Increased ectodomain shedding of CADM1 in T2DM pancreata.

<p>(<b>a</b>) Western blot analysis of CADM1 expression in control and T2DM pancreata. Cases are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100988#pone.0100988.s005" target="_blank">Table S1</a>. Arrowheads indicate bands corresponding to the full-length, αCTF, and βCTF forms of CADM1. The blot was reprobed with an anti-β-actin antibody to show protein loading. (<b>b</b>) CADM1 expression per islet cell in each patient sample. For each lane in <b>a</b>, the intensities of the full-length CADM1, αCTF, βCTF, and β-actin bands were quantified, and CADM1 levels were expressed relative to β-actin and the islet cell count (/cm² of tissue). Statistical significance was analyzed with the Mann-Whitney <i>U</i>-test. <i>P</i>-values are shown. (<b>c</b>) CADM1 ectodomain shedding rates (relative amounts of CTFs to the full-length CADM1). Statistical significance was analyzed with the Mann-Whitney <i>U</i>-test. <i>P</i>-values are shown. (<b>d</b>) Correlation of HbA1c levels with full-length CADM1 expression per islet cell (left) or the CADM1 shedding rate (αCTF/full-length; right). In each graph, the dot distribution approximated a linear function (dotted lines). Correlations and statistical significance were analyzed with Spearman’s rank test. <i>R²</i> and <i>P</i>-values are shown.</p

Representative photomicrographs of CADM1 immunofluorescence in pancreatic islets.

<p>Pancreatic sections from control (case 7; left) and T2DM (case 3; right) patients were double stained with antibodies against CADM1 (red) and glucagon (green; top) or insulin (green; bottom) and then counterstained with DAPI (blue). Merged images are shown. Bar  = 50 µm.</p

Subcellular localization of endogenous and exogenous αCTF in MIN6-m9 cells.

<p>MIN6-m9 cells were untreated (−), treated with PMA (200 nM) and trypsin (0.025% w/v), or transfected with pCX4bsr-SP-αCTF. After 45 min of treatment and 2 days of transfection, CADM1 levels were assessed in western blot (<b>a</b>) and immunofluorescence (<b>b</b>) analyses using a CADM1 antibody. In <b>a</b>, arrowheads indicate full-length CADM and αCTF. The blot was reprobed with an anti-β-actin antibody to show the protein loading. In <b>b</b>, cells were double stained with CADM1 antibody (green; top) and MitoTracker (red; middle). Merged images are also shown (bottom). Data are representative of three independent experiments. Bar  = 10 µm.</p

Increased Ectodomain Shedding of Cell Adhesion Molecule 1 from Pancreatic Islets in Type 2 Diabetic Pancreata: Correlation with Hemoglobin A1c Levels

<div><p>Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (<i>P</i> = 0.041) and positively with ectodomain shedding rates (<i>P</i> = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.</p></div

Characteristics of the patient groups^a.

a<p>Data for individual patients are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100988#pone.0100988.s005" target="_blank">Table S1</a>.</p>b<p>Data are expressed as the mean ± SE.</p>c<p>Control, n = 3; T2DM, n = 10.</p

Differences in the temporal changes in liver lesions between the WT and plasminogen KO mice.

<p><b>A.</b> CECT scans showing time-dependent recovery from PIT-LD liver injury in wild-type (WT), but not in the plasminogen-knockout (KO) mouse. The arrows indicate areas of photochemically induced injury. Scale bars indicate 2 mm. <b>B.</b> Quantitative evaluation of the recovery process from the liver damage in the WT mice. Changes in lesion volumes in WT PIT-BD mice from day 1 through day 14 are shown (n = 4). Kruskal-Wallis test with Dunn's post-hoc tests indicated a significant difference in the lesion volumes between days 1 and 14 (***, <i>p</i><0.001). Two-way ANOVA with Bonferroni post-hoc tests revealed significant differences in the lesion volumes between WT and KO mice on days 4 (†, <i>P</i><0.001), 7 (‡, <i>P</i><0.0001) and 14 (§, <i>P</i><0.0001). <b>C.</b> Changes in the lesion volume in livers of the plasminogen KO mice over the two weeks with a significant reduction in lesion size between days 1 and 14 (*, <i>p</i><0.05 by Kruskal-Wallis test with Dunn's post-hoc tests). <b>D.</b> H&E-stained vertical sections at the center of the liver lesion in WT (upper panels) and plasminogen-KO mice (lower panels). Arrowheads indicate granulation tissue, which was not observed in the KO mouse liver. Scale bars: 1 mm.</p

