Pathological Extracellular Matrix Research Articles

A human stem cell-derived model reveals pathologic extracellular matrix remodeling in diabetic podocyte injury

Diabetic nephropathy results from chronic (or uncontrolled) hyperglycemia and is the leading cause of kidney failure. The kidney’s glomerular podocytes are highly susceptible to diabetic injury and subsequent non-reversible degeneration. We generated a human induced pluripotent stem (iPS) cell-derived model of diabetic podocytopathy to investigate disease pathogenesis and progression. The model recapitulated hallmarks of podocytopathy that precede proteinuria including retraction of foot processes and podocytopenia (detachment from the extracellular matrix (ECM)). Moreover, hyperglycemia-induced injury to podocytes exacerbated remodeling of the ECM. Specifically, mature podocytes aberrantly increased expression and excessively deposited collagen (IV)α1α1α2 that is normally abundant in the embryonic glomerulus. This collagen (IV) imbalance coincided with dysregulation of lineage-specific proteins, structural abnormalities of the ECM, and podocytopenia – a mechanism not shared with endothelium and is distinct from drug-induced injury. Intriguingly, repopulation of hyperglycemia-injured podocytes on decellularized ECM scaffolds isolated from healthy podocytes attenuated the loss of synaptopodin (a mechanosensitive protein associated with podocyte health). These results demonstrate that human iPS cell-derived podocytes can facilitate in vitro studies to uncover the mechanisms of chronic hyperglycemia and ECM remodeling and guide disease target identification.

Characterizing the human intestinal chondroitin sulfate glycosaminoglycan sulfation signature in inflammatory bowel disease

The intestinal extracellular matrix (ECM) helps maintain appropriate tissue barrier function and regulate host-microbial interactions. Chondroitin sulfate- and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) are integral components of the intestinal ECM, and alterations in CS/DS-GAGs have been shown to significantly influence biological functions. Although pathologic ECM remodeling is implicated in inflammatory bowel disease (IBD), it is unknown whether changes in the intestinal CS/DS-GAG composition are also linked to IBD in humans. Our aim was to characterize changes in the intestinal ECM CS/DS-GAG composition in intestinal biopsy samples from patients with IBD using mass spectrometry. We characterized intestinal CS/DS-GAGs in 69 pediatric and young adult patients (n = 13 control, n = 32 active IBD, n = 24 IBD in remission) and 6 adult patients. Here, we report that patients with active IBD exhibit a significant decrease in the relative abundance of CS/DS isomers associated with matrix stability (CS-A and DS) compared to controls, while isomers implicated in matrix instability and inflammation (CS-C and CS-E) were significantly increased. This imbalance of intestinal CS/DS isomers was restored among patients in clinical remission. Moreover, the abundance of pro-stabilizing CS/DS isomers negatively correlated with clinical disease activity scores, whereas both pro-inflammatory CS-C and CS-E content positively correlated with disease activity scores. Thus, pediatric patients with active IBD exhibited increased pro-inflammatory and decreased pro-stabilizing CS/DS isomer composition, and future studies are needed to determine whether changes in the CS/DS-GAG composition play a pathogenic role in IBD.

Genetic modification of cardiac fibroblasts in adult rats using adeno-associated virus serotype 9

