A prediction model for 5-year cardiac mortality in patients with chronic heart failure using ¹²³I-metaiodobenzylguanidine imaging.

PURPOSE: Prediction of mortality risk is important in the management of chronic heart failure (CHF). The aim of this study was to create a prediction model for 5-year cardiac death including assessment of cardiac sympathetic innervation using data from a multicenter cohort study in Japan. METHODS: The original pooled database consisted of cohort studies from six sites in Japan. A total of 933 CHF patients who underwent (123)I-metaiodobenzylguanidine (MIBG) imaging and whose 5-year outcomes were known were selected from this database. The late MIBG heart-to-mediastinum ratio (HMR) was used for quantification of cardiac uptake. Cox proportional hazard and logistic regression analyses were used to select appropriate variables for predicting 5-year cardiac mortality. The formula for predicting 5-year mortality was created using a logistic regression model. RESULTS: During the 5-year follow-up, 205 patients (22 %) died of a cardiac event including heart failure death, sudden cardiac death and fatal acute myocardial infarction (64 %, 30 % and 6 %, respectively). Multivariate logistic analysis selected four parameters, including New York Heart Association (NYHA) functional class, age, gender and left ventricular ejection fraction, without HMR (model 1) and five parameters with the addition of HMR (model 2). The net reclassification improvement analysis for all subjects was 13.8 % (p < 0.0001) by including HMR and its inclusion was most effective in the downward reclassification of low-risk patients. Nomograms for predicting 5-year cardiac mortality were created from the five-parameter regression model. CONCLUSION: Cardiac MIBG imaging had a significant additive value for predicting cardiac mortality. The prediction formula and nomograms can be used for risk stratifying in patients with CHF.

Transcriptome-based systematic identification of extracellular matrix proteins.

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.

Cellular dynamics of epithelial clefting during branching morphogenesis of the mouse submandibular gland.

We cultured the rudimental submandibular gland (SMG) of mice with a non-cell-permeable fluorescent tracer, and observed cell behavior during epithelial branching morphogenesis using confocal time-lapse microscopy. We traced movements of individual cells as shadowgraph movies. Individual epithelial cells migrated dynamically but erratically. The epithelial cleft extended by wiggling and separated a cluster of cells into two buds during branching. We examined the ultrastructure of the clefts in SMG rudiments treated with the laminin peptide A5G77f, which induces epithelial clefting. A short cytoplasmic shelf with a core of microfilaments was found at the deep end of the cleft. We propose that epithelial clefting involves a dynamic movement of cells at the base of the cleft, and the formation of a shelf within a cleft cell. The shelf might form a matrix attachment point at the base of the cleft with a core of microfilaments driving cleft elongation.

Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth.

Nonsteroidal antiinflammatories are known to suppress incidence and progression of malignancies including colorectal cancers. However, the precise mechanism of this action remains unknown. Using prostaglandin (PG) receptor knockout mice, we have evaluated a role of PGs in tumor-associated angiogenesis and tumor growth, and identified PG receptors involved. Sarcoma-180 cells implanted in wild-type (WT) mice formed a tumor with extensive angiogenesis, which was greatly suppressed by specific inhibitors for cyclooxygenase (COX)-2 but not for COX-1. Angiogenesis in sponge implantation model, which can mimic tumor-stromal angiogenesis, was markedly suppressed in mice lacking EP3 (EP3(-/-)) with reduced expression of vascular endothelial growth factor (VEGF) around the sponge implants. Further, implanted tumor growth (sarcoma-180, Lewis lung carcinoma) was markedly suppressed in EP3(-/-), in which tumor-associated angiogenesis was also reduced. Immunohistochemical analysis revealed that major VEGF-expressing cells in the stroma were CD3/Mac-1 double-negative fibroblasts, and that VEGF-expression in the stroma was markedly reduced in EP3(-/-), compared with WT. Application of an EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT, but not in EP3(-/-). These results demonstrate significance of host stromal PGE(2)-EP3 receptor signaling in tumor development and angiogenesis. An EP3 receptor antagonist may be a candidate of chemopreventive agents effective for malignant tumors.

[Gross anatomy dissection and the legal control].

