Original Article

Comparative Characterization of VaginalCells Derived From PremenopausalWomen With and Without SeverePelvic Organ Prolapse

Hala Kufaishi, MD, MSc1, May Alarab, MD, MSc2,3, Harold Drutz, MD2,3,Stephen Lye, PhD1,3, and Oksana Shynlova, PhD1,3

AbstractBackground: This study tested a hypothesis that primary human vaginal cells derived from tissue of premenopausal women withsevere pelvic organ prolapse (POP-HVCs) would display differential functional characteristics as compared to vaginal cells derivedfrom asymptomatic women with normal pelvic floor support (control-HVCs). Methods: Vaginal tissue biopsies were collectedfrom premenopausal patients with POP (n¼ 8) and asymptomatic controls (n¼ 7) during vaginal hysterectomy or repair. Primaryvaginal cells were isolated by enzymatic digestion and characterized by immunocytochemistry. Cell attachment and proliferationon different matrices (collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, and vitronectin) were compared betweenPOP-HVCs and control-HVCs. RNA was extracted, and the expression of 84 genes was screened using Human ExtracellularMatrix and Adhesion Molecules RT2 Profiler PCR array. The expression of selected genes was verified by quantitative reversetranscription-polymerase chain reaction. Results: (1) Control-HVCs attached to collagen IV more efficiently than POP-HVCs; (2)control-HVCs and POP-HVCs show a similar proliferation rate when plated on proNectin and collagen I; (3) when seeded oncollagen I, resting POP-HVCs expressed significantly (P < .05) increased transcript levels of collagen VII, multiple matrix metal-loproteinases (MMP3, MMP7, MMP10, MMP12, MMP13, and MMP14), integrins (ITGA1, ITGA4, ITGA6, ITGA8, ITGB1, ITGB2,and ITGB3), and cell adhesion molecules as compared to control-HVCs. Collagen XV and tissue inhibitors of MMPs (TIMP1 andTIMP2) as well as genes involved in the biogenesis and maturation of collagen and elastin fibers (LOX, LOXL1-LOXL3, BMP1, andADAMTS2) were significantly downregulated in POP-HVCs versus control-HVCs (P < .05). Conclusions: Resting primary POP-HVCs in vitro show altered cellular characteristics as compared to control-HVCs, which may influence their dynamic responsesto external mechanical or hormonal stimuli.

KeywordsPOP, fibroblasts, vagina, premenopausal, ECM

Introduction

Pelvic floor disorders (PFDs) are widely underrecognized con-

ditions that have a significant impact on the quality of women’s

life. They include stress urinary incontinence (SUI), anal

incontinence, and pelvic organ prolapse (POP). Pelvic floor

disorders have a substantial emotional impact on the affected

women, which results in social isolation, psychological dis-

tress, anxiety, and depression.1,2 A recent cross-sectional study

concluded that 24% of women older than 20 years have at least1 PFD.3 For instance, POP affects one-third of premenopausal

women and nearly half of postmenopausal women.4 There are

multiple risk factors for the development of POP, with vaginal

delivery being the greatest independent inciting risk factor.5,6

Other factors include age, race, family history and genetics, and

most importantly abnormalities in their connective tissue his-

tology and morphology.7-9 Caucasian women have a 5 times

higher risk of developing a symptomatic prolapse in compari-

son to African American women and 1.4-fold increase in the

relative risk of developing severe POP.8

Currently, treatment of POP is dominated by surgical repair

of native tissue with vaginal meshes and generally results in

1 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto,

Ontario, Canada2 Division of Urogynecology and Reconstructive Pelvic Surgery, Department of

Obstetrics and Gynecology, Mount Sinai Hospital, Toronto, Ontario, Canada3 Department of Obstetrics and Gynecology, University of Toronto, Toronto,

Ontario, Canada

Corresponding Author:

Oksana Shynlova, Lunenfeld-Tanenbaum Research Institute, Mount Sinai

Hospital, 25 Orde Street, Suite 6-1019, Toronto, Ontario, Canada M5G 1X5.

Email: shynlova@lunenfeld.ca

Reproductive Sciences2016, Vol. 23(7) 931-943ª The Author(s) 2016Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/1933719115625840rs.sagepub.com

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good anatomic and subjective outcomes.10 However, the use of

meshes is associated with considerable risk of erosion, pain,

infection, and vaginal stenosis.11 More recently, a tissue engi-

neering approach was suggested to be a good alternative to

POP repair surgery.12 The goal is to develop a bioresorbable

scaffold in combination with autologous cells (fibroblasts,

myocytes, or myofibroblasts) that will be able to contribute

to tissue regeneration, thereby forming the functional pelvic

tissue. It is suggested that the successful outcome of vaginal

reconstructive surgery will depend on utilizing an optimized

scaffold substrate, such as collagen, to promote the growth and

distribution of cells with high proliferation potential that would

exhibit rates of protein synthesis similar to cells from aged-

matched controls.13 Research is urgently needed to character-

ize the potential autologous cellular source for use in tissue

engineering to improve surgical outcome in POP.

The pelvic floor comprises a highly interconnected system

of striated muscle, smooth muscle, and connective tissue. The

connective tissue that supports the pelvic floor is mainly com-

posed of the fibrous elements of extracellular matrix (ECM),

collagen and elastin, and cellular components, fibroblasts, and

smooth muscle cells (SMCs). Collagen is the major structural

protein in connective tissue. There are 29 members of the col-

lagen family, including fibril-forming, microfibrillar, fibril-

associated collagens with interrupted triple helices (FACITs),

basement membrane, and transmembrane collagens. The bio-

genesis of structural proteins is controlled by enzymes from the

LOX family proteins, BMP1/PCP, and ADAMTS2/PNP.14,15

Connective tissue matrix also contains a variety of multiadhe-

sive glycoproteins, fibronectin, laminins, vitronectin, throm-

bospondin, fibrinogen, and others, which allow cells to

adhere to the ECM. Cells in the tissue bind to the ECM and

to other cells through transmembrane integrin receptors con-

sisting of a and b subunits.16 Integrins also transduce biochem-ical signals across the cell membrane, which influence survival,

metabolism, and cell fate.17 Extracellular matrix proteins exist

under a constant state of remodeling and turnover. This process

is mediated by a family of cell-derived proteolytic enzymes

known as matrix metalloproteinases (MMPs). A group of pro-

teins named tissue inhibitors of MMPs (TIMPs) strictly control

these enzymes to maintain tight balance between ECM biogen-

esis and breakdown.18 Numerous studies using samples from

different pelvic floor tissues (uterosacral ligaments [USL],

pubocervical fascia, and vaginal wall) reported differences in

tissue composition and remodeling proteins between patients

presenting with severe POP and their asymptomatic counter-

parts.19-21 This combined with the ease of access makes the

vagina a good model for use as a source of cells for in vitro

studies.