Cardiac fibrosis is a process involving pathological extracellular matrix remodeling as well as cardiac fibroblast (CF) activation in response to cardiac injury and stress, which is frequently associated with cardiac dysfunction. Therefore, understanding the molecular mechanisms underlying CF activation is critical to develop successful therapies for treating cardiac fibrosis. CF-specific gene modifications in mouse models have been reported as key tools for cardiac fibrosis research. Although rat cardiac disease models also offer many of the same advantages as the mouse models, there are no rat models that employ CF-specific conditional gene modification. Here, we attempted CF-specific gene introduction in rats using adeno-associated virus serotype 9 (AAV9), allowing us to develop fast and simple rat cardiac disease models rather than establishing transgenic rat lines. AAV9 carrying a CF-specific human TCF21 (hTCF21) promoter and a transgene of interest was generated and introduced into adult rats via tail vein injections. Since we previously reported in cultured CFs that protein kinase D (PKD) activation at the outer mitochondrial membrane (OMM) induces mitochondrial fragmentation via phosphorylation of a mitochondrial fission protein DRP1, we generated an OMM-targeted dominant-negative mutant of PKD (termed mt-PKD-DN) to specifically inhibit PKD activity at the OMM in CFs in vivo. In vivo cardiac function, gene expression, protein expression, and mitochondrial morphology were assessed 2-10 weeks after AAV9 injection by echocardiography, quantitative RT-PCR, immunohistochemistry, and transmission electron microscopy (TEM), respectively. AAV9-hTCF21-injected rats maintained normal cardiac dimensions and contractile function compared to aged-matched control rats. Using AAV9-hTCF21-EGFP, we confirmed organ-specific (i.e., heart) and cell type-specific (i.e., CF-specific) expression of GFP. Next, we injected AAV9-hTCF21-mt-PKD-DN or -luciferase (Luc) as a control in adult rats. TEM images from fixed rat ventricular tissues revealed a significant increase in mitochondrial aspect ratio and mitochondrial perimeter in CFs with AAV9-hTCF21-mt-PKD-DN compared to AAV9-hTCF21-Luc, indicating that PKD inhibition at the OMM induces mitochondrial elongation in CFs in vivo. In contrast, no significant change in mitochondrial morphology was observed in the cardiomyocyte area. In summary, we successfully achieved a CF-specific gene delivery to the adult rat hearts in vivo, demonstrating the potential of a simplified CF-specific rat transgenic model using the AAV9-hTCF21 system. NIH/NHLBI R01HL160699. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

P003 Metabolic Reprogramming in Intestinal Fibrosis: Integrating Metabolomics, Single-Cell Transcriptomics and Isotope Tracing

Abstract Background This study aims to systematically elucidate the metabolic landscape of intestinal fibrosis in Crohn's disease, revealing the key reprogrammed metabolic pathways in the fibrotic microenvironment. Methods We collected normal and fibrosis tissues from 122 Crohn's disease patients undergoing surgery for fibrosis-induced intestinal obstruction, and established DSS/TNBS-induced murine intestinal fibrosis model. To measure metabolites level in normal and fibrotic tissues, untargeted metabolomics was performed. C13-labeled glutamine was used for metabolic tracing to delineate metabolic fluxes in vitro and in vivo. We performed integration analysis by merging metabolomics and metabolic tracing data with established single-cell transcriptomic profiles. Isolation and further validation of human intestinal stromal cells were carried out, and gene expression was assessed using qRT-PCR and immunofluorescence. Results Significant metabolic heterogeneity was observed between normal and fibrotic tissues, with differential metabolites enriched in glucose and amino acid metabolism. Notably, intermediates of glycolysis were significantly increased, while tricarboxylic acid (TCA) cycle intermediates were decreased in fibrotic regions (p<0.05). Amino acids were broadly downregulated in fibrosis, whereas glutamate and glutamine were highly enriched (p<0.05). Mechanistic insights into these changes were provided by isotope tracing, revealing enhanced glycolysis and weakened TCA flux attributed to transition from TCA intermediate α-KG to glutamate. Moreover, catabolism of glutamine to glutamate was enhanced. Glutamate from above pathways converted to proline, which is the ingredient of extracellular matrix (ECM). Single-cell transcriptomic analysis indicated a high enrichment of genes related to glutamine metabolism in stromal cells, with significant upregulation of enzymes such as GLS (Glutaminase) and PYCR1 (Pyrroline-5-carboxylate Reductase 1) involved in glutamine and proline synthesis. Immunofluorescence and qRT-PCR confirmed the upregulation of GLS and PYCR1. Further experiments using primary stromal cells and murine models demonstrated that inhibiting GLS activity reduced ECM synthesis, alleviating intestinal fibrosis. Conclusion In intestinal fibrosis, glycolysis and glutamine catabolism enhanced. Stromal cells utilized the reprogrammed metabolic pathway to synthesize glutamate, thereby promoting pathological ECM anabolism. Targeting the key enzyme GLS represent a potential therapeutic strategy for intestinal fibrosis.

Extracellular Matrix Influences Gene Expression and Differentiation of Mouse Trophoblast Stem Cells.

