Source Of Extracellular Matrix Research Articles

Diacetoxy-6-gingerdiol protects the extracellular matrix of nucleus pulposus cells and ameliorates intervertebral disc degeneration by inhibiting the IL-1β-mediated NLRP3 pathway

Intervertebral disc degeneration (IDD) is a common cause of low back pain, causing a huge emotional and economic burden on patients and society. Reduction of nucleus pulposus cells (NPC) and extracellular matrix (ECM) is the main feature of IDD, and NPC is the main source of ECM. Thermal apoptosis is a newly discovered form of cell death in recent years that differs significantly from apoptosis in terms of molecular mechanisms and cellular morphological changes. Diacetoxy-6-gingerdiol(D-6-G), a type of gingerol, has anti-inflammatory and antioxidant effects, but whether it has an inhibitory effect on cellular pyroptosis is not clear. Therefore, in the present study, we investigated the effect of D-6-G on the ECM of the nucleus pulposus oblongata under IL-1β treatment, as well as the mechanism of its effect on NLRP3 inflammasome and cellular focal death. In vitro cellular experiments demonstrated that D-6-G could bind to and inhibit the activity of NLRP3 inflammasome, and interestingly, D-6-G could also inhibit cellular pyroptosis and protect the nucleus pulposusry cellular microenvironment by activating the Nrf2/HO-1 axis. In conclusion, we found that D-6-G could inhibit NLRP3 inflammatory vesicle activity as well as cellular pyroptosis in NPCs and protect the ECM, suggesting the potential of D-6-G to delay IDD.

Involvement of lysophosphatidic acid-LPA1-YAP signaling in healthy and pathological FAPs migration

Skeletal muscle fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) components and is a hallmark of muscular dystrophies. Fibro-adipogenic progenitors (FAPs) are the main source of ECM, and thus have been strongly implicated in fibrogenesis. In skeletal muscle fibrotic models, including muscular dystrophies, FAPs undergo dysregulations in terms of proliferation, differentiation, and apoptosis, however few studies have explored the impact of FAPs migration.Here, we studied fibroblast and FAPs migration and identified lysophosphatidic acid (LPA), a signaling lipid central to skeletal muscle fibrogenesis, as a significant migration inductor. We identified LPA receptor 1 (LPA1) mediated signaling as crucial for this effect through a mechanism dependent on the Hippo pathway, another pathway implicated in fibrosis across diverse tissues. This cross-talk favors the activation of the Yes-associated protein 1 (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), leading to increased expression of fibrosis-associated genes. This study reveals the role of YAP in LPA-mediated fibrotic responses as inhibition of YAP transcriptional coactivator activity hinders LPA-induced migration in fibroblasts and FAPs.Moreover, we found that FAPs derived from the mdx4cv mice, a murine model of Duchenne muscular dystrophy, display a heightened migratory phenotype due to enhanced LPA signaling compared to wild-type FAPs. Remarkably, we found that the inhibition of LPA1 or YAP transcriptional coactivator activity in mdx4cv FAPs reverts this phenotype. In summary, the identified LPA-LPA1-YAP pathway emerges as a critical driver of skeletal muscle FAPs migration and provides insights into potential novel targets to mitigate fibrosis in muscular dystrophies.

A rule-based multiscale model of hepatic stellate cell plasticity: Critical role of the inactivation loop in fibrosis progression.

