Title Page
ABSTRACT
국문 초록
PREFACE
Contents
NOMENCLATURE(or LIST OF SYMBOLS) 29
CHAPTER 1. INTRODUCTION 32
1.1. Radiation therapy 32
1.1.1. Fundamentals of ionizing radiation 32
1.1.2. Fundamentals of radiation therapy 32
1.1.3. Adverse effect of radiation therapy 35
1.2. Regeneration of vascular injury 37
1.2.1. Structure and function of blood vessel 37
1.2.2. Radiation-induced vascular injury 38
1.2.3. Regenerative angiogenesis 41
1.3. Quantifying 3D angiogenic vasculature on a chip using deep learning-based image processing 43
1.3.1. Suitability of microfluidic chip 43
1.3.2. The need for indices indicating the cell nucleus of a branch or node and 3D structure 44
1.3.3. Quantifying 3D angiogenic morphogenesis from radiation-injured vasculature using deep learning-based image processing 48
CHAPTER 2. Materials and methods 50
2.1. Preparation of microfluidic angiogenesis assay 50
2.2. Preparation of microfluidic vascular network and angiogenic vasculature 51
2.3. Immunofluorescence staining of vasculature models 51
2.4. Quantification of angiogenesis 52
2.5. Hardware and software 57
2.6. Irradiation 57
2.7. Viability and apoptosis of irradiated blood vessels 57
2.8. Quantitative reverse transcription-PCR 58
2.9. Permeability and blockage of vasculature 58
2.10. Statistical analysis 59
CHAPTER 3. Analyzing angiogenesis using deep learning-based image processing 60
3.1. 3D angiogenic morphogenesis in microfluidic angiogenesis assay 60
3.2. Processing of 3D angiogenic vasculature images 62
3.3. Daily progression of 3D angiogenic morphogenesis 66
3.4. 3D angiogenic morphogenesis in relation to the VEGF concentration 70
3.5. 3D angiogenic morphogenesis in presence of S1P 74
CHAPTER 4. Radiation-induced acute vascular injury on 3D vasculature 83
4.1. Development of 3D vasculature on a microfluidic chip 83
4.2. Radiation disrupts the formation of new blood vessels 85
4.3. Radiation induces the destruction of mature vascular networks 90
4.4. Radiation induces DNA-damage, apoptosis, and death in blood vessels 99
CHAPTER 5. Quantifying 3D angiogenic morphogenesis in radiation-injured vasculature using deep learning-based image processing 103
5.1. Radiation inhibits the recovery of blood vessels 103
5.2. SDF-1 induces the regeneration of radiation-injured blood vessels 113
CONCLUSION 121
REFERENCES 125
Table 1. Adverse effects of radiation therapy 36
Table 2. Proportion of patients receiving radiation therapy, and organs affected by radiation 37
Table 3. Radiation-induced vascular injury 41
Table 4. Classification of vascular structure analysis indicators represented in prior studies. 48
Table 5. Evaluate the extent of vascular injury in the irradiated group relative to the control group using nine metrics. 110
Table 6. Increase in 14 metrics in the 4 and 16 Gy groups with and without SDF-1 treatment. 119
Figure 1. DNA damage-induced cell death after irradiation. (a) Schematic of direct and indirect damage to DNA by radiation. (b) Both direct and indirect damage cause... 34
Figure 2. Schematics of the structure of a blood vessel for quantification. Schematics of whole vasculature (a) branch and node (b) cell nuclei comprising branches or nodes (c)... 46
Figure 3. Classification of vascular structure analysis indicators described in previous publications. (a) Papers were categorized on a coordinate plane with the following x, y,... 47
Figure 4. Schematic concepts of the research. Quantifying 3D angiogenic morphogenesis from radiation-injured vasculature using deep learning-based image processing. 49
Figure 5. Image processing based on the structure of 3D angiogenesis images. (a) Procedure for the threshold image (Ith). Gaussian filtering, threshold adjustment, and image...[이미지참조] 55
Figure 6. Image processing based on the cell nucleus of 3D angiogenic vasculature images. The process by which the nucleus image (In) is converted into a cell-segmented...[이미지참조] 56
Figure 7. Angiogenic morphogenesis in three dimensions is developed in the microfluidic angiogenesis assay. (a) Angiogenesis schematics on a chip. One hydrogel... 61
Figure 8. An overview of the process of using stained images to analyze angiogenesis. The two primary algorithms employed were NuSeT and skeletonization. 62
Figure 9. Definition of branches and nodes in vessels and derivation of the pseudo- depth of endothelial cells. (a) Define the branches and nodes of skeletonized vessels. (b)... 64
Figure 10. The definition of sixteen metrics for assessing angiogenesis In order to quantify angiogenesis, sixteen indicators have been developed by applying NuSeT and... 65
Figure 11. Quantification of the daily growth in 3D angiogenic morphogenesis using a heatmap approach. (a) A heatmap displaying the normalized mean values of each indicator... 67
Figure 12. Quantification of the daily changes in 3D angiogenic morphogenesis using a graph-based approach. (a) The quantitative graphs for BLeng, BWidth, BNum, NRadi,... 68
Figure 13. Other indicators indicate the daily change in 3D angiogenic morphogenesis quantified using a graph. Quantitative graphs of BArea, BEucLeng, BWidthDiff,... 69
Figure 14. Quantification of 3D angiogenic morphogenesis using a heatmap with respect to VEGF concentration. (a) A heatmap exhibiting the normalized mean values of... 71
Figure 15. Quantification of 3D angiogenic morphogenesis using a graph approach in regard to VEGF concentration. (a) BLeng, BWidthDiff, BNum, NNum, NRadi, and... 72
Figure 16. Other indicators of graph-based quantification of 3D angiogenic morphogenesis in response to the concentration of VEGF. Quantitative graphs of BArea,... 73
