PPE-induced emphysema combined with chronic low-grade LPS-induced pulmonary inflammation does not affect atherosclerosis development.

<p>After 20 weeks of WTD feeding, mice were sacrificed and the total atherosclerotic lesion area (A) and lesion severity (B) were assessed. Furthermore, the number of monocytes adhering to the vessel wall, (C) macrophage (D), smooth muscle cell (E), collagen content (F) of the atherosclerotic lesions and collagen type I+III of Sirius red-positive area were measured. Significant effects detected by two-way ANOVA analysis are shown as p-value in textboxes above the figure. Values are presented as means ± SEM; n = 10–15; *p<0.05, **p<0.01, ***p<0.001.</p

PPE-induced emphysema does not affect atherosclerosis lesion area.

<p>During the study, blood was drawn to assess plasma cholesterol levels (A). After 20 weeks of WTD feeding mice were sacrificed, hearts were isolated and atherosclerotic lesion area (B) was determined in the aortic root area (representative pictures are shown in C). Also, the number of monocytes adhering to the vessel wall (D) and macrophage- (E), smooth muscle cell- (F) and collagen (G) content of the atherosclerotic lesions were measured. Values are presented as means ± SEM; n = 12–15; *p<0.05.</p

Antibodies used for MSC characterization.

<p>Antibodies used for MSC characterization.</p

LPS exposure or MSC treatment do not affect blood leukocytes.

<p>E3L mice were intranasally instilled with vehicle or 10 μg LPS twice weekly during 20 weeks WTD feeding. Mice received vehicle or 0.5x10⁶ MSC in week 14, 16, 18 and 20. Blood was drawn at 12 and 19 weeks and blood leukocytes, including WBC counts (A and B) were measured using an automated cell counter (Sysmex) and FACS analysis. (C) Levels of SAA were measured in plasma of mice at week 11 and week 16 (before and after MSC treatment). Data for A and B are shown as mean±SEM; n = 6–8; *p<0.05. Data in C are shown as mean±SEM; n = 12–15; ***p<0.001.</p

MSC treatment reduces inflammation in acute LPS-induced pulmonary inflammation.

<p>APOE*3-Leiden (<i>E3L</i>) mice fed a Western-type diet (WTD) were intranasally instilled with vehicle or 10 μg LPS on day 1 and 3. Mice received 0.5x10⁶ MSC 4 h and 24 h after the first LPS instillation. Blood was drawn 3 h after LPS instillation to measure systemic IL-6 response (A). Mice were sacrificed on day 4 after which BAL was collected and total (B) and differential (C) cell number was counted. The number of MPO-positive neutrophils in the lungs was determined immunohistochemically (D). Data are shown as mean±SEM; n = 12–15; *p<0.05, **p<0.01, ***p<0.001.</p

Intratracheal PPE instillation dose-dependently increases alveolar destruction, without affecting pulmonary inflammation.

<p>E3L mice were intratracheally instilled with vehicle, 15 or 30 µg PPE and sacrificed after 24 weeks. Mean linear intercept (B) and air/tissue ratio (C) were determined on sections of the lungs (A). Furthermore, right ventricular (RV) (D) and left ventricular (LV) wall thickness was determined and the ratio was calculated (E). The number of MAC3-positive macrophages (F) and MPO-positive neutrophils (G) in the lung was determined immunohistochemically. Values in B+C are represented as the mean; values in D-G are presented as means ± SEM; n = 12–15; *p<0.05, **p<0.01, ***p<0.001.</p

PPE-induced emphysema combined with low-dose LPS administration induces chronic low-grade pulmonary inflammation, without affecting emphysema.

<p>After intratracheal instillation of vehicle or PPE (30 µg/mouse) in <i>E3L</i> mice, low-dose intranasal LPS (1 µg/mouse) or vehicle was administered during 20 weeks WTD feeding. Total respiratory amplitude was analyzed at 8 and 18 weeks (C and D) after PPE. After sacrifice, mean linear intercept (A) and air/tissue ratio (B) were determined and inflammatory cell influx was determined by immunohistochemical staining of macrophages with F4/80 (E) and neutrophils with MPO (F). Significant effects detected by two-way ANOVA analysis are shown as p-value in textboxes above the figure. Values are presented as means ± SEM; n = 10–15; *p<0.05, **p<0.01, ***p<0.001.</p

Groups and groups sizes.

<p>Groups and groups sizes.</p

Antibodies used for FACS analysis of BAL cells and lung tissue.

<p>Antibodies used for FACS analysis of BAL cells and lung tissue.</p

Chronic LPS instillation and MSC treatment do not affect atherosclerosis.

<p>E3L mice fed a WTD were intranasally instilled with vehicle or 10 μg LPS twice weekly during 20 weeks WTD feeding. Mice received vehicle or 0.5x10⁶ MSC in week 14, 16, 18 and 20. Hearts were isolated and fixed in phosphate-buffered 4% formaldehyde and processed for paraffin embedding. For quantification and classification of atherosclerosis, the hearts were cross-sectioned (5 μm) throughout the entire aortic root area and stained with HPS (A). Lesion area (B) and severity (C) was determined with ImageJ. MAC-3 positive macrophages content was determined (D). Data are shown as mean±SEM; n = 12–15.</p