Promoting Extracellular Matrix Degradation Research Articles

Abstract 3016: Cytoplasmic And Nuclear Nfatc3 Cooperatively Contributes To Vascular Smooth Muscle Cell Dysfunction And Drives Aortic Aneurysm And Dissection

Background: Aortic aneurysm and aortic dissection (AAD) are fatal diseases resulting in catastrophic aortic rupture and sudden death. The degradation of the extracellular matrix (ECM) and the phenotypic transformation of vascular smooth muscle cells (VSMCs) are crucial pathological mechanisms in AAD. We previously found that macrophage NFATc3 inhibits atherosclerosis and foam cell formation. However, the precise role of VSMC-NFATc3 in AAD remains unknown. Aims: This study investigates the role of NFATc3 in VSMCs in AAD progression and the molecular mechanisms. Methods: To investigate the role of NFATc3 in VSMCs in AAD, we used VSMC-NFATc3-specific knockout and knock-in mice. By establishing mouse models for thoracic AAD (TAAD) and abdominal aortic aneurysm (AAA), we employed techniques such as RNA sequencing, mass spectrometry, and chromatin immunoprecipitation to study the mechanistic role of VSMC-NFATc3 in AAD. Results: Cytoplasmic and nuclear NFATc3 levels are elevated in human and mouse AAD. VSMC-NFATc3 deletion reduces TAAD and AAA progression in mice, contrary to VSMC-NFATc3 overexpression. VSMC-NFATc3 deletion reduces ECM degradation and maintains the VSMC contractile phenotype. Nuclear NFATc3 targets and transcriptionally upregulates MMP9 and MMP2, promoting ECM degradation and AAD development. NFATc3 promotes VSMC phenotypic switching by binding to eukaryotic elongation factor 2 (eEF2) and inhibiting its phosphorylation in the VSMC cytoplasm. Restoring eEF2 reverses the beneficial effects in VSMC-specific NFATc3-knockout mice. Cabamiquine—targets eEF2 and inhibits protein synthesis—inhibited AAD development and progression in VSMC-NFATc3-overexpressing mice. Conclusion: VSMC-NFATc3 promotes VSMC switch and ECM degradation while exacerbating AAD development, making it a novel potential therapeutic target for preventing and treating AAD.

Traditional Chinese Medicine formula Bu-Shen-Huo-Xue-Fang (BSHXF) protects nucleus pulposus cells against the inflammatory and oxidative stress-induced degenerative changes

Degeneration of the intervertebral disc is primarily caused by the loss of nucleus pulposus cells (NPCs) and extracellular matrix (ECM) (IDD). Bu-Shen-Huo-Xue-Fang (BSHXF), a traditional Chinese medicine decoction, has been used to treat IDD in clinical; nevertheless, the active components and underlying molecular mechanisms remain unknown. BSHXF improved IL-1β and H2O2 stimulation-induced injuries on NPCs by promoting cell viability, increasing ECM deposition, inhibiting cell senescence, and decreasing the levels of inflammatory factors. The active ingredients in BSHXF were identified by LC-MS/MS analysis; three active ingredients from the principal drugs, Aucubin, Tanshinol, and Tanshinone II A promoted NPC viability; and Aucubin and Tanshinol promoted NPC viability more. Aucubin and Tanshinol, respectively, improved H2O2 stimulation-induced injuries on NPCs by promoting cell viability, increasing ECM deposition, inhibiting cell senescence, and decreasing the levels of inflammatory factors. The activator of NF-κB and Wnt signaling pathways attenuated Aucubin and Tanshinol's protective effects by promoting ECM degradation and NPC senescence. Aucubin, Tanshinol, and Tanshinone II A were identified as the most potent compounds in BSHXF protection against degenerative changes in NPCs. The NF-κB and Wnt signaling pathways might be involved in the protective effects of Aucubin and Tanshinol against H2O2-induced degenerative changes.