Hepatic stellate cells (HSC) are the source of extracellular matrix (ECM) whose overproduction leads to fibrosis, a condition that impairs liver functions in chronic liver diseases. Understanding the dynamics of HSCs will provide insights needed to develop new therapeutic approaches. Few models of hepatic fibrosis have been proposed, and none of them include the heterogeneity of HSC phenotypes recently highlighted by single-cell RNA sequencing analyses. Here, we developed rule-based models to study HSC dynamics during fibrosis progression and reversion. We used the Kappa graph rewriting language, for which we used tokens and counters to overcome temporal explosion. HSCs are modeled as agents that present seven physiological cellular states and that interact with (TGFβ1) molecules which regulate HSC activation and the secretion of type I collagen, the main component of the ECM. Simulation studies revealed the critical role of the HSC inactivation process during fibrosis progression and reversion. While inactivation allows elimination of activated HSCs during reversion steps, reactivation loops of inactivated HSCs (iHSCs) are required to sustain fibrosis. Furthermore, we demonstrated the model's sensitivity to (TGFβ1) parameters, suggesting its adaptability to a variety of pathophysiological conditions for which levels of (TGFβ1) production associated with the inflammatory response differ. Using new experimental data from a mouse model of CCl4-induced liver fibrosis, we validated the predicted ECM dynamics. Our model also predicts the accumulation of iHSCs during chronic liver disease. By analyzing RNA sequencing data from patients with non-alcoholic steatohepatitis (NASH) associated with liver fibrosis, we confirmed this accumulation, identifying iHSCs as novel markers of fibrosis progression. Overall, our study provides the first model of HSC dynamics in chronic liver disease that can be used to explore the regulatory role of iHSCs in liver homeostasis. Moreover, our model can also be generalized to fibroblasts during repair and fibrosis in other tissues.

Nitric oxide donor S‐Nitroso‐N‐acetyl penicillamine for hepatic stellate cells to restore quiescence

AbstractLiver fibrosis is a hepatic scar repair response associated with a wide range of liver injuries, which is mediated by an imbalance between extracellular matrix (ECM) synthesis and degradation, leading to massive ECM deposition and disruption of normal liver architecture. Hepatic stellate cells (HSCs) are the main source of ECM during liver fibrosis and are the first identified cell subpopulation associated with liver fibrosis formation. Various current studies on the mechanism and treatment of liver fibrosis require resting‐state HSCs as study subjects. However, spontaneous activation of primary HSCs occurs after 2–3 days of culture after isolation, and it is common that HSCs cell lines gradually differentiate into fibroblasts with culture time. This study provides an induction medium for quiescent HSCs‐containing all‐trans retinoic acid, sodium oleate, and S‐nitroso‐N‐acetyl penicillamine (SNAP)‐and an induction method. The induction method not only maintains the HSCs cell line in a quiescent state but also restores the activated HSCs to a quiescent state. The method has a good induction effect, short induction time, and convenient operation, which is worth being popularized and used in a wide range of laboratories.

Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

Collagen and Beyond: A Comprehensive Comparison of Human ECM Properties Derived from Various Tissue Sources for Regenerative Medicine Applications.

Collagen, along with proteoglycans, glycosaminoglycans, glycoproteins, and various growth factors, forms the extracellular matrix (ECM) and contributes to the complexity and diversity of different tissues. Herein, we compared the physicochemical and biological properties of ECM hydrogels derived from four different human tissues: skin, bone, fat, and birth. Pure human collagen type I hydrogels were used as control. Physical characterization of ECM hydrogels and assessment of cell response of cord-tissue mesenchymal stem cells (CMSCs) were performed. Decellularization efficiency was found to be >90% for all ECM. Hydroxyproline quantification assay showed that collagen content in birth ECM was comparable to collagen control and significantly greater than other sources of ECM. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of γ, β, α1 and α2 collagen chains in all ECMs. Gelation kinetics of ECM hydrogels was significantly slower than collagen control. Compressive modulus of skin ECM was the highest and birth ECM was the lowest. Skin and birth ECM hydrogels were more stable than bone and fat ECM hydrogels. CMSCs encapsulated in birth ECM hydrogels exhibited the highest metabolic activity. Rheological characterization revealed that all ECM-derived inks exhibited shear thinning properties, and skin-derived ECM inks were most suitable for extrusion-based bioprinting for the concentration and printing conditions used in this study. Overall, results demonstrate that the physicochemical and biological properties of ECM hydrogels vary significantly depending on the tissue source. Therefore, careful selection of tissue source is important for development of ECM-based biomimetic tissue constructs for regenerative medicine applications.

