hypoxia induces apoptosis through hif-1𝛼signaling pathway...

Research ArticleHypoxia Induces Apoptosis through HIF-1120572 Signaling Pathwayin Human Uterosacral Ligaments of Pelvic Organ Prolapse

Xinrui Zhao Congcong Ma Rui Li Jing Xue Lidong Liu and Peishu Liu

Department of Obstetrics and Gynecology Qilu Hospital of Shandong University 107 Wenhua Xi Road JinanShandong 250012 China

Correspondence should be addressed to Peishu Liu peishuliu126com

Received 13 June 2017 Accepted 8 October 2017 Published 2 November 2017

Academic Editor Vasiliki Galani

Copyright copy 2017 Xinrui Zhao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The purpose of this study is to evaluate the expression of hypoxia-inducible factor-1120572 (HIF-1120572) in women uterosacral ligamenttissues with pelvic organ prolapse andwomenwith normal uterine support structures and illuminate its relationship with apoptosisSamples were collected from 38 women with pelvic organ prolapse and 31 age matched controls The expression levels of HIF-1120572 and BNIP3 in the uterosacral ligaments were measured using immunohistochemistry qRT-PCR and Western blot To assessapoptosis we performed TUNEL assay andWestern blot analyses Lastly the short form of the Pelvic Floor Impact Questionnaire-7(PFIQ-7) was used to evaluate prognosis of surgical patients and twenty patients finished the follow-up The expressions of HIF-1120572 and BNIP3 in the uterosacral ligaments were significantly higher in patients with pelvic organ prolapse than in control groupPearsonrsquos correlation test revealed significant positive correlations between HIF-1120572 and apoptosis index Similarly Western blotanalysis showed the expression of proapoptosis proteins (Bax and Bad) Cytochrome-c cleaved caspase-3 and caspase-9 in patientswith pelvic organ prolapse was upregulated The PFIQ-7 scores were higher in HIF-1120572 positive group than in the negative groupHypoxiamay contribute to the pathological process of pelvic organ prolapse by increasing apoptosis via activating HIF-1120572 signalingpathway

1 Introduction

Pelvic organ prolapse (POP) the fall of the female pelvicorgans (vagina uterus bladder andor rectum) into orthrough the vagina is a common and costly condition inelderly women [1] The estimated lifetime risk of surgery forPOP is 126 by the age of 80 years [2] Over 200000surgeries are performed annually in theUnited States for POPcostingmore than 1 billion dollars a year [3 4] Although sev-eral risk factors for POP such as advancing age vaginal child-birth increasing body-mass index family history of POPand chronic intra-abdominal pressure arewell established [5ndash7] the underlying molecular mechanism remains unknown

Pelvic organ support ismainly provided by the connectivetissues (endopelvic fascia and ligaments) and the levator animuscles [5] The uterosacral ligaments (USLs) are importantparts of the support system [8] Traditionally research on thepathophysiology of POP has centered on alteration of extra-cellularmatrix (ECM) such as decreased amount of collagen

altered collagen metabolism and increased breakdown ofcollagen and elastin [9ndash12] Recently studies have suggestedthat apoptosis may be a contributor in the development ofpelvic floor disorders [13 14] Apoptosis programmed celldeath is fundamental to many physiologic processes suchas embryogenesis and tissue remodeling during healing [15]Pelvic trauma related childbirth pelvic surgeries or evenchronic mechanical stress may increase apoptosis and lead toabnormal tissue remodeling Some studies have proposed thehypothesis that hypoxia may cause the apoptosis of the USLs[16]

Hypoxia triggers the expression of hypoxia-induciblefactor-1120572 (HIF-1120572) protein and increases its nuclear translo-cation [17] HIF-1120572 may play a role in cell death or cellsurvival via upregulating downstream proteins such as Bcl-2adenovirus E1B 19-kDa protein (BNIP3) BNIP3 a memberof BH3-only subfamily of Bcl-2 superfamily belongs toproapoptotic proteins It localizes on the outside of themitochondrial membrane Once being stimulated BNIP3

HindawiBioMed Research InternationalVolume 2017 Article ID 8316094 8 pageshttpsdoiorg10115520178316094

2 BioMed Research International

leads to the activation of Bax andor Bak opening of themito-chondrial permeability transition pore and finally apoptosis[18ndash20] Thus we hypothesized that hypoxia may contributeto the pathological process of POP via HIF-1120572 pathway

Our study sought to explore the expression of HIF-1120572 andits target gene BNIP3 in the USLs In addition this studyalso investigated the rate of cell apoptosis and examinedexpression of the apoptosis-related proteins Cytochrome-c(Cyto-c) caspase-3 caspase-9 and Bcl-2 family in order todeterminewhether hypoxia influences cell apoptosis inUSLs

2 Materials and Methods

21 Study Population and Sample Collection This study wasapproved by the ethics committee of the Qilu Hospital ofShandong University Written informed consent was col-lected from each eligible patient All participating womenwere followed up by reviewing their medical records andtelephoning

A total of 69 women were enrolled from the Departmentof Obstetrics and Gynecology Qilu Hospital of ShandongUniversity between April 2010 and December 2016 Thepatient group consisted of 38 women who underwent hys-terectomy as part of pelvic reconstruction surgery for POPthat was stage II III or IV according to the POP quantitative(POP-Q) examination [21] In addition the control groupconsisted of 31 age matched women with stage 0 or IPOP-Q who underwent operations for benign gynecologicindications including cervical intraepithelial neoplasia anddysfunctional uterine bleedingWomenwhohadpelvic infec-tions cancer hormone replacement therapy (HRT) cardio-vascular diseases diabetes mellitus and other endocrine dys-function neurodegenerative disorders and immunologicalor inflammatory disorders were not included in either groupFurthermore menopause was defined as the cessation ofmenses for at least 1 year

