DNA damage foci and Toll like receptor (TLR)-9 expression in human amnion cells.

<p>(A) Immunofluorescence staining of phosphorylated (γ) H2AX, a marker for DNA damage response activation. Top panel = T-oligo treated amnion cells, bottom panel = untreated cells. Left panel = γH2AX. Right panel = merged images with DAPI nuclear stain. The bright nuclear dots represent DNA damage foci and are more pronounced in cells treated with T-oligo. (B) Relative quantification of TLR-9 mRNA expression in amnion cells in the studied groups, untreated cells, Cont-oligo and T-oligo treated cells, respectively. Box plots represent the quantification relative to endogenous 16S RNA. Kruskal-Wallis test, p>0.05.(control oligonucleotides (Cont-oligo, [AATCCC]₂); Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂).</p

Animal models of T-oligo induced senescence.

<p>(A-D) Representative image of 3-nitrotyrosine (3-NT) modified proteins, an oxidative stress marker, in murine fetal membranes. Intrauterine injection of pregnant CD-1 mice were performed with either: A. Saline, B. Cont-oligo, C. T-oligo and D. T-oligo+SB23580 (p38MAPK inhibitor). <b>(</b>E-G) Representative image of Western blot analysis and densitometric quantitation of p38MAPK activation in murine amniotic sac. E. Top panel = phosphorylated (P)-p38MAPK, middle panel = total p38MAPK and bottom panel = β-actin in Cont-oligo, saline, T-oligo+SB203580 (p38MAPK inhibitor) and T-oligo treated mice, respectively. F. Densitometric quantitation P-p38MAPK normalized to total p38MAPK in amniotic sac tissue or G. normalized to β-actin. T-oligo treatment produced a significant (*) increase in P-p38MAPK compared to saline and Cont-oligo treated groups. The co-treatment of T-oligo and SB203580 showed similar results to controls (saline and Cont-oligo). (*ANOVA, p<0.05). Results are representative from 3 animals/ group. (Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂); control oligonucleotides (Cont-oligo, [AATCCC]₂).</p

Senescence and inflammation induced by T-oligos in CD-1 pregnant mice.

<p>(A-D) Senescence associated β-galactosidase (SA-β-gal) staining of murine amniotic sac. Single blue stained cells indicate β-gal activity. A. saline, B. Cont-oligo, C. T-oligo and D. T-oligo+SB203580 (p38MAPK inhibitor) treated mice. SA-β-gal staining is pronounced in T-oligo treated mice. (E) Concentration of interleukin (IL)-8 protein in murine amniotic fluid. Higher levels of IL-8 were found in T-oligo treated animals compared to controls (saline and Cont-oligo). The production of IL-8 was inhibited by simultaneous treatment with SB203580. (*ANOVA, p<0.05). Results are representative from 3 animals/ group. (Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂); control oligonucleotides (Cont-oligo, [AATCCC]₂).</p

Quantitation of telomere fragments in human amniotic fluid.

<p>Scatter plot representing the number of telomere fragments detected in amniotic fluid from normal term not in labor (NIL) and term in labor samples. The distribution of telomere fragments significantly differs between groups (p = 0.04; t-test).</p

Descriptive data from studied CD-1 pregnant mice according to treatments groups (n = 5 animals/group): Saline, Cont-Oligo, T-oligo and T-oligo co-treatment with SB203580.

<p>(g: grams; SB203580: p38MAPK inhibitor)</p><p>*Anova, Tukey's Multiple Comparison Test, p = 0.001.</p><p>Descriptive data from studied CD-1 pregnant mice according to treatments groups (n = 5 animals/group): Saline, Cont-Oligo, T-oligo and T-oligo co-treatment with SB203580.</p

Human amniotic cells primary cultures.

<p>(A) Immunofluorescent staining of cytokeratin positive amnion epithelial cells. Inset, <b>a.</b> cytokeratin positive cells and <b>b.</b> nuclear staining DAPI. Original magnification x40. (B-D) Cell viability. Representative fluorescence photomicrographs of merged propidium iodide and Hoechst 33342-stained amnion cells. <b>B.</b> Untreated cells, <b>C.</b> Cont-oligo treated cells and <b>D.</b> T-oligo treated cells. Original magnification x40. (E-G) Representative image of Western blot analysis and densitometric quantitation of p38MAPK activation in amnion cells. E. Top panel = phosphorylated (P)-p38MAPK, middle panel = total p38MAPK and bottom panel = β-actin in untreated, Cont-oligo and T-oligo treated cells, respectively. F. Quantitation of P-p38MAPK densitometry normalized to total p38MAPK. T-oligo treatment produced significant (*) increase in P-p38MAPK compared to both untreated and Cont-oligo treated cells. G. Densitometric quantitation of P-p38MAPK normalized to β-actin. Post hoc tests indicated that T-oligo treatment produced significant (*) increase in P-p38MAPK compared to untreated control, but was not significant compared to Cont-oligo treatment. (*ANOVA, p<0.05). (H) Representative image of Western blot analysis of p53 activation in human amnion cells. Top panel = P-p53, middle panel = total p53 and bottom panel = β-actin in untreated and etoposide treated amnion cells, respectively. (I-M) Senescence associated β-galactosidase (SA-β-gal) staining of amnion cells. Single blue stained cells indicate positive β-gal activity. I. Untreated cells, J. Cont-oligo treated cells, K. T-oligo treated cells and L. T-oligo+SB203580 (p38MAPK inhibitor) treated cells. M. Quantification of the positive SA-β-gal cells. Bar graphs represent the differences in the percentage of SA-β-gal staining cells in each group. T-oligo treatment produced a significant increase (*) in the number of senescing cells, which was inhibited by SB203580 treatment. (*ANOVA, p<0.001). (N-Q) Senescence associated sterile inflammation in amnion cells. N. Relative quantification of IL-6 mRNA (p>0.05), <b>O.</b> Relative quantification of IL-8 mRNA in amnion cells (*p = 0.02), P. Protein concentration of IL-6 in conditioned media (*p<0.0001), and Q. Protein concentration of IL-8 in conditioned media (*p = 0.001). The production of IL-8 and IL-6 was inhibited by simultaneous treatment with SB203580. * ANOVA, T-oligo treated samples significantly higher compared with untreated, cont-oligo or T-oligo+SB samples. (Results are representative from 5 amnion cultures/ group. Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂); control oligonucleotides (Cont-oligo, [AATCCC]₂); untreated cells (Control CTR).</p