More than missing neurons: Intestinal fibrosis in Hirschsprung disease

More than missing neurons: Intestinal fibrosis in Hirschsprung disease

Diverse effector and regulatory functions of fibro/adipogenic progenitors during skeletal muscle fibrosis in muscular dystrophy

Fibrosis is a prominent pathological feature of skeletal muscle in Duchenne muscular dystrophy (DMD). The commonly used disease mouse model, mdx 5cv , displays progressive fibrosis in the diaphragm but not limb muscles. We use single-cell RNA sequencing to determine the cellular expression of the genes involved in extracellular matrix (ECM) production and degradation in the mdx 5cv diaphragm and quadriceps. We find that fibro/adipogenic progenitors (FAPs) are not only the primary source of ECM but also the predominant cells that express important ECM regulatory genes, including Ccn2, Ltbp4, Mmp2, Mmp14, Timp1, Timp2, and Loxs. The effector and regulatory functions are exerted by diverse FAP clusters which are different between diaphragm and quadriceps, indicating their activation by different tissue microenvironments. FAPs are more abundant in diaphragm than in quadriceps. Our findings suggest that the development of anti-fibrotic therapy for DMD should target not only the ECM production but also the pro-fibrogenic regulatory functions of FAPs.

Personalized Scaffolds for Diabetic Foot Ulcer Healing Using Extracellular Matrix from Induced Pluripotent Stem-Reprogrammed Patient Cells.

Diabetic foot ulcers (DFU) are chronic wounds sustained by pathological fibroblasts and aberrant extracellular matrix (ECM). Porous collagen-based scaffolds (CS) have shown clinical promise for treating DFUs but may benefit from functional enhancements. Our previous work showed fibroblasts differentiated from induced pluripotent stem cells are an effective source of new ECM mimicking fetal matrix, which notably promotes scar-free healing. Likewise, functionalizing CS with this rejuvenated ECM showed potential for DFU healing. Here, we demonstrate for the first time an approach to DFU healing using biopsied cells from DFU patients, reprogramming those cells, and functionalizing CS with patient-specific ECM as a personalized acellular tissue engineered scaffold. We took a two-pronged approach: 1) direct ECM blending into scaffold fabrication; and 2) seeding scaffolds with reprogrammed fibroblasts for ECM deposition followed by decellularization. The decellularization approach reduced cell number requirements and maintained naturally deposited ECM proteins. Both approaches showed enhanced ECM deposition from DFU fibroblasts. Decellularized scaffolds additionally enhanced glycosaminoglycan deposition and subsequent vascularization. Finally, reprogrammed ECM scaffolds from patient-matched DFU fibroblasts outperformed those from healthy fibroblasts in several metrics, suggesting ECM is in fact able to redirect resident pathological fibroblasts in DFUs towards healing, and a patient-specific ECM signature may be beneficial.

Modification of lysine deacetylation regulates curcumol-induced necroptosis through autophagy in hepatic stellate cells.

The excessive deposition of extracellular matrix (ECM) is the main characteristic of liver fibrosis, and hepatic stellate cells (HSCs) are the main source of ECM. The removal of activated HSCs has a reversal effect on liver fibrosis. Western blot and MTT analysis indicated that curcumol could relieve hepatic fibrosis by promoting HSCs receptor-interacting protein kinase 1/3 (RIP1/RIP3)-dependent necroptosis. Importantly, autophagy flow was monitored by constructing the mRFP-GFP-LC3 plasmid, and it was found that curcumol cleared activated HSCs in a necroptosis manner that was dependent on autophagy. Our study suggested that the activation of necrosome formed by RIP1 and RIP3 depended on Atg5, and that autophagosomes were also necessary for curcumol-induced necroptosis. Furthermore, microscale thermophoresis and co-immunoprecipitation assay results proved that curcumol could target Sirt1 to regulate autophagy by reducing the acetylation level of Atg5. The HSCs-specific silencing of Sirt1 exacerbated CCl4 -induced liver fibrosis in mice. The deacetylation of Atg5 not only accelerated the accumulation of autophagosomes but also enhanced the interaction between Atg5 and RIP1/RIP3 to induce necroptosis. Overall, our study indicated that curcumol could activate Sirt1 to promote Atg5 deacetylation and enhanced its protein-protein interaction function, thereby inducing autophagy and promoting the necroptosis of HSCs to reduce liver fibrosis.