Our aim therefore was to derive primary cells from vaginal

biopsies of premenopausal Caucasian women with severe POP

(POP-HVCs) and premenopausal asymptomatic controls (con-

trol-HVCs) and to examine their biological characteristics. We

hypothesized that POP-HVCs will display differences in their

characteristics as compared to control-HVCs due to the intrin-

sic genetic defects or due to the microenvironment of diseased

prolapsed tissue. This study examined the ability of primary

vaginal cells to attach to different protein substrates, to prolif-

erate, and to produce/modulate the expression of native ECM

proteins (collagens, laminins, and adhesive glycoproteins),

their integrin receptors, MMPs/TIMPs/ADAMTSs, and LOX

family enzymes.

Materials and methods

Tissue Collection

The Research Ethics Board of Mount Sinai Hospital, Uni-

versity of Toronto, has approved this study. Strict criteria

were determined to select a homogenous group of preme-

nopausal Caucasian women with severe POP and asympto-

matic control group. Eligible patients were invited to

participate.

Inclusion criteria. Premenopausal adult women undergoing pel-vic surgery for POP equal or greater than stage 3 by Pelvic

Organ Prolapse Quantification System (POP-Q) were identi-

fied as patients. Controls were identified as premenopausal

adult women with POP-Q at stage 0 undergoing abdominal

hysterectomy for indications other than prolapse.

Exclusion criteria. Exclusion criteria were women with a historyof gynecological malignancy, connective tissue disorders,

emphysema, endometriosis, steroid therapy in the past 6

months, and prolapse or incontinence surgery. We rationa-

lized that stage 0 is the ‘‘gold standard’’ for normal pelvic

support.15,22

The principal authors (H.K. or M.A.) obtained written

informed consent from each patient before surgery, performed

the examination for POP staging, and collected clinical and

demographic data and tissue samples. Premenopausal status

was confirmed by endometrial histology report of uterine spe-

cimens. The tissue biopsy technique was reported in previous

publication.22 Briefly, after removal of the uterus or pelvic

floor repair, vaginal tissue specimen (at least 1 cm2) was

obtained by sharp dissection down to the avascular space of

loose areolar connective tissue of the vagina. The site of tissue

collection was standardized at the anterior middle portion of

the vaginal vault to account for variations in stretch conditions.

All collected tissue samples were immediately washed in ice-

cold basic salt solution (Hank balanced salt solution [HBSS]

without Ca2þ and Mg2þ, HBSS�/�, pH 7.4) and transferredfrom operation room (OR) to the cell culture facility for pri-

mary cell derivation.

Derivation of Primary Human Vaginal Cells

The technique for the derivation of primary HVCs was modi-

fied from Srikhajon et al.23 Briefly, human vaginal tissues were

cut into small pieces (1� 1� 1 mm), enzymatically digested at37�C with agitation in buffer supplemented with 2 mg/mLcollagenase type IA (Sigma, Missouri), 0.15 mg/mL DNase I

932 Reproductive Sciences 23(7)

(Roche Applied Science, Québec, Canada), and 1 mg/mL

bovine serum albumin (BSA; Sigma). After 1.5-hour incuba-

tion, the digestion mixture was mechanically disrupted by

pipetting. A suspension of isolated primary cells was centri-

fuged at 250g for 8 minutes at 4�C, and the cell pellet wasresuspended in phenol red-free cell culture media (Dulbecco

modified eagle medium [DMEM]; Thermo Fisher Scientific

Inc, Delaware) supplemented with 20% fetal bovine serum(FBS; Gibco, Burlington, Ontario, Canada), 50 mg/L Normo-

cin (Invivogen, California), and 25 mmol/L Hepes (Invivo-

gen, California). These cells were seeded in tissue culture

dishes and incubated overnight (ON) at 37�C with 5% CO2.Cells were washed with HBSS�/� to remove unattached cellsand cultured in DMEM/20% FBS. Cell culture medium waschanged every 3 days. After 2 to 4 weeks, primary vaginal

cells (passage 0) reached 90% confluence and were passaged.After 3 passages, HVCs were trypsinized, resuspended in the

freezing medium (90% FBS; 10% dimethyl sulfoxide[DMSO]), and frozen in liquid nitrogen.

Cell Morphology and Phenotype Identification

To verify the cellular origin of primary vaginal cells, we used

specific biomarkers of fibroblastic versus myogenic origin via

immunofluorescent staining. Cells derived from the tissue (pas-

sages 0) and passaged cells (passage 1) were cultured in 8-well

cell chamber slides until 50% to 75% confluence, fixed with4% paraformaldehyde for 15 minutes at room temperature(RT), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich,Missouri) for 30 seconds, and blocked with serum-free protein

block (DAKO, Ontario, Canada) for 30 minutes at RT. Human

vaginal cells were then incubated with a specific antibody for

fibroblasts biomarker, mouse antihuman vimentin (Sc-7558;

Santa Cruz, Texas), with antibodies for myogenic biomarker,

mouse anti-human smooth muscle actin (SMA; M0851,

DAKO), diluted 1:100 in phosphate-buffered saline (PBS),

or with endothelial biomarker anti-cytokeratin (M0821, dilu-

tion 1:200; DAKO), for 18 hours at 4�C. Slides were thenwashed in PBS and incubated with fluorochrome-linked sec-

ondary antibody goat anti-mouse Alexa Fluor-488 (A21202,

dilution 1:100; Invitrogen, Burlington, Ontario, Canada) or

donkey anti-goat Alexa Fluor-594 (A11058, dilution 1:200;

Invitrogen, Oregon) plus 4,6-diamidino-2-phenylindole dihy-

drochloride (DAPI) nuclear staining (D-9542, Sigma-Aldrich;

dilution 1:1000) in the dark for 40 minutes. Slides were

mounted with media (Thermo Fisher, Ontario, Canada),

viewed under a fluorescent microscope (Leica Microsystems,

Richmond Hill, Ontario, Canada), and photographed with

Sony DXC-970 MD (Sony Ltd, Toronto, Ontario, Canada)

3CCD color video camera.