Trophoblast stem (TS) cells were first isolated from the mouse placenta; however, little is known about their maintenance and niche in vivo. TS cells, like other stem cells, have a unique microenvironment in which the extracellular matrix (ECM) is a component. Placental pathology is associated with ECM change. However, how these changes and the individual ECM components impact the maintenance or differentiation of TS cells has not been established. This study identified which ECM component(s) maintain the greatest expression of markers associated with undifferentiated mouse trophoblast stem (mTS) cells and which alter the profile of markers of differentiation based on mRNA analysis. mTS cells cultured on individual ECM components and subsequent quantitative polymerase chain reaction analysis revealed that laminin promoted the expression of markers associated with undifferentiated TS cells, fibronectin promoted gene expression associated with syncytiotrophoblast (SynT) layer II cells, and collagen IV promoted the expression of genes associated with differentiated trophoblast. To investigate whether pathological placental ECM influenced the expression of genes associated with different trophoblast subtypes, the mouse model of streptozotocin (STZ)-induced pancreatic β cell ablation and diabetes was used. Female mice administered STZ (blood glucose ≥300 mg/dL) or control (blood glucose ≤150 mg/dL) were mated. Placental pathology at embryonic day (E)14.5 was confirmed with reduced fetal blood space area, reduced expression of the pericyte marker αSMA, and decreased expression of ECM proteins. mTS cells cultured on ECM isolated from STZ placenta were associated with reduced expression of undifferentiated mTS markers and increased expression of genes associated with terminally differentiated trophoblast [Gcm-1 and SynA (SynT) and junctional zone Tpbpa and Prl2c2]. Altogether, these results support the value of using ECM isolated from the placenta as a tool for understanding trophoblast contribution to placental pathology.

The extracellular matrix mechanics in the vasculature.

Mechanical stimuli from the extracellular matrix (ECM) modulate vascular differentiation, morphogenesis and dysfunction of the vasculature. With innovation in measurements, we can better characterize vascular microenvironment mechanics in health and disease. Recent advances in material sciences and stem cell biology enable us to accurately recapitulate the complex and dynamic ECM mechanical microenvironment for in vitro studies. These biomimetic approaches help us understand the signaling pathways in disease pathologies, identify therapeutic targets, build tissue replacement and activate tissue regeneration. This Review analyzes how ECM mechanics regulate vascular homeostasis and dysfunction. We highlight approaches to examine ECM mechanics at tissue and cellular levels, focusing on how mechanical interactions between cells and the ECM regulate vascular phenotype, especially under certain pathological conditions. Finally, we explore the development of biomaterials to emulate, measure and alter the physical microenvironment of pathological ECM to understand cell-ECM mechanical interactions toward the development of therapeutics.

Intervention of modified Shenling Baizhu San on peritoneal fibrosis induced by peritoneal dialysate with different sugar concentration in rats

