Kyoko Imanaka-Yoshida

Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure

Big mitogen-activated protein kinase 1 (BMK1), also known as ERK5, is a member of the MAPK family. Genetic ablation of BMK1 in mice leads to embryonic lethality, precluding the exploration of pathophysiological roles of BMK1 in adult mice. We generated a BMK1 conditional mutation in mice in which disruption of the BMK1 gene is under the control of the inducible Mx1-Cre transgene. Ablation of BMK1 in adult mice led to lethality within 2-4 weeks after the induction of Cre recombinase. Physiological analysis showed that the blood vessels became abnormally leaky after deletion of the BMK1 gene. Histological analysis revealed that, after BMK1 ablation, hemorrhages occurred in multiple organs in which endothelial cells lining the blood vessels became round, irregularly aligned, and, eventually, apoptotic. In vitro removal of BMK1 protein also led to the death of endothelial cells partially due to the deregulation of transcriptional factor MEF2C, which is a direct substrate of BMK1. Additionally, endothelial-specific BMK1-KO leads to cardiovascular defects identical to that of global BMK1-KO mutants, whereas, surprisingly, mice lacking BMK1 in cardiomyocytes developed to term without any apparent defects. Taken together, the data provide direct genetic evidence that the BMK1 pathway is critical for endothelial function and for maintaining blood vessel integrity.

Tenascin-C Regulates Recruitment of Myofibroblasts during Tissue Repair after Myocardial Injury

Tenascin-C (TN-C) is an extracellular matrix molecule that is expressed during wound healing in various tissues. Although not detectable in the normal adult heart, it is expressed under pathological conditions. Previously, using a rat model, we found that TN-C was expressed during the acute stage after myocardial infarction and that alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts appeared in TN-C-positive areas. In the present study, we examined whether TN-C controls the dynamics of myofibroblast recruitment and wound healing after electrical injury to the myocardium of TN-C knockout (TNKO) mice compared with wild-type (WT) mice. In TNKO mice, myocardial repair seemed to proceed normally, but the appearance of myofibroblasts was delayed. With cultured cardiac fibroblasts, TN-C significantly accelerated cell migration, alpha-SMA expression, and collagen gel contraction but did not affect proliferation. Using recombinant fragments of murine TN-C, the functional domain responsible for promoting migration of cardiac fibroblasts was mapped to the conserved fibronectin type III (FNIII)-like repeats and the fibrinogen (Fbg)-like domain. Furthermore, alternatively spliced FNIII and Fbg-like domains proved responsible for the up-regulation of alpha-SMA expression. These results indicate that TN-C promotes recruitment of myofibroblasts in the early stages of myocardial repair by stimulating cell migration and differentiation.

Tenascin-C Modulates Adhesion of Cardiomyocytes to Extracellular Matrix during Tissue Remodeling after Myocardial Infarction

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for α-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization.

Tenascin-C is a useful marker for disease activity in myocarditis

Tenascin‐C (TNC) is an extracellular matrix protein which appears at active sites of tissue remodelling during embryogenesis or cancer invasion. In normal heart, TNC is only present during the early stages of development but reappears in pathological states. This study examined the diagnostic value of TNC for assessing disease activity of myocarditis. Expression of TNC was examined in myosin‐induced autoimmune myocarditis mouse models. Sequential changes in amount, localization and the producing cells were analysed by reverse transcriptase–polymerase chain reaction, western blotting, immunohistochemistry and in situ hybridization and compared with the histological picture. The expression of TNC was upregulated at a very early stage of myocarditis. Immunostaining was detectable before cell infiltration and myocytolysis became histologically apparent, remained during the active stage while cell infiltration and necrosis continued, and disappeared in scar tissue with healing. TNC immunostaining was always observed at the periphery of necrotic or degenerating cardiomyocytes in foci of inflammation, the expression level correlating with histological evidence of inflammatory activity. Interstitial fibroblasts were the major source of TNC, expressing the large isoform containing alternative splicing sites. These data demonstrate that TNC is a useful marker for evaluation of disease activity in myocarditis. Copyright

