The interplay between extracellular matrix and progenitor/stem cells during wound healing: Opportunities and future directions

Skin wound healing, a dynamic physiological process, progresses through coordinated overlapping phases to restore skin integrity. In some pathological conditions such as diabetes, wounds become chronic and hard-to-heal resulting in substantial morbidity and healthcare costs. Despite much advancement in understanding mechanisms of wound healing, chronic and intractable wounds are still a considerable challenge to nations’ health care systems. Extracellular matrix (ECM) components play pivotal roles in all phases of wound healing. Therefore, a better understanding of their roles during wound healing can help improve wound care approaches. The ECM provides a 3D structure and forms the stem cell niche to support stem cell adhesion and survival and to regulate stem cell behavior and fate. Also, this dynamic structure reserves growth factors, regulates their bioavailability and provides biological signals. In various diseases, the composition and stiffness of the ECM is altered, which as a result, disrupts bidirectional cell-ECM interactions and tissue regeneration. Hence, due to the impact of ECM changes on stem cell fate during wound healing and the possibility of exploring new strategies to treat chronic wounds through manipulation of these interactions, in this review, we will discuss the importance/impact of ECM in the regulation of stem cell function and behavior to find ideal wound repair and regeneration strategies. We will also shed light on the necessity of using ECM in future wound therapy and highlight the potential roles of various biomimetic and ECM-based scaffolds as functional ECM preparations to mimic the native stem cell niche.     

Modulation of mesenchymal stem cell genotype and phenotype by extracellular matrix proteins

ABSTRACT

Aim: To investigate the effect of extracellular matrix (ECM) proteins on characteristics of mesenchymal stem cells (MSCs) and tendon-derived cells (TDCs). Materials and Methods: MSCs and TDCs, cultured in a monolayer (2D) or hydrogels (3D), with or without ECM protein supplementation, and on a non-viable native tendon (NNT) matrix were assayed for adhesion, proliferation, gene expression, and integrin expression. Results: MSCs exhibited a fibroblastic, spindle-shaped morphology on 2D matrices except in the presence of fibronectin. In 3D matrices, MSCs displayed a rounded phenotype except when cultured on NNTs where cells aligned along the collagen fibrils but, unlike TDCs, did not form inter-cellular cytoplasmic processes. MSC proliferation was significantly (p < 0.01) increased by collagen type I in 2D culture and fibronectin in 3D culture. TDC proliferation was unaffected by substrata. MSCs and TDCs differentially expressed α2 integrin. Adhesion to substrata was reduced by RGD-blocking peptide and β1 integrin antibody. The presence of collagen I or fibronectin upregulated MSC expression of collagen type I and collagen type III, COMP, decorin, osteopontin, and fibronectin. Conclusions: The morphology, gene expression, and adhesion of both MSCs and TDCs are sensitive to the presence of specific ECM components. Interaction with the ECM is, therefore, likely to affect the mechanism of action of MSCs in vitro and may contribute to phenotypic modulation in vivo.     

Effect of serum and platelet-rich plasma on human early or advanced degenerative meniscus cells

Purpose: The purpose of this in vitro study was to evaluate the migratory, proliferating, and extracellular matrix (ECM) forming effect of human serum (HS) and platelet-rich plasma (PRP) on meniscus cells derived from human knees with early or advanced degenerative changes. Materials and Methods: Medial menisci from knees with early degenerative changes (n = 5; mean Kellgren score of 1) undergoing arthroscopic meniscal surgery and advanced degenerative changes (n = 5; mean Kellgren score of 4) undergoing total knee replacement were collected. Cell migration and proliferation upon stimulation with HS and PRP were assessed by migration and proliferation assays. Induction of meniscal ECM was evaluated histologically by hematoxylin and eosin, collagen type I, and alcian blue staining and by gene expression analysis of meniscus-related genes in pellets that have been stimulated with 10% HS or 5% PRP. Results: Meniscal cells from knees with early and advanced degenerative changes were significantly attracted by 2.5%–30% PRP or 10% HS. Cell proliferation was significantly increased upon stimulation with 10% HS or 5% PRP. Both cell groups showed the formation of a well-structured, meniscus-like ECM after stimulation with 10% HS, whereas stimulation with 5% PRP led to inhomogeneous, more fibrous ECM. Stimulation with 10% HS showed a significant induction of aggrecan and COMP, while 5% PRP showed no inducing effect. Conclusions: Only stimulation with HS showed the formation of meniscal ECM as well as cell proliferating and migratory effects on meniscal cells derived from knees with early or advanced degenerative changes. Thus, we suggest that the selected stimulating factor itself and not the status of the knee may primarily affect repair processes. HS may have a potential to augment in meniscal repair procedures.     

