Characterization of distal vessel remodelling in chronic thromboembolic pulmonary hypertension Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfilment of the requirements for the Degree of Doctor in Human Biology (Doctor biologiae hominis - Dr. biol. hom.) of the Justus Liebig University Giessen by Dijana Iloska-Leyer from Ohrid, Macedonia Gießen 2020 Characterization of distal vessel remodelling in chronic thromboembolic pulmonary hypertension Inauguraldissertation zur Erlangung des Grades eines Doktors der Humanbiologie des Fachbereichs Medizin der Justus-Liebig-Universität Gießen vorgelegt von Dijana Iloska-Leyer aus Ohrid, Mazedonien Gießen 2020 I From the Max-Planck-Institute for Heart and Lung Research. Department IV Lung Development and Remodelling (Prof. Dr. Med. Werner Seeger) of the Faculty for Medicine of the Justus-Liebig-Universität Gießen Supervisor: Prof. Dr. Seeger Supervisor: Prof. Dr. Dorresteijn Day of disputation: 17.07.2020 II Dedicated to: My parents, Ratka and Nikola! На моите родители, Ратка и Никола! III Т’га за југ Орелски крилја как да си метнех, И в наши ст’рни да си прелетнех! На наши места ја да си идам, Да видам Стамбол, Кукуш да видам, Да видам дали с’нце и тамо Мрачно угревјат како и вамо. Ако как овде с’нце ме стретит, Ако пак мрачно с’нцето светит На п’т далечни ја ќе се стегнам И в други ст’рни ќе си побегнам, К’де с’нцето светло угревјат, К’де небото ѕвезди посевјат. Овде је мрачно и мрак м’ обвива И темна м’гла земја покрива: Мразој и снегој и пепелници, Силни ветришча и вијулици; Околу м’гли и мразој земни, А в гр’ди студој и мисли темни. Не, ја не можам овде да седам, Не, ја не можам мразој да гледам! Дајте ми крилја ја да си метнам, И в наши ст’рни да си прелетнам: На наши места ја да си идам, Да видам Охрид, Струга да видам. Тамо зората греит душата И с’нце светло зајдвит в гората: Тамо дарбите природна сила Со с’та роскош ги растурила: Бистро езеро гледаш белеит Или од ветар сино – темнеит; Поле погледниш или планина, Сегде божева је хубавина. Тамо по срце в кавал да свирам, С’нце да зајдвит, ја да умирам. Константин Миладинов Longing for the South If I had an eagle's wings I would rise and fly on them To our shores, to our own parts, To See Stambol, to see Kukus, And to watch the sunrise: is it dim there too, as it is here? If the sun still rises dimly, If it meets me there as here, I'll prepare for further travels, I shall flee to other shores Where the sunrise greets me brightly And the sky is sewn with the stars. It is dark here, dark surrounds me, Dark covers all the earth, Here are frost and snow and ashes, Blizzards and harsh winds abound, Fogs all around, the earth is ice, And in the breast are cold, dark thoughts. No, I cannot stay here, no; I cannot sit upon this frost. Give me wings and I will don them; I will fly to our own shores, Go once more to our own places, Go to Ohrid and to Struga. There the sunrise warms the soul, The sun gets bright in mountain woods: Yonder gifts in great profusion Richly spread by nature's power. See the clear lake stretching white- Or bluely darkened by the wind, Look at the plains or mountains: Beauty everywhere divine. To pipe there to my heart's content. Ah! Let the sun set, let me die. Konstantin Miladinov http://www.mn.mk/pesni-za-makedonija/742-Tga-za-jug Table of contents IV Table of contents List of Tables ........................................................................................................................... X List of Figures ........................................................................................................................ XI List of Abbreviations ........................................................................................................... XV 1. Theoretical background ................................................................................................... 1 1.1. Pulmonary vasculature: composition and function ..................................................... 1 1.2. Pulmonary hypertension .............................................................................................. 3 1.3. Classification of pulmonary hypertension................................................................... 3 1.4. Group 1 PH: Pulmonary arterial hypertension (PAH) ................................................ 5 1.4.1. Pathogenesis of PAH ........................................................................................ 5 1.4.2. Pulmonary vascular remodelling ...................................................................... 7 1.4.3. Vascular inflammation ..................................................................................... 8 1.4.4. Transcription factors in PAH ........................................................................... 9 1.5. Chronic thromboembolic pulmonary hypertension (CTEPH) .................................. 10 1.5.1. CTEPH classification ..................................................................................... 12 1.5.2. Current treatment for CTEPH ........................................................................ 12 1.5.2.1. Pulmonary endarterectomy ......................................................................... 13 1.5.2.2. Balloon angioplasty .................................................................................... 13 1.5.2.3. Medical therapy .......................................................................................... 14 1.5.2.4. Supportive medical therapy ........................................................................ 15 1.5.3. Pathophysiology of CTEPH ........................................................................... 16 1.5.4. Histopathology of CTEPH ............................................................................. 21 1.5.4.1. Proximal major vessel obliterations in CTEPH .......................................... 21 1.5.4.2. Microvascular disease in CTEPH ............................................................... 22 1.5.5. Molecular mechanisms in CTEPH ................................................................. 24 1.5.6. Animal models for CTEPH ............................................................................ 29 2. Aims and objectives ........................................................................................................ 31 3. Materials and Methods .................................................................................................. 32 3.1. Materials .................................................................................................................... 32 3.1.1. Reagents and chemicals ......................................................................................... 32 3.1.2. Kits......................................................................................................................... 33 3.1.3. Cell culture medium and reagents ......................................................................... 34 3.1.4. Other materials ...................................................................................................... 34 3.1.5. Equipment .............................................................................................................. 35 3.2. Methods ..................................................................................................................... 35 Table of contents V 3.2.1. Human tissues ........................................................................................................ 35 3.2.2. Staining .................................................................................................................. 36 3.2.2.1. Weigart – Van Gieson staining .......................................................................... 36 3.2.2.2. Double immunohistological staining ................................................................. 36 3.2.2.3. Masson`s Trichrome staining ............................................................................. 37 3.2.2.4. Sirius Red staining ............................................................................................. 38 3.2.2.5. Double immunofluorescent staining .................................................................. 38 3.2.2.6. Single immunohistological staining ................................................................... 38 3.2.3. Laser capture microdissection of pulmonary vessels ............................................ 39 3.2.4. Cell culture ............................................................................................................ 39 3.2.5. RNA isolation ........................................................................................................ 40 3.2.5.1. RNA isolation from LCM vessels .................................................................. 40 3.2.5.2. RNA isolation from PEA material ................................................................. 40 3.2.5.3. RNA isolation from cells................................................................................ 41 3.2.6. Microarray analysis ............................................................................................... 42 3.2.7. Reverse transcription of RNA ............................................................................... 42 3.2.8. Quantitative real time PCR (qRT-PCR) ................................................................ 43 3.2.9. Sircol collagen assay ............................................................................................. 43 3.2.9.1. Sircol soluble collagen assay.......................................................................... 44 3.2.9.2. Sircol insoluble collagen assay ...................................................................... 44 3.2.10. Protein isolation ..................................................................................................... 45 3.2.10.1. Protein isolation from cells ............................................................................ 45 3.2.10.2. Protein quantification ..................................................................................... 45 3.2.10.3. Western blotting ............................................................................................. 45 3.2.10.4. Densitometric analysis of the Western blots .................................................. 47 3.2.11. Cell proliferation.................................................................................................... 47 3.2.12. Cell apoptosis ........................................................................................................ 48 3.2.13. Macrophages polarization ...................................................................................... 48 3.2.14. Cell migration ........................................................................................................ 49 3.2.15. Animal experiments and genotyping ..................................................................... 49 3.2.16. Contractility assay ................................................................................................. 50 3.2.17. Ink injected CTEPH case study ............................................................................. 51 3.2.18. Precision cut lung slides (PCLS) ........................................................................... 51 3.2.19. Statistical analysis.................................................................................................. 51 Table of contents VI 4. Results .............................................................................................................................. 52 4.1. Chapter I: Histopathological and molecular characterization of distal vessel remodelling in explanted CTEPH, and in comparison, to IPAH and donor lung tissues ......................................................................................................................... 52 4.1.1. Characterization and comparison of vascular remodelling in explanted CTEPH and in comparison to IPAH and donor lung tissues ................................ 