In Japan, dissection of human body is generally prohibited by the Penal Code, i.e. the criminal law. However, the Postmortem Examination and Corpse Preservation Act allows for the dissection of the body in very limited situations, that include gross anatomy dissection and pathological and forensic autopsy in medical and dental schools. Growing numbers of co-medical schools have been founded more recently in Japan, and not a small number of co-medical schools try to adopt human body dissection in the course of anatomy education. The present short communication reminds us of the ways of thinking of the Postmortem Examination and Corpse Preservation Act and the Act on Body Donation for Medical and Dental Education in order that anatomy education in medical as well as co-medical schools takes place under the regulation by these two laws.

A novel cell-adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds.

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide-chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27- and A5G33-chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33-chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and alpha-smooth muscle actin, showed that keratinocyte delivery by the present peptide-chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

Prediction of cardiac events in patients with dilated cardiomyopathy using 123I-BMIPP and 201Tl myocardial scintigraphy.

OBJECTIVE: Various clinical trials for dilated cardiomyopathy (DCM) have demonstrated that the prognosis as well as cardiac function is improved by the administration of beta-blocker therapy. On the other hand, 123I-betamethyl-p-iodophenyl-pentadecanoic acid (BMIPP) reflects myocardial fatty acid metabolism and is considered to be a more sensitive tracer than perfusion tracers. In this study, the efficacy of DCM for the evaluation of myocardial damage and the prediction of cardiac events was studied using 123I-BMIPP and 201TI (Tl) myocardial scintigraphy. METHODS: Study subjects comprised 33 DCM patients, divided into a cardiac event group (event, n = 9) and an event-free group (event free, n = 24). An extent score (ES) and severity score (SS) were calculated for each BMIPP image. BMIPP and Tl images were divided into 17 segments, and total defect scores (TDS) were calculated for each. The TDS of the BMIPP and Tl images were compared with score differences greater than or equal to 4 and less than 4 defined as mismatch and non-mismatch, respectively. RESULTS: The TDS of BMIPP was significantly higher in the event group than in the event-free group (P < 0.05). The ES and SS were significantly higher in the event group than in the event-free group (P < 0.01). The comparison in the 2 x 2 contingency tables showed that the occurrence of non-mismatch was significantly higher in the event-free group (chi2 test; P < 0.01). The ES of BMIPP was a significant predictor of cardiac events in the multivariate analysis (P < 0.01). CONCLUSIONS: These results suggest that the ES for BMIPP is useful as a predictor of cardiac events in DCM.

Creation and characterization of Japanese standards for myocardial perfusion SPECT: database from the Japanese Society of Nuclear Medicine Working Group.

OBJECTIVE: Standards for myocardial single-photon emission computed tomography (SPECT) adapted for a Japanese population were not available. The purpose of this study was to create standard files approved by the Japanese Society of Nuclear Medicine and to make known the characteristics of the myocardial perfusion pattern of this population. METHODS: With the collaboration of nine hospitals, a total of 326 sets of exercise-rest myocardial perfusion images were accumulated from subjects with a low likelihood of cardiac diseases. The normal database included a (99m)Tc-MIBI/tetrofosmin myocardial perfusion study with 360 degrees (n = 80) and 180 degrees (n = 56) rotations, (201)Tl study with 360 degrees (n = 115) and 180 degrees rotations (n = 54) and a dual-isotope study with 360 degrees rotation (n = 27). The projection images were transferred by digital imaging and communications in medicine (DICOM) format and reconstructed and analyzed with polar maps. RESULTS: The projection data from multiple centers were successfully transferred to a common format for SPECT reconstruction. When the average values were analyzed using a 17-segment model, myocardial counts in the septal segment differed significantly between 180 degrees and 360 degrees rotation acquisitions. Regional differences were observed between men and women in the inferior and anterior regions. A tracer difference between (99m)Tc and (201)Tl was also observed in some segments. The attenuation patterns differed significantly between subjects from the United States and those from Japan. CONCLUSIONS: Myocardial perfusion data that were specific for the Japanese population were generated. The normal database can serve a standard for nuclear cardiology work conducted in Japan.

Laminin peptide-conjugated chitosan membrane: Application for keratinocyte delivery in wounded skin.

