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## Meet the editor

Jelena Stojšić, PhD has been a thoracic pathologist for 25 years. Her major interest is in the field of lung pathology, particularly lung cancer and interstitial lung diseases. She wrote her PhD thesis on reconizing the link between molecular pathways in lung cancer and transport pumps involved in reflux of chemotherapeutics from malignant cells. In daily practice, Dr. Stojšić uses morphology and immunoprofiling to diagnose lung cancer and

personalize therapy for her patients.

Contents

*by Jelena Stojšić*

Fibrosis

**Preface III**

**Chapter 1 1**

**Chapter 2 5**

**Chapter 3 23** Lung Transplantation: A Final Option for End-Stage Interstitial Lung Diseases

**Chapter 4 39**

**Chapter 5 55** The Role of Pulmonary Rehabilitation in Patients with Idiopathic Pulmonary

*by Koichi Takagi, Munekazu Yamakuchi, Teruto Hashiguchi and Hiromasa Inoue*

Introductory Chapter: Diagnosis of Interstitial Lung Disease

*by Elena Dantes, Emanuela Tudorache and Milena Adina Man*

The Role of miRNAs in Idiopathic Pulmonary Fibrosis

*by Mohammed Fakhro and Sandra Lindstedt*

Lung Transplant for Interstitial Lung Diseases *by Brandon Nokes, Eugene Golts and Kamyar Afshar*

### Contents

Preface

Interstitial lung diseases are rare diffuse lung diseases characterized by specific radiological and pathohistological findings. This book is an attempt to better understand these very specific lung diseases at the molecular level and thus understand and interpret changes in the lung parenchyma and correlate with radiological and pathological findings and therapeutic modalities. This book also includes information on lung transplantation as the most current approach to the treatment of interstitial lung diseases in all stages including the end stage. A special chapter is devoted to the physical therapy of patients with these lung diseases. This book is intended for all doctors who deal with diagnosis and treatment of interstitial lung

I would like to thank all my colleagues who devoted their effort and time in shaping

**Jelena Stojšić, MD PhD**

Service of Pathology, Clinical Center of Serbia,

Belgrade, Serbia

Department of Thoracopulmonary Pathology,

diseases, as a manual for everyday and practical literature.

and writing their chapters. I wish them great success in their work.

## Preface

Interstitial lung diseases are rare diffuse lung diseases characterized by specific radiological and pathohistological findings. This book is an attempt to better understand these very specific lung diseases at the molecular level and thus understand and interpret changes in the lung parenchyma and correlate with radiological and pathological findings and therapeutic modalities. This book also includes information on lung transplantation as the most current approach to the treatment of interstitial lung diseases in all stages including the end stage. A special chapter is devoted to the physical therapy of patients with these lung diseases. This book is intended for all doctors who deal with diagnosis and treatment of interstitial lung diseases, as a manual for everyday and practical literature.

I would like to thank all my colleagues who devoted their effort and time in shaping and writing their chapters. I wish them great success in their work.

> **Jelena Stojšić, MD PhD** Department of Thoracopulmonary Pathology, Service of Pathology, Clinical Center of Serbia, Belgrade, Serbia

**1**

**Chapter 1**

*Jelena Stojšić*

**1. Introduction**

the diagnosis [5].

**biopsy**

diagnostic procedure [2–4].

Introductory Chapter: Diagnosis

Interstitial lung diseases are rare diffuse lung disease characterized by a specific

clinical picture and radiological (imaging) and pathohistological findings. It is considered that these diseases represent about 15% of all respiratory diseases [1]. Diffuse changes of the lung parenchyma in each type of these diseases are characterized by various morphological patterns which are reflected by a different imaging finding and a specific clinical picture [2–4]. The clinical picture at an early stage of the disease is not specific, and it is hard to suspect interstitial lung disease. Symptoms of interstitial lung disease are dry cough, short breath, fever, and fatigue. A specific high resolution - computed tomographyn (HR-CT) finding indicates an interstitial lung disease which is proven by biopsy. Transbronchial biopsy primarily excludes specific granulomatous lung diseases, primary malignancy and metastatic as well as eosinophilic pneumonia, alveolar proteinosis, and pulmonary histiocytosis. If a bioptized lung sample has nonspecific morphological pattern, it is necessary to perform an open lung biopsy or video-assisted thoracoscopic surgery (VATS). Open lung biopsy procedure requires a multidisciplinary approach that includes a chest surgeon. An agreement on taking large number of lung tissue samples characterized by change evolution increases the efficiency and accuracy of

Integrated clinical and radiological data help the pathologist to establish an accurate diagnosis of the type of interstitial lung disease. Besides pulmonologist, radiologist, and pathologist, microbiologist and immunologist also participate in

**2. Pathohistological diagnosis of interstitial lung disease in open lung**

analysis. Before all, it is necessary to completely instill 10% buffered formalin into the lung parenchyma with a needle for injection but in a moderate amount, in order to not provoke artifacts in the lung parenchyma. In such a manner, alveolar spaces are as if they were in the air-inspiration phase, and also damages on the lung parenchyma caused by a forceps during biopsy performed by a surgeon are avoided. After fixation that lasts 24 h, the entire material is sampled into thin sections which are fixed in the buffered formalin, alcohol, and xylol. After that, they are embedded into paraffin and then cut and stained with a classical hematoxylin-eosin staining. The pathologist at first performs microscopic examination on a low-poweredfield microscope in order to see the distribution of changes, that is, whether they are confluent or not. Then he observes localization of changes and their peribronchial or

It is necessary to take the entire surgical material for processing and microscopic

of Interstitial Lung Disease

#### **Chapter 1**

### Introductory Chapter: Diagnosis of Interstitial Lung Disease

*Jelena Stojšić*

#### **1. Introduction**

Interstitial lung diseases are rare diffuse lung disease characterized by a specific clinical picture and radiological (imaging) and pathohistological findings. It is considered that these diseases represent about 15% of all respiratory diseases [1].

Diffuse changes of the lung parenchyma in each type of these diseases are characterized by various morphological patterns which are reflected by a different imaging finding and a specific clinical picture [2–4]. The clinical picture at an early stage of the disease is not specific, and it is hard to suspect interstitial lung disease. Symptoms of interstitial lung disease are dry cough, short breath, fever, and fatigue. A specific high resolution - computed tomographyn (HR-CT) finding indicates an interstitial lung disease which is proven by biopsy. Transbronchial biopsy primarily excludes specific granulomatous lung diseases, primary malignancy and metastatic as well as eosinophilic pneumonia, alveolar proteinosis, and pulmonary histiocytosis. If a bioptized lung sample has nonspecific morphological pattern, it is necessary to perform an open lung biopsy or video-assisted thoracoscopic surgery (VATS). Open lung biopsy procedure requires a multidisciplinary approach that includes a chest surgeon. An agreement on taking large number of lung tissue samples characterized by change evolution increases the efficiency and accuracy of the diagnosis [5].

Integrated clinical and radiological data help the pathologist to establish an accurate diagnosis of the type of interstitial lung disease. Besides pulmonologist, radiologist, and pathologist, microbiologist and immunologist also participate in diagnostic procedure [2–4].

#### **2. Pathohistological diagnosis of interstitial lung disease in open lung biopsy**

It is necessary to take the entire surgical material for processing and microscopic analysis. Before all, it is necessary to completely instill 10% buffered formalin into the lung parenchyma with a needle for injection but in a moderate amount, in order to not provoke artifacts in the lung parenchyma. In such a manner, alveolar spaces are as if they were in the air-inspiration phase, and also damages on the lung parenchyma caused by a forceps during biopsy performed by a surgeon are avoided. After fixation that lasts 24 h, the entire material is sampled into thin sections which are fixed in the buffered formalin, alcohol, and xylol. After that, they are embedded into paraffin and then cut and stained with a classical hematoxylin-eosin staining.

The pathologist at first performs microscopic examination on a low-poweredfield microscope in order to see the distribution of changes, that is, whether they are confluent or not. Then he observes localization of changes and their peribronchial or perivascular presence or connective subpleural, interlobular, or interalveolar septa. On highermicrosopic microscopic field enlargements, the cellularity of changes is noted, as well as the type of cells that contain changes and then the presence and degree of fibrosis. By connective tissue staining, it is desirable to confirm the degree of fibrosis and with other methods to determine type of cells and presence of microorganisms. Immunohistochemical technique can detect the type of cells in the analyzed changes [5].