Involvement of Large Tenascin-C Splice Variants in Breast Cancer Progression

Alternative splicing of fibronectin-like type III (FNIII) repeats of tenascin-C (Tn-C) generates a number of splice variants. The distribution of large variants, typical components of provisional extracellular matrices that are up-regulated during tumor stroma remodeling, was here studied by immunoblotting and immunohistochemistry using a monoclonal antibody against the FNIII B domain (named 4C8MS) in a series of human breast cancers. Large Tn-C variants were found at only low levels in normal breast tissues, but were highly expressed at invading sites of intraductal cancers and in the stroma of invasive ductal cancers, especially at invasion fronts. There was a positive correlation between the expression of large Tn-C variants and the cell proliferation rate determined by immunolabeling of the Ki-67 antigen. Of the Tn-C recombinant fragments (all FNIII repeats or mFNIII FL, the conserved FNIII domain only, the epidermal growth factor-like domain, and the fibrinogen-like domain) which were expressed by CHO-K1 cells transfected with mouse Tn-C cDNAs, only the mFNIII FL enhanced in vitro migration and mitotic activity of mammary cancer cells derived from a Tn-C-null mouse. Addition of 4C8MS blocked the function of mFNIII FL. These findings provide strong evidence that the FNIII alternatively spliced region has important roles in tumor progression of breast cancer.

Tenascin-C upregulates matrix metalloproteinase-9 in breast cancer cells: Direct and synergistic effects with transforming growth factor β1

Tenascin‐C (TN‐C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN‐C and MMPs are often co‐localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN‐C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN‐C‐deficient mice to TN‐C and examined MMPs expression. TN‐C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b‐FGF) and transforming growth factor‐beta1 (TGF‐β1). Results of endpoint RT‐PCR, quantitative real‐time RT‐PCR and gelatin zymography demonstrated that TN‐C, strongly and dose dependently, upregulates MMP‐9 expression in murine mammary cancer cells. TN‐C weakly induced MMP‐2, MMP‐3 and MMP‐13. FN and LN induced MMP‐9 to lesser extents compared with TN‐C. b‐FGF had no effect on MMP‐9 expression. TGF‐β1 induced MMP‐9 expression in a dose‐dependent manner, and this induction was significantly enhanced by addition of TN‐C. TN‐C and TGF‐β1 also upregulated MMP‐9 expression in the human breast cancer cell line MDA‐MB‐231. Neutralization with specific anti‐TGF‐β1 antibody showed decreased expression of MMP‐9, indicating that TGF‐β controls the baseline MMP‐9 expression by a direct autocrine mechanism. Under neutralization of TGF‐β, addition of TN‐C still upregulated MMP‐9. Conversely, neutralization of endogenous TN‐C (in a TN‐C‐positive mammary cancer cell line) downregulated TGF‐β‐induced MMP‐9 expression. Thus, TN‐C induces MMP‐9 expression directly and by collaboration with TGF‐β. These findings reveal a novel role of TN‐C in breast cancer progression.

Tenascin-C may aggravate left ventricular remodeling and function after myocardial infarction in mice

