Elastic Extracellular Matrix Research Articles

Abstract 18280: Mechanosensitive Trpv4 Channels Mediate Cardiac Myofibroblast Differentitation

Cardiac fibroblasts play critical role in cardiac remodeling and during development. Cardiac remodeling following myocardial infarction involves the migration, proliferation and differentiation of cardiac fibroblasts into hypersecretory myofibroblasts. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extra cellular matrix (ECM) and fibrosis. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. Because the infarcted/ischemic myocardium is known to undergo mechanical stretch and the muscle phenotypic modulator TGF-β is also increased in ischemia, we hypothesized the mechanotransduction and TGF-β signaling pathway play active role in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF-β-induced differentiation of cardiac fibroblasts into myofibroblasts. We found that, the TRPV4-specific antagonist, AB159908 significantly inhibited TGFβ-induced differentiation as measured by incorporation of α-SMA in actin stress fibers (p < 0.05). In contrast, AB159908 did not affect the proliferation of cardiac fibroblasts. Importantly, calcium imaging experiments with Fluo-4 revealed that TGF-β treated fibroblasts exhibit enhanced TRPV4-dependent calcium influx compared to untreated controls (3 fold; p< 0.05). Further, we found that TGF-β-induced myofibroblast differentiation is regulated by the variations in ECM elasticity in a TRPV4 dependent manner. Finally, TGF-β induced myofibroblast differentiation is significantly reduced in cardiac fibroblasts from TRPV4 KO mice compared to controls. Taken together these results suggest, for the first time, that a mechanosensitive ion channel, TRPV4 regulates cardiac fibroblast differentiation to myofibroblasts which could be used as a novel therapeutic target for the treatment of ischemia and myocardial infarction.

Smooth Muscle Biomechanics and Plasticity: Relevance for Vascular Calibre and Remodelling

Blood vessel structure and calibre are not static. Rather, vessels remodel continuously in response to their biomechanical environment. Vascular calibre is dictated by the amount, composition and organization of the elastic extracellular matrix. In addition, the amount and organization of contractile smooth muscle cell (SMC) also need to be regulated. The SMCs are organized such that maximum contractile force generally occurs at diameters slightly below the diameter at full dilation and physiological pressure. Thus, in a remodelling vessel, not only the matrix but also the SMCs need to undergo structural adaptation. Surprisingly little is known on the adaptation of SMC contractile properties in the vasculature. The purpose of this review is to explore this SMC plasticity in the context of vascular remodelling. While not much work on this has been carried out on blood vessels, SMC plasticity is more extensively studied on other hollow structures such as airway and bladder. We therefore include studies on bladder and airway SMCs because of their possible relevance for vascular SMC behaviour. Here, plasticity is thought to form an adaptation allowing maintained function despite large volume changes. In blood vessels, the general match of active and passive diameter-tension relations suggests that SMC plasticity is part of normal vascular physiological adaptation. Vascular SMCs display similar processes and forms of adaptation as seen in nonvascular SMCs. This may become particularly relevant under strong vasoconstriction, when inward cytoskeletal adaptation possibly prevents immediate full dilation. This may contribute to structural inward remodelling as seen in hypertension and flow reduction.

Elasticity-Dependent Modulation of TGF-β Responses in Human Trabecular Meshwork Cells

To gain further insight into a possible role of biomechanical cues in glaucoma, the authors assessed the influence of extracellular matrix (ECM) elasticity on TGF-β2-induced changes in trabecular meshwork (TM) cells. Human TM cells derived from donor cornea rings were plated on rigid collagen-coated tissue culture plastic or polyacrylamide gels of different elasticity and treated with vehicle or TGF-β2. Activation of Smad-2/3, ERK, and AKT signaling and expression of α-SMA and PAI-1 proteins were assessed by Western blot analysis. Subcellular localization of α-SMA was determined by confocal immunofluorescence microscopy. Transcription of collagen-I, -IV, and -VI, α-SMA, PAI-1, fibronectin, fibrillin-1, cochlin, and MGP-1 was studied by RT-qPCR. The contribution of non-Smad signaling pathways to TGF-β-induced α-SMA and PAI-1 expression was assessed using the small molecule inhibitors U0126 and LY294002. TGF-β2-induced activation of Smad-2/3, ERK, and AKT signaling as well as expression of collagen-1, α-SMA, fibulin, and MGP-1 were attenuated with increasing elasticity. In contrast, TGF-β2-induced collagen-6, fibronectin, PAI-1, and cochlin expression were enhanced on elastic substrates. The MEK-inhibitor U0126 blocked TGF-β-induced PAI-1 expression, whereas α-SMA expression was enhanced. PI3K inhibition with LY294002 reduced α-SMA expression. ECM elasticity modulates TGF-β-induced signaling and protein expression in human TM cells. Non-Smad signaling contributes to TGF-β-induced alterations. Increasing ECM elasticity in vitro promotes protein expression patterns reminiscent of patterns reported in primary open-angle glaucoma. Therefore, changes in TM elasticity and mechanical load may have a significant role in primary open-angle glaucoma.

