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Interstitial fibrosis is a histopathological hallmark of hypertrophic cardiomyopathy (HCM). Although extracellular matrix (ECM) biomarkers, including matrix metalloproteinases, are overexpressed in HCM patients, they do not correlate with sudden cardiac death (SCD) risk. The objective of this study was to determine whether scleraxis, a transcription factor that regulates collagen gene expression, is detectable in HCM patients and correlates with disease burden. Between 2017 and 2018, a total of 46 HCM patients were enrolled (58 [+ or -] 14 years (31 males, 15 females)) with a mean 5 year SCD risk of 2.3% [+ or -] 1.3%. Cardiac MRI confirmed HCM in all patients with a mean interventricular septal thickness of 20 [+ or -] 2 mm. Late gadolinium enhancement (LGE) was present in 32 (70%) study participants occupying 18% [+ or -] 7% of the left ventricular (LV) myocardium. Serum scleraxis levels were significantly higher in the HCM patients by approximately twofold as compared to controls (0.76 [+ or -] 0.06 versus 0.32 [+ or -] 0.02 ng/mL, p < 0.05). No correlation was demonstrated between serum scleraxis levels and markers of disease severity in HCM patients, including maximum LV wall thickness, %LGE, and SCD risk factors. Serum scleraxis is elevated in the HCM population. Future studies are warranted to evaluate the prognostic value of scleraxis in identifying high-risk HCM patients who require aggressive management for prevention of SCD. Key words: hypertrophic cardiomyopathy, scleraxis, collagen, cardiac fibrosis, transthoracic echocardiography, cardiac MRI, extracellular matrix. La fibrose interstitielle est la marque histopathologique de la cardiomyopathie hypertrophique (CMH). Bien que des biomarqueurs de la matrice extracellulaire (MEC), y compris les metalloproteinases matricielles, soient surexprimes chez les patients atteints de CMH, ils ne sont pas en correlation avec le risque de mort subite cardiaque (MSC). Cette etude avait pour objectif d'etablir si le facteur scleraxis, qui participe a la transcription regulant l'expression des genes du collagene, est detectable chez les patients atteints de CMH, et s'il est en correlation avec le fardeau de la maladie. De 2017 a 2018, nous avons inscrit a l'etude 46 patients atteints de CMH (ages de 58 [+ or -] 14 ans (31 hommes, 15 femmes)) presentant un risque de MSC a 5 ans moyen de 2,3 % [+ or -] 1,3 %. L'examen d'IRM cardiaque a permis de confirmer que tous les patients atteints de CMH presentaient un septum interventriculaire d'une epaisseur moyenne de 20 [+ or -] 2 mm. Le rehaussement tardif apres gadolinium (RTG) etait present chez 32 (70 %) des participants a l'etude avec une occupation du myocarde ventriculaire gauche (VG) de 18 % [+ or -] 7 %. Les taux seriques du facteur scleraxis etaient nettement plus eleves chez les patients atteints de CMH, d'environ le double par rapport aux temoins (0,76 [+ or -] 0,06 par rapport a 0,32 [+ or -] 0,02 ng/mL, p < 0,05). Chez les patients atteints de CMH, nous n'avons observe aucune correlation entre les taux seriques du facteur scleraxis et des marqueurs de la gravite de la maladie, y compris en matiere d'epaisseur maximale du VG, de pourcentage de RTG et de facteurs de risque de MSC. Les taux seriques de facteur scleraxis sont eleves chez les patients atteints de CMH. De futures etudes seraient necessaires en vue d'evaluer la valeur pronostique du facteur scleraxis en vue de depister les patients atteints de CMH a risque eleve, qui necessitent une prise en charge energique en vue de prevenir la MSC. Mots-des : cardiomyopathie hypertrophique, facteur scleraxis, collagene, fibrose cardiaque, echocardiographie transthoracique, IRM cardiaque, matrice extracellulaire., Introduction Hypertrophic cardiomyopathy (HCM) is a heritable disorder of the heart muscle with a well-described genetic, pathological, and clinical profile (Loar et al. 2015). As an autosomal dominant disease caused [...]

The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome., Introduction Hennekam syndrome (OMIM #235510 and #616006) is an autosomal-recessive disorder characterized by congenital lymphedema and lymphangiectasia, unusual facial morphology, attributed at least in part to intrauterine facial lymphedema, and [...]

Poster Board No. 001 - EXAMINING DIFFERENTIAL EXPRESSION OF COL9A3 BETWEEN CAVE AND SURFACE MORPHOTYPES OF ASTYANAX MEXICANUS TO EXPLAIN PHENOTYPIC DIFFERENCES. Michael A. Warden (1), mawarden2@gmail.com, 1410 Blackstone Place, [...]

