||Downregulation of caveolin-1 in keloid is responsible for
cell softening and its mechanical activation of RUNX2 and excessive extracellular matrix production
||Institute of Clinical Medicine
蟹足腫是一種會擴展到原傷口大小外的病態性疤痕，其致病機轉尚未完全了解。蟹足腫經常發生在身上皮膚具有比較大張力與硬度的地方。因此，我們假設蟹足腫來自蟹足腫纖維母細胞對於機械力的過度反應。本論文的目的是要了解蟹足腫的臨床表現與機械力的關係，蟹足腫的生物機械特性，並探討蟹足腫纖維母細胞對於機械力過度反應的分子機轉。具體目標包括 (I) 確認蟹足腫產生的位置與機械力之間的關係 (II) 探討蟹足腫纖維母細胞的機械力特性以及對於機械力刺激的反應 (III) 研究蟹足腫纖維母細胞對於機械力具過度反應的分子機轉 (IV) 釐清造成蟹足腫纖維母細胞具有獨特機械力特性的分子機轉。臨床上我們分析664位台灣蟹足腫患者的3978個蟹足腫病灶 (258位男性, 406位女性, 平均年紀33.7歲)。病患出現蟹足腫的年紀最多分布在20歲至29歲之間 (278位患者；41.9%)，有1809個 (45.5%) 蟹足腫病灶發生在前胸，有864個 (21.7%)病灶發生在肩膀與後背。此結果顯示蟹足腫傾向發生於年輕族群且皮膚張力大的地方。在實驗室的研究方面，我們利用原子力學顯微鏡檢測蟹足腫母細胞與組織的軟硬度，並培養纖維母細胞在具有不同軟硬度且膠原蛋白覆蓋的聚丙烯醯胺膠體培養系統上，以了解基質軟硬度對於纖維母細胞所造成的影響。我們發現蟹足腫纖維母細胞比正常纖維母細胞軟，而且在機械力的刺激下製造比較多的細胞外基質。利用生物學路徑分析軟體(Ingenuity Pathway Analysis)分析蟹足腫組織的RNA表現，我們發現與成骨細胞分化相關的轉錄因子—RUNX2在蟹足腫生成過程中可能扮演重要的調節因子。RUNX2異位表現於蟹足腫組織，且機械力刺激會增加RUNX2mRNA表現量並促進核轉位。Caveolin-1 (CAV1) 是caveolae上的主要結構蛋白，過去已被發現與細胞機械力學相關。我們發現CAV1蛋白質在蟹足腫組織的表現量下降，且調控蟹足腫纖維母細胞的軟硬度，RUNX2的活化以及細胞爬行能力。我們進一步發現組織蛋白去乙醯酶抑制劑—trichostatin A (TSA) 會增加CAV1蛋白的表現量，並降低RUNX2與纖維連接蛋白的表現量。此外，TSA也可以讓蟹足腫纖維母細胞的細胞變硬並降低細胞爬行的能力。總結，本研究結果顯示機械力的刺激在蟹足腫的致病機轉中扮演重要的角色。CAV1在蟹足腫纖維母細胞對於機械力刺激的過度反應與過度製造細胞外基質的過程中扮演重要的角色。此結果讓我們有機會在預防與治療蟹足腫的發展上有新的突破。
Keloids are pathologic scars and the exact pathogenesis remains unclear. Keloids are more likely to form in areas of the body subjected to increased skin tension or stiffness. We hypothesize that keloid results from the hyper-responsiveness of keloid fibroblasts (KF) to mechanical stimulation. The overall goal of this thesis is to understand the clinical manifestation of keloid and its relationship with mechanical effects, the biomechanical properties of keloid, and elucidate the molecular mechanism(s) regulating the hyper-responsiveness of KF to mechanical stimulation. The four specific aims are: (I) to confirm the preferred site of keloid formation is associated with mechanical tension(II) to investigate the mechanical properties of KF and its response to mechanical stimulation, (III) to identify the molecular mechanisms involved in the hyper-responsiveness of KF, and (IV) to elucidate the possible mechanisms contributing to the mechanical properties of KF.Clinically, we analyzed 3978 keloid lesionsof 664 Taiwanese keloid patients (258 males, 406 females, average age 33.7 years). The largest number of cases was in the age period 20 to 29 years (278 patients; 41.9%), and there were only 55 (8.3%) patients who were more than 60 years old. There were 1809 (45.5%) located on chest region and 864 (21.7%) on shoulder and back. The age and site distributions indicated that keloid tends to occur in the skin area with high skin tension of the young people.In our experimental design, we applied atomic force microscopy to detect the stiffness of KF and keloid tissue, and cultured fibroblasts on collagen-coated polyacrylamide gels with different stiffness to understand the effects of mechanical stimulation on KF. We found that KF were softer and produced more extracellular matrix (fibronectin) than NF under the mechanical stimulation (substratum stiffness). Using Ingenuity Pathway Analysis on microarray data, we identified RUNX2 as a potential key regulator in the pathogenesis of keloid. RUNX2 is ectopically expressed in keloid tissue. In addition, under mechanical stimulation, the upregulation of mRNA level and the nuclear translocation of RUNX2 were noted. Caveolin-1 (CAV1), the principal coat protein of caveolae, has been associated with the regulation of cell mechanics. We found that CAV1 was downregulated in keloid, and responsible for cell softening, the activation of RUNX2 and increased migratory ability in KFs. Furthermore, we found that histone deacetylase (HDAC) inhibitor, trichostatin A (TSA)increased CAV1 and decreased RUNX2 and fibronectin. TSA treatment also resulted in cell stiffening and decreased migratory ability in KFs.Collectively,these results suggest mechanical stimulation plays an important role in the pathogenesis of keloid formation. We found the novel role for CAV1 downregulation in linking the aberrant responsiveness to mechanical stimulation and extracellular matrix accumulation with the progression of keloids, findings that may lead to new developments in the prevention and treatment of keloid scar.