52 4.1.1.1. Assessment of the medial hypertrophy in explanted CTEPH and in comparison to IPAH and donor lung tissues.................................................. 52 4.1.1.2. Evaluation of the neointima/media ratio in explanted CTEPH and in comparison to IPAH and donor lung tissues.................................................. 54 4.1.1.3. Evaluation of the degree of muscularization in explanted CTEPH and in comparison to IPAH and donor lung tissues.................................................. 56 4.1.1.4. Collagen deposition in explanted CTEPH and in comparison to IPAH and donor lung tissues .................................................................................... 59 4.1.1.5. Differential gene expression of laser capture microdissected vessels from explanted CTEPH and in comparison to IPAH and donor lung tissues ........ 60 4.1.1.6. KEGG pathway analysis of the differential gene expression of LCM vessels from explanted CTEPH and in comparison to IPAH and donor lung tissues ..................................................................................................... 64 4.2. Chapter II: Histopathological and molecular characterization and comparison of distal vessel remodelling in explanted central and peripheral CTEPH in comparison to donor lung tissues .............................................................................. 67 4.2.1. Characterization and comparison of vascular remodelling in explanted central and peripheral CTEPH in comparison to donor lung tissues ................................ 67 4.2.1.1. Assessment of the medial hypertrophy in explanted central and peripheral CTEPH in comparison to donor lung tissues ................................ 67 4.2.1.2. Evaluation of the ratio neointima/media in explanted central and peripheral CTEPH in comparison to donor lung tissues ................................ 70 4.2.1.3. Collagen distribution in explanted central and peripheral CTEPH in comparison to donor lung tissues ................................................................... 71 4.2.2. Genome wide expression profiling of explanted central and peripheral CTEPH in comparison to donor lung tissues ...................................................................... 72 4.2.2.1. Differential gene expression of LCM vessels from explanted central and peripheral CTEPH in comparison to donor lung tissues ................................ 72 4.2.2.2. KEGG pathway analysis of the differential gene expression of LCM vessels from explanted central and peripheral CTEPH in comparison to donor lung tissues .......................................................................................... 76 4.3. Chapter III: Histopathological characterization of distal vessel remodelling in a case study of a patient with central (proximal) and recurrent CTEPH ................... 78 Table of contents VII 4.3.1. Characterization of distal vascular lesions in central CTEPH – ink-injected case study .............................................................................................................. 78 4.3.1.1. Assessment of the distal medial hypertrophy in pre-thrombus, thrombus and post-thrombus area of a central and recurrent CTEPH ........................... 80 4.3.1.2. Ratio neointima media evaluation in pre-thrombus, thrombus and post- thrombus area of a central and recurrent CTEPH .......................................... 81 4.3.1.3. Collagen deposition in pre-thrombus, thrombus and post-thrombus area of a central and recurrent CTEPH .................................................................. 82 4.4. Chapter IV: Histopathological characterization of distal vessel remodelling in central and peripheral lobes from central CTEPH in comparison to donor lung tissues ......................................................................................................................... 83 4.4.1. Characterization of vascular remodelling in central and peripheral lobes from central CTEPH in comparison to donor lung tissues ............................................ 83 4.4.1.1. Assessment of medial hypertrophy in central and peripheral lobes from central CTEPH in comparison to donor lung tissues ..................................... 84 4.4.1.2. Ratio neointima/media evaluation in central and peripheral lobes from central CTEPH in comparison to donor lung tissues ..................................... 85 4.4.1.3. Collagen distribution in central and peripheral lobes from central CTEPH in comparison to donor lung tissues............................................................... 87 4.5. Chapter V: Histopathological characterization of distal vessel remodelling in sarcoma-CTEPH patients in comparison to donors ................................................... 88 4.5.1. Characterization of vascular remodelling in sarcoma-CTEPH patients in comparison to donors ............................................................................................ 88 4.5.1.1. Assessment of medial hypertrophy in sarcoma-CTEPH patients in comparison to donors ..................................................................................... 88 4.5.1.2. Ratio neointima/media evaluation in sarcoma-CTEPH patients in comparison to donors ..................................................................................... 89 4.5.1.3. Collagen distribution in sarcoma-CTEPH patients in comparison to donors 90 4.6. Chapter VI: Histopathological and molecular characterization of CTEPH patients undergoing pulmonary endarterectomy (PEA) .......................................................... 91 4.6.1. Structural and cellular evaluation of PEA biorepository form CTEPH with ongoing course of the disease ............................................................................... 92 4.6.2. Recanalization in proximal and distal PEA biorepository..................................... 92 4.6.3. Deposition of soluble and insoluble collagen in proximal and distal PEA biorepository ......................................................................................................... 94 4.6.4. Genome wide expression profiling of proximal and distal PEA material ............. 96 4.7. Chapter VI: Chitinase-3-like-1 (CHI3L1), expression and their functional effects on vascular cells ............................................................................................. 98 4.7.1. Screening of CHI3L1 expression in donor, CTEPH and IPAH patients and their basal expression in human vascular and tumour lung cells ................... 98 Table of contents VIII 4.7.2. CHI3L1 expression in human naïve (M0) and polarized-activated (M1 and M2) macrophages ................................................................................... 99 4.7.3. Effect of different PH-associated growth factors in the expression of CHI3L1 in lung vascular cells ........................................................................................... 100 4.7.3.1. Effect of different growth factors on the expression of CHI3L1 in human pulmonary microvascular endothelial cells (hPMECs) ............................... 100 4.7.3.2. Effect of different growth factors on the expression CHI3L1 in human pulmonary artery smooth muscle cells (hPASMCs) .................................... 101 4.7.3.3. Effect of different growth factors on the expression CHI3L1 in human pulmonary artery adventitial fibroblasts (hPAAFs) ..................................... 102 4.7.4. Effect of recombinant CHI3L1 on the proliferation and apoptosis of vascular cells ....................................................................................................... 103 4.7.5. Effect of recombinant CHI3L1 on migration of vascular cells ........................... 105 4.7.6. Effect of CHI3L1 on contractility of hPASMCs ................................................. 106 4.7.7. Effect of CHI3L1 on VEGF signalling pathway in hPMECs ............................. 107 4.7.8. Ex-vivo assessment of the vessel number in end-stage CTEPH .......................... 109 4.7.9. Ex-vivo effect of CHI3L1 on human precision cut lung sections in terms of vascular remodelling ........................................................................................... 110 4.8. Chapter VII: ENPP2-LPA axis expression and their biological effects on vascular cells ............................................................................................................ 113 4.8.1. Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) expression in donors, CTEPH and IPAH patients and their basal expression in human vascular and tumour lung cells ............................................................................ 113 4.8.2. ENPP2 expression in human naïve (M0) and polarized (M1,M2) macrophages 114 4.8.3. Effect of different PH-associated growth factors in the expression of CHI3L1 in lung vascular cells ........................................................................................... 115 4.8.3.1. Effect of different growth factors on the expression of ENPP2 in human pulmonary microvascular endothelial cells (hPMECs) ............................... 115 4.8.3.2. Effect of different growth factors on the expression ENPP2 in human pulmonary artery smooth muscle cells (hPASMCs) .................................... 116 4.8.3.3. Effect of different growth factors on the expression of ENPP2 in human pulmonary artery adventitial fibroblasts ...................................................... 117 4.8.4. Pharmacological effect of LPA on the proliferation and apoptosis of vascular cells ....................................................................................................... 118 4.8.5. Effect of LPA on migration of hPASMCs and hPAAFs ..................................... 120 4.8.6. Ex-vivo effect of LPA on human precision cut lung slides (PCLS) .................... 122 4.8.7. The effect of ENPP2 reduction in mouse thrombosis model .............................. 124 5. Discussion ...................................................................................................................... 127 5.1. CTEPH exhibits distal histopathological resemblance to IPAH ............................. 127 Table of contents IX 5.2. CTEPH manifests significantly divergent global transcriptional regulatory landscape in comparison to IPAH ........................................................................... 130 5.3. Central CTEPH manifest similar histopathological changes as peripheral CTEPH ..................................................................................................................... 133 5.4. Genome wide expression profiling of central CTEPH manifests similar and unique global transcriptional regulatory landscape compared to peripheral CTEPH ..................................................................................................................... 134 5.5. Distal vessel remodelling in CTEPH is evenly distributed and independent on the disease site ......................................................................................................... 137 5.6. Different lung lobes from patients with CTEPH dispense a uniform distribution of distal vessel remodelling ..................................................................................... 138 5.7. Sarcoma-CTEPH patients present comprehensive vascular remodelling, similar to end-stage CTEPH patients ....................................................................... 139 5.8. PEA biorepository presents with tissue repair phenotype ....................................... 139 5.9. CHI3L1 is expressed in end-stage CTEPH and regulates vast varieties of cellular processes ..................................................................................................... 141 5.10. ENPP2 is expressed in end-stage CTEPH and regulates some cellular processes .................................................................................................................. 