Manipulation of the early events in post-operative adhesion formation

150 Pages·2016·5.47 MB·English

Manipulation of the early events in post-operative
adhesion formation

A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy (PhD) in the Faculty of Biology, Medicine and
Health

2016

Akshita Patil

School of Biological Sciences
Table of Contents
Table of contents ................................................................................................. 2
List of Figures ...................................................................................................... 5
List of tables ........................................................................................................ 7
List of abbreviations ............................................................................................ 8
Abstract ............................................................................................................. 10
Declaration ........................................................................................................ 11
Copyright statement .......................................................................................... 12
Acknowledgements ........................................................................................... 13
Dedication ......................................................................................................... 13
1. Introduction ................................................................................................... 15
1.1 Peritoneal adhesions and mesothelial regeneration ........................................ 15
1.1.1 Incidence of peritoneal adhesion formation ............................................... 15
1.1.2 The peritoneum .......................................................................................... 16
1.1.3 Mesothelial cells ........................................................................................ 17
1.1.3.1 Mesothelial cell morphology ............................................................................. 17
1.1.3.2 Mesothelial cell function ................................................................................... 18
1.1.4 Peritoneal injury and repair ........................................................................ 19
1.1.4.1 Events in wound healing ................................................................................... 20
1.1.4.2 Peritoneal repair and mesothelial regeneration ................................................ 22
1.1.5 Pathogenesis of adhesion formation ......................................................... 23
1.1.5.1 Factors contributing to adhesion formation ...................................................... 24
1.1.5.2 Models of adhesion formation .......................................................................... 26
1.1.5.3 Prevention of adhesions ................................................................................... 27
1.2 Coagulation and fibrinolysis processes in adhesion formation ........................ 30
1.2.1 Coagulation cascade ................................................................................. 30
1.2.1.1 Extrinsic pathway .............................................................................................. 30
1.2.1.2 Intrinsic pathway ............................................................................................... 31
1.2.1.3 Common pathway ............................................................................................. 31
1.2.1.4 Key coagulation factors .................................................................................... 31
1.2.2 Fibrinolytic system ..................................................................................... 34
1.2.2.1 Plasminogen, plasminogen activators and plasminogen activator inhibitors .... 35
1.2.3 Role of coagulation and fibrinolysis in adhesion formation ........................ 36
1.2.3.1 Tissue factor and tissue factor pathway inhibitor .............................................. 37
1.2.3.2 Tissue plasminogen activator and plasminogen activator-1 ............................. 37
1.3 Fibroblasts and matrix remodelling during adhesion formation ....................... 38
1.3.1 Sources of fibroblasts ................................................................................ 39
1.3.2 Mesothelial-to-mesenchymal transition ..................................................... 40
1.3.3 Myofibroblast differentiation ....................................................................... 40
1.3.4 Collagen synthesis, remodelling and stabilisation ..................................... 42
1.3.5 Fibroblast and extracellular matrix interactions ......................................... 43
1.3.6 Myofibroblasts and ECM remodelling during adhesion formation ............. 44
1.4 Objectives ........................................................................................................ 46
1.5 Hypothesis and Aims ....................................................................................... 46
2. Materials and Methods .................................................................................. 47
2.1 Cell culture ....................................................................................................... 48
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2.1.1 Commercially available cells ...................................................................... 48
2.1.2 Primary human omental cell culture .......................................................... 49
2.1.3 Cell passaging ........................................................................................... 51
2.1.4 Freezing and thawing cells ........................................................................ 51
2.1.5 Cell seeding on fibrin film and fibrin gel ..................................................... 51
2.1.6 Addition of TGF-β1 to cultures ................................................................... 52
2.1.7 CRISPR transfection .................................................................................. 52
2.2 Sample harvesting ........................................................................................... 52
2.3 Assays ............................................................................................................. 53
2.3.1 MTS assay ................................................................................................. 53
2.3.2 Sircol assay ............................................................................................... 54
2.3.3 Enzyme-linked immunosorbent assay (ELISA) ......................................... 55
2.3.4 Protein assay ............................................................................................. 55