Extracellular matrix internalization links nutrient signalling to invasive migration

Integrins are the key mediators of cell–extracellular matrix (ECM) interaction, linking the ECM to the actin cytoskeleton. Besides localizing at the cell surface, they can be internalized and transported back to the plasma membrane (recycled) or delivered to the late endosomes/lysosomes for degradation. We and others have shown that integrin can be endocytosed together with their ECM ligands. In this short review, I will highlight how extracellular protein (including ECM) endocytosis impinges on the activation of the mechanistic target of rapamycin (mTOR) pathway, a master regulator of cell metabolism and growth. This supports the intriguing hypothesis that ECM components may be considered as nutrient sources, primarily under soluble nutrient‐depleted conditions.     

Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State-of-the-art imaging techniques were used to construct a three-dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63alpha and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies. Disclosure of potential conflicts of interest is found at the end of this article.     

Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue

Biochemical and biomechanical extracellular matrix (ECM) cues have recently been shown to play a role in stimulating stem cell differentiation towards several lineages, though how they combine to induce adipogenesis has been less well studied. The objective of this study was to recapitulate both the ECM composition and mechanical properties of adipose tissue in vitro to stimulate adipogenesis of human adipose-derived stem cells (ASCs) in the absence of exogenous adipogenic growth factors and small molecules. Adipose specific ECM biochemical cues have been previously shown to influence adipogenic differentiation; however, the ability of biomechanical cues to promote adipogenesis has been less defined. Decellularized human lipoaspirate was used to functionalize polyacrylamide gels of varying stiffness to allow the cells to interact with adipose-specific ECM components. Culturing ASCs on gels that mimicked the native stiffness of adipose tissue (2 kPa) significantly upregulated adipogenic markers, in the absence of exogenous adipogenic growth factors and small molecules. As substrate stiffness increased, the cells became more spread, lost their rounded morphology, and failed to upregulate adipogenic markers. Together these data imply that as with other lineages, mechanical cues are capable of regulating adipogenesis in ASCs.     

The impact of low levels of collagen IX and pyridinoline on the mechanical properties of in vitro engineered cartilage

The application of in vitro engineered cartilage has become a promising approach to repair cartilage defects. Nevertheless, the poor mechanical properties of in vitro engineered cartilage limit its potential for clinical applications. Studies have shown that the extracellular matrix (ECM) components are strongly correlated with the mechanical strength of engineered cartilage, but it remains unclear which components play a key role in determining the mechanical property of engineered cartilage. To address this issue, quantitative analyses of cartilage-specific components among native cartilage, in vivo and in vitro engineered cartilages were performed, and the correlation between various ECM molecules and Young's modulus was further analyzed. The results showed that many ECM molecules, such as highly sulphated glycosaminoglycan (GAG), collagens II, IX, and pyridinoline (PYR), contributed to the mechanical strength of cartilages. Further comparison between in vitro engineered cartilage and stress-stimulated in vitro engineered cartilage, known to have stronger mechanical properties, showed that only collagen IX and PYR, but not GAG and collagen II, were the key factors determining the mechanical properties of in vitro engineered cartilage. These results indicate that in vitro environment lacks the niche for enhancing collagen crosslinking that is mediated by collagen IX and PYR during cartilage formation. Thus, the discovery provides a clue for engineering strong cartilage in vitro in the future by enhancing the levels of these two molecules.     

