ApoA-II hepatic expression and plasma levels in wild type, <i>Apoe</i> knockout, and <i>Apoa1/Apoe</i> double knockout C57 and FVB mice.

<p>(<b>A</b>) ApoA-II hepatic RNA expression by real-time PCR. (<b>B</b>) Plasma apoA-II by western blot. @, p<0.05; apoE−/− vs EA−/−. Data represents mean +/− standard deviation for 2–5 samples. (<b>C</b>) FPLC fractionated plasma apoA-II by western blot along with percent of total apoA-II associated with each fraction. (<b>D</b>) FPLC fractionated plasma cholesterol.</p

Circular dichroism of recombinant mouse apoA-II.

<p>Three accumulations of each sample were run at room temperature and the spectra shown as the overall mean. CD spectra were fitted using a Continll protein fit to derive protein secondary structure values.</p

ApoA-II as an acceptor for cholesterol efflux from (A) mouse J774 macrophages, and (B) ABCA1- and ABCG1- overexpressing BHK cells.

<p>(<b>A</b>) J774 cells were incubated 24 hrs with 25 µg/ml Acetylated LDL+2 µg/ml Sandoz ACAT inhibitor+1 µCi/ml [³H]-cholesterol +/− the presence of 0.3 mM 8-(4-chlorophenyl-thio)-cAMP (CPT-cAMP). Cells were washed and incubated 4 hrs with varying concentrations of C57 or FVB apoA-II and the [³H]-cholesterol counted in the media and cells. (<b>B</b>) Mock, ABCA1, or ABCG1 transfected BHK cells were loaded overnight with 1 µCi/ml [³H]-cholesterol. Cells were washed and induced 24 hrs with 1 mg/ml BSA +/− 20 mM Mifepristone. Cells were washed and incubated 4 hrs with 0 or 5 µg of C57 or FVB apoA-II and the [³H]-cholesterol counted in the media and cells. Data represents mean +/− standard deviation for triplicate samples. @, p<0.05;%, p<0.01; #, p<0.005.</p

DMPC vesicle clearance by mouse apoA-II. DMPC multi-lamellar vesicles (0.25 mg/ml final concentration) were incubated at 25°C with 0.0–0.1 mg/ml of each recombinant protein (n = 2–3).

<p>The decrease in absorbance over time at OD490 was measured every 30(no protein control). Data represents mean +/− standard deviation.</p

Purification of recombinant mouse apoA-II.

<p>(<b>A</b>) Expression and cleavage of apoA-II-Intein-Chitin binding domain fusion protein. Lanes 1: Post lysis pellet, 2: Post lysis supernatant, 3: Post Chitin unbound elution, 4: Bound fusion protein, 5: Post DTT cleave/pre elution Chitin bead, 6: Post DTT cleave eluted protein, 7: Post cleave/post elution Chitin bead, 8: molecular weight marker. (<b>B</b>) Purified recombinant mouse apoA-II post HPLC purification. Lane 9: recombinant C57 apoA-II, 10: recombinant FVB apoA-II. ApoA-II-Intein-Chitin binding domain = 37.6 kd, Intein-Chitin binding domain = 28.7 kd, apoA-II = 9.0 kd.</p

Decreased HL expression in Ad-LIGHT infected mice is independent of T cells and IL-1β.

<p>Mice were injected with PBS or adenoviral vectors (1.25×10⁹ pfu/mouse) and sacrificed on the 7^th day. (A) Liver HL mRNA expression in LIGHT adenovirus (Ad-L), human apoA-I adenovirus (Ad-AI) or PBS (P) injected LDLR^−/− mice was analyzed by real time PCR. (B) Morphology of liver from PBS and adenoviral infected mice (H & E staining, 20x objective). (C) Liver HL mRNA expression in Ad-L and P injected RAG^−/−LDLR^−/− and LIGHT^−/−LDLR^−/− mice. (D) Liver HL and IL-1β mRNA expression in Ad-L injected LDLR^−/− mice treated with control (C) or clodronate (CL) liposomes. The virus was injected 2 days after clodronate injection. (n = 3; *p<0.05, **p<0.01; for panels A and C: vs. PBS treated mice; for Panel D: vs. control liposome treated mice.).</p

LIGHT-mediated HL regulation in mice is independent of the presence of Kupffer cells.

<p>To deplete Kupffer cells, liposomes containing clodronate were injected through the tail vein into wild type C57BL/6 mice or Tg-LIGHT mice every 5^th day for 14 days. Control liposomes did not contain clodronate. (A) F4/80 staining for liver Kupffer cells in control liposome and clodronate liposome injected Tg-LIGHT mice. (B) Real-time PCR data for HL mRNA expression in the liver of control (C) and clodronate (CL) liposome injected wild type (WT) and Tg-LIGHT mice. (n = 3; *p<0.01 WT vs. Tg-LIGHT).</p

Evidence for trans-regulation by LIGHT and lack of a role of liver non-parenchymal cells.

<p>(A) HL mRNA expression in hepatocytes from LDLR^−/− (WT) and LTβR^−/−LDLR^−/− mice cocultured with Ad-LIGHT (Ad-L) or Ad-Null (Ad-N) infected FL83B cells. (B) HL mRNA expression in hepatocytes from LTβR^−/−LDLR^−/− mice cocultured with Ad-N or Ad-L infected FL83B cells and Tg-LIGHT mouse liver non-parenchymal cells (NPC). (n = 3; *<0.05 vs. Ad-N infected FL83B coculture).</p

LTβR expression on hepatocytes is sufficient for HL regulation by Ad-LIGHT.

<p>(A) Liver LTβR and HVEM expression in LDLR^−/−, HVEM^−/−LDLR^−/− and hepatocyte-specific knockout of LTβR (H-LTβR^−/−) LDLR^−/− mice. (B and C) Mice were injected with PBS or adenovirus and sacrificed on the 7^th day. Liver HL mRNA expression in Ad-LIGHT (Ad-L) and PBS (P) injected LTβR^−/−LDLR^−/− mice (B) and (H-LTβR^−/−LDLR^−/− and HVEM^−/−LDLR^−/− mice C). (n = 3; **p<0.01 vs. PBS injected mice).</p

LIGHT and HL expression in Ad-LIGHT transduced primary hepatocytes in vitro.

<p>(A) HL mRNA expression in Ad-LIGHT (Ad-L) and Null adenovirus (Ad-N) infected primary hepatocytes from wild type mice 18, 24 and 36 hours post-infection. The numbers indicate the % decrease in expression in the Ad-LIGHT infected cells relative to the Ad-N infected cells. (B) Total protein from 1×10⁶ Ad-LIGHT infected hepatocytes (in triplicate) at various times post-infection were immunoblotted with anti-mouse LIGHT antibody. Lane 12 is from non-infected hepatocytes and lane 13 is from Tg-LIGHT mouse spleen. (C) Liver HL mRNA expression in Ad-N and Ad-L infected hepatocytes from LDLR^−/− and LTβR^−/−LDLR^−/− mice. (n = 3; *p<0.05, **p<0.01 vs. Ad-N).</p