Extracellular Matrix Gene Expression Research Articles

126 Adenomyosis is associated with the changes in the expression of extracellular matrix and adhesion molecule genes in bovine uterus

126 Adenomyosis is associated with the changes in the expression of extracellular matrix and adhesion molecule genes in bovine uterus

Abstract 532: Deficiency of CCN3 in Smooth Muscle Cells Perturbs Vascular Homeostasis and Exacerbates Thoracic Aortic Dissection

Introduction: Thoracic aortic dissection (TAD) is a life-threatening aortic disease with a high mortality rate in the event of rupture. Key mechanisms underlying the development of TAD include extracellular matrix (ECM) degradation and vascular smooth muscle cell (SMC) reprograming. Cellular communication network factor 3 (CCN3), a matricellular protein highly expressed in SMC, exerts a myriad of context-dependent biological functions. However, its role in SMC homeostasis and in the development of TAD remains largely undefined. Methods: A single-cell sequencing (Sc-seq) dataset (GSE213740) from human aortic dissection patients was analyzed to determine the association between CCN3 expression and TAD. In newly weaned male CCN3-floxed mice harboring Myh11-driven inducible Cre, 4 consecutive tamoxifen injections induced CCN3 deletion in SMCs. Mice without Cre expression were subjected to the same procedures and were used as controls. After 1 week of washout, 4-week-old mice were given 0.5% BAPN (β-aminopropionitrile monofumarate) in their drinking water to induce TAD. Histologic and protein measurements were performed on TAD tissues from control and CCN3-SMC-KO mice. Sc-seq analysis was performed to recapitulate cell heterogeneities and transcriptome profile changes. The regulatory role of SMC-derived CCN3 in TGFβ signaling was evaluated by western blot and colocalization analyses. Results: Analysis of the human Sc-seq dataset revealed downregulation of CCN3 in aortas from patients with TAD. CCN3-positive SMCs have elevated ECM and elastin gene expression in comparison to CCN3-null SMCs. In vivo, concordant with increased mortality in CCN3-SMC-KO animals, SMC-specific deletion of CCN3 promotes BAPN-induced elastin fibers fragmentation and degradation. In vitro, SMC deficiency of CCN3 diminished TGFβ-induced elastogenesis. CCN3 binds to the TGFβ receptor on SMCs and augments TGFβ receptor phosphorylation, thereby activating Smads, upregulating SMC elastogenesis, and as a consequence, alleviates elastin fiber fragmentation and TAD development. Conclusion: CCN3 is a key determinate of SMC reprogramming and sustains vascular homeostasis. Our findings suggest a novel target for the prevention and treatment of TAD.

Expression of Adipose Tissue Extracellular Matrix-Related Genes Predicts Weight Loss after Bariatric Surgery.

We evaluated the association between white adipose tissue parameters before bariatric surgery (BS) and post-surgical weight loss, with an especial focus on extracellular matrix (ECM) gene expression. Paired samples from subcutaneous (SAT) and visceral adipose tissue (VAT) were obtained from 144 subjects undergoing BS. The association between total body weight loss (%TBWL) at 12 months after BS and the histological characteristics and gene expression of selected genes in SAT and VAT was analyzed. Fat cell area, size-frequency distribution, and fibrosis in SAT or VAT prior to surgery were not associated with %TBWL. On the contrary, the SAT expression of COL5A1 and COL6A3 was associated with %TBWL after BS (both p < 0.001), even after adjusting for age, gender, baseline BMI, and type 2 diabetes status (T2D). Furthermore, in logistic regression analyses, the expression of these genes was significantly associated with insufficient WL (IWL = TBWL < 20%) after BS (respectively, p = 0.030 and p = 0.031). Indeed, in ROC analysis, the prediction of IWL based on sex, age, BMI, T2D, and the type of surgery (AUC = 0.71) was significantly improved with the addition of SAT-COL5A1 gene expression (AUC = 0.88, Z = 2.13, p = 0.032). Our data suggest that the expression of SAT ECM-related genes may help explain the variability in TBWL following BS.

