Extracellular Matrix Collagen Research Articles

Knockdown of LRCH4 Remodels Tumor Microenvironment Through Inhibiting YAP and TGF-β/Smad Signaling Pathway in Colorectal Cancer.

Colorectal cancer is one of the most common gastrointestinal malignancies worldwide. LRCH4 is the top 1 gene associated with an unfavorable prognosis in colorectal cancer. Here, we reported that the knockdown of LRCH4 inhibited the proliferation, migration and invasion in HT29 cells. The activity of Yes-Associated Protein (YAP), a transcription factor in the Hppo-YAP signaling pathway, was significantly inhibited by LRCH4-siRNA. LRCH4 knockdown also reversed the EMT and regulated the expression of extracellular matrix (ECM) protein, Fibronectin and Collagen IV in HT29 cells. In addition, the TGF-β/Smad signaling pathway, as the downstream pathway of Yap, was also inhibited by LRCH4 knockdown. Knockdown of LRCH4 involved in the regulation of ECM and EMT and inhibited YAP and the TGF-β/Smad signaling pathway in colorectal cancer cells. Our study provided a mechanism of LRCH4 on colorectal cancer cells, and a new potential target for clinical tumor treatment.

Spatial transcriptome mapping of the desmoplastic growth pattern of colorectal liver metastases by in situ sequencing reveals a biologically relevant zonation of the desmoplastic rim.

We describe the fibrotic rim formed in the desmoplastic growth pattern (DHGP) of colorectal cancer liver metastasis (CLM) using in situ sequencing (ISS). The origin of the desmoplastic rim is still a matter of debate, and the detailed cellular organization has not yet been fully elucidated. Understanding the biology of the DHGP in CLM can explore targeted treatment to improve survival. We used ISS, targeting 150 genes, to characterize the desmoplastic rim by unsupervised clustering of gene co-expression patterns. The cohort comprised 10 chemo-naïve liver metastasis resection samples with a DHGP. Unsupervised clustering of spatially mapped genes revealed molecular and cellular diversity within the desmoplastic rim. We confirmed the presence of the ductular reaction and cancer-associated fibroblasts. Importantly, we discovered angiogenesis and outer and inner zonation in the rim, characterized by NGFR and POSTN expression. ISS enabled the analysis of the cellular organization of the fibrous rim surrounding CLM with a DHGP and suggests a transition from the outer part of the rim, with nonspecific liver injury response, into the inner part, with gene expression indicating collagen synthesis and extracellular matrix remodeling influenced by the interaction with cancer cells, creating a cancer cell supportive environment. Moreover, we found angiogenic processes in the rim. Our results provide a potential explanation of the origin of the rim in DHGP and lead to exploring novel targeted treatments for patients with CLM to improve survival.

Intercellular interaction between FAP+ fibroblasts and CD150+ inflammatory monocytes mediates fibro-stenosis in Crohn's disease.

Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibro-stenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate to the mechanisms underlying fibro-stenosis in CD, we analysed the transcriptome of cells isolated from the transmural ileum of CD patients, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from non-CD patients. Our computational analysis revealed that pro-fibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibro-stenosis in CD.

Topical amlodipine-loaded solid lipid nanoparticles for enhanced burn wound healing: A repurposed approach

Burn wounds are a complicated process with ongoing psychological and physical issues for the affected individuals. Wound healing consists of multifactorial molecular mechanisms and interactions involving; inflammation, proliferation, angiogenesis, and matrix remodeling. Amlodipine (ADB), widely used in cardiovascular disorders, demonstrated antioxidant and anti-inflammatory effects in some non-cardiovascular studies. It was reported that amlodipine is capable of promoting the healing process by regulation of collagen production, extracellular matrix, re-epithelialization and wound healing through its vasodilation and angiogenic activity. The objective of the current study is to appraise the wound healing capacity of amlodipine-loaded SLN (ADB-SLN) integrated into a hydrogel. The in-vitro characterization revealed that the optimized formulation was nanometric (190.4 ± 1.6 nm) with sufficiently high entrapment efficiency (88 % ± 1.4) and sustained ADB release (85.45 ± 4.45 % after 12 h). Furthermore, in-vivo evaluation was conducted on second-degree burns induced in male Sprague-Dawley rats. ADB-SLN gel revealed a high wound contraction rate and a significant improvement in skin regeneration and inflammatory biomarkers levels, confirming its efficiency in enhancing wound healing compared to other tested and commercial formulations. To conclude, the present findings proved that ADB-SLN integrated hydrogel offers a promising novel therapy for burn wound healing with a maximum therapeutic value.