生体内細胞の力学環境場に対する応答性(セミナー「生物のストレス受容のメカニズム-分子レベルから細胞レベルまで-」)

生体内細胞の力学環境場に対する応答性(セミナー「生物のストレス受容のメカニズム-分子レベルから細胞レベルまで-」)

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

0433 細胞外マトリクスの力学特性を利用した生体外胆管様組織の再構築(OS23:再生医療工学)

0433 細胞外マトリクスの力学特性を利用した生体外胆管様組織の再構築(OS23:再生医療工学)

Emilin1 gene and essential hypertension: a two-stage association study in northern Han Chinese population

BackgroundElastogenesis of elastic extracellular matrix (ECM) which was recognized as a major component of blood vessels has been believed for a long time to play only a passive role in the dynamic vascular changes of typical hypertension. Emilin1 gene participated in the transcription of ECM's formation and was recognized to modulate links TGF-β maturation to blood pressure homeostasis in animal study. Recently relevant advances urge further researches to investigate the role of Emilin1 gene in regulating TGF-β signals involved in elastogenesis and vascular cell defects of essential hypertension (EH).MethodsWe designed a two-stage case-control study and selected three single nucleotide polymorphisms (SNPs), rs3754734, rs2011616 and rs2304682 from the HapMap database, which covered Emilin1 gene. Totally 2,586 subjects were recruited from the International Collaborative Study of Cardiovascular Disease in Asia (InterASIA). In stage 1, all the three SNPs of the Emilin1 gene were genotyped and tested within a subsample including 503 cases and 490 controls, significant SNPs would enter into stage 2 including 814 cases with hypertension and 779 controls and analyze on the basis of testing total 2,586 subjects.ResultsIn stage 1, single locus analyses showed that SNPs rs3754734 and rs2011616 had significant association with EH (P < 0.05). In stage 2, weak association for dominant model were observed by age stratification and odds ratio (ORs) of TG+GG vs. TT of rs3754734 were 0.768 (0.584-1.009), 0.985 (0.735-1.320) and 1.346 (1.003-1.806) in < 50, 50-59 and ≥ 60 years group and ORs of GA+AA vs. GG of rs2011616 were 0.745 (0.568-0.977), 1.013 (0.758-1.353) and 1.437 (1.072-1.926) in < 50, 50-59 and ≥ 60 years group respectively. Accordingly, significant interactions were detected between genotypes of rs3754734 and rs2011616 and age for EH, and ORs were 1.758 (1.180-2.620), P = 0.006 and 1.903 (1.281-2.825), P = 0.001, respectively. Results of haplotypes analysis showed that there weren't any haplotypes associated with EH directly, but the interaction of hap2 (GA) and age-group found to be significant after being adjusted for the covariates, OR was 1.220 (1.031-1.444), P value was 0.020.ConclusionOur findings don't support positive association of Emilin1 gene with EH, but the interaction of age and genotype variation of rs3754734 and rs2011616 might increase the risk to hypertension.

Probing cellular mechanobiology in three-dimensional culture with collagen–agarose matrices

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Does Substrate Stiffness Guide Neutrophils During An Inflammation Response?