Profibrotic role of the SOX9–MMP10–ECM biosynthesis axis in the tracheal fibrosis after injury and repair

Fibroblast activation and extracellular matrix (ECM) deposition play an important role in the tracheal abnormal repair process and fibrosis. As a transcription factor, SOX9 is involved in fibroblast activation and ECM deposition. However, the mechanism of how SOX9 regulates fibrosis after tracheal injury remains unclear. We investigated the role of SOX9 in TGF-β1-induced fibroblast activation and ECM deposition in rat tracheal fibroblast (RTF) cells. SOX9 overexpression adenovirus (Ad-SOX9) and siRNA were transfected into RTF cells. We found that SOX9 expression was up-regulated in RTF cells treated with TGF-β1. SOX9 overexpression activated fibroblasts and promoted ECM deposition. Silencing SOX9 inhibited cell proliferation, migration, and ECM deposition, induced G2 arrest, and increased apoptosis in RTF cells. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) assays identified MMP10, a matrix metalloproteinase involved in ECM deposition, as a direct target of SOX9, which promotes ECM degradation by increasing MMP10 expression through the Wnt/β-catenin signaling pathway. Furthermore, in vivo, SOX9 knockdown ameliorated granulation proliferation and tracheal fibrosis, as manifested by reduced tracheal stenosis. In conclusion, our findings indicate that SOX9 can drive fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/β-catenin signaling pathway. The SOX9–MMP10–ECM biosynthesis axis plays an important role in tracheal injury and repair. Targeting SOX9 and its downstream target MMP10 may represent a promising therapeutic approach for tracheal fibrosis.

Research hotspots and trends of microRNAs in intervertebral disc degeneration: a comprehensive bibliometric analysis

BackgroundMicroRNAs (miRNAs) are involved in various pathological processes, such as proliferation, growth, and apoptosis, of intervertebral disc (IVD) cells and play an important role in the development of intervertebral disc degeneration (IDD). Although some studies have reported the role of miRNAs in IDD, scientific econometric analysis in this field is not available.ObjectivesWe designed this study to describe the current research trends and potential mechanisms associated with the role of miRNAs in IDD and to provide new ideas for future research in this field.MethodsWe conducted a bibliometric analysis of the publications on the role of miRNAs in IDD included in the Web of Science core collection database to elucidate the current research trends in this field. The potential mechanisms were constructed using the Arrowsmith project.ResultsWe found that the number of miRNAs and IDD-related publications increased over the years. China was the most important contributor to research in this field. The top three institutions in terms of number of articles published were Huazhong University of Science and Technology, Shanghai Jiao Tong University, and Xi’an Jiao Tong University. Shanghai Jiao Tong University had the highest number of citations. Experimental and thermal medicine had the maximum number of documents, and Cell promotion had the most citations. The journal with the most mean times cited per study was Annals of the Rheumatic Diseases. The author Wang K had the highest number of publications, and Wang HQ had the highest number of citations. These two authors made important contributions to the research in this field. The keyword analysis showed that recent studies have focused on miRNAs regulating nucleus pulposus cell apoptosis and proliferation. Moreover, we revealed the potential mechanisms of miRNAs associated with IDD, including miRNAs regulating the extracellular matrix (ECM) degradation, mediating cartilage endplate (CEP) degeneration, and participating in inflammatory responses.ConclusionWe demonstrated the knowledge map of miRNAs and IDD-related research through bibliometric analysis and elucidated the current research status and hotspots in this field. The mechanisms by which miRNAs regulate the apoptosis and proliferation of degenerated IVDs, promote ECM degradation, mediate CEP degeneration, and participate in inflammatory responses should be explored in further studies.

Senescent cell population with ZEB1 transcription factor as its main regulator promotes osteoarthritis in cartilage and meniscus

ObjectivesSingle-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees.MethodsSingle-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy...

2-O-β-d-Glucopyranosyl-4,6-dihydroxybenzaldehyde Isolated from Morus alba (Mulberry) Fruits Suppresses Damage by Regulating Oxidative and Inflammatory Responses in TNF-α-Induced Human Dermal Fibroblasts.