SA-β-Gal staining.

<p><b>A–B.</b> Pp38 MAPK mediated senescence phenotype (SP) development was determined using Senescence Associated β -Galactosidase (SA β-Gal) assay. SP was seen as blue staining cells and the number of these cells were higher after 50 ng/ml of HMGB1 treatment (<b>B</b>) compared to control (<b>A</b>). <b>C.</b> Bar graph on the right shows percentage of SA β-Gal staining cells after 50 ng/ml of HMGB1 treatment compared to untreated control.</p

HMGB1 Expression in Fetal Membrane and Amniotic Fluid Cases and Controls A.

<p>Bar graph shows HMGB1 expression in fetal membranes as the mean and standard error of relative number of transcripts as determined by real time PCR. GAPDH was used as an internal control. Comparison among the clinical samples from women at term not in labor (Term Birth), with preterm premature rupture of membranes (pPROM), and with preterm birth with intact membranes (PTB). (ANOVA, *p<0.05). <b>B-E.</b> HMGB1 and acetylated lysine in human amniotic fluid (AF). <b>B.</b> pPROM AF without steroids has a greater amount of HMGB1 than term AF. <b>C</b>. Term AF has a greater amount of total protein acetylation than pPROM AF without steroids. <b>D</b>. pPROM AF with glucocorticoids has a greater amount of HMGB1 than term AF. <b>E</b>. Term AF has a greater amount of total protein acetylation than pPROM AF with steroids. However, HMGB1 retains acetylation in pPROM despite deacetylation of proteins by administered steroids (<i>D vs. E</i>) as seen by the preserved HMGB1 pattern. Interestingly, more proteins are acetylated in pPROM AF without steroid use than with steroid administration (<i>C vs. E</i>). In both term and pPROM, HMGB1 shows a consistent expression pattern (<i>B vs. D</i>).</p

HMGB1 SASP Cytokine Production.

<p>Bar graphs show cytokine concentrations in p38MAPK inhibitor SB203580, HMGB1 inhibitor (GA), and HMGB1treated, and simultaneous HMGB1 with SB203580 (30 uM), GA (100 uM), or a combination of both SB203580 and GA treated fetal membrane culture media. <b>A.</b> Comparison of IL-6 concentration among control, SB 203580 alone, GA alone, HMGB1alone, HMGB1+SB203580, HMGB1+GA, and HMGB1+SB+GA cultures. <b>B.</b> Comparison of IL-8 concentration among control, SB 203580 alone, GA alone, HMGB1alone, HMGB1+SB203580, HMGB1+GA, and HMGB1+SB+GA cultures. (ANOVA; *p <0.05).</p

HMGB1, Receptor, and Proinflammatory Cytokine Expression in Fetal Membranes.

<p>Bar graphs show <b>A.</b> HMGB1 expression as the mean and standard error of relative number of transcripts as determined by real time PCR. GAPDH was used as an internal control. Comparison among control, cigarette smoke extract-stimulated (CSE), and lipopolysaccharide-stimulated (LPS) fetal membranes at term. <b>B.</b> HMGB1 concentration as the mean and standard error of relative intensity as determined by competitive enzyme immunoassay ELISA. Note: Intensity of color detected is inversely proportional to the HMGB1 concentration. Comparison among control, cigarette smoke extract-stimulated (CSE), and lipopolysaccharide-stimulated (LPS) culture media at term. <b>C–E.</b> HMGB1 receptor expression in HMGB1-stimulated (1, 5, 10, 50 ng/ml) fetal membranes as the mean and standard error of relative number of transcripts as determined by real time PCR. GAPDH was used as an internal control. <b>C.</b> Comparison of RAGE expression among HMGB1 treated fetal membrane samples. <b>D.</b> Comparison of TLR2 expression among HMGB1 treated fetal membrane samples. <b>E.</b> Comparison of TLR4 expression among HMGB1 treated fetal membrane samples. <b>F–H.</b> Cytokine concentration in HMGB1-stimulated (1, 5, 10, 50 ng/ml) fetal membrane culture media as the mean and standard error of relative concentration as determined by multiplex human cytokine panel analysis. <b>F.</b> Comparison of IL-1β concentration among HMGB1 treated culture media samples. <b>G.</b> Comparison of TNFα concentration among HMGB1 treated culture media samples. <b>H.</b> Comparison of IL-6 concentration among HMGB1 treated culture media samples. (ANOVA, *p<0.05).</p