Key Extracellular Matrix Components Research Articles

Tyrosinase-crosslinked, tissue adhesive and biomimetic alginate sulfate hydrogels for cartilage repair

The native cartilage extracellular matrix (ECM) is enriched in sulfated glycosaminoglycans with important roles in the signaling and phenotype of resident chondrocytes. Recapitulating the key ECM components within engineered tissues through biomimicking strategies has potential to improve the regenerative capacity of encapsulated cells and lead to better clinical outcome. Here, we developed a double-modified, biomimetic and tissue adhesive hydrogel for cartilage engineering. We demonstrated sequential modification of alginate with first sulfate moieties to mimic the high glycosaminoglycan content of native cartilage and then tyramine moieties to allow in situ enzymatic crosslinking with tyrosinase under physiological conditions. Tyrosinase-crosslinked alginate sulfate tyramine (ASTA) hydrogels showed strong adhesion to native cartilage tissue with higher bond strength compared to alginate tyramine (AlgTA). Both ASTA and AlgTA hydrogels supported the viability of encapsulated bovine chondrocytes and induced a strong increase in the expression of chondrogenic genes such as collagen 2, aggrecan and Sox9. Aggrecan and Sox9 gene expression of chondrocytes in ASTA hydrogels were significantly higher than those in AlgTA. Chondrocytes in both ASTA and AlgTA hydrogels showed potent deposition of cartilage matrix components collagen 2 and aggrecan after 3 weeks of culture whereas a decreased collagen 1 deposition was observed in the sulfated hydrogels. ASTA and AlgTA hydrogels with encapsulated human chondrocytes showed in vivo stability as well as cartilage matrix deposition upon subcutaneous implantation into mice for 4 weeks. Our data is the first demonstration of a double-modified alginate with sulfation and tyramination that allows in situ enzymatic crosslinking, strong adhesion to native cartilage and chondrogenic re-differentiation.

Implications of Skeletal Muscle Extracellular Matrix Remodeling in Metabolic Disorders: Diabetes Perspective.

The extracellular matrix (ECM) provides a scaffold for cells, controlling biological processes and providing structural as well as mechanical support to surrounding cells. Disruption of ECM homeostasis results in several pathological conditions. Skeletal muscle ECM is a complex network comprising collagens, proteoglycans, glycoproteins, and elastin. Recent therapeutic approaches targeting ECM remodeling have been extensively deliberated. Various ECM components are typically found to be augmented in the skeletal muscle of obese and/or diabetic humans. Skeletal muscle ECM remodeling is thought to be a feature of the pathogenic milieu allied with metabolic dysregulation, obesity, and eventual diabetes. This narrative review explores the current understanding of key components of skeletal muscle ECM and their specific roles in the regulation of metabolic diseases. Additionally, we discuss muscle-specific integrins and their role in the regulation of insulin sensitivity. A better understanding of the importance of skeletal muscle ECM remodeling, integrin signaling, and other factors that regulate insulin activity may help in the development of novel therapeutics for managing diabetes and other metabolic disorders.

Coronary artery tortuosity: a narrative review.

Coronary artery tortuosity (CAT) is a prevalent angiographic finding commonly associated with aging, hypertension, atherosclerosis and other conditions. Preliminary evidence suggests that degradation of elastin, a key component of extracellular matrix in the vascular wall, may be responsible for the development of CAT. The clinical significance of CAT should be considered in several aspects. First, coronary flow alteration associated with CAT may result in myocardial ischemia owing to reduced perfusion pressure distal to the tortuous segment. Second, increased and oscillatory shear stress in the tortuous vessel may promote atherosclerotic plaque formation and acute coronary syndrome. Third, as one of the criteria for coronary lesion complexity, the presence of severe tortuosity proximal to the culprit lesion may pose a challenge to wiring and stent or balloon delivery, thereby increasing the risk of periprocedural complications. Last, the presence of CAT may serve as a diagnostic clue of concurrent vasculopathy such as fibromuscular dysplasia or spontaneous coronary artery dissection. In general, CAT represents a benign entity that does not require specific treatment or intervention. Further research is warranted to elucidate the pathogenesis and prognostic effect of coronary tortuosity.

