Deletion of Tak1 in Fibroblast-specific Protein 1 Positive Cells Reduces Tendon Adhesion ...

171 Pages·2014·6.43 MB·English

Deletion of Tak1 in Fibroblast-specific Protein 1 Positive
Cells Reduces Tendon Adhesion Formation Via Reduced
Myofibroblast Activation

by
Shanshan Shi

Submitted in Partial Fulfillment of the
Requirements for the Degree
Doctor of Philosophy

Supervised by
Professor Regis James O’Keefe

Department of Pathology and Laboratory Medicine
School of Medicine and Dentistry
University of Rochester
Rochester, NY

2015
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Biographical Sketch
The author attended Ohio Wesleyan University where she graduated with a
Bachelor of Arts degree in Genetics in 2008. She began her study in Pathology and
Laboratory Medicine Program in University of Rochester Medical Center in 2008 and
received her Master of Science degree in 2011. She pursued her research in Center for
Musculoskeletal Research under the direction of Dr. Regis J. O’Keefe.

List of Publications

Fabrication of three-dimensional porous scaffold based on collagen fiber and bioglass
for bone tissue engineering.
Shi S, Long T, Yang J, Guo Y, Ke Q, Zhu Z. J. Biomed. Mater. Res. Part B Appl.
Biomater. 2014.

TIMP2 deficient mice develop accelerated osteoarthritis via promotion of angiogenesis
upon destabilization of the medial meniscus.
Mi M, Shi S, Li T, Holz J, Lee YJ, Sheu TJ, Liao Q, Xiao T. Biochem Biophys Res
Commun. 2012 Jun 29;423(2):366-72. 1.

Impaired angiogenesis during fracture healing in GPCR kinase 2 interacting protein-1
(GIT1) knock out mice.
Yin G, Sheu TJ, Menon P, Pang J, Ho HC, Shi S, Xie C, Smolock E, Yan C, Zuscik
MJ, Berk BC.
PLoS One. 2014 Feb 19;9(2):e89127.
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Acknowledgements
I owe deepest gratitude to my advisor Dr. Regis O’Keefe for his generous
guidance, encouragement, and unbelievable patience throughout my years of graduate
study. His intellectual rigor and gift for conceptualization continue to be an exceptional
inspiration to me.
I am thankful to my committee members, Dr. Hani Awad, Dr. Michael Zuscik,
Dr. Catherine Ovitt, and Dr. Margot Mayor-Proschel, for their scholarly input and
encouraging feedback. Their provocative questions pushed me to pursue my own ideas
to their limits.
This current thesis would not have been possible without innumerous enabling
people. I would like to thank, Dr. Jennifer Jonason, Dr. Tzong-jen Sheu, and Dr. Jie
Shen, from whom I have gleaned insights to propel my study. I would also like to thank
Sarah Mack, Kathy Malby and Jayne Gavrity for their technical expertise. Center for
Musculoskeletal Research offered me a congenial environment to nurture my growth as
a student of mind and science. It was a great honor to be in the cohort of brilliant minds
and work along side the liked minded scientists.
I have had privilege of faith and friendship from many people, who encouraged
my pursuit for academic excellence. I would like to thank Dr. Teng Long, for his
relentless support and reassurance; Dr. Danielle Hamill, who cultivated my appreciation
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for the beauty of science and the coherence of theoretical work; Neha, with whom I
shared my formative years, you are a constant inspiration behind my endeavor; Ahmad,
for sharing my passion for the art of cognitive work, and stimulated my intellectual
creativity.
I cannot adequately express my gratitude to my parents for their unconditional
supports over the years. Their acceptance of my choices and accommodation of my
goals are the greatest gifts they have bestowed upon me.
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Abstract
The clinical outcomes of flexor tendon injury repairs are frequently complicated
by extensive scar formation that impairs hand functions. Fibrotic scars contain
myofibroblasts that produce excessive extracellular matrix proteins such as collagen I
and collagen III. TGFβ, a potent activator of fibroblasts has been implicated in tendon
adhesion formation. Though studies targeting components of the TGFβ signaling
pathway have shown effective reduction in adhesion formations, little progress has been
made in translating these findings from bench to bedside due to our limited knowledge
of the precise role of TGFβ signaling during tendon healing. TGFβ regulates target
genes through multiple mechanisms. In particular, TGFβ-Activated Kinase 1 (TAK1), a
MAP Kinase Kinase Kinase which is a component of the TGFβ receptor complex, can
activate p38 MAP Kinase and JNK. In current study, we employed both in vitro and in
vivo approaches to investigate the role of TAK1 in regulation of tendon injury repair.
Specifically, we utilized a murine model of primary tendon repair in which Tak1 is
conditionally deleted in fibroblasts using the Fsp1-Cre transgene with the Tak1 floxed
allele. In vitro, we investigated the role of TAK1 in regulation of fibroblast
proliferation, migration, contraction, and collagen production. We found that upon
injury, Fsp1-lineage cells populated the reparative tissues at the injury site while native
tendon tissue did not have any Fsp1-positive cells. Histology showed reduced scar
formation in Fsp1cre;Tak1fx/fx mice during tendon healing beginning at day 7.
Consistent with histology, Fsp1cre;Tak1fx/fx mice had improved range of motion in
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tendon repairs and reduced expression of collagen I and III throughout the repair
process. Tendon repairs in Fsp1cre;Tak1fx/fx mice also had reduced expression of
Acta2, a marker for myofibroblasts, suggesting that TAK1 is involved in fibroblast
activation. Inhibition of TAK1 reduces the transformation of fibroblasts to
myofibroblasts following TGFβ1 treatment of fibroblasts in vitro. Tak1 deletion in
TGFβ1-treated fibroblasts resulted in decreased expression of Acta2. Several other key
functions of activated myofibroblasts, such as proliferation, migration, contraction, and
collagen production were also inhibited by the loss of Tak1. Additionally, we found
that addition of TGFβ1 to fibroblast cell cultures enhanced the activation and nuclear
accumulation of β-catenin. In contrast, Tak1 deletion in MEF cultures resulted in
decreased myofibroblast gene expression and reduced activation of Wnt/β-catenin
signaling. These analyses demonstrate that TAK1 is a critical mediator in tendon
adhesion formation and fibroblast to myofibroblast conversion. Inhibition of Tak1
could serve as novel approach for therapeutic intervention for the treatment of adhesion
formation. TAK1 links TGFβ and β-catenin signaling during the development of
fibrotic responses after tendon injury.
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Contributors and Funding
Sources
This work was supported by a dissertation committee consisting of: Dr. Regis J.
O’Keefe (advisor), Dr. Hani A. Awad of the Department of Bioengineering, Dr.
Catherine Ovitt and Dr. Margot Mayer-Proschel of the Department of Genetics, and Dr.
Michael Zuscick of the Department of Orthorpeadics.
The analyses depicted with human patient sample were conducted in part by Dr.
Teng Long. The author completed all other work in the dissertation independently.
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Table of Contents