On the basis of the overall finding, descriptive diagnosis that correlates with the disease entity is made. Diagnosis must correlate with the clinical and imaging picture by the type of change. For this purpose, current classification of interstitial lung diseases is used [5].

The first used classification of interstitial diseases in different subtypes is according to Liebow and Carrington [6]. By consensus of the American Thoracic Society and European Respiratory Society (ATS/ERS), current classification of interstitial lung diseases is determined. This classification defines different clinicalpathological entities according to clinical, radiological, and histological criteria. By using this classification, it is possible to predict the course of the disease, treatment, and outcome. Therapy and monitoring of the disease course require consultative decisions. The medical advisory board consists of experienced pulmonologists, radiologists, and pulmonary pathologists. If the course of the disease is not satisfactory even with applied therapy, treatment may be evaluated, and in exceptional circumstances, archived tissue samples can be reexamined and diagnosis can be changed [1]. In addition to the therapy, in order to improve lung function which is tested by various functional tests, physical therapy is also desirable.

The ATS/ERS classification of these diffuse lung diseases divides them into those of known and those of unknown etiologies [1]. According to ATS/ERS classification, morphological image in the lung parenchyma should correspond to clinicalpathological diagnosis (in this part of the text, insert **Table 1** from the bottom of the text).

Diffuse diseases of known etiology, such as connective tissue diseases or diseases caused by taking some drugs, are less common. Allergic alveolitis or hypersensitivity pneumonitis is a specific entity of generally known etiology. Diseases of unknown etiology, that is, idiopathic diseases such as idiopathic lung disease, nonspecific interstitial lung disease, desquamative interstitial pneumonia, respiratory bronchiolitis, organized pneumonia, acute interstitial pneumonia, and lymphoid interstitial pneumonia, are more frequent. This group of diseases includes granulomatosis of which the most common is sarcoidosis characterized by the presence of epithelial, non-necrotizing granulomas [1].

#### **Table 1.**

*Morphological image which corresponds to a certain clinical-pathological diagnosis according to the ATS/ERS classification [1].*

**3**

**Author details**

of Serbia, Belgrade, Serbia

Jelena Stojšić

*Introductory Chapter: Diagnosis of Interstitial Lung Disease*

microbiological tests and transbronchial biopsy [7–9].

By using the said formulations, uniformity of the findings is achieved and therapy is specified. If good treatment results fail, it comes to a stage known as "end-stage lung disease." In such a case, lung transplantation is proposed which lately requires a special chapter in each textbook about these diseases. Selection of the transplantation type (single, bilateral, or heart-lungs) depends on the type of interstitial lung disease and other factors. The success of transplantation depends on the type of organism reaction on the presence of the transplanted lung or the type of rejection which can be subacute, acute, and chronic. Changes in rejection reactions are localized around the blood vessels of the lungs or bronchi. The intensity of rejection reaction depends on the extent of the changes spreading in the pulmonary interstitium and the type of inflammatory infiltrate. Prevention and treatment of rejection are regulated by immunosuppressive drugs. Therefore, it is necessary to control the presence of microorganisms in the transplanted lung by

However, the future of the treatment of interstitial lung diseases depends on the molecular changes in the cells found in the lesions. miRNAs profiling in lung tissue in various interstitial lung diseases in a variety of cells that contain lung lesions: endothelium, alveolar epithelium, inflammatory cells, fibroblast and myofibroblast. This profiling can determine the type of therapy and their outcome or success.

Department of Thoracopulmonary Pathology, Service of Pathology, Clinical Center

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.87243*

#### *Introductory Chapter: Diagnosis of Interstitial Lung Disease DOI: http://dx.doi.org/10.5772/intechopen.87243*

*Interstitial Lung Diseases*

lung diseases is used [5].

the text).

perivascular presence or connective subpleural, interlobular, or interalveolar septa. On highermicrosopic microscopic field enlargements, the cellularity of changes is noted, as well as the type of cells that contain changes and then the presence and degree of fibrosis. By connective tissue staining, it is desirable to confirm the degree of fibrosis and with other methods to determine type of cells and presence of microorganisms. Immunohistochemical technique can detect the type of cells in the analyzed changes [5]. On the basis of the overall finding, descriptive diagnosis that correlates with the disease entity is made. Diagnosis must correlate with the clinical and imaging picture by the type of change. For this purpose, current classification of interstitial

The first used classification of interstitial diseases in different subtypes is according to Liebow and Carrington [6]. By consensus of the American Thoracic Society and European Respiratory Society (ATS/ERS), current classification of interstitial lung diseases is determined. This classification defines different clinicalpathological entities according to clinical, radiological, and histological criteria. By using this classification, it is possible to predict the course of the disease, treatment, and outcome. Therapy and monitoring of the disease course require consultative decisions. The medical advisory board consists of experienced pulmonologists, radiologists, and pulmonary pathologists. If the course of the disease is not satisfactory even with applied therapy, treatment may be evaluated, and in exceptional circumstances, archived tissue samples can be reexamined and diagnosis can be changed [1]. In addition to the therapy, in order to improve lung function which is

tested by various functional tests, physical therapy is also desirable.

**Histological pattern Clinicopathological diagnosis**

Organizing pneumonia Cryptogenic organizing pneumonia Diffuse alveolar damage (DAD) Acute interstitial pneumonia Desquamative interstitial pneumonia (DIP) Desquamative interstitial pneumonia

Lymphocytic interstitial pneumonia (LIP) Lymphocytic interstitial pneumonia

Respiratory bronchiolitis Respiratory bronchiolitis-interstitial lung disease

*Morphological image which corresponds to a certain clinical-pathological diagnosis according to the ATS/ERS*

(RB-ILD)

Nonspecific interstitial pneumonia (NSIP) Nonspecific pneumonia

epithelial, non-necrotizing granulomas [1].

The ATS/ERS classification of these diffuse lung diseases divides them into those of known and those of unknown etiologies [1]. According to ATS/ERS classification, morphological image in the lung parenchyma should correspond to clinicalpathological diagnosis (in this part of the text, insert **Table 1** from the bottom of

Diffuse diseases of known etiology, such as connective tissue diseases or diseases

Usual interstitial pneumonia (UIP) Idiopathic pulmonary fibrosis (IPF)/cryptogenic fibrosing alveolitis (CFA)

caused by taking some drugs, are less common. Allergic alveolitis or hypersensitivity pneumonitis is a specific entity of generally known etiology. Diseases of unknown etiology, that is, idiopathic diseases such as idiopathic lung disease, nonspecific interstitial lung disease, desquamative interstitial pneumonia, respiratory bronchiolitis, organized pneumonia, acute interstitial pneumonia, and lymphoid interstitial pneumonia, are more frequent. This group of diseases includes granulomatosis of which the most common is sarcoidosis characterized by the presence of

**2**

**Table 1.**

*classification [1].*

By using the said formulations, uniformity of the findings is achieved and therapy is specified. If good treatment results fail, it comes to a stage known as "end-stage lung disease." In such a case, lung transplantation is proposed which lately requires a special chapter in each textbook about these diseases. Selection of the transplantation type (single, bilateral, or heart-lungs) depends on the type of interstitial lung disease and other factors. The success of transplantation depends on the type of organism reaction on the presence of the transplanted lung or the type of rejection which can be subacute, acute, and chronic. Changes in rejection reactions are localized around the blood vessels of the lungs or bronchi. The intensity of rejection reaction depends on the extent of the changes spreading in the pulmonary interstitium and the type of inflammatory infiltrate. Prevention and treatment of rejection are regulated by immunosuppressive drugs. Therefore, it is necessary to control the presence of microorganisms in the transplanted lung by microbiological tests and transbronchial biopsy [7–9].

However, the future of the treatment of interstitial lung diseases depends on the molecular changes in the cells found in the lesions. miRNAs profiling in lung tissue in various interstitial lung diseases in a variety of cells that contain lung lesions: endothelium, alveolar epithelium, inflammatory cells, fibroblast and myofibroblast. This profiling can determine the type of therapy and their outcome or success.