Cell Attachment to Different Extracellular Matrices

The attachment of HVCs was studied using the Human ECM

Cell Adhesion Array Kit (colorimetric, ECM540; Millipore,

Massachusetts) according to the manufacturer’s instruction.

The kit is comprised of 8 well strips coated with 7 different

human ECM proteins (collagen [COL] I, COL II, COL IV,

fibronectin, laminin, tenascin, and vitronectin) and BSA (neg-

ative control). Briefly, 13 confluent POP-HVCs and control-

HVCs at passage 2 (7 POP-HVCs and 6 control-HVCs) were

trypsinized (Gibco), centrifuged, washed 3 times in HBSS�/�

counted on CASY Cell Counter (Roche Applied Science, Qué-

bec, Canada), and diluted to a final concentration of 1 � 106cells/ mL. Hundred microliters of each cell suspension

(100 000 cells) was added in triplicates to 7 ECM-coated wells

and the control well. The plate was incubated for 1 hour at 37�Cin a CO2 chamber, and then cell stain solution was added to

each well, followed by extraction buffer, and incubated in the

dark under gentle rotation at RT for 10 minutes; the absorbance

was determined at 560 nm on a microplate reader (mQuantBiotek, Vermont, USA).

Cell Proliferation on Different Extracellular Matrices

Thiazolyl blue tetrazolium bromide (MTT) assay was used to

assess the proliferation ability of 10 HVCs lines (5 POP-HVCs

and 5 control-HVCs) as was described previously.24 All HVC

lines used for the proliferation study were at passage 2. Briefly,

24-well black high-throughput (HT) BioFlex culture plates

coated with COL I (HTPB-3001C; Flexcell Inc, Burlington,

NC, USA) or proNectin (HTPB-3001P; Flexcell Inc) were

used. Human vaginal cells were trypsinized, pelleted, and

washed in HBSS�/� as described previously. The final pelletswere resuspended in DMEM/20% FBS to a final concentrationof 5000 cells/mL. Human vaginal cells (5000 cells/well) were

plated in quadruplicates on the HT BioFlex culture plate, and

the control well contained only media. Identical plates for both

ECM substrates were prepared for each cell line to study the

proliferation of POP-HVCs and control-HVCs over the span of

5 days. After 1, 2, 3, 4, or 5 days in culture, 500 mL of MTTsolution (5 mg/mL MTT in DMEM/20% FBS; Sigma-Aldrich)was added to each well including the control (media only)

wells. The plate was incubated at 37�C for 3.5 hours in aCO2 chamber, then the media was carefully aspirated, and 1

mL of DMSO/well (Sigma) was added and incubated in the

dark at RT for 10 minutes. The absorbance was determined at

590 nm on a microplate reader (mQuant) at the same time every24 hours for the duration of experiment.

RNA Extraction

Human vaginal cells were washed twice with ice-cold

HBSS�/�, scraped, and lysed by TRIzol (Gibco). Total RNAwas extracted according to the manufacturer’s protocol,

treated with 2.5 mL DNase I (2.73 Kunitz unit/mL; Qiagen,Mississauga, Ontario, Canada) to remove genomic DNA con-

tamination, and column purified using RNeasy MiniElute

Cleanup kit (Qiagen). RNA concentration was then measured

by the NanoDrop 1000 spectrophotometer (Thermo Fisher).

Stock complementary DNA (cDNA) solutions (50 ng/mL)were generated with iScript Reverse Transcription (RT)

Kufaishi et al 933

Supermix (Bio-Rad Laboratories Inc, California) from 1 mg ofRNA following the manufacturer’s recommended protocol.

All polymerase chain reactions (PCRs) were carried out on

the CFX96 or CFX384 Touch Real-Time PCR Detection Sys-

tems (Bio-Rad).

Gene Expression Analysis: Quantitative Profiler PCR Array

We screened for the expression of 84 genes (see Table 1) in

HVCs using the Human ECM and Adhesion Molecules RT2

Profiler PCR array (SABiosciences Corp, Frederick, Maryland)

according to the manufacturer’s instructions. Two pooled sam-

ples were prepared with equal amounts of RNA from (1) POP-

HVCs (n ¼ 8) and (2) Control-HVCs (n ¼ 7). ComplementaryDNA was prepared from 1 mg total pooled RNA using iScriptRT Supermix (Bio-Rad). The PCR amplification was con-

ducted with an initial 10-minute step at 95�C followed by 40cycles of 95�C for 15 seconds and 60�C for 1 minute. Theexperiment was repeated 3 times. Data were imported into the

integrated Web-based software package (http://pcrdataanaly-

sis.sabiosciences.com/pcr/arrayanalysis.php; Qiagen GmbH).

Quantitative analysis was based on the DDCt method, withnormalization of the raw data and the geometric mean of 5

housekeeping genes (GAPDH, HPRT1, ACTB, B2M, and

RPLP0). The Web portal automatically performs all DDCt-based calculations from the uploaded raw threshold cycle

(Ct) data.

Real-Time RT-PCR

To confirm the results of the RT2 Profiler PCR array, we

conducted quantitative reverse transcription-polymerase chain

reaction (qRT-PCR) on individual cDNA samples produced

from 8 individual cell lines derived from patients with POP

and 7 individual cell lines derived from asymptomatic controls

using CFX384 Touch Real-Time PCR Detection Systems (Bio-

Rad). Specific sequences of oligonucleotide primers for indi-

vidual genes were obtained from Gene Bank Database of the

National Centre for Biotechnology Information (NCBI) of the

National Institutes of Health using Primer-BLAST (Supple-

mental Table 1). Gene expression levels in all studies were

normalized against the geometric mean of the following 3

housekeeping genes: YWHAZ, TBP, and ACTB. Reverse

transcription-PCR reactions were carried out in triplicates,

where each reaction contained 10 ng of cDNA, LuminoCt

SYBR Green qPCR ReadyMix (Sigma), and forward and

reverse primers at a final concentration of 300 nmol/L in a total

of 10 ml per well reaction mix. The cycling protocol startedwith an initial denaturation 95�C for 30 seconds, then 40 cyclesof denaturation 95�C for 5 seconds, and annealing/extension60�C for 20 seconds. Each qRT-PCR run was followed by amelting curve analysis to confirm the specificity of the primers

used. Gene expression values were analyzed using the DDCqmode on the CFX Manager software 2.0 (Bio-Rad). Sample

replicates with a Cq > 35 were excluded from data analysis,

and the cutoff quantification cycle standard deviation (DCq)

was set at �0.2. A no-template control of each primer master-mix was also loaded on every plate to ensure the absence of

contaminations.