Fibrosis, which is characterized by the excessive production of matrix proteins, occurs in multiple tissues and is associated with increased morbidity and mortality. Despite its significant negative impact on patient outcomes, therapies targeted to treat fibrosis are currently lacking. Screening for inhibitors of the expression of collagen, the primary component of fibrotic lesions, represents an option for the identification of novel lead compounds for therapeutic development with potentially fewer off-target effects compared with the targeting of multifunctional cell signaling pathways. Here we report on the generation of a stable luciferase reporter system using a fibroblast cell line, which can be used for rapidly screening both activators and repressors of human collagen COL1A2 gene transcription in a high throughput setting. This in vitro screening tool was validated using known agonists (scleraxis, TGF-β, angiotensin II, CTGF) and antagonists (TNF-α, pirfenidone) of COL1A2 gene expression. The COL1A2-luc NIH-3T3 fibroblast system provides a useful and effective screen for potential lead compounds with pro- or anti-fibrotic properties. Key words: collagen, luciferase reporter, stable cell line, in vitro screening assay, fibrosis, high-throughput assay, fibroblast. La fibrose, caracterisee par une production excessive de proteines de la matrice extracellulaire, survient dans une multitude de tissus, et elle est associee avec un accroissement de morbidite et de mortalite a l'echelle mondiale. Malgre son impact negatif significatif sur le patient, des therapies visant a traiter la fibrose font actuellement defaut. Le criblage d'inhibiteurs de l'expression du collagene, la premiere composante des lesions fibreuses, constitue une option afin d'identifier de nouveaux medicaments tetes de serie en vue d'un developpement therapeutique qui presenteraient potentiellement moins d'effets hors cible, comparativement a cibler des voies de signalisation cellulaire multifonctionnelles. Les auteurs rapportent ici la production d'un systeme rapporteur a la luciferase stable, a l'aide d'une lignee de fibroblastes qui peut etre utilisee pour cribler rapidement tant les activateurs que les represseurs de la transcription du gene COL1A2 codant le collagene humain, dans un procede a haut debit. Cet outil de criblage in vitro a ete valide a l'aide d'agonistes (scleraxis, TGF-β, angiotensine II, CTGF) et d'antagonistes (TNF-α, pirfenidone) connus de l'expression genique de COL1A2. Le systeme COL1A2- luciferase/fibroblastes NIH-3T3 constitue un outil de criblage utile et efficace afin d'identifier des composes tetes de serie potentiels, possedant des proprietes pro- et anti-fibrogenes. [Traduit par la Redaction] Mots-cles: collagene, rapporteur luciferase, lignee cellulaire stable, test de criblage in vitro, fibrose, test a haut- debit, fibroblaste., Introduction The extracellular matrix (ECM) is a complex and dynamic structure that provides cells and organs with mechanical and functional support. This compartment of our body's architecture is primarily composed [...]

The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation. Previous investigators have induced ATDC5 cells to differentiate by exposing them to insulin at high concentrations. We have observed spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing [alpha]-MEM. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X. We took advantage of the action of ascorbic acid to examine signaling events associated with differentiation. In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation. Furthermore, ascorbic acid exposure was associated with ERK activation, and ERK inhibition attenuated ascorbic acid-induced differentiation. This was in contrast to the inhibitory effect of ERK activation during IGF-I-induced differentiation. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. Our results indicate that matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation. We conclude that ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway, which promotes the differentiation program. ATDC5 cells; chondrocyte differentiation; collagen; extracellular matrix doi: 10.1152/ajpendo.00145.2010.

Mohawk (Mkx) is a member of the Three Amino acid Loop Extension superclass of atypical homeobox genes that is expressed in developing tendons. To investigate the in vivo functions of Mkx, we generated [Mkx.sup.-/-] mice. These mice had hypoplastic tendons throughout the body. Despite the reduction in tendon mass, the cell number in tail tendon fiber bundles was similar between wild-type and [Mkx.sup.-/-] mice. We also observed small collagen fibril diameters and a down-regulation of type I collagen in [Mkx.sup.-/-] tendons. These data indicate that Mkx plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells. doi/ 10.1073/pnas.1000525107