Cancer Metastasis: The Role of the Extracellular Matrix and the Heparan Sulfate Proteoglycan Perlecan.

Cancer metastasis is the dissemination of tumor cells to new sites, resulting in the formation of secondary tumors. This process is complex and is spatially and temporally regulated by intrinsic and extrinsic factors. One important extrinsic factor is the extracellular matrix, the non-cellular component of tissues. Heparan sulfate proteoglycans (HSPGs) are constituents of the extracellular matrix, and through their heparan sulfate chains and protein core, modulate multiple events that occur during the metastatic cascade. This review will provide an overview of the role of the extracellular matrix in the events that occur during cancer metastasis, primarily focusing on perlecan. Perlecan, a basement membrane HSPG is a key component of the vascular extracellular matrix and is commonly associated with events that occur during the metastatic cascade. Its contradictory role in these events will be discussed and we will highlight the recent advances in cancer therapies that target HSPGs and their modifying enzymes.

Expression of Heparanase in uterosacral ligaments of women with or without uterine prolapse

ObjectivePelvic organ prolapse (POP) is a global health problem for which the pathophysiological mechanism remains unclear. The loss of extracellular matrix proteins is considered an important molecular basis for this pathology. Heparanase is a heparin sulfate degrading endoglycosidase that has an important role in various biological processes and is a key component of extracellular matrix. The aim of this study was to compare expression of Heparanase in connective tissue of uterosacral ligaments in women with or without uterine prolapse. Study designThirty-nine women who underwent hysterectomy for benign reasons were enrolled in the study. Twenty-three women with uterine prolapse (stage ≥3) who underwent vaginal hysterectomy (VH) - POP group, were compared to sixteen women without uterine prolapse who underwent abdominal hysterectomy (stage <2) - control group. Uterosacral ligaments (USL) biopsies were obtained from all uterine specimens near their origin. All tissue samples were analyzed by immunohistochemistry and tested for the presence of Heparanase using antiheparanse antibody 733. ResultsHeparanse positive staining was more common in the connective tissue of uterosacral ligaments in women with uterine prolapse. Positive staining was seen in 17/23 (73.9 %) women with uterine prolapse compared to 4/16 (25 %) without uterine prolapse (p = 0.003). On multivariate logistic regression analysis, positive staining displayed a trend for an independent association with POP, after controlling for menopausal status and parity (OR 13.57, 95 %CI 0.82–224.4, p = 0.06). ConclusionHeparanase expression is more common in the connective tissue of uterosacral ligaments in women with uterine prolapse compared to women with no prolapse.

Maintenance and Acceleration of Pericellular Matrix Formation within 3D Cartilage Cell Culture Models.

In native articular cartilage, chondrocytes are surrounded by a thin pericellular matrix (PCM) forming chondrons. The PCM is exclusively rich in type VI collagen. The retention of the PCM has a significant influence on the metabolic activity of the chondrocytes. This study investigated the influence of 2 hydrogels (hyaluronic acid [HA] and agarose) and 2 media compositions (basal and chondrogenic) on the preservation/maintenance and acceleration of PCM formation over a 21-day time course. Different combinations of chondrocytes, chondrons, and mesenchymal stem cells (MSCs) were studied. Both hydrogels preserved chondrons PCM from day 1 up to 21-day culture regardless of media composition. Type VI collagen immunostaining of the cultured chondrons appeared both dense and homogenous. The presence of MSCs did not influence this outcome. At day 1, type VI collagen was not present around chondrocytes alone or their co-culture with MSCs. In the HA hydrogel, type VI collagen was located within the PCM after 7 days in both mono- and co-cultures. In the agarose hydrogel, collagen VI was located within the PCM at 7 days (co-cultures) and 14 days (monocultures). In both hydrogel systems, chondrogenic media enhanced the production of key extracellular matrix components in both mono- and co-cultures in comparison to basal media (11.5% and 14% more in glycosaminoglycans and type II collagen for chondrocytes samples at day 21 culture samples, respectively). However, the media types did not enhance type VI collagen synthesis. Altogether, a 3D chondrogenic hydrogel environment is the primary condition for maintenance and acceleration of PCM formation.

