung inflexibility, disruption of gas exchange, respiratory failure, and death. Sodium Hydrogen Exchanger Isoform 1 (NHE1) is an ion transporter protein found in the membrane that mediates cellular pH, cell migration, and cytoskeletal anchoring. NHE1 has also been found to impact the pathophysiological development of cancer and other diseases. Interestingly, a number of signaling pathways which contribute to the activation of tumor cells are also involved in profibrotic behavior in fibroblasts and myofibroblast, the purpose of this study was to investigate if NHE1 worsened IPF and if inhibiting NHE1 would decrease the profibrotic activity of fibrosis. We measured two of the indicators of progressing fibrosis: the remodeling of skeletal proteins through formation of actin stress fibers, and the production and secretion of ECM proteins such as collagen and fibronectin. Actin stress fibers have an important role in cell migration and contractility. To stimulate this formation, cells were treated with three agonists that have all been found to increase fibrotic activity: Transforming Growth Factor‐β (TGF‐β), Serotonin (5HT), and Lysophosphatidic Acid (LPA) in the presence and absence of Ethyl‐Isopropyl Amiloride (EIPA), a NHE1 inhibitor. LPA induced stress fibers 43.94 +/‐ 4.4 % over the control, and both TGF‐β and 5HT also stimulated the formation of stress fibers by 32.08 +/‐ 2.1 and 39.1 +/‐ 4.2 %, respectively, over the control non‐treated cells. In each case, the addition of EIPA blocked stress fibers indicating that all three profibrotic factors required NHE1 activity for cytoskeletal remodeling. Additionally, when any combination of agonists were added in combination or a mixture of all three, it was observed that these combinations seemed to block stress fiber formation in comparison to the control. An initial interpretation hints at a competitive or feed‐back loop to controlling fibrosis. The role of NHE1 in TGF‐ β induced fibroblast‐myofibroblast differentiation is also determined and is identified with expression of alpha smooth muscle actin (α‐SMA) with and without EIPA. Finally, the role of NHE1 in production and secretion of ECM proteins, such as collagen and fibronectin, were investigated to detect changes in these proteins in the presence and absence of agonists and EIPA. This work highlights that NHE1 has a role in supporting profibrotic behavior and may be a novel target to fight IPF.

Prognostic value of microRNAs in heart failure: A meta-analysis.

Background:Reported studies have shown that expression levels of microRNAs (miRNAs) are related to survival time of patients with heart failure (HF). A systematic review and meta-analysis were conducted to study circulating miRNAs expression and patient outcome.Methods:Meta-analysis estimating expression levels of circulating miRNAs in HF patients from January 2010 until June 30, 2018, through conducting online searches in Pub Med, Cochrane Database of Systematic, EMBASE and Web of Science and reviewed by 2 independent researchers. Using pooled hazard ratio with a 95% confidence interval to assess the correlation between miRNAs expression levels and overall survival.Results:Four relevant articles assessing 19 circulating miRNAs in 867 patients were included. In conclusion, the meta-analysis results suggest that HF patients with low expression of serum miR-1, miR-423-5p, miR-126, miR-21, miR-23, miR-30d, miR-18a-5p, miR-16-5p, miR-18b-5p, miR-27a-3p, miR-26b-5p, miR-30e-5p, miR-106a-5p, miR-233-3P, miR-301a-3p, miR-423-3P, and miR-128 have significantly worse overall survival (P < .05). Among them, miR-18a-5p, miR-18b-5p, miR-30d, miR-30e-5p, and miR-423-5p are strong biomarkers of prognosis in HF.

