Data_Sheet_1_Extracellular Vesicles Work as a Functional Inflammatory Mediator Between Vascular Endothelial Cells and Immune Cells.docx

<p>Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes.</p

Detection of NO produced in explanted murine carotid arteries <i>ex</i><i>vivo</i> using Cu ₂FL2E (20 µM) after precontraction.

<p>(<b>a</b>) Detection of NO in response to NA (ECs and SMCs are not apparent), (<b>b</b>) Detection of NO in post NA and ACh stimulation (2.5min) (ECs and SMCs are apparent), (<b>c</b>) Syto 41 staining of nucleus of ECs and SMCs, (<b>d</b>) plot of fluorescence intensities of the ECs and SMCs (from carotid artery) measured with NA and ACh stimulation for 15min.</p

Functional imaging of NO in pre-contracted arteries.

<p>3D reconstruction and luminal diameter measured from explanted murine carotid arteries <i>ex </i><i>vivo</i> using Cu ₂FL2E (20 µM) (<b>a</b>) before precontraction (<b>b</b>) after precontraction with NA, (<b>c</b>) in post NA and ACh stimulation (2.5min), error bars indicate s.d. (n=3), (<b>d</b>) luminal diameter measured from arteries with conditions mentioned in a, b and c, error bars indicate s.d. (n=3).</p

Detection of NO produced in explanted murine carotid arteries <i>ex</i><i>vivo</i> using Cu ₂FL2E (20 µM).

<p>(<b>a</b>) & (<b>b</b>) Magnified images of vessel showing basal NO signal detected after 5 min incubation of Cu ₂FL2E without any stimulus at medial and intimal focal planes, respectively. (<b>c</b>) NO signal detected in smooth muscle cells (SMCs) and (<b>d</b>) endothelial cells (ECs) of the tissue with 5 min incubation of Cu ₂FL2Eand, subsequently 45min incubation of H₂O₂ (150 µM). Scale bar is 50 µm, (<b>e</b>) & (<b>f</b>) Magnified images of vessel showing NO signal detected after 5 min incubation of Cu ₂FL2E and subsequently, 45 min incubation of H₂O₂ (150 µM) in SMCs at medial plane and in ECs at intimal plane respectively, (<b>g</b>) Quantification of spatial distribution of fluorescence intensity as measure of NO in cells of vessel wall stimulated with H₂O₂ (n = 5). (<b>h</b>) Quantification of spatial distribution of fluorescence intensity as measure of NO in cells of vessel wall stimulated with flow (flow rate= 2.1 Pa, time=45min), (n = 5).</p

Sensitivity and specificity of Cu ₂FL2E.

<p>(<b>a</b>) Fluorescence response of Cu ₂FL2E (2 µM) to various concentrations of NO after 1 min of SNAP administration. n = 5 for each concentration, (<b>b</b>) Linear regression curve plotted from (a), (<b>c</b>) Fluorescence response of Cu ₂FL2E to NO (50 µM SNAP in PBS at 37°C, pH 7.4) and H₂O₂ (150 µM). The spectra were obtained 1 min after SNAP addition n = 5. Error bars indicate s.d., (<b>d</b>) Cytotoxicity assay with different concentrations Cu ₂FL2E.</p

Scheme of NO detection by Cu ₂FL2E in endothelial cells, showing the probe is trapped in the cell via hydrolysis of the pendant ester groups by intracellular esterases to give Cu ₂FL2A since the ester (E) is hydrolyzed to the acid (A).

<p>Scheme of NO detection by Cu ₂FL2E in endothelial cells, showing the probe is trapped in the cell via hydrolysis of the pendant ester groups by intracellular esterases to give Cu ₂FL2A since the ester (E) is hydrolyzed to the acid (A).</p

Detection of NO with Cu ₂FL2E produced by endothelial cells <i>in</i><i>vitro</i>.

<p>(<b>a</b>) NO detection in porcine aortic endothelial cells (PAECs); Left: 45 min incubation of Cu ₂FL2E (20 µM). Right: 45 min incubation of Cu ₂FL2E (20 µM) and H₂O₂ (150 µM). Top: bright-field images of cells. Bottom: fluorescence images of cells. Scale bar is50 µm. (<b>b</b>) Quantification of fluorescence intensity plotted against incubation time. (<b>c</b>) Detection of NO with Cu ₂FL2E in HCAECs cells, with or without NO-inhibitor (L-NAME). Shown are the fluorescence images after 45min co-incubation of the probe (Cu ₂FL2E =2 µM) with H₂O₂ (150 µM), L-NAME (100 µM), and/or ACh (10 µM) according to scheme. Scale bar is 75 µm. (<b>d</b>) Quantification of fluorescence intensity from (c) plotted against each condition mentioned in (c) (n = 5). Error bars indicate s.d.</p

Functional imaging of NO.

<p>(<b>a</b>) 3D reconstruction of vessels with Cu ₂FL2E (20 µM) without/ with stimulus (here ACh), (<b>b</b>) luminal diameter measured from arteries with conditions mentioned in (a), (<b>c</b>) normalized fluorescence intensities of the arteries with conditions mentioned in (a), (<b>d</b>) 3D reconstruction of vessels with Cu ₂FL2E without/ with stimulus (here ACh) and also in combination with L-NAME, (<b>e</b>) luminal diameter measured from arteries with conditions mentioned in (d), (<b>f</b>) normalized fluorescence intensities of the arteries with conditions mentioned in (c), error bars indicate s.d. (n=5).</p

Relative mRNA expression in control and <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice under physiological and endotoxemic conditions.

<p>(A) Relative <i>Arg1</i> mRNA expression was significantly higher in the control + LPS group than in basal and <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> + LPS groups. As expected, <i>Arg1</i> expression was low in <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice. (B) Interestingly, the relative <i>Arg2</i> mRNA expression was significantly higher in the <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice after LPS infusion compared to control mice during endotoxemic conditions. (C) LPS infusion resulted in increased <i>Nos2</i> expression in both mouse strains compared to basal conditions. (D) <i>Nos3</i> mRNA expression was significantly higher in the <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice compared to control animals under basal and LPS-infused conditions (p<0.05). The relative expression of the two household genes <i>Actb</i> and <i>Ppia</i> were used to calculate the geometric mean, which served as a normalization factor.</p

LPS treatment increases density of MPO-positive cells in <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice.

<p>(A) LPS infusion increased the influx of MPO-positive cells into jejunal tissue of control and <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice, but more so in <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice (P<0.05). Magnification 200x. (B) Jejunal villi of H&E- (upper panel) and MPO-stained (lower panel) villi of LPS-treated control (left) and <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice (right). Arrows indicate the MPO positive cells. Magnification 200x.</p