Decellularized extracellular matrix gelloids support mesenchymal stem cell growth and function in vitro.

Volumetric muscle loss (VML) injuries are irrecoverable due to a significant loss of regenerative elements, persistent inflammation, extensive fibrosis, and functional impairment. When used in isolation, previous stem cell and biomaterial-based therapies have failed to regenerate skeletal muscle at clinically relevant levels. The extracellular matrix (ECM) microenvironment is crucial for the viability, stemness, and differentiation of stem cells. Decellularized-ECM (D-ECM) scaffolds are at the forefront of ongoing research to develop a viable therapy for VML. Due to the retention of key ECM components, D-ECM scaffolds provide an excellent substrate for the adhesion and migration of several cell types. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and are currently under investigation in clinical trials for a wide range of medical conditions. However, a major limitation to the use of MSCs in clinical applications is their poor viability at the site of transplantation. In this study, we have fabricated spherical scaffolds composed of gelatin and skeletal muscle D-ECM for the adhesion and delivery of MSCs to the site of VML injury. These spherical scaffolds termed "gelloids" supported MSC survival, expansion, trophic factor secretion, immunomodulation, and myogenic protein expression in vitro. Future studies would determine the therapeutic efficacy of this approach in a murine model of VML injury.

CD24 expression indicates healthier phenotype and less tendency of cellular senescence in human nucleus pulposus cells

Identification of specific cell markers is crucial for recognizing functionally healthy nucleus pulposus (NP) cells. The objective of this study was to investigate the role of CD24 expression in adult human NP cells. Cells were retrieved from NP tissues of 20 patients (aged 17–44) operated on for lumbar disc herniation. Based on CD24 expression, NP cells were separated by sorting and then used to examine phenotypic behavior, the effects of culture conditions and cellular senescence pathway related proteins. CD24 expression was positive in 35.5 ± 3.7% (range 9.1–65.2%) of NP cells. Consistently, normoxic expansion and serial passages in monolayers decreased percentage positivity for CD24 in NP cells. CD24– NP cells showed a markedly decreased GSK-3β activity and increased mitogen-activated protein kinase phosphorylation accompanying by an increased β-catenin expression. Higher levels of matrix metalloproteinases, as well as lower levels of ACAN and COL2 in CD24– cells, indicated the breakdown and reduced the formation of key extracellular matrix components. CD24+ NP cells presented a more favorable phenotype while CD24– cells showed a more prominent cellular senescence fate. CD24 in NP cells may be a surrogate marker of healthy cells, in the cell-based therapeutic treatment of degenerative disc disorders.

Fibronectin regulates the self-renewal of rabbit limbal epithelial stem cells by stimulating the Wnt11/Fzd7/ROCK non-canonical Wnt pathway

Microenvironmental factors regulate stem cell fate. Fibronectin (FN), a key extracellular matrix component of the microenvironment, has been linked to various stem cell behaviors. However, how FN controls self-renewal, proliferation, and homeostasis of limbal stem cells remains unclear. Our study investigated the roles of FN in the self-renewal of rabbit limbal epithelial stem cells (rLESCs) by assessing rLESC proliferation and stemness in the presence and absence of FN. We further examined the effect of FN on non-canonical Wnt signaling during rLESC proliferation by evaluating the expression of cell cycle regulators. We found that rLESC proliferation increased after FN treatment and that 12.5 μg/cm2 FN maintained rLESC stemness. FN facilitated rLESC self-renewal by promoting Wnt11 and Fzd7 interaction. Furthermore, FN modulated cell cycle regulators to enhance rLESC proliferation via the upregulation of ROCK1 and ROCK2. Our study provides new insights into the mechanism through which FN regulates the self-renewal of rLESCs; specifically, this occurs via stimulation of the Wnt11/Fzd7/ROCK non-canonical Wnt pathway. The roles of FN in the self-renewal of limbal epithelial stem cells should be further investigated for the potential treatment of limbal deficiency.