2.4 Western blot analysis ....................................................................................... 56
2.4.1 Densitometry .............................................................................................. 57
2.5 Molecular Biology ............................................................................................ 57
2.5.1 RNA extraction ........................................................................................... 57
2.5.2 Reverse transcription ................................................................................. 58
2.5.3 Selection of reference gene ....................................................................... 58
2.5.4 Real-time PCR ........................................................................................... 58
2.6 Flow cytometry ................................................................................................. 60
2.7 Immunofluorescent staining ............................................................................. 60
2.8 Statistical analysis ........................................................................................... 61
3. Establishment of an in vitro assay system to investigate the early fibrogenic
response of mesothelial cells and fibroblasts to fibrin substrates and exogenous
TGF-β1 ................................................................................................................ 62
3.1 Introduction ...................................................................................................... 63
3.2 Results ............................................................................................................. 65
3.2.1 MeT-5As morphology remained unaltered with TGF-β1 treatment and fibrin
film………. ............................................................................................................. 65
3.2.2 Increased metabolic activity of HPFs induced by TGF-β1 treatment and
fibrin film but not MeT-5As .................................................................................... 66
3.2.3 Increased α-SMA production by HPFs induced by TGF-β1 treatment and
fibrin film but not by MeT-5As ............................................................................... 68
3.2.4 Increased collagen production by HPFs induced by TGF-β1 treatment and
fibrin film but not with MeT-5As ............................................................................. 70
3.2.5 Increased α-SMA expression and production by HPFs induced by TGF-β1
treatment and fibrin film but not fibrin gel .............................................................. 72
3.2.6 Increased procollagens (type I and type III) expression and production by
HPFs induced by TGF-β1 treatment and fibrin ..................................................... 74
3.3 Discussion ....................................................................................................... 76
4. Epithelial-mesenchymal transition of primary omental cells in response to
fibrin substrates and exogenous TGF-β1 .......................................................... 79
4.1 Introduction ...................................................................................................... 80
4.2 Results ............................................................................................................. 82
4.2.1 Characterisation of isolated human omental cells ..................................... 82
4.2.1.1 Primary omental cell populations exhibit phenotypic variation ......................... 82
4.2.1.2 HOMCs express cytokeratin 8+18 and vimentin .............................................. 82
4.2.1.3 HOMCs express panel of mesothelial cell markers .......................................... 85
4.2.2 TGF-β1 treatment induced altered HOMCs morphology ........................... 86
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4.2.3 Upregulated expression of mesothelial-mesenchymal transition markers by
HOMCs induced by TGF-β1 treatment ................................................................. 87
4.2.4 Variable production of mesothelial-mesenchymal transition markers by
HOMCs induced by TGF-β1 .................................................................................. 92
4.2.5 HOMCs exhibit variable fibrogenic response with TGF-β1 treatment and in
the presence of fibrin ............................................................................................. 93
4.3 Discussion ....................................................................................................... 94
55. Coagulation and fibrinolytic response of omental cells and fibroblasts to
fibrin substrates and exogenous TGF-β1 .......................................................... 97
5.1 Introduction ...................................................................................................... 98
5.2 Results ............................................................................................................. 99
5.2.1 Human omental cells express coagulation factors, fibrinolytic factors and
matrix cross-linking enzymes in response to TGF-β1 and fibrin ........................... 99
5.2.1.1 Increased TF and variable TFPI mRNA expression by HOMCs ...................... 99
5.2.1.2 Variable tPA and PAI-1 mRNA expression by HOMCs .................................. 101
5.2.1.3 Unaltered LOX and increased TG2 mRNA expression by HOMCs ................ 103
5.2.2 Fibroblasts express coagulation factors, fibrinolytic factors and matrix
cross-linking enzymes in response to TGF-β1 and fibrin .................................... 105
5.2.2.1 Variable TF and TFPI mRNA expression by HPFs ........................................ 105
5.2.2.2 Variable tPA and increased PAI-1 mRNA expression by HPFs ..................... 107
5.2.2.3 Increased LOX and unaltered TG2 mRNA expression by HPFs .................... 109
5.2.3 HOMCs produce variable levels of TFPI in response to TGF-β1 ............ 111
5.2.4 HOMCs produce increased levels of PAI-1 in response to TGF-β1 ........ 112
5.2.5 Effect of CRISPR TFPI transfection on HOMCs ...................................... 113
5.3 Discussion ..................................................................................................... 115
6. Discussion ................................................................................................... 120
6.1 Overall findings .............................................................................................. 121
6.2 Future work .................................................................................................... 125
Bibliography .................................................................................................... 127