Comparisons of the CT and MR images of stroke lesions in the SHRSP rats.

<p><b>A.</b> Images acquired with CECT (left), T2-weighted MR (T2WI, middle), and H&E staining (right) of the same stroke areas in an SHRSP rat brain two weeks after the onset. The areas indicated by arrows are suspected loci of hemorrhagic infarction. Infarct lesions with gliosis and inflammatory cell infiltration were observed in the upper and lower H&E images (left and right panels are the same lesion at low and high magnifications, respectively). Hemorrhage (or congestion) is also seen in the lower panels. Scale bars: 2 mm (CECT); 400 µm (H&E). <b>B.</b> Images acquired by CECT, T2WI, and T1-weighted gradient echo (GRE), along with an H&E image of identical stroke areas in an SHRSP rat brain after 3 weeks of stroke onset. Arrows indicate suspected hemorrhagic infarction of about 0.5 mm in diameter. Gliosis and small-sized cystic cavity formation are observed in the infarct area (H&E image). Scale bars: 2 mm (CECT), 100 µm (H&E). <b>C.</b> Images obtained with CECT, T2WI, T1-weighed GRE, and an H&E stain of identical stroke areas in an SHRSP rat brain 4 weeks after onset. Bold arrows (red and white) in upper panels indicate suspected hemorrhagic infarction. A suspected hemorrhage of about 0.1 mm in diameter was also detected (thin arrows). The corresponding areas were analyzed by H&E staining (upper right and lower panels). The lesion areas indicated by both red and white bold arrows in the H&E images are wedge-shaped infarcts with inflammatory cell infiltration, gliosis, and cystic cavities (lower panels at low and high magnifications). Scale bars: 2 mm (CECT), 500 µm (H&E, white bars) and 100 µm (H&E, black bars). <b>D.</b> Images of the posterior brain of the same SHRSP rat shown in C. The bold arrow indicates a suspected hemorrhagic infarction. Scale bars indicate 2 mm (CECT) and 500 µm (H&E, lower panel).</p

Temporal changes in the infarct area in the PIT-BD mouse model.

<p><b>A.</b> Serial CECT images taken from a representative PIT-BD mouse brain. <b>B.</b> Time-dependent changes in %HAA and total volume of lesion. Changes in lesion volumes of three MCAO mice through days 1 and 14 are shown. <b>C.</b> Temporal changes in %HAA in the same PIT-BD mice shown in B. There were significant differences in %HAA between day 1 and days 7 and 14 (Kruskal-Wallis test with Dunn's post-hoc tests, *<i>P</i><0.05). <b>D.</b> A TTC-stained coronal section of a PIT-BD mouse brain on day 7. The arrow indicates the ischemic lesion. <b>E.</b> Leakage of Evans blue indicating elevated vascular permeability in the infarct area. <b>F.</b> A 3-D image of the PIT-BD lesion (white area) reconstructed from sequential CECT images. Scale bars: 1 mm (A, D, E).</p

Expression of NOSs in xenografts of HeLa cells.