Tissue engineering requires the delivery and survival of cells to organ sites needing repair. Previously, we showed that an active laminin peptide (AG73: RKR-LQVQLSIRT)-conjugated chitosan membrane promoted cell adhesion and spreading in vitro. Here, we seeded human keratinocytes onto AG73-chitosan membranes and found that nearly 80% of the cells were attached to the membranes within 2 h. The membranes carrying the keratinocytes were inverted and placed onto exposed muscle fascia on the backs of nude mice. After 3 days, the keratinocytes had migrated from the membrane and established a stratified epidermis-like structure on the fascia. Cells recognize the AG73 through transmembrane proteoglycan syndecans, which recognition system has not previously been tested in tissue engineering applications. We suggest that the AG73-chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system such as delivering keratinocytes to a wound bed.

Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.

We evaluated the significance of the host kallikrein-kinin system in tumor angiogenesis and tumor growth using two rodent models genetically deficient in a kallikrein-kinin system. Inoculation of Walker 256 carcinoma cells into the s.c. tissues of the back of normal Brown Norway Kitasato rats (BN-Ki rats) resulted in the rapid development of solid tumors with marked angiogenesis. By contrast, in kininogen-deficient Brown Norway Katholiek rats (BN-Ka rats), which cannot generate intrinsic bradykinin (BK), the weights of the tumors and the extent of angiogenesis were significantly less than those in BN-Ki rats. Daily administration of B(2) receptor antagonists significantly reduced angiogenesis and tumor weights in BN-Ki rats to levels similar to those in BN-Ka rats but did not do so in BN-Ka rats. Angiogenesis and tumor growth were significantly suppressed in B(2) receptor knockout mice bearing sarcoma 180 compared with their wild-type counterparts. Immunoreactive vascular endothelial growth factor (VEGF) was localized in Walker tumor stroma more extensively in BN-Ki rats than in BN-Ka rats, although immunoreactive B(2) receptor also was detected in the stroma to the same extent in both types of rats. Cultured stromal fibroblasts isolated from BN-Ki rats and BN-Ka rats produced VEGF in response to BK (10(-8)-10(-6) m), and this stimulatory effect of BK was abolished with a B(2) receptor antagonist, Hoe140 (10(-5) m). These results suggest that BK generated from kininogens supplied from the host may facilitate tumor-associated angiogenesis and tumor growth by stimulating stromal B(2) signaling to up-regulate VEGF production mainly in fibroblasts.

Salivary gland morphogenesis and basement membranes.

The basement membrane separates the epithelium from the surrounding mesenchyme and plays an essential role in the development of various epithelial-mesenchymal organs. Among these, the submandibular salivary gland (SMG) has been chosen to review the expression patterns and roles of the epithelial basement membrane and its components, in particular the laminins, during SMG morphogenesis. At the outset, a brief description of SMG development is provided with special reference to changes in the epithelial architecture and the epithelial basement membrane. The restricted expression patterns of various laminin isoforms in the developing SMGs are also summarized. Furthermore, an overview is given of several lines of experimental evidence that indicate significant but distinct roles for laminin-1 and laminin-10, their individual domains and their receptor-mediated signaling in SMG morphogenesis.

Usefulness of meta-[123I]iodobenzylguanidine myocardial scintigraphy for predicting cardiac events in patients with dilated cardiomyopathy who receive long-term beta blocker treatment.

OBJECTIVE: Large-scale clinical trials have demonstrated that beta blocker therapy improves the prognosis of dilated cardiomyopathy (DCM), but cardiac events sometimes occur even in patients showing favourable response to the therapy. In this study, the usefulness of meta-iodobenzyguanidine (MIBG) in predicting cardiac events was investigated in DCM patients successfully receiving long-term treatment with beta blockers. METHODS: The subjects were 53 patients with DCM (including 10 women; mean age, 56.5+/-10.9 years) who could continue beta blocker therapy for more than 6 months. MIBG was performed within 1 year of commencing the therapy. The extent score, severity score and washout rate were obtained from single photon emission computed tomography images. At the same time, left ventricular ejection fraction (LVEF) and left ventricular end diastolic diameters were measured by echocardiography. The endpoints were cardiac events (cardiac death and admission to hospital due to heart failure or arrhythmia), and patients were observed for an average of 1314+/-986 days (150-4100 days). RESULTS: Cardiac events occurred in nine patients during the observation period. The multivariate statistical analysis demonstrated that the delayed extent score was the strongest significant predictive factor, (hazard ratio 1.036, P<0.01). while LVEF was not a predictive factor. Both the improvement of LVEF and MIBG were significant predictive factors. The improvement of washout rate was the strongest. CONCLUSION: Parameters of MIBG but not of LVEF were useful in predicting cardiac events in DCM patients whose condition had been successfully stabilized by the introduction of beta blockers.