MicroRNA 27B-3P: role in extracellular matrix regulation in osteoarthritis synovial fibroblasts

Purpose: Synovial fibrosis is a key pathological event that contributes towards joint destruction, pain and stiffness associated with osteoarthritis (OA). Fibrosis is associated with the accumulation of excessive amount of extra-cellular matrix (ECM) resulting in tissue damage. Currently, endogenous mechanisms that play a key role in the initiation and progression of synovial fibrosis during OA are not well understood. We have previously shown that the expression of a small non-coding RNA called microRNA27b-3p (miR-27b-3p) is elevated in the synovial fluid of late-stage (Kellgren-Lawrence, KL score 3 & 4) compared to early-stage (KL score 1 & 2) radiographic knee OA patients. Furthermore, we also observed that miR-27b-3p is abundantly expressed in OA synovium (Y-H. Li, G. Tavallaee, et al. 2016). Since the role of miR-27b-3p in synovial pathology associated with OA has never been studied; we investigated if miR-27b-3p plays any role in ECM regulation in the OA synovium. Methods: Synovial tissues were obtained from OA patients undergoing total knee replacement with informed written consent. Primary OA synovial fibroblasts (major cell types present in the synovium) were isolated, cultured and transfected with miR-27b-3p mimic/inhibitor and subjected to qPCR arrays specific for ECM markers. Results obtained from arrays were further validated using quantitative-PCR analysis. For in vivo studies, mice were also subjected to DMM model of OA to determine the expression of miR-27b-3p during the time course of OA development in knee joint using in situ hybridization (ISH). Results: ECM-specific qPCR-arrays followed by validation of responsive genes by quantitative-PCR analysis revealed that miR-27b-3p mimic treatment resulted in a significant upregulation of key ECM genes including pro-collagen type-I (COL1A1), A disintegrin and metalloproteinase with thrombospondin motifs 8 (ADAMTS-8), Thrombospondin 1 (THBS1), fibronectin1 (FN1), Collagen 5 A 1 (COL5A1) and Collagen 14 A 1 (COL14A1) in OA Synovial fibroblasts. Furthermore, miR-27b-3p mimic resulted in a marked increase in type-I collagen protein expression; this effect was inhibited by miR-27b-3p inhibitor. Overall, these results suggest that miR-27b-3p may play a key role in the regulation of key ECM components in OA synovial fibroblasts. Next, we employed “Watson Discovery tool” to identify direct targets of miR-27b-3p to further elucidate its signalling pathway. This tool identified a panel of 11 genes including peroxisome proliferator-activated receptor gamma (PPARγ) as direct targets for miR-27b-3p. Indeed, we confirmed that miR-27b-3p mimic treatment results in a marked suppression of PPARγ expression, while inhibition of miR-27b-3p upregulates PPARγ, suggesting that miR-27b-3p may impart its ECM regulatory effect through modulation of PPARγ. Furthermore, ISH of DMM-induced OA mice knee joints further show that miR-27b-3p expression is prominent in the synovial lining and fibrocartilage compared to sham control mice in vivo. Conclusions: Our results thus far suggest that, in OA synovial fibroblasts, miR-27b-3p is involved in the regulation of key ECM components. Our data also suggest that miR-27b-3p may signal via negative regulation of PPARγ. Our ongoing studies are confirming if miR-27b-3p signalling via PPARγ controls ECM regulation in OA synovial fibroblasts. Our in vivo studies show that miR-27b-3p expression is elevated in the synovium during OA. To comprehensively investigate the endogenous role of miR-27b-3p in synovial pathology, we have intra-articularly injected mouse knee joints with the in-vivo grade mimic of miR-27b-3p to determine any changes in the synovium upon miR-27b-3p treatment in vivo.

Periostin in Bone Regeneration.