Human skin is composed of three layers, of which the dermis is composed of an extracellular matrix (ECM) comprising collagen, elastin, and other proteins. These proteins are reduced due to skin aging caused by intrinsic and extrinsic factors. Among various internal and external factors related to aging, ultraviolet (UV) radiation is the main cause of photoaging of the skin. UV radiation stimulates DNA damage, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine production such as tumor necrosis factor-alpha (TNF-α), and promotes ECM degradation. Stimulation with ROS and TNF-α upregulates mitogen-activated protein kinases (MAPKs), nuclear factor kappa B (NF-κB), and activator protein 1 (AP-1) transcription factors that induce the expression of the collagenase matrix metalloproteinase-1 (MMP-1). Moreover, TNF-α induces intracellular ROS production and several molecular pathways. Skin aging progresses through various processes and can be prevented through ROS generation and TNF-α inhibition. In our previous study, 2-O-β-d-glucopyranosyl-4,6-dihydroxybenzaldehyde (GDHBA) was isolated from the Morus alba (mulberry) fruits and its inhibitory effect on MMP-1 secretion was revealed. In this study, we focused on the effect of GDHBA on TNF-α-induced human dermal fibroblasts (HDFs). GDHBA (50 μM) inhibited ROS generation (18.8%) and decreased NO (58.4%) and PGE2 levels (53.8%), significantly. Moreover, it decreased MMP-1 secretion (55.3%) and increased pro-collagen type I secretion (207.7%). GDHBA (50 μM) decreased the expression of different MAPKs as per western blotting; p-38: 35.9%; ERK: 47.9%; JNK: 49.5%; c-Jun: 32.1%; NF-κB: 55.9%; and cyclooxygenase-2 (COX-2): 31%. This study elucidated a novel role of GDHBA in protecting against skin inflammation and damage through external stimuli, such as UV radiation.

GSK3β-dependent lysosome biogenesis: An effective pathway to mitigate renal fibrosis with LM49.

Renal fibrosis is an incurable disorder characterised by an imbalance of the extracellular matrix (ECM) favouring excess production over degradation. The identification of actionable pathways and agents that promote ECM degradation to restore ECM homeostasis may help mitigate renal fibrosis. In this study, we identified 5,2′-dibromo-2,4′,5′-trihydroxydiphenylmethanone (LM49), a compound we previously synthesised, as a small-molecule inducer of ECM degradation. LM49 administration efficiently reduced ECM deposition in renal tissue of diabetic nephropathy rats and in transforming growth factor β-treated renal fibroblast cells. LM49 promoted the cytosol-to-nucleus translocation of transcription factor EB (TFEB) to increase lysosome biogenesis, leading to lysosome-based degradation of the ECM. TFEB-mediated lysosome biogenesis was induced by LM49 directly inhibiting the activity of glycogen synthase kinase 3β (GSK3β) rather than mammalian target of rapamycin complex 1. LM49 inhibited GSK3β kinase activity concentration-dependently via competing with ATP. Direct binding between LM49 and GSK3β was confirmed by the bio-layer interferometry assay, cellular thermal shift assay, and drug affinity responsive target stability. A molecular docking and molecular dynamic simulation revealed that LM49 occupied the ATP pocket of GSK3β, which was consistent with the kinase activity assay. In summary, LM49 enhances TFEB-mediated lysosome biogenesis by directly inhibiting GSK3β, leading to the degradation of the ECM by lysosomes. The enhancement of GSK3β-dependent lysosome biogenesis to rebalance the ECM may be a novel strategy to counteract renal fibrosis, and LM49 may be a viable clinical candidate for treating this disorder.

