Extraembryonic Endoderm cells as a model of endoderm development

Signal transduction in aging related disease

Glycosylated Cell Surface Markers for the Isolation of Human Cardiac Progenitors

Therapeutic cell differentiatio

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction

<div><p>Aims</p><p>Hereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFβ co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery.</p><p>Methods and results</p><p>After inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (<i>Eng</i>^+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4⁺ MNCs residing in the infarcted <i>Eng</i>^+/- hearts (<i>Eng</i>^+/- 73.17±12.67 vs. <i>Eng</i>^+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (<i>Eng</i>^+/- 46.60±9.33% vs. <i>Eng</i>^+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in <i>Eng</i>^+/- mice. An increased number of capillaries (<i>Eng</i>^+/- 61.63±1.43 vs. <i>Eng</i>^+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (<i>Eng</i>^+/- 11.88±0.63 vs. <i>Eng</i>^+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in <i>Eng</i>^+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. <i>Eng</i>^+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels.</p><p>Conclusions</p><p>In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in <i>Eng</i>^+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.</p></div

DPP4 inhibitor treatment of <i>Eng</i>^<i>+/-</i> mice restores homing of MNCs to injured myocardium at 4 days post-MI.

<p><b>(A)</b> Representative microscopy images of CXCR4 expression in the infarct border zone. White = surviving myocardium, black/grey area = infarcted myocardium. Photos taken at 30x magnification. Scale bar: 50μm. CXCR4 = red, cTnI = white, DAPI nuclear staining = blue. <b>(B)</b> Quantification of CXCR4 expressing cells in the infarct border zone (n = 6–8). Data shown are mean ± SEM, *P<0.05. <b>(C)</b> Quantification of MAC3 positive cells in the infarct border zone (n = 6–7). Data shown are mean ± SEM, *P<0.05. <b>(D)</b> Representative microscopy images of MAC3⁺/CD206^- (%M1) and MAC3⁺/CD206⁺ (%M2) expressing cells in the infarct border zone. Smaller panels: Top panel is the MAC3 signal, lower panel is the CD206 signal. Photos taken at 50x magnification. Scale bar: 20μm MAC3 = red, CD206 = green, DAPI = blue. <b>(E)</b> Quantification of the ratio of MAC3⁺/CD206^- (%M1) and MAC3⁺/CD206⁺ (%M2) expressing cells in the infarct border zone (n = 6–7). <b>(F)</b> Flow cytometric analysis of the macrophage population in the infarct area, ratio of inflammatory M1(Ly6G^-/CD11b⁺/Ly6C^high) versus regenerative M2 macrophages (Ly6G^-/CD11b⁺/Ly6C^low) (n = 3–6, non-parametric ANOVA testing). Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p

Monocyte specific knock-out of endoglin does not recapitulate the <i>Eng</i>^<i>+/-</i> phenotype.

<p><b>(A)</b> Western blot analysis of endoglin protein expression in LysM-Cre-<i>Eng</i>^+/+, LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl cultured macrophages. A representative experiment is shown. <b>(B)</b> Quantification of the Western blots for endoglin protein in LysM-Cre-<i>Eng</i>^+/+, LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl cultured macrophages in two independent experiments(macrophage cultures from 3 individual mice of each genotype were pooled per western blot). <b>(C)</b> Kaplan-Meier survival curve of wild type (WT), LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl mice 28 days post-MI (n = 6–10). <b>(D)</b> Cardiac function in percentage ejection fraction (ï) 14 days post-MI. Cardiac function was measured by ultrasound in long axis view (n = 5–9). Data are shown as mean ± SEM, *P<0.05.</p

DPP4 inhibitor treatment reduces infarct size.

<p><b>(A)</b> Experimental protocol and treatment overview. At day 0, MI is induced and DPP4 inhibition is started (shown in green) till day 5 post-MI by intraperitoneal (i.p.) injection of DipA (treatment group) or distilled water (control group). Cardiac echography was performed at day 7 and 14 post-MI. <b>(B)</b> Histological analysis of the infarct size by Picrosirius red staining for collagen (n = 5–9). Transverse sections of left ventricle, photos taken at 1.0x magnification. Infarct area = dark pink, healthy myocardium = light pink, blood cells = yellow. <b>(C)</b> Quantification of Picrosirius red staining in left ventricle (LV). Mice were subjected to MI and treated with either distilled water or DipA from day 0 till day 5 by daily i.p. injection (n = 5–9). Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p

DPP4 inhibition affects angiogenesis in the infarct border zone post-MI.

<p><b>(A)</b> Capillaries in infarct border zone, day 14 post-MI. Quantification of PECAM-1 positive vessels per area (n = 4–6). Data shown are mean ± SEM, *P<0.05. <b>(B)</b> Arteries in infarct border zone, day 14 post-MI. Quantification of PECAM-1/αSMA positive vessels per area (n = 4–6). Data shown are mean ± SEM, *P<0.05. <b>(C)</b> Quantification of total macrophage (MAC3) number in day 14 post-MI hearts (n = 4–6). Data shown are mean ± SEM, *P<0.05. <b>(D)</b> Quantification of MAC3⁺/CD206⁺ (%M2) expressing cells in the infarct border zone 14 days post-MI (n = 3–6). Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p

DipA treatment does not maintain improved cardiac function of <i>Eng</i>^<i>+/-</i> mice after MI.

<p><b>(A)</b> Experimental overview and percentage EF at 7 and 14 days post-MI of control treated mice, measured by ultrasound via left ventricle tracing (n = 5–9). Data shown are mean ± SEM, *P<0.05. <b>(B)</b> Percentage EF 7 and 14 days post-MI of DipA treated mice, measured by ultrasound via left ventricle tracing. Note that the control WT and <i>Eng</i>^<i>+/-</i> groups are the repeat of measurements used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189805#pone.0189805.g001" target="_blank">Fig 1D</a> (n = 9–11). Data shown are mean ± SEM, *P<0.05. <b>(C)</b> Long term treatment overview and Δï. DipA treatment (shown in green) up to 14 days post-MI and cardiac function with extended follow-up of 6 months (n = 5–11). Data depicted as ΔEF are the EF at the time point indicated on the x-axis compared to EF measured at day 7 post-MI. Cardiac function was measured by ultrasound via left ventricle tracing. DipA = DPP4 inhibitor Diprotin A, US = Ultrasound measurement. Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p