Fibronectin-rich Extracellular Matrix Research Articles

Abstract 2979: Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to create a fibronectin-rich extracellular matrix niche and promote tumor initiation

Abstract Tumor initiation is an inefficient process in which tumor cells must develop cell-autonomous mechanisms to survive harsh microenvironments, establish colonies, and thereby overcome “isolation stress.” We report that pancreatic cancer cells transiently upregulate lysophosphatidic acid receptor 4 (LPAR4), a G-protein coupled receptor, in response to microenvironmental stress or chemotherapy, which promotes stress tolerance, drug resistance, and tumor initiation. Even without exogenous LPA, LPAR4 drives the expression of a subset of extracellular matrix (ECM) genes via an AKT/CREB axis, including fibronectin and versican, which are established drivers of cancer stemness. The upregulation of fibronectin is indispensable for LPAR4-induced tumor initiation in vivo and anchorage-independent growth in vitro. Furthermore, the ligation of this fibronectin-containing ECM created by LPAR4-positive cells via integrins α5β1 or αvβ3 can transfer cancer stemness and stress tolerance to LPAR4-negative cells by activating Yes-associated protein 1 (YAP). Our findings reveal that in response to isolation stress, pancreatic tumor cells that upregulate LPAR4 can autonomously generate a fibronectin-enriched ECM niche and thereby initiate tumor formation. Citation Format: Chengsheng Wu, Taha Rakhshandehroo, Hiromi I. Wettersten, Alejandro Campos, Tami Von Schalscha, Shashi Jain, Ziqi Yu, Jiali Tan, Evangeline Mose, Betzaira G. Childers, Andrew M. Lowy, Sara M. Weis, David A. Cheresh. Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to create a fibronectin-rich extracellular matrix niche and promote tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2979.

A TGFβ-ECM-integrin signaling axis drives structural reconfiguration of the bile duct to promote polycystic liver disease.

The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-β (TGFβ) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFβ signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery.

Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to promote an ECM-enriched niche and support tumour initiation.

Defining drivers of tumour initiation can provide opportunities to control cancer progression. Here we report that lysophosphatidic acid receptor 4 (LPAR4) becomes transiently upregulated on pancreatic cancer cells exposed to environmental stress or chemotherapy where it promotes stress tolerance, drug resistance, self-renewal and tumour initiation. Pancreatic cancer cells gain LPAR4 expression in response to stress by downregulating a tumour suppressor, miR-139-5p. Even in the absence of exogenous lysophosphatidic acid, LPAR4-expressing tumour cells display an enrichment of extracellular matrix genes that are established drivers of cancer stemness. Mechanistically, upregulation of fibronectin via an LPAR4/AKT/CREB axis is indispensable for LPAR4-induced tumour initiation and stress tolerance. Moreover, ligation of this fibronectin-containing matrix via integrins α5β1 or αVβ3 can transfer stress tolerance to LPAR4-negative cells. Therefore, stress- or drug-induced LPAR4 enhances cell-autonomous production of a fibronectin-rich extracellular matrix, allowing cells to survive 'isolation stress' and compensate for the absence of stromal-derived factors by creating their own tumour-initiating niche.

The IRE1α-XBP1s Arm of the Unfolded Protein Response Activates N-Glycosylation to Remodel the Subepithelial Basement Membrane in Paramyxovirus Infection.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections (LRTI) associated with decreased pulmonary function, asthma, and allergy. Recently, we demonstrated that RSV induces the hexosamine biosynthetic pathway via the unfolded protein response (UPR), which is a pathway controlling protein glycosylation and secretion of the extracellular matrix (ECM). Because the presence of matrix metalloproteinases and matricellular growth factors (TGF) is associated with severe LRTI, we studied the effect of RSV on ECM remodeling and found that RSV enhances the deposition of fibronectin-rich ECM by small airway epithelial cells in a manner highly dependent on the inositol requiring kinase (IRE1α)–XBP1 arm of the UPR. To understand this effect comprehensively, we applied pharmacoproteomics to understand the effect of the UPR on N-glycosylation and ECM secretion in RSV infection. We observe that RSV induces N-glycosylation and the secretion of proteins related to ECM organization, secretion, or proteins integral to plasma membranes, such as integrins, laminins, collagens, and ECM-modifying enzymes, in an IRE1α–XBP1 dependent manner. Using a murine paramyxovirus model that activates the UPR in vivo, we validate the IRE1α–XBP1-dependent secretion of ECM to alveolar space. This study extends understanding of the IRE1α–XBP1 pathway in regulating N-glycosylation coupled to structural remodeling of the epithelial basement membrane in RSV infection.

