ANGIOTENSIN II REGULATION OF EXTRACELLULAR MATRIX PROTEINS: LYSYL OXIDASE AND OSTEOPONTIN Open Access

Remus, Ebony Renee Washington (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/70795853r?locale=en
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Abstract


Abstract

ANGIOTENSIN II REGULATION OF EXTRACELLULAR MATRIX PROTEINS:
LYSYL OXIDASE AND OSTEOPONTIN

By

Ebony Washington Remus
The renin angiotensin system has been implicated in the pathogenesis of cardiovascular disease through its primary effector molecule Angiotensin II (AngII). Ang II is multifunctional hormone that acts as a potent vasoconstrictor, mediating its action on cells within the vasculature primarily through the angiotensin I receptor. Vascular smooth muscle cells (VSMCs) are a prominent cell type within the vasculature on which Ang II exerts its effects. Ang II bound to its receptor initiates intracellular signaling events that lead to hypertrophy, hyperplasia, migration, adhesion and an increased oxidative state of vascular smooth muscle cells. These changes are mediated in part changes to the extracellular matrix environment, leading to the progression of atherosclerosis and abdominal aortic aneurysms. Within this thesis, we will explore Ang II regulation on two important extracellular matrix components implicated in vessel stability: lysyl oxidase and osteopontin. The lysyl oxidase family of extracellular matrix enzymes crosslink collagen and elastin, thereby enhancing vessel wall stability. Osteopontin is an extracellular matrix protein that facilitates adhesion, migration and inflammation in vascular smooth muscle cells. While lysyl oxidase contributes to the stability of the vessel wall, osteopontin represents an extracellular matrix protein that decreases vessel integrity. This thesis aims to understand the regulation of these extracellular matrix proteins using both in vivo disease models and primary cell culture. The results from these experiments support the notion that lysyl oxidase stabilizes vessel structure in a mouse model of abdominal aortic aneurysms; however Ang II does not directly regulate its activity or expression in vascular smooth muscle cells. Conversely, Ang II does increase osteopontin expression in a mouse model of hypertension that precedes atherosclerosis formation. Furthermore, by uncovering a previously unreported translation dependent induction of osteopontin protein expression were able to identify a novel mechanism for inhibiting Ang II induced osteopontin expression using microRNAs.

Table of Contents



Table of Contents

Figure legend ...................................................................................................... x
Abbreviations ................................................................................................... xiv
Chapter 1: Background ...................................................................................... 1
Angiotensin II and Cardiovascular Disease ................................................................2
Hypertension ......................................................................................................................................... 4
Atherosclerosis ..................................................................................................................................... 5
Abdominal Aortic Aneurysms ............................................................................................................. 8
Angiotensin II ................................................................................................................ 11
Renin Angiotensin System ............................................................................................................... 11
In vitro angiotensin II signaling ......................................................................................................... 12
Migration and adhesion of vascular smooth muscle cells ............................................................ 15
Extracellular Matrix ...................................................................................................... 19
Lysyl Oxidase and Lysyl Oxidase Like Isoenzymes ..................................................................... 19
Osteopontin ......................................................................................................................................... 25
Scope of Dissertation .................................................................................................. 27
Chapter 2: The role of Angiotensin II on Lysyl Oxidase Family Members in
the regulation of Abdominal Aortic Aneurysms and in cultured Vascular
Smooth Muscle Cells. ...................................................................................... 29

Part 1. The Role of Lysyl Oxidase Family Members in the Stabilization of
Abdominal Aortic Aneurysms .................................................................................... 30

Introduction ......................................................................................................................................... 32
Methods ............................................................................................................................................... 34
Animals ........................................................................................................................................... 34
Immunostaining .............................................................................................................................. 35
Quantitative Real Time - PCR ..................................................................................................... 35
Western Blot ................................................................................................................................... 38
Additional Reagents ...................................................................................................................... 38
Statistical Analysis ......................................................................................................................... 38
Results ................................................................................................................................................. 39
LOXL3 mRNA expression is significantly upregulated in the aortas of Ang II treated ApoE-/- mice fed a high fat diet. .............................................................................................................. 39
LOX/LOXL proteins are expressed in the aortic wall ............................................................... 44
LOX/LOXL activity protects against abdominal aortic aneurysm formation .......................... 46
LOX/LOXL activity protects against Ang II induced atherosclerotic lesion formation .......... 49
Discussion ........................................................................................................................................... 55
Part II. The Regulation of LOX/LOXL family member expression in Vascular
Smooth Muscle Cells by Angiotensin II .................................................................... 59

