DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility.

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

A new cytosolic pathway from a Parkinson disease-associated kinase, BRPK/PINK1: activation of AKT via mTORC2.

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

Expression of REIC/Dkk-3 in normal and hyperproliferative epidermis.

Dickkopf (Dkk) family members are known as Wnt modulators involved in the development, cell growth/differentiation and cancer. REIC/Dkk-3, which does not interfere with Wnt signalling, has been proposed to be a tumor suppressor gene, but its physiological function has remained unclear. In this study, we analysed the expression of REIC/Dkk-3 in normal interfollicular epidermis (IFE) and hyperproliferative epidermis. REIC/Dkk-3 was expressed in human and mouse IFE, being localized at the interface of upper spinous layer and granular layer. Skin cancer cell lines lost REIC/Dkk-3 expression as reported previously. When we analysed patient samples, REIC/Dkk-3 expression was down-regulated in the hyperproliferative epidermis including skin cancers and non-cancerous proliferative diseases. REIC/Dkk-3 expression was also suppressed in the regenerative and inflammative epidermis of model mice. These findings will certainly contribute to the extension of studies on REIC/Dkk-3.

Down-regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene-therapeutic overexpression of REIC/Dkk-3.

We have recently shown that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) exhibits a potent tumor-specific cell-killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad-REIC-induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad-REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad-REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk-3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad-REIC-induced apoptosis, we found that BiP/GRP78, an ER-residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad-REIC were inversely correlated. Down-regulation of BiP with siRNA sensitized the resistant cells to Ad-REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad-REIC-induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad-REIC.

Transcriptional regulation of a brown adipocyte-specific gene, UCP1, by KLF11 and KLF15.

Several growth factors and transcription factors have been reported to play important roles in brown adipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1.

Rh(II)-Catalyzed skeletal reorganization of 1,6- and 1,7-enynes through electrophilic activation of alkynes.

The skeletal reorganization of 1,6- and 1,7-enynes leading to 1-vinylcycloalkenes using Rh(II) as a catalyst is reported. Two possible isomers of 1-vinylcycloalkenes, type I and type II, can be obtained, the ratio of which are highly dependent on the substitution pattern of the enynes used. Formation of type I compounds involves a single cleavage of a C-C double bond, the product of which is identical to that of enyne metathesis. In contrast, the formation of type II compounds involves the double cleavage of both the C-C double and triple bonds, which has an anomalous bond connection. The presence of both a phenyl group at the alkyne carbon and a methyl group at the internal alkene carbon facilitates the formation of type II compounds. The electronic and steric nature of the substituents on the aromatic ring also affects the ratio of type I and type II. The nature of a tether also has a significant effect on the course of the reaction. Experimental evidence for the intermediacy of a cyclopropyl rhodium carbenoid, a key intermediate in the skeletal reorganization of enynes, is also reported. In addition to the skeletal reorganization of enynes, Rh(II) complexes were found to have a high catalytic activity for some cycloisomerization reactions of alkyne derivatives, including the bicyclization of enynes to bicyclo[4.1.0]heptene derivatives and the cyclization of alkynylfurans to phenol derivatives.

Targeted disruption of Bcl-xL in mouse keratinocytes inhibits both UVB- and chemically induced skin carcinogenesis.

Bcl-x(L) is one of several antiapoptotic proteins regulated by signal transducer and activator of transcription 3 (Stat3). We have recently shown that Stat3 is required for chemically induced and ultraviolet B (UVB)-induced skin carcinogenesis. In this study, the functional role of Bcl-x(L) in skin carcinogenesis was investigated using skin-specific Bcl-x(L)-deficient mice. In this model, Bcl-x(L) expression is disrupted in the basal compartment of mouse epidermis using the bovine keratin 5 (K5) promoter to drive expression of Cre recombinase (K5.Cre x Bcl-x(L) (fl/fl) mice). A significant increase in apoptosis induced by either UVB irradiation or 7,12-dimethylbenz[a]anthracene (DMBA) treatment was observed in the epidermis of Bcl-x(L)-deficient mice. Furthermore, an increase in apoptotic cells was noted in hair follicle keratinocytes, including those located in the bulge region. Cell proliferation was not affected by Bcl-x(L) deficiency following exposure to either UVB or 12-O-tetradecanoylphorbol-13-acetate (TPA). Bcl-x(L)-deficient mice were more resistant than wild-type controls to skin tumor development with delayed onset and reduced number of tumors using either UVB or the DMBA/TPA two-stage regimen. Moreover, Bcl-2, Mcl-1, and survivin protein levels were increased in the epidermis of Bcl-x(L)-deficient mice in the absence of stimuli. Furthermore, levels of these antiapoptotic proteins were also high in skin tumors from Bcl-x(L)-deficient mice that developed in response to either UVB or two-stage carcinogenesis protocols. Collectively, these studies demonstrate that Bcl-x(L) plays a role early in skin carcinogenesis through its anti-apoptotic functions to enhance survival of keratinocytes, including bulge region keratinocyte stem cells, following DNA damage.

Suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow.

Transplantation of hepatocytes or bone marrow-derived cells has been shown to ameliorate liver fibrosis in animal models, but no direct comparison of relative efficiency has been made. The aim of this study was to compare the efficiency of a bone marrow-derived clonal mesenchymal stem cell line established by us (rBM25/S3) with that of its adipogenic or hepatogenic differentiation derivative for suppression of rat liver fibrosis. After induction of differentiation of rBM25/S3 cells into adipogenic or hepatogenic cells in culture, we intrasplenically transplanted the three types of cells into rats (3 x 10(7) cells/rat) before and 4 weeks after initiation of carbon tetrachloride treatment (1 ml/kg body weight twice a week for 8 weeks) to induce liver fibrosis. Undifferentiated rBM25/S3 cells were the most effective for suppression of liver fibrosis, followed by the adipogenic cells and hepatogenic cells. Expression levels of MMP-2 and MMP-9 were also highest in undifferentiated rBM25/S3 cells. These results indicate that bone marrow-derived clonal mesenchymal stem cell lines are useful for further mechanistic studies on cell-mediated suppression of liver fibrosis and that such cell lines will provide information on an appropriate cell source for transplantation therapy for cirrhosis.

Stage-specific disruption of Stat3 demonstrates a direct requirement during both the initiation and promotion stages of mouse skin tumorigenesis.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss-of-function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER(T2) x Stat3(fl/fl)) that show epidermal-specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by intraperitoneal injection) led to inhibition of tumor growth compared with tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series.

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC(50) 3.2 nM; MCP-1-Induced Chemotaxis IC(50) 0.83 nM; Ca(2+) Flux IC(50) 7.5 nM].

Derivation of hepato-pancreatic intermediate progenitor cells from a clonal mesenchymal stem cell line of rat bone marrow origin.

We have recently established a clonal mesenchymal stem cell line (rBM25/S3) from adult rat bone marrow. The cells have practically unlimited proliferation capacity (over 300 PDL), maintaining multipotency for differentiation. In the present study, we examined the potential for rBM25/S3 cells to differentiate into insulin-secreting cells. When cultured in the presence of HGF and FGF-4 on Matrigel, rBM25/S3 cells expressed genes specific to pancreatic beta-cells as well as those specific to hepatocytes. They still maintained proliferation capacity with a doubling time of approximately 30 h. These hepato-pancreatic intermediate progenitor cells, but not the original undifferentiated rBM25/S3 cells, were induced by the overexpression of PDX-1 to produce significant amounts of insulin in a manner responding to glucose concentration in medium. The present culture system indicates a direction for further studies aimed at the realization of cell transplantation therapy for type I diabetes mellitus.

Mechanistic analysis of resistance to REIC/Dkk-3-induced apoptosis in human bladder cancer cells.

We have recently shown that a new therapeutic modality using the REIC/Dkk-3 gene (Ad-REIC) is effective against various human cancers, including those of prostate, testis and breast origins. The aim of the present study was to examine the sensitivity of bladder cancers to Ad-REIC and to clarify the molecular mechanisms that determine sensitivity/resistance. We found that 2 human bladder cancer cell lines, T24 and J82, are resistant to Ad-REIC. In T24 and J82 cells, the ER stress response and activation of JNK were observed in a manner similar to that in the sensitive PC3 cells. Translocation of Bax to mitochondria occurred in PC3 cells but not in T24 and J82 cells. Bcl-2 was remarkably overexpressed in T24 and J82 compared with the expression levels in sensitive cell lines. Treatment of T24 and J82 cells with a Bcl-2 inhibitor sensitized the cells to Ad-REIC-induced apoptosis. The results indicate that some human bladder cancers are resistant to apoptosis induced by overexpression of REIC/Dkk-3, which is at least in part due to up-regulation of Bcl-2. These results provide a basis for possible use of Bcl-2 as a marker of sensitive cancers and to try to sensitize resistant cancers to Ad-REIC by down-regulation of Bcl-2.

Tumor necrosis factor-α downregulates the REIC/Dkk-3 tumor suppressor gene in normal human skin keratinocytes.

