Extracellular Matrix Gene Expression Research Articles

Integrative analysis of transcriptome, DNA methylome and chromatin accessibility reveals candidate therapeutic targets in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM. DNA methylation and chromatin accessibility, as well as gene expression, were assessed by nucleosome occupancy and methylome sequencing (NOMe-seq) and RNA-seq, respectively, using the cardiac tissues of HCM patients. Compared with those of the controls, the transcriptome, DNA methylome and chromatin accessibility of the HCM myocardium showed multifaceted differences. At the transcriptome level, HCM hearts returned to the fetal gene program through decreased sarcomeric and metabolic gene expression and increased extracellular matrix gene expression. In the DNA methylome, hypermethylated and hypomethylated differentially methylated regions (DMRs) were identified in HCM. At the chromatin accessibility level, HCM hearts showed changes in different genome elements. Several transcription factors (TFs), including SP1 and EGR1, exhibited a fetal-like pattern of binding motifs in nucleosome-depleted regions (NDRs) in HCM. In particular, the inhibition of SP1 or EGR1 in an HCM mouse model harboring sarcomere mutations markedly alleviated the HCM phenotype of the mutant mice and reversed fetal gene reprogramming. Overall, this study not only provides a high precision multi-omics map of HCM heart tissue but also sheds light on the therapeutic strategy by intervening the fetal gene reprogramming in HCM.

Identification of established and novel extracellular matrix components in glioblastoma as targets for angiogenesis and prognosis.

Glioblastomas (GBM) are aggressive tumors known for their heterogeneity, rapid proliferation, treatment resistance, and extensive vasculature. Angiogenesis, the formation of new vessels, involves endothelial cell (EC) migration and proliferation. Various extracellular matrix (ECM) molecules regulate EC survival, migration, and proliferation. Culturing human brain EC (HBMEC) on GBM-derived ECM revealed a decrease in EC numbers compared to controls. Through in silico analysis, we explored ECM gene expression differences between GBM and brain normal glia cells and the impact of GBM microenvironment on EC ECM transcripts. ECM molecules such as collagen alpha chains (COL4A1, COL4A2, p < 0.0001); laminin alpha (LAMA4), beta (LAMB2), and gamma (LAMC1) chains (p < 0.0005); neurocan (NCAN), brevican (BCAN) and versican (VCAN) (p < 0.0005); hyaluronan synthase (HAS) 2 and metalloprotease (MMP) 2 (p < 0.005); MMP inhibitors (TIMP1-4, p < 0.0005), transforming growth factor beta-1 (TGFB1) and integrin alpha (ITGA3/5) (p < 0.05) and beta (ITGB1, p < 0.0005) chains showed increased expression in GBM. Additionally, GBM-influenced EC exhibited elevated expression of COL5A3, COL6A1, COL22A1 and COL27A1 (p < 0.01); LAMA1, LAMB1 (p < 0.001); fibulins (FBLN1/2, p < 0.01); MMP9, HAS1, ITGA3, TGFB1, and wingless-related integration site 9B (WNT9B) (p < 0.01) compared to normal EC. Some of these molecules: COL5A1/3, COL6A1, COL22/27A1, FBLN1/2, ITGA3/5, ITGB1 and LAMA1/B1 (p < 0.01); NCAN, HAS1, MMP2/9, TIMP1/2 and TGFB1 (p < 0.05) correlated with GBM patient survival. In conclusion, this study identified both established and novel ECM molecules regulating GBM angiogenesis, suggesting NCAN and COL27A1 are new potential prognostic biomarkers for GBM.

Mechanical properties, collagen and glycosaminoglycan content of equine superficial digital flexor tendons are not affected by training.

Physical activity can activate extracellular matrix (ECM) protein synthesis and influence the size and mechanical properties of tendon. In this study, we aimed to investigate whether different training histories of horses would influence the synthesis of collagen and other matrix proteins and alter the mechanical properties of tendon. Samples from superficial digital flexor tendon (SDFT) from horses that were either (a) currently race trained (n = 5), (b) previously race trained (n = 5) or (c) untrained (n = 4) were analysed for matrix protein abundance (mass spectrometry), collagen and glycosaminoglycan (GAG) content, ECM gene expression and mechanical properties. It was found that ECM synthesis by tendon fibroblasts invitro varied depending upon the previous training history. In contrast, fascicle morphology, collagen and GAG content, mechanical properties and ECM gene expression of the tendon did not reveal any significant differences between groups. In conclusion, although we could not identify any direct impact of the physical training history on the mechanical properties or major ECM components of the tendon, it is evident that horse tendon cells are responsive to loading invivo, and the training background may lead to a modification in the composition of newly synthesised matrix.

