Growth factor secretion and the cytoprotective effect of amnion and chorion MSCs.

<p>(A–D) Cytoprotective effect of FM MSC-derived conditioned medium was analyzed by the MTS assay (A, B) and caspase-3 activity (C, D) in HUVECs (A, C) and cardiomyocytes (B, D). Values are mean ± SEM. *p<0.05 vs. serum-free. (E–H) Conditioned medium obtained from FM-derived MSCs was collected after incubation for 24 h. The concentration of HGF (E), IGF-1 (F), bFGF (G), and VEGF (H) in serum free conditioned medium was measured by ELISA. *p<0.05 and ***p<0.001.</p

Immunosuppressive property of amnion and chorion MSCs.

<p>(A) Inhibition of human CD4+ T cell proliferation upon co-culture with human amnion, chorion, and bone marrow MSCs. (B) The concentration of PGE2 in FM-MSC-conditioned medium was measured by ELISA. Amnion MSCs secreted a significant amount of PGE2 compared with chorion MSCs. (C, D) Effect of human amnion (C) or chorion (D) MSC transplantation in a murine GVHD model. Treatment with amnion MSCs significantly reduced recipient weight loss in a mouse model of GVHD. *p<0.05, **p<0.01 and ***p<0.001.</p

Characterization of human amnion- and chorion-derived MSCs.

<p>(A) Representative photographs of human amnion and chorion. (B) Photographs of cultured MSCs obtained from human amnion and chorion at passage 3. Scale bars = 500 µm. (C) Relative cell number of amnion- and chorion-derived MSCs at each passage. (D) FACS analysis of amnion and chorion MSCs. (E, F) Differentiation of amnion and chorion MSCs into adipocytes (E) and osteocytes (F). Scale bars = 100 (E) and 50 (F) µm.</p

Hemodynamic studies.

<p>LVEDV  =  left ventricular end-diastolic volume, LVESV  =  left ventricular end-systolic volume, LVEDP  =  left ventricular end-diastolic pressure, LVESP  =  left ventricular end-systolic pressure, EF  =  ejection fraction. Values are means ± SD.</p><p>*<i>p</i> = 0.05 (<i>sik2^+/+</i> vs. <i>sik2^−/−</i> under normal salt),</p><p>***<i>p</i> = 0.001 (<i>sik2^+/+</i> vs. <i>sik2^−/−</i> under high salt).</p

Acting via SIK2, adducin controls the expression of transcription factors and gene expression associated with CH.

<p>A) Presence of SIK1 and SIK2 isoforms in HL-1 cells in culture. An equal amount of protein (150 µg) was loaded in each condition. SDS-PAGE and Western blot was performed and a representative blot is shown. B) SIK2 mRNA expression levels in HL-1 cells expressing either the normotensive (NT) or hypertensive (HT) variant of α-adducin. C) SIK2 activity in HL-1 cells expressing either the normotensive (NT) or hypertensive (HT) variant of α-adducin. Bars represent the mean + s.e.m. of 6 experiments. D) β-myosin heavy chain (β-MHC) luciferase activity in HL-1 cells co-expressing either variant (NT or HT) of α-adducin with the SIK2 wild-type or a negative mutant (SIK2-K49M). E) Brain natriuretic peptide (BNP) luciferase activity in HL-1 cells expressing co-expressing either variant (NT or HT) of α-adducin with the SIK2 wild-type or a negative mutant (SIK2-K49M). F) MEF2C luciferase activity in HL-1 cells co-expressing either variant (NT or HT) of α-adducin with the SIK2 wild-type or a negative mutant (SIK2-K49M). Mean + s.e.m. of 6 experiments performed in duplicate are shown.</p

Adducin controls cellular SIK2 expression.

<p>A) Correlations between α-adducin (<i>left panel</i>) and γ-adducin (<i>right panel</i>) and SIK2 mRNA expression in biopsies obtained from human hearts. B) Representative Western blots of α- (<i>left panel</i>) and γ- adducin (<i>right panel</i>) in HL-1 cells transiently (48 h) transfected with α-add- (α-add.) or γ-add- siRNAs (γ-add.) and compared to control cells transfected with scrambled-siRNAs (scr.). Equal amount of protein (75 µg) was loaded in each condition. Antibodies used: α-adducin (1∶1000), γ-adducin (1∶1000) and loading control, β-tubulin (1∶1000). C) Expression levels (mRNA) of α-, γ- adducin, and SIK2 in cells transiently transfected with α-add- (<i>left panel</i>) or γ-add-siRNAs (<i>right panel</i>) vs. cells transfected with scrambled RNAs. Each bar represents the mean ± s.e.m., n = 4.</p

Expression of cellular markers of CH in normotensive- and pre-hypertensive Milan rats.

<p>Studies were performed in normotensive (MNS) and pre-hypertensive (MHS-pre) Milan rats at 30 days of age. Each bar represents the mean ± s.e.m., n = 6 animals in each group. <b>A)</b> Body weight; <b>B)</b> Left ventricular (LV) weight/body weight (BW) ratio; <b>C)</b> Systolic blood pressure (SBP); <b>D)</b> Brain natriuretic peptide (NPPB) mRNA expression levels; <b>E)</b> β-myosin heavy chain (β-MHC) mRNA expression levels; <b>F)</b> α-myosin heavy chain (α-MHC) mRNA expression levels; <b>G)</b> Representative hematoxylin and eosin stained cross-sections from the left ventricle MNS and MHS-pre rats. Bar: 50 µm; <b>H)</b> Expression levels of mRNA for SIK isoforms.</p

Correlations between mRNA levels of adducin and SIK2 with markers of CH in 139 biopsies from human hearts.

<p>A) Correlation between SIK2 and β-myosin heavy chain mRNA (MYH7) (<i>left panel</i>), cardiac specific β-myosin heavy chain mRNA (MYH7B) (<i>middle panel</i>) and sarcomere β-myosin heavy chain mRNA (MYH7.2) (<i>right panel</i>). B) Correlation between the mRNA expression levels of SIK2 and skeletal actin gene (ACTA1). C) Correlation between the mRNA expression levels of α-adducin (<i>left panel</i>) and γ-adducin (<i>right panel</i>) and sarcomere β-myosin heavy chain (MYH7.2). D) Correlation between the mRNA expression levels of α-adducin (<i>left panel</i>) and γ-adducin (<i>right panel</i>) and skeletal actin gene (ACTA1). E) Correlation between the mRNA expression levels of SIK2 (<i>left panel</i>), α-adducin (<i>middle panel</i>), γ-adducin (<i>right panel</i>) and MEF2C.</p

Serum electrolytes.

<p>mE/L =  milliequivalents per liter. Values are means ± SD. *<i>p</i> = 0.038 (<i>sik2^+/+</i> vs. <i>sik2^−/−</i> under high salt).</p

Effects of high salt diet on blood pressure and left ventricle size in <i>sik2^+/+</i> and <i>sik2^−/−</i> mice.

<p>Black bars correspond to mice on normal salt diet, whereas blue bars correspond to mice on high salt diet. A) Body weight, B) Systolic blood pressure (SBP), C) Diastolic blood pressure (DBP) and D) Heart weight/body weight (HW/BW) ratio. E) Representative gross images of whole hearts, and F) Representative ultrasound scans. Quantitative analysis of G) left ventricular anterior wall (LVAW) thickness, H) left ventricular posterior wall (LVPW) thickness, I) Left ventricular end-diastolic diameter (LVDd) and J) Fractional shortening (FS). Mean ± s.e.m. of 7–9 mice in each group.</p