145 6. Future outlook............................................................................................................... 148 Summary ............................................................................................................................... 150 Zusammenfassung................................................................................................................ 153 References .......................................................................................................................... XVII Appendix ........................................................................................................................ XXXIV Publication list ..................................................................................................................... XLI Acknowledgements .......................................................................................................... XLIV List of Tables X List of Tables Table 1: Comprehensive clinical classification of pulmonary hypertension (PH) .................. 4 Table 2: A proposed new surgical classification of CTEPH ................................................. 12 Table 3: List of reagents and chemicals ................................................................................ 32 Table 4: List of the used kits ................................................................................................. 33 Table 5: List of cell culture medium and reagents ................................................................ 34 Table 6: List of additionally used materials .......................................................................... 34 Table 7: List of used equipment ............................................................................................ 35 Table 8: Compounds of mastermix for reverse transcription and their concentration .......... 42 Table 9: qRT PCR reaction mix composition ....................................................................... 43 Table 10: qRT PCR reaction steps .......................................................................................... 43 Table 11: Composition of the 5X loading buffer .................................................................... 45 Table 12: Resolving and stacking gel constituents .................................................................. 46 Table 13: Running buffer composition .................................................................................... 46 Table 14: Blotting buffer composition .................................................................................... 47 Table 15: TBST constituents ................................................................................................... 47 Table 16: Primer sequences for genotyping protocol .............................................................. 50 Table 17: Mastermix composition ........................................................................................... 50 Table 18: PCR protocol ........................................................................................................... 50 Table 19: List of genes regulated in CTEPH with opposite direction to IPAH ...................... 63 List of Figures XI List of Figures Figure 1: Architecture of the arterial wall and its extracellular matrix (ECM) components2 Figure 2: CTEPH as a dual compartment disease. ............................................................. 11 Figure 3: The treatment options for chronic thromboembolic pulmonary hypertension (CTEPH) ....................................................................................... 13 Figure 4: Schematic representation of the current pathophysiological concept of CTEPH ............................................................................................................................ 17 Figure 5: Microvascular disease in CTEPH affecting the pulmonary arterioles, venules and capillaries . ...................................................................................... 23 Figure 6: Pre- and post- elements of pathobiology of CTEPH .......................................... 25 Figure 7: Visualization of severe vascular wall thickening in human lung samples from CTEPH and IPAH patients compared to donors ....................................... 53 Figure 8: Medial hypertrophy quantification of donor, CTEPH and IPAH patients following the Weigert–van Gieson staining ....................................................... 54 Figure 9: Computational assessment of the neointima/media ratio of pulmonary vessels from donor, CTEPH and IPAH lung samples ........................................ 55 Figure 10: Visualization and quantification of the degree of muscularization for patients with CTEPH and IPAH, in comparison to control (donor samples) .................................................................................................. 58 Figure 11: Visualization and quantification of total collagen area in explanted CTEPH and IPAH lungs in comparison to donor ............................................... 60 Figure 12: Gene regulation patterns of CTEPH, IPAH and donor lung samples employing laser capture microdissection and microarray screening .................. 61 Figure 13: Transcriptional similarities and differences between CTEPH and IPAH .......... 64 Figure 14: In silico KEGG pathway analysis of CTEPH and IPAH in contrast to donor samples and their comparison .................................................................. 66 Figure 15: Visualization of severe vascular wall thickening in human lungs from central CTEPH and peripheral CTEPH and respective controls ........................ 68 Figure 16: Medial hypertrophy quantification of donor, central CTEPH and peripheral CTEPH following the Weigert–van Gieson staining ........................ 69 Figure 17: Computational assessment of the ratio neointima/media of pulmonary vessels from donor, central CTEPH and peripheral CTEPH lung samples ............................................................................................................... 70 List of Figures XII Figure 18: Visualization and quantification of total collagen area in human donor and explanted central and peripheral CTEPH .................................................... 72 Figure 19: Gene regulation patterns of central and peripheral CTEPH in contrast to donor lung samples based on combined LCM-microarray approach ............. 73 Figure 20: Transcriptional similarities and differences between central and peripheral CTEPH .............................................................................................. 74 Figure 21: Genes with opposite direction of regulation in central and peripheral CTEPH, both in contrast to donor ..................................................................................... 75 Figure 22: In silico KEGG pathway analysis of central and peripheral CTEPH in contrast to donors and their correlation ............................................................................ 77 Figure 23: Labeling of pulmonary arteries through blue color in a fresh lung CTEPH explant ................................................................................................... 78 Figure 24: Thromboembolism of a pulmonary artery in a fresh lung explant of a patient suffering from chronic thromboembolic pulmonary hypertension (CTEPH) ....................................................................................... 79 Figure 25: Visualization of severe vascular wall remodelling in pre-, thrombus and post- thrombus of case study explanted CTEPH ......................................... 80 Figure 26: Quantification of the medial hypertrophy of pulmonary vessels from pre-, thrombus and post-thrombus area of explanted CTEPH lung in comparison to a mean value of 12 donor samples .............................................. 81 Figure 27: Computational assessment of the ratio neointima/media of pulmonary vessels from pre-, thrombus and post-thrombus area of explanted CTEPH lung in comparison to a mean value of 12 donor samples .................... 82 Figure 28: Visualization and quantification of total collagen area from pre-, thrombus and post-thrombus part of explanted, ink-injected CTEPH lung in comparison to a mean value of five donor samples ............................... 83 Figure 29: Visualization of severe vascular wall thickening in human lungs from central and peripheral lobes of central CTEPH and respective controls ............ 84 Figure 30: Medial hypertrophy quantification of donor, central and peripheral CTEPH lobes following the Weigert–van Gieson staining ................................ 85 Figure 31: Computational assessment of the ratio neointima/media of pulmonary vessels from donor, central and peripheral CTEPH lung lobes .......................... 86 Figure 32: Visualization and quantification of total collagen area in human donor and explanted central and peripheral CTEPH lobes ........................................... 87 List of Figures XIII Figure 33: Visualization of severe vascular wall thickening in human lungs from central CTEPH and peripheral CTEPH and respective controls ........................ 88 Figure 34: Medial hypertrophy quantification of donor and sarcoma- CTEPH lobes following the Weigert–van Gieson staining ....................................................... 89 Figure 35: Computational assessment of the ratio neointima/media of pulmonary vessels from donor and sarcoma-CTEPH patients ............................................. 90 Figure 36: Visualization and quantification of total collagen area in human donor and Sarcoma CTEPH .......................................................................................... 91 Figure 37: Representative images of healthy pulmonary artery and surgical material from PEA .............................................................................................. 92 Figure 38: Proximal and distal PEA biorepository with recanalized regions ...................... 93 Figure 39: Characterization of the deposited collagen in PEA biorepository ...................... 95 Figure 40: Gene regulation patterns of central, patent and completely occluded PEA repository in comparison to their respective contols. ................................ 96 Figure 41: CHI3L1 expression in pulmonary vessels of donors, CTEPH and IPAH patients ................................................................................................................ 98 Figure 42: Evaluation of CHI3L1 basal mRNA and protein expression in human vascular and tumour lung cells ........................................................................... 99 Figure 43: Evaluation of CHI3L1 basal mRNA and protein expression in human naïve (M0) and polarized (M1, M2) macrophages ........................................... 100 Figure 44: Growth factors effect on the CHI3L1 expression in human pulmonary microvascular endothelial cells ........................................................................ 101 Figure 45: Growth factors effect on the CHI3L1 expression in human pulmonary artery smooth muscle cells. .............................................................................. 102 Figure 46: Growth factors effect on the CHI3L1 expression in human pulmonary artery adventitial fibroblasts ............................................................................. 103 Figure 47: CHI3L1 effect on proliferation and apoptosis of vascular cells ....................... 105 Figure 48: Effect of CHI3L1 on migration of hPASMCS and hPAAFs ............................ 106 Figure 49: CHI3L1 induced contractility of hPASMCs .................................................... 107 Figure 50: Effect of CHI3L1 on VEGF signalling pathway .............................................. 108 Figure 51: Total vessel density in explanted end-stage CTEPH in comparison to donors ............................................................................................................... 