At surgery tissue samples measuring 05 times 10 times 10 cmwere obtained from the right side USLs closed to the cervixwhere the ligament is consistently identifiable Half of thespecimens were immediately snapped frozen in liquid nitro-gen and kept at minus80∘C until RNA extraction andWestern blotanalysis were processedThe other half of the specimens werefixed in formalin and embedded in paraffin for immunohis-tochemistry and TUNEL assay

22 Immunohistochemical Staining and Evaluation Immun-ohistochemical analysis of all subjects was performed on 4-120583m thick tissue sections using the standard PV two-stepstaining kit (ZSGB-BIO Beijing China) According to themanufacturerrsquos instruction the primary mouse anti-HIF-1120572antibody (1 200 ab150595 Abcam Cambridge UK) andrabbit anti-BNIP3 (1 100 ab150595 Abcam Cambridge UK)antibody incubated tissue sections overnight at 4∘C Theprimary antibody was replaced with Tris-buffered saline toact as the negative control Subsequently these sections wereexposed by DAB and hematoxylin Staining intensity wasscored as follows absent (0) weak (1) moderate (2) or strong(3) Quantifications were recorded as follows lt25 positivecells (0) 25ndash50 (1) 51ndash75 (2) andgt75 (3)Thefinal score

was the multiplication of the staining intensity and quantifi-cation A final score of 0-1 was classified as negative and gt1was considered positive

23 TUNELAssay TheTUNEL (TdT-mediated digoxigenin-dUTPnick-end labeling)methodwas carried out with a com-mercially available in situ apoptosis detection kit (SolarbioBeijing China) Staining was performed according to themanufacturerrsquos protocol DAPI was used to visualize allnuclei Cells with TUNEL-positive nuclei (red) were detectedby fluorescence microscopy (Olympus) The percentage ofapoptotic cells was calculated by dividing the number ofTUNEL-positive cells by the total number of cells visualizedin the same field

24 Quantitative Real-Time Reverse Transcription PolymeraseChain Reaction (Real-Time RT-PCR) The total RNA of 12ligament specimens obtained from patients with POP and 12from patients with non-POP controls was extracted usingTRIzol reagent (Life Technologies Carlsbad CA USA)Then the RNA was transcripted into cDNA using M-MLVreverse transcriptase (Invitrogen Shanghai China) accord-ing to the manufacturerrsquos instructions PCR was performedon StepOne (Applied Biosystems Shanghai China) anddata quantification using the 2minusΔΔCt method The followingprimers (BioSune Biotechnology Co Ltd Shanghai China)were used GAPDH (used for normalization) forward 51015840-GCA CCG TCA AGG CTG AGA AC-31015840 and reverse 51015840-TGGTGA GGT AGA CGC CAG TGG A-31015840 HIF-1120572 forward 51015840-ATC CAT GTG ACC ATG AGG AAA TG-31015840 and reverse 51015840-TCG GCT AGT TAG GGT ACA CTT C-31015840 BNIP3 forward51015840-CAG GGC TCC TGG GTA GAA CT-31015840 and reverse 51015840-CTA CTC CGT CCA GAC TCA TGC-31015840

25 Western Blot Analysis Twenty-four samples selectedfrom the patient and control groups were homogenized inRIPA buffer (Beyotime Institute of Biotechnology HaimenChina) with 1 phenylmethylsulfonyl fluoride (PMSF) and1 NaF for 1 h and centrifuged at 12000 gravities for 15 min-utes at 4∘C Equal amounts of protein (30120583g) were loaded andseparated by 10 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and transferred to polyvinylidene fluoridemembranes (Millipore) The membranes were blocked with5 nonfat dry milk for 1 hour and incubated with anti-HIF-1120572 and anti-BNIP3 (Abcam) anti-Bcl-2 anti-Bcl-xl anti-Baxand anti-Bad (Santa Cruz Biotechnology) anti-Cytochrome-c anti-caspase-3 anti-cleaved caspase-3 anti-caspase-9 anti-cleaved caspase-9 and anti-GAPDH [Cell Signaling Tech-nology (CST)] antibodies overnight at 4∘C The membraneswere then incubated with respective secondary antibodies(Millipore) and visualized by enhanced chemiluminescence(ECL) using ImageQuant LAS 4000 (GE Healthcare LifeSciences Logan UT USA) The GAPDH band served ascontrol The results were analyzed by ImageJ software

26 Postoperative Follow-Up We selected patients to follow-up who underwent transvaginal total hysterectomy pluscolporrhaphy anterior-posterior surgery about 3 years agoTwenty women were divided into two groups according to

BioMed Research International 3

Table 1 Clinical characteristic of patients with POP and control groups

POP (119899 = 38) Control (119899 = 31) 119875 valueAge (years mean plusmn SD) 5661 plusmn 741 5365 plusmn 654 009BMI (mean plusmn SD) 2426 plusmn 311 2348 plusmn 344 033Parity (mean plusmn SD) 224 plusmn 097 187 plusmn 088 011Menopause () 6316 4516 013POP-Q stage0-I 0 31II 10 0III 20 0IV 8 0

the immunoreactivity of HIF-1120572 (including 8 cases of HIF-1120572 negative 12 cases of HIF-1120572 positive) All women filled inthe short forms of the Pelvic Floor Impact Questionnaire-7(PFIQ-7) [22]