Neutrophils play a critical role in host response to infection and injury. To reach points of inflammation, they execute a series of adhesion and migration events that allow them to move from the blood stream, through the endothelial cells lining the blood vessels, and into the tissue and surrounding extracellular matrix. Within these few minutes of time, the neutrophil experiences drastically different physical environments, ranging from the viscous fluid in the blood vessel, to the elastic extracellular matrix, to the highly variable points of stiffness at sites of inflammation. Each of these physical environments offers its own unique mechanical cues which can affect a neutrophil's function and guide its behavior. Traditional studies have relied upon glass and plastic substrates, despite the fact that they are orders of magnitude stiffer than blood vessels and most tissues in the body. Turning instead to a physiologically relevant range of elasticity of 5 to 50 kPa in Young's modulus, we tested how neutrophil adhesion, spreading and migration were affected by substrate stiffness. We find that a dramatic and immediate difference is seen in the neutrophil's ability to spread on softer substrates. During migration we find that both speed and directionality are influenced by the substrate stiffness, with more efficient migration occurring on stiffer gels. We also find that these adherent neutrophils pull significantly harder on stiffer gels. These findings demonstrate that neutrophils respond and are sensitive to mechanical cues in the microenvironment, and suggest a possible novel mechanism for neutrophil guidance during injury and inflammation.

A mechanosensitive transcriptional mechanism that controls angiogenesis

Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.

Abstract 1604: Improved Cardiac Function after Expression of the Human Elastin Gene in the Infarcted Myocardium

Rationale: After a myocardial infarction, necrosed cardiomyocytes are replaced by fibrotic tissue. Subsequent thinning and expansion of this non-elastic scar contributes to cardiac dilatation and congestive heart failure. We attempted to restore the elastic properties of the scar by implanting the infarct area with cells expressing elastin. Methods: Full-length elastin cDNA was generated by PCR using the human lung cDNA library, and then transfected (in adenoviral vector) into rat bone marrow stromal cells (BMSCs). Western blot and immuno-staining confirmed expression of elastin protein. Seven days after coronary artery ligation, BMSCs transfected with genes for either adeno-elastin (BMSC+Elastin) or adeno-green fluorescence protein (BMSC), or media (control group) were injected into the infarct area of adult rats (n=8/group). Results: Over 8 weeks following cell or media implantation, cardiac function (fractional shortening by echocardiography), ventricular volumes, and load independent indices of cardiac function (end systolic and diastolic volumes, preload recruitable stroke work and end systolic elastance by pressure-volume catheter), were all significantly better preserved in both BMSC groups (p&lt;0.05 vs. media), with further improvements in the BMSC+Elastin group (p&lt;0.05 vs. BMSC group). Biochemical analysis detected over-expression of recombinant elastin, and histological examination revealed that collagen fiber length and diameter were preserved in the scar tissue. Myocardial birefringence determined by polarized light (linear retardance of the tissue structure) was significantly highest in the infarct area of the BMSC+Elastin group (p&lt;0.05 compared to BMSC and media groups). Elastin over-expression by cell-based gene therapy restored the elastic matrix structure in the scar tissue, stabilized the infarct and prevented ventricular dilatation, producing the smallest (p&lt;0.05) scar size and surface area in the BMSC+Elastin group. Conclusions: Over-expression of elastin in transplanted BMSCs contributed to the alignment and organization of an elastic extracellular matrix structure, which prevented cardiac dilatation and dysfunction. Elastin plays a key role in the remodeling of the extracellular matrix.

Tumor-derived endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal angiogenesis in vitro

Tumor blood vessels exhibit abnormal structure and function that cause disturbed blood flow and high interstitial pressure, which impair delivery of anti-cancer agents. Past efforts to normalize the tumor vasculature have focused on inhibition of soluble angiogenic factors, such as VEGF; however, capillary endothelial (CE) cell growth and differentiation during angiogenesis are also influenced by mechanical forces conveyed by the extracellular matrix (ECM). Here, we explored the possibility that tumor CE cells form abnormal vessels because they lose their ability to sense and respond to these physical cues. These studies reveal that, in contrast to normal CE cells, tumor-derived CE cells fail to reorient their actin cytoskeleton when exposed to uniaxial cyclic strain, exhibit distinct shape sensitivity to variations in ECM elasticity, exert greater traction force, and display an enhanced ability to retract flexible ECM substrates and reorganize into tubular networks in vitro. These behaviors correlate with a constitutively high level of baseline activity of the small GTPase Rho and its downstream effector, Rho-associated kinase (ROCK). Moreover, decreasing Rho-mediated tension by using the ROCK inhibitor, Y27632, can reprogram the tumor CE cells so that they normalize their reorientation response to uniaxial cyclic strain and their ability to form tubular networks on ECM gels. Abnormal Rho-mediated sensing of mechanical cues in the tumor microenvironment may therefore contribute to the aberrant behaviors of tumor CE cells that result in the development of structural abnormalities in the cancer microvasculature.