Plasma proteomic biomarkers and the association between poor cardiovascular health and incident dementia: The UK Biobank study

BackgroundThe study examined how plasma proteome indicators may explain the link between poor cardiovascular health (CVH) and dementia risk. MethodsThe present study involved 28,974 UK Biobank participants aged 50-74y (2006–2010) at baseline (2006–2010) who were followed-up for ≤ 15 y for incidence of dementia. CVH was calculated using Life’s Essential 8 (LE8) total scores. The scores were standardized and reverse coded to reflect poor CVH (LE8z_rev). OLINK proteomics was available on this sample (k = 1,463 plasma proteins). The study primarily tested the mediating effects of the plasma proteome in LE8z_rev-dementia effect. The total effect was decomposed into “mediation only” or pure indirect effect (PIE), “interaction only” or interaction referent (INTREF), “neither mediation nor interaction” or controlled direct effect (CDE), and “both mediation and interaction” or mediated interaction (INTMED). ResultsThe study found poorer CVH assessed by LE8z_rev increased the risk of all-cause dementia by 11 % [per 1 SD, hazard ratio, (HR) = 1.11, 95 % CI: 1.03–1.20, p = 0.005). The study identified 11 plasma proteins with strong mediating effects, with GDF15 having the strongest association with dementia risk (per 1 SD, HR = 1.24, 95 % CI: 1.16, 1.33, P < 0.001 when LE8z_rev is set at its mean value) and the largest proportion mediated combining PIE and INTMED (62.6 %; 48 % of TE is PIE), followed by adrenomedullin or ADM. A first principal component with 10 top mediators (TNFRSF1A, GDF15, FSTL3, COL6A3, PLAUR, ADM, GFRAL, ACVRL1, TNFRSF6B, TGFA) mediated 53.6 % of the LE8z_rev-dementia effect. Using all the significant PIE (k = 526) proteins, we used OLINK Insight pathway analysis to identify key pathways, which revealed the involvement of the immune system, signal transduction, metabolism, disease, protein metabolism, hemostasis, membrane trafficking, extracellular matrix organization, developmental biology, and gene expression among others. STRING analysis revealed that five top consistent proteomic mediators were represented in two larger clusters reflecting numerous interconnected biological gene ontology pathways, most notably cytokine-mediated signaling pathway for GDF15 cluster (GO:0019221) and regulation of peptidyl-tyrosine phosphorylation for the ADM cluster (GO:0050730). ConclusionDementia is linked to poor CVH mediated by GDF15 and ADM among several key proteomic markers which collectively explained ∼ 54 % of the total effect.

NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in HSCs.

Fibrosis is the common end point for all forms of chronic liver injury, and the progression of fibrosis leads to the development of end-stage liver disease. Activation of HSCs and their transdifferentiation into myofibroblasts results in the accumulation of extracellular matrix proteins that form the fibrotic scar. Long noncoding RNAs regulate the activity of HSCs and provide targets for fibrotic therapies. We identified long noncoding RNA TILAM located near COL1A1 , expressed in HSCs, and induced with liver fibrosis in humans and mice. Loss-of-function studies in human HSCs and human liver organoids revealed that TILAM regulates the expression of COL1A1 and other extracellular matrix genes. To determine the role of TILAM in vivo, we annotated the mouse ortholog ( Tilam ), generated Tilam- deficient green fluorescent protein-reporter mice, and challenged these mice in 2 different models of liver fibrosis. Single-cell data and analysis of single-data and analysis of Tilam-deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Tilam -deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Furthermore, loss of Tilam expression attenuated the development of fibrosis in the setting of in vivo liver injury. Finally, we found that TILAM interacts with promyelocytic leukemia nuclear body scaffold protein to regulate a feedback loop by which TGF-β2 reinforces TILAM expression and nuclear localization of promyelocytic leukemia nuclear body scaffold protein to promote the fibrotic activity of HSCs. TILAM is activated in HSCs with liver injury and interacts with promyelocytic leukemia nuclear body scaffold protein to drive the development of fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end-stage liver disease.