Cysteine-rich protein 2 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in mice

BackgroundAbdominal aortic aneurysm (AAA) is a relatively common and often fatal condition. A major histopathological hallmark of AAA is the severe degeneration of aortic media with loss of vascular smooth muscle cells (VSMCs), which are the main source of extracellular matrix (ECM) proteins. VSMCs and ECM homeostasis are essential in maintaining structural integrity of the aorta. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed protein; however, the role of CRP2 in AAA formation is unclear.MethodsTo investigate the function of CRP2 in AAA formation, mice deficient in Apoe (Apoe−/−) or both CRP2 (gene name Csrp2) and Apoe (Csrp2−/−Apoe−/−) were subjected to an angiotensin II (Ang II) infusion model of AAA formation. Aortas were harvested at different time points and histological analysis was performed. Primary VSMCs were generated from Apoe−/− and Csrp2−/−Apoe−/− mouse aortas for in vitro mechanistic studies.ResultsLoss of CRP2 attenuated Ang II-induced AAA incidence and severity, accompanied by preserved smooth muscle α-actin expression and reduced elastin degradation, matrix metalloproteinase 2 (MMP2) activity, deposition of collagen, particularly collagen III (Col III), aortic tensile strength, and blood pressure. CRP2 deficiency decreased the baseline MMP2 and Col III expression in VSMCs and mitigated Ang II-induced increases of MMP2 and Col III via blunting Erk1/2 signaling. Rescue experiments were performed by reintroducing CRP2 into Csrp2−/−Apoe−/− VSMCs restored Ang II-induced Erk1/2 activation, MMP2 expression and activity, and Col III levels.ConclusionsOur results indicate that in response to Ang II stimulation, CRP2 deficiency maintains aortic VSMC density, ECM homeostasis, and structural integrity through Erk1/2–Col III and MMP2 axis and reduces AAA formation. Thus, targeting CRP2 provides a potential therapeutic strategy for AAA.

Bioinks Enriched with ECM Components Obtained by Supercritical Extraction.

Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell’s own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO2 protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extract’s composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.

Optimal delipidation solvent to secure extracellular matrix from human perirenal adipose tissue.

The objective of this study was to select the optimal delipidation solvent for preparation of human perirenal adipose tissue-derived extracellular matrix (ECM). Human perirenal adipose tissue can be obtained in large amounts during surgery, and it can be an alternative source of human ECM. Delipidation is an essential procedure for the ECM preparation, because lipid strongly inhibits regeneration of target tissue. Isopropanol has been widely used as a delipidation solvent for adipose tissue. However, because adipose tissue is mostly composed of nonpolar lipid, a nonpolar solvent might be more effective for delipidation. We evaluated the delipidation efficiency of acetone, chloroform, methanol, ether, ethanol, isopropanol, water, chloroform/methanol, ethanol/heptane, ether/methanol, hexane/ethanol, and butanol/methanol solvents for ECM extraction from human perirenal adipose tissue. Among them, acetone-treated adipose tissue showed the greatest delipidation efficiency (93.05%), significantly lower residual DNA content, and the greatest residual collagen concentration (42.49 ± 0.05 μg/g). In addition, acetone-treated tissue also had well-preserved ultrastructure with high porosity and significantly low in vitro cytotoxicity. These results suggested that acetone may be an optimal delipidation solvent for extraction of ECM from human perirenal adipose tissue.

Initiation of hepatic stellate cell activation extends into chronic liver disease

Activated hepatic stellate cells (aHSC) are the main source of extra cellular matrix in liver fibrosis. Activation is classically divided in two phases: initiation and perpetuation. Currently, HSC-based therapeutic candidates largely focus on targeting the aHSCs in the perpetuation phase. However, the importance of HSC initiation during chronic liver disease (CLD) remains unclear. Here, we identified transcriptional programs of initiating and activated HSCs by RNA sequencing, using in vitro and in vivo mouse models of fibrosis. Importantly, we show that both programs are active in HSCs during murine and human CLD. In human cirrhotic livers, scar associated mesenchymal cells employ both transcriptional programs at the single cell level. Our results indicate that the transcriptional programs that drive the initiation of HSCs are still active in humans suffering from CLD. We conclude that molecules involved in the initiation of HSC activation, or in the maintenance of aHSCs can be considered equally important in the search for druggable targets of chronic liver disease.