#### **Author details**

Jelena Stojšić

Department of Thoracopulmonary Pathology, Service of Pathology, Clinical Center of Serbia, Belgrade, Serbia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the idiopathic inetrstitial pneumonias. American Journal of Respiratory and Critical Care Medicine. 2002;**165**:277-304

[2] Cottin V. Interstitial lung disease. European Respiratory Review. 2013;**22**:26-32

[3] Flaherty KR, King TE Jr, Raghu G, Lynch JP 3rd, Colby TV, Travis WD, et al. Idiopathic interstitial pneumonia: What is the effect of a multidisciplinary approach to diagnosis? American Journal of Respiratory and Critical Care Medicine. 2004;**170**:904-910

[4] Thomeer M, Demedts M, Behr J, Buhl R, Costabel U, Flower CD, et al. Multidisciplinary observer agreement in the diagnosis and of idiopathic pulmonary fibrosis. The European Respiratory Journal. 2008;**31**:585-591

[5] Leslie KO. Pathology of interstitial lung disease. Clinics in Chest Medicine. 2004;**25**:657-703

[6] Liebow AA, Carringotn CB. In: Simon M, Potchen EJ, Lemay M, editors. The Interstitial Pneumonias, Frontiers of Pulmonary Radiology. New York: Grune and Strattin; 1969. pp. 102-141

[7] Roden AC, Aisner DL, Allen TC, Aubry MC, Barrios RJ, Beasley MB, et al. Diagnosis of acute cellular rejection and antibody-mediated rejection on lung transplant biopsies: A perspective from members of the pulmonary pathology society. Archives of Pathology & Laboratory Medicine. 2017;**141**(3):437-444

[8] Roden AC, Maleszewski JJ, Yi ES, Jenkins SM, Gandhi MJ, Scott JP, et al. Reproducibility of complement 4d deposition by immunofluorescence and immunohistochemistry in lung allograft biopsies. The Journal of Heart and Lung Transplantation. 2014;**33**(12):1223-1232

[9] Calabrese F, Alessandrini L, Loy M, Marulli G, Balestro E, Perissinotto E, et al. Comparison between referral diagnosis of patients requiring transplantation and pathologic diagnosis of native lungs. The Journal of Heart and Lung Transplantation. 2009;**28**(11):1135-1140

**5**

**Chapter 2**

**Abstract**

The Role of miRNAs in Idiopathic

*Koichi Takagi, Munekazu Yamakuchi, Teruto Hashiguchi*

Idiopathic pulmonary fibrosis (IPF) is a genetic heterogeneous disease with high mortality and poor prognosis. IPF is characterized by persistent fibroblasts and relentless accumulation of collagen matrix. Epithelial-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) contribute to the progression of the fibrotic process. There are some therapeutic drugs that delay this progress, but eradicative medicine does not exist yet. MicroRNAs (miRNAs) are short single-stranded RNAs that regulate posttranscriptional silencing. Recent reports have shown that miRNAs play important roles in the development of IPF, as different expression levels of miRNAs in blood and lung tissue from IPF patients were closely associated with the occurrence of IPF disease. In this chapter, we will discuss the role of miRNAs in the pathogenesis, diagnosis, and treatment of IPF. In

particular, we will focus on the regulation of EMT/EndoMT by miRNAs.

Idiopathic pulmonary fibrosis (IPF) is a genetic heterogeneous disease with high mortality and poor prognosis that is characterized by progressive scarring of the pulmonary parenchyma, which leads to progressive loss of lung function with dyspnea and hypoxemia, and ultimately respiratory failure and death. IPF is a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown cause. Disease pathogenesis involves airway tissue remodeling through the excess deposition of extracellular matrix (ECM) proteins and the formation of fibroblastic foci, evident by histology [1]. These events are occurred mainly by continuous damage to the lung epithelium, which destroys the basement membrane and activates myofibroblasts. Myofibroblasts may not only come from resident or circulating fibroblasts but also be brought by epithelial cells transdifferentiated through epithelial-mesenchymal transition (EMT) [2]. Signaling through the TGF-β path-

According to the 2018 joint statement by the American Thoracic Society (ATS), European Respiratory Society (ERS), Latin America Thoracic Association (ALAT), and Japanese Respiratory Society (JRS) [4], the diagnosis of IPF can be secured by exclusion of other known causes of interstitial lung disease (ILD) and either the

Pulmonary Fibrosis

*and Hiromasa Inoue*

**Keywords:** IPF, miRNA, EndoMT, EMT

**1.1 Overview of idiopathic pulmonary fibrosis**

way is thought to underlie many of these changes [3].

**1. Introduction**

#### **Chapter 2**

## The Role of miRNAs in Idiopathic Pulmonary Fibrosis

*Koichi Takagi, Munekazu Yamakuchi, Teruto Hashiguchi and Hiromasa Inoue*

#### **Abstract**

Idiopathic pulmonary fibrosis (IPF) is a genetic heterogeneous disease with high mortality and poor prognosis. IPF is characterized by persistent fibroblasts and relentless accumulation of collagen matrix. Epithelial-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) contribute to the progression of the fibrotic process. There are some therapeutic drugs that delay this progress, but eradicative medicine does not exist yet. MicroRNAs (miRNAs) are short single-stranded RNAs that regulate posttranscriptional silencing. Recent reports have shown that miRNAs play important roles in the development of IPF, as different expression levels of miRNAs in blood and lung tissue from IPF patients were closely associated with the occurrence of IPF disease. In this chapter, we will discuss the role of miRNAs in the pathogenesis, diagnosis, and treatment of IPF. In particular, we will focus on the regulation of EMT/EndoMT by miRNAs.

**Keywords:** IPF, miRNA, EndoMT, EMT

#### **1. Introduction**

#### **1.1 Overview of idiopathic pulmonary fibrosis**

Idiopathic pulmonary fibrosis (IPF) is a genetic heterogeneous disease with high mortality and poor prognosis that is characterized by progressive scarring of the pulmonary parenchyma, which leads to progressive loss of lung function with dyspnea and hypoxemia, and ultimately respiratory failure and death. IPF is a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown cause. Disease pathogenesis involves airway tissue remodeling through the excess deposition of extracellular matrix (ECM) proteins and the formation of fibroblastic foci, evident by histology [1]. These events are occurred mainly by continuous damage to the lung epithelium, which destroys the basement membrane and activates myofibroblasts. Myofibroblasts may not only come from resident or circulating fibroblasts but also be brought by epithelial cells transdifferentiated through epithelial-mesenchymal transition (EMT) [2]. Signaling through the TGF-β pathway is thought to underlie many of these changes [3].

According to the 2018 joint statement by the American Thoracic Society (ATS), European Respiratory Society (ERS), Latin America Thoracic Association (ALAT), and Japanese Respiratory Society (JRS) [4], the diagnosis of IPF can be secured by exclusion of other known causes of interstitial lung disease (ILD) and either the

**4**

*Interstitial Lung Diseases*

**References**

Medicine. 2002;**165**:277-304

Medicine. 2004;**170**:904-910

[4] Thomeer M, Demedts M, Behr J, Buhl R, Costabel U, Flower CD, et al. Multidisciplinary observer agreement in the diagnosis and of idiopathic pulmonary fibrosis. The European Respiratory Journal. 2008;**31**:585-591

[5] Leslie KO. Pathology of interstitial lung disease. Clinics in Chest Medicine.

[6] Liebow AA, Carringotn CB. In: Simon M, Potchen EJ, Lemay M, editors. The Interstitial Pneumonias, Frontiers of Pulmonary Radiology. New York: Grune and Strattin; 1969. pp. 102-141

[7] Roden AC, Aisner DL, Allen TC, Aubry MC, Barrios RJ, Beasley MB, et al. Diagnosis of acute cellular rejection and antibody-mediated rejection on lung transplant biopsies: A perspective from members of the pulmonary pathology society. Archives of Pathology & Laboratory Medicine.

[8] Roden AC, Maleszewski JJ, Yi ES, Jenkins SM, Gandhi MJ, Scott JP, et al. Reproducibility of complement 4d deposition by immunofluorescence and

2004;**25**:657-703

2017;**141**(3):437-444

2013;**22**:26-32

[2] Cottin V. Interstitial lung disease. European Respiratory Review.