110 Figure 52: CHI3L1 ex-vivo induced vascular remodelling ............................................... 111 List of Figures XIV Figure 53: Gene regulation patterns of CHI3L1 stimulated PCLS and in silico KEGG pathway analysis ................................................................................... 112 Figure 54: ENPP2 expression in pulmonary vessels of donors, CTEPH and IPAH patients .............................................................................................................. 113 Figure 55: Evaluation of ENPP2 basal mRNA and protein expression in human vascular and tumour lung cells ......................................................................... 114 Figure 56: Evaluation of ENPP2 basal mRNA and protein expression in human naïve (M0) and polarized (M1, M2) macrophages ........................................... 114 Figure 57: Growth factors effect on the ENPP2 expression in human pulmonary microvascular endothelial cells ........................................................................ 116 Figure 58: Growth factors effect on the ENPP2 expression in human pulmonary artery smooth muscle cells ............................................................................... 117 Figure 59: Growth factors effect on the ENPP2 expression in human pulmonary artery adventitial fibroblasts ............................................................................. 118 Figure 60: LPA effect on proliferation and apoptosis of vascular cells ............................. 120 Figure 61: Effect of LPA on migration of hPASMCS and hPAAFs ................................. 121 Figure 62: LPA ex-vivo induced vascular remodelling and effect of LPA specific inhibition ........................................................................................................... 123 Figure 63: Schematic representation of the experimental plan of CTEPH thrombosis model ............................................................................................. 124 Figure 64: The effect of Enpp2 partial deficiency in thrombosis model of CTEPH ......... 125 Figure 65: Schematic representation of the experimental plan of hypoxia-CTEPH thrombosis model ............................................................................................. 149 List of Abbreviations XV List of Abbreviations ACTB β-actin AF Adventitial fibroblast ANGPT1 Angiopoetin 2 ANGPT2 Angiopoetin 2 APA Antiphospholipid antibody ATX Autotaxin BPA Balloon angioplasty BSA Bovine serum albumin BrdU Bromodeoxyuridine B2M Beta-2-Microglobulin CCL2 C-C Motif Chemokine Ligand 2 cGMP cyclic Guanosine monophosphate CHI3L1 Chitinase-3-like-1 CTEPH Chronic thromboembolic pulmonary hypertension CTE Chronic thromboembolism CRP C-reactive protein CKS1B Cyclin-Dependent Kinases Regulatory Subunit 1 DAPI 4′,6-Diamidin-2-phenylindol DMSO Dimethyl sulfoxide DVT Deep vein thrombosis EC Endothelial cell EEL External lamina elastica ELISA Enzyme-linked immunosorbent assay ENA-78 Neutrophil-activating protein ENPP2 Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 eNOS Endothelial Nitric Oxide Synthase ET-1 Endothelin-1 FCS Fetal calf serum FGF Fibroblast growth factor FOXO Forkhead box O GAPDH Glyceraldehyde 3-phosphate dehydrogenase GF Growth factor HIF1-α Hypoxia Inducible Factor 1α List of Abbreviations XVI HOXC6 Homeobox C6 IBS Inflammatory bowel disease IEL Internal elastic lamina IL Interleukin IPAH Idiopathic pulmonary arterial hypertension IP-10 Interferon-γ-inducible 10 kDa KDR Kinase insert domain protein receptor KEGG Kyoto Encyclopedia of Genes and Genomes KLF2 Kruppel-like factor 2 LCM Laser capture microdissection LPA Lysophosphatidic acid MCEMP1 Mast Cell-Expressed Membrane Protein mRNA messenger Ribonucleic Acid NFATC2 Nuclear factor of activated T-cells, cytoplasmic 2 NO Nitric oxide NOS 3 Nitric Oxide Synthase 3 PA Pulmonary artery PAI-1 Plasma plasminogen activator inhibitor PAAF Pulmonary adventitial fibroblast PACP Pulmonary artery capillary pressure PAEC Pulmonary artery endothelial cell PAH Pulmonary arterial hypertension PASMC Pulmonary arterial smooth muscle cell PAP Pulmonary artery pressure PAWP Pulmonary artery wedge pressure PCLS Precision cur-lung slide PDGF Platelet Derived Growth Factor PDPN Podoplanin PECAM 1 Platelet endothelial cell adhesion molecule PF4 Platelet factor 4 PE Pulmonary embolism PEA Pulmonary endarterectomy PH Pulmonary hypertension RHC Right heart catheterization List of Abbreviations XVII PMEC Pulmonary microvascular endothelial cells PRPG6 Pre-MRNA Processing Factor 6 PVR Pulmonary vascular resistance PVOD Pulmonary veno-occlusive disease RV Right ventricle sGC Soluble guanylate cyclase t-PA Tissue plasminogen activator TNF-α Tumour Necrosis Factor-α Tyr Tyrosine VEGF Vascular endothelial growth factor VE-catherin Vascular endothelial cadherin VTE Venous thromboembolism vWf von Willebrand factor WT Wildtype α-SMA alpha- smooth muscle actin Theoretical background 1 1. Theoretical background 1.1. Pulmonary vasculature: composition and function The pulmonary vasculature is exclusive in its structure, volume and function. The lung is the only organ displaying two different types of circulation: the pulmonary circulation, with main function of gas exchange, and the bronchial circulation, a systemic vascular supply that provides oxygenated blood to the walls of the conducting airways, pulmonary arteries and veins. (Suresh & Shimoda, 2016). During embryonic stage of development, pulmonary circulation is a low compliance and high resistance system (Suresh & Shimoda, 2016), while postnatally, it is directed towards high compliance and low resistance in order to effectively accomplish the gas exchange (Townsley, 2013). In fact, the pulmonary vascular resistance (PVR) is approximately one-tenth of the one of systemic circulation. In comparison to their systemic counterparts, the pulmonary arteries have thinner walls, smaller smooth muscle layer, balancing between high production of endogenous vasodilators and low production of vasoconstrictors (Suresh & Shimoda, 2016) in order to accommodate a large volume of blood with little increase in mean pulmonary arterial pressure (mPAP) (Lammers et al., 2012). The pulmonary circulation arises from the right ventricle (RV), while the bronchial vessels usually originate from the aorta or the intercostal arteries, draining into the right heart. Bronchial vessels also supply the intrapulmonary airways, at a level of terminal bronchioles, where they anastomose with the pulmonary vasculature (Baile, 1996). The pulmonary vasculature is composed of three anatomic compartments associated in a sequence: the arterial tree, an extensive capillary bed and the venular tree (Townsley, 2013). Further on, the arterial wall comprises of three layers, tunica intima, tunica media and tunica adventitia, each contributing in a different extent to the total vessel thickness (Figure 1). Theoretical background 2 Figure 1: Architecture of the arterial wall and its extracellular matrix (ECM) components. The major components of the vessel wall are: tunica intima (TI), tunica media (TM) and tunica adventitia (TA). TI is represented by endothelial cells (ECs), further supported by basal membrane (BM) and internal elastic lamina (IEL); TM corresponds to the smooth muscles cells (SMCs) layer connected to the TA, defined by adventitial fibroblasts (AF) via external elastic lamina (EEL) (Chelladurai, Seeger, & Pullamsetti, 2012). Reproduced with permission of the © ERS 2019: European Respiratory Journal 40 (3) 766-782; DOI: 10.1183/09031936.00209911 Published 31 August 2012 Tunica intima is the innermost layer of the vascular wall, under direct influence of the blood flow. It is comprised of continuous monolayer of endothelial cells (ECs), supported by a sub- endothelial layer of connective tissue and supportive cells, ultimately forming a tightly regulated semipermeable barrier. The gas exchange takes place in the capillary segment (normally around 3-4µm in size), dispersed from the small arteries and comprised only of ECs, surrounded by the alveolar epithelial cells. In addition to tunica intima towards the tunica media, in muscular arteries, the internal elastic lamina can be identified as a concentric layer of elastic tissue. Tunica media is comprised of a single layer (arterioles) or multiple layers (pulmonary arteries) of smooth muscle cells (SMCs) and elastic and connective fibres arranged circumferentially around the vessel. It is a substantial part of the vessel, playing a role in contraction and relaxation of the blood vessel, in order to decrease and increase the diameter of the vessel lumen, respectively. The media is demarcated from the adventitia with a thick layer of external elastic lamina. Theoretical background 3 Tunica adventitia is the outermost layer composed of adventitial fibroblasts (AFs) as a predominant cell type, interstitial cells, connective fibres, a vasa vasorum and neuronal network, providing a structural integrity and a damage protection of the vessel. Tunica adventitia as well hosts resident progenitor stem cells and infiltrating immune cells (Chelladurai et al., 2012). Any kind of mechanical or structural alterations in the vascular wall can affect the pressure in the pulmonary circulation, contributing to initiation and maintenance of pulmonary hypertensive state. 1.2. Pulmonary hypertension Pulmonary hypertension (PH) is a progressive, multi-etiological disease of the pulmonary vasculature characterized by pathological remodelling of the resistance pulmonary arteries, resulting in RV failure and ultimately death (Farber & Loscalzo, 2004). Clinically, PH confers mPAP greater or equal to 25 mmHg and PVR greater or equal to 3 Wood units at rest, measured by right heart catheterization (RHC), which is a reference standard for assessing PH patients. During the RHC, an additional measurement of pulmonary artery wedge pressure (PAWP) or pulmonary artery capillary pressure (PACP) is performed to differentiate between pre-capillary and post-capillary PH (M. M. Hoeper et al., 2013). Recently, a new definition has been proposed by the 6th WSPH Task Force on PH diagnosis and classification, suggesting that pre- capillary PH is best defined by the concomitant presence of mPAP >20 mmHg, PAWP ≤15 mmHg and PVR ≥3 WU (Gérald Simonneau et al., 2019). All forms of PH are estimated to affect over 100 million people worldwide. Once diagnosed and left untreated, the median survival rate of patients is only 2.8 years (D'Alonzo et al., 1991). 