High expression of the extracellular matrix component tenascin-C in the tumor microenvironment correlates with decreased patient survival. Tenascin-C promotes cancer progression and a disrupted tumor vasculature through an unclear mechanism. Here, we examine the angiomodulatory role of tenascin-C. We find that direct contact of endothelial cells with tenascin-C disrupts actin polymerization, resulting in cytoplasmic retention of the transcriptional coactivator YAP. Tenascin-C also downregulates YAP pro-angiogenic target genes, thus reducing endothelial cell survival, proliferation, and tubulogenesis. Glioblastoma cells exposed to tenascin-C secrete pro-angiogenic factors that promote endothelial cell survival and tubulogenesis. Proteomic analysis of their secretome reveals a signature, including ephrin-B2, that predicts decreased survival of glioma patients. We find that ephrin-B2 is an important pro-angiogenic tenascin-C effector. Thus, we demonstrate dual activities for tenascin-C in glioblastoma angiogenesis and uncover potential targeting and prediction opportunities.

BACKGROUND:The clinical implications of the 2010-2012 low-end shift of high-sensitivity cardiac troponin T (hs-cTnT) regarding possible misdiagnosis of acute myocardial infarction are largely unknown.METHODS:We aimed to quantify the impact of the 2010-2012 low-end shift and adjustment issue in 857 patients presenting to the emergency department with suspected acute myocardial infarction by comparing measurements performed with affected 2010-2012 lots with recalculated 2010-2012 values using a linear regression formula (provided by the manufacturer) and the corrected assay (re-measured in 2013). The final diagnosis was adjudicated by two independent cardiologists using all information including coronary angiography, echocardiography and serial hs-cTnT levels (with the corrected 2013 assay).RESULTS:Acute myocardial infarction was the adjudicated diagnosis in 195 patients (22.7%). Median hs-TnT values were 8.5 ng/l for affected lots, 11.1 ng/l with recalculated and 10 ng/l with the corrected assay (P<0.001 for all comparisons). Spearman correlation coefficient was 0.937 (<0.001) for correct and affected respective correct and recalculated values. The Cusum test indicated significant deviation from linearity (P<0.01) for both correlations. Deviations nearly exclusively affected hs-cTnT levels below the 99th percentile (14 ng/L). Among the 195 patients with an adjudicated diagnosis of acute myocardial infarction, no patient was misclassified using affected lots if using conventional serial sampling. In contrast, misdiagnosis of acute myocardial infarction was significantly increased by affected lots if applying the novel ESC 0 h/1 h algorithm for the early rule-out of acute myocardial infarction (negative predictive value with affected lots 97.7% versus 99.7% with corrected lots).CONCLUSION:The 2010-2012 hs-cTnT low-end shift affected nearly exclusively levels below the 99th percentile cut-off. While it did not affect the diagnosis of acute myocardial infarction when using conventional serial sampling as done in 2010-2012, it would impact on new early rule-out strategies using very low levels of hs-cTnT such as the ESC 0 h/1 h algorithm.CLINICAL TRIALS REGISTRATION:NCT0047058, NCT00470587.

The extracellular matrix (ECM) molecule tenascin-C (TNC) promotes tumor progression. This has recently been demonstrated in the stochastic murine RIP1-Tag2 insulinoma model, engineered to either express TNC abundantly or to be devoid of TNC. However, our knowledge about organization of the TNC microenvironment is scant. Here we determined the spatial distribution of TNC together with other ECM molecules in murine RIP1-Tag2 insulinoma and human cancer tissue (insulinoma and colorectal carcinoma). We found that TNC is organized in matrix tracks together with other ECM molecules of the AngioMatrix signature, a previously described gene expression profile that characterizes the angiogenic switch. Moreover, stromal cells including endothelial cells, fibroblasts and leukocytes were enriched in the TNC tracks. Thus, TNC tracks may provide niches for stromal cells and regulate their behavior. Given similarities of TNC rich niches for stromal cells in human insulinoma and colon cancer, we propose that the RIP1-Tag2 model may be useful for providing insights into the contribution of the tumor stroma specific ECM as promoter of cancer progression.