Inhibition of hair follicle growth by a laminin-1 G-domain peptide, RKRLQVQLSIRT, in an organ culture of isolated vibrissa rudiment.

We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.

Co-expression of de novo DNA methyltransferases Dnmt3a2 and Dnmt3L in gonocytes of mouse embryos.

In mouse male germ cells, global DNA methylation occurs in gonocytes at 16-18 days postcoitum. In the present study, we examined which de novo-type DNA methyltransferase, Dnmt3a, Dnmt3a2 or Dnmt3b is expressed in gonocytes at these stages. Immuno-histochemical and Western blot analyses revealed that Dnmt3a2 was the major DNA methyltransferase expressed in gonocytes at 14-18 day postcoitum. Dnmt3L, which is necessary for spermatogenesis, was co-expressed in gonocytes at identical stages to Dnmt3a2. On the other hand, Dnmt3a was expressed not in germ cells but in the Sertoli cells and connective tissue cells that surround gonocytes and spermatogonia. Dnmt3b2, an isoform of Dnmt3b, was expressed faintly but significantly in gonocytes at 16 days postcoitum, and increased in spermatogonia at 4 and 6 days postpartum. The expression of Dnmt3a2, Dnmt3L, and Dnmt3b2 at 14-18 dpc was confirmed by reverse transcriptase-coupled polymerase chain reaction amplification and nucleotide sequencing of the amplified fragments. The results strongly suggest that Dnmt3a2 and Dnmt3L are responsible for the global DNA methylation in mouse male germ cells.

Structural integrity of the Golgi stack is essential for normal secretory functions of rat parotid acinar cells: effects of brefeldin A and okadaic acid.

We examined the effects of specific inhibitors, brefeldin A (BFA) and okadaic acid (OA), on the ultrastructural organization of the Golgi apparatus and distributions of amylase, Golgi-associated proteins, and cathepsin D in the rat parotid acinar cells. BFA induced a rapid regression of the Golgi stack into rudimentary Golgi clusters composed of tubulovesicules, in parallel with a redistribution of the Golgi-resident proteins and a coat protein (beta-COP) into the region of the rough endoplasmic reticulum (rER) or cytosol. The rapid disruption of the Golgi stack could also be induced by the effect of OA. However, redistribution of the Golgi proteins in rER or cytosol could not be observed and beta-COP was not dispersed but was retained on the rudimentary Golgi apparatus. These findings suggested that the mechanism of OA in inducing degeneration of the Golgi stack was markedly different from that of BFA. In addition, missorting of amylase, a Golgi protein, and cathepsin D into incorrect transport pathways is apparent in the course of the disruption of the Golgi stack by OA. These Golgi-disrupting effects are reversible and the reconstruction of the stacked structure of the Golgi apparatus started immediately after the removal of inhibitors. In the recovery processes, missorting was also observed until the integrated structure of the Golgi apparatus was completely reconstructed. This suggested that the integrated structure of the Golgi apparatus was quite necessary for the occurrence of normal secretory events, including proper sorting of molecules.

Disorganization of actin bundles of ectoplasmic specialization.

The degradation of ectoplasmic specialization consisting of bundles of actin sandwiched between the plasma membrane and endoplasmic reticulum of the Sertoli cell, occurs just before spermiation. For elucidation of the processes involved in this degradation, changes in fibrous actin of the rat testis were analyzed using BODIPY-phalloidin by fluorescence and electron microscopy. Before step 17, the fluorescence of BODIPY-phalloidin was evenly distributed around the spermatid head. When the spermatids became positioned at the luminal surface, the fluorescence had condensed on the concave side of the spermatid head. At step 19, lines of fluorescence distributed at regular intervals projected at right angles from the head. Ultrastructural observation showed that the tubulobulbar complex was formed at step 19 and electron-dense material accumulated around thin tubules of the tubulobulbar complex. Immunohistochemical examination of BODIPY-phalloidin showed that the electron dense materials around the thin tubules of the tubulobulbar complex had the capacity to bind to phallotoxin. Therefore the pattern of fluorescence in the spermatid at step 19 corresponds to that of the tubulobulbar complex. Actin bundles of the ectoplasmic specialization would thus appear to de-polymerize into actin monomers via electron dense materials around the thin tubules of the tubulobulbar complex. The tubulobulbar complex may contribute to the disorganization of actin bundles.