Extracellular control of TGFβ signalling in vascular development and disease

The intracellular mechanism of transforming growth factor-beta (TGFbeta) signalling via kinase receptors and SMAD effectors is firmly established, but recent studies of human cardiovascular syndromes such as Marfan syndrome and pre-eclampsia have refocused attention on the importance of regulating the availability of active extracellular TGFbeta. It seems that elastic extracellular matrix (ECM) components have a crucial role in controlling TGFbeta signalling, while soluble and membrane bound forms of TGFbeta co-receptors add further layers of regulation. Together, these extracellular interactions determine the final bioavailability of TGFbeta to vascular cells, and dysregulation is associated with an increasing number of vascular pathologies.

Idiopathic and Collagen Vascular Disease Nonspecific Interstitial Pneumonia: Clinical Significance of Remodeling Process

Recently, active remodeling may indicate a good prognosis in idiopathic interstitial pneumonias. In this study we sought to validate the importance of the collagen/elastic system in the extracellular matrix remodeling and to study the relationships between the collagen/elastic system in nonspecific interstitial idiopathic pneumonia (NSIP) and collagen vascular disease associated with nonspecific interstitial idiopathic pneumonia (CVD-NSIP). We examined collagen/elastic system fibers in open lung biopsies of 20 idiopathic NSIP and 21 CVD-NSIP patients. The clinical features were analyzed with respect to age, gender, pulmonary functional tests, chest X-ray and computed tomography, treatment, and survival. We used the picrosirius polarization method and Weigert's resorcin-fuchsin histochemistry and morphometric analysis to evaluate the amount of collagen/elastic system fibers and their association with the NSIP histologic pattern. No differences in clinical features and pulmonary function tests were observed between idiopathic NSIP and CVD-NSIP, but a significantly higher collagen and elastic fiber proliferation was detected in CVD-NSIP lungs and fibrosing NSIP histologic pattern. Multivariate Cox model analysis demonstrated that sex and quantitative elastic fiber staining added important prognostic information (p=0.01) and was indicative of a worse prognosis than collagen staining. A cut point at the mean staining of 1.5% for elastic fibers divided the patients into two groups with distinctive survival times. Those with elastic fibers greater than 1.5% had a median survival time of just 52 months. We concluded that idiopathic NSIP and CVD-NSIP were clinically similar but pathologically different, suggesting that different remodeling profiles in NSIP may represent evolutionary adapted responses to injury grade, which depend, at least in part, on the extent of elastic extracellular matrix deposition. Patients with greater than 1.5% of elastic fibers comprise a subset with a high risk for dying due to NSIP and may be an appropriate target for prospective studies.

Molecular mechanisms of cellular mechanics

Mechanical forces play an essential role in cellular processes as input, output, and signals. Various protein complexes in the cell are designed to handle, transform and use such forces. For instance, proteins of muscle and the extracellular matrix can withstand considerable stretching forces, hearing-related and mechanosensory proteins can transform weak mechanical stimuli into electrical signals, and regulatory proteins are suited to forcing DNA into loops to control gene expression. Here we review the structure-function relationship of four protein complexes with well defined and representative mechanical functions. The first example is titin, a protein that confers passive elasticity on muscle. The second system is the elastic extracellular matrix protein, fibronectin, and its cellular receptor integrin. The third protein system is the transduction apparatus in hearing and other mechanical senses, likely containing cadherin and ankyrin repeats. The last system is the lac repressor protein, which regulates gene expression by looping DNA. This review focuses on atomic level descriptions of the physical mechanisms underlying the various mechanical functions of the stated proteins.

227 細胞外基質の弾性が毛細血管ネットワーク形成に与える影響(OS1-11 : セル・エンジニアリング,マイクロ・ナノバイオメカニクス)

227 細胞外基質の弾性が毛細血管ネットワーク形成に与える影響(OS1-11 : セル・エンジニアリング,マイクロ・ナノバイオメカニクス)

Fibulins: physiological and disease perspectives.

The fibulins are a family of proteins that are associated with basement membranes and elastic extracellular matrix fibres. This review summarizes findings from studies of animal models of fibulin deficiency, human fibulin gene mutations, human tumours and injury models that have advanced our understanding of the normal and pathological roles of members of this formerly obscure family.