27 Statistical Analysis GraphPad Prism 501 (GraphPadSoftware USA)was used for statistical analysis In the presentstudy data were expressed as mean plusmn standard deviation(SD) and statistical comparisons were performed usingStudentrsquos 119905-test or the Chi-squared test Correlation analyseswere performed with Pearsonrsquos correlation test 119875 values lt005 were considered statistically significant

3 Results

31 Clinical Characteristics of Patients with POP and ControlGroups As listed in Table 1 the clinical characteristics ofpatients with POP and control groups were matched in termsof demographic and clinical characteristics No significantdifferences were observed in age body-mass index parity ormenopausal status between the two groups

32 The Expression of HIF-1120572 and BNIP3 in USL Tissuesof POP and Control Groups Using immunohistochemicalassay we found that the immunoreactivity of HIF-1120572 in USLtissues of the POP group was significantly higher than thecontrol group (1477 plusmn 0779 versus 2668 plusmn 1542 resp119875 lt 001) BNIP3 showed the same results (1523 plusmn 0615versus 3084plusmn1618 resp119875 lt 0001 Figure 1(a)) Correlationanalyses revealed a significant positive correlation betweenHIF-1120572 and BNIP3 (Pearson correlation coefficient 077119875 lt 0001) Similarly qRT-PCR and Western blot analysisrevealed the expression of HIF-1120572 and BNIP3 was increasedin the level of mRNA and protein respectively (Figures1(b)ndash1(d))

33 Relationship between the Expression ofHIF-1120572 in POPUSLTissues and Clinical Characteristics Next we performed theimmunohistochemical staining results of HIF-1120572 in Table 2We found that the immunoreactivity of HIF-1120572 in stage IIIgroup was significantly higher than stage II group (119875 =0001) and no significant differences were found betweenstage III and stage IV group (119875 = 0053) In addition the

expression level of HIF-1120572 in USL tissues was not correlatedwith age (119875 = 0259) and menopausal status (119875 = 0311)

34 The Level of Apoptosis in USLs of POP Patients and Con-trols The TUNEL assay demonstrated apoptosis in the USLcells in POP and control groups (Figure 2(a))The percentageof apoptosis cells was significantly higher in POP group thanin controls (1642plusmn794 versus 791plusmn556 119875 lt 001)and significant positive correlation was detected between theimmunoreactivity of HIF-1120572 and the percentage of apoptosiscells (119903 = 060 119875 lt 001)

The expression of Bax and Bad in USL tissues fromthe POP group was higher than that in the control group(119875 lt 005) However the expression of Bcl-2 and Bcl-xl inthe POP group was not significantly lower than the controlones (Figures 2(b) and 2(c)) We also examined the ratioof antiapoptotic proteins to proapoptotic proteins The Bcl-2Bax Bcl-2Bad Bcl-xlBax and Bcl-xlBad ratios in USLtissues of the POP group were lower than the control groupbut only the Bcl-2 Bax ratio (119875 lt 001) and Bcl-xlBax ratio(119875 lt 005) were significantly different between two groups(Figure 2(d)) Similarly Western blot analysis showed theexpression of procaspase-3 and procaspase-9 decreased andthe expression of Cyto-c cleaved caspase-3 and caspase-9increased in patients with POP (Figures 2(e) and 2(f))

35 PFIQ-7 Scores of Postoperative Patients The PFIQ-7presents seven questions with urinary impact colorectal-analimpact and pelvic organ prolapse impact Each aspect hasfour answers (not at all = 0 somewhat = 1 moderate = 2 alot = 3) The final score was calculated when average of everycolumn was multiplied by 333 and total score ranged from0 to 300 [23] The total scores were significantly differentbetween groups with higher scores in HIF-1120572 positive group(3849 plusmn 1963 versus 1726 plusmn 1078 119875 lt 005)

4 Discussion

Female pelvic floor tissues are subject to multiple mechanicalstresses including pregnancy childbirth defecation coughand normal gravity which contribute to the increase inabdominal pressure [24] This pressure extension of pelvic


Control group POP groupBN

IP3

HIF

-1

(a)

ControlPOP

lowast

lowast

0

5

10

15

20

Relat

ive m

RNA

leve

l of H

IF-1

BN

IP3

BNIP3HIF-1

(b)

C P C P

BNIP3

GAPDH

HIF-1

(c)

ControlPOP

lowast lowastlowast

00

05

10

15

20

Prot

ein

expr

essio

n le

vel o

f HIF

-1

BN

IP3

BNIP3HIF-1

(d)

Figure 1 The expression of HIF-1120572 and BNIP3 are upregulated in USL tissues of the POP group (a) Representative immunohistochemicalstaining of HIF-1120572 and BNIP3 in the uterosacral ligaments in control and POP group (magnification times200 scale bar = 10 120583m) (b)Quantification of the expression of HIF-1120572 and BNIP3 between the POP group and the control group at the mRNA level lowast119875 lt 005 (cand d) HIF-1120572 and BNIP3 were detected by Western blot analysis at the protein level lowast119875 lt 005 lowastlowast119875 lt 001

floor expansion is thought to result in hypoxia and produc-tion of free radicals and reactive oxygen species [25]