RSjP40 Inhibited the Activity of Collagen Type I Promoter via Ets-1 in HSCs.

Liver fibrosis is a severe disease characterized by excessive deposition of extracellular matrix (ECM) components in the liver. Activated hepatic stellate cells (HSCs) are a major source of ECM and a key regulator of liver fibrosis. Collagen type I alpha I (COL1A1) is one of the main components of ECM and is a major component in fibrotic tissues. Previously, we demonstrated that soluble egg antigen from Schistosoma japonicum could inhibit the expression of COL1A1 in activated HSCs. In addition, studies have found that Ets proto-oncogene 1 (Ets-1) suppresses the production of ECM by down-regulating matrix related genes such as COL1A1 induced by transforming growth factor β, and ultimately inhibits liver fibrosis. In this study, the major aim was to investigate the effect and mechanism of Ets-1 on inhibiting COL1A1 gene promoter activity in HSCs by recombinant Schistosoma japonicum protein P40 (rSjP40). We observed the rSjP40 inhibited the expression of COL1A1 by inhibiting the activity of the COL1A1 promoter, and the core region of rSjP40 acting on COL1A1 promoter was located at -1,722/-1,592. In addition, we also demonstrated that rSjP40 could promote the expression of Ets-1, and Ets-1 has a negative regulation effect on the COL1A1 promoter in human LX-2 cells. These data suggest that rSjP40 might inhibit the activity of COL1A1 promoter and inhibit the activation of HSCs by increasing the expression of transcription factor Ets-1, which will provide a new experimental basis for the prevention and treatment of liver fibrosis.

DNA Methylation of Fibroblast Phenotypes and Contributions to Lung Fibrosis

Fibroblasts are an integral part of connective tissue and play a crucial role in developing and modulating the structural framework of tissues by acting as the primary source of extracellular matrix (ECM). A precise definition of the fibroblast remains elusive. Lung fibroblasts orchestrate the assembly and turnover of ECM to facilitate gas exchange alongside performing immune functions including the secretion of bioactive molecules and antigen presentation. DNA methylation is the covalent attachment of a methyl group to primarily cytosines within DNA. DNA methylation contributes to diverse cellular phenotypes from the same underlying genetic sequence, with DNA methylation profiles providing a memory of cellular origin. The lung fibroblast population is increasingly viewed as heterogeneous with between 6 and 11 mesenchymal populations identified across health and lung disease to date. DNA methylation has been associated with different lung fibroblast populations in health and with alterations in lung disease, but to varying extents. In this review, we will discuss lung fibroblast heterogeneity and the evidence for a contribution from DNA methylation to defining cell populations and alterations in disease.

Inhibition of longevity regulator PAPP-A modulates tissue homeostasis via restraint of mesenchymal stromal cells.

Pregnancy‐associated plasma protein‐A (PAPP‐A) is a secreted metalloprotease that increases insulin‐like growth factor (IGF) availability by cleaving IGF‐binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP‐A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP‐A’s role, we developed a PAPP‐A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti‐PAPP‐A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP‐A in bone marrow and primary responders to PAPP‐A inhibition. Mice treated with anti‐PAPP‐A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM‐producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP‐A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC‐like cells are the major source of PAPP‐A in other tissues also, suggesting that reduced MSC‐like cell activity drives the system‐wide reduction in ECM gene expression due to PAPP‐A inhibition. Dysregulated ECM production is associated with aging and drives age‐related diseases, and thus, this may be a mechanism by which PAPP‐A deficiency enhances longevity.

Cell-free scaffolds functionalized with bionic cartilage acellular matrix microspheres to enhance the microfracture treatment of articular cartilage defects.

