Representative examples of histological and immunohistochemical cross-section (CHE, MAC2, SMA).

<p>A–D) Elastase treated aortae after 7 weeks: (A) Cholinesterase (CHE) staining (x100) shows absence of neutrophils, (B) MAC2 (x100) staining shows absence of macrophages, (C) alpha-Smooth Muscle Actin (SMA) staining (x100) shows abundant content of smooth muscle cells, (D) DAPI staining. E–H) Control grafted aortae after 7 weeks: (E) Cholinesterase (CHE) staining (x100) shows absence of neutrophils (F), MAC2 (x100) staining shows no accumulation of macrophages, (G) alpha-Smooth Muscle Actin (SMA) staining (x100) shows minimal loss of smooth muscle cell actin, (H) DAPI staining.</p

Parameters measured by high resolution ultrasound.

<p>Indicated parameters were measured in control grafted mice and mice transplanted with elastase-pretreated aortae at 4 and 7 weeks after surgery. * p≤0,05; ** p ≤0,01; *** p ≤0,001 <i>versus</i> controls.</p

Representative examples of histological cross-section (EVG, SIR).

<p>A,B) Elastase treated aortae after 7 weeks: (A) Elastic van Gieson (EVG) staining (×100, ×200) reveals destruction of medial elastin layer, neointima formation, but no existence of pseudoaneurysms. (B) Sirius Red Staining (SIR) (x100) shows substantial collagen content. C,D) Control grafted aortae after 7 weeks: (C) Elastic van Gieson (EVG) staining (x100) shows no destruction of the medial elastin layer, no existence of aneurysms or no neoitimal formation, (D) Sirius Red Staining (SIR) (x100) shows only low collagen content.</p

Anterior δa/Dd and posterior δp/Dd wall displacement ratio.

<p>(A) Anterior and (B) posterior wall displacement ratios were measured in control grafted mice and mice transplanted with elastase-pretreated aortae at 4 and 7 weeks after surgery using high resolution ultrasound. *p<0,05, ***p<0,001.</p

Schematic presentation of the main steps of the procedure.

<p>(A, D) Step 1: Mouse jugular catheter introduced through an aortotomy and secured with a silk tie. (B, E) Step 2: Aorta filled with saline containing type I porcine pancreatic elastase. (C, F, G) Step 3: Aortic transplantation using sleeve technique/F: Proximal anastomosis, G: Distal anastomosis/. (H) Aorta after transplantation.</p

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

<div><p>Purpose</p><p>Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.</p><p>Methods</p><p>To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.</p><p>Results</p><p>Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.</p><p>Conclusion</p><p>Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.</p></div

BC−/EC+ exhibited increased end-systolic and end-diastolic volume and decreased left ventricular function 3 weeks post MI.

<p>BC−/EC+ exhibited an increase in end-systolic (<b>A</b>) and end-diastolic volume (<b>B</b>), a significantly more pronounced decrease in stroke volume (<b>C</b>) (BC+/EC+ n = 8 and BC−/EC+ n = 5; two-way ANOVA and Bonferroni’s post hoc test or student’s t-test; * p<0.05, ** p≤ 0.01 BC+/EC+ vs. BC−/EC+; # p<0.05, ## p≤ 0.01, ### p≤ 0.001 BC+/EC+ at different time points; $p<0.05,$$p≤ 0.01,$ $$ p≤ 0.001 BC−/EC+ at different time points), and decreased left ventricular developed pressure (<b>D</b>) 3 weeks post MI compared to BC+/EC+ (BC+/EC+ n = 24 and BC−/EC+ n = 18; student‘s t-test; ***p≤0.001).</p

Flow chart of the presented study.

<p>In a closed chest model, animals were subjected to reperfused myocardial infarction. After 60 min of ischemia, animals were divided into two different groups: 1) 72 h post MI 2) 3 weeks post MI. Further analysis followed as depicted.</p

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

<div><p>We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.</p></div

Caffeine effects in the heart depend on p27.

<p><b>(A)</b> The mouse cardiomyocyte cell line HL-1 was lentivirally transduced with an empty vector (“EV”) or an expression vector for mitochondrially targeted p27 (“mito p27”) and treated with 500 μM H₂O₂ for 48 hours. Apoptosis was measured as annexin V positive/7-PI negative cells by flow cytometry. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus EV −H₂O₂, ^#<i>p</i> < 0.05 versus EV +H₂O₂ (one-way ANOVA). <b>(B)</b> Respiration was determined in isolated heart mitochondria of adult wild-type mice (“wt”) and p27-deficient littermates (“p27ko”), who had received drinking water without caffeine or water supplemented with 0.05% caffeine for 10 days. Respiration was measured as O₂ consumption without the addition of substrates (“mito”) and after the successive addition of malate/glutamate (“M/G”), ADP, rotenone (“rot”), and succinate (“succ”) (left panel). The right panel shows a magnification of O₂ consumption after the addition of M/G and ADP, respectively. Data are mean ± SEM, <i>n</i> = 5–8 per group, *<i>p</i> < 0.05 versus wt without caffeine (one-way ANOVA). <b>(C)</b> Adult p27-deficient animals and their wild-type littermates received drinking water or water supplemented with 0.05% caffeine for 10 days. RNAs were isolated from the hearts of those mice, and microarray analyses were conducted. Data are represented as a Venn diagram. The numbers in the circles indicate the number of transcripts regulated in the two genotypes (<i>n</i> = 3 animals per genotype and treatment, <i>p</i> < 0.05). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. ADP, adenosine diphosphate; n.s., not significant; PI, propidium iodide.</p