We demonstrated that the expression of HIF-1120572 and itstarget gene BNIP3 was significantly upregulated in USLtissues in the POP group Furthermore there was a positivecorrelation between the expression of HIF-1120572 and apoptosisindex in USLs We also preliminarily demonstrated the HIF-1120572 expression is associated with life quality after operationTo the best of our knowledge the present study is the first toreport the effects of hypoxia on apoptosis in USL tissues withPOP

When cells are exposed to chronic or extreme hypoxiathe expression of HIF-1120572 was increased and resulted in

apoptosis As a transcription factor HIF-1120572 is involved incell death by activating prodeath genes such as BNIP3 [26]BNIP3 is a BH3-only Bcl-2 family member regulated by HIF-1120572 and it has been identified as one of the most prominenthypoxia responsive genes [27] The expression of BNIP3 canbe upregulated under hypoxia in cell lines such as carcinomas[28 29] fibroblasts [30] and macrophages [31] We have alsofound that expression of HIF-1120572 and BNIP3 in USLs withPOP was significantly increased both in mRNA and in pro-tein levels Although the results of immunohistochemistryrevealed that the expression of HIF-1120572 was not associatedwith age and menopausal status expression level was higherin the severe prolapse group compared to the mild prolapse


Table 2 Relationship between HIF-1120572 expression in POP USL tissues and clinical characteristics

Clinical factors Number Expression of HIF-1120572119875 value

Negative PositiveAge (years)le50 9 5 4gt50 29 10 19 0259Menopausal statusBefore menopause 14 7 7Menopause 24 8 16 0311POP-Q stageII 10 8 2III 20 3 17 0001IV 8 4 4 0053

groupWe could see a significant difference in theHIF-1120572 lev-els between stage II and stage III group but not between stageIII and stage IV group In general the patients of stage IV hadstopped developing when they underwent the operationsThe patients of stage II and stage III however are in a periodof aggravation It also proves that HIF-1120572 has participated inthe development of POP

Takacs et al compared 12 womenwho underwent surgeryfor either POP repair or benign gynecological disease andfound a significant increased apoptosis in smooth musclecells [32] Comparison between the smoothmuscle and apop-totic cells in the USL tissues of women with and without POPdemonstrated an increased apoptosis in women with POP[33] Consistently our results exhibited an increase in apop-totic index by TUNEL analysis in the USLs of patients withPOP In addition correlation analyses revealed a significantcorrelation between the expression of HIF-1120572 and apoptosisindex indicating that the increased apoptosis noted in thepelvic connective tissues of POP patients was closely relatedto hypoxia

The BH3 domain of BNIP3 plays an important role incell death as well as in mediating heterodimerization withantiapoptotic proteins or proapoptotic proteins which reg-ulate cell death [34] We found that the expression of theproapoptotic proteins was Bax and Bad was increased andthe ratios of Bcl-2Bax and Bcl-xlBax were reduced in USLtissues in the POP group compared with the non-POP groupLower ratios indicate higher sensitivity to apoptosis Thesefindings were consistent with those of Wen et al who foundhigher expression of proapoptotic proteins in vaginal tissuesfrom women with POP [14] However Saatli et al foundthat the expression of both the antiapoptotic genes and theproapoptotic genes was increased in vaginal and USL tissuesin the POP group compared with the control group [35]Diversity of age menopausal status and histologic type in thedifferent patient groups may provide the difference

In recent years the mechanisms underlying the intrinsic[36] and extrinsic [37] apoptotic pathways have been clarified[38] The binding of death-inducing ligands to cell-surfacedeath receptors can stimulate the extrinsic pathway DNAdamage growth factor deprivation and oxidative stress can

activate the intrinsic pathway Initiation of the intrinsicpathway leads to mitochondrial depolarization which allowsthe release of Cytochrome-c In turn Cytochrome-c engagesapoptotic protease activating factor 1 (APAF 1) and forms theapoptosome in the presence of deoxyadenosine triphosphateThe apoptosome activates caspase-9 which activates caspase-3 and induces apoptosis [39] We investigated the expressionof the Bcl-2 family key regulators ofmitochondrial apoptosisand demonstrated the expression of proapoptotic proteinswas increased and the ratio of antiapoptotic to proapoptoticproteins was downregulated in the USL tissues of patientswith POP compared to females with non-POP Consistentwith that our results exhibited the expression of Cyto-ccleaved caspase-3 and caspase-9 in the USLs of patientswith POP was increased Therefore we believed that hypoxiainduced intrinsic apoptotic pathway which led to attenuationof pelvic support tissues as a reasonable mechanism inpathogenesis and prognosis of POP

Limitations to our study include small sample size ofUSL tissues and follow-up patients During the follow-upperiod many patients were lost for various reasons such asdeath Therefore the sample size of follow-up was furtherreduced At present there is no established animal model orfibroblasts cell line of uterine ligaments to be experimentedon which limited the development of POP research We willtrain primary fibroblasts from the USL ligament tissues tofurther clarify the pathogenesis of POP

In conclusion hypoxia resulted in cell death with apop-tosis in human USL tissues via activating HIF-1120572 signalingpathwayThepathogenesis andprognosis of POPmaydependon HIF-1120572 signaling pathway which further modulated theexpression of the Bcl-2 family proteins and the caspasefamily proteins such as BNIP3 Bax Bad Cyto-c caspase-3and caspase-9 Therefore approaches to modulate this unde-sirable condition seem to be useful in preventing or delayingthe progression of POP

Conflicts of Interest

The authors declare that they have no conflicts of interest


TUNEL DAPI Merge

Con

trol

POP

(a)

