Expression of Fbln5 in targeted mice.

<p>rtTA/<i>Fbln5</i>^f/f/SMA⁺⁺/Cre^-, rtTA/<i>Fbln5</i>^f/-/SMA/Cre+, or rtTA/<i>Fbln5</i>^f/f/SMA⁺⁺/Cre⁺ mice were treated with doxycycline as outlined in Materials and Methods. Thereafter, Fbln5 was quantified in the fibromuscular layer of the vaginal wall. <b>A.</b> Representative immunoblot and coomassie stained protein gel. <b>B.</b> Quantification of Fbln5 in vaginal urea extracts from <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^- (n = 7, solid bar), <i>Fbln5</i>^f/f/SMA/Cre+ (n = 9, open bar), or <i>Fbln5</i>^f/-/SMA⁺⁺/Cre+ (n = 7, grey bar) normalized to protein content. Tissue extracts from wild type (<b>WT)</b> animals were used as a standard on each gel. *p < 0.05 compared with Cre- animals, ANOVA followed by Dunnett’s test using WT as control. <b>C.</b> Gelatin zymography of soluble protein extracts from <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^-, <i>Fbln5</i>^f/-/SMA/Cre+, or <i>Fbln5</i>^f/f/SMA⁺⁺/Cre+. Purified proMMP (<b>+ctl</b>), protein from <b>Fbln5</b>^<b>-/</b>-, and <b>Mmp9</b>^<b>-/-</b> vaginal tissues were used as positive and negative controls. <b>D.</b> Hart’s stain of mid-vagina from <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^- (<b>a. Cre-</b>) or <i>Fbln5</i>^f/f/SMA⁺⁺/Cre+ (<b>b. Cre+</b>) mice in basal conditions. Vaginal wall from age-matched Fbln5^-/- mouse shown in <b>c</b> (<b>Fbln5</b>^<b>-/-</b>). Arrows indicate elastic fibers. <b>epi</b>, vaginal epithelium; <b>lp</b>, lamina propria; <b>m</b>, muscularis. Bar = 40 μm. Note decreased magnification in <b>c</b> to illustrate paucity of fibers. <b>E.</b> Effect of Fbln5 cKO on spontaneous development of prolapse as a function of age. Perineal body length was measured at 10–12, 20, and 52 weeks of age in <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^- (<b>Ctl</b>, n = 3) and <i>Fbln5</i>^f/f/SMA⁺⁺/Cre⁺ (<b>cKO</b>, n = 4) mice. Magnitude of bulge did not differ among genotypes (not shown). All animals were treated with doxycycline at 6 weeks of age.</p

Expression of Fbln5 and urea-extractable tropoelastin (TE) in tissues from adult and postnatal (P5-6) rats.

<p><b>A. Representative immunoblot of Fbln5 (upper blot) and tropoelastin (TE, lower) of adult and postnatal tissues</b>. Coomassie-stained gel of protein extracts shown as an index of protein loading. Tissues were homogenized directly in urea buffer and extracted overnight. <b>Ao</b>, aorta; <b>Lu</b>, lung; <b>Sk</b>, skin; <b>Va</b>, vagina; <b>Bl</b>, bladder; <b>Ut</b>, uterus; <b>Cx</b>, cervix. Quantification of Fbln5 (<b>B</b>) and tropoelastin (<b>C</b>) in urea extracts of adult tissues. Data represent mean ± SEM of 3–4 tissues in each group. *p<0.05; **p<0.05 compared with vagina as the control, ANOVA with Dunnett’s post hoc testing</p

Expression of Fbln5 and urea-extractable soluble tropoelastin (TE) in tissues as a function of development in rats.

<p>Quantitative amounts of Fbln5 (PANEL A) and extracted tropoelastin <b>(B)</b> normalized to coomassie-stained protein in aorta, lung, vagina, and skin. Data represent mean ± SEM of 3–4 tissues in each group. <b>RU</b>, Relative Units, <b>E20</b>, embryonic day 20, <b>P1</b>, postnatal day 1; <b>P5</b>, postnatal day 5 *p<0.05 ANOVA with Dunnett’s post hoc testing with E20 as the control</p

Pelvic Organ Support in Animals with Partial Loss of Fibulin-5 in the Vaginal Wall - Fig 1

<p><b>A. Targeting strategy.</b> The exons 1a through 4 are numbered. Wild-type, targeting vector, targeted alleles (<i>neo-loxP</i>, <i>loxP</i>, <i>and KO</i>) are shown. <i>FLPe</i>-mediated excision removes a <i>Neo</i>^<i>r</i> cassette to generate <i>Fbln5</i>^<i>loxP</i> allele and Cre-mediated excision removes exons 1a and 1b to generate a null allele. DTA: Diphtheria Toxin A; H: Hind III; Xb: Xba I; Xo: Xho; Kp: Kpn I; B: Bam HI; RI: Eco RI, Sc: Sac I. <b>B.</b> Confirmation of mutant alleles by genomic PCR. Amplification with primer set A distinguishes <i>Fbln5</i>^<i>loxP</i> allele from wild-type (+/+) allele. Primer set B only amplifies <i>Fbln5</i>^<i>KO</i> allele. * non-specific band.</p

Effect of pregnancy and vaginal delivery on <i>Fbln5</i> cKO mice.

