Heme induces MMP3 promoter activity in HBVEC cells.

<p>A, Sequence of the 5′-flanking region of the human MMP3 promoter. The primers used to generate MMP3 promoter fragment are shown in blue. The putative STAT3 binding sites, TT(N4–6)AA, are shown in red. Underlined sequences are the primers used to amplify the putative human STAT3 binding sites in MMP3 promoter region in ChIP assay. The “C” shaded in yellow denotes the transcription start site (TSS). To further delineate the transcriptional activity of MMP3 affected by STAT3, HBVEC cells were co-transfected with the MMP3 construct and STAT3-siRNA (siSTAT3) and incubated with Heme. The MMP3 promoter activity was proportional to amounts of MMP3 luc within the 50ng to 1000ng range when treated with Heme (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g003" target="_blank">Figure 3B</a>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g003" target="_blank">Figure 3C</a> showed that Heme enhances the MMP3 promoter activity in a dose-dependent manner within a 5 µM to 30 µM range. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g003" target="_blank">Figure 3D</a>, co-transfection with siSTAT3 significantly reduced the Heme-induced luciferase activity of MMP3.</p

STAT3 induces apoptosis through MMP3 in HBVEC cells.

<p>HBVEC were treated with 30 µM of Heme for 24 h followed by evaluation of cell death and apoptosis using MTT and TUNEL assay. Cell death progression in HBVEC cultured were conducted by treating HBVEC cells with AG490 (50 µmol/L) or siSTAT3 as well as corresponding controls followed by incubation with Heme for 24 h, then assayed by MTT measurement. The curves correspond to 3 experiments run in parallel. Heme induced 20%–50% of cell death when treated with 10 to 40 µM of Heme for 24 h (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g006" target="_blank">Figure 6A</a>) with 20–30 µM causing maximum effects. The cell death can be rescued by JAK inhibitor AG490 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g006" target="_blank">Figure 6A, left panel</a>) and siSTAT3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g006" target="_blank">Figure 6A, right panel</a>, *<i>p<</i>0.05, n = 3 triplicate experiments). The reduced cell viability by Heme was further discovered to be caused by cell apoptosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g006" target="_blank">Figure 6B and 6C</a>). The apoptotic cells were found to be increased by Heme treatment (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g006" target="_blank">Figure 6B</a>–d vs. 6B–a, 6B–f vs. 6B–c and 6C) using TUNEL assay. When HBVEC cells were transfected with siMMP3 followed by treatment of Heme for 24 h, number of apoptotic cells were reduced (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g006" target="_blank">Figure 6D</a>–d vs. 6D–a, 6D–f vs. 6D–c and 6E). Upper panel of panel E confirmed specific MMP3 down regulation by siMMP3 by Western blot.</p

Heme phophorylates STAT3 and upregulates MMP3 protein levels.

<p>The effects of Heme on HBVEC as well as activated signaling pathways in HBVEC were examined. HBVEC was treated with different concentrations of Heme as indicated for 24 h. STAT3 activation achieved the maximum effects in HBVEC when treated with Heme with 30 µM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2A</a>). We performed a time course analysis of Heme treatment to identify when peak STAT3, JAK2 (Tyr1007/1008) activation and endogenous MMP3 induction occurs by Western blot. We found that STAT3 activation peaked at 24 hours. Both of JAK2 (Tyr1007/1008) activation and endogenous MMP3 induction exhibit comparable kinetics in response to Heme (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2B</a>). MMP3 protein was induced by Heme with a similar pattern as HO-1, which appears to be induced later than pSTAT3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2A</a>). MMP3 mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2E</a>) and protein levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2C</a>) were up regulated after C57BL/6 mice were infected with <i>P. berghei</i> ANKA- PbA (WT, In) at day 8 compared to non-infected controls (WT,C), with a similar trend as observed for STAT3 activation. Interestingly, PbA infection did not up-regulate MMP3 protein in CXCL10-deficient mice, where STAT3 is not activated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2D</a>). Heme treatment also induced expression of CXCL10 and HO-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2F, G</a>). Corresponding densitometric analyses of the bands performed with the ImageQuant program were shown on the right sides for each panel of Western blot result.</p

Human JAK/STAT signaling pathway is activated by Heme.

<p>Total RNA was extracted and cDNA was synthesized and then used to screen the human JAK/STAT signaling pathway PCR array (SABiosciences, PAHS-039A). Fold changes and p values were calculated using Student’s t-test. A p value<0.05 and a fold change greater than 2.0 were considered to be a significant dysregulation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g001" target="_blank">Figure 1A</a> is a list of up regulated and down regulated genes with fold-change greater than 2. The heat-map (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g001" target="_blank">Figure 1B</a>) and scatter plot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g001" target="_blank">Figure 1C</a>) generated are also shown.</p

STAT3 transcribes MMP3 and induces MMP3 protein production in HBVEC cells.