Background— Misdiagnosis of acute myocardial infarction (AMI) may significantly harm patients and may result from inappropriate clinical decision values (CDVs) for cardiac troponin (cTn) owing to limitations in the current regulatory process. Methods and Results— In an international, prospective, multicenter study, we quantified the incidence of inconsistencies in the diagnosis of AMI using fully characterized and clinically available high-sensitivity (hs) cTn assays (hs-cTnI, Abbott; hs-cTnT, Roche) among 2300 consecutive patients with suspected AMI. We hypothesized that the approved CDVs for the 2 assays are not biologically equivalent and might therefore contribute to inconsistencies in the diagnosis of AMI. Findings were validated by use of sex-specific CDVs and parallel measurements of other hs-cTnI assays. AMI was the adjudicated diagnosis in 473 patients (21%). Among these, 86 patients (18.2%) had inconsistent diagnoses when the approved uniform CDV was used. When sex-specific CDVs were used, 14.1% of female and 22.7% of male AMI patients had inconsistent diagnoses. Using biologically equivalent CDV reduced inconsistencies to 10% (P<0.001). These findings were confirmed with parallel measurements of other hs-cTn assays. The incidence of inconsistencies was only 7.0% for assays with CDVs that were nearly biologically equivalent. Patients with inconsistent AMI had long-term mortality comparable to that of patients with consistent diagnoses (P=NS) and a trend toward higher long-term mortality than patients diagnosed with unstable angina (P=0.05). Conclusions— Currently approved CDVs are not biologically equivalent and contribute to major inconsistencies in the diagnosis of AMI. One of 5 AMI patients will receive a diagnosis other than AMI if managed with the alternative hs-cTn assay. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00470587.

Angiogenesis represents a rate-limiting step during tumor progression. Targeting angiogenesis is already applied in cancer treatment, yet limits of anti-angiogenic therapies have emerged, notably because tumors adapt and recur after treatment. Therefore, there is a strong need to better understand the molecular and cellular mechanisms underlying tumor angiogenesis. Using the RIP1-Tag2 transgenic murine model, we identified 298 genes that are deregulated during the angiogenic switch, revealing an ingression/expansion of specific stromal cell types including endothelial cells and pericytes, but also macrophages and perivascular mesenchymal cells. Canonical TGF-β signaling is up-regulated during the angiogenic switch, especially in tumor-associated macrophages and fibroblasts. The matrisome, comprising extracellular matrix (ECM) and ECM-associated molecules, is significantly enriched, which allowed us to define the AngioMatrix signature as the 110 matrisomal genes induced during the RIP1-Tag2 angiogenic switch. Several AngioMatrix molecules were validated at expression level. Ablation of tenascin-C, one of the most highly induced ECM molecules during the switch, resulted in reduced angiogenesis confirming its important role. In human glioma and colorectal samples, the AngioMatrix signature correlates with the expression of endothelial cell markers, is increased with tumor progression and finally correlates with poor prognosis demonstrating its diagnostic and therapeutic potential.

Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LMα1 and LMα5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LMα1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LMα1 or LMα5 specifically in the intestine. We demonstrated that high LMα1 or LMα5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LMα1 and LMα5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach.

Objective Copeptin, a quantitative marker of endogenous stress, seems to provide incremental value in addition to cardiac troponin in the early rule-out of acute myocardial infarction (AMI). Prevalence, characteristics and outcome of acute chest pain patients with causes other than AMI and elevated copeptin are poorly understood.Methods A total of 984 consecutive patients with non-cardiac chest pain were selected from a prospective multicentre study of acute chest pain patients presenting to the emergency department. Levels of copeptin were determined in a blinded fashion and considered elevated if above 13 pmol/L (the 97,5th centile of healthy individuals). The final diagnosis was adjudicated by two independent cardiologists. Median duration of follow-up was 756 days.Results Elevated copeptin levels were seen in 215 patients (22%). In comparison to patients with normal copeptin levels, patients with elevated levels were older, had more pre-existing cardiac and non-cardiac disorders, more silent cardiomyocyte injury and increased haemodynamic stress as quantified by levels of high-sensitivity cardiac troponin T (9.6 ng/L (3.6-18.3) vs 5.8 ng/L (2.9-9.4)) and B-type natriuretic peptide (75 ng/L (37-187) vs 35 ng/L (15-77)) (both p<0.001), more electrocardiographic abnormalities, more often an adjudicated diagnosis of gastroesophageal reflux or bronchitis/pneumonia and higher 2-year mortality (HR 2.9, 95% CI 1.5 to 5.7). The increased mortality rate seemed to be largely explained by age and comorbidities.Conclusions Elevated levels of copeptin are present in about one in five patients with non-cardiac chest pain and are associated with aging, cardiac and non-cardiac comorbidities as well as mortality.