Dual-functional hydrogel system for spinal cord regeneration with sustained release of arylsulfatase B alleviates fibrotic microenvironment and promotes axonal regeneration

Traumatic damage to the spinal cord does not spontaneously heal, often leading to permanent tissue defects. We have shown that injection of imidazole-poly(organophosphazene) hydrogel (I-5) bridges cystic cavities with the newly assembled fibronectin-rich extracellular matrix (ECM). The hydrogel-created ECM contains chondroitin sulfate proteoglycans (CSPGs), collagenous fibrils together with perivascular fibroblasts, and various fibrotic proteins, all of which could hinder axonal growth in the matrix. In an in vitro fibrotic scar model, fibroblasts exhibited enhanced sensitivity to TGF-β1 when grown on CSPGs. To alleviate the fibrotic microenvironment, the I-5 hydrogel was equipped with an additional function by making a complex with ARSB, a human enzyme degrading CSPGs, via hydrophobic interaction. Delivery of the I-5/ARSB complex significantly diminished the fibrotic ECM components. The complex promoted serotonergic axonal growth into the hydrogel-induced matrix and enhanced serotonergic innervation of the lumbar motor neurons. Regeneration of the propriospinal axons deep into the matrix and to the lumbar spinal cord was robustly increased accompanied by improved locomotor recovery. Therefore, our dual-functional system upgraded the functionality of the hydrogel for spinal cord regeneration by creating ECM to bridge tissue defects and concurrently facilitating axonal connections through the newly assembled ECM.

Engineering aligned human cardiac muscle using developmentally inspired fibronectin micropatterns

Cardiac two-dimensional tissues were engineered using biomimetic micropatterns based on the fibronectin-rich extracellular matrix (ECM) of the embryonic heart. The goal of this developmentally-inspired, in vitro approach was to identify cell–cell and cell-ECM interactions in the microenvironment of the early 4-chambered vertebrate heart that drive cardiomyocyte organization and alignment. To test this, biomimetic micropatterns based on confocal imaging of fibronectin in embryonic chick myocardium were created and compared to control micropatterns designed with 2 or 20 µm wide fibronectin lines. Results show that embryonic chick cardiomyocytes have a unique density-dependent alignment on the biomimetic micropattern that is mediated in part by N-cadherin, suggesting that both cell–cell and cell-ECM interactions play an important role in the formation of aligned myocardium. Human induced pluripotent stem cell-derived cardiomyocytes also showed density-dependent alignment on the biomimetic micropattern but were overall less well organized. Interestingly, the addition of human adult cardiac fibroblasts and conditioning with T3 hormone were both shown to increase human cardiomyocyte alignment. In total, these results show that cardiomyocyte maturation state, cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblast interactions, and cardiomyocyte-ECM interactions can all play a role when engineering anisotropic cardiac tissues in vitro and provides insight as to how these factors may influence cardiogenesis in vivo.

Substrate Resistance to Traction Forces Controls Fibroblast Polarization

The mechanics of fibronectin-rich extracellular matrix regulate cell physiology in a number of diseases, prompting efforts to elucidate cell mechanosensing mechanisms at the molecular and cellular scale. Here, the use of fibronectin-functionalized silicone elastomers that exhibit considerable frequency dependence in viscoelastic properties unveiled the presence of two cellular processes that respond discreetly to substrate mechanical properties. Weakly cross-linked elastomers supported efficient focal adhesion maturation and fibroblast spreading because of an apparent stiff surface layer. However, they did not enable cytoskeletal and fibroblast polarization; elastomers with high cross-linking and low deformability were required for polarization. Our results suggest as an underlying reason for this behavior the inability of soft elastomer substrates to resist traction forces rather than a lack of sufficient traction force generation. Accordingly, mild inhibition of actomyosin contractility rescued fibroblast polarization even on the softer elastomers. Our findings demonstrate differential dependence of substrate physical properties on distinct mechanosensitive processes and provide a premise to reconcile previously proposed local and global models of cell mechanosensing.