Statistical Analysis

Patient’s demographics were subjected to Fisher exact test for

categorical data and Student t test for continuous data. To

determine the differences in the attachment to different ECM

substrates between POP-HVCs and control-HVCs, 2-way

analysis of variance (ANOVA) was used. Results of prolifera-

tion assay were subjected to 2-way ANOVA to determine

levels of significant difference. Real-time PCR results were

subjected to Student t test. Experimental error was reported as

standard error of the mean (SEM). The statistical program

Prism (version 4.0; Graph Pad Software Inc, San Diego, Cali-

fornia) was used, with the level of significance for comparison

set at P < .05.

Results

Patients’ Demographics

Vaginal vault biopsy samples were obtained from 15 Cau-

casian premenopausal women (8 in the ‘‘patient’’ group 1

and 7 in the ‘‘control’’ group 2) matched for age and body

mass index. The majority of patients with POP (88%) andcontrols (57%) reported symptoms of SUI, and the differ-ence between the 2 groups was not significant. There was

however a statistical difference between patients with POP

and asymptomatic controls with respect to the family history

of POP (P ¼ .019). The mean parity and the number ofvaginal deliveries were significantly higher in patients with

POP (P < .05; Table 2).

Isolation and Characterization of Primary Vaginal Cells

We derived cells from 8 patients with POP and 7 women with

clinically normal pelvic floor support using collagen digestion

and then used specific biomarkers of fibroblastic versus myo-

genic origin via immunoflourescent staining to identify the

phenotype of these cells. Our results show that immediately

after derivation (passage 0-2), the majority of DAPI-stained

cells were positive for vimentin (fibroblastic marker) and myo-

genic marker a-SMA in both POP and control groups (Figure1A). To verify these results, in passage 2, we used antibodies

against desmin, a specific marker of differentiated SMCs, and

found only 5% to 6% desmin-positive cells in both POP andcontrol groups (Figure 1B). Furthermore, we noted no cells

stained for the antiepithelial cell marker, cytokeratin, in pas-

sages 0 to 2. We concluded that the majority of primary vaginal

cells in culture were myofibroblasts, with a minor presence of

myocytes and no epithelial contamination.

To determine whether POP-HVCs and control-HVCs pre-

ferentially attach to different ECM proteins, we used the com-

mercially available Human Cell Adhesion Array Kit. Our

results indicate no differential attachment of HVCs on COL

934 Reproductive Sciences 23(7)

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Table 1. List of 84 ECM and Cell Adhesion Genes Per Functional Group.a

Reference Sequences Symbol Description

CollagensNM_000088 COL1A1 Collagen, type I, a1NM_001846 COL4A2 Collagen, type IV, a2NM_000093 COL5A1 Collagen, type V, a1NM_001848 COL6A1 Collagen, type VI, a1NM_001849 COL6A2 Collagen, type VI, a2NM_000094 COL7A1 Collagen, type VII, a1NM_001850 COL8A1 Collagen, type VIII, a1NM_080629 COL11A1 Collagen, type XI, a1NM_004370 COL12A1 Collagen, type XII, a1NM_021110 COL14A1 Collagen, type XIV, a1NM_001855 COL15A1 Collagen, type XV, a1NM_001856 COL16A1 Collagen, type XVI, a1

ECM proteases and protease inhibitorsNM_002421 MMP1 Matrix metallopeptidase 1 (interstitial collagenase)NM_004530 MMP2 Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)NM_002422 MMP3 Matrix metallopeptidase 3 (stromelysin 1, progelatinase)NM_002423 MMP7 Matrix metallopeptidase 7 (matrilysin, uterine)NM_002424 MMP8 Matrix metallopeptidase 8 (neutrophil collagenase)NM_004994 MMP9 Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)NM_002425 MMP10 Matrix metallopeptidase 10 (stromelysin 2)NM_005940 MMP11 Matrix metallopeptidase 11 (stromelysin 3)NM_002426 MMP12 Matrix metallopeptidase 12 (macrophage elastase)NM_002427 MMP13 Matrix metallopeptidase 13 (collagenase 3)NM_004995 MMP14 Matrix metallopeptidase 14 (membrane-inserted)NM_002428 MMP15 Matrix metallopeptidase 15 (membrane inserted)NM_005941 MMP16 Matrix metallopeptidase 16 (membrane inserted)NM_006988 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1NM_139025 ADAMTS13 ADAM metallopeptidase with thrombospondin type 1 motif, 13NM_007037 ADAMTS8 ADAM metallopeptidase with thrombospondin type 1 motif, 8NM_003254 TIMP1 TIMP metallopeptidase inhibitor 1NM_003255 TIMP2 TIMP metallopeptidase inhibitor 2NM_000362 TIMP3 TIMP metallopeptidase inhibitor 3

Transmembrane and cell–matrix adhesion moleculesNM_001843 CNTN1 Contactin 1NM_001903 CTNNA1 Catenin (cadherin-associated protein), a1, 102 kDaNM_001904 CTNNB1 Catenin (cadherin-associated protein), b1, 88 kDaNM_001331 CTNND1 Catenin (cadherin-associated protein), d1NM_001332 CTNND2 Catenin (cadherin-associated protein), d2 (neural plakophilin-related arm-repeat protein)NM_181501 ITGA1 Integrin, a1NM_002203 ITGA2 Integrin, a2 (CD49B, alpha 2 subunit of VLA-2 receptor)NM_002204 ITGA3 Integrin, a3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)NM_000885 ITGA4 Integrin, a4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)NM_002205 ITGA5 Integrin, a5 (fibronectin receptor, alpha polypeptide)NM_000210 ITGA6 Integrin, a6NM_002206 ITGA7 Integrin, a7NM_003638 ITGA8 Integrin, a8NM_002209 ITGAL Integrin, a L (antigen CD11A [p180], lymphocyte function-associated antigen 1; a polypeptide)NM_000632 ITGAM Integrin, a M (complement component 3 receptor 3 subunit)NM_002210 ITGAV Integrin, a V (vitronectin receptor, a polypeptide, antigen CD51)NM_002211 ITGB1 Integrin, b 1 (fibronectin receptor, b polypeptide, antigen CD29 includes MDF2, MSK12)NM_000211 ITGB2 Integrin, b 2 (complement component 3 receptor 3 and 4 subunit)NM_000212 ITGB3 Integrin, b 3 (platelet glycoprotein IIIa, antigen CD61)NM_000213 ITGB4 Integrin, b 4NM_002213 ITGB5 Integrin, b 5NM_000201 ICAM1 Intercellular adhesion molecule 1NM_000615 NCAM1 Neural cell adhesion molecule 1NM_000442 PECAM1 Platelet/endothelial cell adhesion moleculeNM_001078 VCAM1 Vascular cell adhesion molecule 1