Long-Lasting Tissue Inflammatory Processes Trigger Autoimmune Responses to Extracellular Matrix Molecules

Extracellular matrix (ECM) remodeling is strongly favored by the conditions occurring in the inflammatory microenvironment and can lead to pathogenic reactions directed toward the ECM itself. Several reports have described autoimmune responses to ECM molecules in patients with inflammatory diseases whose pathogenesis is not primarily related to ECM autoimmunity. This review will focus on the molecular interplay that governs ECM remodeling during tissue inflammation and will discuss how chronic inflammation can act as a driving force for the induction of autoimmune responses to ECM components as well as how the elicited autoimmunity can sustain local or induce distant tissue damage.     

Long-lasting tissue inflammatory processes trigger autoimmune responses to extracellular matrix molecules

Extracellular matrix (ECM) remodeling is strongly favored by the conditions occurring in the inflammatory microenvironment and can lead to pathogenic reactions directed toward the ECM itself. Several reports have described autoimmune responses to ECM molecules in patients with inflammatory diseases whose pathogenesis is not primarily related to ECM autoimmunity. This review will focus on the molecular interplay that governs ECM remodeling during tissue inflammation and will discuss how chronic inflammation can act as a driving force for the induction of autoimmune responses to ECM components as well as how the elicited autoimmunity can sustain local or induce distant tissue damage.     

Substantially elevated TSH,not traditional clinical subclinical thyroid disorder groupings,are associated with smaller LDL-P mean size: ELSA-Brasil

BackgroundLDL appears to drive atherogenesis in overt hypothyroidism, but in subclinical dysfunction, its role is not completely elucidated.ObjectiveThe aim of this study was to evaluate subfractions of LDL in subclinical (SC) thyroid disorders.MethodsIndividuals were divided into three groups by baseline thyroid function (SC hypothyroidism, euthyroidism, and SC hyperthyroidism). LDL particle (LDL-P) subfractions were analyzed by Nuclear Magnetic Resonance (NMR) spectroscopy. The association between LDL-P subfractions and thyroid groups and quintiles was evaluated by linear regression models.ResultsWe evaluated 3304 participants (54.1% women, 51.2% white, mean age 50.6 ± 8.7 years). In the univariate analysis, small LDL particle concentrations (SLDL-P) were not different between SC hypo- and hyperthyroidism compared to euthyroid individuals (p = 0.485 and p = 0.314, respectively). Large LDL-P (LDL-P) levels also did not differ in SC hyperthyroidism and SC hypothyroidism compared to euthyroidism (p = 0.698 and 0.788 respectively). Intermediate LDL-P levels were not different across the groups. These numbers did not materially change in multivariate analysis. However, we also analyzed LDL subfractions according to quintiles of TSH. We showed that in the higher TSH quintile LDL subfractions presented a significantly smaller mean size of LDL subfractions compared to the first quintile.ConclusionsSC thyroid disorders are not associated with significant changes in LDL-P subfractions measured by NMR spectroscopy. However, it seems that the LDL mean size decreases as TSH levels increase, which may represent a more atherogenic lipid profile.     

Novel understanding of high mobility group box-1 in the immunopathogenesis of incisional hernias

ABSTRACT

Introduction: Incisional hernias (IH) arise as a complication of patients undergoing laparotomy. Current literature has assessed the role of extracellular matrix (ECM) disorganization, alterations in type I and type III collagen, matrix metalloproteinases, and tissue inhibitors of metalloproteases on IH. However, there is limited information on the underlying molecular mechanisms that lead to ECM disorganization.

Areas covered: We critically reviewed the literature surrounding IH and ECM disorganization and offer a novel pathway that may be the underlying mechanism resulting in ECM disorganization and the immunopathogenesis of IH.

Expert opinion: High mobility group box-1 (HMGB-1), a damage-associated molecular pattern, plays an important role in the sterile inflammatory pathway and has been linked to ECM disorganization and the triggering of the NLRP3 inflammasome. Further research to investigate the role of HMGB-1 in the molecular pathogenesis of IH would be critical in identifying novel therapeutic targets in the management of IH formation.     