Small extracellular vesicles encapsulating lefty1 mRNA inhibit hepatic fibrosis

Liver fibrosis is the deposition of extracellular matrix (ECM) in the liver caused by persistent chronic injury, which can lead to more serious diseases such as cirrhosis or cancer. Blocking the effect of transforming growth factor β1 (TGF-β1), one of the most important cytokines in liver fibrosis, may be one of the effective ways to inhibit liver fibrosis. As a kind of natural nano-scale vesicles, small extracellular vesicles (sEvs) have displayed excellent delivery vehicle properties. Herein, we prepared hepatic stellate cell (HSC)-derived sEvs loading left-right determination factor 1 (lefty1) mRNA (sEvLs) and we wanted to verify whether they can inhibit fibrosis by blocking the TGF-β1 signaling pathway. The results showed that sEvLs had effective cell uptake and reduced activation of HSCs. Rats that were injected with CCl4 by intraperitoneal injection for 6 weeks exhibited obvious symptoms of liver fibrosis and were treated with systemically administered sEvLs and free sEvs for 4 weeks. Rats injected with olive oil alone served as sham controls. Administration of sEvLs significantly reduced the area of fibrosis compared with free sEvs. We demonstrated that sEvLs inhibited HSCs activation and ECM production, and promote ECM degradation by downregulating α-smooth muscle actin (α-SMA), collagen I, tissue inhibitor of metalloproteinase (TIMP) -1 and upregulating matrix metalloprotease (MMP) -1. In summary, as an endogenous delivery vehicle, sEvs could deliver mRNA to attenuate hepatic fibrosis by blocking the TGF-β/Smad signaling pathway.

Endothelial cells induce degradation of ECM through enhanced secretion of MMP14 carried on extracellular vesicles in venous malformation.

Venous malformations (VMs), featuring localized dilated veins, are the most common developmental vascular anomalies. Aberrantly organized perivascular extracellular matrix (ECM) is one of the prominent pathological hallmarks of VMs, accounting for vascular dysfunction. Although previous studies have revealed various proteins involved in ECM remodeling, the detailed pattern and molecular mechanisms underlying the endothelium-ECMinterplay have not been fully elucidated. Our previous studies revealed drastically elevated extracellular vesicle (EV) secretion in VM lesions. Here, we identified increased EV-carried MMP14 in lesion fluids of VMs and culture medium of TIE2-L914F mutant endothelial cells (ECs), along with stronger ECM degradation. Knockdown of RAB27A, a required regulator for vesicle docking and fusion, led to decreased secretion of EV-carried MMP14 in vitro. Histochemical analysis further demonstrated a highly positive correlation between RAB27A in the endothelium and MMP14 in the perivascular environment. Therefore, our results proved that RAB27A-regulated secretion of EV-MMP14, as a new pattern ofendothelium-ECM interplay, contributed to the development of VMs by promoting ECM degradation.

Degenerative Nucleus Pulposus Cells Derived Exosomes Promoted Cartilage Endplate Cells Apoptosis and Aggravated Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is a complex multifactorial disease model, which pathogenesis has not been fully defined. There are few studies on the information interaction between nucleus pulposus (NP) cells and cartilage endplate (CEP) cells. Exosomes, as a carrier of information communication between cells, have become a research hotspot recently. The purpose of this study was to explore whether degenerative NP cells-derived exosomes promoted CEP cells apoptosis and aggravated IVD degeneration. The degenerative NP cells model was induced by TNFα. NPC exosomes were isolated from the supernatant of the NP cell culture medium. The viability of NP cells and CEP cells was examined by CCK-8 assays. The exosomes were identified by TEM, NTA, and western blot. Extracellular matrix (ECM) metabolism was measured by cellular immunofluorescence and qRT-PCR. Apoptosis was detected by flow cytometry and TUNEL. X-ray and magnetic resonance imaging (MRI), as well as hematoxylin-eosin (H&E), Safranine O-Green staining was adopted to evaluate IVD degeneration grades. TNFα had a minor impact on NPC viability but inhibited ECM synthesis and promoted ECM degradation. TNFα-NPC-Exo had less effect on CEPC proliferation but promoted CEPC apoptosis and affect ECM metabolism, inhibiting aggrecan and collagen II expression and enhancing MMP-3 expression. TNFα-NPC-Exo aggravates IVD degeneration in a rat model and promoted CEPC apoptosis. In conclusion, this study demonstrated that degenerated NPC-exosome could induce apoptosis of CEPCs, inhibit ECM synthesis, and promote ECM degradation. In addition, it was proved that degenerated NPC-exosome aggravates IVD degeneration.

Wenfei Buqi Tongluo Formula Against Bleomycin-Induced Pulmonary Fibrosis by Inhibiting TGF-β/Smad3 Pathway.