Extracellular Matrix Regulation in Physiology and in Brain Disease.

The extracellular matrix (ECM) surrounds cells in the brain, providing structural and functional support. Emerging studies demonstrate that the ECM plays important roles during development, in the healthy adult brain, and in brain diseases. The aim of this review is to briefly discuss the physiological roles of the ECM and its contribution to the pathogenesis of brain disease, highlighting the gene expression changes, transcriptional factors involved, and a role for microglia in ECM regulation. Much of the research conducted thus far on disease states has focused on "omic" approaches that reveal differences in gene expression related to the ECM. Here, we review recent findings on alterations in the expression of ECM-associated genes in seizure, neuropathic pain, cerebellar ataxia, and age-related neurodegenerative disorders. Next, we discuss evidence implicating the transcription factor hypoxia-inducible factor 1 (HIF-1) in regulating the expression of ECM genes. HIF-1 is induced in response to hypoxia, and also targets genes involved in ECM remodeling, suggesting that hypoxia could contribute to ECM remodeling in disease conditions. We conclude by discussing the role microglia play in the regulation of the perineuronal nets (PNNs), a specialized form of ECM in the central nervous system. We show evidence that microglia can modulate PNNs in healthy and diseased brain states. Altogether, these findings suggest that ECM regulation is altered in brain disease, and highlight the role of HIF-1 and microglia in ECM remodeling.

Analysis of biomechanical properties of mouse skin dermis through atomic force microscopy: Application to demonstrate a sexual dimorphism.

An in-depth understanding of the mechanical properties of the dermis is indispensable to improve wound healing or slow-down skin ageing. Despite crucial research issues for dermatological and cosmetic industries, very little is known about the mechanical behaviour of the dermis at nanoscale level. This knowledge is relevant not only to human skin but also to mouse skin since this animal model is widely used in basic and preclinical studies for skin biology and health. Here, we describe an original protocol that we developed to specifically measure the mechanical properties of mouse dermis using atomic force microscopy-based nano-indentation approach. Using horizontal cryosections (i.e. parallel to the skin surface) performed at different depths through the dermis of dorsal skin, our protocol allowed us to detect nanoscale mechanical changes between female and male dermis samples. We found that the dermis was softer (i) in females than in males and (ii) with depth within the dermis of male mice. We also quantified compositional differences between female and male skin dermis and found that increased extracellular matrix gene expression and type V collagen staining were associated with increased dermal stiffness in male mice, compared with females. Our results demonstrating a sexual dimorphism in the nanomechanical properties and molecular composition of mouse dermis, open the way to better consider sex-related cutaneous differences to understand skin disease and to stimulate the development of female versus male-specific products with more appropriate dermatological treatments and cosmetic interventions.

Keloids, hypertrophic scars, and uterine fibroid development: a prospective ultrasound study of Black and African American women

To examine the association between keloids, hypertrophic scars, and uterine fibroid incidence as well as growth. Both keloids and fibroids are fibroproliferative conditions that have been reported to be more prevalent among Blacks than Whites, and they share similar fibrotic tissue structures, including extracellular matrix composition, gene expression, and protein profiles. We hypothesized that women with a history of keloids would have greater uterine fibroid development. A prospective community cohort study (enrollment 2010-2012) with 4 study visits over 5 years to conduct standardized ultrasounds to detect and measure fibroids ≥0.5 cm in diameter, assess the history of keloid and hypertrophic scars, and update covariates. Detroit, Michigan area. A total of 1,610 self-identified Black and/or African American women aged 23-35 years at enrollment without a previous clinical diagnosis of fibroids. Keloids (raised scars that grow beyond the margins of the original injury) and hypertrophic scars (raised scars that stay within the bounds of the original injury). Because of the difficulties in distinguishing keloids and hypertrophic scars, we separately examined the history of keloids and the history of either keloids or hypertrophic scars (any abnormal scarring) and their associations with fibroid incidence and growth. Fibroid incidence (new fibroid after a fibroid-free ultrasound at enrollment) was assessed using Cox proportional-hazards regression. Fibroid growth was assessed using linear mixed models. The estimates for the change in log volume per 18 months were converted to the estimated percentage difference in volume for scarring vs. no-scarring. Both incidence and growth models were adjusted for time-varying demographic, reproductive, and anthropometric factors. Of the 1,230 fibroid-free participants, 199 (16%) reported ever having keloids, 578 (47%) reported keloids or hypertrophic scars, and 293 (24%) developed incident fibroids. Neither keloids (adjusted hazard ratio = 1.04; 95% confidence interval: 0.77, 1.40) nor any abnormal scarring (adjusted hazard ratio = 1.10; 95% confidence interval: 0.88, 1.38) were associated with fibroid incidence. Fibroid growth differed little by scarring status. Despite molecular similarities, self-reported keloid and hypertrophic scars did not show an association with fibroid development. Future research may benefit from the examination of dermatologist-confirmed keloids or hypertrophic scars; however, our data suggest little shared susceptibility for these 2 types of fibrotic conditions.