The extracellular matrix molecule tenascin-C (TNC) is a major component of the cancer-specific matrix, and high TNC expression is linked to poor prognosis in several cancers. To provide a comprehensive understanding of TNC's functions in cancer, we established an immune-competent transgenic mouse model of pancreatic β-cell carcinogenesis with varying levels of TNC expression and compared stochastic neuroendocrine tumor formation in abundance or absence of TNC. We show that TNC promotes tumor cell survival, the angiogenic switch, more and leaky vessels, carcinoma progression, and lung micrometastasis. TNC downregulates Dickkopf-1 (DKK1) promoter activity through the blocking of actin stress fiber formation, activates Wnt signaling, and induces Wnt target genes in tumor and endothelial cells. Our results implicate DKK1 downregulation as an important mechanism underlying TNC-enhanced tumor progression through the provision of a proangiogenic tumor microenvironment.

Background— Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α 1 β 1 γ 1 ) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α 2 , α 4 , or α 5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. Methods and Results— Using an in vitro flow assay, we show that laminin-411 (α 4 β 1 γ 1 ), laminin-511 (α 5 β 1 γ 1 ), and laminin-521 (α 5 β 2 γ 1 ), but not laminin-211 (α 2 β 1 γ 1 ), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α 6 β 1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α 5 -containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α 6 . Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α 6 β 1 -Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. Conclusions— This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α 6 β 1 , could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here.

Hirschsprung disease (HD), a developmental disorder, is associated with failure of enteric ganglia formation. Signaling molecules, including secreted basement membrane molecules, derived from the mesenchyme of the gut wall play an important role in the colonization and/or differentiation of the enteric nervous system. The current study aims to define the possible alterations of laminins involved in the pathogenesis of HD. Expression of the various laminin α, β, and γ chains, was assessed in the aganglionic, transitional, and ganglionic bowel segments of patients with HD or with other motor disorders. Cytoskeletal, neuronal, and glial markers were also included in this study. The major finding highlighted by the present work concerns the clear identification and location of myenteric aganglionic plexuses in HD with some of the laminin antibodies, which reveal a peripheral nerve type of differentiation. Furthermore, we could show an increase of laminin α5 chain immunostaining in the dilated muscle of the ganglionic bowel upstream the distal aganglionic region in a subgroup of patients with HD, as well as a relocalization of laminin α2 chain in the subepithelial basement membrane. Overall, these basement membrane molecules could provide useful markers for diagnosis of aganglionosis or hypoganglionosis.

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (α1, β1, γ1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin α1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed‐allele‐carrying mice (Lama1 flox/flox ) display no overt phenotype. Lama1 flox/flox mice were crossed with transgenic deleter mice (CMV‐Cre) to generate Lama1‐deficient mice (Lama1 Δ/Δ ). Lama1 Δ/Δ embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin α1 is normally highly expressed. In parallel, Lama1 Δ/Δ embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time‐ and tissue‐specific inactivation of the Lama1 gene in various organs. genesis 43:59–70, 2005. © 2005 Wiley‐Liss, Inc.

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the lammin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that lammin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelia] basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent. (C) 2003 Elsevier Science (USA). All rights reserved.

Epithelial cell proliferation and differentiation occur concomitant with striking remodeling of the cytoskeleton. Microtubules (MTs) play important roles in these processes, during which the MTs themselves are reorganized and stabilized by microtubule-associated proteins (MAPs). Among the proteins classified as structural MAPs, E-MAP-115 (also named ensconsin) is preferentially expressed in cells of epithelial origin. The aims of this study were, first, to determine if E-MAP-115, like other MAPs, is expressed as different isoforms during differentiation and, second, to perform a detailed analysis of the expression and distribution of any E-MAP-115 variants detected in intestinal epithelial cells during their polarization/differentiation. It was our expectation that these data would help us to develop hypotheses concerning the role of this MAP in epithelial development. We report the expression of three E-MAP-115 transcripts encoding isoforms of 115, 105, and 95 kDa; two display an expression gradient inverse to the third one as Caco-2 cells progress from proliferation through the stages of differentiation. To monitor the proteins produced from each transcript, we used purified polyclonal antibodies against synthetic peptides contained within the 115, 105, and 95 kDa isoforms to assay proliferating and differentiating CaCo-2 cells. Our results indicate that the expression and MT-binding capacity of the 115, 105, and 95 kDa isoforms vary upon proliferation/differentiation of the cells. E-MAP-115 proteins colocalize with MTs in proliferative and differentiated Caco-2 cells; in vivo, they are expressed in both crypt and villus epithelial cells where they are mainly concentrated at the apical pole of the cells.

Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.