Spermiation in the Mouse: Contribution of the Invading Sertoli Cell Process to Adluminal Displacement of the Spermatid Head: (Spermiogenesis/Spermiation/Sertoli cell/Ultrastructure/Cell movement).

At the maturation phase of spermiogenesis in mice, the spermatid heads that are embedded deeply in the epithelium of the seminiferous tubules dislocate toward the luminal surface. In the present study, to clarify the manner in which the spermatid head is displaced toward the lumen, morphological changes in spermatids and Sertoli cells were examined on ultrathin and thick sections stained with adenosine triphosphatase cytochemistry. During adluminal displacement, the spermatid head is surrounded by an invading process of Sertoli cell which invaginates into the spermatid cytoplasm to form complicated passages called the canal complex. At the site of the spermatid head, the wall of an invading Sertoli cell process folds to form a sheath in which the spermatid head is located. The sheath correspond to a structure known as ectoplasmic specialization. The invading Sertoli cell process also shows branching and swelling at the site where spermatid heads are present. The present results suggest that the canal complex is directly involved in the adluminal displacement of the spermatid head. Dynamic changes of invading Sertoli cell processes may produce the motive force for adluminal displacment of the spermatid head. Also, ectoplasmic specialization may contribute to the adluminal displacement of the spermatid possibly by mediating cell to cell interaction between the spermatid nucleus and the invading Sertoli cell process.

Cyclooxygenase-2 inhibitors attenuate increased blood pressure in renovascular hypertensive models, but not in deoxycorticosterone-salt hypertension.

COX-2 is an inducible cyclooxygenase (COX) that has been reported to be expressed in the macula densa and surrounding cortical thick ascending limb in normotensive rats. The present study assessed the contribution of COX-2 in three different rat models of hypertension, each characterized by a different activation of the renal renin-angiotensin system. Mean blood pressure (MBP) in the rat 2 kidney-1 clip (2K1C) model was significantly reduced with a COX-2 selective inhibitor, NS-398 (10 mg/kg, p.o., twice a day) (vehicle-administered rats (n = 8): 154 +/- 6 mmHg; NS-398-administered rats (n = 5): 128 +/- 10 mmHg). By contrast, a COX-1 selective inhibitor, mofezolac, did not lower MBP. Increased plasma renin activity (23 +/- 8 ng/kg/h (n = 6) vs. sham operation, 2.4 +/- 0.9 ng/kg/h (n = 4)) was markedly reduced to 6.8 +/- 2.7 ng/ml/h (n = 5) by NS-398, but not by mofezolac. The development of 1 kidney-1 clip (1K1C) hypertension was also inhibited by NS-398 (vehicle (n = 12): 133 +/- 1 mmHg; NS-398 (n = 7): 122 +/- 3 mmHg) accompanied by a reduction in plasma renin activity (3.0 +/- 0.3 ng/ml/h, n = 4) to 1.0 +/- 0.2 ng/ml/h (n = 5). The COX-2 inhibitor increased urinary excretions in the 1K1C model, but not in the 2K1C model. In a deoxycorticosterone acetate (DOCA)-salt model, plasma renin activity was markedly suppressed to less than 0.3 ng/ml/h. The COX-2 inhibitor caused no significant changes in MBP, plasma renin activity, or urinary excretion, suggesting that COX-2 made a lesser contribution in this model. Increased expression of COX-2 mRNA and protein was observed in the kidneys of 1K1C and 2K1C rats, but not in DOCA-salt rats. These results suggest that COX-2 plays a significant role in the development of 2K1C and 1K1C renovascular hypertension, in addition to making a substantial contribution to the diuretic effect in the 1K1C model.