To investigate the effect of modified Shenling Baizhu San on the pathological changes and extracellular matrix (ECM) in rats with peritoneal fibrosis induced by peritoneal dialysate fluid (PDF) with different sugar concentration and its mechanism. Seventy male Sprague-Dawley (SD) rats were randomly divided into control group, different sugar content PDF model groups and corresponding traditional Chinese medicine intervention groups, with 10 rats in each group. Peritoneal fibrosis model was reproduced by intraperitoneal injection of 100 mL×kg-1×d-1 PDF containing 1.5%, 2.5% and 4.25% sugar once a day for 8 weeks. The rats in the control group were given the same amount of normal saline. The rats in the traditional Chinese medicine intervention groups were treated with gavage of 10 mL/kg of modified Shenling Baizhu San (containing 2.014 g crude drug per liter) immediately after modeling. The PDF model groups and the control group were given the same amount of normal saline by gavage. After 8 weeks, the peritoneal ultrafiltration volume of rats in each group was measured. The peritoneal tissues were collected and stained with hematoxylin-eosin (HE), and the structural changes and thickness of the parietal peritoneum were observed under a light microscope. After Masson staining, the deposition of collagen fibers was observed under a light microscope. Western blotting was used to detect the protein expressions of E-cadherin, α-smooth muscle actin (α-SMA) and Vimentin, the main components of ECM in parietal peritoneum. The positive expressions of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-β1 (TGF-β1) were detected by immunohistochemical staining. Compared with the control group, PDF with different sugar contents could induce peritoneal fibrosis in rats, and the degree of fibrosis was gradually aggravated with the increase of sugar content, which was manifested as peritoneal thickening, increased collagen fiber deposition, decreased peritoneal ultrafiltration volume, down-regulated expressions of E-cadherin and MMP-9 in peritoneal tissue, and up-regulated expressions of α-SMA, Vimentin, TIMP-1 and TGF-β1, and the pathological changes and ECM accumulation in peritoneal tissues were more serious in 4.25% PDF model group. After the intervention of modified Shenling Baizhu San, compared with the corresponding PDF model groups, the peritoneal fibrosis of rats was improved to varying degrees, and the effect of the 4.25% PDF+traditional Chinese medicine intervention group was more significant, the parietal peritoneum was significantly thinner (μm: 101.86±16.01 vs. 140.65±10.13, P < 0.05), collagen fiber deposition was significantly reduced, peritoneal ultrafiltration volume was significantly increased (mL: -0.01±3.45 vs. -3.53±1.84, P < 0.05), the expressions of E-cadherin and MMP-9 in peritoneal tissues were significantly up-regulated [E-cadherin protein (E-cadherin/β-actin): 0.84±0.08 vs. 0.28±0.05, MMP-9 (A value): 0.60±0.15 vs. 0.37±0.01, both P < 0.05], and the expressions of α-SMA, Vimentin, TIMP-1 and TGF-β1 were significantly down-regulated [α-SMA protein (α-SMA/β-actin): 0.36±0.08 vs. 1.05±0.09, Vimentin protein (Vimentin/β-actin): 0.53±0.07 vs. 1.19±0.04, TIMP-1 (A value): 0.49±0.06 vs. 0.87±0.02, TGF-β1 (A value): 0.67±0.04 vs. 0.89±0.10, all P < 0.05]. The degree of peritoneal fibrosis gradually increased with the increase of PDF sugar content in rats. Modified Shenling Baizhu San can improve peritoneal fibrosis induced by PDF with different sugar contents in rats, and the mechanism is related to the changes in the expression of fibrosis markers and ECM accumulation.

Immunology of human fibrosis.

Fibrosis, defined by the excess deposition of structural and matricellular proteins in the extracellular space, underlies tissue dysfunction in multiple chronic diseases. Approved antifibrotics have proven modest in efficacy, and the immune compartment remains, for the most part, an untapped therapeutic opportunity. Recent single-cell analyses have interrogated human fibrotic tissues, including immune cells. These studies have revealed a conserved profile of scar-associated macrophages, which localize to the fibrotic niche and interact with mesenchymal cells that produce pathological extracellular matrix. Here we review recent advances in the understanding of the fibrotic microenvironment in human diseases, with a focus on immune cell profiles and functional immune-stromal interactions. We also discuss the key role of the immune system in mediating fibrosis regression and highlight avenues for future study to elucidate potential approaches to targeting inflammatory cells in fibrotic disorders.

Matrix disequilibrium in Alzheimer's disease and conditions that increase Alzheimer's disease risk.

Alzheimer's Disease (AD) and related dementias are a leading cause of death globally and are predicted to increase in prevalence. Despite this expected increase in the prevalence of AD, we have yet to elucidate the causality of the neurodegeneration observed in AD and we lack effective therapeutics to combat the progressive neuronal loss. Throughout the past 30 years, several non-mutually exclusive hypotheses have arisen to explain the causative pathologies in AD: amyloid cascade, hyper-phosphorylated tau accumulation, cholinergic loss, chronic neuroinflammation, oxidative stress, and mitochondrial and cerebrovascular dysfunction. Published studies in this field have also focused on changes in neuronal extracellular matrix (ECM), which is critical to synaptic formation, function, and stability. Two of the greatest non-modifiable risk factors for development of AD (aside from autosomal dominant familial AD gene mutations) are aging and APOE status, and two of the greatest modifiable risk factors for AD and related dementias are untreated major depressive disorder (MDD) and obesity. Indeed, the risk of developing AD doubles for every 5 years after ≥ 65, and the APOE4 allele increases AD risk with the greatest risk in homozygous APOE4 carriers. In this review, we will describe mechanisms by which excess ECM accumulation may contribute to AD pathology and discuss pathological ECM alterations that occur in AD as well as conditions that increase the AD risk. We will discuss the relationship of AD risk factors to chronic central nervous system and peripheral inflammation and detail ECM changes that may follow. In addition, we will discuss recent data our lab has obtained on ECM components and effectors in APOE4/4 and APOE3/3 expressing murine brain lysates, as well as human cerebrospinal fluid (CSF) samples from APOE3 and APOE4 expressing AD individuals. We will describe the principal molecules that function in ECM turnover as well as abnormalities in these molecular systems that have been observed in AD. Finally, we will communicate therapeutic interventions that have the potential to modulate ECM deposition and turnover in vivo.