<p>STAT3 activation of MMP3 has not been previously reported. Therefore, we sought to detect whether STAT3 transcribes MMP3. We stimulated HBVEC cells with Heme, and determined mRNA and protein levels of MMP3 by using qRT-PCR and Western blot. Consistent with the observation that Heme upregulated protein levels of MMP3 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g002" target="_blank">Figure 2A</a>, Heme upregulated mRNA levels of MMP3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g005" target="_blank">Figure 5A</a>. We then determined whether STAT3 regulates MMP3 using a STAT3-inducible model in HBVEC cells. To this end, HBVEC was transfected with 1 µg of constitutively active STAT3 (caSTAT3), dominant negative STAT3 (dnSTAT3), wild type STAT3 (wtSTAT3) as well as control vector for 24 h. Protein lysates were then made to probe with anti-MMP3 antibody. The results indicate that wtSTAT3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g005" target="_blank">Figure 5B</a>) and caSTAT3 increased MMP3 expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g005" target="_blank">Figure 5C</a>) whereas dnSTAT3 reduced MMP3 expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g005" target="_blank">Figure 5D</a>). When pSTAT3 is reduced by siSTAT3, MMP3 protein expression was inhibited (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071366#pone-0071366-g005" target="_blank">Figure 5E</a>).</p

Primer sequences used.

<p>Primer sequences used.</p

Inhibition of inflammation in the brain of P. berghei-infected mice with ECM after treatment.

<p>(A) The number of intravascular leukocytes per mm2 of brain area was markedly decreased after treatment. Parenchymal vessel of (B) untreated mice with ECM on day 5 and (C) saline-treated mice on day 11, plugged with leukocytes (black arrows). Parenchymal vessels of (D) ARM-treated mice, (E) ATV-treated mice and (F) ATV/ARM-treated mice showing remnant adherent leukocytes (black arrows) on day 11. The leukocytes counts are mean ± standard error. A p value of <0.05 was considered significant. Asterisks (*) denote statistically significant change compared with ctrl D5 and section sign (§) denote statistically significant change compared with ctrl D11. Ctrl = control; D = day; ARM = Artemether; ATV = Atorvastatin.</p

Modulating CXCL10 levels improves survival in mice with late stage ECM.

<p>(A) Survival curves: The Kaplan-Meier curves are shown for efficacy of ARM at 25 mg/kg/day ( = 11), ATV at 25 mg/kg/day ( = 11) and ATV/ARM at 25 mg/kg/day ( = 11) in rescuing mice with late stage ECM. All treatments started no day 6 and ends on day 9 post infection. ARM-treated and ATV-treated mice showed survival rates of 75% and 50% respectively compared to controls (p<0.05). The survival rates improved with ATV/ARM-treated mice (100%) which are significantly superior to ARM-treated, ATV-treated or control mice (p<0.001). Survival was assessed twice daily. Significant differences in survival were assessed by Log rank test. (B) Parasitemia of <i>P. berghei</i> ANKA-infected mice after treatment. Parasitemia was monitored by Giemsa-stained blood smears using light microscopy at 100 magnifications with an oil immersion lens. Parasitemia was checked and quantified by counting the number of parasitized red blood cells in at least 1,000 red blood cells. The experiment is a representative of three independent infections. (C) Serum CXCL10 levels of <i>P. berghei</i> ANKA-infected mice after treatment. Serum CXCL10 was measured on samples collected on day 5 and day 11 post infection ( = 5 per group). The results in panels B and C are mean the standard deviation. Mean values were determined to be significantly different using the student <i>t</i>-test. A p value of<0.05 was considered significant. ARM = artemether; ATV = atorvastatin.</p

Dose-response apoptotic effect of recombinant human CXCL10 on HBVECs and neuroglia cells.

<p>(A) Shown are results of TUNEL assay of percentage of apoptotic HBVECs and neuroglia cells incubated with different concentrations of recombinant human CXCL10 for 24 hour at 37°C. (B) Homogeneous caspase assay showing HBVECs and Neuroglia cells exposed to different concentrations of recombinant human CXCL10 for 24 hours at 37°C. Subsequently, the cells were directly incubated with substrate solution for 2.5 hours at 37°C. The Relative Fluorescence Units (RFU) signal is converted to nM free Rhodamine via standard curve. The increase of the caspase activity was calculated as difference of the RFU signal of the induced cells to the RFU signal of non-induced cells. Bars represent standard deviations of three experiments.</p

Schematic model of signaling pathways ATV utilize to modulate the expression of CXCL10 identified by Ingenuity Pathway Analysis.

<p>In response to various stimulus, transcription factors, STAT1, NFκB, and RELA, are activated resulting in the transcription of CXCL10 gene to mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Qiu1" target="_blank">[57]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Penafuerte1" target="_blank">[64]</a>. Atorvastatin inhibit activation of RELA (NFκB3/p65) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Moreno1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Qiu1" target="_blank">[57]</a>, NFκB complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Li1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Wagner1" target="_blank">[59]</a> and STAT1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Wagner1" target="_blank">[59]</a>. Atorvastatin increases expression of HO-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Gueler1" target="_blank">[68]</a> which inhibit activation of NFκB complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Drechsler1" target="_blank">[69]</a>, STAT1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Vareille1" target="_blank">[70]</a> as well as expression of CXCL10 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Mandal1" target="_blank">[71]</a>. Atorvastatin stimulates production of nitric oxide (NO) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Sasmazel1" target="_blank">[72]</a>, which inhibit active NFκB/p65 and NFκB complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Marshall1" target="_blank">[73]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060898#pone.0060898-Franek1" target="_blank">[76]</a>. ATV = atorvastatin; IRBC = infected red blood cells; HO-1 = heme oxygenase-1; NO = nitric oxide; RELA = v-rel reticuloendotheliosis viral oncogene homolog A (avian); NFκB = nuclear factor of kappa light polypeptide gene enhancer in B-cells; STAT1 = signal transducer and activator of transcription 1.</p