Cell viability.

<p>MTT assay was performed to evaluate mouse choroidal endothelial cells (ECs) viability after treatment with different concentrations of α1(IV)NC1 and its N- and C- terminal domains α1S1(IV)NC1 and α1S2(IV)NC1. ECs grown with and without FCS as positive and negative controls. Experiments were performed with three replicates and data in the graphs are represented as mean±SD.</p

FasL activation (A and B).

<p>Mouse choroidal endothelial cells were incubated with and without different doses of α1S1(IV)NC1 and α1S2(IV)NC1 domains for 6 and 18-hrs, and total cells were collected, lysed for 30-min in ice-cold RIPA lysis buffer and about 25 µg of total cytosolic extract per lane was separated and immunoblotted with primary antibodies against FasL and Fas. In panel A and B, β-catenin was shown as loading control.</p

Caspase-8 activation (A and B).

<p>Mouse choroidal endothelial cells (ECs) were incubated with and without different doses of α1S1(IV)NC1 and α1S2(IV)NC1 domains for 6 and 18-hrs, and total cells lysed for 30-min in ice-cold RIPA lysis buffer and about 25 µg of cytosolic extract per lane was separated and immunoblotted with primary antibodies against caspase-8. <b>Caspase-3 activation (C and D)</b>. ECs were incubated with and without different doses of α1S1(IV)NC1 and α1S2(IV)NC1 domains and total cytosolic extract immunoblotted with primary antibodies against caspase-3.</p

Attenuation of antitumor activity and tumor vasculature apoptosis by caspase-3 inhibitor.

<p>α1(IV)NC1 alone or together with DEVD was injected into SCC-PSA1 tumor bearing mice daily for 15-days. Frozen sections (4.0 µm) of tumors were examined through immunohistochemistry. Apoptotic TUNEL positive cells (green, left), and CD-31 positive tumor blood vessel counts were scored from 10-microscopic fields and four tumors for each experimental condition (red, middle) and co-localization (yellow, right) was shown at 100× magnification. Scale bar corresponds to 50 µm.</p

Proliferation (A).

<p>Graph summarizes relative levels of methylene blue incorporation in mouse choroidal endothelial cells (ECs) treating with different concentrations of α1(IV)NC1 and its N- and C terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains compared with and without FCS controls after 48-hrs. All groups represent triplicate samples and data in the graphs are represented as mean±SD. <b>Migration (B)</b>. Number of ECs with and without α1S1(IV)NC1 and α1S2(IV)NC1 domains migrated towards VEGF on the underside of Boyden chamber membrane were shown and cells viewed using a light microscope after 48-hrs representative fields (100× magnification) shown. <b>Tube formation (C)</b>. ECs were plated on Matrigel coated plates in endothelial cell medium as control or with 1.0 µM α1(IV)NC1, α1S1(IV)NC1 and α1S2(IV)NC1 proteins. Tube formation was assessed using a light microscope after 48-hrs, and representative fields at 100× magnification were shown.</p

Immunohistochemistry of adventitial layer in abdominal aorta from ApoE^−/− mice with <i>P. gingivalis</i> infection.

<p>(A) Saline treated mouse tissue (B) demonstrates reduced macrophage invasion with Serp-1 treatment. (C) Bar graph demonstrates significantly reduced counts of positively stained macrophage in the aortic adventitia in Serp-1 treated mice with <i>P. gingivalis</i> infection + BA (C; <i>P</i>≤0.009).</p

Histology of aortic plaque with <i>P. gingivalis</i> infection.

<p>Hematoxylin and eosin stained cross sections of abdominal aorta from ApoE^−/− mice 24 weeks after BA. <i>P. gingivalis</i> induced increase plaque thickness is significantly reduced by M-T7, with Serp-1 showing a trend towards reduction. BA with saline treatment and no <i>P. gingivalis</i> infection (A). BA with saline treatment and <i>P. gingivalis</i> infection (B). M-T7 treatment with BA and <i>P. gingivalis</i> (C). Serp-1 treatment with BA and <i>P. gingivalis</i> (D). Arrows indicate margins of intimal plaque. Arrow heads point to inflammatory mononuclear cell invasion. Magnification 200X.</p

Anti-inflammatory protein treatment reduced TLR4 and MyD88 staining in mice after <i>P. gingivalis</i> infection + BA.

<p>(A) Immunohistochemical staining of TLR4 in sham-infected mice (left). (B) Increased TLR4 staining in mice infected with <i>P. gingivalis</i> + BA injury. (C) Increased MyD88 staining in mice infected with <i>P. gingivalis</i> + BA injury. (D) Decreased MyD88 staining in mice with <i>P. gingivalis</i> + BA after Serp-1 treatment. (E) Bar graph of MyD88 expression showing increased MyD88 in aortic adventitia in <i>P. gingivalis</i> infected mice + BA (24 weeks) compared to <i>P. gingivalis</i> + BA with Serp-1 (<i>P</i>≤0.056) or M-T7 treatment (<i>P</i>≤0.013). (F) Bar graph of TLR4 expression showing increased TLR4 in abdominal aortic adventitial in <i>P. gingivalis</i> + BA (24 weeks) compared to <i>P. gingivalis</i> + BA in Serp-1 (<i>P</i> = 0.0004) and in M-T7 treated mice (<i>P</i>≤0.0001).</p

Morphometric evaluation of horizontal area alveolar bone resorption induced by <i>P. gingivalis</i>.

<p>Alveolar bone resorption (ABR) area measured between cementoenamel junction (CEJ) to the alveolar bone crest (ABC) on the buccal and palatal surfaces of the roots of all molars in mice (mean value in mm²± SD).</p><p>*Mean value in mm² and standard deviation from 3–5 mice per group measured using AxioVision line tool software. Mice jaw images captured at 10 x magnification and measured between CEJ to the ABC on the buccal and palatal surfaces of the roots of all molars.</p><p>Morphometric evaluation of horizontal area alveolar bone resorption induced by <i>P. gingivalis</i>.</p

Experimental scheme and serum IgG levels in mice.

<p>(A) Schematic diagram illustrating experimental design and time course. (B) Serum <i>P. gingivalis</i> IgG antibody levels in ApoE^−/− mice following 24 weeks oral infection (n = 3–5). Bar graphs show the mean ± SD IgG levels in serum from mice infected with <i>P. gingivalis</i> alone, <i>P. gingivalis</i> + BA with Serp-1 or M-T7, or from BA mice (P = 0.34 and 0.37, respectively). <i>Pg</i> - <i>P. gingivalis</i>; BA - balloon angioplasty.</p