1.3. Classification of pulmonary hypertension Based on their clinical presentation, pathological findings, haemodynamic characteristics and treatment strategy (Galiè et al., 2016), updated clinical classification from the 6th World PH symposium, PH has been classified into five main groups, Group 1 to 5 (Galiè et al., 2016; Gérald Simonneau et al., 2019). As presented in Table 1, the Group 1 is Pulmonary Arterial Hypertension (PAH), represented by wide range of clinical manifestations, including the pulmonary veno-occlusive disease. The other clinical groups are as following: Group 2: PH due to left heart disease; Group 3: PH due to lung diseases and/or hypoxia; Group 4: Chronic thromboembolic pulmonary hypertension and Group 5: PH with unclear multifactorial mechanisms. Theoretical background 4 Table 1: Comprehensive clinical classification of pulmonary hypertension (PH) (Gérald Simonneau et al., 2019) 1. Pulmonary arterial hypertension (PAH) 1.1. Idiopathic PAH 1.2. Heritable PAH 1.2.1 BMPR2 mutation 1.2.2 ALK-1, ENG, SMAD9, CAV1, KCNK3 1.2.3 Unknown 1.3. Drugs and toxins induced 1.4. PH associated with: 1.4.1 Connective tissue disease 1.4.2 Human immunodeficiency virus (HIV) infection 1.4.3 Portal hypertension 1.4.4 Congenital heart disease 1.4.5 Schistosomiasis 1.5. PAH long-term responders to calcium channel blockers 1.6. PAH with overt features of venous/capillaries (PVOD/PCH) involvement 1.7. Persistent PH of the newborn syndrome 2. Pulmonary hypertension due to left heart disease 2.1. PH due to heart failure with preserved left ventricular ejection fraction 2.2. PH due to heart failure with reduced left ventricular ejection fraction 2.3. Valvular heart disease 2.4. Congenital/acquired left cardiovascular conditions leading to post-capillary PH 3. Pulmonary hypertension due to lung diseases and/or hypoxia 3.1. Obstructive lung disease 3.2. Restrictive lung disease 3.3. Other lung disease with mixed restrictive/obstructive pattern 3.4. Hypoxia without lung disease 3.5. Developmental lung diseases 4. Pulmonary hypertension due to pulmonary artery obstructions 4.1. Chronic thromboembolic pulmonary hypertension 4.2. Other pulmonary artery obstructions 4.2.1. Sarcoma (high or intermediate grade) or angiosarcoma Theoretical background 5 4.2.2. Other intravascular tumours (renal, uterine carcinoma, germ cell tumours of the testis, other tumours) 4.2.3. Non-malignant tumours (Uterine leiomyoma) 4.2.4. Arteritis without connective tissue disease 4.2.5. Congenital pulmonary arteries stenoses 4.2.6. Parasites (hydatidosis) 5. Pulmonary hypertension with unclear and/or multifactorial mechanisms 5.1. Hematologic disorders: chronic hemolytic anemia, myeloproliferative disorders, splenectomy 5.2. Systemic and metabolic disorders: pulmonary Langerhans cell histiocytosis, Gaucher disease, glycogen storage disease, neurofibromatosis, Sarcoidosis 5.3. Others: fibrosing mediastinitis, chronic renal failure with or without haemodialysis 5.4. Complex congenital heart disease 1.4. Group 1 PH: Pulmonary arterial hypertension (PAH) In spite of the classification, featured by the clinical parameters, every PH group (or subgroup), regardless of the clinical manifestations and histopathological characteristics, exhibits pulmonary vasoconstriction and excessive pulmonary vascular remodelling, leading to increased PVR, RV overload and failure. PAH as the first group in the classification system of PH, appears to involve multiple aetiologies, including the idiopathic PAH (IPAH), heritable PAH (BMPR2 mutation, ALK-1, ENG, SMAD9 etc.), drugs and toxins induced PAH, as well PAH associated with several other diseases such as collagen vascular disease, PA shunts, portal hypertension, HIV infection and other conditions that include congenital heart disease, hemoglobinopathy, and hereditary haemorrhagic telangiectasia. 1.4.1. Pathogenesis of PAH Sustained pulmonary vasoconstriction, lumen obliteration of small- and medium-sized arteries and arterioles, formation of plexiform lesions and in situ thrombosis, and concentric thickening of pulmonary arteries are the main cause for elevated mPAP and PVR in patients with PAH regardless of the initial pathogenic trigger (J. X. Yuan & Rubin, 2005). It is well appreciated that patients with PAH exhibit imbalanced levels of vasodilators and vasoconstrictors. Endothelin-1 (ET-1) levels are elevated in PH patients (Giaid et al., 1993), as a major player in the vasodilator/vasoconstrictor imbalance. It is primarily produced by pulmonary artery ECs (PAECs). Through the action on its receptor expressed on pulmonary Theoretical background 6 artery SMCs (PASMCs), it induces vasoconstriction, proliferation and the production of cytokines and growth factors (Luscher & Barton, 2000; Pollock, Keith, & Highsmith, 1995). Similarly, PH patients have reduced circulating levels of the vasodilators such as prostacyclin and nitric oxide (NO) relative to levels of the vasoconstrictor such as thromboxane (Christman et al., 1992). It was shown that prostacyclin induces vasodilation, inhibits platelet activity and exhibits anti-proliferative effects on PASMCs, therefore it is considered as a therapeutic target for the treatment of PH (Olschewski et al., 2004). On the other hand, NO levels are as well decreased in PAH patients due to high arginase levels (substrate of nitric oxide synthase) or increased asymmetric dimethyl arginine (competitive inhibitor of nitric oxide synthase) (S. Pullamsetti et al., 2005). NO stimulates soluble guanylate cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase G in PASMCs, causing vasodilatory and anti-proliferative effects as well as PASMC relaxation. Phosphosdiesterase-5 (PDE-5) is the enzyme responsible for breakdown of cGMP in response to increase of NO. Consequently, PDE-5 inhibitors are used effectively in treatment of PAH, as they can increase intracellular cGMP and enhancing PASMCs relaxation and vasodilation (Wilkins, Wharton, Grimminger, & Ghofrani, 2008). Circulating levels of serotonin (5-Hydroxy tryptamine) are elevated in PH patients. Serotonin is mainly produced in PAECs and via the serotonin transporter, expressed in PASMCs and pulmonary artery adventitial Fibroblasts (PAAFs), leading to vasoconstriction and proliferation (Naeije & Eddahibi, 2004). Voltage gated potassium channels (Kv channels), Kv1.2 and Kv1.5 are known to be downregulated in PASMCs from PH patients (X.-J. Yuan, Wang, Juhaszova, Gaine, & Rubin, 1998). Downregulation of these channels, along with subsequent membrane depolarization and opening of the voltage-gated Ca2+ channels results in vasoconstriction (Archer et al., 2008). Mitochondrial abnormalities such as pathological activation of pyruvate dehydrogenase kinase (PDK) are known to inhibit Kv channels and activate vasoconstrictive, pro-proliferative and anti-apoptotic signalling cascades (Moudgil, Michelakis, & Archer, 2006). Although the causal PAH pathomechanisms are still largely unclear, recently, a cancer-like concept for PAH has emerged, rationalized by in situ and in vitro observations (Rai et al., 2008; Sakao et al., 2011). According to this concept, a monoclonal expansion of ECs has been found in IPAH when compared with ECs found in lungs of patients with congenital heart malformations (S. D. Lee et al., 1998). Next, instability of short DNA microsatellite sequences within plexiform lesions in IPAH has been evident (Yeager, Halley, Golpon, Voelkel, & Tuder, Theoretical background 7 2001) and the presence of somatic chromosome abnormalities in the lungs of patients with PAH and cultured cells has been reported (Aldred et al., 2010). PAECs and PASMCs derived from patients with PAH maintain their abnormal hyperproliferative, apoptosis-resistant phenotype (Tu et al., 2012; Tu et al., 2011) and human pulmonary vascular cells derived from PAH patients exhibit an altered energy metabolism in situ and in vitro (Michelakis et al., 2002; Sutendra et al., 2010; Tuder, Davis, & Graham, 2012; Xu et al., 2007). Yet, one should consider that there are crucial differences between PAH pathogenesis and carcinogenesis. 1.4.2. Pulmonary vascular remodelling PAH pathophysiology is represented by endothelial dysfunction and a persistent vasoconstriction, resulting in obliterative remodelling of small pulmonary arteries (< 500 μm diameter) and pre-capillary (< 70µm diameter) due to proliferation of PAECs and PASMCs (K. R. Stenmark, Meyrick, Galie, Mooi, & McMurtry, 2009; Tuder et al., 2009). Indeed, the remodelling is primarily a result of excessive proliferation of vascular cells and affects all the layers (intima, media and adventitia), leading to intimal thickening, media hypertrophy, adventitial thickening, plexiform lesions and vascular pruning (S. S. Pullamsetti et al., 2014). Recruitment of inflammatory and progenitor cells within the vessel wall is also contributing to the vascular remodelling (A. L. Firth, Mandel, & Yuan, 2010). Endothelial cells regulate the vascular tone and their injury or dysfunction can cause a switch from quiescent phenotype towards a pro-proliferative and apoptotic-resistant phenotype, leading to neointima formation (Meyrick, Clarke, Symons, Woodgate, & Reid, 1974; Sakao et al., 2005; Tuder, Groves, Badesch, & Voelkel, 1994). Dysfunctional endothelium then releases growth factors (fibroblast growth factor (FGF)-2 and angipoetin-1 and 2 (ANGPT-1; ANGPT- 2)), vasoconstrictors (ET-1), pro-thrombotic mediators (thromboxane-A2) and pro- inflammatory cytokines (bone morphogenetic proteins (BMPs)) that stimulate proliferation of PASMCs (Morrell et al., 2009; Thompson & Rabinovitch, 1996). A network of vascular channels, called plexiform lesions, is formed at later stage of the disease evolving from monoclonal expansion of apoptosis-resistant PAECs, migration and proliferation of PASMCs and accumulation of circulating cells such as macrophages and endothelial progenitor cells (S. D. Lee et al., 1998; Rabinovitch, 2012). Medial hypertrophy and hyperplasia is featured by expansion of apoptosis-resistant smooth muscle cells, as well as differentiating cells present in the vessels or migration and de- differentiation of PAAFs (Davie et al., 2006). In larger pulmonary arteries, hypertrophy of PASMCs and extracellular matrix deposition are most effective in enlarging the vessel wall, Theoretical background 8 while hyperplasia is prevalent in smaller resistance vessels (Jeffery & Morrell, 2002). As mentioned before, several growth factors are released after endothelial injury affecting the SMCs as one of the remodelling effectors. Platelet derived growth factor (PDGF), is shown to induce proliferative and migratory phenotype of PASMCs. Pulmonary arteries of IPAH patients are shown to highly express PDGF ligands and the receptors (PDGFR-α, PDGFR-β) (Perros et al., 2008). Administration of imatinib, a PDGFR antagonist, reversed the vascular remodelling in hypoxia model of PH (Schermuly et al., 2005). Furthermore, a transcription factor FoxO1, is downregulated in IPAH, negatively regulating pro-proliferative and anti- apoptotic PASMC phenotype by affecting some proliferative genes as Cyclin D1, p27, and apoptotic genes like BCL6 and GADD45a (Savai et al., 2014). Tunica adventitia is collagen-enriched vessel platform containing mainly fibroblast and adrenergic nerves. Additionally, it accommodates many immunomodulatory cells, such as macrophages, mast cells, T-cells, dendritic cells, CD34+/Sca1+ progenitor cells, ECs of vasa vasorum, pericytes and adipocytes (Majesky, Dong, Hoglund, Mahoney, & Daum, 2011). The vascular remodelling also includes thickening of the adventitia, as PAAFs proliferate and secrete chemokines thus facilitating the recruitment of inflammatory cells in response to environmental stress (Kurt R. Stenmark et al., 2012). In fact, increased levels of tumour necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, and IL-8 have been reported in severe hypoxic pulmonary hypertension (Soon et al., 2010). Several of these cytokines are multifunctional, directly regulating proliferation, migration and differentiation of vascular cells. IL-6, for instance, induces PASMCs proliferation via elevation of FGF-2. When overexpressed in mice it caused exaggerated pulmonary hypertensive response to hypoxia (Golembeski, West, Tada, & Fagan, 2005). Elevated TFG-β in PH can induce PAAFs to differentiate into myofibroblasts, leading to increased production of extracellular matrix proteins like collagen or can further migrate to the medial or intimal layer supporting the extensive pathological remodelling (Davie et al., 2006). 1.4.3. Vascular inflammation One of the features observed from histopathologic specimens and serum/ plasma samples from patients with PAH is the presence of inflammation. Inflammation represents a complex series of interactions among soluble factors and cells that can arise in response to traumatic, infectious, post-ischemic, toxic, or autoimmune injury (Chatelain & Dardik, 1988). Theoretical background 9 Patients with IPAH have elevated serum levels of cytokines, including IL-1- β, IL-6, and IL-8 (Humbert et al., 1995; Soon et al., 2010) and chemokines such as chemokine (C-C motif) ligand (CCL)2/monocyte chemotactic protein (MCP)-1 (Sanchez et al., 2007), CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES) (Peter Dorfmüller et al., 2002) and CXC3CL1/fractalkine (Balabanian et al., 2002). TNF-α, IL-6, MCP-1, and C-reactive protein (CRP) are also increased in congenital heart disease associated PAH (Diller et al., 2008). However, increased levels of such mediators are common to the pathology of PAH per se and are not restricted to one particular subtype. 