Distinct subpopulations of human subcutaneous adipose tissue precursor cells revealed by single-cell RNA sequencing.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women.NEW & NOTEWORTHY Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women.

Abstract 5475: Development of high-throughput 3D bioprinted pediatric models for precision medicine

Abstract Childhood cancer patients with high-risk disease have poor prognosis. Therapies given to treat these patients are often highly toxic and determined empirically, exposing children to damaging and ineffective therapies. Precision medicine is a promising strategy for these patients. However, tumor biopsies often lack sufficient sample for phenotypic drug screening, requiring cell expansion via primary cell culture or development of patient-derived xenograft models - a slow process with variable success rates. There is an unmet need for sensitive, predictive, and timely drug testing models. Dynamic interactions between cells and the extracellular matrix (ECM) impact cellular signaling and response to cancer therapy. The development of patient-derived tumor models that are reflective of the patient sample and achieved in a clinically relevant timeframe will be game changing. In-silico analysis of 265 ECM gene expression from a high-risk neuroblastoma and sarcoma patient cohort (n=145) identified collagens and fibronectin to be the most abundantly expressed ECM genes in the tumor samples. To reflect this environment, we developed tuneable tissue ECM-like bioinks where cells are embedded within the bioink that mimics growth constraints within a tumor [1]. Specifically, we functionalized the bioinks with peptides of collagen I, fibronectin, and laminin to mimic the ECM environment and combined this with HTP 3D bioprinting technology to create patient-derived tumoroids. In this proof-of-concept study, we identified conditions that enable the growth and expansion of the neuroblastoma and sarcoma cells in a 3D ECM-like environment. The tumor samples proliferate in the bioinks and HTP drug screening was used to determine drug sensitivity. Importantly, the bioprinted cells reflect the genetic and phenotypic characteristics of the original patient tumors and retained their tumorigenic capacity in vivo. Collectively, we have successfully generated preclinical models that reflect patient tumors and are directly compatible with preclinical drug testing. Importantly, our 3D bioprinting platform has the potential to advance cancer precision medicine in a clinically relevant timeframe. These findings have broader applications for a range of cancer types for preclinical testing, drug discovery and cancer biology.1. Utama, RH, et al. Macromolecular Bioscience, 2021. 21(9):e2100125 Citation Format: Valentina Poltavets, MoonSun Jung, Joanna Skhinas, Kathleen Kimpton, Alvin Kamili, Gabe Tax, Jie Mao, Louise Cui, Marie Wong, Mark J. Cowley, Loretta Lau, Emmy ME Dolman, John J. Gooding, Maria Kavallaris. Development of high-throughput 3D bioprinted pediatric models for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5475.

Abstract B078: Insulin-like growth factor signaling contributes to omental preadipocyte-mediated tumorigenesis in ovarian cancer by regulating expression of extracellular matrix genes

Abstract B078: Insulin-like growth factor signaling contributes to omental preadipocyte-mediated tumorigenesis in ovarian cancer by regulating expression of extracellular matrix genes

Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

Targeting RORγ inhibits the growth and metastasis of hepatocellular carcinoma

Approximately 80%-90% of hepatocellular carcinomas (HCC) occur in a premalignant environment of fibrosis and abnormal extracellular matrix (ECM), highlighting an essential role of ECM in the tumorigenesis and progress of HCC. However, the determinants of ECM in HCC are poorly defined. Here, we show that nuclear receptor RORγ is highly expressed and amplified in HCC tumors. RORγ functions as an essential activator of the matrisome program via directly driving the expression of major ECM genes in HCC cells. Elevated RORγ increases fibronectin-1 deposition, cell-matrix adhesion, and collagen production, creating a favorable microenvironment to boost liver cancer metastasis. Moreover, RORγ antagonists effectively inhibit tumor growth and metastasis in multiple HCC xenografts and immune-intact models, and they effectively sensitize HCC tumors to sorafenib therapy in mice. Notably, elevated RORγ expression is associated with ECM remodeling and metastasis in patients with HCC. Taken together, we identify RORγ as a key player of ECM remodeling in HCC and as an attractive therapeutic target for advanced HCC.