Bone regeneration is an efficient regenerative process depending on the recruitment and activation of skeletal stem cells that allowcartilage and bone formation leading to fracture consolidation. Periosteum, the tissue located at the outer surface of bone is now recognized as an essential player in the bone repair process and contains skeletal stem cells with high regenerative potential. The matrix composition of the periosteum defines its roles in bone growth, in cortical bone modeling and remodeling in response to mechanical strain, and in bone repair. Periostinis a key extracellular matrix component of the periosteum involved in periosteum functions. In this chapter, we summarize the current knowledge on the bone regeneration process, the role of the periosteum and skeletal stem cells, and Periostin functions in this context. The matricellular protein Periostinhas several roles through all stages of bone repair: in the early days of repair during the initial activation of stem cells within periosteum, in the active phase of cartilage and bone deposition in the facture callus, and in the final phase of bone bridging and reconstitution of the stem cell pool within periosteum.

Spectrum-Effect Relationships between High-Performance Liquid Chromatography (HPLC) Fingerprints and the Antioxidant and Anti-Inflammatory Activities of Collagen Peptides.

A total of 13 batches of collagen peptide samples were extracted, isolated, and purified from chicken sternal cartilage under various process parameters. The fingerprint profiles of 13 batches of collagen peptides were established by high-performance liquid chromatography (HPLC). In addition, the amino acid profiles and molecular weight distributions of collagen peptides were investigated. The in vitro antioxidant activities of the peptide samples were measured using the 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assay, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the ferric-reducing antioxidant power (FRAP) assay and an assay of the oxidative damage induced by hydrogen peroxide (H2O2) in the degenerative cartilage cells from the knee joint of rat C518 (C518 cell line). The anti-inflammatory activities of the peptide samples were assessed by measuring the inflammatory responses induced by lipopolysaccharides (LPS) in C518 cells. Subsequently, the spectrum-effect relationships between HPLC fingerprints and the antioxidant and anti-inflammatory activities of collagen peptides were investigated using grey relational analysis (GRA). Fifteen common peaks were obtained from the HPLC fingerprints of collagen peptides. Each collagen peptide sample had a characteristic set of amino acid types and contents. All of the hydrolysates of the collagen peptides were primarily composed of fractions II (500–1000 Da) and III (1000–3000 Da). Collagen peptides exhibited good scavenging activity on ABTS radical, DPPH radical, and ferric-reducing antioxidant power. Collagen peptides were also effective against H2O2-induced cellular oxidative damage in C518 cells. The antioxidant activity of collagen peptides was due to the low molecular weight and the presence of antioxidant and hydrophobic amino acid residues within its sequence. Collagen peptides significantly inhibited the secretion of inflammatory cytokines IL-1β, TNF-α, and PGE2 in C518 cells. The anti-inflammatory activity of collagen peptides may include increased synthesis of the key components of extracellular matrix (ECM) and inhibited apoptosis of chondrocytes. The GRA results showed that peaks 2, 3, and 8 were the main components contributing to the antioxidant activity of the collagen peptides, whereas peaks 11 and 14 were the main components contributing to the anti-inflammatory activity of the collagen peptides. The components of peaks 8 and 14 were identified as GPRGPPGPVGP and VAIQAVLSLYASGR by UPLC-MS/MS. Those identified collagen peptides offer a potential therapeutic strategy for the treatment of osteoarthritis (OA) due to their antioxidative stress and due to them disturbing the catabolism and anabolism processes in arthrodial cartilage.

The hippocampal extracellular matrix regulates pain and memory after injury

Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.

Incorporation of Collagen and Hyaluronic Acid to Enhance the Bioactivity of Fibrin-Based Hydrogels for Nucleus Pulposus Regeneration.