(continued)

Kufaishi et al 935

I, COL II, fibronectin, laminin, tenascin, or vitronectin pro-

teins. A significant decrease in attachment ability of POP-

HVCs compared to control-HVCs was observed on basement

membrane protein COL IV (Figure 2A, P < .05). Since we did

not detect differential attachment on COL I, the most abundant

fibrillar collagen in the ECM, or on the major linker protein

fibronectin, we selected these proteins as substrates for the

proliferation study. The ability of primary HVCs derived from

asymptomatic controls and patients with POP to proliferate was

assessed using COL I-coated or proNectin-coated plates over a

period of 5 days. The MTT assay was carried out to assess a

daily proliferation rate of 5 cell lines derived from prolapsed

tissue and 5 cell lines derived from normal vaginal biopsies.

Our results indicate no significant difference in the prolifera-

tion rate of POP-HVCs plated on COL I or proNectin (a syn-

thetic analogue of fibronectin) in comparison to the control-

HVCs (Figure 2B).

Extracellular Matrix and Adhesion MoleculeQuantitative PCR Arrays

This PCR array detects the expression of 84 genes involved

in cell adhesion and communication, ECM remodeling, ECM

ligands, and their transmembrane receptors (see Table 1). In

particular, we compared the gene expression of multiple col-

lagen molecules (Figure 3A), ECM proteases and protease

inhibitors (Figure 3B), transmembrane integrin receptors and

cell–matrix adhesion molecules (Figure 3C), cell–cell adhe-

sion molecules, and basement membrane constituents (Figure

3D) in resting primary HVCs derived from patients with POP

and controls. The summary of the PCR results presented in

the Supplemental Table 2 indicate that 36 genes were

Table 1. (continued)

Reference Sequences Symbol Description

Cell–cell adhesion molecules and basement membrane constituentsNM_004425 ECM1 Extracellular matrix protein 1NM_002026 FN1 Fibronectin 1NM_001523 HAS1 Hyaluronan synthase 1NM_000216 KAL1 Kallmann syndrome 1 sequenceNM_005559 LAMA1 Laminin, a 1NM_000426 LAMA2 Laminin, a 2NM_000227 LAMA3 Laminin, a 3NM_002291 LAMB1 Laminin, b 1NM_000228 LAMB3 Laminin, b 3NM_002293 LAMC1 Laminin, g 1 (formerly LAMB2)NM_001901 CTGF Connective tissue growth factorNM_003919 SGCE Sarcoglycan, epsilonNM_000582 SPP1 Secreted phosphoprotein 1NM_003118 SPARC Secreted protein, acidic, cysteine-rich (osteonectin)NM_000450 SELE Selectin ENM_000655 SELL Selectin LNM_003005 SELP Selectin P (granule membrane protein 140 kDa, antigen CD62)NM_003119 SPG7 Spastic paraplegia 7 (pure and complicated autosomal recessive)NM_002160 TNC Tenascin CNM_003246 THBS1 Thrombospondin 1NM_003247 THBS2 Thrombospondin 2NM_007112 THBS3 Thrombospondin 3NM_000358 TGFBI Transforming growth factor, beta-induced, 68kDaNM_004385 VCAN VersicanNM_000638 VTN VitronectinNM_003278 CLEC3B C-type lectin domain family 3, member BNM_004360 CDH1 Cadherin 1, type 1, E-cadherin (epithelial)NM_000610 CD44 CD44 molecule (Indian blood group)

aAdapted from http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-013Z.html.

Table 2. Summary of Patient Demographics.a

Study GroupsGroup 1 (Patient

With POP)Group 2

(Non-POP Control)

n 8 7Ageb 43.5 (37-50) 45.0 (42-49)BMIb 27.8 (20.6-41.6) 27.5 (21.6-32.8)Parityb 3 (1-6)c 1 (0-3)Vaginal deliveriesb 3 (1-3)c 1 (0-1)SUI 88% 57%Family history of POP (%) 63%c 0%Stage III-IV of POP (n) 8 0No POP (n) 0 7

Abbreviations: BMI, body mass index; POP, pelvic organ prolapse; SUI, stressurinary incontinence.aFisher exact test; level of significance: P < .05.bData are presented as median (range).cStatistical difference between groups 1 and 2.

936 Reproductive Sciences 23(7)

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differentially expressed between the 2 groups (marked red,

P < .05). These results demonstrate that under resting culture

conditions and in comparison to control-HVCs, (1) POP-

HVCs expressed significantly higher levels of COL7A1,

numerous ECM integrin receptors (ITGA1, ITGA4, ITGA6,

ITGA8, ITGAAV, ITGB1, ITGB2, and ITGB3), catenins

(CTNNA1, CTNNB1, and CTNND1), cadherin 1 (CDH1),

contactin 1 (CNTN1), hyaluronic acid receptor (CD44), cell

adhesion molecules (NCAM1), thrombospondin-2 (THBS2)

and laminins (LAMB1 and LAMB13), and ECM remodeling

genes (MMP3, MMP7, MMP10, MMP12, MMP13, MMP14,

and TIMP2; P < .05), (2) whereas messenger RNA (mRNA)

0 1 2 (passage)

VIM VIM VIM

ASMA ASMA DESMIN

NEG CONTROL NEG CONTROL

CK CK CK

SM Ac�n

0

20

40

60

80

100

Perc

enta

ge o

f (+)

cel

ls

Desmin

CONTROL

Vimen�n

POP

B

A

Figure 1. Characterization of primary human cells derived from vaginal biopsies. A, Representative immunofluorescence images of primary cellsderived from premenopausal woman at passage 0, 1, and 2. Cells were fixed in paraformaldehyde (PFA) and incubated with antibodies againstvimentin (red), a-smooth muscle actin (a-SMA), desmin (only passage 2), and cytokeratin (CK; all green). Nuclei were stained with4,6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Negative controls show staining with secondary antibodies only. Original magnifica-tions: �100. B, Quantitative analysis of human vaginal cells (HVCs) from passage 1 derived from control (black bars) and patients with pelvicorgan prolapse (POP; white bars), which expressed vimentin, desmin, and a-SMA, respectively (percentage of positive cells versus all cells). (Thecolor version of this figure is available in the online version at http://rs.sagepub.com/.)