Microfracture surgery remains the most popular treatment for articular cartilage lesions in the clinic, but often leads to the formation of inferior fibrocartilage tissue and damage to subchondral bone. To overcome these problems, extracellular matrix (ECM) scaffolds derived from decellularized natural cartilaginous tissues were introduced and showed excellent biological properties to direct the differentiation of bone marrow stem cells. However, besides the limited allogenic/allogenic supply and the risk of disease transfer from xenogeneic tissues, the effectiveness of ECM scaffolds always varied with a high variability of natural tissue quality. In this study, we developed composite scaffolds functionalized with a cell-derived ECM source, namely, bionic cartilage acellular matrix microspheres (BCAMMs), that support the chondrogenic differentiation of bone marrow cells released from microfracture. The scaffolds with BCAMMs at different developmental stages were investigated in articular cartilage regeneration and subchondral bone repair. Compared to microfracture, the addition of cell-free BCAMM scaffolds has demonstrated a great improvement of regenerated cartilage tissue quality in a rabbit model as characterized by a semi-quantitative analysis of cells, histology and biochemical assays as well as micro-CT images. Moreover, the variation in ECM properties was found to significantly affect the cartilage regeneration, highlighting the challenges of homogenous scaffolds in working with microfracture. Together, our results demonstrate that the biofunctionalized BCAMM scaffold with cell-derived ECM shows great potential to combine with microfracture for clinical translation to repair cartilage defects.

Attenuation of fibrogenesis by PDE4 inhibitor via decreased cytoskeleton remodeling, activation and contractility of hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix (ECM) proteins that occurs due to chronic liver injury. Activated hepatic stellate cells (HSCs) are main source of ECM in the liver. Rho‐associated kinase (Rho‐kinase/ROCK) promotes the assembly of focal adhesions and actin stress fibers by regulating the organization of the cytoskeleton in HSCs. Exchange protein directly regulated by cAMP 1 (EPAC1) attenuated Rho kinase signaling in HSCs and alleviated the development of liver fibrosis. We previously showed that cAMP degrading phosphodiesterase 4 (PDE4) enzymes play a critical role in the pathogenesis of liver fibrosis suggesting that hepatic cAMP signaling is compromised during fibrogenesis. Based on these observations, we hypothesize that preserving cAMP/EPAC1 signaling by inhibiting PDE4 will attenuate HSC activation and fibrotic signaling.C57Bl/6 mice were subjected to repeated carbon tetrachloride (CCl4) injection for 4 weeks to estabilesh liver fibrosis. One group of mice received PDE4 inhibitor, Rolipram (targeted to the liver) twice a week. To examine the direct effect of EPAC1/PDE4 pathway on HSCs, in vitro studies using human HSC cell line (LX2) were also performed. Activation of LX2 HSCs was achieved by using recombinant TGFβ1. Liver tissue proteomic analysis was performed. Pathway enrichment analysis was done using Clarivate analytics software, MetaCore. Sirius Red staining and hydroxyproline (HYP) assay was performed to assess collagen deposition. Real time qPCR and Western blot were performed to validate the findings from proteomic analysis. Statistical analysis was performed using ANOVA.CCl4 administration resulted in a significant accumulation of collagen confirmed by Sirius red staining and hydroxyproline (HYP) content in mice. Importantly, Rolipram prevented HYP accumulation and the expression of genes involved in collagen synthesis (Serpinh1) and crosslinking (Loxl1 and Lox). Additionally, Smad3 (central modulator of canonical TGFβ1 signaling) activation and mRNA levels of Mmp2 and Timp2 were markedly decreased by Rolipram. Pathway enrichment analysis of proteomic data identified several fibrotic pathways impacted by Rolipram, including HSC activation, integrin signaling and cytoskeleton remodeling regulated by Rho‐kinase. Western blot analyses confirmed that Rolipram significantly decreased phosphorylation of myosin light chain (MLC), target of Rho‐associated protein kinase. The effect of PDE4 inhibition on TGFβ1 inducible Smad3 and MLC phosphorylation was recapitulated in LX2 cells by using Rolipram and EPAC1 specific agonist. Moreover, Rolipram decreased Endothelin 1 (ET1) levels both in vivo and in vitro LX2 cells indicating a reduced contractility of HSCs. In LX2 cells TGFβ1 inducible nuclear translocation of Yes‐associated protein (YAP) was also inhibited by Rolipram suggesting that PDE4 inhibition promotes the crosstalk between TGFβ1 and YAP pathway to repress TGFβ1 signaling.These results demonstrate that PDE4 inhibition attenuates liver fibrosis by affecting multiple pathways involved in HSC activation and perpetuation of fibrosis.Support or Funding InformationNIH P20GM113226