Transforming growth factor (TGF)-[beta] is a central mediator in the progression of glomerulosclerosis, leading to accumulation of aberrant extracellular matrix proteins and inappropriate expression of smooth muscle [alpha]-actin in the kidney. Previously, we reported that disrupting the cytoskeleton diminished TGF-[beta]-stimulated type I collagen accumulation in human mesangial cells. As cytoskeletal signaling molecules, including the Rho-family GTPases, have been implicated in fibrogenesis, we sought to determine the respective roles of RhoA and Rac1 in HMC collagen I expression. TGF-[beta]1 activated both RhoA and Rac1 within 5 min of treatment, and this activation was dependent on the kinase activity of the type I TGF-[beta] receptor. TGF-[beta]1-stimulated induction of type I collagen mRNA expression and promoter activity was diminished by inhibiting Rac1 activity and was increased by a constitutively active Rac1 mutant, whereas inhibiting RhoA activity had no such effect. Rac1 activation required phosphatidylinositol-3-kinase (PI3K) activity. Furthermore, the PI3K antagonist, LY294002, reduced TGF-[beta]1-stimulated COL1A2 promoter activity and Rac1 activation. It also partially blocked active Rac1-stimulated collagen promoter activity, suggesting that PI3K activity contributes to both TGF-[beta] activation of Rac1 and signal propagation downstream of Rac1. Thus, while both Rac1 and RhoA are rapidly activated in response to TGF-[beta]1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving PI3K. kidney; fibrosis; Rho-GTPase doi: 10.1152/ajprenal.00345.2009.

Physical forces generated by cells drive morphologic changes during development and can feedback to regulate cellular phenotypes. Because these phenomena typically occur within a 3-dimensional (3D) matrix in vivo, we used microelectromechanical systems (MEMS) technology to generate arrays of microtissues consisting of cells encapsulated within 3D micropatterned matrices. Microcantilevers were used to simultaneously constrain the remodeling of a collagen gel and to report forces generated during this process. By concurrently measuring forces and observing matrix remodeling at cellular length scales, we report an initial correlation and later decoupling between cellular contractile forces and changes in tissue morphology. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that cellular forces increased with boundary or matrix rigidity whereas levels of cytoskeletal and extracellular matrix (ECM) proteins correlated with levels of mechanical stress. By mapping these relationships between cellular and matrix mechanics, cellular forces, and protein expression onto a bio-chemo-mechanical model of microtissue contractility, we demonstrate how intratissue gradients of mechanical stress can emerge from collective cellular contractility and finally, how such gradients can be used to engineer protein composition and organization within a 3D tissue. Together, these findings highlight a complex and dynamic relationship between cellular forces, ECM remodeling, and cellular phenotype and describe a system to study and apply this relationship within engineered 3D microtissues. biomechanics | cell mechanics | collagen | morphogenesis | PDMS

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.08.039 Byline: Roy Morello (1), Terry K. Bertin (1), Yuqing Chen (1)(11), John Hicks (2), Laura Tonachini (3), Massimiliano Monticone (3), Patrizio Castagnola (3), Frank Rauch (4)(5), Francis H. Glorieux (4)(5), Janice Vranka (6)(7), Hans Peter Bachinger (6)(7), James M. Pace (8), Ulrike Schwarze (8), Peter H. Byers (8), MaryAnn Weis (9), Russell J. Fernandes (9), David R. Eyre (9), Zhenqiang Yao (10), Brendan F. Boyce (10), Brendan Lee (1)(11)(12) Abstract: Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease. Author Affiliation: (1) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA (2) Department of Pathology, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030, USA (3) Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy (4) Genetics Unit, Shriners Hospital for Children, Montreal, Canada (5) McGill University, Montreal, Canada (6) Shriners Hospital for Children, Portland, OR 97201, USA (7) Department of Biochemistry and Molecular Biology, Oregon Health & Science University, OR 97239, USA (8) Department of Pathology, University of Washington, Seattle, WA 98195, USA (9) Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA 98195, USA (10) Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14627, USA (11) Howard Hughes Medical Institute, Houston, TX 77030, USA (12) One Baylor Plaza, Room 635E, Houston, TX 77030, USA Article History: Received 31 January 2006; Revised 4 May 2006; Accepted 18 August 2006 Article Note: (miscellaneous) Published: October 19, 2006

The nematode exoskeleton is a multilayered structure secreted by the underlying hypodermal cells and mainly composed of small collagens, which are encoded by a large gene family. In previous work, we reported analysis of the C. elegans dpy-31 locus, encoding a hypodermally expressed zinc-metalloprotease of the BMP-1/TOLLOID family essential for viability and cuticle deposition. We have generated a large set of extragenic suppressors of dpy-31 lethality, most of which we show here to be allelic to tile cuticle collagen genes sqt-3 and dpy-17. We analyzed the interaction among dpy,-31, sqt-3, and dpy-17 using a SQT-3-specific antiserum, which was employed in immunofluorescence experiments. Our results support a role for DPY-31 in SQT-3 extracellular processing and suggest that the SQT-3 C-terminal nontrimeric region serves multiple roles during SQT-3 assembly. Different missense mutations of tiffs region have diverse phenotypic consequences, including cold-sensitive lethality. Furthermore, the biochemical and genetic data indicate that the extracellular assemblies of DPY-17 and SQT-3 are interdependent, most likely because the collagens are incorporated into the same cuticular substructure. We find that absence of DPY-17 causes extensive intracellular retention of SQT-3, indicating that formation of the SQT-3-DPY-17 polymer could begin in the intracellular environment before secretion.