Kufaishi et al 937

http://rs.sagepub.com/

levels of COL15A1, TIMP3, VCAM1, and ICAM1 were sig-

nificantly (P < .05) downregulated (Supplemental Table 2;

Figure 4).

The results of the PCR array for selected ECM proteins, their

integrin receptors, proteases, and protease inhibitors that showed

significant difference were chosen for verification by quantita-

tive RT-PCR using individual RNA samples isolated from POP-

HVCs (n ¼ 8) and control-HVCs (n ¼ 7). Overall, the RT-PCRanalysis confirmed the PCR array results, in particular, the basal

expression of multiple integrins (ITGA1, ITGA4, ITGA8, and

ITGB1), COL7A1, and MMP3 was significantly higher,

whereas COL15A1, MMP2, TIMP2, and TIMP3 genes were

significantly lower in cells isolated from patients with POP than

in control vaginal cells (Figure 5A and B, P < .05).

Our previous in vivo study found differential mRNA expres-

sion of proteins involved in the processing and maturation of

collagen and elastin polymers, namely LOX, LOXL1 to

LOXL4, BMP1/PCP, and ADAMTS2/PNP, in vaginal biopsy

samples from premenopausal patients with POP and asympto-

matic controls.14,15,22 Unfortunately, these genes were not

included in the PCR array. Hence, to understand whether these

in vivo changes occur due to the differences in the cellular

expression, we examined mRNA levels of those genes by

qRT-PCR in the unstimulated (resting) HVC cultures derived

from patients with POP and asymptomatic controls. Similar to

the earlier in vivo data, a significant decrease in LOX, LOXL1

to LOXL3, ADAMTS2, and BMP1 mRNA expression was

noted in vitro in cultured POP-HVCs compared to resting

control-HVCs (Figure 5C)

Discussion

In an attempt to improve the outcome of POP surgery, urogy-

necologists have extensively used surgical implants to rein-

force the weakened pelvic floor tissue. The efficacy of this

approach is questionable, as many women report serious com-

plications, such as erosion, pain, vaginal stenosis, and infec-

tion.11,25 Simultaneously, the need for improved long-lasting

repair therapies for POP is growing due to the increased life

expectancy and activity levels of modern women. Because of

their accessibility, muscle-derived stem cells were the first

explored in cell-based therapy for the treatment of POP in the

animal model.26,27 The use of human fibroblasts derived from

the endometrium, vagina, foreskin, and buccal membranes in

conjunction with synthetic scaffolds has also been suggested as

a treatment for POP.12,13,28,29 To understand the reason for

unsuccessful outcomes after POP repair surgery and to deter-

mine whether the cause of failure might be a defective pro-

lapsed tissue, we designed this study in which we compare

characteristics of vaginal cells originated from POP and non-

POP tissue.

Our current data indicate that human vaginal cells derived

from patients with POP and asymptomatic controls were

mostly myofibroblasts with a small presence of SMCs. Cells

from diseased and healthy tissue showed a similarity in the

attachment to different ECM components, except for COL

IV, the protein that present ubiquitously in basement mem-

branes. COL IV provides a scaffold for cellular assembly and

mechanical stability and plays an important role in cell adhe-

sion, growth, migration, proliferation, and differentiation. It is

the main ECM protein excreted from fibroblasts following

trauma and is induced during scar formation.30 Attachment of

cells to COL IV is mediated via multiple binding sites and

involves several integrins and nonintegrin receptors.31 There-

fore, we speculate that decreased attachment of POP-HVCs to

COL IV may contribute to the weakening of the pelvic floor in

patients with POP. On the other hand, cellular proliferation of

HVCs derived from patients with POP in comparison to control

HVCs was similar on major fibrillar COL I and proNectin

(which contains the Arg-Gly-Asp attachment domain of

fibronectin).

0

0.05

0.1

0.15

0.2

0 1 2 3 4 5

OD

Day

Control Collagen 1

Control proNec�nPOP Collagen 1

0

0.1

0.2

0.3

0.4

0.5

Col I Col II Col IV FN LN TN VN

OD

ECM Substrate

ControlA

B

POP

POP proNec�n

Figure 2. Cellular characteristics of primary human vaginal cells(HVCs) derived from premenopausal women. A, Attachment of HVCsfrom asymptomatic controls (n ¼ 6, black bars) and patients withsevere pelvic organ prolapse (POP; n ¼ 7, white bars) on 7 differenthuman extracellular matrix (ECM) proteins: collagen (Col) I, Col II,Col IV, fibronectin (FN), laminin (LN), tenascin (TN), and vitronectin(VN). The results shown are the mean + standard error of the mean(SEM) of absorbance relative to the control protein, bovine serumalbumin (BSA). A significant difference is indicated by *P < .05. B,Proliferation of HVCs derived from premenopausal asymptomaticcontrols (solid line) and patients with severe POP (dashed line) oncollagen I (black) and proNectin (gray)-coated plates, respectively(n ¼ 5/group). The results shown are the optical density per well,mean + SEM for each patient group. No significant difference wasdetected by analysis of variance (ANOVA).