RUNX1/NPM1/H3K4me3 complex contributes to extracellular matrix remodeling via enhancing FOSL2 transcriptional activation in glioblastoma

Extracellular matrix (ECM) remodeling has been implicated in the tumor malignant progression and immune escape in glioblastoma (GBM). Runt-related transcription factor 1 (RUNX1) is a vital transcriptional factor for promoting tumorigenesis and invasion in mesenchymal subtype of GBM. But the correlation between RUNX1 and ECM genes expression and regulatory mechanism of RUNX1 on ECM genes expression remain poorly understood to date. In this study, by using integral analysis of chromatin immunoprecipitation-sequencing and RNA sequencing, we reported that RUNX1 positively regulated the expression of various ECM-related genes, including Fibronectin 1 (FN1), Collagen type IV alpha 1 chain (COL4A1), and Lumican (LUM), in GBM. Mechanistically, we demonstrated that RUNX1 interacted with Nucleophosmin 1 (NPM1) to maintain the chromatin accessibility and facilitate FOS Like 2, AP-1 Transcription Factor Subunit (FOSL2)-mediated transcriptional activation of ECM-related genes, which was independent of RUNX1’s transcriptional function. ECM remodeling driven by RUNX1 promoted immunosuppressive microenvironment in GBM. In conclusion, this study provides a novel mechanism of RUNX1 binding to NPM1 in driving the ECM remodeling and GBM progression.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

In Vivo and In Vitro Pro-Fibrotic Response of Lung-Resident Mesenchymal Stem Cells from Patients with Idiopathic Pulmonary Fibrosis.

Lung-resident mesenchymal stem cells (LR-MSC) are thought to participate in idiopathic pulmonary fibrosis (IPF) by differentiating into myofibroblasts. On the other hand, LR-MSC in IPF patients present senescence-related features. It is unclear how they respond to a profibrotic environment. Here, we investigated the profibrotic response of LR-MSC isolated from IPF and control (CON) patients. LR-MSC were inoculated in mice 48 h after bleomycin (BLM) instillation to analyze their contribution to lung damage. In vitro, LR-MSC were exposed to TGFβ. Mice inoculated with IPF LR-MSC exhibited worse maintenance of their body weight. The instillation of either IPF or CON LR-MSC sustained BLM-induced histological lung damage, bronchoalveolar lavage fluid cell count, and the expression of the myofibroblast marker, extracellular matrix (ECM) proteins, and proinflammatory cytokines in the lungs. In vitro, IPF LR-MSC displayed higher basal protein levels of aSMA and fibronectin than CON LR-MSC. However, the TGFβ response in the expression of TGFβ, aSMA, and ECM genes was attenuated in IPF LR-MSC. In conclusion, IPF LR-MSC have acquired myofibroblastic features, but their capacity to further respond to profibrotic stimuli seems to be attenuated. In an advanced stage of the disease, LR-MSC may participate in disease progression owing to their limited ability to repair epithelial damage.

Viable tendon neotissue from adult adipose-derived multipotent stromal cells.

Background: Tendon healing is frequently prolonged, unpredictable, and results in poor tissue quality. Neotissue formed by adult multipotent stromal cells has the potential to guide healthy tendon tissue formation. Objectives: The objective of this study was to characterize tendon neotissue generated by equine adult adipose-derived multipotent stromal cells (ASCs) on collagen type I (COLI) templates under 10% strain in a novel bioreactor. The tested hypothesis was that ASCs assume a tendon progenitor cell-like morphology, express tendon-related genes, and produce more organized extracellular matrix (ECM) in tenogenic versus stromal medium with perfusion and centrifugal fluid motion. Methods: Equine ASCs on COLI sponge cylinders were cultured in stromal or tenogenic medium within bioreactors during combined perfusion and centrifugal fluid motion for 7, 14, or 21days under 10% strain. Viable cell distribution and number, tendon-related gene expression, and micro- and ultra-structure were evaluated with calcein-AM/EthD-1 staining, resazurin reduction, RT-PCR, and light, transmission, and scanning electron microscopy. Fibromodulin was localized with immunohistochemistry. Cell number and gene expression were compared between culture media and among culture periods (p < 0.05). Results: Viable cells were distributed throughout constructs for up to 21days of culture, and cell numbers were higher in tenogenic medium. Individual cells had a round or rhomboid shape with scant ECM in stromal medium in contrast to clusters of parallel, elongated cells surrounded by highly organized ECM in tenogenic medium after 21days of culture. Transcription factor, extracellular matrix, and mature tendon gene expression profiles confirmed ASC differentiation to a tendon progenitor-like cell in tenogenic medium. Construct micro- and ultra-structure were consistent with tendon neotissue and fibromodulin was present in the ECM after culture in tenogenic medium. Conclusion: Long-term culture in custom bioreactors with combined perfusion and centrifugal tenogenic medium circulation supports differentiation of equine adult ASCs into tendon progenitor-like cells capable of neotissue formation.