Restriction fragment length polymorphisms are good anthropological markers for discriminating geographically distinct populations at both the allele and the haplotype level. Two communities of African ancestry and ladinos, mestizos, and mulattoes living in the Esmeraldas province in northwestern Ecuador were analyzed for three RFLPs (EcoRI, RsaI, and MspI) of the COL1A2 gene. Also, the same markers were studied in a population sample from Spain to compare the allele and haplotype frequencies of the Esmeraldas populations with those of their representative European parental population. Data for the native American and sub-Saharan African founder components were available from the literature. No significant levels of differentiation between the two African Ecuadoran communities emerged from either the frequency analysis of each single marker and all three RFLP markers together or from the AMOVA. The ladinos and mestizos also showed a rather similar distribution of allele and haplotype frequencies, confirming that the two ethnic terms do not correspond to genetically different populations. The comparison with the supposed founding European, sub-Saharan African, and native American populations indicated a large presence of African genes in the gene pool of both communities, with a higher proportion of the Amerindian component in Viche than in Rio Cayapas. The present findings confirm the previous genetic admixture estimates based on nuclear and mitochondrial DNA markers and the demographic data. KEY WORDS: COL1A2, EcoRI, RsaI, MspI, ECUADORAN MIXED POPULATIONS, SPANISH POPULATION, ECUADOR., The population of Ecuador includes ethnically and genetically distinct groups besides native Americans, who make up nearly half the total population. Other groups include the ladinos, who are descended from [...]

EPh kinases and their ephrin ligands are widely expressed in epithelial cells in vitro and in vivo. Our results show that activation of endogenous EphA kinases in Madin-Darby canine kidney (MDCK) cells negatively regulates hepatocyte growth factor/scatter factor (HGF)-induced branching morphogenesis in collagen gel. Cotreatment with HGF and ephrin-A1 reduced sprouting of cell protrusions, an early step in branching morphogenesis. Moreover, addition of ephrin-A1 after HGF stimulation resulted in collapse and retraction of preexisting cell protrusions. In a newly developed assay that simulates the localized interactions between Ephs and ephrins in vivo, immobilized ephrin-A1 suppressed HGF-induced MDCK cell scattering. Ephrin-A1 inhibited basal ERK1/2 mitogen-activated protein kinase activity; however, the ephrin-A1 effect on cell protrusion was independent of the mitogen-activated protein kinase pathway. Ephrin-A1 suppressed HGF-induced activation of Rac1 and p21-activated kinase, whereas RhoA activation was retained, leading to the preservation of stress fibers. Moreover, dominant-negative RhoA or inhibitor of Rho-associated kinase (Y27632) substantially negated the inhibitory effects of ephrin-A1. These data suggest that interfering with c-Met signaling to Rho GTPases represents a major mechanism by which EphA kinase activation inhibits HGF-induced MDCK branching morphogenesis.

Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet--collagen interaction, adhesion is mediated via the inside-out regulation of integrin [[alpha].sub.2][[beta].sub.1] and activation through the glycoprotein VI (GPVI)--Fc receptor (FcR) [gamma]-chain complex. The present study extends this model by demonstrating that engagement of [[alpha].sub.2][[beta].sub.1] by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase--based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI-FcR [gamma]-chain complex. The same events do not occur in platelet suspensions. [[alpha].sub.2][[beta].sub.1] activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI-FcR [gamma]-chain cascade, including Src, Syk, SLP-76, and PLC[gamma]2 as well as plasma membrane calcium ATPase and focal adhesion kinase. [[alpha].sub.2][[beta].sub.1]-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLC[gamma]2-deficient platelets. Spreading is abolished by chelation of intracellular [Ca.sup.2+]. Demonstration that adhesion of platelets to collagen via [[alpha].sub.2][[beta].sub.1] generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of [[beta].sub.1]-deficient thrombi and why loss of the GPVI-FcR [gamma]-chain complex has a relatively minor effect on bleeding.