[3] Flaherty KR, King TE Jr, Raghu G, Lynch JP 3rd, Colby TV, Travis WD, et al. Idiopathic interstitial pneumonia: What is the effect of a multidisciplinary approach to diagnosis? American Journal of Respiratory and Critical Care

[1] American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the idiopathic inetrstitial pneumonias. American Journal of Respiratory and Critical Care

immunohistochemistry in lung allograft biopsies. The Journal of Heart and Lung Transplantation. 2014;**33**(12):1223-1232

[9] Calabrese F, Alessandrini L, Loy M, Marulli G, Balestro E, Perissinotto E, et al. Comparison between referral diagnosis of patients requiring transplantation and pathologic diagnosis of native lungs. The Journal of Heart and Lung Transplantation.

2009;**28**(11):1135-1140

presence of high resolution computed tomography (HRCT) pattern of usual interstitial pneumonia (UIP) or specific combinations of HRCT patterns and histopathology patterns in patients subjected to lung tissue sampling. Any plausible cause of secondary interstitial involvement should be excluded by a thorough medical history and other procedures such as laboratory tests or bronchoalveolar lavage (BAL) when necessary. If a specific diagnosis is not made or no potential cause for ILD is identified, then clinical findings and HRCT are considered during multidisciplinary discussion among different experts (including clinicians, radiologists, pathologists, rheumatologists, and thoracic surgeons in selected cases) [4].

#### **1.2 Overview of miRNAs**

MicroRNAs (miRNAs) are short single-stranded RNA molecules of 19–25 nucleotides in length that mediate posttranscriptional gene silencing of target genes [5]. MiRNAs have been recognized as a distinct class of small regulatory RNAs in multiple species that regulate a wide variety of functions such as cell proliferation, differentiation, apoptosis, stress response, and immune response [6–8]. MiRNAs regulate the onset and progress of a variety of diseases, such as cancer, infections, and atherosclerosis [9–11]. In lung diseases, many miRNAs contribute to the pathogenesis. For instance, miR-424/503 regulates the progression of pulmonary hypertension (PH) [12, 13]. miR-424/503 expression is reduced in the pulmonary artery of PH patients [13]. One of the targets of miR-424/503 is *Rictor*, and inhibition of miR-424/503 expression increased the expression of *Rictor*, leading to endothelialto-mesenchymal transition (EndoMT) and migration of pulmonary artery endothelial cells [12]. In allergic asthma, miR-21, miR-126, and miR-145 are upregulated in multiple experimental asthma models [14–16], and miR-221 and miR-485-3p are upregulated in the peripheral blood of pediatric patients with asthma, compared with controls [17].

#### **2. IPF and miRNAs**

Profiling of miRNAs in the lung tissue from IPF patients was performed by different groups [18–22]. In IPF lungs, the expression of miR-21 and miR-155 was increased, whereas the expression of let-7a, miR-29, miR-30, and miR-101 was decreased [23–25]. MiR-21 expression was increased in the serum of IPF patients, and its levels correlated with a decrease in lung function [26]. In the miRNA expression profiling of sputum-derived exosomes from IPF patients, 21 miRNAs were differentially expressed, among which seven (e.g., miR-142-3p and miR-33a-5p) were upregulated and 14 (e.g., Let-7d-5p) were downregulated [27]. In bronchoalveolar lavage fluid (BALF), miR-29a and miR-185 are downregulated in IPF patients [28, 29]. However, little is known about miRNA functions in IPF; much of the functional data has been inferred from chemically induced mouse IPF models. In the lungs of mouse IPF models, miR-21, miR-9, and miR-155 were increased [19, 21, 30], and miR-26, miR-101, miR-139, miR-200, miR-326, miR-489, miR-503, and miR-708 were decreased [24, 31–37]. Furthermore, miR-21 expression was increased in myofibroblasts from IPF lungs, and its inhibition in mouse IPF models attenuated the disease severity [19]. The expression of miR-200 family members was reduced in mouse IPF models and restoration of miR-200c inhibited fibrosis [33]. Similarly, miR-26a was decreased in mouse IPF models and its inhibition caused pulmonary fibrosis, while overexpression repressed fibrotic disease [38]. Finally, miR-326 was decreased in mouse IPF models, and its administration mimicked inhibited TGF-β expression and attenuated fibrosis [34].

**7**

regulatory.

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

**3. The role of miRNAs in IPF-contributing cells**

plays an important role in the development of IPF.

elucidated and requires further research.

of alveolar architecture.

Epithelial cells communicate each other via adhesion molecules under physiological condition. Among many adhesion molecules, E-cadherin plays an important role in maintaining tight junctions of epithelial cells. Recurrent and nonresolving injury to lung epithelial cells appears to be a key driver of pulmonary fibrosis. Recent studies have supported the concept that altered alveolar epithelial cell phenotypes, including those produced by increased endoplasmic reticulum stress, predispose to further epithelial cell injury and abnormal repair, facilitating the development of pulmonary fibrosis [39, 40], and thus suggested that the epithelial abnormalities/dysfunction underlying fibrosis should be referred to as reprogramming. The ability of epithelial cells to change into mesenchymal cells through EMT

Myofibroblasts and related mesenchymal cells are generally accepted as the cells predominantly responsible for fibrotic destruction/distortion of the lung in IPF [41]. Proliferation of fibroblasts and their differentiation into myofibroblasts are important in the development of IPF. The stage of mesenchymal cell differentiation at which IPF myofibroblasts acquire hallmark pathological properties, the mechanisms underlying their pathological differentiation, and the roles of signals from epithelial cells, immune cells, and the matrix in this process has not been fully

The lung extracellular matrix (ECM) is comprised of collagens, elastin, glycoproteins, and proteoglycans, which serve as structural scaffolding for cells and provide the mechanical stability and elastic recoil necessary for proper lung function. In normal wound healing, myofibroblasts that produce ECM proteins are recruited during the active phase of repair, but are quickly removed via apoptosis once the repair process is underway. Most of the scar matrix subsequently resolves and is replaced by a more permanent tissue. In IPF, myofibroblasts are relatively resistant to apoptosis, and their persistence leads to excessive scarring [42]. A hallmark of IPF is the accumulation of myofibroblasts in clusters called fibroblastic foci and extensive ECM deposition within the interstitium, resulting in destruction

Vascular muscularization driven by repeated small injury is the main pathogenesis of PH, leading to the sustained fibrotic process of intima [43]. Different cellular processes have been described during pulmonary artery remodeling of PH-associated IPF, including endothelial dysfunction [44], EndoMT as a source of myofibroblasts [45], and pulmonary artery smooth muscle proliferation [46]. Monocytes and macrophages have a key role in regulating tissue repair and fibrosis, and several molecular pathways regulating their activity are being investigated as potential treatments for IPF. Macrophages possess many functions that would be expected to promote fibrosis, including fibroblast proliferation [47], regulation of ECM components [48], and secretion of profibrotic cytokines and growth factors. However, macrophages may also possess antifibrotic properties [47, 49]. Several studies have attributed these opposing functions to different macrophage populations distinguished along the lines of classical (M1) and alternative (M2) macrophage activation [50–52], with M1 macrophages being antifibrotic and M2 macrophages being either profibrotic or

Adaptive immune responses are involved in the pathological process in IPF patients. Infiltration of activated CD4+ T-cells into IPF lung is often observed before symptoms are developed [53]. Mature dendritic cells also infiltrate in the pulmonary parenchyma of IPF patients [54]. FoxP3 positive regulatory T-cells are

diminished in both the circulation and BALF of IPF patients [55].

*Interstitial Lung Diseases*

**1.2 Overview of miRNAs**

with controls [17].

