Angiogenesis, apoptosis and re-epithelialization at the foci of recent injury in usual interstitial pneumonia and bronchiolitis obliterans organizing pneumonia

Elisa Lappi-Blanco Sequeiros

Department of Pathology, University of Oulu

Abstract

Idiopathic usual interstitial pneumonia (UIP) and bronchiolitis obliterans organizing pneumonia (BOOP) are fibrous pulmonary disorders in both of which there is newly formed connective tissue in distal air spaces. UIP is a progressive and usually fatal lung disease without any efficient treatment, while the prognosis of BOOP is good. In both diseases, an injury of the alveolar epithelium and its basement membrane (BM) leads to migration of fibroblasts and myofibroblasts into air spaces and production of extracellular matrix by these cells. In UIP, the newly formed intraluminal connective tissue lesions cause fusion of alveolar structures and interstitial remodeling, while in BOOP the newly formed connective tissue may resolve completely.

One of the major aims of the research on pulmonary fibrosis is to define the mechanisms that lead to persistence of the newly formed connective tissue and thus to irreversible fibrosis in UIP. The aim of the present study was to compare the extent of capillarization, apoptotic activity and re-epithelialization of the newly formed connective tissue in BOOP and UIP. The number of capillaries per tissue surface area was measured. Furthermore, the expression of angiogenic growth factors vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) was evaluated in the same areas, in addition to the expression of Flt-1 and Flk-1, which serve as receptors for VEGF. Apoptotic activity was analyzed using TUNEL-method, and the immunohistochemical expression of apoptosis regulating proteins bcl-2, mcl-1, and bax was studied. Finally, the extent of re-epithelialization was studied with the immunohistochemical and ultrastructural localization of laminin-5 γ 2 chain, and the sites of synthesis of laminin-5 γ 2 chain mRNA.

In BOOP, an efficient repair process with good capillarization along with high expression of VEGF and bFGF, and orderly re-epithelialization of the newly formed connective tissue takes place after lung injury. The apoptotic activity of the newly formed connective tissue is also high, presumably leading to resolution of the intraluminal connective tissue in BOOP. In UIP, the newly formed connective tissue showed poor capillarization, inadequate re-epithelialization and low apoptotic activity. The results suggest disturbed or delayed repair process in UIP, contributing to irreversible interstitial fibrosis and remodeling.

Dedication

To my family

Table of Contents

Acknowledgements

Abbreviations

List of original articles

1. Introduction

2. Review of the literature

2.1. Function and anatomy of the lung

2.1.1. Normal structure and function of alveoli

2.2. Pulmonary fibrosis

2.2.1. Idiopathic interstitial pneumonias
2.2.2. Idiopathic usual interstitial pneumonia
2.2.3. Bronchiolitis obliterans organizing pneumonia
2.2.4. Pathogenesis of pulmonary fibrosis
2.2.5. Pathogenesis of intraluminar fibrosis in BOOP and UIP

2.3. Angiogenesis

2.3.1. Vascular endothelial growth factor-A
2.3.2. Flt-1 and Flk-1
2.3.3. Basic fibroblast growth factor
2.3.4. Angiogenesis in pulmonary fibrosis

2.4. Apoptosis

2.4.1. Pathways of apoptotic signaling
2.4.2. Analysis of apoptosis
2.4.3. Apoptosis in pulmonary fibrosis

2.5. Re-epithelialization

2.5.1. Laminins

2.6. Evolving theory of the pathogenesis of idiopathic UIP

3. Aims of the study

4. Materials and methods

4.1. Tissue specimens, patients and follow-up information

4.2. Methods

4.2.1. Immunohistochemistry for paraffin sections (I–IV)
4.2.2. Immunohistochemistry for frozen sections (Mab 6C12) (IV)
4.2.3. Assessment of vascular density in newly formed intraluminal connective tissue (I)
4.2.4. Analysis of the results of the immunostainings (II–IV)
4.2.5. 3´-end labeling of DNA in apoptotic cells and assessment of the apoptotic index (III)
4.2.6. In situ hybridization for laminin-5 γ 2 chain mRNA (IV)
4.2.7. Immunoelectron microscopy of laminin-5 γ 2 chain (IV)
4.2.8. Statistical analyses

5.1. Capillarization in the newly formed connective tissue (I)

5.1.1. General findings
5.1.2. Number of capillaries in newly formed connective tissue

5.2. Immunohistochemical expression of VEGF and bFGF (II)

5.2.1. VEGF and bFGF expression in intraluminal connective tissue
5.2.2. VEGF and bFGF expression outside the intraluminal connective tissue

5.3. Immunohistochemical expression of Flt-1 and Flk-1 (II)

5.3.1. Flt-1 and Flk-1 expression in intraluminal connective tissue lesions
5.3.2. Flt-1 and Flk-1 expression outside intraluminal connective tissue

5.4. Apoptotic index (III)

5.5. Immunohistochemistry for bcl-2 family proteins (III)

5.6. Immunohistochemical expression of cytokeratin (clone MNF 116): re-epithelialization (IV)

5.7. Immunohistochemical expression of laminin γ 2 chain (IV)

5.8. In situ hybridization for laminin-5 γ 2 chain mRNA (IV)

5.9. Immunoelectron microscopy of laminin-5 γ 2 chain (IV)

6. Discussion

6.1. Capillarization of newly formed intraluminal connective tissue in BOOP and UIP

6.1.1. Expression of VEGF, bFGF and receptors of VEGF in newly formed intraluminal connective tissue in BOOP and UIP

6.2. Apoptotic activity in newly formed intraluminal connective tissue in BOOP and UIP

6.2.1. Expression of apoptosis regulating proteins bcl-2, mcl-1 and bax in newly formed intraluminal connective tissue in BOOP and UIP

6.3. Re-epithelialization of newly formed intraluminal connective tissue with laminin-5 γ 2 chain in BOOP and UIP

7. Conclusions

List of Tables
1. Previous classifications of idiopathic interstitial pneumonias.
2. Histologic and clinical classification of idiopathic interstitial pneumonias (ATS/ERS 2002).
3. The bcl-2 protein family. Modified from Antonsson 2001 and Zhang et al. 2001.
4. Antigens and their respective antibodies used in the studies.
5. The number of capillary cross sections per square millimeter (no/mm²) of the newly formed connective tissue in BOOP and in UIP.
6. The number of VEGF and bFGF positive cells as percentage of the whole cell number in the newly formed connective tissue in BOOP and in UIP.
7. Staining pattern of VEGF in BOOP and in UIP.
8. Staining for bcl-2 family proteins in BOOP and UIP used in the study. No statistical difference was found between BOOP and UIP and therefore the staining patterns for BOOP and UIP were combined.

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