Chronic liver disease and fibrosis: A review of emerging biomarkers and therapeutic targets

Chronic liver disease (CLD) and fibrosis represent a significant global health burden, driven by a range of etiologies including viral hepatitis, alcohol abuse, and non-alcoholic fatty liver disease (NAFLD). These conditions often progress to cirrhosis, liver failure, and hepatocellular carcinoma (HCC), underscoring the urgent need for effective diagnostic and therapeutic strategies. Emerging biomarkers and therapeutic targets offer promising avenues for early diagnosis and intervention in CLD and fibrosis. Biomarkers are crucial for the early detection and monitoring of CLD and fibrosis, allowing for timely therapeutic intervention. Serum biomarkers such as liver enzymes (ALT, AST), bilirubin, and platelet count have traditionally been used, but they lack specificity and sensitivity. Recent advances have identified novel biomarkers with improved diagnostic performance. For instance, serum levels of fibrosis markers like hyaluronic acid, procollagen type III N-terminal peptide (P3NP), and tissue inhibitor of metalloproteinases-1 (TIMP-1) have shown potential in assessing liver fibrosis. Additionally, non-invasive imaging techniques such as transient elastography and magnetic resonance elastography provide quantitative measures of liver stiffness, correlating with fibrosis stage. The pathogenesis of liver fibrosis involves complex interactions between hepatocytes, hepatic stellate cells (HSCs), and the extracellular matrix (ECM). HSCs play a central role in fibrogenesis by transforming into myofibroblasts that secrete collagen and other ECM components. Targeting the activation and proliferation of HSCs has emerged as a promising therapeutic strategy. Small molecule inhibitors, such as those targeting the PDGF and TGF-? signaling pathways, have shown efficacy in preclinical models. Furthermore, antifibrotic agents like simtuzumab, an anti-LOXL2 monoclonal antibody, are being evaluated in clinical trials for their potential to halt or reverse fibrosis progression. Another promising approach involves the modulation of the gut-liver axis. Dysbiosis and increased intestinal permeability contribute to liver inflammation and fibrosis. Probiotics, prebiotics, and fecal microbiota transplantation (FMT) are being explored for their potential to restore gut homeostasis and mitigate liver injury. Additionally, the role of the immune system in fibrosis has gained attention, with immune checkpoint inhibitors and anti-inflammatory agents being investigated for their ability to modulate immune responses and reduce fibrosis. Keywords: Chronic Liver Disease, Fibrosis, Biomarkers, Therapeutic Targets.

Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

More than 90% of hepatocellular carcinoma (HCC) cases develop in the presence of fibrosis or cirrhosis, making the tumor microenvironment (TME) of HCC distinctive due to the intricate interplay between cancer-associated fibroblasts (CAFs) and cancer stem cells (CSCs), which collectively regulate HCC progression. However, the mechanisms through which CSCs orchestrate the dynamics of the tumor stroma during HCC development remain elusive. Our study unveils a significant upregulation of Sema3C in fibrotic liver, HCC tissues, peripheral blood of HCC patients, as well as sorafenib-resistant tissues and cells, with its overexpression correlating with the acquisition of stemness properties in HCC. We further identify NRP1 and ITGB1 as pivotal functional receptors of Sema3C, activating downstream AKT/Gli1/c-Myc signaling pathways to bolster HCC self-renewal and tumor initiation. Additionally, HCC cells-derived Sema3C facilitated extracellular matrix (ECM) contraction and collagen deposition in vivo, while also promoting the proliferation and activation of hepatic stellate cells (HSCs). Mechanistically, Sema3C interacted with NRP1 and ITGB1 in HSCs, activating downstream NF-kB signaling, thereby stimulating the release of IL-6 and upregulating HMGCR expression, consequently enhancing cholesterol synthesis in HSCs. Furthermore, CAF-secreted TGF-β1 activates AP1 signaling to augment Sema3C expression in HCC cells, establishing a positive feedback loop that accelerates HCC progression. Notably, blockade of Sema3C effectively inhibits tumor growth and sensitizes HCC cells to sorafenib in vivo. In sum, our findings spotlight Sema3C as a novel biomarker facilitating the crosstalk between CSCs and stroma during hepatocarcinogenesis, thereby offering a promising avenue for enhancing treatment efficacy and overcoming drug resistance in HCC.

