Placental membrane aging and HMGB1 signaling associated with human parturition.

Aging is associated with the onset of several diseases in various organ systems; however, different tissues may age differently, rendering some of them dysfunctional sooner than others. Placental membranes (fetal amniochorionic membranes) protect the fetus throughout pregnancy, but their longevity is limited to the duration of pregnancy. The age-associated dysfunction of these membranes is postulated to trigger parturition. Here, we investigated whether cellular senescence-the loss of cell division potential as a consequence of stress-is involved in placental membrane function at term. We show telomere reduction, p38 MAPK activation, increase in p21 expression, loss of lamin B1 loss, increase in SA-β-galactosidase , and senescence-associated secretory phenotype (SASP) gene expression in placental membranes after labor and delivery (term labor [TL]) compared to membranes prior to labor at term (term, not-in-labor [TNIL]). Exposing TNIL placental membranes to cigarette smoke extract, an oxidative stress inducer, also induced markers of cellular senescence similar to those in TL placental membranes. Bioinformatics analysis of differentially expressed SASP genes revealed HMGB1 signaling among the top pathways involved in labor. Further, we show that recombinant HMGB1 upregulates the expression of genes associated with parturition in myometrial cells. These data suggest that the natural physiologic aging of placental tissues is associated with cellular senescence and human parturition

Placental membrane aging and HMGB1 signaling associated with human parturition.

Aging is associated with the onset of several diseases in various organ systems; however, different tissues may age differently, rendering some of them dysfunctional sooner than others. Placental membranes (fetal amniochorionic membranes) protect the fetus throughout pregnancy, but their longevity is limited to the duration of pregnancy. The age-associated dysfunction of these membranes is postulated to trigger parturition. Here, we investigated whether cellular senescence-the loss of cell division potential as a consequence of stress-is involved in placental membrane function at term. We show telomere reduction, p38 MAPK activation, increase in p21 expression, loss of lamin B1 loss, increase in SA-β-galactosidase , and senescence-associated secretory phenotype (SASP) gene expression in placental membranes after labor and delivery (term labor [TL]) compared to membranes prior to labor at term (term, not-in-labor [TNIL]). Exposing TNIL placental membranes to cigarette smoke extract, an oxidative stress inducer, also induced markers of cellular senescence similar to those in TL placental membranes. Bioinformatics analysis of differentially expressed SASP genes revealed HMGB1 signaling among the top pathways involved in labor. Further, we show that recombinant HMGB1 upregulates the expression of genes associated with parturition in myometrial cells. These data suggest that the natural physiologic aging of placental tissues is associated with cellular senescence and human parturition

Telomere fragment induced amnion cell senescence: a contributor to parturition?

Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]2) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]2). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

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

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

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

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

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