1.4.4. Transcription factors in PAH Growing evidence indicates that transcription factors (TFs) have been implicated in the pathogenesis of PH and RV dysfunction (S. S. Pullamsetti et al., 2017). The activity of the TFs can be targeted by different stimuli converging the PH vascular phenotype (S. S. Pullamsetti et al., 2017). TFs are sequence-specific DNA-binding proteins controlling the process of transcription. TFs regulate the gene expression by direct binding to the core promoter elements in proximity to transcriptional start sites to recruit transcriptional machinery and regulate gene expression (Goodrich & Tjian, 2010) or by utilizing regulatory DNA elements called enhancers, the general transcriptional machinery, and Pol II (RNA polymerase II) complexes (Goodrich & Tjian, 2010; Lelli, Slattery, & Mann, 2012). As known, several TFs are key regulators of cellular proliferation and may directly be involved in the pathogenesis of PH and most likely CTEPH. Evidence that Forkhead box O1 (FoxO1) transcription factor, among all FoxO isoforms, is centrally involved in the hyperproliferative and apoptosis-resistant phenotype of PASMCs, the hallmark of PAH, has been recently presented by Savai et al. (2014). FoxO TFs belong to a family of transcriptional regulators characterized by a conserved DNA-binding domain termed the Forkhead box (Eijkelenboom & Burgering, 2013) and can act as transcriptional activators and repressors. In mammals, four FoxO isoforms have been identified: FoxO1, FoxO3, FoxO4 and FoxO6. FoxOs control various cellular responses (Eijkelenboom & Burgering, 2013) and have been implicated in vascular structural maintenance (Mahajan et al., 2012; Oellerich & Potente, 2012). FoxO1 and FoxO3 are described in the literature to regulate essential set of genes involved in angiogenesis as well as vascular remodelling (Potente et al., 2005). GATA-6 is a member of GATA family of zink-finger TFs, expressed in a wide spectrum of tissues, heart, lung, liver, kidney, pancreas, spleen etc. maintaining the differential cell Theoretical background 10 composition (Suzuki et al., 1996). It is highly expressed in quiescent vasculature, particularly in vascular SMCs, maintaining their contractile phenotype and lost upon vascular injury (Mano, Luo, Malendowicz, Evans, & Walsh, 2015; Nishida et al., 2002). GATA-6 may play a role in the pathogenesis of PH by regulating ET-1, plasminogen activator inhibitor-1 (PAI-1), matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10) etc (Ghatnekar et al., 2013). KLF-2 belongs to the Kruppel-like factors (KLFs), a subclass of the zinc finger family of TFs which regulates cellular differentiation and tissue development (Bieker, 2001). Within the vessel wall it is exclusive to EC and induced when cultured ECs were exposed to sustained shear stress (Dekker et al., 2002). Share stress induced KLF-2 is directly affecting the eNOS levels, as an essential regulator of vascular reactivity and tone (Huang et al., 1995). 1.5. Chronic thromboembolic pulmonary hypertension (CTEPH) Chronic thromboembolic pulmonary hypertension (CTEPH) is a rear, complex, multifactorial and severe vascular disease that is life threatening if untreated (I. M. Lang, Dorfmuller, & Vonk Noordegraaf, 2016; J. Pepke-Zaba, 2010). It represents the group 4 of the current clinical classification of PH. CTEPH has a distinguishable nature when compared to other types of PH, with regard to the pathophysiological features, as well as the treatment options (I. Lang, 2015). Extensive clinical evidence indicates that it might be initiated by a spectrum of thrombotic or inflammatory lesions of the pulmonary vasculature, although the acute pulmonary embolism (PE) emerge as a most common reason (J. Pepke-Zaba, 2010). A large international registry confirmed that approximately around 75% of the patients had a history of acute PE, despite the fact that there were some reservations in regard to the thromboembolic nature of the disease (J. Pepke-Zaba et al., 2011). Several ongoing studies suggest that CTEPH is a disease of dual vascular compartments, presented by major vessel remodelling and small vessel arteriopathy, characterized by medial hypertrophy, obstructive intimal thickening, microthrombi formation and plexiform lesions (Figure 2) (Galiè & Kim, 2006; I. Lang, 2010; Piazza & Goldhaber, 2011). CTEPH usually starts with a persistent thrombi obstruction in the proximal (main, lobar and segmental) arteries which fails to resolve (G. Simonneau, Torbicki, Dorfmuller, & Kim, 2017). Small vessel disease occurs distally to the obstructed areas and most likely due to excessive collateral blood supply from high pressure pulmonary and systemic circulation (G. Simonneau et al., 2017). Theoretical background 11 Figure 2: CTEPH as a dual compartment disease. Major vessel remodelling and microvascular arteriopathy characterized by medial hypertrophy, intimal thickening, intimal fibrosis and plexiform lesions CTEPH diagnosis is confirmed if after 3 months of effective anticoagulation, the patient has mPAP greater or equal to 20 mmHg, pulmonary capillary wedge pressure (PCWP) smaller or equal to 15 mmHg and at least one mismatched segmental perfusion defect (I. M. Lang et al., 2016; I. M. Lang, Pesavento, Bonderman, & Yuan, 2013; Gérald Simonneau et al., 2019). The cut-off inclusion criteria is based on different scanning tests, such as multidetector computed tomography angiography or pulmonary angiography and most importantly ventilation/perfusion (V′/Q′) scanning (Galiè et al., 2009). V′/Q′ scanning is used to distinguish between CTEPH and PAH, as PAH patients will have normal or small peripheral perfusion defects (N. H. Kim et al., 2013). The clinical signs of CTEPH are not specific (I. M. Lang & Madani, 2014), therefore setting on the early diagnosis is challenging (P. F. Fedullo, Augur, Kerr, & Rubin, 2001). Clinically significant pulmonary hypertension can manifest months or years later, after the “honeymoon period” for the majority of the patients (McNeil & Dunning, 2007). After the disease has been established, as in other PH forms, patients experience progressive dyspnoea on exertion as a prevailing symptom. Additionally, patients might present fatigue, syncope, haemoptysis, and signs of right-heart failure (Marius M. Hoeper et al., 2014). Despite the fact that estimating the overall incidence and prevalence of CTEPH correctly has been a challenging process, prospective studies are reporting the incidence of CTEPH after acute PE between 0.4 and 9.1% (I. M. Lang & Madani, 2014). The prevalence of CTEPH in overall population is estimated at 17-20 per million (I. M. Lang et al., 2016). Theoretical background 12 1.5.1. CTEPH classification The treatment of patients with CTEPH is largely contingent on eligibility of the patient for surgical excision and/or balloon angioplasty. Recently, a refined classification of surgical CTEPH has been introduced and proposed by University of California, San Diego - (UCSD) (unpublished data), presented at the International CTEPH conference 2017 (Jenkins, Madani, Fadel, D'Armini, & Mayer, 2017). According to this classification, based on location of the disease and degree of difficulty in dissection/excision/resection, there are “levels” of thromboembolic disease (Table 2). Table 2: A proposed new surgical classification of CTEPH, University of California, San Diego, preliminary results (M. Madani, Ogo, & Simonneau, 2017) Surgical level Location of the thromboembolic disease 0 No evidence of CTE (chronic thromboembolic disease) I CTE at the level of main pulmonary arteries II CTE at the level of lobar or intermediate arteries III CTE at the segmental level IV CTE at the subsegmental level 1.5.2. Current treatment for CTEPH The management of the therapeutic strategies for each individual patient is proceeded in expert centres, supported by a multidisciplinary team of surgeons, cardiologists, pulmonologists and radiologists (Dartevelle et al., 2004; M. M. Hoeper, Mayer, Simonneau, & Rubin, 2006). Depending on the clinical manifestation, as CTEPH represents dual compartment disease, patients with major vessel obliterations are endorsed for surgical therapy; while patients experiencing distal vascular arteriopathy, not accessible to surgery are referred to medical therapy or non-invasive interventions (Figure 3). Theoretical background 13 Figure 3: The treatment options for chronic thromboembolic pulmonary hypertension (CTEPH) (M. Madani et al., 2017, p. 4) Reproduced with permission of the © ERS 2019: European Respiratory Review 26 (146) 170105; DOI: 10.1183/16000617.0105-2017 Published 20 December 2017 1.5.2.1. Pulmonary endarterectomy The surgical resection known as pulmonary endarterectomy (PEA) comprises of full bilateral endarterectomy in order to remove the scaring obstruction at the level of intima-media, proximally in the main, lobar and segmental arteries; while mid-segmental and sub-segmental affected areas require experienced surgeon or are technically inoperable (Galiè et al., 2016; Jenkins, 2015; M. Madani et al., 2017). The procedure is performed under deep hypothermic circulatory arrest to provide clear operating field and protect the deterioration of the brain function (Galiè et al., 2016; M. Madani et al., 2017). Many parameters influence whether patients are available for surgery, at first their age and general health (Jenkins et al., 2017). Furthermore, considering the hemodynamic measures, assessed by right heart catheterization, patients with high PVR, have greater mortality risks, yet, show better improvement after PEA (Jenkins et al., 2017). Once the right place for resection is identified, the surgeon “cleaves/scrap” the obstruction till smooth vessel wall, accessible for continual blood flow (M. M. Madani & Jamieson, 2006). Data regarding the clinical outcomes after PEA, indicate not only hemodynamic enhancement, but higher 10- year post-surgical survival rates as well as improvement of the WHO (World Health Organization) functional class (Cannon et al., 2016; I. M. Lang et al., 2013; Mayer et al., 2011; Skoro-Sajer et al., 2007). 1.5.2.2. Balloon angioplasty Emerging therapeutic strategy, termed as percutaneous balloon pulmonary angioplasty (BPA) has been proven recently as a considerable measure for patients with inoperable CTEPH, Theoretical background 14 patients with limited benefit from PEA or with persistent or recurrent PH after PEA (I. Lang et al., 2017). BPA is usually accompanied by a medicated therapy. BPA as non-invasive, catheter based intervention in comparison to PEA and is performed to open distal obstructed vessels or widen stenotic lesions, in order to improve haemodynamics and pulmonary perfusion, ultimately preventing right ventricular failure (I. Lang et al., 2017). A balloon is inserted via the femoral or jugular vein at the site of the obstruction using a guide wire with a help of imaging techniques and inflated to break the webs and bands (Ogawa & Matsubara, 2015; Ogo et al., 2017). Improvements in clinical parameters and haemodynamics, as well as the functional class and the exercise capacity are notable after successful single or multiple session/s of BPA (Ogo et al., 2017). BPA is not a risk-free procedure as pulmonary artery injury and haemorrhage can occur very easily (Inami et al., 2015). Despite that, further development of BPA and/or bridging it with PEA allows expanded application for delicate patients experiencing both proximal and distal vascular lesions (I. Lang et al., 2017). 