938 Reproductive Sciences 23(7)

Our previous in vivo studies demonstrated impairment in the

expression and activity of ECM homeostasis-related proteins

(enzymes regulating ECM assembly, synthesis, and degrada-

tion) in vaginal tissue biopsies derived from patients with POP

in comparison to asymptomatic controls.14,15,32 Other research

groups assessed collagen content in pelvic floor tissue of

women with and without POP and concluded that the overall

collagen content is decreased in the pelvic floor tissue of

women with POP in comparison to asymptomatic controls.33,34

However, there are limited data on the metabolism of collagen

and elastin and the lack of detailed analysis of different col-

lagen subtypes in HVCs. Makinen et al found that the rate of

collagen synthesis and procollagen mRNA levels in fibroblasts

derived from patients with POP was similar or slightly higher

than those from age-matched asymptomatic controls.35

Recently, Kerkhof et al compared tissue sample from non-

POP sites and POP sites in the same patient and found that

there was a tendency toward an increase in the COL III and

elastin content and a significantly increased number of collagen

cross-links in the POP site.36 We report here key differences in

mRNA expression levels of multiple collagens and integrins

between POP-HVCs and control-HVCs. In particular, under

resting culture conditions, POP-HVCs in comparison with

control-HVCs expressed lower mRNA levels of FACIT COL

XV that codistributes with COL I. Interestingly, COL XV has

the strongest expression in basement membrane zones and

functions to adhere basement membranes to underlying con-

nective tissue stroma. It was reported in mouse that COL XV

deficiency is associated with muscle and microvessel deteriora-

tion.37 In contrast, COL VII, which functions to anchor fibril

between the external stratified epithelia and the underlying

stroma, was upregulated by POP-HVF. The expression of COL

VII was shown to be restricted to the basement zone beneath

stratified squamous epithelia, and its mutations were associated

Figure 3. Gene expression heat map from polymerase chain reaction (PCR) array analysis. In the resting human vaginal cells (HVCs) derivedfrom asymptomatic controls (CONTROL) and patients with severe pelvic organ prolapse (POP), gene expression levels of (A) collagens andextracellular matrix (ECM) structural constituents; (B) ECM proteases and protease inhibitors; (C) cell–cell adhesion molecules and basementmembrane constituents; and (D) transmembrane and cell–matrix adhesion molecules were different between groups. Pooled RNA samples ofCONTROL (n ¼ 7) and patients with POP (n ¼ 8) were used. Shown are results of 3 separate PCR arrays per group. A significant difference isindicated in red (see Supplemental Table 2, P < .05).

Kufaishi et al 939

with chronic subepidermal blistering disease.38 We also noted

differential expression of basement membrane proteins

(LAMB1 and LAMB3) between POP-HVCs and control-

HVCs. Basement membranes separate cell from the underlying

connective tissue, stabilize and maintain the tissue architecture.

Laminins are very large cross-shaped molecules constituted by

the association of a, b, and g chains. They have the ability toself-associate into polymers forming a high-equimolecular

complex, which in combination with COL IV creates a stable

scaffold anchoring cells and other components of the basement

membrane (ie, fibronectin). Changes in the expression of ECM

constituents (in particular, basement membranes) show a gen-

eralized dysregulation in the prolapsed vaginal tissue also man-

ifested by the decreased attachment capability of POP-HVCs

probably indicating misbalance of pelvic floor tissue morphol-

ogy on a micromolecular level.

Specific interactions between cells and the ECM are

mediated by integrin molecules. Our results also demonstrate

that POP-HVCs expressed higher mRNA levels of numerous

integrins (a1, a4, a6, a8, av, b1, b2, and b3) and cell–celladhesion molecules (contactin 1 and catenins a, b, d). Integrinsare transmembrane a/b noncovalently-associated heterodimersignal transduction molecules, sensing physical forces and

transducing mechanical signals (stretch) into a biochemical

response. It is possible that the expression of integrins is upre-

gulated in POP-HVCs due to an increased mechanical stimula-

tion of the prolapsed tissue and may reflect the secondary

effects of POP. Signaling through integrins involves the assem-

bly of a complex of multiple proteins inside the cell, which may

implicate the altered cell–matrix interaction and potential dif-

ference in mechanotransduction mechanism, especially when

external stimuli are applied. For instance, integrin duplex a1b1is a cell surface receptor for collagen and laminin, and avb3 is acell surface receptor for vitronectin. Integrin b1 in combinationwith integrin a4 forms integrin VLA-4 that act as a receptor forfibronectin,39 with integrin a6–VLA-6 and integrin a8 receptorfor tenascin, fibronectin, and vitronectin. These heterodimeric

receptors are involved in cell–cell adhesion and may play a role

in inflammation and fibrosis. It was reported that the integrin

a8 gene may be a major effector of HOXA11,40 a homeoboxtranscription factor that orchestrates embryonic development

C

A

0

0.5

1

1.5

LOX LOXL1 LOXL2 LOXL3 LOXL4 BMP1 ADAMTS2

* * * *** **

Gen

e Ex

pres

sion

(Rel

a�ve

Fol

d Ch

ange

)

0

0.5

1

1.5

2

2.5

3

3.5

COL7A1 COL15A1 MMP2 MMP3 TIMP 2 TIMP 3

CONTROLPOP

** ** **

*

*

B

0

2

4

6

8

CNTN1 ITGA1 ITGA4 ITGA8 ITGB1 ITGB2

***

**

**

Figure 5. Expression of selected genes by human vaginal cells (HVCs)from asymptomatic controls (n ¼ 7, black bars) and patients withpelvic organ prolapse (POP; n ¼ 8, white bars) validated by quantita-tive reverse transcription-polymerase chain reaction (qRT-PCR) andnormalized to 3 housekeeping genes YWHAZ, TBP and ACTB: (A)extracellular matrix (ECM) and ECM-degrading genes; (B) ECM integ-rin receptors and cell adhesion genes; and (C) ECM maturation pro-teins. The results shown are fold change relative to asymptomaticCONTROL (mean + standard error of the mean [SEM]). A sig-nificant difference is indicated by *P < .05 and **P < .01.

Gen

e Ex

pres

sion

(Rel

a�ve

Fol

d Ch

ange

)

0

0.5

1

1.5

2

2.5

3

3.5

CNTN1 ITGA1 ITGA4 ITGA8 ITGB1 ITGB2

A

B

0

0.5

1

1.5

2

2.5

COL7A1 COL15A1 MMP2 MMP3 TIMP2 TIMP3

CONTROL

POP

Figure 4. Relative expression of extracellular matrix (ECM) genes byresting human vaginal cells (HVCs) derived from asymptomatic con-trols (CONTROL) and patients with severe pelvic organ prolapse(POP) determined using a reverse transcription-polymerase chainreaction (RT-PCR)-based mRNA expression array. The resultsshown are fold change relative to asymptomatic controls (mean +standard error of the mean [SEM]). A significant difference is indicatedby *P < .05, **P < .01 and ***P < 0.001.