Abstract 5177: Nilotinib suppresses the myofibroblastic cancer-associated fibroblast phenotype

Abstract Background: Cancer-associated fibroblasts (CAFs) modulate the tumor immune microenvironment and are an exciting target for improving response to immunotherapy. Major roles of CAFs in the immune microenvironment include deposition of extracellular matrix (ECM) to prevent immune cell infiltration, a function associated with myofibroblastic CAFs (myCAFs), and production of cytokines to alter the immune milieu, associated with inflammatory CAFs (iCAFs). Recently we have demonstrated the potential for tyrosine kinase inhibitors (TKIs) to alter CAF phenotypes. Here we investigate the ability of dasatinib and nilotinib to alter the immune regulatory functions of CAFs. Methods: Bulk RNA sequencing comparing the effects of nilotinib and dasatinib on primary-derived patient CAFs was performed. CAFs from two rectal cancer patient tumors were isolated, cultured, and treated for 96 hours with 2.5μM nilotinib, 100nM dasatinib, or control feeding media. RNA isolation, library preparation, sequencing, data processing, and differential expression analysis was done through the University of Wisconsin - Madison Gene Expression Center, Biotechnology Center, and Bioinformatics Resource. Gene set enrichment analyses were done using GSEA 4.2.3 (Broad Institute) and all other analyses done in R. Results: The primary variance in samples is between CAF lines (RC1 and RC2), followed by treatments. RNA expression markers for myCAFs significantly associate with the Gene Ontology molecular function ECM structural constituent (q &amp;lt; 0.001). Nilotinib decreased expression of this gene set in RC1 (q &amp;lt; 0.001), while dasatinib treatment increased these genes in RC2, though not significantly (q = 0.2). Both dasatinib and nilotinib downregulated immune-related hallmark gene sets including IL6/JAK/STAT3 signaling (RC2 dasatinib q &amp;lt; 0.05, q &amp;lt; 0.001 for others) and TNFα signaling through NFκB (RC1 dasatinib q &amp;lt; 0.001, RC2 niltoinib q &amp;lt; 0.05, others q = 0.001,). Additionally, dasatinib induced increased expression of genes involved in myogenesis in both CAF lines (RC1 q = 0.001, RC2 q &amp;lt; 0.001), while nilotinib significantly decreased these genes in RC1 (q = 0.001). MYOCD is a transcription factor involved in myogenesis that regulates important myCAF genes such as ACTA2 and TAGLN. Nilotinib treatment decreased expression of MYOCD (RC1 log2FC -1.3 q &amp;lt; 0.001, RC2 -0.75 q &amp;lt; 0.05) and its target genes in both lines, whereas dasatinib did not significantly alter expression. Conclusions: CAFs derived from different cancers harbor different transcriptional profiles and have different responses to TKIs. Dasatinib treatment led to an increase in expression of ECM genes associated with the myCAF phenotype, while nilotinib decreased this phenotype, potentially by inhibiting MYOCD expression. Further investigation into the mechanisms by which nilotinib treatment decreases expression of ECM genes and whether these trends continue in vivo are warranted. Citation Format: Katherine Anne Johnson, Yousef Gadalla, Cheri A. Pasch, Dustin A. Deming. Nilotinib suppresses the myofibroblastic cancer-associated fibroblast phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5177.