Final word count: 43,842

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List of Figures
Figure 1.1 Adhesion formation between loops of bowel……………………………………………15
Figure 1.2 Morphology of mesothelial cells…………………………………………………………..18
Figure 1.3 Phases of cutaneous wound healing…………………………………………………….20
Figure 1.4 Proposed origins of mesothelial cells during peritoneal repair……......………………22
Figure 1.5 Events leading to adhesion formation after trauma to the peritoneum……………….24
Figure 1.6 Extrinsic and intrinsic pathways of the coagulation cascade………………………….30
Figure 1.7 Fibrinogen and fibrin formation.……………………………………………………..……33
Figure 1.8 Process of epithelial-to-mesenchymal transition………………………………………..40
Figure 1.9 Stages of myofibroblast differentiation.…………………………………………………..41
Figure 2.1 Example of omental samples prior to enzymatic digestion..…………………………..49
Figure 2.2 Schematic showing cells seeded on tissue culture plastic and fibrin substrates…....51
Figure 2.3 Metabolic activity of MeT-5As on tissue culture plastic and fibrin film………….........53
Figure 2.4 Sircol assay standard curve using collagen reference standards…………………….54
Figure 3.1 TGF-β1 treatment does not induce morphological changes in MeT-5A cells on tissue
culture plastic or fibrin film….………………………………………………………………………….65
Figure 3.2 TGF-β1 treatment and fibrin film induced increased metabolic activity of HPFs but not
MeT-5As…...…………………………………………………..………………………………………...67
Figure 3.3 TGF-β1 treatment and fibrin film induced increased α-SMA production of HPFs but
not MeT-5As….……………………………………………..………………………………………......69
Figure 3.4 TGF-β1 treatment and fibrin film induced increased collagen production of HPFs but
not MeT-5As. ….…………………………………………………..……………………………………71
Figure 3.5 TGF-β1 treatment and fibrin induced increased α-SMA expression and production by
HPFs.….…………………………………………………..……………………………………….........73
Figure 3.6 TGF-β1 treatment and fibrin induced increased procollagens expression and
collagen production by HPFs…….……………………..……………………………………….........75
Figure 4.1 Photomicrographs of monolayers of HOMCs…………………………………………...82
Figure 4.2 HOMCs express cytokeratin 8+18 and vimentin…..……………………………………83
Figure 4.3 Flow cytometry analysis of HOMCs………………………………………………………85
Figure 4.4 TGF-β1 treatment induces morphological changes in HOMCs……………………….86
Figure 4.5 TGF-β1 treatment and fibrin induced MMT in patient 7 HOMCs………………...……88
Figure 4.6 TGF-β1 treatment and fibrin induced MMT in patient 8 HOMCs……………………...89
Figure 4.7 TGF-β1 treatment and fibrin induced MMT in patient 9 HOMCs……………………...90
Figure 4.8 Combined MMT data from three HOMCs isolates……………………………………...91
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Figure 4.9 TGF-β1 treatment and fibrin film induced MMT at protein level in HOMCs; combined
data from 3 patients..………………….………………………...……………………………………..92
Figure 4.10 Variable fibrogenic response of HOMCs treated with and without TGF-β1 on TCP,
FF and FG.…………………..……………………...…………………………………………………...93
Figure 5.1 Increased expression of TF and variable expression of TFPI by HOMCs treated with
TGF-β1 for 24h………………………………………………………………………………………...100
Figure 5.2 Variable expression of tPA and PAI-1 by HOMCs treated with TGF-β1 for 24h…..102
Figure 5.3 Unaltered expression of LOX and increased expression of TG2 by HOMCs treated
with TGF-β1 for 24h…………………………………………………………………………………...104
Figure 5.4 Variable expression of TF and TFPI by HPFs treated with TGF-β1 for 24h………..106
Figure 5.5 Variable expression of tPA and increased expression of PAI-1 by HPFs treated with
TGF-β1 for 24h.…………………………………………………………………………...…………...108
Figure 5.6 Increased expression of LOX and variable expression of TG2 by HPFs treated with
TGF-β1 for 24h………………………………………………………………………………………...110
Figure 5.7 TFPI production by HOMCs was highly variable in response to 24h treatment with
TGF-β1…………………………………………………………………………………......................111
Figure 5.8 PAI-1 production by HOMCs on TCP and FF was highly upregulated in response to
24h treatment with TGF-β1…………………………………………………………………………...112
Figure 5.9 HOMCs on TCP after CRISPR transfection with either control or TFPI plasmid and
TGF-β1 treatment for 24h………………………………………………………………………….....113
Figure 5.10 Variable expression of TFPI by HOMCs after CRISPR transfection with either
control or TFPI plasmid and TGF-β1 treatment for 24h and 48h…………………………………114
Figure 5.3.1 Balance between coagulation cascade factors and fibrinolytic enzymes activity..118
Figure 6.1.1 Proposed roles of mesothelial cells and fibroblasts during fibrin remodeling and
peritoneal adhesion formation………………………………………………………………………..124

