LPS challenge to hepatocytes and resident macrophages.

<p>To investigate the role played by hepatocytes and resident macrophages following LPS challenge, we used chimeric mice that expressed TLR4 either in the liver or in peripheral blood mononuclear cells. Chimerism was confirmed 8-10 weeks following transplantation when at least 95% of the transplanted bone marrow was present. Representative samples of the 3 transplanted groups: WT/WT, TLR4KO/WT, WT/TLR4KO mice are presented in Figure A1 –A3. Following LPS challenge, TLR4 was highly expressed in the leukocyte–immune bone marrow derived cells in both chimeric mice that were transplanted with WT BM, as analyzed by FACS and presented in Figures B1 and B3. However, TLR4 expression was not induced in the leukocytes following LPS in chimeric mice that were transplanted with TLR4KO BM, since the hematopoietic system lacks TLR4 as presented in B2. TLR4 expression in the different groups are stated as mean ±SEM (n=3 in each group) and presented in Figure C. Histological analysis of chimeric mice challenged with LPS is presented in Figure D1-D2. There was diffuse hepatic congestion and focal necrosis associated with diffused neutrophil infiltration in LPS administered WT/WT, TLR4KO/WT mice compared to WT/TLR4KO mice administered LPS in which few neutrophils were observed (D3). Values are mean±SEM (n=3 mice per group) *p>0.05 compared with other groups.</p

CoPP increases HO-1,AKT and GSK3β expression in cardiomyocytes culture.

<p>A: Western blot analysis of HO-1 protein in the cardiomyocyte subjected to hypoxia CoPP treatment increased HO-1 expression levels in cells subjected to hypoxia compared to non-treated cells subjected to hypoxia. Means ± SD, n = 3 in 3 different experiments (*<i>p</i><0.05 vs. normoxia, ^#<i>p</i><0.005 vs. hypoxia). B: Western blot analysis of AKT protein in the cardiomyocytes subjected to hypoxia: CoPP treatment increased AKT phosphorylation in cells subjected to hypoxia compared to non-treated cells subjected to hypoxia Means ± SD, n = 3 in 3 different experiments. (*<i>p</i><0.01 vs. normoxia, ^#<i>p</i><0.05 vs. hypoxia, ^{p<0.05 vs. hyp+CoPP). C: Western blot analysis of pGSK3β protein in the cardiomyocytes subjected to hypoxia CoPP treatment increased GSK3β phosphorylation in cells subjected to hypoxia compared to non-treated cells subjected to hypoxia. Means ± SD, n = 3 in 3 different experiments (*p<0.01 vs. normoxia, ^#p<0.01 vs. hypoxia,}<i>p</i><0.05 vs. hyp+CoPP).</p

A: Kaplan-Meier survival curve.

<p>While 5/6 mice (83%) were treated with LPS died in less than 8 hours and the sixth died within the first 24 hours, all TLR4KO mice (6/6) survived following LPS (Kaplan-Meier, p < 0.001). <b>B: Real-time PCR analysis of hepatic TLR4 mRNA expression:</b> The results are shown as the fold-change of the relative quantity of TLR4 mRNA normalized to TATA-box mRNA values. TLR4 mRNA expression significantly increased 3-fold compared with saline administration in WT mice, 4 hours following LPS administration (*p ≤ 0.005); results are expressed as mean ±SE, n=4 in each group.</p

experimental protocol: Schematic illustration of the protocols used in the in vivo experiments.

<p>experimental protocol: Schematic illustration of the protocols used in the in vivo experiments.</p

Serum biochemical analysis.

<p>Figs. A, D.: The serum LDH and AST level increased significantly in LPS-administered WT mice compared with saline administered mice. In LPS administered TLR4KO, the increase in serum enzyme levels was much lower compared with saline administered and compared with LPS administered WT mice (4 and 24 hours). Values are mean±SEM with n=6 mice per group *p<0.01 compared with other groups. Figs. B, E: The serum and hepatic Il-1β level increased significantly in WT mice following LPS compared with saline treated mice (p< 0.01). TLR4KO mice hepatic IL-1β level was significantly lower compared with WT LPS treated mice (p< 0.05). Values are mean±SEM (n=6 mice per group). Figs. C, F: The serum and hepatic TNFα level increased significantly in LPS WT-treated mice (p< 0.01). In TLR4KO mice the hepatic TNFα level was significantly lower compared with WT LPS treated mice (p< 0.005) and serum TNFα rose by a very small amount. Values are mean±SEM (n=6 mice per group). </p

Changes in fractional shortening and left ventricle dimensions in Diabetic Mice.