Ginkgetin Alleviates Intervertebral Disc Degeneration by Inhibiting Apoptosis, Inflammation, and Disturbance of Extracellular Matrix Synthesis and Catabolism via Inactivation of NLRP3 Inflammasome

ABSTRACT Background Apoptosis, inflammation, and the extracellular matrix (ECM) synthesis and catabolism are compromised with intervertebral disc degeneration (IDD). Ginkgetin (GK) has been demonstrated to alleviate several diseases; however, its effect on IDD remains unknown. Methods The nucleus pulposus cells (NPCs) were stimulated with interleukin (IL)-1β to construct the IDD models in vitro. Rats were used for the construction of the IDD models in vivo via the fibrous ring puncture method. The effect and mechanism of GK on IDD were determined by cell counting kit-8 (CCK-8), flow cytometry, western blot, real-time quantitative polymerase chain reaction (RT-qPCR), enzyme‑linked immunosorbent assay (ELISA), hematoxylin and eosin (HE) and safranine O staining, and immunohistochemistry (IHC) assays, respectively. Results GK increased the cell viability and upregulated the expressions of anti-apoptosis and ECM synthesis markers in NPCs treated with IL-1β. GK also decreased apoptosis rate, and downregulated the expressions of proteins related to pro-apoptosis, ECM catabolism, and inflammation in vitro. Mechanically, GK reduced the expression of nucleotide binding oligomeric domain like receptor protein 3 (NLRP3) inflammasome-related proteins. Overexpression of NLRP3 reversed the effect of GK on the proliferation, apoptosis, inflammation, and ECM degradation in IL-1β-induced NPCs. Moreover, GK attenuated the pathological manifestations, inflammation, ECM degradation, and NLRP3 inflammasome expression in IDD rats. Conclusion GK suppressed apoptosis, inflammation, and ECM degradation to alleviate IDD via the inactivation of NLRP3 inflammasome.

Single-cell RNA-seq of heart reveals intercellular communication drivers of myocardial fibrosis in diabetic cardiomyopathy.

Myocardial fibrosis is the characteristic pathology of diabetes-induced cardiomyopathy. Therefore, an in-depth study of cardiac heterogeneity and cell-to-cell interactions can help elucidate the pathogenesis of diabetic myocardial fibrosis and identify treatment targets for the treatment of this disease. In this study, we investigated intercellular communication drivers of myocardial fibrosis in mouse heart with high-fat-diet/streptozotocin-induced diabetes at single-cell resolution. Intercellular and protein-protein interaction networks of fibroblasts and macrophages, endothelial cells, as well as fibroblasts and epicardial cells revealed critical changes in ligand-receptor interactions such as Pdgf(s)-Pdgfra and Efemp1-Egfr, which promote the development of a profibrotic microenvironment during the progression of and confirmed that the specific inhibition of the Pdgfra axis could significantly improve diabetic myocardial fibrosis. We also identified phenotypically distinct Hrchi and Postnhi fibroblast subpopulations associated with pathological extracellular matrix remodeling, of which the Hrchi fibroblasts were found to be the most profibrogenic under diabetic conditions. Finally, we validated the role of the Itgb1 hub gene-mediated intercellular communication drivers of diabetic myocardial fibrosis in Hrchi fibroblasts, and confirmed the results through AAV9-mediated Itgb1 knockdown in the heart of diabetic mice. In summary, cardiac cell mapping provides novel insights into intercellular communication drivers involved in pathological extracellular matrix remodeling during diabetic myocardial fibrosis.