Human cardiac fibroblast extracellular matrix remodeling: dual effects of tissue inhibitor of metalloproteinase-2

ObjectiveTissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinases (MMPs) that attenuates maladaptive cardiac remodeling in ischemic heart failure. We examined the effects of TIMP-2 on human cardiac fibroblast activation and extracellular matrix (ECM) remodeling.MethodsHuman cardiac fibroblasts within a three-dimensional collagen matrix were assessed for phenotype conversion, ECM architecture and key molecular regulators of ECM remodeling after differential exposure to TIMP-2 and Ala+TIMP-2 (a modified TIMP-2 analogue devoid of MMP inhibitory activity).ResultsTIMP-2 induced opposite effects on human cardiac fibroblast activation and ECM remodeling depending on concentration. TIMP-2 activated fibroblasts into contractile myofibroblasts that remodeled ECM. At higher concentrations (> 10 nM), TIMP-2 inhibited fibroblast activation and prevented ECM remodeling. As compared to profibrotic cytokine transforming growth factor (TGF)-beta1, TIMP-2 activated fibroblasts and remodeled ECM without a net accumulation of matrix elements. TIMP-2 increased total protease activity as compared to TGF-beta1. Ala+TIMP-2 exposure revealed that the actions of TIMP-2 on cardiac fibroblast activation are independent of its effects on MMP inhibition. In the presence of GM6001, a broad-spectrum MMP inhibitor, TIMP-2-mediated ECM contraction was completely abolished, indicating that TIMP-2-mediated fibroblast activation is MMP dependent.ConclusionTIMP-2 functions in a contextual fashion such that the effect on cardiac fibroblasts depends on the tissue microenvironment. These observations highlight potential clinical challenges in using TIMP-2 as a therapeutic strategy to attenuate postinjury cardiac remodeling.     

The regulation of growth and metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney

Native extracellular matrix (ECM) that is secreted and maintained by resident cells is of great interest for cell culture and cell delivery. We hypothesized that specialized bioengineered niches for stem cells can be established using ECM-derived scaffolding materials. Kidney was selected as a model system because of the high regional diversification of renal tissue matrix. By preparing the ECM from three specialized regions of the kidney (cortex, medulla, and papilla; whole kidney, heart, and bladder as controls) in three forms: (i) intact sheets of decellularized ECM, (ii) ECM hydrogels, and (iii) solubilized ECM, we investigated how the structure and composition of ECM affect the function of kidney stem cells (with mesenchymal stem cells, MSCs, as controls). All three forms of the ECM regulated KSC function, with differential structural and compositional effects. KSCs cultured on papilla ECM consistently displayed lower proliferation, higher metabolic activity, and differences in cell morphology, alignment, and structure formation as compared to KSCs on cortex and medulla ECM, effects not observed in corresponding MSC cultures. These data suggest that tissue- and region-specific ECM can provide an effective substrate for in vitro studies of therapeutic stem cells.     

Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation

Corneal epithelial stem cells (SCs) are an ideal model for investigating how adult lineage-committed epithelial SCs are regulated by an anatomically defined and accessible niche, that is, limbal palisades of Vogt, located between the cornea and the conjunctiva. We have used collagenase digestion to isolate the entire limbal epithelial SCs and subjacent mesenchymal cells, and we have demonstrated that their close association is crucial for promoting epithelial clonal growth, implying that the latter serves as niche cells (NCs). After their close association was disrupted by trypsin/EDTA, single SCs and NCs could reunite to generate sphere growth in three-dimensional Matrigel in the embryonic SC medium, and that such sphere growth initiated by SC-NC reunion was mediated by SDF-1 uniquely expressed by limbal epithelial progenitor cells and its receptor CXCR4, but not CXCR7, strongly expressed by limbal stromal NCs. Inhibition of CXCR4 by AMD3100 or a blocking antibody to CXCR4 but not CXCR7 disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones. For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4 signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche.     