Abstract 2360: Digital spatial profiling of RNA and protein in the tumor microenvironment of invasive lobular breast cancer

Abstract Invasive lobular breast cancer (ILC) is an understudied breast cancer subtype with late recurrence, metastasis to serosal surfaces including the peritoneum, and dismal long-term outcome. The interaction between a tumor and its microenvironment leads to phenotypic changes in stromal cells and the ECM that promote proliferation and invasion of malignant cells. ILC is histologically distinct from invasive ductal carcinoma, characterized by discohesive E-cadherin-negative tumor cells that grow in single file. We expect the TME to be unique in ILC. We hypothesized that differing levels of nuclear receptor expression in tumor cells would impact stromal cell composition, presumably through paracrine signaling. There is a range of glucocorticoid receptor (GR) expression in ILC. We sought to determine how crosstalk between GR+ or GR- ILC cells and their respective TME differentially impact stromal cell gene and protein expression, as well as the immune cell milieu. To profile the tumor and stroma separately, we performed nanoString GeoMx digital spatial profiling (DSP) of RNA and protein expression in GR-positive (+) and GR-negative (-) primary ILC. We performed RNA DSP for 12 primary ILCs: 5 strongly positive for GR, 5 GR- and 2 tumors with mixed GR expression. We also completed DSP of proteins on adjacent tissue sections for a total of 87 proteins. To profile tumor and TME independently we segmented regions into PanCK+ tumor and PanCK- stroma. RNA expression analyses revealed striking differences between GR+ and GR- ILC in both tumor cells and TME. Pathway analyses showed enrichment of genes encoding eukaryotic translation initiation and control factors and ribosomal proteins in GR+ tumor cells, as well as downregulation of immune response and antigen presentation pathways. In the TME of the GR- tumors, we saw significantly higher expression of collagen biosynthesis and extracellular matrix genes. Using a spatial deconvolution tool, we observed gene expression indicating markedly higher abundance of macrophages and other myeloid subsets, endothelial cells and fibroblasts in GR+ ILC stroma. There was also evidence for more regulatory T-cells, suggestive of an immune suppressive microenvironment in GR+ ILC. Protein DSP revealed upregulation of proteins involved in DNA repair and cell survival, and reduction of some immune checkpoint proteins in the GR+ tumor cells. In the TME of GR+ ILC, we observed more Ki-67 and ER-alpha as well as proteins expressed on T-regs, as well as downregulation of cytotoxic proteins such as granzyme A. We conclude that in-depth examination of molecular profiles in both the tumor cells and the TME of ILCs will give insight into how tumor GR expression/activation influences the crosstalk between ILC cells and their microenvironment. As expected, we observed heterogeneity between patients, but DSP will allow in-depth characterization of individual tumors at the molecular level. Citation Format: Lynda B. Bennett, Sunati Sahoo, Cheryl M. Lewis, Indu Raman, Candace Frerich, Guanchun Chen, Min Xu, Suzanne D. Conzen. Digital spatial profiling of RNA and protein in the tumor microenvironment of invasive lobular breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2360.

PI3K/AKT/mTOR inhibitors as potential extracellular matrix modulators for targeting EMT subtype gastric tumors.