Digital image analysis using video microscopy of human-derived prostate cancer vs normal prostate organoids to assess migratory behavior on extracellular matrix proteins.

The advent of perpetuating living organoids derived from patient tissue is a promising avenue for cancer research but is limited by difficulties with precise characterization. In this brief communication, we demonstrate via time-lapse imaging distinct phenotypes of prostate organoids derived from patient material- without confirmation of cellular identity. We show that organoids derived from histologically normal tissue more readily spread on a physiologic extracellular matrix (ECM) than on pathologic ECM (p<0.0001), while tumor-derived organoids spread equally on either substrate (p=0.2406). This study is an important proof-of-concept to defer precise characterization of organoids and still glean information into disease pathology.

YAP/Smad3 promotes pathological extracellular matrix microenviroment-induced bladder smooth muscle proliferation in bladder fibrosis progression.

Fibrosis is a chronic inflammation process with excess extracellular matrix (ECM) deposition that cannot be reversed. Patients suffer from bladder dysfunction caused by bladder fibrosis. Moreover, the interactive mechanisms between ECM and bladder fibrosis are still obscure. Hence, we assessed the pivotal effect of Yes‐associated protein (YAP) on the proliferation of bladder smooth muscle in fibrosis process. We identified that stiff ECM increased the expression and translocation of YAP in the nucleus of human bladder smooth muscle cell (hBdSMC). Sequencings and proteomics revealed that YAP bound to Smad3 and promoted the proliferation of hBdSMC via MAPK/ERK signaling pathway in stiff ECM. Moreover, CUT and TAG sequencing and dual‐luciferase assays demonstrated that Smad3 inhibited the transcription of JUN. The YAP inhibitor CA3 was used in a partial bladder outlet obstruction (pBOO) rat model. The results showed that CA3 attenuated bladder smooth muscle proliferation. Collectively, YAP binding with Smad3 in the nucleus inhibited the transcription of JUN, and promoted the proliferation of bladder smooth muscle through the MAPK/ERK signaling pathway. The current study identified a novel mechanism of mechanical force induced bladder fibrosis that provided insights in YAP‐associated organ fibrosis.

Fibronectin-EDA accumulates via reduced ubiquitination downstream of Toll-like receptor 9 activation in SSc-ILD fibroblasts.

Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of systemic sclerosis-associated interstitial lung disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-like receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several damage-associated molecular pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform fibronectin-extra domain A (FN-EDA) are prominent in pathological extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.

The Extracellular Matrix: A Key Accomplice of Cancer Stem Cell Migration, Metastasis Formation, and Drug Resistance in PDAC.

Simple SummaryThis review takes the extracellular matrix (ECM) as the starting point, and describes its influence and related mechanisms on the biological behavior of pancreatic ductal carcinoma (PDAC). It focuses on how the ECM regulates cancer stem cells and thus affects the metastasis and drug resistance of PDAC. Finally, current and ongoing treatment strategies for ECM are presented. ECM-related factors and mechanisms are both the focus and difficulty of current therapeutic strategy research, but further exploration is crucial for effectively eradicating the disease and improving patient survival.Pancreatic ductal adenocarcinoma (PDAC) is rich in dense fibrotic stroma that are composed of extracellular matrix (ECM) proteins. A disruption of the balance between ECM synthesis and secretion and the altered expression of matrix remodeling enzymes lead to abnormal ECM dynamics in PDAC. This pathological ECM promotes cancer growth, survival, invasion, and alters the behavior of fibroblasts and immune cells leading to metastasis formation and chemotherapy resistance, which contribute to the high lethality of PDAC. Additionally, recent evidence highlights that ECM, as a major structural component of the tumor microenvironment, is a highly dynamic structure in which ECM proteins establish a physical and biochemical niche for cancer stem cells (CSCs). CSCs are characterized by self-renewal, tumor initiation, and resistance to chemotherapeutics. In this review, we will discuss the effects of the ECM on tumor biological behavior and its molecular impact on the fundamental signaling pathways in PDAC. We will also provide an overview of how the different ECM components are able to modulate CSCs properties and finally discuss the current and ongoing therapeutic strategies targeting the ECM. Given the many challenges facing current targeted therapies for PDAC, a better understanding of molecular events involving the interplay of ECM and CSC will be key in identifying more effective therapeutic strategies to eliminate CSCs and ultimately to improve survival in patients that are suffering from this deadly disease.