C P C P

Bcl-2

Bcl-xl

Bax

Bad

GAPDH

(b)

ControlPOP

Bcl-xlBcl-2 BadBax

lowastlowast

0

1

2

3

4

Bcl-2

fam

ilyG

APD

H

(c)

POPControl

lowastlowastlowast

Bcl-2Bax Bcl-2Bad Bcl-xlBax Bcl-xlBad0

1

2

3

4

5

Ratio

of B

cl-2

fam

ily p

rote

in

(d)

C P C P

Cyto-c

Caspase-3

Cleavedcaspase-3

Caspase-9

Cleavedcaspase-9

GAPDH

(e)

ControlPOP

caspase-9Cyto-c Cleaved Cleaved Caspase-9Caspase-3

caspase-3

lowastlowastlowastlowastlowastlowastlowastlowast

00

05

10

15

20

Relat

ive p

rote

in le

vel

(f)

Figure 2 TUNEL assay and expression of apoptotic proteins in USL tissues of POP patients and control group (a) TUNEL assay (b) Theprotein expression of the Bcl-2 family in the POP group and control group (c) Quantification of the expression of Bcl-2 family in USLtissues showed in (b) lowast119875 lt 005 (d) Quantification of the antiapoptoticproapoptotic proteins expression ratio showed in (b) lowast119875 lt 005 andlowastlowast119875 lt 001 (e) The protein expression of the caspase family in the POP group and control group (f) Quantification of the expression of

Cyto-c and caspase family showed in (e) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 The results are presented as the mean plusmn SD


References

[1] M D Barber ldquoPelvic organ prolapserdquo BMJ vol 354 Article IDi3853 2016

[2] J M Wu C A Matthews M M Conover V Pate and MJonsson Funk ldquoLifetime risk of stress urinary incontinence orpelvic organ prolapse surgeryrdquoObstetricsampGynecology vol 123no 6 pp 1201ndash1206 2014

[3] F J Smith C D J Holman R E Moorin and N TsokosldquoLifetime risk of undergoing surgery for pelvic organ prolapserdquoObstetrics amp Gynecology vol 116 no 5 pp 1096ndash1100 2010

[4] L L Subak L E Waetjen S van den Eeden D H ThomE Vittinghoff and J S Brown ldquoCost of pelvic organ prolapsesurgery in the United Statesrdquo Obstetrics amp Gynecology vol 98no 4 pp 646ndash651 2001

[5] J E Jelovsek C Maher and M D Barber ldquoPelvic organprolapserdquoThe Lancet vol 369 no 9566 pp 1027ndash1038 2007

[6] S L Hendrix A Clark I Nygaard A Aragaki V Barnabei andA McTiernan ldquoPelvic organ prolapse in the Womenrsquos HealthInitiative gravity and gravidityrdquo American Journal of Obstetricsamp Gynecology vol 186 no 6 pp 1160ndash1166 2002

[7] K S Kenton ldquoWhat is new in pelvic organ prolapse bestarticles from the past yearrdquo Obstetrics amp Gynecology vol 126no 5 pp 1102ndash1105 2015

[8] J O L DeLancey ldquoAnatomic aspects of vaginal eversion afterhysterectomyrdquo American Journal of Obstetrics amp Gynecologyvol 166 no 6 pp 1717ndash1728 1992

[9] S R Jackson N C Avery J F Tarlton S D Eckford PAbrams and A J Bailey ldquoChanges inmetabolism of collagen ingenitourinary prolapserdquoTheLancet vol 347 no 9016 pp 1658ndash1661 1996

[10] L de Landsheere S Blacher C Munaut et al ldquoChanges inelastin density in different locations of the vaginal wall inwomen with pelvic organ prolapserdquo International Urogynecol-ogy Journal and Pelvic Floor Dysfunction vol 25 no 12 pp1673ndash1681 2014

[11] T Strinic M Vulic S Tomic V Capkun I Stipic and IAlujevic ldquoIncreased expression of matrix metalloproteinase-1in uterosacral ligament tissue from women with pelvic organprolapserdquoActaObstetricia et Gynecologica Scandinavica vol 89no 6 pp 832ndash834 2010

[12] M Dviri E Leron J Dreiher M Mazor and R Shaco-LevyldquoIncreased matrix metalloproteinases-1-9 in the uterosacralligaments and vaginal tissue from women with pelvic organprolapserdquo European Journal of Obstetrics amp Gynecology andReproductive Biology vol 156 no 1 pp 113ndash117 2011

[13] E J Kim N Chung S H Park et al ldquoInvolvement of oxidativestress andmitochondrial apoptosis in the pathogenesis of pelvicorgan prolapserdquoThe Journal of Urology vol 189 no 2 pp 588ndash594 2013

[14] Y Wen J Y-P Ho M L Polan and B Chen ldquoExpression ofapoptotic factors in vaginal tissues fromwomenwith urogenitalprolapserdquo Neurourology and Urodynamics vol 30 no 8 pp1627ndash1632 2011

[15] T R Martin M Nakamura and G Matute-Bello ldquoThe role ofapoptosis in acute lung injuryrdquo Critical Care Medicine vol 31no 4 pp S184ndashS188 2003

[16] N Yucel A Usta K Guzin et al ldquoImmunohistochemicalanalysis of connective tissue in patients with pelvic organprolapserdquo Journal of Molecular Histology vol 44 no 1 pp 97ndash102 2013