Aesculus hippocastanum L. Extract Does Not Induce Fibroblast to Myofibroblast Conversion but Increases Extracellular Matrix Production In Vitro Leading to Increased Wound Tensile Strength in Rats.

The ability of horse chestnut extract (HCE) to induce contraction force in fibroblasts, a process with remarkable significance in skin repair, motivated us to evaluate its wound healing potential in a series of experiments. In the in vitro study of the ability of human dermal fibroblasts to form myofibroblast-like cells was evaluated at the protein level (Western blot and immunofluorescence). The in vivo study was conducted on male Sprague-Dawley rats with inflicted wounds (one open circular and one sutured incision) on their backs. Rats were topically treated with two tested HCE concentrations (0.1% and 1%) or sterile water. The control group remained untreated. The incisions were processed for wound tensile strength (TS) measurement whereas the open wounds were subjected to histological examination. On the in vitro level the HCE extract induced fibronectin-rich extracellular matrix formation, but did not induced α-smooth muscle actin (SMA) expression in dermal fibroblasts. The animal study revealed that HCE increased wound TS and improved collagen organization. In conclusion, the direct comparison of both basic wound models demonstrated that the healing was significantly increased following HCE, thus this extract may be found useful to improve healing of acute wounds. Nevertheless, the use of an experimental rat model warrants a direct extrapolation to the human clinical situation.

Fibronectin in the Tumor Microenvironment Activates a TLR4-dependent Inflammatory Response in Lung Cancer Cells.

The microenvironment of solid tumors plays an essential role in tumor progression. In lung cancer, the stromal cells produce a fibronectin rich extracellular matrix which is known to contribute to both tumor metastasis and drug resistance. Due to its conformational lability, fibronectin is considerably remodeled by the contractile forces of the fibrotic microenvironment within the tumor stroma. As a result, the secondary structure of fibronectin's Type III domains is disrupted and the molecule becomes highly stretched. The contribution/impact of these strained forms of fibronectin on tumor growth and metastasis is not known. In the current study we show that the partially unfolded first Type III domain of fibronectin, III-1c, activates a toll-receptor/NF-κB pathway leading to an increase in the expression of IL-8. Using a 3-D model of tumor-associated extracellular matrix, we demonstrate that lung cancer cells seeded onto this matrix activate a TLR4/NF-κB signaling pathway leading to a robust increase in the release of IL-8. Cytokine release by these cells is completely dependent on the presence of fibronectin in the extracellular matrix. These findings suggest that paracrine signaling between the tumor and the stromal myofibroblasts causes a remodeling of the matrix fibronectin into a strained conformation which supports the activation of a TLR4/NF-κB signaling pathway resulting in the upregulation of fibro-inflammatory cytokines.

Stepwise Adipogenesis of Decellularized Cellular Extracellular Matrix Regulates Adipose Tissue-Derived Stem Cell Migration and Differentiation.

Microenvironmental factors can modulate the cellular status of adipose tissue-derived stem cells (ASCs). In response to microenvironmental changes, cells can remodel extracellular matrix (ECM) proteins, which play an important role in regulating cell behaviors. During adipogenic differentiation, ECM components secreted from ASCs remodel dramatically. To evaluate the role of stepwise adipogenesis-induced cellular secretion of ECM on the behavior of ASCs, we cultured ASCs in growth and adipogenic media, and ECM secreted from cells was characterized and decellularized. The ASCs were then reseeded on decellularized ECM (d-ECM) to determine the regulatory effects of ECM on cellular behaviors. During adipogenesis, cell-secreted ECM underwent remodeling characterized by conversion from fibronectin-rich ECM to laminin-rich ECM. The cellular status of ASCs was tested after reseeding on decellularized ECM. When reseeded on growth d-ECM, ASCs exhibited greater migration ability. In contrast, ASCs seeded on adipogenic d-ECM underwent adipogenic differentiation. In addition, integrin subunit αv and integrins α6 and α7 were detected at significantly greater levels in ASCs cultured on growth and adipogenic d-ECM, respectively, suggesting that integrins play an important role in ASC migration and adipogenesis. This study demonstrated that stepwise adipogenesis-induced ECM production plays an important role in ASC migration and differentiation. In addition, this study provided a strategy to achieve precise regulation of stem cell function in adipose tissue engineering.