Figure 17. Quantitative analysis of the 3D angiogenic morphogenesis in the absence or presence of S1P. (a) A heatmap representing the normalized mean values of each indicator... 75
Figure 18. An in-depth look at 3D angiogenic morphogenesis while S1P is present. (a) Quantitative plots of BLeng, BWidth, BCellNum, NCellNum, BDepthDistri, and... 76
Figure 19. Representative images of 3D angiogenic morphogenesis with S1P present. (a) Representative angiogenesis images corresponding to BLeng, BWidth, BCellNum,... 77
Figure 20. Additional indicators of 3D angiogenic morphogenesis when S1P is present. Quantitative plots of BCurv, BArea, BEucLeng, BWidthDiff, NRadi, and... 78
Figure 21. Analyses of angiogenesis are reproducible. A heatmap displays the results of quantifying the variations in the presence or absence of S1P using HUVECs with the... 79
Figure 22. An overview of angiogenesis with regard to progression, VEGF concentration, and S1P presence. Angiogenesis analysis results are categorized according... 80
Figure 23. Variations in angiogenic sprouting with respect to VEGF concentration. (a) Phase-contrast images showing the response of angiogenic morphogenesis to 100 and 200... 81
Figure 24. Distribution of branches along the z-axis with S1P present. (a) The representative sprouted branches are shown as 3D confocal images. The scale bar is 50 μm.... 82
Figure 25. Developing a 3D vascular network within a microfluidic chip. (a) A schematic of a 3D vascular network on a chip. (b) Phase contrast images showing how the... 84
Figure 26. Radiation disrupts neovascularization. (a) Illustration showing the experimental setup used to examine how radiation affects neovascularization. (b) The... 87
Figure 27. Quantification of radiation-disrupted neovascularization. To examine the vascular structure of control and irradiated vessels, quantitative indicators like vasculature... 88
Figure 28. Variations in gene expression in irradiated versus control vasculatures. (a) Expression of control and irradiated vessel vascular genes. VEGF receptor 2 (Flk1) and... 89
Figure 29. Variations in viability in control versus irradiated vessels. (a) Quantitative viability plots for irradiated and control vessels (mean ± SD, n=12). (b) Fluorescent... 90
Figure 30. The structure of the vascular network is destroyed by radiation. (a) Illustration of the experimental setup used for assessing how radiation affects the vascular... 93
Figure 31. Analyzing the radiation-damaged mature vascular network structure. In order to assess the vascular structure of the control and irradiated vessels, quantitative... 94
Figure 32. Endothelial cells become detached from the basement membrane beneath them as a result of irradiation. (a) Laminin, DAPI, and F-actin fluorescent images of... 95
Figure 33. Tight junctions are disturbed by radiation. When 16 Gy of radiation is applied to vessels, junctions are damaged. Adherens junctions (VE-cad) are not... 96
Figure 34. The vascular network becomes permeabile and blocked as a result of irradiation. (a) Perfusion status of FITC-dextran in irradiated and control vessels. Images... 97
Figure 35. The blockage of irradiated vessels with an intensity of 16 Gy. The perfusion status of vessels exposed to 16 Gy irradiation was measured using 70 kDa FITC-... 98
Figure 36. Radiation induces double-strand breaks in vasculature. (a) Fluorescent images of control and irradiated vessels showing DAPI and γH2AX, a marker for double-... 100
Figure 37. Apoptosis and cell death are induced in irradiated blood vessels. (a) Nucleus and caspase-3 fluorescent images in irradiated and control vessels. The scale bar... 101
Figure 38. Vascular and apoptotic gene expression in irradiated and control vascular networks. (a) Gene expression of vascular markers and inflammatory marker in the... 102
Figure 39. The regeneration of 3D vasculature is inhibited by radiation. (a) Schematic illustrating the experimental setup used for assessing how radiation affects blood vessel... 106
Figure 40. Radiation inhibits the 3D angiogenic morphogenesis in vasculature. DAPI and F-actin images in control and irradiated blood vessels on days (a) 6 and (b) 17. Scale... 107
Figure 41. Heatmap-based quantification of the 3D angiogenic morphogenesis in radiation-injured vasculature. Heatmap depiction of normalized angiogenic morphology... 108
Figure 42. Graph-based quantification of the 3D angiogenic morphogenesis in radiation-injured vasculature. BLeng, branch length; BWidth, branch width; BNum, branch... 109
Figure 43. Other indicators of graph-based quantification of the 3D angiogenic morphogenesis in radiation-injured vasculature. BArea, branch area; BEucLeng, branch... 111
Figure 44. Sensitive indices in Radiation-injured vasculature. (a) The heatmap illustrates the normalized vasculature indices for days 6 and 17, with values of 4 and 16 Gy... 112
Figure 45. SDF-1 induces angiogenic morphogenesis in radiation-injured 3D vasculature. (a) Schematics of experimental procedure. (b) Nucleus (blue) and F-actin (red)... 115
Figure 46. Quantitative analysis of 3D angiogenic morphogenesis induced by SDF-1 in radiation-injured vasculature. The normalized mean values of each indicator at 4 Gy (a)... 116
Figure 47. An in-depth look at SDF-1-induced 3D angiogenic morphogenesis in radiation-injured vasculature. Quantitative graphs of BLeng, BWidth, BNum, BCellNum,... 117
Figure 48. Other indicators of 3D angiogenic morphogenesis induced by SDF-1 in radiation-injured vasculature. Quantitative plots of BArea, BEucLeng, BWidthDiff,... 118
Figure 49. Evaluation of vascular regeneration degree through multiple metrics. (a) Regenerated four indicators, while four indicators were less regenerated in vessels... 120