[17] C-C Feng C-C Lin Y-P Lai et al ldquoHypoxia suppresses myo-cardial survival pathway through HIF-1120572-IGFBP-3-dependentsignaling and enhances cardiomyocyte autophagic and apop-totic effects mainly via FoxO3a-induced BNIP3 expressionrdquoGrowth Factors vol 34 no 3-4 pp 73ndash86 2016

[18] K A Webster R M Graham and N H Bishopric ldquoBNip3 andsignal-specific programmed death in the heartrdquo Journal ofMolecular andCellular Cardiology vol 38 no 1 pp 35ndash45 2005

[19] K M Regula K Ens and L A Kirshenbaum ldquoInducibleexpression of BNIP3 provokes mitochondrial defects andhypoxia-mediated cell death of ventricular myocytesrdquo Circula-tion Research vol 91 no 3 pp 226ndash231 2002

[20] X Feng X Liu W Zhang and W Xiao ldquoP53 directly sup-presses BNIP3 expression to protect against hypoxia-inducedcell deathrdquo EMBO Journal vol 30 no 16 pp 3397ndash3415 2011

[21] AMWeber P Abrams L Brubaker et al ldquoThe standardizationof terminology for researchers in female pelvic floor disordersrdquoInternational Urogynecology Journal and Pelvic Floor Dysfunc-tion vol 12 no 3 pp 178ndash186 2001

[22] M D Barber M D Walters and R C Bump ldquoShort forms oftwo condition-specific quality-of-life questionnaires for womenwith pelvic floor disorders (PFDI-20 and PFIQ-7)rdquo AmericanJournal of Obstetrics amp Gynecology vol 193 no 1 pp 103ndash1132005

[23] S Swift P Woodman A OrsquoBoyle et al ldquoPelvic Organ SupportStudy (POSST) the distribution clinical definition and epi-demiologic condition of pelvic organ support defectsrdquo Ameri-can Journal of Obstetrics amp Gynecology vol 192 no 3 pp 795ndash806 2005

[24] D Chow and L V Rodrıguez ldquoEpidemiology and prevalence ofpelvic organ prolapserdquo Current Opinion in Urology vol 23 no4 pp 293ndash298 2013

[25] K Chin C Wieslander H Shi et al ldquoPelvic organ support inanimals with partial loss of fibulin-5 in the vaginal wallrdquo PLoSONE vol 11 no 4 Article ID e0152793 2016

[26] H M Sowter P J Ratcliffe P Watson A H Greenberg and AL Harris ldquoHIF-1-dependent regulation of hypoxic induction ofthe cell death factors BNIP3 andNIX in human tumorsrdquoCancerResearch vol 61 no 18 pp 6669ndash6673 2001

[27] G Chinnadurai S Vijayalingam and S B Gibson ldquoBNIP3subfamily BH3-only proteins Mitochondrial stress sensors innormal and pathological functionsrdquoOncogene vol 27 pp S114ndashS127 2008

[28] P Secades I S De Santa-Marıa A Merlo C Suarez and M-D Chiara ldquoIn vitro study of normoxic epidermal growth fac-tor receptor-induced hypoxia-inducible factor-1-alpha vascularendothelial growth factor and BNIP3 expression in head andneck squamous cell carcinoma cell lines Implications for anti-epidermal growth factor receptor therapyrdquoHeadampNeck vol 37no 8 pp 1150ndash1162 2015

[29] A H Chourasia K Tracy C Frankenberger et al ldquoMitophagydefects arising from BNip3 loss promote mammary tumorprogression to metastasisrdquo EMBO Reports vol 16 no 9 pp1145ndash1163 2015

[30] A Vengellur and J J LaPres ldquoThe role of hypoxia induciblefactor 1120572 in cobalt chloride induced cell death in mouseembryonic fibroblastsrdquo Toxicological Sciences vol 82 no 2 pp638ndash646 2004

[31] S-D Ha D Ng J Lamothe M A Valvano J Han and O KSung ldquoMitochondrial proteins Bnip3 and Bnip3L are involvedin anthrax lethal toxin-induced macrophage cell deathrdquo The


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[32] P Takacs M Gualtieri M Nassiri K Candiotti and C AMedina ldquoVaginal smooth muscle cell apoptosis is increased inwomen with pelvic organ prolapserdquo International Urogynecol-ogy Journal vol 19 no 11 pp 1559ndash1564 2008

[33] P Takacs M Nassiri M Gualtieri K Candiotti and C AMedina ldquoUterosacral ligament smooth muscle cell apoptosisis increased in women with uterine prolapserdquo ReproductiveSciences vol 16 no 5 pp 447ndash452 2009

[34] KWang X Yin D T Chao C LMilliman and S J KorsmeyerldquoBID a novel BH3 domain-only death agonistrdquo Genes ampDevelopment vol 10 no 22 pp 2859ndash2869 1996

[35] B Saatli S Kizildag E Cagliyan E Dogan and U Say-gili ldquoAlteration of apoptosis-related genes in postmenopausalwomen with uterine prolapserdquo International UrogynecologyJournal and Pelvic Floor Dysfunction vol 25 no 7 pp 971ndash9772014

[36] N N Danial and S J Korsmeyer ldquoCell death critical controlpointsrdquo Cell vol 116 no 2 pp 205ndash219 2004

[37] A Ashkenazi and V M Dixit ldquoDeath receptors signaling andmodulationrdquo Science vol 281 no 5381 pp 1305ndash1308 1998

[38] Z Jin and W S El-Deiry ldquoOverview of cell death signalingpathwaysrdquo Cancer Biology ampTherapy vol 4 no 2 pp 139ndash1632005