**2. IPF and miRNAs**

presence of high resolution computed tomography (HRCT) pattern of usual interstitial pneumonia (UIP) or specific combinations of HRCT patterns and histopathology patterns in patients subjected to lung tissue sampling. Any plausible cause of secondary interstitial involvement should be excluded by a thorough medical history and other procedures such as laboratory tests or bronchoalveolar lavage (BAL) when necessary. If a specific diagnosis is not made or no potential cause for ILD is identified, then clinical findings and HRCT are considered during multidisciplinary discussion among different experts (including clinicians, radiologists, pathologists,

MicroRNAs (miRNAs) are short single-stranded RNA molecules of 19–25 nucleotides in length that mediate posttranscriptional gene silencing of target genes [5]. MiRNAs have been recognized as a distinct class of small regulatory RNAs in multiple species that regulate a wide variety of functions such as cell proliferation, differentiation, apoptosis, stress response, and immune response [6–8]. MiRNAs regulate the onset and progress of a variety of diseases, such as cancer, infections, and atherosclerosis [9–11]. In lung diseases, many miRNAs contribute to the pathogenesis. For instance, miR-424/503 regulates the progression of pulmonary hypertension (PH) [12, 13]. miR-424/503 expression is reduced in the pulmonary artery of PH patients [13]. One of the targets of miR-424/503 is *Rictor*, and inhibition of miR-424/503 expression increased the expression of *Rictor*, leading to endothelialto-mesenchymal transition (EndoMT) and migration of pulmonary artery endothelial cells [12]. In allergic asthma, miR-21, miR-126, and miR-145 are upregulated in multiple experimental asthma models [14–16], and miR-221 and miR-485-3p are upregulated in the peripheral blood of pediatric patients with asthma, compared

Profiling of miRNAs in the lung tissue from IPF patients was performed by different groups [18–22]. In IPF lungs, the expression of miR-21 and miR-155 was increased, whereas the expression of let-7a, miR-29, miR-30, and miR-101 was decreased [23–25]. MiR-21 expression was increased in the serum of IPF patients, and its levels correlated with a decrease in lung function [26]. In the miRNA expression profiling of sputum-derived exosomes from IPF patients, 21 miRNAs were differentially expressed, among which seven (e.g., miR-142-3p and miR-33a-5p) were upregulated and 14 (e.g., Let-7d-5p) were downregulated [27]. In bronchoalveolar lavage fluid (BALF), miR-29a and miR-185 are downregulated in IPF patients [28, 29]. However, little is known about miRNA functions in IPF; much of the functional data has been inferred from chemically induced mouse IPF models. In the lungs of mouse IPF models, miR-21, miR-9, and miR-155 were increased [19, 21, 30], and miR-26, miR-101, miR-139, miR-200, miR-326, miR-489, miR-503, and miR-708 were decreased [24, 31–37]. Furthermore, miR-21 expression was increased in myofibroblasts from IPF lungs, and its inhibition in mouse IPF models attenuated the disease severity [19]. The expression of miR-200 family members was reduced in mouse IPF models and restoration of miR-200c inhibited fibrosis [33]. Similarly, miR-26a was decreased in mouse IPF models and its inhibition caused pulmonary fibrosis, while overexpression repressed fibrotic disease [38]. Finally, miR-326 was decreased in mouse IPF models, and its administration mimicked inhibited TGF-β

rheumatologists, and thoracic surgeons in selected cases) [4].

**6**

expression and attenuated fibrosis [34].

#### **3. The role of miRNAs in IPF-contributing cells**

Epithelial cells communicate each other via adhesion molecules under physiological condition. Among many adhesion molecules, E-cadherin plays an important role in maintaining tight junctions of epithelial cells. Recurrent and nonresolving injury to lung epithelial cells appears to be a key driver of pulmonary fibrosis. Recent studies have supported the concept that altered alveolar epithelial cell phenotypes, including those produced by increased endoplasmic reticulum stress, predispose to further epithelial cell injury and abnormal repair, facilitating the development of pulmonary fibrosis [39, 40], and thus suggested that the epithelial abnormalities/dysfunction underlying fibrosis should be referred to as reprogramming. The ability of epithelial cells to change into mesenchymal cells through EMT plays an important role in the development of IPF.

Myofibroblasts and related mesenchymal cells are generally accepted as the cells predominantly responsible for fibrotic destruction/distortion of the lung in IPF [41]. Proliferation of fibroblasts and their differentiation into myofibroblasts are important in the development of IPF. The stage of mesenchymal cell differentiation at which IPF myofibroblasts acquire hallmark pathological properties, the mechanisms underlying their pathological differentiation, and the roles of signals from epithelial cells, immune cells, and the matrix in this process has not been fully elucidated and requires further research.

The lung extracellular matrix (ECM) is comprised of collagens, elastin, glycoproteins, and proteoglycans, which serve as structural scaffolding for cells and provide the mechanical stability and elastic recoil necessary for proper lung function. In normal wound healing, myofibroblasts that produce ECM proteins are recruited during the active phase of repair, but are quickly removed via apoptosis once the repair process is underway. Most of the scar matrix subsequently resolves and is replaced by a more permanent tissue. In IPF, myofibroblasts are relatively resistant to apoptosis, and their persistence leads to excessive scarring [42]. A hallmark of IPF is the accumulation of myofibroblasts in clusters called fibroblastic foci and extensive ECM deposition within the interstitium, resulting in destruction of alveolar architecture.

Vascular muscularization driven by repeated small injury is the main pathogenesis of PH, leading to the sustained fibrotic process of intima [43]. Different cellular processes have been described during pulmonary artery remodeling of PH-associated IPF, including endothelial dysfunction [44], EndoMT as a source of myofibroblasts [45], and pulmonary artery smooth muscle proliferation [46].

Monocytes and macrophages have a key role in regulating tissue repair and fibrosis, and several molecular pathways regulating their activity are being investigated as potential treatments for IPF. Macrophages possess many functions that would be expected to promote fibrosis, including fibroblast proliferation [47], regulation of ECM components [48], and secretion of profibrotic cytokines and growth factors. However, macrophages may also possess antifibrotic properties [47, 49]. Several studies have attributed these opposing functions to different macrophage populations distinguished along the lines of classical (M1) and alternative (M2) macrophage activation [50–52], with M1 macrophages being antifibrotic and M2 macrophages being either profibrotic or regulatory.

Adaptive immune responses are involved in the pathological process in IPF patients. Infiltration of activated CD4+ T-cells into IPF lung is often observed before symptoms are developed [53]. Mature dendritic cells also infiltrate in the pulmonary parenchyma of IPF patients [54]. FoxP3 positive regulatory T-cells are diminished in both the circulation and BALF of IPF patients [55].

#### **3.1 Alveolar epithelial cells**

miRNA microarray analysis of lung tissue from healthy controls and IPF patients showed that the expression of several let-7 family members was lower in IPF patients [18]. The human let-7 family includes 12 members (let-7-a1, a2, a3, b, c, d, e, f1, f2, g, 1, and miR-98), located on eight different homologous chromosomes [56]. *In vitro* inhibition of let-7d induced an increase in mesenchymal markers in lung epithelial cell lines. The High Mobility Group A2 (*HMGA2*) 3′ UTR has seven conserved sites complementary to the let-7 miRNA [57], and HMGA2 was significantly overexpressed in IPF lungs compared with controls [58]. In lung epithelial cell lines, let-7d regulated *HMGA2* expression, and the increase in *HMGA2* after TGF-β stimulation depended on inhibition of let-7 [18]. HMGA2 facilitates the transcription of *SNAI1* and *TWIST*, the transcriptional repressors of adherens, which are tight and desmosomal junction components, leading to reduced intracellular adhesion. Moreover, it is a mediator of TGF-β-induced EMT. Downregulation of let-7d also led to upregulation of other fibrosis-relevant targets, such as RAS, insulin-like growth factor-1 (IGF-1), and IGF-1 receptor, and thus to the profound and sustained changes in cellular phenotype that are indeed observed in IPF [18] (**Figure 1**).

The expression of miR-26a was significantly decreased in the lungs of mice following the administration of bleomycin [59]. Alignment of miR-26a with the *HMGA2* 3′UTR sequence revealed one potential conserved seed site. Transient introduction of miR-26a into A549 cells, a pulmonary epithelial cell line, led to a significant decrease in HMGA2. However, the reduction in miR-26a expression induced an obvious EMT phenotype in A549 cells together with a clear alteration in certain related proteins, including E-cadherin, vimentin, and α-SMA [59]. Loss of function of miR-26a facilitated lung epithelial cells to transform into myofibroblasts and induce pulmonary fibrosis in mice. Lin28D is also one of the conserved putative target genes of miR-26a. Inhibition of miR-26a induced Lin28D expression and caused an obvious EMT phenotype in A549 cells [31].

**9**

miR-21 ↑

**Figure 2.**

miR-29 ↓

*The role of miRNAs in fibroblasts for IPF.*

Collagen

Smad7

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

expression [33].

**3.2 Fibroblasts and myofibroblasts**

correlates with miR-21 in pulmonary fibroblasts [19].

Transdifferenaon Proliferaon ECM deposion

miR-101 ↓

miR-153 ↓

miR-154 ↓

miR-155 ↓

miR-210 ↓

MNT

LXRα

CDKN2B

TGFβR2

**Myofibroblasts**

NFATc2

in the lungs (**Figure 2**).