<p>A: LV end-systolic diameters 24 hour post -MI. The LV end-systolic diameter increased in diabetic mice compared with non-diabetic values (*<i>p</i><0 05 vs. non diabetic # <i>p</i><0.05 vs. diabetes values are presented as means ± SD). B: Fractional shortening (FS): CoPP treatment increased FS compared to the untreated diabetic ones SnPP abolished the beneficial effect (*<i>p</i><0.05 vs. non diabetic # <i>p</i><0.01 vs. diabetes, ^$<i>p</i><0.05 vs. diabetes + CoPP values are presented as means ± SD; n = 7 in control and CoPP group; n = 5 in other groups values are presented as means ± SD).</p

Gold Nanorods as Absorption Contrast Agents for the Noninvasive Detection of Arterial Vascular Disorders Based on Diffusion Reflection Measurements

In this study we report the use of gold nanorods (GNRs) as absorption contrast agents in the diffusion reflection (DR) method for the in vivo detection of atherosclerotic injury. The early detection and characterization of atherosclerotic vascular disease is considered to be one of the greatest medical challenges today. We show that macrophage cells, which are major components of unstable active atherosclerotic plaques, uptake gold nanoparticles, resulting in a change in the optical properties of tissue-like phantoms and a unique DR profile. In vivo DR measurements of rats that underwent injury of the carotid artery showed a clear difference between the DR profiles of the injured compared with healthy arteries. The results suggest that DR measurements following GNRs administration represent a potential novel method for the early detection of atherosclerotic vascular disease

Gold Nanorods as Absorption Contrast Agents for the Noninvasive Detection of Arterial Vascular Disorders Based on Diffusion Reflection Measurements

In this study we report the use of gold nanorods (GNRs) as absorption contrast agents in the diffusion reflection (DR) method for the in vivo detection of atherosclerotic injury. The early detection and characterization of atherosclerotic vascular disease is considered to be one of the greatest medical challenges today. We show that macrophage cells, which are major components of unstable active atherosclerotic plaques, uptake gold nanoparticles, resulting in a change in the optical properties of tissue-like phantoms and a unique DR profile. In vivo DR measurements of rats that underwent injury of the carotid artery showed a clear difference between the DR profiles of the injured compared with healthy arteries. The results suggest that DR measurements following GNRs administration represent a potential novel method for the early detection of atherosclerotic vascular disease

A,C: TNFα and IL-1β protein expression in chimeras in serum and liver.

<p>In all chimeras, both TNFα and IL-1β increased significantly at 4 hours post LPS challenge. The proinflammatory cytokine levels in chimeras expressing TLR4 in the liver were found to be significantly higher than the levels expressed in TLR4KO chimera livers ( * p<0.05 vs. WT/WT LPS treated ; + p<0.01 vs. + WT/WT saline-treated). Values are mean±SEM ( n=6 mice per group). <b>B,D: Hepatic TNFα and IL-1β expression:</b> No differences were observed between hepatic TNFα and IL-1β whether the livers expressed TLR4 or KO for TLR4. Values are mean±SEM (n=3 mice per group). <b>E: TLR2 and TLR4 mRNA expression level in a Huh7 cell line:</b> Macrophages expressed a significantly higher level of TLR4 and TLR2 mRNA compared with the hepatocyte cell line (p < 0.0001). <b>F: TLR2 and TLR4 mRNA expression following LPS treatment:</b> LPS induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while there was no change in TLR2 mRNA expression. The results are shown as the fold-change of the relative quantity of TLR mRNA normalized to GAPDH mRNA values; the control cells were assigned a value of 1 and results are normalized to controls. Each point represents the mean ±SEM of a set of data determined by at least 5 experiments.</p

Anti-apoptotic effects of HO-1 on cardiomyocytes in diabetic mice.

<p>A: Western blot analysis of Bax B: Bcl-2 C: and Bcl-2/Bax ratio, in mice subjected to LAD ligation. CoPP treatment increased the levels of Bcl-2 expression but it did not change Bax expression to shift the Bcl-2/Bax ratio towards the antiapoptotic Bcl-2 (Means ± SD, n = 5. *<i>p</i><0.05 vs. diabetes+MI, ^#<i>p</i><0.05 vs. diabetes+CoPP, Table, *<i>p</i><0.05 vs. diabetes+MI, ^#<i>p</i><0.05 vs. diabetes+CoPP).</p