Pulmonary fibrosis (PF) is the end stage of various chronic and progressive interstitial lung diseases. TGF-β, a profibrotic cytokine, can promote epithelial–mesenchymal transition (EMT), extracellular matrix (ECM) accumulation, and fibroblast proliferation, which contribute to progressive lung remodeling in PF. The Wenfei Buqi Tongluo (WBT) formula has been certified to be effective in the prevention and treatment of PF in clinical practice and has inhibitory effects on EMT, inflammation, and profibrotic factors. However, the pharmacological mechanisms of WBT against PF need to be further explored. In this study, we first analyzed the chemical components of the WBT formula using the UHPLC/Q-TOF-MS analysis. The potential targets of the identified compounds from WBT were predicted by the network pharmacology, which was confirmed by in vivo and in vitro study. After screening by the PubChem database, we first identified the 36 compounds of WBT and predicted the TGF-β signaling pathway, with ECM degradation as potential mechanism of WBT against PF by the network pharmacology. Furthermore, WBT treatment inhibited the levels of TGF-β and Smad3 phosphorylation and subsequently alleviated EMT and ECM accumulation in the bleomycin-induced mouse model and TGF-β1–induced cell model. These findings indicate that WBT can block the progressive process of PF by inhibiting EMT and promoting ECM degradation via the TGF-β/Smad3 pathway. This study may provide new insights into the molecular mechanism of WBT for the prevention and treatment of PF in the clinical application.

Delayed Administration of Nintedanib Ameliorates Fibrosis Progression in CG-Induced Peritoneal Fibrosis Mouse Model

Background: A multiple-target tyrosine kinase inhibitor, nintedanib, which is approved for treatment of interstitial pulmonary disease, has been demonstrated to have anti-fibrotic activity outside of the lungs. We explored its therapeutic effect in a murine model of peritoneal fibrosis. Methods: Daily intraperitoneal injections of chlorhexidine gluconate (CG) induced peritoneal fibrosis in mice. The effects of delayed administration of nintedanib (given at day 21 after CG injection and then given daily for 14 days) were determined by immunohistochemical staining, ELISA, and immunoblot analysis. Results: Delayed administration of nintedanib significantly inhibited peritoneal fibrosis progression as indicated by decreasing deposition and expression of extracellular matrix (ECM) proteins (fibronectin and type I collagen). Treatment with nintedanib also upregulated MMP-2 and reciprocally downregulated TIMP-2, along with reducing expression of α-SMA, β-vimentin, and two transcription factors (Snail and Twist), and retaining E-cadherin expression. Nintedanib also inhibited co-expression of β-vimentin with Snail or Twist as shown by immunofluorescent staining. Moreover, nintedanib decreased the number of CD31-positive blood vessels and CD31 expression in the injured peritoneum. Moreover, delayed application of nintedanib inhibited the expression of several cytokines/chemokines, including monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6, and infiltration of CD68⁺ macrophages to the injured peritoneum. Finally, nintedanib blocked phosphorylation of STAT3, NF-κB, and Smad3 during the development of peritoneal fibrosis. Conclusions: Delayed administration of nintedanib inhibits progression of peritoneal fibrosis and partially reverses established peritoneal fibrosis by attenuating epithelial-mesenchymal transition, inflammation, and angiogenesis, as well as promoting ECM degradation. We conclude that nintedanib has a therapeutic potential to treat peritoneal fibrosis.

Network Pharmacology and Experimental Assessment to Explore the Pharmacological Mechanism of Qimai Feiluoping Decoction Against Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is one of the pathologic changes in COVID-19 patients in convalescence, and it is also a potential long-term sequela in severe COVID-19 patients. Qimai Feiluoping decoction (QM) is a traditional Chinese medicine formula recommended in the Chinese national medical program for COVID-19 convalescent patients, and PF is one of its indications. Through clinical observation, QM was found to improve the clinical symptoms and pulmonary function and reduce the degree of PF of COVID-19 convalescent patients. To further explore the pharmacological mechanisms and possible active components of QM in anti-PF effect, UHPLC/Q-TOF-MS was used to analyze the composition of the QM extract and the active components that can be absorbed into the blood, leading to the identification of 56 chemical compounds and 10 active components. Then, network pharmacology was used to predict the potential mechanisms and targets of QM; it predicted that QM exerts its anti-PF effects via the regulation of the epithelial–mesenchymal transition (EMT), extracellular matrix (ECM) degradation, and TGF-β signaling pathway. Finally, TGF-β1–induced A549 cells were used to verify and explore the pharmacological effects of QM and found that QM could inhibit the proliferation of TGF-β1–induced A549 cells, attenuate EMT, and promote ECM degradation by inhibiting the TGF-β/Smad3 pathway.

