Presentation at British Menopause Society Annual Scientific Meeting, July 2006 - Dr Ayman Ewies

1. 1
Effect of Mechanical Stretch & Levormeloxifene on
Gene Expression and Actin Morphology of Cardinal
Ligament Fibroblasts
Ayman A. A. Ewies, MBChB, MSc, MROCG, MD
Consultant in Obstetrics and Gynaecology
The Ipswich Hospital NHS Trust
July 2006
British Menopause Society Annual Meeting

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Background
 Failure of ligamentous support
of the genital tract to resist
intra-abdominal pressure is the
underlying cause for pelvic
organ prolapse (POP).
 However, the nature of
molecular response of pelvic
supportive structures remains
unknown.

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Background – Hypoestrogenic State
 The high incidence of POP observed in postmenopausal
women suggested an aetiological role for the
hypoestrogenic state.
 This role was further elucidated by the observation of
“Drug-Induced POP”!!
Can it happen?

4. 4
Background – Drug Induced Prolapse
 2 selective oestrogen receptor modulators “SERMs”:
-Levormeloxifene
-Idoxifene
 The clinical trials used them in postmenopausal women
to treat & prevent osteoporosis were discontinued in
late 1990s because of ↑ incidence of:
-POP
-Urinary incontinence

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Background – There is no in vitro model to study POP?
 “The Stretch Model” was developed to
explore the mystery of POP.
 The assumption being:
In vitro long duration mechanical stretch of primary
culture cardinal ligament fibroblasts grown on collagen
matrix
=
The chronic stretch that the fibroblasts, of the prolapsed
ligaments, are exposed to In vivo

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Stretch Experiment
12 hourly 4 Days
Chronic cyclical mechanical stretch:
• The cells were exposed to cycles of: 21% stretch & 0% relaxation
• 12 hours each
• 4 days.
-BioFlex® plates with flexible Collagen-I coated membrane bottoms.
-The Flexercell Stress Unit.
-Plates were kept in the same incubator at 370C and humedified atmosphere with 5% CO2
.

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Hypothesis - 1
 The expression of genes coding for proteins involved in
maintaining the cellular integrity would be altered in cases of
POP.
 The changes in the gene expression of cardinal ligament
fibroblasts in primary culture in response to stretch, using
cDNA microarray technology.
To test this hypothesis, I examined

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Genes Alterations (n =3)
 6 mechano-responsive genes involved in the regulation of
actin cytoskeleton remodelling:
1. ↑ Phosphatidyl inositol-4-phosphate 5-kinase (PIP5K1C).
2. ↑ The human signal-induced proliferation associated gene-1
(SIPA-1).
3. ↑ TNFRSF1A-associated via death domain (TRADD).
4. ↑ Deoxyribonuclease 1-like 1 (DNase 1-L1).
5. ↑ Matrix metalloproteinase-20 (MMP-20).
6. ↓ Transforming growth factor-β3 (TGF-β3).

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Actin Filaments
-Enables cells to resist tractional forces, and maintain their shape.
-Exist in bundles or networks (The larger are called stress fibres).
-Dynamic structures (the rapid conversion between polymerised
filamentous (F) and globular monomeric (G) forms facilitates the
dynamic changes of cell shape).

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Hypothesis - 2
1. The mechanical strain of cardinal ligaments, e.g. in cases of increased intra-abdominal pressure,
could cause POP by disrupting the configuration of the actin cytoskeleton of the fibroblasts.
2. SERMs induced POP in healthy asymptomatic women by the same mechanism.
3. Oestrogen would have an ameliorating effect.
 The effect of mechanical stretch, levormeloxifene, and 17β-oestradiol on the morphology of the F-
actin of those fibroblasts grown in primary cultures.
To test this hypothesis, I examined

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Actin Experiment (n = 8)
Stretch No Stretch
17β-oestradiol 10-6M 17β-oestradiol 10-6M
17β-oestradiol 10-9M 17β-oestradiol 10-9M
Levormeloxifene 10-6M Levormeloxifene 10-6M
Levormeloxifene 2 X 10-7M Levormeloxifene 2 X 10-7M
17α-oestradiol 10-6M 17α-oestradiol 10-6M
Ethanol Ethanol
 F-actin Texas Red®-X Phalloidin
Treatment
f
Fluorescent Staining

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Results
 The percentage of cells with abnormal actin configuration was
significantly higher in fibroblasts:
-Subjected to stretch when compared with the static model
(P=0.0001), irrespective of treatment.
-Treated with both doses of Levormeloxifene when compared
with ethanol (P=0.0001), irrespective of stretch.
 There were no significant differences between oestradiol and ethanol.
Nonetheless, it significantly (P=0.02) increased the rate of fibroblast
proliferation.