<p>A, B. Expression levels of nNOS mRNA (A) and iNOS mRNA (B) in HeLaXs. *, **and *** indicate significant differences between experimental and control xenografts, and between HeLaXs and HeLa cells by ANOVA-test (A) and by Student's t-test (B), at *, P<0.05, **, P<0.01, ***, P<0.001, respectively. The letter a means significant differences between average levels of nNOS mRNA and iNOS mRNA in HeLa cells by Student's t-test, at P<0.001. C—H. Respective NOS expression, nNOS (C, F), iNOS (D, G) and eNOS (E, H), in HeLaX-E9s (C, D, E) and in HeLaX-Ss (F, G, H). Arrows in C, D, F, G and H point to tumor cells with pores (C, D) or without pores, and in E, fibrous cells; middle arrows in H, indicate DCs; small arrows in D, F and G point to presumptive immune cells, and in E and H, processes of DCs. Open arrows in C and E point to dying cells; open arrowheads in F point to immunopositive nNOS; and asterisks in D and H indicate remnant of dying cells. I—K. Corresponding regions to C, D and E stained with anti-cytochrome C indicating apoptotic death. Small arrows in I and long arrows in J and K point to tumor cells in apoptotic death. Open arrowheads in J point immune cells in apoptotic death. Asterisks in J, K indicate remnant of dying cells. L. Negative control for NOSs antibodies stained with rabbit IgG in HeLaX-E9-9s. Arrow points to tumor cells. M, N. Immunoblots for nNOS (M) and eNOS (N) proteins in HeLaX-E9s and their controls. Each band intensity is shown adjusted value by the density of β-actin in the HeLaX-Ss as 1.0. SM indicates safety margin of lung cancer which is the remainder used on 2010 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122458#pone.0122458.ref040" target="_blank">40</a>]. O. Localization of iNOS in macrophages (arrows) in HeLaX-E9-9s (a) and in NK cells (arrows) in HeLaX-E9-4.5s (b).</p

How xenografts were destroyed and resolved through EMP9 treatment in nude mice.

<p>A. Various types of degenerating lobule (a—d), and their magnified figures (e—h). Arrows in a—d indicate intervening fibrous cords; Three types of characteristic degenerating region are shown in a and e as Type II, in b and f as Type III, in c and g as Type I and in d and h as Type IV (necrotic figures). Arrows in e—g indicate tumor cells, arrowheads and open arrows point to presumptive macrophage and NK cells, respectively. Double open arrows in e and f point to dying tumor cells with large pores. Arrows in h point to pyknotic nuclei or nuclear fragments. Asterisk in c shows calcified masses. a—h, Scale bar, 10 μm. Incidence of destructive lobules (B) and each degenerating type (C) in HeLaX-E9s, HeLaX-Ss and HeLaX-NTs. *, ** and ***, and a—d indicate significant differences by Chi-square test, at *, P<0.05, **, P<0.01, ***, P<0.001, respectively. a, b, c and d indicate significant difference from the value of NT (a), from the value of HeLaX-Ss or HeLaX-NTs (b), from the value of HeLaX-Ss or HeLaX-NTs (c), and between the same letter (d), at P<0.001. D. Type II region of HeLaX-E9-9s showing macrophages (small arrows) attached to tumor cells with pores (arrows). E. Type III region of HeLaX-E9-9s showing NK cells (small arrows) attached to tumor cells (arrows). F, G. Non-degenerating region of HeLaX-Ss showing macrophages (small arrows, F) and NK cells (small arrows, G) near tumor cells (arrows, F, G). H, I. Negative control for anti-F4/80 antibody stained with rat-IgG in HeLaX-E9-9s (H) and for anti-mouse NK1.1 antibody stained with rat IgG in HeLaX-E9-4.5s. Arrows point to tumor cells (H) and to presumptive NK cell (I). Scale bar, 10 μm. J. Vascular density in each HeLaXs. Individual vessels were counted as described in "Materials and Methods". **and *** indicate significant differences by ANOVA-test, at **, P<0.01 and ***, P<0.001, respectively. K—M. Population of proliferating tumor cells (K), perforating tumor cells (L) and apoptotic cells (M). *, **and *** indicate significant differences by Chi-square test, at *, P<0.05, **, P<0.01 and ***, P<0.001.</p