Human Liver Stem Cell Transplantation Alleviates Liver Fibrosis in a Rat Model of CCl4-Induced Liver Fibrosis.

Background and ObjectivesMesenchymal stem cells (MSCs) elicit therapeutic effects against liver fibrosis in animal models. Human liver stem cells (HLSCs) are cells isolated from human liver tissue that have mesenchymal morphology and express MSC markers. HLSCs also possess intrahepatic stem cell properties. We introduce a rat model of liver fibrosis and trans-portal transplantation of HLSC to demonstrate alleviation of liver fibrosis.Methods and ResultsLiver fibrosis was induced by intraperitoneal injection of Carbon tetrachloride (CCl4). Sprague Dawley rats underwent simultaneous partial hepatectomy of the left hepatic lobe and HLSC transplantation via the portal vein. Gross appearance of the liver observed following CCl4 injection showed cholestasis and surface nodularity. Sirius red staining revealed deposition of collagen fibers in the extracellular matrix (ECM). Following HLSC transplantation, human albumin secreting cells were detected by immunohistochemistry in liver specimens. Quantitative measurements of fibrosis area stained by Sirius red were compared between baseline and post-HLSC transplant (1×107 cells) following 10 weeks of CCl4 treatment liver specimens. Fibrosis area (p<0.05), serum markers of liver inflammation and fibrosis (AST, ALT levels and APRI, p<0.05) significantly decreased from baseline after HLSC transplantation. RNA expression in liver tissues revealed significant decrease in tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 expression and increase in hepatocyte growth factor expression following HLSC transplantation (p<0.05).ConclusionsHLSC transplantation effectively reduced the area of liver fibrosis with increased expression of factors promoting ECM degradation. These findings suggest the potential therapeutic role of HLSCs in various liver diseases presenting with liver fibrosis.

Xanthohumol Inhibited Mechanical Stimulation-Induced Articular ECM Degradation by Mediating lncRNA GAS5/miR-27a Axis.

Osteoarthritis (OA) is histopathologically marked by extracellular matrix (ECM) degradation in joint cartilage. Abnormal mechanical stimulation on joint cartilage may result in ECM degeneration and OA development. Matrix metalloproteinase 13 (MMP-13) is one of the catabolic enzymes contributing to the degradation of ECM, and it has become the potential biomarker for the therapeutic management of OA. Xanthohumol (XH), a naturally occurring prenylflavonoid derived from hops and beer, shows the protective activity against OA development. However, the potential mechanisms still need great effort. In this article, mechanical stimulation could significantly increase the expression of MMP-13 and lncRNA GAS5 (GAS5) and promoting ECM degradation. These could be effectively reversed by XH administration. Suppressed expression GAS5 ameliorated mechanical stimulation-induced MMP-13 expression. MiR-27a was predicted and verified as a target of GAS5, and overexpression of miR-27a down regulated the expression of MMP-13. Collectively, XH exhibited protective effects against mechanical stimulation-induced ECM degradation by mediating the GAS5/miR-27a signaling pathway in OA chondrocytes.

Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is the prevailing spine disorder and is associated with musculoskeletal disease. The extracellular matrix (ECM) degradation is an essential hallmark of IDD progression. Circular RNAs (circRNAs), as crucial cellular regulators, participate in multiple pathological processes including IDD. Here, we tried to explore the effect of circITCH on the ECM degradation of IDD and the underlying mechanism. Significantly, the expression levels of circITCH were elevated in the IDD patients’ nucleus pulposus (NP) tissues relative to that of normal cases. CircITCH promoted apoptosis and decreased proliferation of NP cells. CircITCH contributed to ECM degradation, as demonstrated by increased ADAMTS4 and MMP13 expression and decreased aggrecan and collagen II expression. Mechanically, miR-17-5p could be sponged by circITCH and miR-17-5p inhibited ECM degradation by repressing SOX4 in degenerative NP cells. CircITCH could activate Wnt/β-catenin pathway by targeting miR-17-5p/SOX4 signaling. SOX4 overexpression, miR-17-5p inhibitor, or Wnt/β-catenin signaling activator LiCl was able to reverse circITCH knockdown-inhibited apoptosis and ECM degradation, and circITCH knockdown-enhanced proliferation in NP cells. Thus, we conclude that circITCH promotes ECM degradation in IDD by activating Wnt/β-catenin through miR-17-5p/SOX4 signaling. Our finding presents novel insight into the mechanism that circITCH modulates the IDD progression. CircITCH and SOX4 may serve as potential targets for IDD therapy.

Legumain is a predictor of all-cause mortality and potential therapeutic target in acute myocardial infarction

The prognostic impact of extracellular matrix (ECM) modulation and its regulatory mechanism post-acute myocardial infarction (AMI), require further clarification. Herein, we explore the predictive role of legumain—which showed the ability in ECM degradation—in an AMI patient cohort and investigate the underlying mechanisms. A total of 212 AMI patients and 323 healthy controls were enrolled in the study. Moreover, AMI was induced in mice by permanent ligation of the left anterior descending artery and fibroblasts were adopted for mechanism analysis. Based on the cut-off value for the receiver-operating characteristics curve, AMI patients were stratified into low (n = 168) and high (n = 44) plasma legumain concentration (PLG) groups. However, PLG was significantly higher in AMI patients than that in the healthy controls (median 5.9 μg/L [interquartile range: 4.2–9.3 μg/L] vs. median 4.4 μg/L [interquartile range: 3.2–6.1 μg/L], P < 0.001). All-cause mortality was significantly higher in the high PLG group compared to that in the low PLG group (median follow-up period, 39.2 months; 31.8% vs. 12.5%; P = 0.002). Multivariate Cox regression analysis showed that high PLG was associated with increased all-cause mortality after adjusting for clinical confounders (HR = 3.1, 95% confidence interval (CI) = 1.4–7.0, P = 0.005). In accordance with the clinical observations, legumain concentration was also increased in peripheral blood, and infarcted cardiac tissue from experimental AMI mice. Pharmacological blockade of legumain with RR-11a, improved cardiac function, decreased cardiac rupture rate, and attenuated left chamber dilation and wall thinning post-AMI. Hence, plasma legumain concentration is of prognostic value in AMI patients. Moreover, legumain aggravates cardiac remodelling through promoting ECM degradation which occurs, at least partially, via activation of the MMP-2 pathway.

AA9. Macrophage-Derived Netrin 1 Promotes Abdominal Aortic Aneurysms by Activating Matrix Metalloproteinase 3 in Vascular Smooth Muscle Cells