Extracellular matrix in intervertebral disc: basic and translational implications.

Intervertebral disc (IVD) degeneration (IVDD) is the most common spinal disorder, which can lead to the symptoms of neck pain or low back pain. In healthy mature IVD tissues, extracellular matrix (ECM) complex possesses favorable biochemical and biomechanical properties, withstanding compression and torsion forces. IVD cells and ECM associate with each other to form a coordinated functional system. IVD cells are the main producers of ECM components, while ECM could modulate the viability and phenotype of IVD cells via direct interactions or indirect regulations. However, with the process of IVDD and ageing, ECM of IVD undergoes content loss and structure degeneration. Moreover, the accumulation of catabolic products may further deteriorate the IVD microenvironment. A better understanding of the physiology and the pathology of ECM within the IVD provides new insight into potential IVD regeneration strategies. Natural ECM components, functional motifs, or mimetic peptides are widely used in IVD repair by not only restoring structural support but also regulating cell fate and tissue microenvironment. Herein, we reviewed recent advances in the involvement of ECM in IVD health and disease, with an emphasis on ECM composition and organization, cell-matrix interactions, pathological ECM degradation, and promising matrix-based biomaterials for IVD regeneration.

Connective Tissue Growth Factor in Idiopathic Pulmonary Fibrosis: Breaking the Bridge.

CTGF is upregulated in patients with idiopathic pulmonary fibrosis (IPF), characterized by the deposition of a pathological extracellular matrix (ECM). Additionally, many omics studies confirmed that aberrant cellular senescence-associated mitochondria dysfunction and metabolic reprogramming had been identified in different IPF lung cells (alveolar epithelial cells, alveolar endothelial cells, fibroblasts, and macrophages). Here, we reviewed the role of the CTGF in IPF lung cells to mediate anomalous senescence-related metabolic mechanisms that support the fibrotic environment in IPF.

Cardiac Fibrosis in the Pressure Overloaded Left and Right Ventricle as a Therapeutic Target.

Myocardial fibrosis is a remodeling process of the extracellular matrix (ECM) following cardiac stress. “Replacement fibrosis” is a term used to describe wound healing in the acute phase of an injury, such as myocardial infarction. In striking contrast, ECM remodeling following chronic pressure overload insidiously develops over time as “reactive fibrosis” leading to diffuse interstitial and perivascular collagen deposition that continuously perturbs the function of the left (L) or the right ventricle (RV). Examples for pressure-overload conditions resulting in reactive fibrosis in the LV are systemic hypertension or aortic stenosis, whereas pulmonary arterial hypertension (PAH) or congenital heart disease with right sided obstructive lesions such as pulmonary stenosis result in RV reactive fibrosis. In-depth phenotyping of cardiac fibrosis has made it increasingly clear that both forms, replacement and reactive fibrosis co-exist in various etiologies of heart failure. While the role of fibrosis in the pathogenesis of RV heart failure needs further assessment, reactive fibrosis in the LV is a pathological hallmark of adverse cardiac remodeling that is correlated with or potentially might even drive both development and progression of heart failure (HF). Further, LV reactive fibrosis predicts adverse outcome in various myocardial diseases and contributes to arrhythmias. The ability to effectively block pathological ECM remodeling of the LV is therefore an important medical need. At a cellular level, the cardiac fibroblast takes center stage in reactive fibrotic remodeling of the heart. Activation and proliferation of endogenous fibroblast populations are the major source of synthesis, secretion, and deposition of collagens in response to a variety of stimuli. Enzymes residing in the ECM are responsible for collagen maturation and cross-linking. Highly cross-linked type I collagen stiffens the ventricles and predominates over more elastic type III collagen in pressure-overloaded conditions. Research has attempted to identify pro-fibrotic drivers causing fibrotic remodeling. Single key factors such as Transforming Growth Factor β (TGFβ) have been described and subsequently targeted to test their usefulness in inhibiting fibrosis in cultured fibroblasts of the ventricles, and in animal models of cardiac fibrosis. More recently, modulation of phenotypic behaviors like inhibition of proliferating fibroblasts has emerged as a strategy to reduce pathogenic cardiac fibroblast numbers in the heart. Some studies targeting LV reactive fibrosis as outlined above have successfully led to improvements of cardiac structure and function in relevant animal models. For the RV, fibrosis research is needed to better understand the evolution and roles of fibrosis in RV failure. RV fibrosis is seen as an integral part of RV remodeling and presents at varying degrees in patients with PAH and animal models replicating the disease of RV afterload. The extent to which ECM remodeling impacts RV function and thus patient survival is less clear. In this review, we describe differences as well as common characteristics and key players in ECM remodeling of the LV vs. the RV in response to pressure overload. We review pre-clinical studies assessing the effect of anti-fibrotic drug candidates on LV and RV function and their premise for clinical testing. Finally, we discuss the mode of action, safety and efficacy of anti-fibrotic drugs currently tested for the treatment of left HF in clinical trials, which might guide development of new approaches to target right heart failure. We touch upon important considerations and knowledge gaps to be addressed for future clinical testing of anti-fibrotic cardiac therapies.

