Western blot analysis showing inhibition of diabetic induction of Angpt1, Angpt2 and Tie-2 by PCE.

<p>HRMC were incubated in 5.5(A). HUVEC were incubated in HRMC conditioned media [with (w/) 27.5 mM mannitol and w/33 mM glucose] for 6 h in the absence and presence of 1–20 µg/ml PCE (B). Cell lysates were subjected to Western blot analysis with a primary antibody of Angpt1, Angpt2 and Tie-2. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the bottom panels represent quantitative results obtained from a densitometer. Respective values not sharing a common letter differ, <i>P</i><0.05.</p

Attenuation of diabetic induction of PECAM-1, integrin β3, VE-cadherin and TSP-1 by PCE.

<p>HRMC were incubated in 5.5[with (w/) 27.5 mM mannitol and w/33 mM glucose] for 6 h in the absence and presence of 1–20 µg/ml PCE (A). Cell lysates were subjected to Western blot analysis with a primary antibody of PECAM-1 and integrin β3 (A). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 5) in the bottom panels represent quantitative results obtained from a densitometer. The db/db mice were orally supplemented with 10 mg/kg PCE daily for 8 weeks (B and C). The db/m mice were employed as diabetic controls. The VE-cadherin induction in histological sections of mouse kidneys was immunohistochemically determined by using anti-mouse VE-cadherin and FITC-conjugated IgG (B). Each photograph is representative of four animals. Magnification: x400. The bar graphs (mean ± SEM, n = 4) in the right panel represent quantitative staining intensity. Plasma level of TSP-1 in mice was measured with an ELISA kit (C). Means not sharing a common letter differ, <i>P</i><0.05.</p

Aortic ring assay showing the diminution of diabetic angiogenesis by PCE.

<p>Aorta ring pieces were placed in a martrigel-pre-coated 24-well plate and treated with HRMC conditioned media in the presence of PCE or 10 ng/ml VEGF for 6 d. The microvessel outgrowth from aorta ring pieces was photographed and quantified. Respective values in bar graphs (mean ± SEM, n = 3) not sharing a letter are different at <i>P</i><0.05.</p

Inhibition of induction of VEGFR2, phospho-VEGFR2, Angpt1, Angpt2, Tie-2 and phospho-Tie-2 in diabetic kidneys by PCE.

<p>The db/db mice were orally supplemented with 10 mg/kg PCE daily for 8 weeks. The db/m mice were employed as diabetic controls. For the measurements of the tissue levels of VEGFR2 (A), histological sections of mouse kidneys were immunohistochemically stained by using anti-mouse VEGFR2 and color-fixed with substrate chromogens of 3,3′-diaminobenzidine. The sections were counter-stained with hematoxylin. The VEGFR2 level was identified as brown staining. Each photograph is representative of four animals. Magnification: x400. The bar graphs (mean ± SEM, n = 3) in the right panel represent quantitative staining intensity. For the Western blot analysis (B and C), tissue extracts were subjected to Western blot analysis with a primary antibody of phospho-VEGFR2, Angpt1, Angpt2, Tie-2 and phospho-Tie-2. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the bottom panels represent quantitative results obtained from a densitometer. Respective values not sharing a common letter differ, <i>P</i><0.05.</p

Double-immunofluorescence staining showing PECAM-1 (green) and Ki-67 (red) in db/db mice supplemented with PCE.

<p>The db/db mice were orally supplemented with 10 mg/kg PCE daily for 8 weeks. The db/m mice were employed as diabetic controls. For the measurements of the tissue level of Ki-67, histological sections of mouse kidneys were immunohistochemically stained by using anti-mouse Ki-67 and Cy3-conjugated IgG. The Ki-67 level was identified as reddish staining. The sections were also stained with anti-mouse PECAM-1 and FITC-conjugated IgG. Each merged image is representative of four animals. Magnification: x400. The bar graphs (mean ± SEM, n = 3) in the right panel represent quantitative staining intensity of fluorescent Ki-67. Respective values not sharing a letter are different at <i>P</i><0.05.</p

Inhibition of diabetic up-regulation of VEGF and HIF-1α by PCE.

<p>HRMC were incubated in 5.5[with (w/) 27.5 mM mannitol and w/33 mM glucose] for 6 h in the absence and presence of 1–20 µg/ml PCE (A). The db/db mice were orally supplemented with 10 mg/kg PCE daily for 8 weeks. The db/m mice were employed as diabetic controls (B and C). Cell lysates and tissue extracts were subjected to Western blot analysis with a primary antibody of VEGF and HIF-1α (A and D). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the bottom panels represent quantitative results obtained from a densitometer. Plasma level of VEGF in mice was measured with an ELISA kits (B). Means not sharing a common letter differ, <i>P</i><0.05.</p

Time course responses of ATF6, BiP/GRP78 and XBP1 (A) and their inhibition by 1–10 µg/ml PPE (B) in 1 µM tunicamycin-treated macrophages.

<p>For the measurement of induction of ATF6, BiP/GRP78 and XBP1 (B), total cell lysates were subject to Western blot analysis with a primary antibody against ATF6, BiP/GRP78 or XBP1. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05.</p

Time course responses of ATF6 and BiP/GRP78 induction (A) and their inhibition by 1–10 µg/ml PPE (B) in 1 µM tunicamycin-treated macrophages.

<p>For the measurement of induction of ATF6 and BiP/GRP78 (B), total cell lysates were subject to Western blot analysis with a primary antibody against ATF6 or BiP/GRP78. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05.</p

Inhibition of nuclear translocation (A and B), and temporal response of XBP1 transcription and its inhibition by PPE (C and D).

<p>J774A.1 macrophages were treated with 50 µg/ml oxidized LDL for 18 h in the absence and presence of 1–10 µg/ml PPE. Nuclear extracts were isolated by nuclear fraction assay and Western blot analysis was conducted by using primary anti-XBP1 (A). Lamin B was used as a nuclear control. Values (mean ± SEM, n = 3) not sharing a common letter are different at P<0.05. Nuclear translocation of XBP1 was also detected by immunofluorocytochemical staining with Cy3-conjugated XBP1 antibody and nuclear counter-staining was carried out with DAPI (B). Microscopic observation was done by fluorescent microscopy. Magnification: 200-fold. XBP1 mRNA levels were measured by quantitative RT-PCR assay (C and D). GAPDH was used for the internal control (n = 3).</p

PPE restoration of induction of ABCA1(A), SR-B1 (B) and ICAM-1 (C) demoted in tunicamycin-treated J774A1 murine macrophages.

<p>Cells were stimulated with 1 µM T091317 for 12 h, 50 µg/ml Cu²⁺-oxidized LDL for 6 h, and 10 ng/ml TNF-α for 6 h in the absence and presence of 1 µM tunicamycin and 10 µg/ml PPE. For the measurement of expression of ABCA1, SR-B1 and ICAM-1, total cell lysates were subject to Western blot analysis with a primary antibody against ABCA1, SR-B1 and ICAM-1. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative densitometric results of upper bands. Means without a common letter differ, P<0.05. Values (mean ± SEM, n = 3) not sharing a common letter are different at P<0.05.</p