Our previous studies revealed that REIC/Dkk-3 was expressed various tissues, including skin keratinocytes. The aim of the present study was to identify the factors that regulate the expression of the dickkopf Wnt signaling pathway inhibitor 3 (REIC/Dkk­3) tumor suppressor gene in normal human skin keratinocytes (NHKs). Several growth factors and cytokines that have previously been reported to be involved in the growth and differentiation of keratinocytes were screened as potential regulators. Western blot analysis was performed using protein from NHKs cultured with/without various factors including the epidermal growth factor, tumor necrosis factor­α, transforming growth factor­ß, interleukin (IL)­1F9, IL­6, IL­8 and Ca2+. The results indicated that only TNF­α downregulated REIC/Dkk­3 expression in NHKs. Subsequently, TNF­α was confirmed to reduce the expression levels of REIC/Dkk­3 in mouse skin tissue and hair culture models. TNF­α­mediated downregulation of REIC/Dkk­3 expression in NHKs was abrogated by the addition of a TNF­α­specific antibody. In conclusion, the results indicate that TNF­α downregulates REIC/Dkk­3 expression in normal skin keratinocytes.

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine.

Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

Signal transducer and activator of transcription 3 is a key regulator of keratinocyte survival and proliferation following UV irradiation.

UVB irradiation of signal transducer and activator of transcription 3 (Stat3)-deficient keratinocytes resulted in a high incidence of apoptosis compared with controls. Conversely, forced expression of Stat3 desensitized keratinocytes to UVB-induced apoptosis. Upon UVB exposure, keratinocyte Stat3 was rapidly dephosphorylated, followed by decreases of both Stat3 mRNA and protein levels in a p53-independent manner. Vanadate treatment reversed the UVB-induced down-regulation of Stat3 and generation of apoptotic keratinocytes, suggesting the involvement of a tyrosine phosphatase. Furthermore, Stat3 was required for UVB-induced proliferation of follicular keratinocytes, leading to epidermal thickening. Finally, constitutive activation of Stat3 was observed in UVB-induced squamous cell carcinomas of either mice or human origin. These data suggest that Stat3 is required for survival and proliferation of keratinocytes following UVB exposure and that Stat3 is tightly regulated as part of a novel protective mechanism against UVB-induced skin cancer.

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene.

Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

Efficient differentiation into skin cells of bone marrow cells recovered in a pellet after density gradient fractionation.

We had previously demonstrated the participation of whole bone marrow cells from adult mice in the reconstitution of skin, including the epidermis and hair follicles. To get an insight into cell populations that give rise to the epithelial components of the reconstituted skin, we fractionated bone marrow cells derived from green fluorescent protein-transgenic mice by density gradient. Unexpectedly, we found that a substantial amount of mononucleated cells (approximately 30%) was recovered in the pellet fraction and that the cells in the pellet fraction preferentially differentiated into epithelial components of skin, rather than the cells in the mononuclear cell fraction. The pellet fraction contained more CD45-negative (thus uncommitted to the hematopoietic cell lineage) cells than the mononuclear cell fraction. These results indicate that density gradient fractionation results in significant loss of specific progenitor cells into the usually discarded pellet fraction.

Immediate administration of mineralocorticoid receptor antagonist spironolactone prevents post-infarct left ventricular remodeling associated with suppression of a marker of myocardial collagen synthesis in patients with first anterior acute myocardial infarction.

BACKGROUND: Aldosterone (ALD) has been shown to stimulate cardiac collagen synthesis and fibroblast proliferation via activation of local mineralocorticoid receptors. In patients with acute myocardial infarction, we demonstrated that ALD was extracted through the infarct heart and extracting ALD-stimulated post-infarct left ventricular (LV) remodeling. METHODS AND RESULTS: To evaluate the effect of mineralocorticoid receptor antagonist (MRA) spironolactone on post-infarct LV remodeling, 134 patients with first anterior acute myocardial infarction were randomly divided into the MRA (n=65) or non-MRA (n=69) groups after revascularization. All patients were administered angiotensin-converting enzyme (ACE) inhibitor and study drug just after revascularization. Left ventriculography with contrast medium was performed at the acute stage and after 1 month to evaluate LV remodeling. ALD was measured at aortic root and coronary sinus. There was no difference in the baseline characteristics including infarct size and LV performance between the two groups. However, LV ejection fraction was significantly improved in the MRA group compared with that in the non-MRA group (46.0+/-0.6% to 53.2+/-0.8% versus 46.5+/-0.8% to 51.0+/-0.8%, Pinteraction=0.012). LV end-diastolic volume index was significantly suppressed in the MRA group compared with that in non-MRA group (86.5+/-1.0 to 90.6+/-2.4 versus 87.5+/-1.3 to 106.8+/-3.5 mL/m2, Pinteraction=0.002). Transcardiac extraction of ALD through the heart was significantly suppressed in the MRA group (Pinteraction=0.001), and plasma procollagen type III aminoterminal peptide level, a biochemical marker of fibrosis, was significant lower in the MRA group compared with the non-MRA group (Pinteraction=0.002). CONCLUSIONS: These findings indicate that MRA combined with ACE inhibitor can prevent post-infarct LV remodeling better than ACE inhibitor alone in association with the suppression of a marker of collagen synthesis.