**Fibroblasts**

The miR-200 family has been suggested to regulate the progression of pulmonary fibrosis by suppressing the EMT of alveolar epithelial cells (AECs). MiR-200 family members correlated with E-cadherin expression in epithelial cells [60, 61] and negatively correlated with ZEB family expression, which has been implicated in EMT [62]. Furthermore, the miR-200 family members have been identified as EMT suppressors through direct targeting of ZEB1 and ZEB2 [63]. MiR-200 is downregulated in the lungs of IPF patients and mouse IPF models [33]. In AECs, overexpression of miR-200, particularly miR-200b and miR-200c, attenuated TGF-β1-induced mesenchymal cell morphological characteristics and TGF-β1-repressed E-cadherin

In fibrotic lungs, TGF-β1 is overexpressed in a broad range of cells, including epithelial cells, leading to the dysregulation of normal lung homeostasis by increasing the synthesis and altering the balance of matrix metalloproteinases and their inhibitors [64]. MiR-326 targets the *TGF-β1* 3′UTR, and miR-326 levels inversely correlate with TGF-β1 protein levels in multiple human cell lines and in the lungs of mouse IPF models [34]. Restoration of miR-326 levels by intranasal delivery of miR-326 mimics was sufficient to inhibit TGF-β1 expression and attenuate the fibrotic response in the lungs of mouse IPF models [34]. Furthermore, miR-1343 targets the 3′UTRs of *TGFBR1* and *TGFBR2* and attenuates EMT and fibrogenesis

Several studies have shown that some miRNAs including miR-21, miR-29, miR-101, miR-153, miR-154, miR-155, and miR-210 regulate the function of fibroblasts

The expression of miR-21 is increased in the lungs of mouse IPF models and of patients with IPF [19]. In mouse IPF models, miR-21 inhibition by lentiviruses encoding anti-miR-21 suppressed morphological markers of pulmonary fibrosis compared with controls [23]. TGF-β1 is the most important profibrogenic cytokine, which increases the expression of miR-21 through SMAD2/3/4 in lung fibroblasts [19] [66]. MiR-21 inhibition inversely regulates TGF-β1-induced ECM protein expression in human pulmonary fibroblast cell lines [23]. Smad7, which inhibits the proliferation of interstitial fibroblasts, is a direct target of miR-21 and negatively

by inhibiting the TGF-β1 pathway in alveolar epithelial cell lines [65].

**Figure 1.** *The role of miRNAs in alveolar epithelial cells for IPF.*

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

*Interstitial Lung Diseases*

**3.1 Alveolar epithelial cells**

miRNA microarray analysis of lung tissue from healthy controls and IPF patients showed that the expression of several let-7 family members was lower in IPF patients [18]. The human let-7 family includes 12 members (let-7-a1, a2, a3, b, c, d, e, f1, f2, g, 1, and miR-98), located on eight different homologous chromosomes [56]. *In vitro* inhibition of let-7d induced an increase in mesenchymal markers in lung epithelial cell lines. The High Mobility Group A2 (*HMGA2*) 3′ UTR has seven conserved sites complementary to the let-7 miRNA [57], and HMGA2 was significantly overexpressed in IPF lungs compared with controls [58]. In lung epithelial cell lines, let-7d regulated *HMGA2* expression, and the increase in *HMGA2* after TGF-β stimulation depended on inhibition of let-7 [18]. HMGA2 facilitates the transcription of *SNAI1* and *TWIST*, the transcriptional repressors of adherens, which are tight and desmosomal junction components, leading to reduced intracellular adhesion. Moreover, it is a mediator of TGF-β-induced EMT. Downregulation of let-7d also led to upregulation of other fibrosis-relevant targets, such as RAS, insulin-like growth factor-1 (IGF-1), and IGF-1 receptor, and thus to the profound and sustained changes in

cellular phenotype that are indeed observed in IPF [18] (**Figure 1**).

caused an obvious EMT phenotype in A549 cells [31].

The expression of miR-26a was significantly decreased in the lungs of mice following the administration of bleomycin [59]. Alignment of miR-26a with the *HMGA2* 3′UTR sequence revealed one potential conserved seed site. Transient introduction of miR-26a into A549 cells, a pulmonary epithelial cell line, led to a significant decrease in HMGA2. However, the reduction in miR-26a expression induced an obvious EMT phenotype in A549 cells together with a clear alteration in certain related proteins, including E-cadherin, vimentin, and α-SMA [59]. Loss of function of miR-26a facilitated lung epithelial cells to transform into myofibroblasts and induce pulmonary fibrosis in mice. Lin28D is also one of the conserved putative target genes of miR-26a. Inhibition of miR-26a induced Lin28D expression and

**8**

**Figure 1.**

*The role of miRNAs in alveolar epithelial cells for IPF.*

The miR-200 family has been suggested to regulate the progression of pulmonary fibrosis by suppressing the EMT of alveolar epithelial cells (AECs). MiR-200 family members correlated with E-cadherin expression in epithelial cells [60, 61] and negatively correlated with ZEB family expression, which has been implicated in EMT [62]. Furthermore, the miR-200 family members have been identified as EMT suppressors through direct targeting of ZEB1 and ZEB2 [63]. MiR-200 is downregulated in the lungs of IPF patients and mouse IPF models [33]. In AECs, overexpression of miR-200, particularly miR-200b and miR-200c, attenuated TGF-β1-induced mesenchymal cell morphological characteristics and TGF-β1-repressed E-cadherin expression [33].

In fibrotic lungs, TGF-β1 is overexpressed in a broad range of cells, including epithelial cells, leading to the dysregulation of normal lung homeostasis by increasing the synthesis and altering the balance of matrix metalloproteinases and their inhibitors [64]. MiR-326 targets the *TGF-β1* 3′UTR, and miR-326 levels inversely correlate with TGF-β1 protein levels in multiple human cell lines and in the lungs of mouse IPF models [34]. Restoration of miR-326 levels by intranasal delivery of miR-326 mimics was sufficient to inhibit TGF-β1 expression and attenuate the fibrotic response in the lungs of mouse IPF models [34]. Furthermore, miR-1343 targets the 3′UTRs of *TGFBR1* and *TGFBR2* and attenuates EMT and fibrogenesis by inhibiting the TGF-β1 pathway in alveolar epithelial cell lines [65].

#### **3.2 Fibroblasts and myofibroblasts**

Several studies have shown that some miRNAs including miR-21, miR-29, miR-101, miR-153, miR-154, miR-155, and miR-210 regulate the function of fibroblasts in the lungs (**Figure 2**).

The expression of miR-21 is increased in the lungs of mouse IPF models and of patients with IPF [19]. In mouse IPF models, miR-21 inhibition by lentiviruses encoding anti-miR-21 suppressed morphological markers of pulmonary fibrosis compared with controls [23]. TGF-β1 is the most important profibrogenic cytokine, which increases the expression of miR-21 through SMAD2/3/4 in lung fibroblasts [19] [66]. MiR-21 inhibition inversely regulates TGF-β1-induced ECM protein expression in human pulmonary fibroblast cell lines [23]. Smad7, which inhibits the proliferation of interstitial fibroblasts, is a direct target of miR-21 and negatively correlates with miR-21 in pulmonary fibroblasts [19].

**Figure 2.**

*The role of miRNAs in fibroblasts for IPF.*

Members of the miR-29 family, which consists of three mature members, miR-29a, miR-29b, and miR-29c, are among the most downregulated miRNAs in IPF lungs [66]. The expression of miR-29c is also decreased in the lungs of mouse IPF models [67]. MiR-29 has many target genes involved in the composition and regulation of ECM, thus downregulation of miR-29 is capable of contributing to excessive ECM deposition [20, 68]. In human fetal lung fibroblasts, miR-29 suppression upregulated a series of genes, such as TGF-β1 regulated genes and adhesion molecules associated with the fibrotic phenotype [18].

MiR-101 is also one of the most downregulated miRNAs in fibrotic lungs from IPF patients and mouse IPF models [24]. Hypoxia-inducible factors (HIFs) contribute to the pathogenesis of pulmonary fibrosis through Nuclear Factor-Activated T-cell (NFAT) c2 [69]. MiR-101 suppressed WNT5a-induced proliferation of lung fibroblasts via inhibition of NFATc2 signaling by targeting Frizzled receptor 4/6 and attenuated bleomycin-induced pulmonary fibrosis in mouse IPF models [24].