High Resolution Structural Changes of Schwann Cell and Endothelial Cells in Peripheral Nerves Across Leprosy Spectrum

ABSTRACT

A systematic ultrastructure of peripheral nerves across the spectrum of leprosy was studied with an aim to better understanding the pathogenesis of nerve involvement in leprosy using light and electron microscope. The pathogenesis of nerve destruction varies in leprosy considerably along the spectrum. The study has begun to shed new light on some aspects of the infection of Mycobacterium leprae (M. lepare) and phenomenon has opened new avenue of research and possible mechanism of pathogenesis in TT/BT/BL/LL leprosy. In tuberculoid type (TT) and borderline tuberculoid (BT) leprosy, the degenerative changes of Schwann cells (SCs) and presence of perineural and perivascular cuffing by mononuclear cells. The endoneurial blood vessel (EBV) showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen and causing ischemia. However, borderline lepromatous (BL) and lepromatous leprosy (LL) foamy macrophages and vacuolated SC contain numerous small dense materials, irregular in shape and size was prominent and, considered to be degenerated and fragmented M. Leprae. The dense materials were also found in the cytoplasm of vascular EC. It was revealed that besides SC, the EC of EBV frequently harbor M. leprae in LL. The lumen of the EBV was wide open with enlarged nucleus. In the present study, the ultrastructural characteristics suggest that hypersensitivity mechanisms are possibly responsible for nerve damage in TT/BT leprosy. However, the study indicates that the mechanisms of nerve damage in BL/LL are basically different wherein hypersensitivity appears to play a very limited role.     

Hypoxia Regulates the Extracellular Matrix via Mitogen-Activated Protein Kinases Pathway in Cells Retrieved from the Human Intervertebral Disc

PurposeThe present study aimed to identify the physiological characteristics of cells by investigating the change in gene expression and protein levels during extracellular matrix (ECM) synthesis in the intervertebral disc (IVD) under hypoxic conditions.Materials and MethodsTo test the effect of oxygen on cell growth and ECM synthesis of chondrocyte-like cells, the cells from IVD were separated and cultured in two hypoxia-mimicking systems: chemical hypoxic conditions using deferoxamine (DFO), and physiological hypoxic conditions using a hypoxic chamber for 7 days. Chondrocyte like cells cultured without DFO and under the normal oxygen concentration (21% O₂ and 5% CO₂, 37°C) served as the controls.ResultsChondrocyte-like cells cultured in the presence of 6% oxygen demonstrated a 100% increase in cellular proliferation compared to the control. The cells treated with chemical hypoxic conditions demonstrated a dose-dependent increase in the mRNA expression of glucose transporter-1, GAPDH, aggrecan, and type II collagen on Day 1. When treated with 100 µM DFO, the cells showed a 50% increase in the levels of proteoglycan protein on Day 7. The cells treated with chemical hypoxic condition demonstrated increase in sulfated glycosaminoglycan (GAG) protein levels on Day 7. Moreover, the cells cultured in the presence of 6% oxygen showed a 120% increase in sulfated GAG levels on Day 7.ConclusionThe oxygen concentration had an important role in the viability, proliferation, and maturation of chondrocyte-like cells in IVD. In addition, chondrocyte-like cells are sensitive to the concentration of oxygen.     

Genetic Disorders of the Extracellular Matrix

Mutations in the genes for extracellular matrix (ECM) components cause a wide range of genetic connective tissues disorders throughout the body. The elucidation of mutations and their correlation with pathology has been instrumental in understanding the roles of many ECM components. The pathological consequences of ECM protein mutations depend on its tissue distribution, tissue function, and on the nature of the mutation. The prevalent paradigm for the molecular pathology has been that there are two global mechanisms. First, mutations that reduce the production of ECM proteins impair matrix integrity largely due to quantitative ECM defects. Second, mutations altering protein structure may reduce protein secretion but also introduce dominant negative effects in ECM formation, structure and/or stability. Recent studies show that endoplasmic reticulum (ER) stress, caused by mutant misfolded ECM proteins, makes a significant contribution to the pathophysiology. This suggests that targeting ER-stress may offer a new therapeutic strategy in a range of ECM disorders caused by protein misfolding mutations. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.