Aberrant extracellular matrix (ECM) fragmentation and sustained inflammation are hallmarks of abdominal aortic aneurysms (AAAs). However, the mechanisms by which these events are coupled, thereby fueling focal vascular damage, are undefined. We have previously demonstrated critical immunoregulatory functions of the neuronal guidance cue netrin 1 in regulating macrophage accumulation in the vasculature. Here, we investigate the novel role of netrin 1 in the pathogenesis of AAA and test the hypothesis that netrin 1 mediates crosstalk between infiltrating macrophages and vascular smooth muscle cells (VSMCs), thereby promoting ECM degradation in AAA. AAA was experimentally induced in apolipoprotein E-deficient (ApoE−/−) C57Bl/6 mice infused with angiotensin II for 28 days. Aneurysm progression and severity were monitored by ultrasound Doppler imaging and elastin staining. A t-distributed stochastic neighbor embedding analysis of data sets from single-cell RNA sequencing of diseased aortas profiled the distribution of netrin 1 among the aortic cellular subpopulations. Conditional deletion of netrin 1 in macrophages was obtained with Ntn1flox/floxLysMCre+/− transgenic mouse models, and AAA was induced. RNA sequencing identified the specific regulation of matrix metalloprotease 3 (MMP3) by netrin 1 in VSMCs. MMP3-deficient mice were exposed to angiotensin II, and the contribution to ECM degradation in response to netrin 1 signaling was analyzed. Netrin 1-induced calcium influx and nuclear translocation of nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) were measured in VSMCs. Netrin 1 expression peaked in human and murine aneurysmal macrophages. Targeted deletion of netrin 1 macrophage protected mice from disease occurrence and complications compared with control mice. Macrophage-derived netrin 1 induced robust intracellular calcium flux necessary for the nuclear localization of the transcription factor NFATc3, leading to transcriptional regulation and sustained catalytic activity of MMP3 by adjacent VSMCs. The deficiency of MMP3 reduced ECM damage and the susceptibility of mice to develop AAA. Here, we established netrin 1 as a major signal that mediates the crosstalk between inflammation and chronic erosion of the ECM in AAA. Strategies to target netrin 1 could be of potential therapeutic interest to reduce the progression and morbidity of AAA.

PO-251 High density fibrillar collagen promotes breast cancer cell invasion by decreasing actin dynamics at invadopodia

IntroductionDuring metastasis, cancer cells migrate through the extracellular matrix (ECM) to reach blood and lymphatic vessels and spread into distant sites. Tumour cell invasiveness is associated with the formation of actin-rich protrusions termed invadopodia. These structures promotes ECM degradation and thereby increase cancer cell ability to cross physical barriers. An increased deposition of dense fibrillar collagen has been associated with higher risks of metastasis. In this regard, high-density fibrillar collagen (HDFC), a condition that mimics the dense microenvironment of breast cancer, was reported to be a potent inducer of invadopodia. Invadopodia formation and activity critically rely on the actin cytoskeleton. To get a better insight into the mechanism by which the desmoplastic stroma stimulates invadopodia formation, we explored actin cytoskeleton dynamics in HDFC-induced breast cancer cell invadopodia.Material and methodsFluorescent Recovery After Photobleaching (FRAP) was used to quantify actin dynamics at invadopodia from cancer cells expressing GFP-fused actin and plated on HDFC-coated slides. In addition, the role of two invadopodial actin-bundling proteins associated with metastatic breast cancer (Fascin and CRP2) was evaluated using a RNAi strategy, and their biochemical activities were compared in in vitro reconstituted assays using TIRF microscopy.Results and discussionsOur FRAP analyses revealed that a gelatin matrix induces the formation of invadopodia with identical actin dynamics. In contrast, HDFC induced the formation of an additionnal population of invadopodia with significantly decreased actin dynamics. The proportion of this stable invadopodia population increased over time and was associated with MT1-MMP accumulation. Knocking down actin bundling proteins, such as fascin or CRP2, induced an increase in the mobile actin fraction and a shift toward the dynamic subpopulation. Such effects were associated with a decrease in MT1-MMP levels at invadopodia, and a reduction in the invasive potential of breast cancer cells. TIRF microscopy analyses revealed that only CRP2 protects actin bundles against cofilin-mediated severing, suggesting only partial redundancy between the two actin regulators.ConclusionIn this study we found that HDFC decreases actin dynamics at invadopodia, which in turn increases tumour cell invasive capacity. In addition, our data suggest that such decrease in actin dynamics involves functionally similar, but not identical, actin bundling factors.

Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation.

In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction 1 . Defective trabeculation leads to embryonic lethality2-4 or non-compaction cardiomyopathy (NCC) 5 . There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium 6 , whereas in chicks, chamber wall thickening occurs before overt trabeculation 7 . In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs 2 . Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch2,8 and Neuregulin (NRG) 4 . Late disruption of the Notch pathway causes NCC 5 . Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice9,10 and the matrix metalloprotease ADAMTS1 promotes trabecular termination 3 , the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.