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

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

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

Number of perfused vessels per villus under basal conditions and after LPS treatment in control, <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> and <i>Nos2^−/−</i> mice.

<p>Number of perfused vessels was determined with SDF-imaging. Fewer vessels per villus were perfused in <i>Arg1^fl/fl/Tie2-Cre^tg/−</i> mice than in control and <i>Nos2^−/−</i> mice, both under basal conditions and after LPS treatment. LPS infusion decreased the number of perfused vessels per villus in all three mouse strains (all n = 7).</p