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Results - Effect of Stretch and levormeloxifene on Actin Morphology
0
1
2
3
4
5
6
7
8
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10
1
S v NS
Ratios%and95%CIforthe%ofcellswithabnormalactin
17-β E 10-6 v Ethanol 17-β E 10-9 v Ethanol L 10- 6 v Ethanol L 2x10-7 v Ethanol 17-α E 10-6 v Ethanol
The estimated ratios and 95% confidence intervals for the percentages of cells with abnormal
actin morphology. The plot represents the comparison between the effect of stretch (regardless
of treatment) versus no stretch, and the comparison between the effect of individual treatment
versus ethanol. S = Stretch NS = No Stretch E = Oestradiol L = Levormeloxifene *p=0.0001
*
*
*

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Results – Normal Actin Morphology
F-actin appeared as well defined, brightly fluorescent, discrete filaments

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Results – Normal Actin Morphology
Stress fibres run from one edge of the cell to the other

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Results – Normal Actin Morphology
Sometimes continue into very long filopodia

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Results – Normal Actin Morphology
Sometimes crossed one another

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Results – Abnormal Actin Morphology
1- partial disintegration of the
filaments into small spots and
micro-aggregates especially at
the cell periphery
(the most frequent abnormality)

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Results – Abnormal Actin Morphology
2- Microfilaments dissolution

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Results – Abnormal Actin Morphology
3- Microaggregates coalescence &
alteration of the cell shape

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Results – Abnormal Actin Morphology
4- Diminished number of stress fibres

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Results – Abnormal Actin Morphology
5- Alteration of cell shape into arborized configuration

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Concluding Remarks
 Both mechanical stretch and levormeloxifene,
independently, destroyed the actin cytoskeleton of
the fibroblasts.
 Oestrogen treatment did not prevent or reverse the
damage, and failed to maintain the integrity of
fibroblasts.
 Nonetheless, oestrogen significantly increased the
rate of fibroblast proliferation, suggesting a
beneficial role in healing process.

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Concluding Remarks
Stretch or Levormeloxifene
Abnormal configuration of F-actin
Disturbance of the integrity of fibroblasts
Interference with the quality of attachment to ECM
Failure of ligamentous support
POP
POP

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Concluding Remarks
 The use of this model may represent an exciting starting
point to contribute to the understanding of the
pathogenesis of POP.
 It is also an important example of translation of clinical
observation into basic sciences research & important
laboratory findings were documented.

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Acknowledgment
 Dr. Mona El-Shafie (The Histologist/Morphologist)
 Prof. John Thompson (The Statistician)
 Dr. Adrian Stanley (Lecturer, helped with the stretch)
 Dr. Jin Lee (Post doc, helped with RNA extraction)
 Dr. Jerry Styles (Post doc, cDNA Microarray)
 Mr. Farook Al-Azzawi (The Supervisor)

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Thank You
Thank You
Thank You

31. 31
Pelvic Support
The principal support of the uterus arises from the dynamic actions of:
-Levator ani muscle
-Uterosacral-cardinal ligament complex

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Materials & Methods – choice of doses
 The two doses of 17β-oestradiol represented
the low physiological and the high
pharmacological doses as previously described
in the literature (Liu et al., 1997, Yu et al., 1999).
 The two doses of levomeloxifene represented
the highest and lowest serum levels in women
treated with this drug to prevent osteoporosis
(Novo Nordisk' study protocol).

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Materials & Methods - Specimens
 8 cardinal ligament samples
 Abdominal hysterectomy
 Premenopausal Caucasian women
 No history of POP
 The supra-cervical area was cut
(5 mm thick slices) with a scalpel to
obtain the medial ends of the
ligament