Comparative Transcriptome Analysis of Human and Mouse Canalicular Lungs in Fetal Diaphragmatic Hernia

BackgroundThe nitrofen model of congenital diaphragmatic hernia (CDH) is widely used in translational research. However, the molecular pathways associated with pulmonary hypoplasia in this model compared to the human CDH phenotype have not been well described. The aim of this study was to investigate differentially expressed genes (DEG) and signaling pathways in early stage fetal lungs in mouse and human CDH. MethodsCDH lung tissue was obtained from human fetuses (21-23 weeks gestation) and nitrofen mouse pups (E15.5). NovaSeq Flowcell RNA-seq was performed to evaluate differentially expressed transcriptional and molecular pathways (DEGs) in fetal mice with CDH, compared with age-matched normal mouse lungs and human CDH samples. ResultsThere were thirteen overlapping DEGs in human and mouse CDH lung samples compared to controls. These genes were involved in extracellular matrix, myogenesis, cilia, and immune modulation pathways. Human CDH was associated with an upregulation of collagen formation and extracellular matrix reorganization whereas mouse CDH was associated with an increase in muscular contraction. The most common cell types upregulated in human and mouse CDH samples were ciliated airway cells. ConclusionsThis study highlights the unique gene transcriptional patterns in early fetal mouse and human lungs with CDH. These data have implications when determining the translational potential of novel therapies in CDH using nitrofen-based animal models. Level of EvidenceLevel IV Study TypeBasic science/case series

Unprocessed snRNAs Are a Prognostic Biomarker and Correlate with a Poorer Prognosis in Colorectal Cancer.

The human Integrator complex is a set of 15 subunits that mediates processing of small nuclear RNAs (snRNAs), and which later participates in splicing messenger RNAs (mRNAs). In addition, it controls the pause and release of RNA polymerase II (RNA pol II) at specific gene promoters in response to growth factors. Mutations in Integrator-complex subunit 6 (INTS6) are associated with different types of tumors. However, the INTS6 gene product does not have a significant prognostic value as a biomarker for tumor progression. Here we show that Integrator-complex deregulation is involved in 8.3% of the colorectal cancer cases diagnosed from the population screen carried out in La Rioja (Spain) from the years 2017 to 2019. Lack of Integrator-complex function, measured by an increased level of unprocessed snRNA, is a prognostic biomarker and correlates with a poorer prognosis in colorectal-cancer patients. The transcriptomic profile of all analyzed colorectal tumors shows a strong alteration of the metabolic state of tumor cells, which compromises standard energy production through mitochondrial respiration, known as the Warburg effect. Furthermore, there is a significant upregulation of genes involved in extracellular matrix organization and collagen rearrangement. This is consistent with tissue reorganization in a growing tumor forming a polyp. Crossing the molecular data generated in this study with the follow-up of patients from population screening indicates that population screening combined with early typing of tumors appears to be the most efficient way to increase patient survival.

Type VI Collagen Deficiency Causes Enhanced Periodontal Tissue Destruction.

The periodontal ligament (PDL) is a fibrillar connective tissue that lies between the alveolar bone and the tooth and is composed of highly specialized extracellular matrix (ECM) molecules and a heterogeneous population of cells that are responsible for collagen formation, immune response, bone formation, and chewing force sensation. Type VI collagen (COL6), a widely distributed ECM molecule, plays a critical role in the structural integrity and mechanical properties of various tissues including muscle, tendon, bone, cartilage, and skin. However, its role in the PDL remains largely unknown. Our study shows that deficiency of COL6 impairs PDL fibrillogenesis and exacerbates tissue destruction in ligature-induced periodontitis (LIP). We found that COL6-deficient mice exhibited increased bone loss and degraded PDL in LIP and that fibroblasts expressing high levels of Col6α2 are pivotal in ECM organization and cell-ECM interactions. Moreover, COL6 deficiency in the PDL led to an increased number of fibroblasts geared toward the inflammatory response. We also observed that cultured COL6-deficient fibroblasts from the PDL exhibited decreased expression of genes related to collagen fiber turnover and ECM organization as well as migration and proliferation. Our findings suggest that COL6 plays a crucial role in the PDL, influencing fibroblast function in fibrillogenesis and affecting the immune response in periodontitis. These insights advance our understanding of the molecular mechanisms underlying PDL maturation and periodontal disease.