Decellularized normal and cancer tissues as tools for cancer research.

Today it is widely accepted that molecular mechanisms triggering cancer initiate with a genetic modification. However, a genetic alteration providing the aberrant clone with a growing advantage over neighboring cells is not sufficient to develop cancer. Currently, tumors are considered a heterogeneous population of cells and an extracellular matrix (ECM) that make up a characteristic microenvironment. Interactions between tumor cells and cancer microenvironment define cancer progression and therapeutic response. To investigate and clarify the role of ECM in the regulation of cancer cell behavior and response to therapy, the decellularization of ECM, a widely used technique in tissue engineering, has been recently employed to develop 3D culture model of disease. In this review, we briefly explore the different components of healthy and pathological ECM and the methods to obtain and characterize the ECM from native bioptic tissue. Finally, we highlight the most relevant applications of ECM in translational cancer research strategies: decellularized ECM, ECM-hydrogel and 3D bioprinting.

Abstract 122: Pharmacological Inhibition Of Macrophage Infiltration Prevents Myxomatous Valve Degeneration In Marfan Syndrome

Introduction: Myxomatous valve degeneration (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. Currently, there is no medical therapy for the treatment of MVD. MVD in a mouse model of Marfan syndrome (MFS) is characterized leaflet thickening and increased macrophage infiltration, which are reduced with loss of C-C chemokine receptor type 2 (CCR2). However, the specific contributions of macrophages to pathological extracellular matrix (ECM) remodeling and underlying mechanisms are unknown. Hypothesis: Inhibition of macrophage infiltration by a CCR2 inhibitor blocks ECM abnormalities and MVD progression in mitral valves of MFS mice by suppressing the response to cytokine/chemokines. Methods: Mice with the mutation of Fibrillin 1 (Fbn1 C1039G/+ ) recapitulate histopathological features of MFS. Here, we tested the efficacy of a selective CCR2 antagonist RS504393 in the valves of MFS mice in the initiation (1-month-old) and the progression (2-month-old) of MVD, respectively. MFS mice were intraperitoneally injected with RS504393 at 2 mg/kg/d for 30 days. Histological evaluation and immunofluorescence for macrophages and ECM were performed. RNAseq was performed in mitral valves from 2-month old Fbn1 C1039G/+ mice with CCR2 knockout (CCR2 RFP/RFP ). Results: MFS valves revealed ECM abnormalities characterized by collagen fragmentation and proteoglycan accumulation. RS504393 treatment reduced infiltrating macrophages (MHCII+, CCR2+) in myxomatous valves. Remarkably, RS504393 was protective against both the initiation and the progression of MVD, detected by decreased mitral valve thickness and prevention of pathological ECM remodeling in MFS mice. RNAseq data confirmed increased leukocyte activation involved in immune response and abnormal extracellular matrix remodeling in MFS valves. CCR2 deficiency blocked macrophage infiltration and inhibited the response to cytokines in Fbn1 C1039G/+ valves. Conclusions: Our results show that macrophage infiltration is critical for progressive MVD. Moreover, CCR2 inhibition ameliorates MVD progression by preventing immune response. Thus, the CCR2 inhibitor RS504393 is a potential pharmacological candidate to treat MVD in MFS.