Tenascin-C (TN-C) is an extracellular matrix glycoprotein with high bioactivity. It is expressed at low levels in normal adult heart, but upregulated under pathological conditions, such as myocardial infarction (MI). Recently, we (Ref. 34) reported that MI patients with high serum levels of TN-C have a greater incidence of maladaptive cardiac remodeling and a worse prognosis. We hypothesized that TN-C may aggravate left ventricular remodeling. To examine the effects of TN-C, MI was induced by ligating coronary arteries of TN-C knockout (KO) mice under anesthesia and comparing them with sibling wild-type (WT) mice. In WT+MI mice, TN-C expression was upregulated at day 1, peaked at day 5, downregulated and disappeared by day 28, and the molecule was localized in the border zone between intact myocardium and infarct lesions. The morphometrically determined infarct size and survival rate on day 28 were comparable between the WT+MI and KO+MI groups. Echocardiography and hemodynamic analyses demonstrated left ventricular end-diastolic diameter, myocardial stiffness, and left ventricular end-diastolic pressure to be significantly increased in both WT+MI and KO+MI mice compared with sham-operated mice. However, end-diastolic pressure and dimension and myocardial stiffness of KO+MI were lower than those of the WT+MI mice. Histological examination revealed normal tissue healing, but interstitial fibrosis in the residual myocardium in peri-infarcted areas was significantly less pronounced in KO+MI mice than in WT+MI mice. TN-C may thus accelerate adverse ventricular remodeling, cardiac failure, and fibrosis in the residual myocardium after MI.

Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice

Tenascin‐C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune‐mediated hepatitis in wild‐type (WT) and TNC‐null (TNKO) mice. Eight‐week‐old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd‐6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon‐γ, tumour necrosis factor‐α, and interleukin‐4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)‐β1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF‐β expression during progression of hepatitis to fibrosis. Copyright

β1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal α-actin promoter

Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G‐actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differen¬tiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF‐dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA‐dependent skeletal α‐actin promoter ac¬tivation requires βl integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activa¬tion of the α‐actin promoter by RhoA is greatly potenti¬ated (up to 15‐fold) by co‐expression of the integrin βlA or βlD isoform but is significantly reduced by 70% with a co‐expressed dominant negative mutant of βl integrin. Furthermore, clustering of βl integrin with anti‐βl integrin antibodies potentiates synergistic RhoA and βl integrin activation of the α‐actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA‐induced activation of the α‐actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and βl integrin on the actin promoter. These observations sup¬port the concept that RhoA regulates SRF‐dependent cardiac gene expression through cross‐talk with βl integrin signal pathway via an organized actin cytoskeleton.— Wei, L., Wang, L., Carson, J. A., Agan, J. E., ImanakaYoshida, K., and Schwartz, R. J. βl integrin and organized actin filaments facilitate cardiomyocyte‐specific RhoA‐de¬pendent activation of the skeletal α‐actin promoter. FASEBJ. 15, 785‐796 (2001)

Effects of Aging on Left Ventricular Relaxation in Humans Analysis of Left Ventricular Isovolumic Pressure Decay

BACKGROUND Some experimental studies in animals have shown that myocardial relaxation is prolonged with aging. However, it is not known whether aging alters ventricular isovolumic relaxation in human subjects. METHODS AND RESULTS We analyzed high-fidelity left ventricular pressures, measured by use of a catheter-tipped manometer, and biplane left ventriculograms in 55 normal subjects who underwent diagnostic cardiac catheterization but who were found to have normal cardiac anatomy and function. There were 38 men and 17 women, ranging in age from 20 to 77 years. Left ventricular isovolumic relaxation was assessed by the exponential time constants of isovolumic pressure decay with (Tb) and without (Tw) an asymptote pressure. Left ventricular volume, ejection fraction, and wall thickness or mass were calculated from left ventricular angiograms. Neither of the time constants of left ventricular relaxation correlated with age (Tb: r = .001 to .10, P = NS: Tw: r = .02 to .05, P = NS). Left ventricular systolic function (ie, ejection fraction and end-systolic volume index), heart rate, and left ventricular wall thickness or mass, which are major hemodynamic determinants of left ventricular relaxation, were not significantly affected by aging. The multivariate analysis of age and hemodynamic variables against the time constants of left ventricular relaxation also indicated that no significant relation was found between age and left ventricular relaxation. CONCLUSIONS In the absence of coronary artery disease, systemic hypertension, left ventricular systolic dysfunction, or hypertrophy, left ventricular relaxation assessed by the time constant of isovolumic pressure decay remains essentially unchanged with normal adult aging, at least until the eighth decade.