S-Glutathionylation Influences the Property of Fibronectin

Fibronectin is a key extracellular matrix (ECM) protein that plays a role in cell growth, migration and differentiation through its engagement of cell surface receptors, known as integrins, in a spatiotemporal manner. Fibronectin is a mechanically sensitive protein displaying force-induced conformational changes that impact its multiple growth factor, integrin, and ECM protein binding domains. Fibronectin is a unique ECM protein in that it contains 4 free cysteine residues that are protected in cryptic sites, but that can be exposed under force. S-glutathionylation is a post-translational modification of cysteines that occurs in oxidative environments like the extracellular space during pathologies like cancer and fibrosis. Here, we show for the first time that fibronectin's cysteines can be glutathionylated when exposed either through chemical unfolding in its soluble form or mechanical unfolding in its fibrillar form (both on the single fiber and matrix level). Moreover, we proved that S-glutathionylation impacts the mechanical properties of fibronectin fibers. Specifically, our results reveal that glutathionylated fibronectin fibers are more extensible and less stiff compared to unmodified fibronectin. This is true for both single fibronectin fibers and fibronectin matrices. Thus S-glutathionylation of mechanically exposed cysteines on fibronectin may act as a mechanism of mechanical memory within fibronectin-rich ECMs, regulating subsequent mechanotransduction. One possible realism of this mechanism could be the enhancement fibronectin's integrin binding mechanoswitch due to fibronectin fibers being more easily stretched under lower force regimes.

An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling

The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle for tissue repair in central nervous system (CNS). Here we report that injection of imidazole-poly(organophosphazenes) (I-5), a hydrogel with thermosensitive sol–gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model. Cystic cavities are bridged by fibronectin-rich extracellular matrix. The fibrotic extracellular matrix remodeling is mediated by matrix metalloproteinase-9 expressed in macrophages within the fibrotic extracellular matrix. A poly(organophosphazenes) hydrogel lacking the imidazole moiety, which physically interacts with macrophages via histamine receptors, exhibits substantially diminished bridging effects. I-5 injection improves coordinated locomotion, and this functional recovery is accompanied by preservation of myelinated white matter and motor neurons and an increase in axonal reinnervation of the lumbar motor neurons. Our study demonstrates that dynamic interactions between inflammatory cells and injectable biomaterials can induce beneficial extracellular matrix remodeling to stimulate tissue repair following CNS injuries.

Abstract 4336: Human mesodermal-derived CNS metastasis-associated stromal cells induce a fibrotic response to limit tumor growth

Abstract Metastasis to the central nervous system (CNS) remains a major cause of mortality and morbidity in patients with systemic cancer. However, the mechanistic interactions of the neural niche with disseminated tumors cells in CNS metastases (CM) are still poorly understood. To better understand the cross-talk between the neural niche and metastatic tumors, we generated five different patient-derived cell lines (PDCs) originating from surgically resected CM. To assess the genetic and epigenetic characteristics of each PDC, DNA and RNA sequencing, and DNA methylation analysis was performed. Non-tumoral PDCs revealed normal copy number profiles, and retention of germline mutations as seen in patient-matched germline DNA. In contrast, one PDC (CM04) resembled its patient tumor, showing numerous copy number and somatic alterations. RNA-seq and DNA methylation analysis demonstrated that non-tumoral PDCs highly resembled each other, suggestive of a common cell of origin. Additionally, PDCs revealed gene expression signatures associated with cancer associated fibroblasts, epithelial to mesenchymal transition, and mesenchymal stem cells. Further in vivo studies demonstrated that CM04 cells were tumorigenic, whereas non-tumoral PDC (CM08) cells were unable to form tumors in mice. However, CM04:CM08 mixed tumors were significantly smaller than CM04 only tumors and revealed induction of a fibrotic response by immunohistochemistry. These data offer the first evidence that CNS metastasis-associated stromal cells (cMASCs) produce a collagen and fibronectin-rich extracellular matrix constituting a protective host response, which impedes growth of tumor cells. The therapeutic potential of these cells merits further exploration. Citation Format: Christophe Legendre, Gerald C. Gooden, Kyle N. Johnson, Rae Anne Martinez, Mark Bernstein, Jennifer Glen, Jeffrey Kiefer, Aleksander Hinek, Steven A. Toms, Bodour Salhia. Human mesodermal-derived CNS metastasis-associated stromal cells induce a fibrotic response to limit tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2017-4336

Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization.