Table Content IX
Figure Content X
Chapter 1 Introduction 1
1.1 Skin and mechanobiology 1
1.1.1 Skin structure 1
1.1.2 Mechanobiology of skin 2
1.2 Cutaneous wound healing 3
1.2.1 Inflammation phase 4
1.2.2 Tissue formation (cell proliferation) phase 7
1.2.3 Matrix remodeling phase 9
1.3 Overview of scars 11
1.4 Pathogenesis of keloid 13
1.4.1 Cytokines/growth factors dysregulation 14
1.4.2 Aberrant extracellular matrix turnover 15
1.4.3 Genetic susceptibility 16
1.4.4 Mechanical force 17
1.5 The role of caveolin-1 in fibrosis 19
1.6 Epigenetic control of wound healing and skin fibrosis 20
1.7 Object of thesis 22
Chapter 2 The clinical distribution and morphology of keloid lesions suggest mechanical force plays an important role in the pathogenesis of keloid 24
2.1 Backgrounds and Aims 24
2.2 Material and Methods 25
2.2.1 Patient database and inclusion/exclusion criteria 25
2.2.2 Analysis of keloid distribution 25
2.3 Results 26
2.3.1 The distribution of ageand the location of keloid lesions 26
2.3.2 Two illustrative cases of keloid in view of mechanobiology 26
2.4 Discussion 27
2.5 Figures and Tables 30
Chapter 3 Downregulation of caveolin-1 in keloid is responsible for cell softening and its mechanical activation of RUNX2 and excessive extracellular matrix production 35
3.1 Backgrounds and Aims 35
3.2 Material and Methods 37
3.2.1 Human samples 37
3.2.2 Primary culture of fibroblasts 37
3.2.3 Measurements of cell/tissue mechanical properties by AFM 38
3.2.4 Preparation and fabrication of Polyacrylamide (PA) gels 39
3.2.5 cDNA microarrays analysis and IPA 39
3.2.6 Western blot analyses 41
3.2.7 Reverse transcription-PCR (RT-PCR) 42
3.2.8 Immunofluorescence staining and confocal microscopy 43
3.2.9 Immunohistochemistry (IHC) 43
3.2.10 Cell transfection with siRNA 43
3.2.11 Wound migration assay 44
3.2.12 Fabrication of micropost arrays and quantification of traction force 44
3.2.13 Statistical analysis 45
3.3.1 Keloid tissues are stiffer than normal dermal tissues, whereas keloid fibroblasts (KFs) are softer than normal dermal fibroblasts (NFs). 46
3.3.2 RUNX2 is a potential key regulator for ECM overproduction in keloids 46
3.3.3 Hyperresponsiveness of KFs to dermal tissue-equivalent matrix stiffness causes increased expression of RUNX2 and Col11A1 47
3.3.4 Decreased CAV1 is associated with cell softening and the upregulation of fibrogenesis-associated RUNX2 and migratory ability in KFs 48
3.3.5 TSA, an HDAC inhibitor, inhibited histone deacetylase, increased CAV1 and decreased RUNX2in KFs 50
3.4 Discussion 51
3.5 Figures and Tables 55
Chapter 4:Discussion, Conclusion and Prospects 71
4.1 The non-invasive quantificationof human skin tension is still warranted 71
4.2 The age distribution of keloid indicates that skin tension plays a role in the pathogenesis of keloid. 72
4.3 RUNX2 could not explain the overall pathological features of keloid 72
4.4 The regulation of HDAC2 expression in normal scar and keloid lesions is still unknown 74
4.5 Prospects 75
Curriculum Vitae 94
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