Dynamic changes in mitral valve extracellular matrix, tissue mechanics and function in a mouse model of Marfan syndrome

ObjectiveMouse models of Marfan syndrome (MFS) with Fibrillin 1 (Fbn1) variant C1041G exhibit cardiovascular abnormalities, including myxomatous valve disease (MVD) and aortic aneurism, with structural extracellular matrix (ECM) dysregulation. In this study, we examine the structure-function-mechanics relations of the mitral valve related to specific transitions in ECM composition and organization in progressive MVD in MFS mice from Postnatal day (P)7 to 1 year-of-age. Approach and resultsMechanistic links between mechanical forces and biological changes in MVD progression were examined in Fbn1C1041G/+ MFS mice. By echocardiography, mitral valve dysfunction is prevalent at 2 months with a decrease in cardiac function at 6 months, followed by a preserved cardiac function at 12 months. Mitral valve (MV) regurgitation occurs in a subset of mice at 2–6 months, while progressive dilatation of the aorta occurs from 2 to 12 months. Mitral valve tissue mechanical assessments using a uniaxial Permeabilizable Fiber System demonstrate decreased stiffness of MFS MVs at all stages. Histological and microscopic analysis of ECM content, structure, and fiber orientation demonstrate that alterations in ECM mechanics, composition, and organization precede functional abnormalities in Fbn1C1041G/+MFS MVs. At 2 months, ECM abnormalities are detected with an increase in proteoglycans and decreased stiffness of the mitral valve. By 6–12 months, collagen fiber remodeling is increased with abnormal fiber organization in MFS mitral valve leaflets. At the same time, matrifibrocyte gene expression characteristic of collagen-rich connective tissue is increased, as detected by RNA in situ hybridization and qPCR. Together, these studies demonstrate early prevalence of proteoglycans at 2 months followed by upregulation of collagen structure and organization with age in MVs of MFS mice. ConclusionsAltogether, our data indicate dynamic regulation of mitral valve structure, tissue mechanics, and function that reflect changes in ECM composition, organization, and gene expression in progressive MVD. Notably, increased collagen fiber organization and orientation, potentially dependent on increased matrifibrocyte cell activity, is apparent with altered mitral valve mechanics and function in aging MFS mice.

Kidney Injury by Unilateral Ureteral Obstruction in Mice Lacks Sex Differences.