SummaryHematopoietic stem/progenitor cells (HSPCs) are formed during ontogeny from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). Critically, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Matrix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracellular matrix (ECM) remodeling, cellular interactions, and signaling. Here, inhibition of vascular-associated Mmp2 function caused accumulation of fibronectin-rich ECM, retention of runx1/cmyb+ HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. Significantly, these migration defects were mimicked by overexpression and blocked by knockdown of C-X-C motif chemokine-12 (cxcl12), suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Our findings indicate Mmp2 and Mmp9 play distinct but complementary roles in developmental HSPC production and migration.

Extracellular matrix and \u03b15\u03b21 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion.

Syndecan-1-Induced ECM Fiber Alignment Requires Integrin αvβ3 and Syndecan-1 Ectodomain and Heparan Sulfate Chains.

Expression of the cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in stromal fibroblasts of invasive breast carcinomas. We have recently identified a correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber alignment, both in vitro and in vivo. ECMs derived from Sdc1-positive human mammary fibroblasts (HMF) showed an aligned fiber architecture, which contrasted markedly with the more random fiber arrangement in the ECM produced by Sdc1-negative HMFs. We further demonstrated that aligned fiber architecture promotes the directional migration and invasion of breast carcinoma cells. To decipher the molecular mechanisms governing the formation of an aligned, invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. We found that both the ectodomain and heparan sulfate chains of Sdc1 were required for full activity of Sdc1 in regulating ECM alignment, while transmembrane and cytoplasmic domains were dispensable. Sdc1 regulates the activities of several integrins via its ectodomain. Integrins are key players in the assembly of fibronectin-rich ECM. In addition, integrins are capable of regulating cell morphology and cell shape and orientation may affect ECM architecture. Therefore, we investigated the role of integrins in Sdc1-mediated ECM fiber alignment. Sdc1-overexpressing HMF gained an enhanced spindle-shaped morphology when cultured in an overconfluent state under conditions permissive for ECM production, which was partially reversed by siRNA-mediated silencing of β3 integrin expression. Moreover, suppression of αvβ3 integrin activity by a function-blocking antibody or β3 knockdown largely abolished the aligned ECM fiber architecture and consequently the invasion-permissive properties of the ECM induced by Sdc1. The results suggest that Sdc1 may modulate fibronectin fibrillogenesis and/or alter cell morphology during ECM production through αvβ3 integrin, thereby mediating ECM fiber alignment. Understanding the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer, aiming at converting an invasion-permissive to an invasion-restrictive microenvironment.

The complexity of microenvironment-mediated drug resistance.

The complexity of microenvironment-mediated drug resistance.

The α4β1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts

Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C–C′ loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4β1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition.

Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis.

Chordin forms a self-organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo.

The vertebrate body plan follows stereotypical dorsal-ventral (D-V) tissue differentiation controlled by bone morphogenetic proteins (BMPs) and secreted BMP antagonists, such as Chordin. The three germ layers--ectoderm, mesoderm, and endoderm--are affected coordinately by the Chordin-BMP morphogen system. However, extracellular morphogen gradients of endogenous proteins have not been directly visualized in vertebrate embryos to date. In this study, we improved immunolocalization methods in Xenopus embryos and analyzed the distribution of endogenous Chordin using a specific antibody. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from the anterior endoderm and mesoderm. This Fibronectin-rich extracellular matrix is called "Brachet's cleft" in the Xenopus gastrula and is present in all vertebrate embryos. Chordin protein formed a smooth gradient that encircled the embryo, reaching the ventral-most Brachet cleft. Depletion with morpholino oligos showed that this extracellular gradient was regulated by the Chordin protease Tolloid and its inhibitor Sizzled. The Chordin gradient, as well as the BMP signaling gradient, was self-regulating and, importantly, was able to rescale in dorsal half-embryos. Transplantation of Spemann organizer tissue showed that Chordin diffused over long distances along this signaling highway between the ectoderm and mesoderm. Chordin protein must reach very high concentrations in this narrow region. We suggest that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information coordinately from a single morphogen gradient located in Brachet's cleft.