O-GlcNAcylation controls pro-fibrotic transcriptional regulatory signaling in myofibroblasts

Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O-linked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O-GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O-GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O-GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O-GlcNAcylation of BNC2 at Thr455 and Ser490 and of TEAD4 at Ser69 and Ser99. Altogether, this study unravels protein O-GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.

Skeletal muscle transcriptomics of sheep acclimated to cold desert and tropical regions identifies genes and pathways accentuating their diversity.

The current study attempts to investigate the differences in gene expression in longissimus thoracis muscles between sheep breeds acclimated to diverse environments. Changthangi sheep inhabits the cold arid plateau of Ladakh, at an altitude above 3000m with prevalence of rarefied atmosphere. Muzzafarnagri sheep, on the other hand is found in the sub-tropical hot and humid plains at an altitude of about 250m. Comparative transcriptomics was used to provide a molecular perspective of the differential adaptation of the two breeds. RNA sequencing data was generated from four biological replicates of the longissimus thoracis muscles from both breeds. The common genes expressed in both breeds were involved in muscle contraction and muscle fibre organization. The most significant pathways enriched in Changthangi muscles were glycogen metabolism, reduction of cytosolic Ca++ levels and NFE2L2 regulating anti-oxidant, while those in Muzzafarnagri were extracellular matrix organization and collagen formation. The hub genes identified in Changthangi were involved in hematopoiesis and HIF signaling pathway, suggesting the molecular acclimatization of Changthangi to the high altitude cold desert of Ladakh. The nodal genes discovered in Muzzafarnagri sheep were associated with the extracellular matrix which accentuates its significance in the development, growth and repair of muscles. The observed transcriptomic differences underscore the morphological and adaptive disparity between the two breeds. The candidate genes and pathways identified in this study will form the basis for future research on adaptation to high altitude and body size in small ruminants.

Identification of Potential lncRNA-miRNA-mRNA Regulatory Network Contributing to Arrhythmogenic Right Ventricular Cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) can lead to sudden cardiac death and life-threatening heart failure. Due to its high fatality rate and limited therapies, the pathogenesis and diagnosis biomarker of ARVC needs to be explored urgently. This study aimed to explore the lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network in ARVC. The mRNA and lncRNA expression datasets obtained from the Gene Expression Omnibus (GEO) database were used to analyze differentially expressed mRNA (DEM) and lncRNA (DElnc) between ARVC and non-failing controls. Differentially expressed miRNAs (DEmiRs) were obtained from the previous profiling work. Using starBase to predict targets of DEmiRs and intersecting with DEM and DElnc, a ceRNA network of lncRNA-miRNA-mRNA was constructed. The DEM and DElnc were validated by real-time quantitative PCR in human heart tissue. Protein-protein interaction network and weighted gene co-expression network analyses were used to identify hub genes. A logistic regression model for ARVC diagnostic prediction was established with the hub genes and their ceRNA pairs in the network. A total of 448 DEMs (282 upregulated and 166 downregulated) were identified, mainly enriched in extracellular matrix and fibrosis-related GO terms and KEGG pathways, such as extracellular matrix organization and collagen fibril organization. Four mRNAs and two lncRNAs, including COL1A1, COL5A1, FBN1, BGN, XIST, and LINC00173 identified through the ceRNA network, were validated by real-time quantitative PCR in human heart tissue and used to construct a logistic regression model. Good ARVC diagnostic prediction performance for the model was shown in both the training set and the validation set. The potential lncRNA-miRNA-mRNA regulatory network and logistic regression model established in our study may provide promising diagnostic methods for ARVC.

Single-cell RNA sequencing reveals different chondrocyte states in femoral cartilage between osteoarthritis and healthy individuals.