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List of tables
Table 1.1 Examples of commonly used barriers with recommended type of surgery for use and
limitations………………………………………………………………...………………………………28
Table 1.2 Examples of coagulation factors involved in fibrin clot formation………………………32
Table 1.3 Fibrinolytic factors, activators and inhibitors……………………………………………..35
Table 2.1 Supplements in SupplementMix (PromoCell) ………………………………...…………48
Table 2.2 Patient demographics for human primary fibroblasts…………………………………...48
Table 2.3 Patient demographics for human omental cells…………………………………............50
Table 2.4 Antibody details for western blot…………………………………...................................57
Table 2.5 Primer Sequences for real-time PCR……………………………………………………..59
Table 5.3.1 Summary of mRNA expression by HOMCs and HPFs in response to TGF-β1 and
fibrin substrates………………………………….…………………………………………………….117

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List of abbreviations
Abbreviation Definition
APS ammonium persulphate
ATCC American type culture collection
BM bone marrow
BMI body mass index
BSA bovine serum albumin
COL1A2 procollagen type I, α2 chain
COL3A1 procollagen type III, α1 chain
CRISPR clustered regularly interspaced short palindromic repeats
DMEM Dulbecco’s modified Eagle’s medium
DMSO dimethyl sulfoxide
ECL electrochemiluminescence
ECM extracellular matrix
EDTA ethylenediaminetetra acetic acid
EGF epidermal growth factor
ELISA enzyme-linked immunosorbent assay
EMT epithelial-to-mesenchymal transition
FACS fluorescence-activated cell sorting
FBS fetal bovine serum
FF fibrin film
FG fibrin gel
FSP-1 fibroblast-specific protein-1
GAPDH glyceraldehyde-3-phosphate dehydrogenase
HBSS Hank’s buffered saline solution
HOMCs human omental cells
HPFs human pulmonary fibroblasts
ICAM-1 intracellular adhesion molecule-1
Il-β1 interleukin-β1
LAP latency associated peptide
LLC large latency complex
LOX lysyl oxidase
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M199 Medium 199
MET mesenchymal-to-epithelial transition
MMT mesothelial-to-mesenchymal transition
mRNA message ribonucleic acid
MSCs mesenchymal stem cells
MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt]
no-RT no-reverse transcriptase
p38MAPK p38 mitogen-activated protein kinase
PAI-1/2 plasminogen activator inhibitor-1/2
PCR polymerase chain reaction
RGD Arginine-glycine-aspartic
rTFPI recombinant tissue factor pathway inhibitor
SDS sodium dodecyl sulphate
SEM standard error of mean
TCP tissue culture plastic
TF tissue factor
TFPI tissue factor pathway inhibitor
TG2 tissue transglutaminase
TGF-β1 transforming growth factor-β1
tPA tissue plasminogen activator
uPA urokinase plasminogen activator
vWF von Willebrand factor
WT1 Wilm’s tumor 1
α-SMA α-smooth muscle actin