Mechanical loading regulates tenascin-C expression in the osteotendinous junction.

Elastic extracellular matrix protein tenascin-C (TN) has very restricted expression in normal tissues, but is expressed in large quantities during embryogenesis and hyperplastic processes. To examine the importance of mechanical stress on the regulation of TN expression in vivo, the effects of various mechanical loading states (immobilization and three forms of subsequent remobilization) on the expression of TN were studied immunohistochemically at the bone-tendon attachment of the rat quadriceps muscle. This osteotendinous junction (OTJ) was selected as study site, since it receives its mechanical stimuli only from muscle contracting activity, which is easy to block by cast immobilization. TN was expressed abundantly in the normal OTJ. Following the removal of the mechanical stress from the junction by cast-immobilization of three weeks, the immunoreactivity of TN was almost completely absent. Normal mechanical stress in the form of free remobilization of eight weeks (free cage activity) resulted in a slight increase in TN expression, but could not restore the expression of TN to the level of the healthy contralateral leg. After the application of the increased mechanical stress (intensified remobilization of the eight weeks by low- or high-intensity treadmill running), the distribution and immunoreactivity of TN reached the level of the healthy contralateral limb in the low-intensity running group or even exceeded that in the high-intensity running group. High TN expression was seen around the chondrocytes and fibroblasts of the OTJ as well as around the collagen fibers of the tendon belly. We conclusively show that mechanical strain regulates the expression of TN in vivo, and propose that mechanical stress is a major regulator of TN expression in fibroblasts and chondrocytes. This may be an important aspect of the regulation of TN expression during embryogenesis, tendon degeneration, wound healing, bone formation, and in the other normal or regenerative morphogenetic processes TN is postulated to take part in.

Identification of chicken and C. elegans fibulin-1 homologs and characterization of the C. elegans fibulin-1 gene.

Fibulin-1, a member of the emerging family of fibulin proteins, is a component of elastic extracellular matrix fibers, basement membranes and blood. Homologs of fibulin-1 have been described in man, mouse and zebrafish. In this study, we describe the isolation and sequencing of chicken fibulin-1C and D cDNA variants. We also describe identification of a C. elegans cDNA encoding fibulin-1D and cosmids containing the C. elegans fibulin-1 gene. Using the cDNA, RT-PCR and computer-based analysis of genomic sequences, the exon/intron organization of the C. elegans fibulin-1 gene was determined. The C. elegans fibulin-1 gene is located on chromosome IV, is approximately 6 kb in length, contains 16 exons and encodes fibulin-1C and D variants. Comparative analysis of the deduced amino acid sequences of nematode and chicken fibulin-1 variants with other known vertebrate fibulin-1 polypeptides showed that the number and organization of structural modules are identical. The results of this study indicate that the structure of the fibulin-1 protein has remained highly conserved over a large period of evolution, suggestive of functional conservation.

Expression of collagens and decorin during aortic arch artery development: Implications for matrix pattern formation

The elastic matrix of the large arteries shows a high level of spatial order. However, the mechanisms by which such order is established and maintained are largely unknown. The embryonic development of the avian heart and great vessels provides an appropriate model to investigate these mechanisms. In control embryos, an elastic matrix with a high level of spatial order develops in the nascent great vessels. But after the normal vascular smooth muscle (VSM) progenitor cells in the great vessels are experimentally replaced by other VSM progenitor cells, the elastic extracellular matrix is congenitally disordered. The present study used this model to test the hypothesis that the proteoglycan decorin was involved in the establishment and maintenance of the normal three-dimensional spatial order of the vascular elastic matrix. The temporospatial expression of decorin was analysed during development of normal vessels and in experimental vessels with surrogate VSM. The results showed the following: (1) the expression of decorin was related in time and space to the establishment of large helical collagen type III fibers that are characteristic of the normal elastic extracellular matrix; (2) in the experimental extracellular matrix there were few helical fibers of collagen type III, but those that were present remained positive for decorin; and (3) in both control and experimental vessels, decorin associated with neither fibers of collagen type I nor fibers of collagen type III in any conformation other than the large helical fibers. These data indicate a previously unrecognized relationship between decorin and the spatial order of the physiologically significant helical fibers of collagen type III.