Targeting the extracellular matrix (ECM) is considered as a promising strategy in cancer therapeutics. This study was designed to identify the potential ECM modulators for gastric cancer therapeutics. Exploration of the expression profiles of gastric tumors revealed the elevated expression of ECM genes in gastric tumor tissues compared to the adjacent normal tissues with increased expression in diffuse subtype gastric tumors and specifically in epithelial to mesenchymal transition (EMT) molecular subtype tumors. Consensus ECM gene set was derived from the expression profiles of gastric tumors. The correlative analysis was performed between the expression pattern of the ECM gene set and the drug sensitivity pattern of a panel of drugs across gastric cancer cell lines. Negative correlation between the expression of ECM genes and sensitivity of a number of drugs targeting PI3K/mTOR signaling, chromatin histone acetylation and ABL signaling was observed. These pathways are known for their role in cell-mediated adhesion, differentiation and epithelial to mesenchymal transition. The current results reveal the possibility of using PI3K/AKT/mTOR modulators for targeted gastric cancer therapy in patients with dysregulated ECM.

Interleukin-1β and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro

To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via real-time polymerase chain reaction (rtPCR). Culture media matrix metalloproteinase (MMP) -3 and -13 concentrations were quantified via ELISA. Total glycosaminoglycan (GAG) content in the cell pellets and culture media was also assessed. IL-1β and IL-1β combined with MPA significantly downregulated ECM gene expression to a greater extent in NBF cells compared with DDFT cells. IL-1β and IL-1β combined with MPA significantly upregulated MMP-3 culture media concentrations in DDFT cells only, and MMP-13 culture media concentrations to a greater extent in NBF cells compared with DDFT cells. NBF cells were more susceptible to IL-1β and MPA-mediated ECM gene expression downregulation in vitro. These results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolve the inflammation as well as take into consideration NBF cell biosynthesis in horses with navicular disease, for which currently no information exists.

Mesenchymal stem cell-derived secretome enhances nucleus pulposus cell metabolism and modulates extracellular matrix gene expression in vitro.

Introduction: Intradiscal mesenchymal stromal cell (MSC) therapies for intervertebral disc degeneration (IDD) have been gaining increasing interest due to their capacity to ameliorate intervertebral disc metabolism and relieve low back pain (LBP). Recently, novel investigations have demonstrated that most of MSC anabolic effects are exerted by secreted growth factors, cytokines, and extracellular vesicles, collectively defined as their secretome. In this study, we aimed to evaluate the effect of bone-marrow-MSCs (BM-MSCs) and adipose-derived stromal cells (ADSCs) secretomes on human nucleus pulposus cells (hNPCs) in vitro. Methods: BM-MSCs and ADSCs were characterized according to surface marker expression by flow cytometry and multilineage differentiation by Alizarin red, Red Oil O and Alcian blue staining. After isolation, hNPCs were treated with either BM-MSC secretome, ADSC secretome, interleukin (IL)-1β followed by BM-MSC secretome or IL-1β followed by ADSC secretome. Cell metabolic activity (MTT assay), cell viability (LIVE/DEAD assay), cell content, glycosaminoglycan production (1,9-dimethylmethylene blue assay), extracellular matrix and catabolic marker gene expression (qPCR) were assessed. Results: 20% BM-MSC and ADSC secretomes (diluted to normal media) showed to exert the highest effect towards cell metabolism and were then used in further experiments. Both BM-MSC and ADSC secretomes improved hNPC viability, increased cell content and enhanced glycosaminoglycan production in basal conditions as well as after IL-1β pretreatment. BM-MSC secretome significantly increased ACAN and SOX9 gene expression, while reducing the levels of IL6, MMP13 and ADAMTS5 both in basal conditions and after in vitro inflammation with IL-1β. Interestingly, under IL-1β stimulation, ADSC secretome showed a catabolic effect with decreased extracellular matrix markers and increased levels of pro-inflammatory mediators. Discussion: Collectively, our results provide new insights on the biological effect of MSC-derived secretomes on hNPCs, with intriguing implications on the development of cell-free approaches to treat IDD.

Origin, prospective identification, and function of circulating endothelial colony-forming cells in mice and humans.

Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony-forming cells (C-ECFCs), also known as blood outgrowth endothelial cells, with proliferative and vasculogenic activity can be cultured; however, the origin and naive function of these C-ECFCs remains obscure. Herein, detailed lineage tracing revealed murine C-ECFCs emerged in the early postnatal period, displayed high vasculogenic potential with enriched frequency of clonal proliferative cells compared with tissue-resident ECFCs, and were not committed to or derived from the BM hematopoietic system but from tissue-resident ECFCs. In humans, C-ECFCs were present in the CD34bright cord blood mononuclear subset, possessed proliferative potential and in vivo vasculogenic function in a naive or cultured state, and displayed a single cell transcriptome sharing some umbilical venous endothelial cell features, such as a higher protein C receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naive function, and antigens to prospectively isolate C-ECFCs for translational studies.

Eight-Week Aerobic Training Activates Extracellular Matrix Biogenesis in Human Skeletal Muscle

We aimed to investigate the effect of 8 weeks of moderate endurance training without considerable mechanical stress on the activation of extracellular matrix (ECM) gene expression in human skeletal muscle. Mechanical stress activates ECM biogenesis in the skeletal muscles, therefore only aerobic exercise on a cycling ergometer with concentric muscle contractions was used in the study. Skeletal muscle samples from m. vastus lateralis were taken from seven young untrained males before and after 8 weeks of aerobic training. Changes in the transcriptome (RNA sequencing) and proteome (shotgun quantitative proteomics analysis) were assessed in the samples; ECM-associated proteins (or matrisome) were determined using the Matrisome DB database. After training period, a change (mainly an increase) in the content of 14 ECM proteins and 134 mRNAs of ECM proteins was found. The largest increase in protein content was found for collagens 1 and 3 (1.7 and 2.2 times, respectively) – the main proteins of the human skeletal muscle’s ECM, which was consistent with an increase in the corresponding mRNA by 10–20 times. In addition, an increase in the expression of more than a hundred mRNAs of collagens, glycoproteins, proteoglycans, and enzymatic regulators of ECM was found, which occurs simultaneously with of an increase in the expression of genes of growth factors (IGF1, PDGFs, TGFB1, MDK, etc.) playing an important role in ECM biogenesis regulation. In conclusion, 8-week aerobic exercise training without considerable mechanical stress is a powerful stimulus for the activation of ECM biogenesis in skeletal muscle.

Microglia refine developing retinal astrocytic and vascular networks through the complement C3/C3aR axis.

Microglia, a resident immune cell of the central nervous system (CNS), play a pivotal role in facilitating neurovascular development through mechanisms that are not fully understood. Previous reports indicate a role for microglia in regulating astrocyte density. This current work resolves the mechanism through which microglia facilitate astrocyte spatial patterning and superficial vascular bed formation in the neuroretina during development. Ablation of microglia increased astrocyte density and altered spatial patterning. Mechanistically, we show that microglia regulate the formation of the spatially organized astrocyte template required for subsequent vascular growth, through the complement C3/C3aR axis during neuroretinal development. Lack of C3 or C3aR hindered the developmental phagocytic removal of astrocyte bodies and resulted in increased astrocyte density. In addition, increased astrocyte density was associated with elevated proangiogenic extracellular matrix gene expression in C3- and C3aR-deficient retinas, resulting in increased vascular density. These data demonstrate that microglia regulate developmental astrocyte and vascular network spatial patterning in the neuroretina via the complement axis.

Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples.

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1β, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFβ did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.

Extracellular matrix-based gene signature for predicting prognosis in colon cancer and immune microenvironment.