Renal fibrosis is a critical event in the development and progression of chronic kidney disease (CKD), and it is considered the final common pathway for all types of CKD. The prevalence of CKD is higher in females; however, males have a greater prevalence of end-stage renal disease. In addition, low birth weight and low nephron number are associated with increased risk for CKD. This study examined the development and severity of unilateral ureter obstruction (UUO)-induced renal fibrosis in male and female wild-type (ROP +/+) and mutant (ROP Os/+) mice, a mouse model of low nephron number. Male and female ROP +/+ and ROP Os/+ mice were subjected to UUO, and kidney tissue was collected at the end of the 10-day experimental period. Kidney histological analysis and mRNA expression determined renal fibrosis, tubular injury, collagen deposition, extracellular matrix proteins, and immune cell infiltration. Male and female UUO mice demonstrated marked renal injury, kidney fibrosis, and renal extracellular matrix production. Renal fibrosis and α-smooth muscle actin were increased to a similar degree in ROP +/+ and ROP Os/+ mice with UUO of either sex. There were also no sex differences in renal tubular cast formation or renal infiltration of macrophage in ROP +/+ and ROP Os/+ UUO mice. Interestingly, renal fibrosis and α-smooth muscle actin were 1.5-3-fold greater in UUO-ROP +/+ compared to UUO-ROP Os/+ mice. Renal inflammation phenotypes following UUO were also 30-45% greater in ROP +/+ compared to ROP Os/+ mice. Likewise, expression of extracellular matrix and renal fibrotic genes was greater in UUO-ROP +/+ mice compared to UUO-ROP Os/+ mice. In contrast to these findings, ROP Os/+ mice with UUO demonstrated glomerular hypertrophy with 50% greater glomerular tuft area compared to ROP +/+ with UUO. Glomerular hypertrophy was not sex-dependent in any of the genotypes of ROP mice. These findings provide evidence that low nephron number contributes to UUO-induced glomerular hypertrophy in ROP Os/+ mice but does not enhance renal fibrosis, inflammation, and renal tubular injury. Taken together, we demonstrate that low nephron number contributes to enhanced glomerular hypertrophy but not kidney fibrosis and tubular injury. We also demonstrate that none of the changes caused by UUO was affected by sex in any of the ROP mice genotypes.

In vitro differentiation of human induced pluripotent stem cells into temporomandibular joint disc like cells

Temporomandibular joint discs (TMJ discs) are unable to repair themselves in disease states, while induced stem cell differentiation is a common method to repair tissue defects. Nowadays, kinds of stem cells are attempted for tissue regeneration of TMJ disc, but these methods have several downsides, which limit their wide application. The proliferation and differentiation ability of human induced pluripotent stem cells (hiPSC) provides a new research direction for TMJ disc tissue regeneration. In this study, we investigated the feasibility of induced differentiation of hiPSC into TMJ disc cells in vitro and the differentiation efficiency of different methods to clarify the possibility and conditions of hiPSC application in TMJ disc tissue engineering. We collected sheep TMJ disc cells cultures for adding in hiPSC culture environment and treated hiPSC by both direct induction and Transwell co-culture for 7 days, 14 days and 21 days. The secretion of extracellular matrix in TMJ disc cells was detected by Sirius Red and Safranin O staining. Collagen Ⅰ and Collagen Ⅱ were qualitatively detected by immunohistochemical staining. The expression of extracellular matrix genes (type I collagen (COL1A1), type II collagen(COL2), glycosaminoglycan (GAG)), chondrogenic differentiation gene SOX9 and pluripotency gene OCT4 were detected by RT-qPCR. Our results showed that hiPSC had the ability to differentiate to TMJ disc cells by direct induction in TMJ disc cell culture medium and by Transwell co-culture method. The highest degree of differentiation was observed after 14 days of direct induction, while Transwell co-culture showed significant differentiation at different times and with different major directions. Meanwhile, Transwell co-culture not only differentiates hiPSC but also promotes the growth and proliferation of TMJ disc cells. Our study is valuable to investigate the possibility of differentiation of hiPSC toward TMJ disc cells and to determine the time of differentiation. It provides new ideas for the selection of seed cells for TMJ disc tissue engineering.

Sex-dimorphic expression of extracellular matrix genes in mouse bone marrow neutrophils.

The mammalian innate immune system is sex-dimorphic. Neutrophils are the most abundant leukocyte in humans and represent innate immunity's first line of defense. We previously found that primary mouse bone marrow neutrophils show widespread sex-dimorphism throughout life, including at the transcriptional level. Extracellular matrix [ECM]-related terms were observed among the top sex-dimorphic genes. Since the ECM is emerging as an important regulator of innate immune responses, we sought to further investigate the transcriptomic profile of primary mouse bone marrow neutrophils at both the bulk and single-cell level to understand how biological sex may influence ECM component expression in neutrophils throughout life. Here, using curated gene lists of ECM components and unbiased weighted gene co-expression network analysis [WGCNA], we find that multiple ECM-related gene sets show widespread female-bias in expression in primary mouse neutrophils. Since many immune-related diseases (e.g., rheumatoid arthritis) are more prevalent in females, our work may provide insights into the pathogenesis of sex-dimorphic inflammatory diseases.