1.5.2.3. Medical therapy Presence of microvascular disease in inoperable CTEPH patients has been a challenging issue when designing the therapeutic strategy of each individual patient. As histopathological changes described in CTEPH are similar to those of PAH, medical treatment with PAH approved drugs targeting nitric oxide, endothelin and prostacyclin pathway have been evaluated in CTEPH patients (Moser & Bioor, 1993; J. Pepke-Zaba, Ghofrani, & Hoeper, 2017). Similar to PAH, NO levels have been reduced in CTEPH patients. Importantly, sGC stimulator, known as riociguat is used to compensate the loss of NO by further activating the production of cyclic guanosine monophosphate (cGMP), a secondary signalling molecule. The final consequence of increased cGMP is decreased intracellular calcium and smooth muscle relaxation (Klinger, Abman, & Gladwin, 2013; Tonelli, Haserodt, Aytekin, & Dweik, 2013). The results of the CHEST-1 study suggest improvement in PVR, N-terminal pro-brain natriuretic peptides and WHO functional class in patients with CTEPH. Furthermore, and most striking, an improvement in the 6-min walking distance in contrast to placebo treated CTEPH patients has been noted (H. A. Ghofrani et al., 2013). Theoretical background 15 Reesink et al. (2006) reported deteriorated endothelin pathway in patients with CTEPH. Plasma levels of ET-1 are augmented and this correlates with the haemodynamic parameters and severity of the disease. ET-1, per se is a potent vasoconstrictor, contributing to proliferation of smooth muscle cells in both, proximal and distal CTEPH, as ET-1 receptors have been identified in PEA surgical dissection (Southwood et al., 2016). Endothelin receptor antagonists are well established for treatment of patients with PAH, while they aren’t licenced yet for CTEPH patients, but rather used “off-label” (Southwood et al., 2016). Bosentan significantly improved the PVR and cardiac index, although it failed because it didn`t meet the criteria for the 6-min walking test. However, another endothelin receptor antagonist, macitentan undoubtedly improved cardiopulmonary haemodynamics and clinical variables in patients with inoperable CTEPH, regardless of the use of PAH therapies at baseline (MERIT-1 study) (H.- A. Ghofrani et al., 2017). Prostacyclin levels in PAH patients are reduced (Irene M. Lang & Gaine, 2015), therefore drugs targeting the prostacyclin pathway have been used. A little less is known on the efficacy of prostacyclin therapies in CTEPH patients, particularly because the lack of defining the subgroup in the clinical trials. However, within a PH study, effect of inhaled iloprost was evaluated in inoperable CTEPH patients and the general outcome confirmed the positive effect in 6 min walking distance and the functional class (Olschweski et al., 2002). 1.5.2.4. Supportive medical therapy Lifelong anticoagulation therapy, use of antidiuretics and oxygen in case of hypoxemia, are indispensable for patients with CTEPH, even after PEA (J. Pepke-Zaba et al., 2017). Despite the fact that, there are no randomized controlled clinical trials, the reasoning behind is to prevent in situ thrombosis or recurrent venous thromboembolism (VTE) (Joanna Pepke-Zaba, Jansa, Kim, Naeije, & Simonneau, 2013; Wilkens et al., 2018). According to the Updated Recommendations from the Cologne Consensus Conference 2018, both coumarin-derived and non-vitamin-K-dependent anticoagulants are used as an additional therapy in the post-acute phase of a PE (Wilkens et al., 2018). So far, as stated in the current guidelines by Wilkens et al. (2018), vitamin K antagonists (especially warfarin) are still considered the standard supportive treatment for CTEPH. The need of bypassing the limitations of vitamin K antagonists, accustomed the usage of new oral anticoagulants such as dabigatran (a direct thrombin inhibitor) and rivaroxaban, apixaban, and edoxaban (direct activated factor X inhibitors). The advantages of the new oral anticoagulants Theoretical background 16 are that they do not require laboratory monitoring, have limited food and drug interactions, and doses are convenient for most of the patients (Roca & Roca, 2015). Furthermore, a study conducted by Gavilanes-Oleas et al. (2018) reported the use of new direct oral anticoagulants on twenty patients with CTEPH monitored for 21 months. In this time period, beyond a major bleeding caused by a traumatic fall there was no recurrence of VTE (Gavilanes-Oleas et al., 2018). In the long run, extensive clinical studies can provide detailed knowledge on the safety and the conventional use of these particular anticoagulants. As mentioned before, CTEPH diagnosis is based on measures of the mPAP > 20 mmHg by the latest suggestion from the 6th WSPH Task Force on PH diagnosis and classification) and PCWP ≤ 15mmHg, after ≥ 3 months effective anticoagulation and one perfusion defect, at most (Galiè et al., 2016; I. M. Lang et al., 2013; Gérald Simonneau et al., 2019). It is well known that patients with surgically assessable obstructions have greater chance in functional and improvement of the hemodynamic parameters (J. Pepke-Zaba et al., 2017). However, according to the international registries data, 10-50% of the CTEPH patients with distal vascular arteriopathy aren`t qualified for a surgical dissection of the organized vascular obstruction. (Mayer et al., 2011; Peacock, Simonneau, & Rubin, 2006; J. Pepke-Zaba et al., 2011). Significant portion of patients, with consideration of approximately 50%, endure a persistent or recurrent pulmonary hypertension, even after successful surgery and may require further treatment (Cannon et al., 2016; J. Pepke-Zaba et al., 2017). In the past few years, the treatment of operable and non-operable CTEPH patients evolved, as surgical techniques advance and the medical and percutaneous interventions expand. Nonetheless, even after supposedly successful PEA or BPA, CTEPH patients can suffer further from persistent or recurrent PH. Persistent PH may evolve from incomplete removal of more distal vascular obstruction or presence of microvascular disease in patients with proximal disease (Jenkins, 2015). Recurrent PH may be a consequence of poor anticoagulation and distal arteriopathy (Cannon et al., 2016). 1.5.3. Pathophysiology of CTEPH Current evidence on the pathogenesis of CTEPH, suggests the unlikelihood that a single factor, but rather a combination of factors causing chronic vascular scaring, that subsequently leads to PH and RV dysfunction (Matthews & Hemnes, 2016). The initial event indeed, is an acute PE, following venous thromboembolism (VTE) (P. Fedullo, Kerr, Kim, & Auger, 2011). The incomplete resolution of the acute PE is advancing into occlusive vascular remodelling of Theoretical background 17 proximal and segmental vessels, displayed by the presence of intraluminal webs and bands, stenotic lesions etc. (Marius M. Hoeper et al., 2014). The “completely occluded” arterial tree, distal to the organized thrombus is not affected by the high pressure, while the non-completely (stenotic, patent, open) occluded arterial tree is exposed to shear stress, leading to an increased PVR and secondary pulmonary arteriopathy and ultimately to CTEPH (Figure 4) (Southwood et al., 2016). Figure 4: Schematic representation of the current pathophysiological concept of CTEPH. Reprinted with permission of the American Thoracic Society. Copyright © 2019 American Thoracic Society (I. M. Lang et al., 2016) Reproduced with permission of the © ERS 2019: European Respiratory Review 26 (143) 160112; DOI: 10.1183/16000617.0112-2016 Published 29 March 2017 The factors affecting the incomplete resolution and the formation of the chronic vascular scaring are as follows: Inherited thrombophias. Mutations of factor V Leiden, the prothrombin, anti-thrombin, protein C and S are inherent in the development of VTE (Cohn, Roshani, & Middeldorp, 2007), although their contribution to the progression to CTEPH is not very well understood. Wong, Szydlo, Gibbs, and Laffan (2010), demonstrated that there are no differences in the prevalence of the indicated mutations, except for a higher incidence of the factor V in a subset of Caucasian CTEPH patients, in comparison to patients within other PH groups. Other studies evaluated statistically non-significant association in CTEPH with the general population for factor V (I. Lang, 2010), suggesting the absence of the cause and effect between inherited thrombophilias and CTEPH. Theoretical background 18 Acquired thrombophilias. Antiphospholipid antibodies (APA) and anticardiolipin antibodies are associated with various medical conditions, being specific for anionic phospholipids, phospholipid-binding proteins, or phospholipid-protein complexes (Parthvi et al., 2017). APA are the most prevalent aberration contemplating approximately 20% of the CTEPH cases, in contrast to 10% found in IPAH patients (Wolf et al., 2000). Given that the CTEPH patients had higher titers of APA than IPAH patients, their prothrombic character is quite significant in the initiation and the development of the disease. A study conducted between four European centres, revealed a prevalence of 10% among the CTEPH patient population, in comparison to 4% of the group of non-thromboembolic PH (Bonderman, Wilkens, Wakounig, Schafers, et al., 2009). Further research on the presence of lupus anticoagulant has confirmed that 10.6% of the CTEPH patients are positive for its presence. Elevated factor VIII levels. Factor VIII (antihemophilic factor) is a protein from the coagulation cascade, which has an ability to facilitate the factor IXa-mediated activation of factor X. The mechanism of action is not very well understood, while the major effects is to increase the rate of the reaction. Factor VIII possess no enzymatic activity, has capacity to be activated by thrombin and factor Xa and inactivated by activated protein C and by human antibodies to factor VIII (Chavin & Weidner, 1984). In plasma, factor VIII forms a complex with von Willebrand factor (vWf) as his carrier protein (Vlot et al., 1996). As a response to an injury, factor VIII dissociates from its carrier, being released in the plasma, contributing to clot formation (Sharma & Lang, 2018). Elevated levels of factor VIII has been described to contribute to a single and/or recurrent venous thromboembolic events (Kyrle et al., 2000). Aligned with that, study has presented that in comparison to healthy controls and non- thromboembolic PH patients, the levels of factor VIII were elevated in CTEPH patients (Bonderman et al., 2003). Whether the elevated factor VIII levels are the cause or the consequence of CTEPH, requires further investigation (Matthews & Hemnes, 2016). Inefficient/defective fibrinolysis and fibrinogen. Several thrombotic disorders, such as coronary artery disease (Hamsten, Wiman, de Faire, & Blombäck, 1985; Paramo, Colucci, & Collen, 1985) and VTE (Nillson, Ljungnér, & Tengborn, 1985) are represented by reduction in the plasma fibrinolysis. This modification is most likely due to an increased plasma plasminogen activator inhibitor (PAI-1) activity or deficient tissue plasminogen activator (t- PA) (Olman et al., 1992). Fibrinolysis as an antagonistic process of fibrin formation, comprises of synergistic cleavage of cross-linked fibrinogen, via plasminogen, t-PA, PAI-1 and other interaction proteins. Tranexamic acid, as an antifibrinolytic agent was used in a canine model Theoretical background 19 of acute PE, where a persistent elevation of PAP and thrombus non-resolution was noted (Moser et al., 1991). On one hand, plasma levels of PAI-1 and t-PA were elevated in patients with CTEPH in contrast to the corresponding controls, although there was no difference in the enzymatic activity between them (Olman et al., 1992). Induction of venous occlusion in CTEPH patients, has been shown to result in increased t-PA antigen and PAI-1 activity, suggesting that the defective or deficient fibrinolysis might not be one of the major contributors to the development of CTEPH (Olman et al., 1992). Another consequence is, once formed, the thromboemboli is resistant to fibrinolysis (Nijkeuter, Hovens, Davidson, & Huismann, 2006). On the other hand, patients with CTEPH often bear a point mutation of fibrinogen Aα- Thr312Ala (Le Gal et al., 2007; Li et al., 2013; Suntharalingam et al., 2008), which leads to formation of fibrin clots