The extracellular matrix (ECM) plays a vital role in progression, expansion, and prognosis of malignancies. In this study, we aimed to explore a novel ECM-based prognostic model for patients with colon cancer (CC). ECM-related genes were obtained from Molecular Signatures database. Differential expression analysis was performed using the CC dataset from The Cancer Genome Atlas (TCGA) database. Four ECM-related genes related to overall survival were identified using the Cox regression and LASSO analysis. Then an ECM-related signature was developed and verified in three independent CC cohorts (GSE33882, GSE39582 and GSE29621) from the Gene Expression Omnibus (GEO). A prognostic nomogram was developed incorporating the ECM-related gene signature with clinical risk factors. CIBERSORT was used to explore the immune cell infiltration level. Human Protein Atlas (HPA) database was utilized to validate the expression levels of identified prognostic ECM genes. Four ECM-related genes (CXCL13, CXCL14, SFRP5 and THBS4) were identified to develop an ECM-based gene signature and demarcated CC patients into the high- and low-risk groups. In training and validation datasets, patients in the low-risk group had better overall survival outcomes than those in the high-risk group (log-rank P<0.001). In addition, ECM-related signature was significantly associated with consensus molecular subtype 4 (CMS4) as well as other known clinical risk factors such as a higher Tumor, Nodal Involvement, Metastasis (TNM) stage. Moreover, the risk score derived from the ECM-based gene signature could be utilized as an independent prognostic factor for CC patients. A nomogram including the ECM-related gene signature, age and stage was developed to serve clinical practice. CIBERSORT analysis showed immune cell infiltration was different between high- and low-risk groups. The immunohistochemical results derived from HPA indicated differential expression of prognosis-related ECM genes in CC and normal tissues. In the present study, a novel risk model based on ECM-signature could effectively reflect individual risk classification and provide potential therapeutic targets for CC patients. Moreover, the prognostic nomogram may help predict individualized survival.

Effects of periodic mechanical stress on cytoskeleton dependent lipid raft-induced integrin ɑ1 activation in rat nucleus pulposus cells

Extracellular matrix (ECM) production and nucleus pulposus (NP) cell migration increase under periodic mechanical stress (PMS), but the underpinning regulatory mechanism remains unclear. This work aimed to examine the regulatory effects of cytoskeleton-lipid raft-integrin α1 signaling in NP cells exposed to PMS. Briefly, In NP cells, cytoskeleton rearrangement, lipid raft aggregation and integrin α1 expression in the stress and control groups were assessed by immunofluorescent staining and immunoblot. In addition, cell migration and ECM gene expression were detected by a scratch test and quantitative reverse transcription polymerase chain reaction (qRT‑PCR), respectively. As a result, PMS up-regulated ECM gene expression and enhanced NP cell migration (both P < 0.05), accompanied by increased integrin α1, lipid raft, caveolin-3, F-actin and β-tubulin amounts. Pretreatment with the lipid raft inhibitor methyl-β-cyclodextrin (MβCD) or small interfering RNA (siRNA) targeting caveolin-3 resulted in decreased ECM mRNA synthesis and cell migration induced by PMS (both P < 0.05); meanwhile, integrin α1 expression was also reduced. F-actin and β-tubulin inhibition by cytochalasin D and colchicine, respectively, not only reduced ECM mRNA synthesis and cell migration (both P < 0.05), but also disrupted lipid raft and caveolin-3 amount increases induced by PMS in NP cells. In conclusion, PMS promotes ECM mRNA up-regulation and cell migration through the cytoskeleton-lipid raft-integrin α1 signaling pathway, inhibiting cytoskeleton and lipid rafts could block the cellular effects.

Micro-fragmented and nanofat adipose tissue derivatives: In vitro qualitative and quantitative analysis.