Abstract 11412: Methyltransferase Smyd2 Mediate Atherosclerotic Plaque Calcification

Introduction: Clinical manifestation of atherosclerosis is marked by calcification of vasculature, including the expression of mineralization regulating proteins (Sox9, Msx2, and Runx2) by vascular smooth muscle cells (VSMCs). This pathogenic remodeling of VSMCs into osteochondrogenic cell types is accompanied by increased proliferation, migration, and secretion of extracellular matrix proteins. This study aimed to determine the spatiotemporal role of SMYD2 in atherosclerotic plaque progression and calcification. Methods: SMC-specific SMYD2 knockout was generated in ApoE -/- mice (SMYD2 ΔSMC ApoE -/- ). Age and body weight-matched mice were fed with Western diet (WD) for 18 or 26 weeks. The distribution of atherosclerotic plaque was examined by En-face Oil-Red-O staining (ORO) of freshly collected aorta. Atherosclerotic plaque burden was determined by Hematoxylin &amp; Eosin (H&amp;E) staining. Collagen content and calcification of atherosclerotic plaque were determined by Masson's trichrome staining and von Kossa staining, respectively. The calcification-regulatory genes (MGP1, AGG and transcription factors SOX9, MSX2, BMP2, and RUNX2) in the aorta were analyzed by RT-qPCR. Results: SMC-specific deletion of SMYD2 protected mice from atherosclerotic plaque calcification. En-face ORO and H&amp;E analysis of Brachiocephalic artery demonstrated a significant decrease in ORO positive area, total plaque size, and necrotic core area in SMYD2 ΔSMC ApoE -/- mice as compared to ApoE-/- mice, with no change in the distribution of collagen fibers. Von Kossa staining showed hydroxyapatite depots at the medial lining of the vasculature (microcalcification) in ApoE -/- mice fed with 18-week Western diet, while a smaller depot of macrocalcification was observed in SMYD2 ΔSMC ApoE -/- mice compared with ApoE -/- mice when fed with 26-week WD. mRNA analysis of key calcification transcription factors showed significant suppression of SOX9 and MSX2 genes in aortas of SMYD2 ΔSMC ApoE -/- mice compared with ApoE -/- mice, with downward trends of BMP2 and RUNX2 in both 18- or 26-week WD. Conclusion: SMYD2 ablation attenuates atherosclerotic plaque formation and calcification, suggests targeting SMYD2 can be a potential therapeutic strategy for atherosclerosis.

Abstract PO-069: Modeling the tumor microenvironment using tissue engineering technologies

Abstract Tissue engineering technologies provide controllable and reproducible approaches to reconstruct the extracellular and cellular elements of pancreatic cancer. In a reductionist approach they allow the modelling of the complex tumor microenvironment (TME) and the study of disease biology and anti-cancer treatments. Our objective is that an engineered TME model, that mimics the tissue and matrix composition, architecture and cell types, will behave like a real tumor and is suitable for preclinical drug testing. Using biomimetic materials, we recreated the desmoplastic tissue characteristics of the pancreatic TME. Our natural and synthetic biomaterials were tailored to achieve the mechanical properties that resembled the stiffness and viscoelasticity of patient-derived tissues, providing a supportive cell-matrix interface for 3D cell culture conditions. Pancreatic cancer cells grown embedded in the matrix scaffolds formed tumor spheroids. Cell-based assays and microscopic analysis indicated a high cell viability and proliferation of the tumor spheroids as well as the expression of cancer-associated markers. Incorporation of cancer-associated fibroblasts and myeloid cells led to a multicellular 3D systems and matrix stiffening due to the secretion of extracellular matrix proteins. Transcriptomic analysis of the 3D cell cultures identified differentially regulated pathways related to cell proliferation, mechano-transduction and the secretion of pro-inflammatory cytokines, indicative of a malignant behavior. Treatment with mechano-modulating inhibitors and anti-cancer compounds increased the efficacy of chemotherapeutics, thereby reducing matrix stiffness and the release of cytokines. Our engineered TME model provides an easily translatable technology to ease the burden of pancreatic cancer, allowing us to characterize combination treatments that slow down or reduce tumor growth. Citation Format: Rodrigo Curvello, Verena Kast, Daniela Loessner. Modeling the tumor microenvironment using tissue engineering technologies [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-069.