MSC Reprogramming and Aberrant Inflammatory Signaling in Myelofibrosis: Insights from Single-Cell RNA-Sequencing of Trephine Bone Marrow Cells

Myelofibrosis (MF) results from mutation-induced activation of JAK/STAT signaling in hematopoietic stem, progenitor and mature cells, and features a progressive, fibrosing stromal reaction, splenomegaly, constitutional symptoms and reduced life expectancy. Identification of disrupted cellular pathways within bone marrow (BM) is pivotal for therapeutic breakthrough, but fibrosis severely limits access to stromal elements for single-cell analysis. To overcome this, we utilized collagenase digestion of fresh BM trephine biopsy material surplus to diagnostic needs and profiled isolated cells using scRNAseq. 9 samples were obtained from 8 patients with MF (4, JAK inhibitor naïve; 4, on ruxolitinib); 4 samples were from 4 patients with MF 4, 10, 12 &amp; 12 months after allogeneic transplant; and 7 samples (termed “normal controls”) were from 6 patients who had received an allograft for myelodysplasia or acute leukemia, and 1 who had received CAR-T cells for B-ALL. Normal controls exhibited normal or near-normal blood counts and lacked BM fibrosis. Each sample was subjected to collagenase digestion and flow sorted to isolate non-erythroid hematopoietic (CD45+GlyA-) and microenvironmental (CD45+GlyA-CD71-) cells for single cell RNA sequencing and downstream comparative analysis. 105,231 transcriptomes met quality control criteria (median 4975/sample, range 1036-13277, no significant difference between sample categories) and 43 transcriptional clusters were identified. Compositional analysis identified the non-hematopoietic stromal cell fraction as harboring the biggest differences with, surprisingly, a proportional relative depletion of mesenchymal stromal cells (MSCs) and enrichment of endothelial cells (ECs) in MF vs. normal controls. While transcriptomes of ECs did not differ substantially, there were extensive transcriptional differences between MSCs recovered from MF vs. normal control samples. Subclustering revealed two subsets, MSC1 &amp; MSC2 asymmetrically distributed between MF and normal controls. Intriguingly, MSCs recovered from MF patients after allograft occupied an intermediate position in principal component space indicating that reversion of disease-related MSC transcriptional reprogramming post-allograft occurs over a timescale of months. The reprogrammed transcriptional state of MF MSCs is defined by reduced expression of hematopoietic support factor genes (eg, KITLG, CXCL12, ANGPTL2, NCAM2, CSF1) and increased expression of extracellular matrix (ECM) genes (eg, FN1, COL1A2, COL3A1, DCN, SPARC), features which suggest an explanation for the increased frequency of graft failure following allograft in MF versus other hematologic malignancies. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) indicated transcription factors with either increased (eg, NR2F2, RUNX2, FOXP2) or decreased (eg, CEBPA, FOXC1, and PPARG) transcriptional activity as candidate master transcriptional regulators of the observed MSC reprogramming in MF. Transcriptional change in MSCs correlated strongly with histopathological assessment of BM reticulin. Within the hematopoietic fraction, and agnostic of JAK inhibitor treatment status, comparison of MF vs. control cells unexpectedly revealed significant upregulation of interferon response genes, especially in CD14 + monocytes, but also in neutrophils and their progenitors, centred on expression of interferon-stimulated gene factor 3 (ISGF3) genes IRF9 and STAT1/2. Comparison of MF samples according to ruxolitinib treatment status indicated that ruxolitinib induced downregulation of an NFκB-centered inflammatory program, particularly in neutrophils. Finally, ligand-receptor analysis using NicheNet identified TGFB1, IL1B, and TNF as top predicted ligands for induction of the MF signature in MSCs. While myeloid cells were top producers of these factors, their expression did not vary between MF vs. controls, suggesting other mechanisms, such as prolonged retention of ligand in the ECM, are important. In summary, our comparative scRNA analysis of BM cells from MF patients and controls - the largest reported to date - reveals (i) transcriptional reprogramming of MSCs leading to reduced expression of hematopoietic support factor and increased expression of ECM genes; and (ii) increased expression of interferon and NFkB inflammatory pathways in myeloid cells.