MiR-153 is an intragenic miRNA that is embedded in genes encoding islet-associated protein (IA)-2 and 2β. It has been shown to participate in the pathogenesis of neurodegenerative diseases [70] and inhibit the proliferation and invasion of many types of malignant cells [71]. Furthermore, TGF-β1 downregulates miR-153 expression in lung fibrosis, and miR-153 was downregulated in the fibrotic phase, which was characterized by excessive proliferation of fibroblasts and deposition of ECM in mouse IPF models [72]. However, miR-153 reduced the contractile and migratory activities of fibroblasts. TGFBR2, a transmembrane serine/threonine kinase receptor for TGF-β, has been identified as a direct target of miR-153; moreover, miR-375 regulates SMAD2/3 phosphorylation and the fibrogenic activity of lung fibroblasts through TGFBR2 inhibition [72].

The majority of increased miRNAs in IPF localizes to chromosome 14q32 and is enriched with members of the miR-154 family. TGF-β1 regulates the expression of miRNAs in the 14q32 cluster and induces the expression changes similar to those seen in IPF. TGF-β1 induces cell proliferation through CDK4 and CDKN2B, a CDK4 inhibitor, which is a miR-154 target. Furthermore, TGF-β1-induced increases in proliferation were significantly reduced in the presence of miR-154 inhibitor in fibroblasts, and miR-154-induced changes in proliferation were mediated through the activation of the WNT/β-catenin pathway in fibroblasts [73].

MiR-155 is required for normal immune function [74]. Its overexpression is associated with inflammation, autoimmunity, and cancer [75], whereas miR-155-deficient mice develop age-related airway fibrosis [76]. Liver X receptor alpha (LXRα), which is an oxysterol-activated transcription factor, has a conserved 3′UTR seed-region sequence complementary to miR-155, and inhibition of LXRα ameliorated lung fibrosis in miR-155-deficient mice [77]. However, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155-deficient fibroblasts [77]. Thus, miR-155 attenuates lung fibrosis through inhibition of LXRα in lung fibroblasts.

The expression of miR-210 has been found to be increased in patients with rapidly progressive IPF, who typically have experienced hypoxia [22]. Consistently, miR-210 was upregulated in IPF fibroblasts in response to hypoxia, and its knockdown suppressed the hypoxia-induced proliferation of IPF fibroblasts. Furthermore, HIF-2α regulated miR-210 expression and miR-210-mediated proliferation of IPF fibroblasts in response to hypoxia. Finally, miR-210 promoted IPF fibroblast proliferation through repression of its downstream target, MNT [78].

#### **3.3 Endothelial cells**

PH is frequently seen in IPF patients and is commonly attributed to hypoxic vasoconstriction and capillary destruction. Pathology findings include endothelial

**11**

**Figure 3.**

*The role of miRNAs in pulmonary endothelial cells for IPF.*

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

proliferation and medial hypertrophy that exceed those expected in the setting of hypoxia [79] (**Figure 3**). HIF-1 activates the transcription of genes encoding angiogenic growth factors, including vascular endothelial growth factor (VEGF), angiopoietin (ANGPT), placental growth factor (PGF), and platelet-derived growth factor (PDGF), which are secreted by hypoxic cells and stimulate endothelial cells, leading to angiogenesis. Furthermore, HIF-1 mediates hypoxia-induced

ANGPT1 is a ligand for receptor tyrosine kinase Tie2 [81], which is expressed in endothelial cells [82]. ANGPT1-Tie2 signaling has been shown to be involved mainly in angiogenic activity and promoting maturation of blood vessels, and it is regulated by Akt and MAPK signaling [83, 84]. The activation of ANGPT1-Tie2 signaling correlates with the severity of PH [85]. Mir-34a is downregulated in rat pulmonary arterial hypertension models [86], and ANGPT1 and Tie2 are potential targets of miR-34a, as they have a conserved miR-34a seed sequence in their 3′UTR [87]. Overexpression of miR-34a also downregulated the expression of PDGFRA, one of therapeutic targets of IPF, in pulmonary artery smooth muscle cells [86]. EndoMT has been proposed to be another potential source of myofibroblasts. In EndoMT, endothelial cells lose their own markers and obtain mesenchymal phenotype. Silencing of miR-155 directly increased RhoA expression and activity in endothelial cells and affected phosphorylation of the downstream LMK. A selective Rho kinase inhibitor partly suppressed EndoMT, strengthening the notion that RhoA plays a central role in EndoMT. Overexpression of miR-155 suppressed EndoMT, hence miR-155 functions as a negative regulator of RhoA signaling in TGF-β-induced EndoMT [88]. EndoMT has been considered as a source of myofibroblasts in PH [45], and miR-155 is downregulated in IPF lungs. Thus, miR-155 has

phenotypic changes typical of EndoMT in endothelial cells [80].

been suggested to affect PH-associated IPF through inducing EndoMT.

Macrophages possess many functions that would be expected to promote fibrosis, including regulation of fibroblast proliferation, recruitment, and activation, direct regulation of ECM components, and secretion of profibrotic cytokines and growth factors. There are two opposite characteristic types of functional macrophages; pro-inflammatory (M1) macrophages induced by toll-like receptor (TLR) and T helper type 1 (Th1) signals and anti-inflammatory (M2) macrophages

**3.4 Inflammatory cells**

#### *The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

*Interstitial Lung Diseases*

Members of the miR-29 family, which consists of three mature members, miR-29a, miR-29b, and miR-29c, are among the most downregulated miRNAs in IPF lungs [66]. The expression of miR-29c is also decreased in the lungs of mouse IPF models [67]. MiR-29 has many target genes involved in the composition and regulation of ECM, thus downregulation of miR-29 is capable of contributing to excessive ECM deposition [20, 68]. In human fetal lung fibroblasts, miR-29 suppression upregulated a series of genes, such as TGF-β1 regulated genes and adhesion

MiR-101 is also one of the most downregulated miRNAs in fibrotic lungs from IPF patients and mouse IPF models [24]. Hypoxia-inducible factors (HIFs) contribute to the pathogenesis of pulmonary fibrosis through Nuclear Factor-Activated T-cell (NFAT) c2 [69]. MiR-101 suppressed WNT5a-induced proliferation of lung fibroblasts via inhibition of NFATc2 signaling by targeting Frizzled receptor 4/6 and attenuated bleomycin-induced pulmonary fibrosis in mouse IPF models [24]. MiR-153 is an intragenic miRNA that is embedded in genes encoding islet-associated protein (IA)-2 and 2β. It has been shown to participate in the pathogenesis of neurodegenerative diseases [70] and inhibit the proliferation and invasion of many types of malignant cells [71]. Furthermore, TGF-β1 downregulates miR-153 expression in lung fibrosis, and miR-153 was downregulated in the fibrotic phase, which was characterized by excessive proliferation of fibroblasts and deposition of ECM in mouse IPF models [72]. However, miR-153 reduced the contractile and migratory activities of fibroblasts. TGFBR2, a transmembrane serine/threonine kinase receptor for TGF-β, has been identified as a direct target of miR-153; moreover, miR-375 regulates SMAD2/3 phosphorylation and the fibrogenic activity of lung fibroblasts

The majority of increased miRNAs in IPF localizes to chromosome 14q32 and is enriched with members of the miR-154 family. TGF-β1 regulates the expression of miRNAs in the 14q32 cluster and induces the expression changes similar to those seen in IPF. TGF-β1 induces cell proliferation through CDK4 and CDKN2B, a CDK4 inhibitor, which is a miR-154 target. Furthermore, TGF-β1-induced increases in proliferation were significantly reduced in the presence of miR-154 inhibitor in fibroblasts, and miR-154-induced changes in proliferation were mediated through

MiR-155 is required for normal immune function [74]. Its overexpression is associated with inflammation, autoimmunity, and cancer [75], whereas miR-155-deficient mice develop age-related airway fibrosis [76]. Liver X receptor alpha (LXRα), which is an oxysterol-activated transcription factor, has a conserved 3′UTR seed-region sequence complementary to miR-155, and inhibition of LXRα ameliorated lung fibrosis in miR-155-deficient mice [77]. However, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155-deficient fibroblasts [77]. Thus, miR-155 attenuates lung fibrosis through inhibition of LXRα in lung fibroblasts. The expression of miR-210 has been found to be increased in patients with rapidly progressive IPF, who typically have experienced hypoxia [22]. Consistently,

the activation of the WNT/β-catenin pathway in fibroblasts [73].

miR-210 was upregulated in IPF fibroblasts in response to hypoxia, and its knockdown suppressed the hypoxia-induced proliferation of IPF fibroblasts. Furthermore, HIF-2α regulated miR-210 expression and miR-210-mediated proliferation of IPF fibroblasts in response to hypoxia. Finally, miR-210 promoted IPF fibroblast proliferation through repression of its downstream target, MNT [78].