Introduction ......................................................................................................................................... 60
Methods ............................................................................................................................................... 62

viii

Cell culture ...................................................................................................................................... 62
Quantitative Real Time-PCR........................................................................................................ 63
Western blotting ............................................................................................................................. 64
LOX/LOXL Activity Assay ............................................................................................................. 64
Results ................................................................................................................................................. 66
Ang II has variable effects of LOX/LOXL family members at the mRNA level in VSMCs .. 66
Ang II has no effect of LOX/LOXL protein expression in cultured VSMCs ........................... 71
Ang II does not change LOX/LOXL activity ............................................................................... 73
Discussion ........................................................................................................................................... 75
Chapter 3: The regulation of miRNAs by Angiotensin II in VSMCs .............. 81
Introduction ......................................................................................................................................... 83
Methods ............................................................................................................................................... 88
Cell culture ...................................................................................................................................... 89
microRNA Array and Analysis ..................................................................................................... 89
miRNA Extraction and qRT-PCR Validation .............................................................................. 91
Overexpressing and Antagonizing mir26b ................................................................................. 92
Flow Cytometry .............................................................................................................................. 93
Protein Isolation ............................................................................................................................. 93
Western blotting ............................................................................................................................. 93
Statistical Analysis ......................................................................................................................... 94
Results ................................................................................................................................................. 96
Ang II modulates several miRNAs in VSMCs ........................................................................... 96
Several miRNAs are predicted to target LOXL1 and LOXL2 .................................................. 99
Ang II has differential effects on the expression of miRNA processing enzymes drosha
and dicer ....................................................................................................................................... 103
miRNAs and potential mRNA targets ....................................................................................... 106
Discussion ......................................................................................................................................... 109
Chapter 4. mir181a Protects Against Ang II-Induced Osteopontin
Expression in Vascular Smooth Muscle Cells ............................................. 111

Introduction ....................................................................................................................................... 113
Methods ............................................................................................................................................. 115
Animals ......................................................................................................................................... 115
Cell Culture ................................................................................................................................... 115
Quantitative Real Time-Polymerase Chain Reaction ............................................................. 116
Western blotting and ELISA ....................................................................................................... 116
miRNA isolation ........................................................................................................................... 117
mir181a Overexpression ............................................................................................................ 117
Adhesion Assay ........................................................................................................................... 118
Statistical Analysis ....................................................................................................................... 118
Results ............................................................................................................................................... 119
Ang II increases OPN protein expression in vivo and in vitro ............................................... 119
Early increases in Ang II-induced OPN expression are transcriptionally independent ..... 123
Ang II decreases mir181a expression ...................................................................................... 125
Overexpression of mir181a inhibits Ang II induced OPN protein expression ..................... 127
mir181a overexpression inhibits Ang II-mediated VSMC Adhesion .................................... 130



Discussion ......................................................................................................................................... 135
Chapter 5: Summary and Future Directions................................................. 142
References ...................................................................................................... 152










x

Figure Legend
FIGURE 1. THE STRUCTURE OF AN ARTERY. .................................................................................. 2
FIGURE 2. THE PROGRESSION OF ATHEROSCLEROSIS. ............................................................ 5
FIGURE 3. INFILTRATION OF LEUKOCYTES INTO THE VASCULAR WALL. .............................. 7
FIGURE 4. ANG II INTRACELLULAR SIGNALING EVENTS ............................................................ 14
FIGURE 5. ADHESION AND MIGRATION ........................................................................................... 18
FIGURE 6. THE LYSYL OXIDASE FAMILY ........................................................................................ 22
FIGURE 7. ACHIEVING VESSEL STABILITY ...................................................................................... 24
FIGURE 8. OSTEOPONTIN HAS EXTENSIVE POSTTRANSLATION MODIFICATIONS. .......... 25
FIGURE 9. ANG II INFUSION LEADS TO INCREASED SYSTOLIC BLOOD PRESSURES. ...... 41
FIGURE 10. LOX/LOXL FAMILY MEMBER EXPRESSION 7 DAYS AFTER ANG II INFUSION IN
THE PRESENCE OF A HIGH FAT DIET. ...................................................................................... 42
FIGURE 11. LOX/LOXL FAMILY MEMBER PROTEIN EXPRESSION 7 DAYS AFTER ANG II
INFUSION IN THE PRESENCE OF A HIGH FAT DIET. ............................................................. 43
FIGURE 12. LOCALIZATION OF LOX/LOXL PROTEINS IN AORTIC TISSUE FROM APOE-/-
MICE AFTER 8 WEEKS. .................................................................................................................. 45
FIGURE 13. EFFECT OF Î'-APN ON AAA INCIDENCE AND ATHEROSCLEROTIC LESION
AREA IN APOE-/- MICE ON A HIGH FAT DIET WITH ANG II INFUSION. .............................. 47
FIGURE 14. EFFECT OF Î'-APN ON AAA INCIDENCE AND ATHEROSCLEROTIC LESION
AREA IN APOE-/- MICE ON A HIGH FAT DIET WITH ANG II INFUSION. .............................. 48
FIGURE 15. EFFECT OF Î'-APN ON AAA INCIDENCE AND ATHEROSCLEROTIC LESION
AREA IN C57BL/6 MICE ON A HIGH FAT DIET WITH ANG II. ................................................. 51
FIGURE 16. EFFECT OF Î'-APN ON AAA INCIDENCE AND ATHEROSCLEROTIC LESION
AREA IN C57BL/6 MICE ON A HIGH FAT DIET WITH ANG II. ................................................. 52
FIGURE 17. EFFECT OF Î'-APN ON AAA INCIDENCE AND ATHEROSCLEROTIC LESION
AREA IN BALBC MICE ON A HIGH FAT DIET WITH ANG II. ................................................... 53

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