and increased cross-linking of α-chains (Toshner & Pepke-Zaba, 2014). Further, other β-chain mutations, P235L/γR357W,P235L/γY114H and P235L, along with α-chain mutations L69H and R554L were found in CTEPH patients (Morris et al., 2009). Taken together, all fibrin defects in CTEPH patients lead to ineffective thrombolysis, thus directly influencing the thrombus non-resolution (I. M. Lang et al., 2016; Marsh, Chiles, Liang, & Morris, 2013). Endothelial dysfunction. Endothelial cells (ECs) isolated from PEA biorepositories, possess cobblestone morphology and are positive for endothelial markers. The same study confirmed their hyperproliferative and apoptosis resistant phenotype, reduced tube formation capacity, lower rates of mitochondrial membrane potential and reduction of mitochondrial content in comparison to control PAECs (Tura-Ceide et al., 2016). In another study, ECs isolated from CTEPH were compared to donor ECs for the production of PAI-1 and t-PA, and there were no significant differences in the basal t-PA levels as well as the PAI-1 activity. Similar response was obtained when the ECs were exposed to thrombin (I. M. Lang, Marsh, Olman, Moser, & Schleef, 1994). As a response to increased pressure, inflammation or acute lung injury, endothelial permeability upsurge (Bogatcheva, Garcia, & Verin, 2002; J. Garcia & Schaphorst, 1995). Share stress is yet another factor affecting the ECs function, influencing their production of vasoactive factors (Sacks et al., 2006). Apart of its function in a thrombus formation, it has been reported that, α-thrombin increased albumin clearance via the endothelial monolayer (J. G. Garcia et al., 1986). In these circumstances, ECs became shorter, which leads to formation Theoretical background 20 of intracellular gaps and ECs permeability, through activation of T-type voltage gated Ca2+ channels and increase of cytosolic calcium (Moser & Bioor, 1993; Wu et al., 2003). ECs isolated from PEA material, indeed confirm the different calcium homeostasis in comparison to control hPAECs. The positive identification of the angiostatic factors, such collagen type I, platelet factor 4 (PF4) and interferon-γ-inducible 10 kDa (IP-10) certainly confers towards endothelial dysfunction (Zabini et al., 2012). Platelets aggregation. Thyroid hormone replacement therapy and splenectomy are platelet- activating conditions, suggesting the platelet involvement in the pathogenesis of CTEPH (Bonderman, Wilkens, Wakounig, Schafers, et al., 2009; J. Pepke-Zaba et al., 2011). Platelet activation after splenectomy, per se, has been identified by an increase of thrombus volume in a mouse model of defective thrombus resolution (Frey et al., 2014). Platelet microparticles has been shown to be generated in splenectomised patients in the same study, in contrast to non- splenectomised CTEPH patients. Further research on the potential role of platelets in the development of CTEPH is presenting that the platelets from CTEPH or PAH were activated compared to non-PH controls by their increased surface expression of p-selectin, PAC-1 binding and the GTP-bound GTPase RhoA, involved in their aggregation (Yaoita et al., 2014). On the other hand, patients with CTEPH have a decreased platelet count, higher mean platelet volume and decreased aggregation in response to agonists (Remková, Šimková, & Valkovičová, 2015). These findings suggest the possible contribution of the dysfunctional platelets in the pathogenesis of CTEPH, given that platelets are also activated in PAH patients. Medical conditions associated with CTEPH. Splenectomy. An increased prevalence of splenectomy as a risk factor for CTEPH has been reported in a retrospective study by Jaïs and colleagues (Jais et al., 2005). History of splenectomy has been detected in 8.6% (95% CI 5.2 to 12.0) of patients with CTEPH, in contrast to 2.5% (96% CI 0.7 to 4.4) and 0.56% (95% CI 0 to 1.6) in IPAH and other pulmonary conditions, respectively. The most common reason for splenectomy is a ruptured spleen, which is often caused by an abdominal injury, some blood disorders, certain cancers, infection, and noncancerous cysts or tumours. The mechanisms associating splenectomy with CTEPH are still not well understood. One of the possible reasons is the loss of splenic filtering which leads to abnormal circulating erythrocytes, thrombin generation (Kuypers, 1998) and pro- inflammatory cytokine expression (Moshtaghi-Kashanian, Gholamhoseinian, Hoseinimoghadam, & Rajabalian, 2006). After splenectomy, platelet count returns to normal, and the risk of thromboembolism and ultimately CTEPH is noticeable from 7 to 25 years later Theoretical background 21 (Jais et al., 2005), suggesting that increased risk of CTEPH cannot be addressed only by elevated platelet count. Research evidence is furthermore, suggesting the phospholipid accumulation in PEA thrombi after splenectomy, as well as in a mouse model of venous thromboembolism undergoing splenectomy (Frey et al., 2014). Taken together, the role of the phospholipid factor, post splenectomy in supplemental development of pathophysiological conditions and CTEPH cannot be undermined. Ventriculoatrial (VA) shunts and infected pacemakers. Staphylococcal infection may play a significant role, as patients with CTEPH have been described to have VA shunts and infected pacemakers with higher frequency than other PH etiologies. Despite the fact that, a precise mechanism connecting VA shunts and CTEPH is lacking, Bonderman et al. (2008) suggested that staphylococcal infection can delay thrombus resolution in addition to upregulation of profibrotic molecules, in a murine model of venous thrombosis. Non-O blood group. Individuals with non-O blood group have been demonstrated to bear increased levels of von Willebrand factor (vWf), factor VIII, p-selectin and TNF-α, all highly susceptible for VTE and CTEPH (Gándara et al., 2013). Although, the underlying mechanisms addressing the association between ABO blood group and factor VIII-vWf are unclear, studies have shown that healthy, O-blood group individuals have 25-30% lower levels of plasma vWf, than non-O carriers (Gill, Endres-Brooks, Bauer, Marks, & Montgomery, 1987; McCallum, Peake, Newcombe, & Bloom, 1983; Mohanty et al., 1984; Orstavik et al., 1985). Another study has suggested that the modulated glycosylation, can affect the vWf clearance and further on the plasma levels of factor VIII-vWf complex (Gallinaro et al., 2008). Chronic inflammatory diseases. Finally, several inflammatory diseases such as inflammatory bowel disease (IBS), osteomyelitis, venous ulcers, thyroid hormone replacement and malignancy have been listed in the literature to have direct effect towards an increased risk of development of CTEPH (Bonderman et al., 2005; Bonderman, Wilkens, Wakounig, Schäfers, et al., 2009; P. Fedullo et al., 2011; N.H. Kim & Lang, 2012). 1.5.4. Histopathology of CTEPH 1.5.4.1. Proximal major vessel obliterations in CTEPH CTEPH is dual vascular disorder, comprising of major and microvascular vessel remodelling. The proximal (major) vessel remodelling represented as persistent organized thrombi at a level of main, lobar and segmental arteries is the initial step in orchestrating the further development of CTEPH (G. Simonneau et al., 2017). The organized thrombi, also called neointima is tightly Theoretical background 22 adhered to the medial layer of the pulmonary arteries, which may completely obstruct the lumen of the artery (I. Lang, 2015). As a consequence, the intimal surface roughens and in order to restore the blood circulation and potentially decrease the PVR, recanalization (bands and webs) takes place (P. F. Fedullo et al., 2001). The intimal layer mainly displays collagen and elastic fibres deposition and presence of α-smooth-muscle actin (α-SMA) positive cells, possibly from SMCs migrated from the media or from circulating progenitor cells (A. L. Firth, Yao, et al., 2010; Owens, Kumar, & Wamhoff, 2004). A clinicopathologic study of 200 consecutive PEA samples revealed the presence of organized thrombi, inflammation, cholesterol clefts, calcification and increased cellularity (Bernard & Yi, 2007). Rozenn Quarck, Wxnants, Verbeken, Meyns, and Delcroix (2015) analysed the major-vessel lesions of CTEPH patients using immunohistochemistry and identified the presence of four types of lesions: neointima, thrombotic, atherosclerotic and recanalized lesions. Regarding the composition of infiltrated cells in the organized thrombi, several studies confirmed presence and similar distribution of macrophages, T- and B- lymphocytes and neutrophils (Arbustini et al., 2002; Bernard & Yi, 2007; Rozenn Quarck et al., 2015). Recanalization, or the neo-angiogenesis is regulated by both pro- and anti-angiogenic factors (Ribatti, 2009). A study reported that several angiostatic factors, such as collagen type I, PF4 and IP-10 are present in PEA material (Zabini et al., 2012). In addition, it is shown that these factors contribute to endothelial dysfunction. In contrast to this study, Naito et al. (2018) has been able to demonstrate that ECs isolated from PEA not only demonstrate high proliferative potential, but also extensive angiogenic capacity. Certain discrepancies appear in regard to the presence of fresh thrombus in PEA material of operable CTEPH patients. Some studies are reporting a high percentage of appearance of fresh clot (Arbustini et al., 2002), while in some, these changes is rarely observed (Blauwet, Edwards, Tazelaar, & McGregor, 2003; Rozenn Quarck et al., 2015), which is most likely a result of different interpretation of the pathologist or the effectiveness of the anticoagulation therapy. 1.5.4.2. Microvascular disease in CTEPH Small vessel arteriopathy (also referred as microvascular disease) in CTEPH was at first described by Moser and Bloor in a lung tissues obtained from biopsy or at autopsy of CTEPH patients (Moser & Bioor, 1993). The histological features of distal pulmonary microvasculopathy include intimal thickening, eccentric intimal fibrosis, intimal fibromuscular Theoretical background 23 proliferation, medial hypertrophy and presence of plexiform lesions (Moser & Bioor, 1993; G. G. Pietra et al., 2004). Distal pulmonary vessels with diameter of 100-500 μm are highly affected by the remodelling, including the vessels with less than 100 μm (I. M. Lang et al., 2016). Hypertrophy and hyperplasia of all three cell types of pulmonary vascular wall (PAECs, PASMCs and PAAFs) and accumulation of endothelial progenitor cells underline the histopathological features of CTEPH (Ogawa et al., 2009; Sakao et al., 2011); (I. Lang, 2015). The vascular remodelling distal to the organized thromboembolic obstruction may be similarly distributed in the lung areas of complete and non-complete occlusion. The areas distal of non- completely occluded arteries are affected by high pressure and share stress, triggering the increase of PVR and at the end leading to CTEPH (G. Simonneau et al., 2017). Figure 5: Microvascular disease in CTEPH affecting the pulmonary arterioles, venules and capillaries. Presence of hypertrophied vasa vasorum and bronchial arteries showing the anastomoses between pulmonary and systemic circulation (G. Simonneau et al., 2017). Furthermore, the importance of the bronchial circulation in pulmonary vascular and airways disease has been appreciated. In patients with CTEPH anastomoses between bronchial and pulmonary arterial circulation appears via hypertrophied bronchial arteries and vasa vasorum (Figure 5) (P. Dorfmüller et al., 2014). These anastomoses are similar lesions as in capillary haemangiomatosis and pulmonary veno-occlusive disease (PVOD). Although their function is not yet fully understood, it is estimated that they help the perfusion and support ischaemic tissue downstream of the proximal obstruction (P. Dorfmüller et al., 2014; G. Simonneau et al., Theoretical background 24 2017). In addition, P. Dorfmüller et al. (2014) confirmed that the disease not only affects the pre-capillary arterioles, but also the post-c