Connective Tissue Formation in Wound Healing - E-thesis

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ABBREVIATION AP-1 activator protein-1 CCN Connective tissue growth factor/Cysteine-rich 61/Nephroblastoma over-expressed CTGF connective tissue growth factor ECM extracellular matrix EGF epidermal growth factor Egr-1 early growth response 1 FACIT fibril-associated collagens with interrupted triple helices FGF fibroblast growth factor GAPDH glyceraldehyde 3-phosphate dehydrogenase ICAM-1 intercellular adhesion molecule 1 IFN γ interferon γ IGF-1 insulin-like growth factor 1 IGF-BP insulin-like growth factor binding protein IL-1, -2, -4, -6 interleukin 1, 2, 4, 6 KGF keratinocyte growth factor LAP latency associated protein LTBP latent TGF-β binding protein MMP matrix metalloproteinase MT-MMP membrane-type matrix metalloproteinase MULTIPLEXIN proteins with multiple triple helix domains and interruptions NF-1 nuclear factor-1 PAGE polyacrylamide gel electrophoresis PARP proline and arginine rich peptide PCR polymerase chain reaction PDGF platelet-derived growth factor PDI protein disulfide isomerase PGE2 prostaglandin E2 SMC smooth muscle cell Sp-1 selective promoter factor 1 TbRE TGF-β-response element TGF-α transforming growth factor-α TGF-β transforming growth factor-β TIMP tissue inhibitor of metalloproteinases TNF-α tumor necrosis factor-α VC viscose cellulose VEGF vascular endothelial growth factor 6
ABSTRACT A tight balance between connective tissue synthesis and breakdown is required for extracellular matrix (ECM) deposition in normal wound healing. The formation of new and regenerating tissue requires the coordinated regulation of various genes, which encode both structural and regulatory molecules. In the present study, the spatial and temporal expression patterns of various key components in the connective tissue formation of normal wound healing were investigated. Type I, III and V collagens belong to the family of fibrillar collagens, which form the important structural component of connective tissue. In the present study, type V collagen, in relation to type I and III collagens, in the formation of new connective tissue was investigated. Type V collagen was actively synthesized by both macrophage and fibroblasts-like cells and its expression was seen at every time point but at a considerably lower level than with type I and III collagens. It had maximum expression level at two weeks, which coincides with the expression of myofibroblasts. At the protein level, its distribution was closely associated with the blood vessel walls, which suggests its association with angiogenesis. From the collagen degrading enzymes, gelatinases of the matrix metalloproteinases (MMP) family, MMP-2 and MMP-9 were investigated. In addition, the expression of MMP-14 and tissue inhibitor of matrix metalloproteinase 2 (TIMP), which are known to be important for the activation of latent MMP-2, were studied. Enzyme analyses revealed that latent MMP-2 was present during the whole period of the granulation formation, whereas active MMP-2 started increase after one week and stayed in high level during the whole period of experiment. MMP- 2 mRNA was expressed throughout the granulation formation and mostly in fibroblast-like cells. The MMP-14 gene was up-regulated during the first week of wound healing, which coincides with an increase in MMP-2 activation. In contrast, the TIMP-2 gene was constitutively expressed, as was the MMP-2 gene. Only after two months was the TIMP-2 mRNA level slightly higher than at other time points, implying the completion of the wound healing process. No active MMP-9 enzyme was found and latent MMP-9 was seen only during the first week of the healing process. Immunohistochemical staining revealed that the cells responsible for this MMP-9 were polymorphonuclear leukocytes and macrophages, which are probably invaders from the surrounding tissues and already have pro-enzyme in their granules. MMP-9 mRNA expression started to increase towards the end of the experiment but with no detectable concomitant enzymatic activity. This suggests that MMP-9 regulation occurs at the 7
Page 1 and 2: Connective Tissue Formation in Woun
Page 3 and 4: CONTENTS ORIGINAL PUBLICATIONS 5 AB
Page 5: ORIGINAL PUBLICATIONS This thesis i
Page 9 and 10: INTRODUCTION The regulation of extr
Page 11 and 12: REVIEW OF THE LITERATURE WOUND HEAL
Page 13 and 14: days, and neutrophils are themselve
Page 15 and 16: Formation of Granulation Tissue New
Page 17 and 18: emodeled by contraction of the woun
Page 19 and 20: Table I Collagen types, their genes
Page 21 and 22: Structure and biosynthesis of colla
Page 23 and 24: A B COL1A1 COL3A1 COL5A1 C COL1A1 C
Page 25 and 26: TATA box GC box (Sp1/Sp3 binding el
Page 27 and 28: y sequences in their 3’ untransla
Page 29 and 30: espectively (160). Liver has as muc
Page 31 and 32: in the matrix as compared to type I
Page 33 and 34: is the need to generate mechanical
Page 35 and 36: and the catalytic domain, which con
Page 37 and 38: specific binding and cleavage of th
Page 39 and 40: constitutively expressed and contro
Page 41 and 42: inflammatory cells (283). MMP-9 may
Page 43 and 44: TG F-β ligand Receptor II Receptor
Page 45 and 46: and the core proteins of the ECM pr
Page 47 and 48: hydrophobic stretch of the C-termin
Page 49 and 50: similarities to insulin-like growth
Page 51 and 52: CTGF and TGF-β distinguishes them
Page 53 and 54: OUTLINE OF THE PRESENT STUDIES The
Page 55 and 56: were determined: reduced hemoglobin
Page 57 and 58:
(Minneapolis, MN, USA). Anti-PDGF A
Page 59 and 60:
solution, 10% dextran sulphate and
Page 61 and 62:
sequencer, Model 373A (Applied Bios
Page 63 and 64:
histology. The hemoglobin content o
Page 65 and 66:
especially at the later stages. The
Page 67 and 68:
owing to the high standard deviatio
Page 69 and 70:
A Staining index A 50 45 40 35 30 2
Page 71 and 72:
the proliferative phase of wound re
Page 73 and 74:
type V collagen expression is assoc
Page 75 and 76:
healing model presented here lacks
Page 77 and 78:
PDGF-A and -B protein was observed
Page 79 and 80:
CTGF functions as a downstream medi
Page 81 and 82:
I am also grateful for all my frien
Page 83 and 84:
19. Brown LF, Yeo KT, Berse B, et a
Page 85 and 86:
56. Phillips C, Wenstrup RJ. Biosyn
Page 87 and 88:
89. Lohi J, Lehti K, Valtanen H, Pa
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124. Wiestner M, Krieg T, Horlein D
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159. Gay S, Vijanto J, Raekallio J,
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196. Saelman EU, Nieuwenhuis HK, He
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237. Knäuper V, Cowell S, Smith B,
Page 97 and 98:
273. Rajavashisth TB, Liao JK, Gali
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307. Geng Y, Weinberg RA. Transform
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346. Heldin CH, Östman A, Rönnstr
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382. Frazier KS, Grotendorst GR. Ex
Page 105 and 106:
416. Glumoff V, Mäkelä JK, Vuorio
Page 107:
454. Pierce GF, Tarpley JE, Tseng J
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Connective Tissue Formation in Wound Healing - E-thesis

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