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Abstract
Abstract of a thesis entitled ‘’Manipulation of the early events in post-operative
adhesion formation,’’ submitted by Akshita Patil for the degree of Doctor of
Philosophy (PhD) to the University of Manchester.
Peritoneal adhesions are fibrous bands of tissue joining normally separated
organs and occur frequently as a side effect of abdominal surgery. Despite
being a major cause of infertility, chronic pelvic pain and bowel obstruction,
there are currently no adequate methods of therapy or prevention. Post-
operative adhesions are known to develop from fibrin clots deposited between
damaged serosa in combination with insufficient fibrinolytic capacity. However,
the contribution of surface mesothelial cells to the regulation of fibrin turnover
and subsequent scar formation is not well understood. This thesis addresses
the hypothesis that mesothelial cells actively participate in adhesion formation
by promoting coagulation, inhibiting fibrinolysis and undergoing epithelial-
mesenchymal transition (EMT) to upregulate extracellular matrix (ECM)
deposition. Primary human omental cells, in addition to primary fibroblasts for
comparison, were incorporated in an in vitro assay system aimed at identifying
potential molecular biomarkers or therapeutic targets for the prevention of
adhesion formation.
We used a combination of exposure to the pro-fibrotic growth factor, TGF-β1,
and culturing on a fibrin film or gel in vitro to mimic the post-injury environment
during peritoneal repair. We identified an exaggerated TGF-β1-induced
fibrogenic response by human pulmonary fibroblasts (HPFs) but not by a
transformed mesothelial cell line, MeT-5A, suggesting primary mesothelial cells
were preferable. Human omental cells (HOMCs) were isolated by protease
digestion and characterised using a panel of mesothelial markers. TGF-β1 and
fibrin substrates induced EMT in HOMCs from three patients however,
expression of α-SMA and procollagens was highly variable. Furthermore,
culturing cells on a fibrin gel enhanced the effects observed with TGF-β1 in
HOMCs and promoted a hypercoagulable state (decreased tissue factor
pathway inhibitor, TFPI) and reduced fibrinolysis (increased plasminogen
activator inhibitor-1, PAI-1). Interestingly, HOMCs also expressed matrix cross-
linking factor, tissue transglutaminase (TG2), in response to TGF-β1 when
grown on a fibrin gel in a similar manner to HPFs suggesting they may be
involved in the stabilisation of a newly formed adhesion. HPFs also promoted
coagulation and inhibited fibrinolysis however the response appeared to be
dependent on TGF-β1 rather than fibrin.
In summary, this thesis indicates that fibrin is a key mediator in modulating
HOMCs behaviour during the post-injury response and adhesion formation.
High patient variation in the fibrogenic response suggests HOMCs undergo
EMT and whilst the transitioned cells do not always contribute to excess
collagen deposition, they may be involved in the stabilisation of newly deposited
ECM. HOMCs potentially play a dual role during peritoneal remodeling by
promoting coagulation and regulating fibrin persistence. TFPI may serve as a
novel potential molecular biomarker or therapeutic target for the prevention of
post-operative adhesions.

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