[39] A Ashkenazi W J Fairbrother J D Leverson and A J SouersldquoFrom basic apoptosis discoveries to advanced selective BCL-2family inhibitorsrdquoNature Reviews Drug Discovery vol 16 no 4pp 273ndash284 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


leads to the activation of Bax andor Bak opening of themito-chondrial permeability transition pore and finally apoptosis[18ndash20] Thus we hypothesized that hypoxia may contributeto the pathological process of POP via HIF-1120572 pathway

Our study sought to explore the expression of HIF-1120572 andits target gene BNIP3 in the USLs In addition this studyalso investigated the rate of cell apoptosis and examinedexpression of the apoptosis-related proteins Cytochrome-c(Cyto-c) caspase-3 caspase-9 and Bcl-2 family in order todeterminewhether hypoxia influences cell apoptosis inUSLs

2 Materials and Methods

21 Study Population and Sample Collection This study wasapproved by the ethics committee of the Qilu Hospital ofShandong University Written informed consent was col-lected from each eligible patient All participating womenwere followed up by reviewing their medical records andtelephoning

A total of 69 women were enrolled from the Departmentof Obstetrics and Gynecology Qilu Hospital of ShandongUniversity between April 2010 and December 2016 Thepatient group consisted of 38 women who underwent hys-terectomy as part of pelvic reconstruction surgery for POPthat was stage II III or IV according to the POP quantitative(POP-Q) examination [21] In addition the control groupconsisted of 31 age matched women with stage 0 or IPOP-Q who underwent operations for benign gynecologicindications including cervical intraepithelial neoplasia anddysfunctional uterine bleedingWomenwhohadpelvic infec-tions cancer hormone replacement therapy (HRT) cardio-vascular diseases diabetes mellitus and other endocrine dys-function neurodegenerative disorders and immunologicalor inflammatory disorders were not included in either groupFurthermore menopause was defined as the cessation ofmenses for at least 1 year

At surgery tissue samples measuring 05 times 10 times 10 cmwere obtained from the right side USLs closed to the cervixwhere the ligament is consistently identifiable Half of thespecimens were immediately snapped frozen in liquid nitro-gen and kept at minus80∘C until RNA extraction andWestern blotanalysis were processedThe other half of the specimens werefixed in formalin and embedded in paraffin for immunohis-tochemistry and TUNEL assay

22 Immunohistochemical Staining and Evaluation Immun-ohistochemical analysis of all subjects was performed on 4-120583m thick tissue sections using the standard PV two-stepstaining kit (ZSGB-BIO Beijing China) According to themanufacturerrsquos instruction the primary mouse anti-HIF-1120572antibody (1 200 ab150595 Abcam Cambridge UK) andrabbit anti-BNIP3 (1 100 ab150595 Abcam Cambridge UK)antibody incubated tissue sections overnight at 4∘C Theprimary antibody was replaced with Tris-buffered saline toact as the negative control Subsequently these sections wereexposed by DAB and hematoxylin Staining intensity wasscored as follows absent (0) weak (1) moderate (2) or strong(3) Quantifications were recorded as follows lt25 positivecells (0) 25ndash50 (1) 51ndash75 (2) andgt75 (3)Thefinal score

was the multiplication of the staining intensity and quantifi-cation A final score of 0-1 was classified as negative and gt1was considered positive

23 TUNELAssay TheTUNEL (TdT-mediated digoxigenin-dUTPnick-end labeling)methodwas carried out with a com-mercially available in situ apoptosis detection kit (SolarbioBeijing China) Staining was performed according to themanufacturerrsquos protocol DAPI was used to visualize allnuclei Cells with TUNEL-positive nuclei (red) were detectedby fluorescence microscopy (Olympus) The percentage ofapoptotic cells was calculated by dividing the number ofTUNEL-positive cells by the total number of cells visualizedin the same field

24 Quantitative Real-Time Reverse Transcription PolymeraseChain Reaction (Real-Time RT-PCR) The total RNA of 12ligament specimens obtained from patients with POP and 12from patients with non-POP controls was extracted usingTRIzol reagent (Life Technologies Carlsbad CA USA)Then the RNA was transcripted into cDNA using M-MLVreverse transcriptase (Invitrogen Shanghai China) accord-ing to the manufacturerrsquos instructions PCR was performedon StepOne (Applied Biosystems Shanghai China) anddata quantification using the 2minusΔΔCt method The followingprimers (BioSune Biotechnology Co Ltd Shanghai China)were used GAPDH (used for normalization) forward 51015840-GCA CCG TCA AGG CTG AGA AC-31015840 and reverse 51015840-TGGTGA GGT AGA CGC CAG TGG A-31015840 HIF-1120572 forward 51015840-ATC CAT GTG ACC ATG AGG AAA TG-31015840 and reverse 51015840-TCG GCT AGT TAG GGT ACA CTT C-31015840 BNIP3 forward51015840-CAG GGC TCC TGG GTA GAA CT-31015840 and reverse 51015840-CTA CTC CGT CCA GAC TCA TGC-31015840