Hydrogels, such as fibrin, offer a promising delivery vehicle to introduce cells into the intervertebral disc (IVD) to regenerate damaged disc tissue as a potential treatment for low back pain. However, fibrin lacks key extracellular matrix (ECM) components, such as collagen (Col) and hyaluronan (HA), normally found in native nucleus pulposus (NP) tissue. The overall aim of this work was to create a fibrin-based hydrogel, by incorporating Col and HA into the matrix to enhance NP-like matrix accumulation using articular chondrocytes (CC). Firstly, we assessed the effect of fibrin concentrations on hydrogel stability, and the viability and proliferation kinetics of articular chondrocytes. Secondly, we investigated the effect of incorporating Col and HA to enhance NP-like matrix accumulation, and finally, examined the influence of various HA concentrations. Results showed that increasing fibrin concentration enhanced cell viability and proliferation. Interestingly, incorporation of HA promoted sGAG accumulation and tended to suppress collagen formation at higher concentrations. Taken together, these results suggest that incorporation of ECM components can enhance the bioactivity of fibrin-based hydrogels, which may help advance the clinical potential of commercial cell and biomaterial ventures in the treatment of IVD regeneration.

Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. Here, we show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin-binding domains (HBDs) located in the α chain laminin-type G (LG) domains. These domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improves retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterial matrices as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration.

Laminin β2 Chain Regulates Retinal Progenitor Cell Mitotic Spindle Orientation via Dystroglycan

Vertebrate retinal development follows a pattern during which retinal progenitor cells (RPCs) give rise to all retinal cell types in a highly conserved temporal sequence. RPC proliferation and cell cycle exit are tightly coordinated to ensure proper and timely production of each of the retinal cell types. Extracellular matrix (ECM) plays an important role in eye development, influencing RPC proliferation and differentiation. In this study, we demonstrate that laminins, key ECM components, in the inner limiting membrane, control mitotic spindle orientation by providing environmental cues to the RPCs. In vivo deletion of laminin β2 in mice of both sexes results in a loss RPC basal processes and contact with the ECM, leading to a shift of the mitotic spindle pole orientation toward asymmetric cell divisions. This leads to decreased proliferation and premature RPC pool depletion, resulting in overproduction of rod photoreceptors at the expense of bipolar cells and Müller glia. Moreover, we show that deletion of laminin β2 leads to disruption and mislocalization of its receptors: dystroglycan and β1-integrin. Addition of exogenous β2-containing laminins to laminin β2-deficient retinal explants stabilizes the RPC basal processes and directs their mitotic spindle orientation toward symmetric divisions, leading to increased RPC proliferation, as well as restores proper receptor localization at the retinal surface. Finally, functional blocking of dystroglycan in wild-type retinal explants phenocopies laminin β2 ablation. Our data suggest that dystroglycan-mediated signaling between RPCs and the ECM is of key importance in controlling critical developmental events during retinogenesis.SIGNIFICANCE STATEMENT The mechanisms governing retinogenesis are subject to both intrinsic and extrinsic signaling cues. Although the role of intrinsic signaling has been the subject of many studies, our understanding of the role of the microenvironment in retinal development remains unclear. Using a combination of in vivo and ex vivo approaches, we demonstrate that laminins, key extracellular matrix components, provide signaling cues that control retinal progenitor cell attachment to the basement membrane, mitotic axis, proliferation, and fate adoption. Moreover, we identify, for the first time, dystroglycan as the receptor responsible for directing retinal progenitor cell mitotic spindle orientation. Our data suggest a mechanism where dystroglycan-mediated signaling between the cell and the extracellular matrix controls the proliferative potential of progenitors in the developing CNS.

Preparation and evaluation of tumour microenvironment response multistage nanoparticles for epirubicin delivery and deep tumour penetration