<p><b>A.</b> Perineal body length was measured at time 0 (prior to pregnancy) and 12 h to 12 wks postpartum in <i>Fbln5</i>^f/f/SMA⁺⁺/Cre⁺ (<b>Preg cKO</b>, n = 7) and <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^- mice (<b>Preg Ctl</b>, n = 6) during 3 pregnancies. <b>B.</b> Gelatin zymography of vaginal tissue extracts from <i>Fbln5</i>^f/f/SMA⁺⁺/Cre⁺ (<b>cKO</b>) and <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^- (<b>Ctl</b>) mice 48 h postpartum. Vaginal tissue extracts from <i>Fblin5</i>^<i>-/-</i> (<b>Fib5 KO</b>) and <i>Mmp9</i>^<i>-/-</i> (<b>MMP9 KO</b>) mice were used as positive and negative controls. Lane 2 is blank. <b>C.</b> Immunoblot of Fbln5 in urea extracts of vaginal muscularis from wild type nonpregnant control <b>(WT NP</b>), <b>cKO</b>, and <b>Ctl</b> animals 48 h postpartum. Arrow denotes cleaved form. All animals were treated with doxycycline at 6 weeks of age, mated, and tissues collected after the first pregnancy.</p

Effect of elastase injection on <i>Fbln5</i> Cre+cKO mice.

<p><b>A.</b> Purified porcine elastase was injected into the posterior vaginal wall in <i>Fbln5</i>^f/f/SMA⁺⁺/Cre^- (<b>Cre-</b>, <b>open bar</b>, n = 5) and <i>Fbln5</i>^f/f/SMA⁺⁺/Cre⁺ or <i>Fbln5</i>^f/-/SMA⁺⁺/Cre⁺ (<b>Cre</b>^<b>+</b>, <b>solid bar</b>, n = 9). MOPQ measurements were recorded 48 h after 5, 10, and 15 U of elastase. Since effects of elastase did not differ among <i>Fbln5</i>^f/f/SMA⁺⁺/Cre⁺ (n = 4) compared with <i>Fbln5</i>^f/-/SMA⁺⁺/Cre⁺ (n = 5), data were combined as mean ± SEM. Magnitude of bulge shown. *p < 0.05 compared with Cre-. <b>B</b>. Immunoblotting of Fbln5 in urea extracts from elastase-injected vaginal muscularis of Cre- and Cre+cKO mice. F5^-/-, negative control; Ctl, nonpregnant wild type; PP, 48 h postpartum wild type control indicating decrease in Fbln5. Coomassie gel indicated even loading (not shown). <b>C.</b> Gelatin zymography of Ctl and cKO vaginal extracts from elastase-treated mice. <b>D.</b> Hart’s stain of posterior vaginal wall of elastase-injected Ctl (<b>a</b>) or cKO (<b>b</b>) mice.</p

Quantification of elastic fibers and morphology in vaginal tissues from Cre- and Cre+cKO animals.

<p>Quantification of elastic fibers and morphology in vaginal tissues from Cre- and Cre+cKO animals.</p

Long-term effects of HG ± fibulin-5 on vaginal MMP-9 in <i>Fbln5</i> KO mice treated for 14–28 d.

<p><b>A.</b> Cumulative results of MMP-9 activity in vaginal tissues from 38 mice treated with HG ± fibulin-5 (10 μg/ml) for 2–4 weeks. *P < 0.05 compared with HG alone, ANOVA. <b>B.</b> Immunoblot analysis of urea-extracted protein from vaginal tissues of Fbln5^-/- mice treated with HG or HG + FBLN5 (10 μg/ml) x 2 weeks. The left blot was incubated with <b>anti- His Tag</b> antibody whereas the right blot was incubated with anti-fibulin-5. Positive controls on each blot are recombinant fibulin-5 (rFBLN5, 200 ng). <b>Bl</b>, blank.</p

Thermosensitive property of the polymer solution.

<p><b>A</b>. Transmittance of the polymer aqueous solution (0.5 wt%) as a function of temperature. <b>B</b>. Thermally-induced gelation of the HG aqueous solution (25 wt%).</p

Effect of HG ± fibulin-5 on vaginal MMP-9 and MMP-2 in <i>Fbln5</i> KO mice treated for 7 d.

<p><b>A.</b> Gelatin zymography with protein extracts (10 μg/lane) from adult <i>Fbln5</i>^<i>-/-</i> mice injected with hydrogel alone (HG), HG incorporated with fibulin-5 (5 ug/ml), or fibulin-5 alone x 7 d. <i>Mmp9</i> KO was used as a neg ctl. <b>B.</b> Cumulative results of 34 mice treated with HG ± various doses of fibulin-5 x 7 d. *P < 0.05 compared with HG alone, ANOVA.</p