Toshimichi Yoshida

Ischemic Delayed Neuronal Death: A Mitochondrial Hypothesis

BACKGROUND A brief period of global brain ischemia causes cell death in hippocampal CA1 pyramidal neurons days after reperfusion in rodents and humans. Other neurons are much less vulnerable. This phenomenon is commonly referred to as delayed neuronal death, but the cause has not been fully understood although many mechanisms have been proposed. SUMMARY OF REVIEW Hippocampal CA1 neuronal death usually occurs 3 to 4 days after an initial ischemic insult. Such a delay is essential for the mechanism of this type of cell death. Previous hypotheses have not well explained the reason for the delay and the exact mechanism of the cell death, but a disturbance of mitochondrial gene expression could be a possibility. Reductions of mitochondrial RNA level and the activity of a mitochondrial protein, encoded partly by mitochondrial DNA, occurred exclusively in CA1 neurons at the early stage of reperfusion and were aggravated over time. In contrast, the activity of a nuclear DNA-encoded mitochondrial enzyme and the level of mitochondrial DNA remained intact in CA1 cells until death. Immunohistochemical staining for cytoplasmic dynein and kinesin, which are involved in the shuttle movement of mitochondria between cell body and the periphery, also showed early and progressive decreases after ischemia, and the decreases were found exclusively in the vulnerable CA1 subfield. CONCLUSIONS A disturbance of mitochondrial DNA expression may be caused by dysfunction of the mitochondrial shuttle system and could cause progressive failure of energy production of CA1 neurons that eventually results in cell death. Thus, the mitochondrial hypothesis could provide a new and exciting potential for elucidating the mechanism of the delayed neuronal death of hippocampal CA1 neurons.

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.

Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow.

The small GTPase Rho and one of its targets, Rho-kinase (also termed ROK or ROCK), are implicated in various cellular functions including stress fiber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rho-kinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was significantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow.

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.

Expression of fibronectin and tenascin-C mRNA by myofibroblasts, vascular cells and epithelial cells in human colon adenomas and carcinomas

To understand the mechanisms of tissue remodeling during cancer progression, it is important to know the type of cells that actively express extracellular matrix (ECM) proteins. Twenty‐nine adenocarcinomas, 5 adenomas and non‐neoplastic mucosa samples were therefore investigated to determine their fibronectin (FN) and tenascin‐C (TN‐C) expression using in situ hybridization (ISH) and immunohistochemical staining. In the non‐neoplastic mucosa, no mRNA signals were found. Two of the adenomas demonstrated positive signals in peri‐cryptal cells and the vessels. In the cancers, TN‐C and FN mRNAs were found in 86% and 96% of the total cases, respectively. The signals were mainly detected in myofibroblasts, labeled with α‐smooth muscle actin, in the cancer stroma. TN‐C mRNA‐positive cells were often observed in localized areas, such as in cancer stroma associated with invading edges and/or in host tissues surrounding the invading cancer front, but rarely in the center of the tumors. FN mRNA‐positive cells were more widely spread throughout the cancer stroma, although they were also frequently observed at invading edges. Vascular cells in cancer tissues were also labeled. In 10 specimens, cancer cells themselves expressed FN and/or TN‐C mRNA. Comparison with histo‐pathological findings revealed positive relationships between the degree of mRNA expression of FN and TN‐C and the depth of invasion as well as the frequency of metastasis to lymph nodes. The expression of FN and TN‐C by myofibroblasts, vascular cells and cancer cells could be important for the remodeling process of neoplastic tissues during cancer development and progression. Int. J. Cancer 73:10–15, 1997.

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.