Introduction: Adipose tissue is widely exploited in regenerative medicine thanks to its trophic properties, mainly based on the presence of adipose-derived stromal cells. Numerous devices have been developed to promote its clinical use, leading to the introduction of one-step surgical procedures to obtain minimally manipulated adipose tissue derivatives. However, only a few studies compared their biological properties. This study aimed to characterize micro-fragmented (MAT) and nanofat adipose tissue (NAT) obtained with two different techniques. Methods: MAT, NAT and unprocessed lipoaspirate were collected from surgical specimens. RNA extraction and collagenase isolation of stromal vascular fraction (SVF) were performed. Tissue sections were analysed by histological and immunohistochemical (collagen type I, CD31, CD34 and PCNA) staining to assess tissue morphology and cell content. qPCR was performed to evaluate the expression of stemness-related (SOX2, NANOG and OCT3/4), extracellular matrix (COL1A1) and inflammatory genes (IL1β, IL6 and iNOS). Furthermore, multilineage differentiation was assessed following culture in adipogenic and osteogenic media and staining with Oil Red O and Alizarin red. ASC immunophenotype was assessed by flow cytometric analysis of CD90, CD105, CD73 and CD45. Results: Histological and immunohistochemical results showed an increased amount of stroma and a reduction of adipocytes in MAT and NAT, with the latter displaying the highest content of collagen type I, CD31, CD34 and PCNA. From LA to MAT and NAT, an increasing expression of NANOG, SOX2, OCT3/4, COL1A1 and IL6 was noted, while no significant differences in terms of IL1β and iNOS emerged. No statistically significant differences were noted between NAT and SVF in terms of stemness-related genes, while the latter demonstrated a significantly higher expression of stress-related markers. SVF cells derived from all three samples (LA, MAT, and NAT) showed a similar ASC immunoprofile as well as osteogenic and adipogenic differentiation. Discussion: Our results showed that both MAT and NAT techniques allowed the rapid isolation of ASC-rich grafts with a high anabolic and proliferative potential. However, NAT showed the highest levels of extracellular matrix content, replicating cells, and stemness gene expression. These results may provide precious clues for the use of adipose tissue derivatives in the clinical setting.

Effects of High Glucose on Human Endothelial Cells Exposed to Simulated Microgravity.

A diabetogenic state induced by spaceflight provokes stress and health problems in astronauts. Microgravity (µg) is one of the main stressors in space causing hyperglycaemia. However, the underlying molecular pathways and synergistic effects of µg and hyperglycaemia are not fully understood. In this study, we investigated the effects of high glucose on EA.hy926 endothelial cells in simulated µg (s-µg) using a 3D clinostat and static normogravity (1g) conditions. After 14 days of cell culture under s-µg and 1g conditions, we compared the expression of extracellular matrix (ECM), inflammation, glucose metabolism, and apoptosis-related genes and proteins through qPCR, immunofluorescence, and Western blot analyses, respectively. Apoptosis was evaluated via TUNEL staining. Gene interactions were examined via STRING analysis. Our results show that glucose concentrations had a weaker effect than altered gravity. µg downregulated the ECM gene and protein expression and had a stronger influence on glucose metabolism than hyperglycaemia. Moreover, hyperglycaemia caused more pronounced changes in 3D cultures than in 2D cultures, including bigger and a greater number of spheroids, upregulation of NOX4 and the apoptotic proteins NF-κB and CASP3, and downregulation of fibronectin and transglutaminase-2. Our findings bring new insights into the possible molecular pathways involved in the diabetogenic vascular effects in µg.

Matrix remodeling associated 7 proteins promote cutaneous wound healing through vimentin in coordinating fibroblast functions

Wound healing depends largely on the remodeling of the extracellular matrix around and reorganization of tissue-resident cells. Matrix remodeling associated 7 (MXRA7) is a member of the matrix remodeling-associated gene family and is involved in matrix remodeling-associated processes, such as inflammatory neovascularization, liver injury, and autoimmune skin disease. To investigate whether and how MXRA7 participate in cutaneous wound healing, an ear-punching model was utilized in wild-type (WT) and MXRA7-deficient mice, and the dermal fibroblasts from these mice were further studied in vitro. Results showed that the MXRA7 deficiency impaired the wound healing process in mice. Quantitative PCR indicated that lack of MXRA7 impaired the expression of several extracellular matrix genes (e.g., MMP-2) and inhibited signaling pathways (e.g., STAT3) in healing ear tissues. In in vitro culture system, migration, contraction, or proliferation of fibroblasts was impaired upon MXRA7 deficiency. Pull-down and mass spectrum assay revealed that vimentin was among the proteins that bound MXRA7 proteins in cells, and further investigations indicate MXRA7 was an autocrine factor in fibroblasts that involved vimentin in certain ways, such as JNK and STAT3/STAT5 signaling pathways in our study. In conclusion, MXRA7 proteins promote wound healing through vimentin in coordinating fibroblast functions.