940 Reproductive Sciences 23(7)

of the urogenital tract, in particular uterosacral ligaments

(USL).41 It has been suggested that this signaling pathway

involving HOXA11, COL III, and MMP2 contributes to the

weakened connective tissue of women with POP.41 Altogether,

these results confirm that fibroblasts derived from patients with

POP show cellular characteristics that are already modified by

their environment as compared to the fibroblasts derived from

non-POP asymptomatic controls. We suggest that when

exposed to pathologic external stimuli42,43 or surgical

trauma,44 POP-HVCs may not be able to properly adhere to

ECM, resulting in loss of tissue strength and subsequent devel-

opment or reoccurrence of prolapse.

Little is known about the active interactions between the cell

and the ECM and how this affects the pathophysiology of POP.

The understanding of the cell–matrix interaction in tissue of

women with POP is necessary to cultivate new therapeutic

approaches, especially in the development of new bioscaffolds

for prolapse repair. There is an evidence that the vaginal con-

nective tissue from patients with POP is phenotypically differ-

ent than the connective tissue derived from asymptomatic

controls, showing diminished COL I and COL III levels,45 and

ECM that is disordered, less dense, and loosely arranged.46

Unfortunately, we cannot comment on the expression levels

of COL III in the HVCs-derived POP and control tissues as

this gene was not included in the analysis. Importantly, here we

demonstrated that under static conditions, cultured HVCs from

patients with POP expressed lower transcript levels of the LOX

family proteins, which parallels with our earlier in vivo

results.14 The key known function of LOX enzymes is to con-

tribute to collagen and elastin fiber cross-linking, however,

they also play a role in fibrotic disorders.47 In contrast to our

in vivo results, we detected a decrease in the expression of PCP

and PNP genes in POP-HVCs in vitro in comparison to asymp-

tomatic controls.15 It has been shown before that mechanical

tension can decrease the transcript levels of LOX, pro-COL I

and III, PNP, and PCP.48 Thus, it is possible that HVCs derived

from severely prolapsed (and therefore stretched) tissues would

express lower levels of LOX, PNP, and PCP. During wound

healing, resting fibroblasts differentiate to a migratory myofi-

broblasts characterized by suppressed expression of MMP2 and

increased expression of the contractile protein, a-SMA.49

Importantly, our RT-PCR results indicate that MMP2 transcript

levels were downregulated in the POP-HVCs. It is in a good

agreement with Ruiz-Zapata and colleagues who reported that

vaginal fibroblasts derived from women with mild POP showed

lower gene expression levels of MMP2 mRNA50 and secreted

less MMP2 protein51 when compared with healthy controls. It

is known that factors that increase myofibroblast contractility,

such as transforming growth factor b and serum, decreaseMMP2 expression. Since in pathological wound healing envir-

onments, such as that of the pelvic floor of patients with POP,

the myofibroblast phenotype persists, this could explain why

MMP2 expression was lower in POP-HVCs in comparison to

control-HVCs. In addition, we detected an increased expres-

sion of MMP3, an enzyme involved in tissue repair and crucial

in connective tissue remodeling, as well as MMP13

(collagenase 3) and membrane-type MMP14 in POP-HVCs;

however, there was no change in the expression of collagenases

MMP1 and MMP8, which was in accordance with our recently

published in vivo data.32 Matrix metalloproteinase 14 is able to

cleave pro-MMP2 to active MMP2 in a trimolecular complex

involving TIMP2.52 The MMP14–mediated MMP2 activation

is upregulated by certain ECM molecules in order to regulate

COL I synthesis and prevent fibrosis53; however, it could be

selectively suppressed by ITGB3,54 which colocalizes with

MMP2 on the surface of different cells.55 Importantly, ITGB3

transcript was significantly upregulated in the POP-HVCs (see

supplemental Table 2). We speculate that overexpression of

ITGB3 on the surface of HVCs may serve as a protective

mechanism to decrease the activity of MMP2 in patients with

POP. We previously reported a reduction in TIMP2 and TIMP3

mRNA in POP vaginal biopsies in comparison to asympto-

matic controls.32 In accordance, current in vitro results showed

that cultured HVCs derived from patients with POP expressed

lower levels of TIMP2 and TIMP3 mRNA, in comparison to

HVCs derived from asymptomatic controls. These studies con-

firm the differential expression of the ECM-degrading proteins

and their inhibitors in POP-HVCs versus non-POP-HVCs,

showing genotypic and phenotypic differences in these cells,

which potentially may predispose them to different modes of

reaction to remodeling of the ECM. Altogether, our results

indicate that phenotypic changes that we and others detected

in vivo on the macro level in the prolapsed tissue are actually a

reflection of changes in a cellular level possibly due to

increased mechanical (stretch) or decreased hormonal (meno-

pause) stimulation.

Our study has limitations. It would be useful to compare our

in vitro results with in vivo studies; however, the size of the

tissue specimen limited us to use it only for primary cell deri-

vation. We also acknowledge that the premenopausal study

groups were not matched for parity and vaginal delivery, a

known factor in POP development. We speculate now that

repetitive trauma from delivery may affect the basement mem-

brane assembly/formation influencing their scaffolding charac-

teristics, which appears critical for the pelvic tissue strength. In

conclusion, we were able to derive a homogenous human vagi-

nal cell population, with similar attachment and proliferative

abilities on COL I, from patients with POP and from asympto-

matic control women. However, we detected key differences in

the biological characteristics of these cells, particularly in their

ability to produce ECM. Thus, we suggest that utilization of

cells from an anatomical site not related to pelvic tissue and not

affected by the prolapse may be a better alternative to mesh-

augmented reconstructive surgery of the pelvic floor in order to

generate sufficiently strong new tissue by replacing defective

cells with healthy autologous cells. Further research is neces-

sary to evaluate the effect of external stimuli mimicking risk

factors for POP on the cellular responses of HVCs.

Acknowledgements

The authors gratefully thank Ms Yaryna Rybak for her assistance in

the RT-PCR validation analysis. The authors would like to thank

Kufaishi et al 941

patients of the Urogynecology Division, Mount Sinai Hospital,

Toronto, for helping in tissues collection.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to

the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for

the research, authorship, and/or publication of this article: This study

was supported by a grant from the Department of Obstetrics and

Gynecology, Mount Sinai Hospital, Toronto, and Dean’s fund, Faculty

of Medicine, University of Toronto, Canada.

Supplemental Material

The online supplemental tables are available at http://rs.sagepub.com/

supplemental.

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