25 Western Blot Analysis Twenty-four samples selectedfrom the patient and control groups were homogenized inRIPA buffer (Beyotime Institute of Biotechnology HaimenChina) with 1 phenylmethylsulfonyl fluoride (PMSF) and1 NaF for 1 h and centrifuged at 12000 gravities for 15 min-utes at 4∘C Equal amounts of protein (30120583g) were loaded andseparated by 10 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and transferred to polyvinylidene fluoridemembranes (Millipore) The membranes were blocked with5 nonfat dry milk for 1 hour and incubated with anti-HIF-1120572 and anti-BNIP3 (Abcam) anti-Bcl-2 anti-Bcl-xl anti-Baxand anti-Bad (Santa Cruz Biotechnology) anti-Cytochrome-c anti-caspase-3 anti-cleaved caspase-3 anti-caspase-9 anti-cleaved caspase-9 and anti-GAPDH [Cell Signaling Tech-nology (CST)] antibodies overnight at 4∘C The membraneswere then incubated with respective secondary antibodies(Millipore) and visualized by enhanced chemiluminescence(ECL) using ImageQuant LAS 4000 (GE Healthcare LifeSciences Logan UT USA) The GAPDH band served ascontrol The results were analyzed by ImageJ software

26 Postoperative Follow-Up We selected patients to follow-up who underwent transvaginal total hysterectomy pluscolporrhaphy anterior-posterior surgery about 3 years agoTwenty women were divided into two groups according to






3 Results








4 Discussion




IP3

HIF

-1

(a)

ControlPOP

lowast

lowast

0

5

10

15

20

Relat

ive m

RNA

leve

l of H

IF-1

BN

IP3

BNIP3HIF-1

(b)

C P C P

BNIP3

GAPDH

HIF-1

(c)

ControlPOP

lowast lowastlowast

00

05

10

15

20

Prot

ein

expr

essio

n le

vel o

f HIF

-1

BN

IP3

BNIP3HIF-1

(d)




















TUNEL DAPI Merge

Con

trol

POP

(a)

C P C P

Bcl-2

Bcl-xl

Bax

Bad

GAPDH

(b)

ControlPOP

Bcl-xlBcl-2 BadBax

lowastlowast

0

1

2

3

4

Bcl-2

fam

ilyG

APD

H

(c)

POPControl

lowastlowastlowast


1

2

3

4

5

Ratio

of B

cl-2

fam

ily p

rote

in

(d)

C P C P

Cyto-c

Caspase-3

Cleavedcaspase-3

Caspase-9

Cleavedcaspase-9

GAPDH

(e)

ControlPOP


caspase-3


00

05

10

15

20

Relat

ive p

rote

in le

vel

(f)




References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results








4 Discussion




IP3

HIF

-1

(a)

ControlPOP

lowast

lowast

0

5

10

15

20

Relat

ive m

RNA

leve

l of H

IF-1

BN

IP3

BNIP3HIF-1

(b)

C P C P

BNIP3

GAPDH

HIF-1

(c)

ControlPOP

lowast lowastlowast

00

05

10

15

20

Prot

ein

expr

essio

n le

vel o

f HIF

-1

BN

IP3

BNIP3HIF-1

(d)




















TUNEL DAPI Merge

Con

trol

POP

(a)

C P C P

Bcl-2

Bcl-xl

Bax

Bad

GAPDH

(b)

ControlPOP

Bcl-xlBcl-2 BadBax

lowastlowast

0

1

2

3

4

Bcl-2

fam

ilyG

APD

H

(c)

POPControl

lowastlowastlowast


1

2

3

4

5

Ratio

of B

cl-2

fam

ily p

rote

in

(d)

C P C P

Cyto-c

Caspase-3

Cleavedcaspase-3

Caspase-9

Cleavedcaspase-9

GAPDH

(e)

ControlPOP


caspase-3


00

05

10

15

20

Relat

ive p

rote

in le

vel

(f)




References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















IP3

HIF

-1

(a)

ControlPOP

lowast

lowast

0

5

10

15

20

Relat

ive m

RNA

leve

l of H

IF-1

BN

IP3

BNIP3HIF-1

(b)

C P C P

BNIP3

GAPDH

HIF-1

(c)

ControlPOP

lowast lowastlowast

00

05

10

15

20

Prot

ein

expr

essio

n le

vel o

f HIF

-1

BN

IP3

BNIP3HIF-1

(d)




















TUNEL DAPI Merge

Con

trol

POP

(a)

C P C P

Bcl-2

Bcl-xl

Bax

Bad

GAPDH

(b)

ControlPOP

Bcl-xlBcl-2 BadBax

lowastlowast

0

1

2

3

4

Bcl-2

fam

ilyG

APD

H

(c)

POPControl

lowastlowastlowast


1

2

3

4

5

Ratio

of B

cl-2

fam

ily p

rote

in

(d)

C P C P

Cyto-c

Caspase-3

Cleavedcaspase-3

Caspase-9

Cleavedcaspase-9

GAPDH

(e)

ControlPOP


caspase-3


00

05

10

15

20

Relat

ive p

rote

in le

vel

(f)




References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























TUNEL DAPI Merge

Con

trol

POP

(a)

C P C P

Bcl-2

Bcl-xl

Bax

Bad

GAPDH

(b)

ControlPOP

Bcl-xlBcl-2 BadBax

lowastlowast

0

1

2

3

4

Bcl-2

fam

ilyG

APD

H

(c)

POPControl

lowastlowastlowast


1

2

3

4

5

Ratio

of B

cl-2

fam

ily p

rote

in

(d)

C P C P

Cyto-c

Caspase-3

Cleavedcaspase-3

Caspase-9

Cleavedcaspase-9

GAPDH

(e)

ControlPOP


caspase-3


00

05

10

15

20

Relat

ive p

rote

in le

vel

(f)




References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















hypoxia induces apoptosis through hif-1𝛼signaling pathway...

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