Cartilage injury is the main pathological manifestation of osteoarthritis (OA). Healthy chondrocyte is a prerequisite for cartilage regeneration and repair. Differences between healthy and OA chondrocyte types and the role these types play in cartilage regeneration and OA progression are unclear. This study conducted single-cell RNA sequencing (scRNA-seq) on the cartilage from normal distal femur of the knee (NC group) and OA femur (OA group) cartilage, the chondrocyte atlas was constructed, and the differences of cell subtypes between the two groups were compared. Pseudo-time and RNA velocity analysis were both performed to verify the possible differentiation sequence of cell subtypes. GO and KEGG pathway enrichment analysis were used to explore the potential functional characteristics of each cell subtype, and to predict the functional changes during cell differentiation. Differences in transcriptional regulation in subtypes were explored by single-cell regulatory network inference and clustering (SCENIC). The distribution of each cell subtype in cartilage tissue was identified by immunohistochemical staining (IHC). A total of 75,104 cells were included, they were divided into 19 clusters and annotated as 11 chondrocyte subtypes, including two new chondrocyte subtypes: METRNL+ and PRG4+ subtype. METRNL+ is in an early stage during chondrocyte differentiation, and RegC-B is in an intermediate state before chondrocyte dedifferentiation. With cell differentiation, cell subtypes shift from genetic expression to extracellular matrix adhesion and collagen remodeling, and signal pathways shift from HIF-1 to Hippo. The 11 subtypes were finally classified as intrinsic chondrocytes, effector chondrocytes, abnormally differentiated chondrocytes and dedifferentiated chondrocytes. IHC was used to verify the presence and distribution of each chondrocyte subtype. This study screened two new chondrocyte subtypes, and a novel classification of each subtype was proposed. METRNL+ subtype is in an early stage during chondrocyte differentiation, and its transcriptomic characteristics and specific pathways provide a foundation for cartilage regeneration. EC-B, PRG4+ RegC-B, and FC are typical subtypes in the OA group, and the HippO-Taz pathway enriched by these cell subtypes may play a role in cartilage repair and OA progression. RegC-B is in the intermediate state before chondrocyte dedifferentiation, and its transcriptomic characteristics may provide a theoretical basis for intervening chondrocyte dedifferentiation.

Salvia hispanica L. (chia) seed improves redox state and reverts extracellular matrix collagen deposition in skeletal muscle of sucrose-rich diet-fed rats.

Skeletal muscle (SkM) is a plastic and dynamic tissue, essential in energy metabolism. Growing evidence suggests a close relationship between intramuscular fat accumulation, oxidative stress (OS), extracellular matrix (ECM) remodeling, and metabolic deregulation in SkM. Nowadays natural products emerge as promising alternatives for the treatment of metabolic disorders. We have previously shown that chia seed administration reverts SkM lipotoxicity and whole-body insulin resistant (IR) in sucrose-rich diet (SRD) fed rats. The purpose of the present study was to assess the involvement of OS and fibrosis in SkM metabolic impairment of insulin-resistant rats fed a long-term SRD and the effects of chia seed upon these mechanisms as therapeutic strategy. Results showed that insulin-resistant SRD-fed rats exhibited sarcopenia, increase in lipid peroxidation, altered redox state, and ECM remodeling-increased collagen deposition and lower activity of the metalloproteinase 2 (MMP-2) in SkM. Chia seed increased ferric ion reducing antioxidant power and glutathione reduced form levels, and the activities of glutathione peroxidase and glutathione reductase enzymes. Moreover, chia seed reversed fibrosis and restored the MMP-2 activity. This work reveals a participation of the OS and ECM remodeling in the metabolic alterations of SkM in our experimental model. Moreover, current data show novel properties of chia seed with the potential to attenuate SkM OS and fibrosis, hallmark of insulin-resistant muscle.

Splicing factor hnRNPA1 regulates alternative splicing of LOXL2 to enhance the production of LOXL2Δ13

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively-spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer (ESS) exists at the 3′ splice site (3′ SS) and 5′ splice site (5′ SS) of LOXL2 exon 13. HnRNPA1 can bind to the ESS and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13.

Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Transcriptomic analysis of AHR wildtype and Knock-out rat livers supports TCDD's role in AHR/ARNT-mediated circadian disruption and hepatotoxicity

Single, high doses of TCDD in rats are known to cause wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD – 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, ≤ 22 ng/kg/day in the CL and ≤ 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and high-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs which showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shift circadian processes away from the resting state, leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth, and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes.

Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-β1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-β1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-β1 signaling axis.

Non-TGFβ profibrotic signaling in ulcerative colitis after in vivo experimental intestinal injury in humans.

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high-resolution endoscopic imaging, histological assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 patients with UC in remission and 20 control subjects. Human ER had a 48-h lag before induction of regenerative epithelial cells [wound-associated epithelial (WAE) and transit amplifying (TA) cells] along with the increase of fibroblast-derived stem cell growth factor gremlin 1 mRNA (GREM1). However, UC deconvolution data showed rapid induction of inflammatory fibroblasts and upregulation of major structural ECM collagen mRNAs along with tissue inhibitor of metalloproteinase 1 (TIMP1), suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor β (TGFβ) mRNA, whereas the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC, suggesting that human postinjury responses could be TGFβ-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end-organ failure, i.e., intestinal damage.NEW & NOTEWORTHY The study reveals the regulatory dynamics of epithelial regeneration and extracellular matrix remodeling after experimental injury of the human colon in vivo and shows that human intestinal regeneration is different from data obtained from animals. A lag phase in epithelial restitution is associated with induction of stromal cell-derived epithelial growth factors. Postinjury regeneration is transforming growth factor β-independent, and we find a profibrotic response in patients with ulcerative colitis despite being in remission.

Abstract PO4-26-03: Collagen deposition in the breast cancer microenvironment correlates with ER-positive, Histological Grade I and low proliferation tumors

Abstract Breast cancer is a highly heterogeneous neoplasm both at the molecular and histological levels. Breast cancer cells present extremely high levels of genetic heterogeneity concerning mutational burden, copy number variations (CNVs) and epigenetic signatures. While this level of heterogeneity is well established, other layers of heterogeneity, such as tumor microenvironment (TME) heterogeneity, remains to be elucidated. TME in breast cancer is an intricate network encompassing tumor cells, “normal” stroma cells such as fibroblasts, lymphocytes, and a plethora of extracellular matrix (ECM) proteins produced either by cancer cells or stromal cells. These three interacting modules (cancer cells, stromal cells and the ECM) can affect each other in a reciprocal way with profound effects on patient prognosis and treatment outcome. Major protein components of the TME are ECM-related collagens, that are produced by breast cancer cells or/and cancer associated fibroblasts. The ECM collagen in breast cancer, that is mainly composed of collagen types I, III and V, has multiple roles modulating tumor growth, cancer cell invasiveness and metastasis. Here by Masson Trichrome and Immunohistochemistry/Aniline Blue co-stains for collagen fibers and proliferation index (Ki-67) in breast cancer FFPE tissue samples, we identified a statistically significant correlation between collagen deposition and Estrogen Receptor (ER)) status (p< 0.05, proliferation index (p< 0.01) and histological Grade (p=0.01). Further, in order to expand and support our findings, we analyzed the METABRIC study through the cBioPortal hub in relevance to collagen genes and multiple clinicopathological parameters. We were able to identify a coregulated subset of collagen genes that presented a positive correlation with ER-positive (p< 10−10, q< 10−10), low proliferating (p< 10−10, q< 10−10), Grade I (p=2.35e−9, q< 1.07e−8) group of breast cancer cases, supporting our findings. In addition, analyses of collagen-high patients from the METABRIC study indicated that most cases presented with PIK3CA, CBFB and MAP3K1 mutations, while through the g.Profiler software we identified collagen-high relevant gene signatures that encompassed gene ontology terms related to tissue development, extracellular matrix organization and cell migration. Collectively, our preliminary results indicate that ECM collagen deposition in breast cancer is characteristic of low-grade tumors with a better differentiation and possibly with a favorable prognosis. Citation Format: Angelos Koutras, Chaido Sirinian, Olga Bogri, Martha Nifora, Maria Theakou, Soren Degn, Ioannis Mouratidis, Dimitrios Chaniotis, Stavros Peroukidis, Ilias Georgakopoulos-Soares, Anastasios Papanastasiou. Collagen deposition in the breast cancer microenvironment correlates with ER-positive, Histological Grade I and low proliferation tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-03.