Poor tumour penetration became a major challenge for the use of nanoparticles in anticancer therapy. To further enhance the tumour penetration efficiency, we developed a tumour-microenvironment-responsive multistage drug delivery system which was formed layer by layer via electrostatic interaction with cationic drug-loaded nanoparticles, hyaluronidase (HAase) and iRGD-modified gelatin (G-iRGD). The drug-loaded nanoparticles were formed by self-assembling mPEG-PDPA-PG and encapsulation with epirubicin (EPI). Due to the protonation of tertiary amine groups of PDPA segment in acid environment, mPEG-PDPA-PG could enhance the lysosomal escape and the intracellular release of EPI. This NPs/HAase/G-iRGD delivery system showed great biocompatibility in vitro, confirmed by MTT method. In vitro spherical tumour model penetration and in vivo tumour permeability investigation showed HAase coated NPs-EPI (NPs-EPI/HAase) could significantly enhance its penetrating efficiency. The NPs-EPI/HAase could assist in breaking down the hyaluronic acid (HA), which was a key component of extracellular matrix and thereby improving mass transport within the solid tumours. The flow cytometry studies showed that G-iRGD coated NPs-EPI (NPs-EPI/G-iRGD) was more easily taken up by HepG2 cells than gelatin coated NPs-EPI (NPs-EPI/G), which revealed the active targeting ability of iRGD. The results proved that this NPs/HAase/G-iRGD delivery system showed promising potential in enhancing tumour penetration efficiency.

Extracellular matrix component expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch’s membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. Statement of SignificanceThe expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch’s membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation.

Collagen VI is required for the structural and functional integrity of the neuromuscular junction.

The synaptic cleft of the neuromuscular junction (NMJ) consists of a highly specialized extracellular matrix (ECM) involved in synapse maturation, in the juxtaposition of pre- to post-synaptic areas, and in ensuring proper synaptic transmission. Key components of synaptic ECM, such as collagen IV, perlecan and biglycan, are binding partners of one of the most abundant ECM protein of skeletal muscle, collagen VI (ColVI), previously never linked to NMJ. Here, we demonstrate that ColVI is itself a component of this specialized ECM and that it is required for the structural and functional integrity of NMJs. In vivo, ColVI deficiency causes fragmentation of acetylcholine receptor (AChR) clusters, with abnormal expression of NMJ-enriched proteins and re-expression of fetal AChRγ subunit, both in Col6a1 null mice and in patients affected by Ullrich congenital muscular dystrophy (UCMD), the most severe form of ColVI-related myopathies. Ex vivo muscle preparations from ColVI null mice revealed altered neuromuscular transmission, with electrophysiological defects and decreased safety factor (i.e., the excess current generated in response to a nerve impulse over that required to reach the action potential threshold). Moreover, in vitro studies in differentiated C2C12 myotubes showed the ability of ColVI to induce AChR clustering and synaptic gene expression. These findings reveal a novel role for ColVI at the NMJ and point to the involvement of NMJ defects in the etiopathology of ColVI-related myopathies.

Effect of Resistance Training on Extracellular Matrix Adaptations in Skeletal Muscle of Older Rats.

Accumulation of connective tissue, particularly extracellular matrix (ECM) proteins, has been observed in skeletal muscles with advancing age. Resistance training (RT) has been widely recommended to attenuate age-induced sarcopenia, even though its effects on the components that control ECM turnover in skeletal muscles remain to be elucidated. Thus, the aim of this study was to determine the effects of RT on connective tissue content and gene expression of key components of ECM in the skeletal muscles of aged rats. Young (3 mo.) and older (21 mo.) adult male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), 3 times a week for 12 weeks. Forty-eight hours post-training, the soleus (SOL) and gastrocnemius (GAS) muscles were dissected for histological and mRNA analysis. RT mitigated the age-associated increase of connective tissue content in both muscles, even though mRNA levels of COL-1 and−3 were elevated in older trained rats. Overall, RT significantly elevated the gene expression of key components of connective tissue deposition (TGFβ and CTGF; MMP-2 and-9; TIMP-1 and−2) in the GAS and SOL muscles of older rats. In conclusion, RT blunted the age-induced accumulation of connective tissue concomitant to the upregulation of genes related to synthesis and degradation of the ECM network in the SOL and GAS muscles of older rats. Although our findings indicate that RT plays a crucial role reducing connective tissue accumulation in aged hindlimb muscles, key components of ECM turnover were paradoxically elevated. The phenotypic responses induced by RT were not accompanied by the gene expression of those components related to ECM turnover.