List of Tables ................................................................................................................ xiii
List of Figures .............................................................................................................. xiv
List of Abbreviations ................................................................................................... xvi
1 Introduction .............................................................................................................. 1
1.1 Structure and biology of tendon ....................................................................... 2
1.1.1 Tendon structure and function ...................................................................... 2
1.1.2 Tendon development ..................................................................................... 3
1.1.3 Anatomy of flexor tendon in the hand .......................................................... 6
1.2 Flexor tendon injury and complications.......................................................... 7
1.2.1 Flexor tendon injury and treatment ............................................................... 7
1.2.2 Clinical complications of flexor tendon repair.............................................. 9
1.2.3 Murine model of flexor tendon healing ...................................................... 10
1.3 Biology of tendon healing ................................................................................ 12
1.3.1 Stages of tendon healing ............................................................................. 12
1.3.2 Cellular mechanisms during tendon healing ............................................... 13
1.3.3 Role of myofibroblast in fibrosis ................................................................ 15
1.3.4 Transforming growth factor β signaling ..................................................... 16
1.4 Scientific questions and structure of current thesis ..................................... 17
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2 Deletion of TAK1 in fibroblast-specific protein 1 lineage cells reduces scar
formation of flexor tendon injury ............................................................................... 30
2.1 Abstract ............................................................................................................ 31
2.2 Introduction ..................................................................................................... 32
2.3 Material and Methods ..................................................................................... 34
2.3.1 Flexor tendon repair surgeries ..................................................................... 34
2.3.2 Generation of transgenic mice .................................................................... 35
2.3.3 Flow cytometry analysis ............................................................................. 35
2.3.4 RNA isolation, cDNA reverse transcription and realtime qPCR ................ 36
2.3.5 Histology and immunohistochemistry ........................................................ 37
2.3.5.1 Histology ............................................................................................... 37
2.3.5.2 Immunohistochemistry .......................................................................... 38
2.3.6 Microscopy .................................................................................................. 38
2.3.7 Biomechanics studies .................................................................................. 38
2.3.7.1 Adhesion testing .................................................................................... 38
2.3.7.2 Tensile strength testing .......................................................................... 39
2.3.8 Statistics ...................................................................................................... 40
2.4 Results ............................................................................................................... 40
2.4.1 Increases in phosphorylated TAK1 in tenolysis patient samples ................ 40
2.4.2 Fsp1 is expressed in multiple organs in mouse. .......................................... 40
2.4.3 Deletion of Tak1 in Fsp1-lineage cells significantly reduces the overall
expression of Tak1 mRNA in healing tendon. ....................................................... 41
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2.4.4 Fsp1-specific deletion of Tak1 results in reduced healing callus during
tendon healing ......................................................................................................... 42
2.4.5 Deletion of Tak1 in Fsp1-lineage cells results in improved range of motion
in healing tendon ..................................................................................................... 43
2.4.6 Reduced collagen expressions in the healing tendon of Fsp1cre;Tak1fx/fx
mice 44
2.4.7 Deletion of Tak1 results in changes of the expression of MMP2 and MMP9
45
2.4.8 Deletion of Tak1 results in changes of the expression of several pro-fibrotic
genes 46
2.5 Conclusion and Discussion .............................................................................. 47
3 Deletion of TAK1 inhibits myofibroblast activation by TGF-β ......................... 71
3.1 Abstract ............................................................................................................ 72
3.2 Introduction ..................................................................................................... 73
3.3 Material and methods ..................................................................................... 75
3.3.1 Cell cultures ................................................................................................ 75
3.3.2 RNA isolation, cDNA transcription and realtime PCR .............................. 75
3.3.3 Collagen gel contraction ............................................................................. 75
3.3.4 Scratch assay ............................................................................................... 76
3.3.5 BrdU Elisa and Caspase 3/7 Luminescence assay ...................................... 76
3.3.6 Immunohistochemistry and immunofluorescence ...................................... 76
3.4 Results ............................................................................................................... 77