PH is frequently seen in IPF patients and is commonly attributed to hypoxic vasoconstriction and capillary destruction. Pathology findings include endothelial

molecules associated with the fibrotic phenotype [18].

through TGFBR2 inhibition [72].

**10**

**3.3 Endothelial cells**

proliferation and medial hypertrophy that exceed those expected in the setting of hypoxia [79] (**Figure 3**). HIF-1 activates the transcription of genes encoding angiogenic growth factors, including vascular endothelial growth factor (VEGF), angiopoietin (ANGPT), placental growth factor (PGF), and platelet-derived growth factor (PDGF), which are secreted by hypoxic cells and stimulate endothelial cells, leading to angiogenesis. Furthermore, HIF-1 mediates hypoxia-induced phenotypic changes typical of EndoMT in endothelial cells [80].

ANGPT1 is a ligand for receptor tyrosine kinase Tie2 [81], which is expressed in endothelial cells [82]. ANGPT1-Tie2 signaling has been shown to be involved mainly in angiogenic activity and promoting maturation of blood vessels, and it is regulated by Akt and MAPK signaling [83, 84]. The activation of ANGPT1-Tie2 signaling correlates with the severity of PH [85]. Mir-34a is downregulated in rat pulmonary arterial hypertension models [86], and ANGPT1 and Tie2 are potential targets of miR-34a, as they have a conserved miR-34a seed sequence in their 3′UTR [87]. Overexpression of miR-34a also downregulated the expression of PDGFRA, one of therapeutic targets of IPF, in pulmonary artery smooth muscle cells [86].

EndoMT has been proposed to be another potential source of myofibroblasts. In EndoMT, endothelial cells lose their own markers and obtain mesenchymal phenotype. Silencing of miR-155 directly increased RhoA expression and activity in endothelial cells and affected phosphorylation of the downstream LMK. A selective Rho kinase inhibitor partly suppressed EndoMT, strengthening the notion that RhoA plays a central role in EndoMT. Overexpression of miR-155 suppressed EndoMT, hence miR-155 functions as a negative regulator of RhoA signaling in TGF-β-induced EndoMT [88]. EndoMT has been considered as a source of myofibroblasts in PH [45], and miR-155 is downregulated in IPF lungs. Thus, miR-155 has been suggested to affect PH-associated IPF through inducing EndoMT.

#### **3.4 Inflammatory cells**

Macrophages possess many functions that would be expected to promote fibrosis, including regulation of fibroblast proliferation, recruitment, and activation, direct regulation of ECM components, and secretion of profibrotic cytokines and growth factors. There are two opposite characteristic types of functional macrophages; pro-inflammatory (M1) macrophages induced by toll-like receptor (TLR) and T helper type 1 (Th1) signals and anti-inflammatory (M2) macrophages

**Figure 3.** *The role of miRNAs in pulmonary endothelial cells for IPF.*

activated by Th2 signals [89] [90]. Th2 cytokines, IL-4 and IL-13 (IL-4/IL-13), contribute to extensive tissue fibrosis in mouse models [91], and IL-4/IL-13-induced macrophages activate fibroblasts by secreting pro-fibrogenic cytokines. IL-4/IL-13 induced CCL18 in lung macrophages of IPF patients, and secreted CCL18 increased the production of collagen from fibroblasts [92]. Many transcriptional factors, such as STAT (signal transducers and activator of transcription) family, regulate macrophage polarization. M1 macrophage polarization is controlled via STAT1, and M2 macrophages activation is mediated by STAT6 [93]. To maintain STAT6 phosphorylation, M2 macrophages overcome strong expression of suppressor of cytokine signaling 1 (SOCS1), resulting in the prolongation of fibrogenesis (**Figure 4**).

IL-4/IL-13 induced miR-142-5p expression and suppressed miR-130a-3p expression in macrophages [94]. Transfection of macrophages with either miR-142-5p anti-sense oligonucleotides or miR-130-3p mimics significantly reduced the expression of M2 markers induced by IL-4 and regulated macrophage pro-fibrogenesis [94]. SOCS1 3′UTR has seven conserved sites complementary to miR-142-5p RNA, and PPARγ 3′UTR has seven conserved sites complementary to miR-130a-3p RNA. The expression of SOCS1 is reduced in IPF lungs, and bleomycin-induced lung fibrosis and inflammation were enhanced in SOCS1-deficient mice compared with WT mice [95]. Thus, miR-142-5p and miR-130a-3p regulate pulmonary fibrosis through SOCS1 and PPARγ in macrophages.

Downregulation of miR-185 has been demonstrated in IPF lung tissue and alveolar macrophages [29, 96]. MiR-185 inhibited cell growth and proliferation by directly targeting AKT1 [97]. Downregulation of miR-185 resulted in AKT activation via increased DNMT1 expression, leading to promoter hypermethylation and silencing of *PTEN* [77]. Moreover, miR-185 directly bound and degraded the 3′UTR of *AKT1* mRNA [98]. Finally, TGF-β1 inhibited the expression of miR-185 in macrophages, and thus TGF-β1 signaling has been suggested to induce proliferation and activation of macrophages through inhibition of miR-185 expression [98].

Some lymphocytic cytokines are considered profibrotic with direct effects on fibroblast and myofibroblast activity in IPF. Th1, Th2, and Th17 have been linked to IPF pathogenesis. The Th1 subset produces IL-1α, TNF-α, PDGF, and TGF-β1. The Th2 subset typical interleukins are IL-4 and IL-13, which are directly involved in fibroblast activation and ECM production. The Th17 subset indirectly promotes fibrosis by increasing TGF-β1 levels, and it is positively regulated by TGF-β1,

**13**

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

elucidate the association between miRNAs and IPF.

**4. Conclusions**

creating a positive feedback loop [99]. In both BALF and the peripheral blood of IPF patients, the number of CD4 + CD25 + FOXP3+ regulatory T-cells (Tregs) is decreased compared with healthy people. Thus, Tregs' numerical and functional deficiency may play a central role in the initial phases of IPF pathogenesis [55].

In CD4+ T cells, the level of miR-155 expression is positively associated with the expression of the Th2 cytokines, IL-5 and IL-13, and miR-155 suppresses inhibitors of cytokine production including SOCS1 [100]. However, Foxp3 inhibition significantly decreased miR-146a expression in Tregs. The target gene of miR-146a is TNF receptor-associated factor 6 (TRAF6). Foxp3 and miR-146a have been suggested to regulate colonic inflammation and fibrosis by negatively regulating TRAF6 [101].

IPF is a heterogeneous disease that is affected and developed by various genetic backgrounds and environments. The recent development and approval of antifibrotic agents such as nintedanib and pirfenidone, both of which reduced the lung function decline rate in IPF patients in clinical trials, offer hope that it may be possible to alter the increased mortality associated with IPF; however, these therapeutic effects are not sufficient for curing the disease. To reveal new therapeutic IPF targets, genetic screening of IPF patients, including miRNAs, is an effective option. Over the past decade, the role of miRNAs in both promoting and alleviating chronic respiratory disease has become increasingly apparent. In IPF, some miRNAs have been indicated to be associated with EMT, EndoMT, fibrocyte differentiation, or ECM deposition. Investigation of miRNAs and their target genes associated with IPF may lead to new therapy. However, there have been some problems with applying miRNAs in the clinical scene: (1) multiple groups performing studies with similar experimental conditions have reported different and often contrasting results. Hence, collection methods and specimen parts need to be standardized, and better assays are needed for high-confidence miRNA measurements. (2) It is not clear whether miRNA misregulation is a direct cause of disease or an indirect result of gene regulatory processes. Most studies did not attempt to establish the necessity and sufficiency of the miRNA under investigation. (3) It is still difficult to interfere with miRNA expression in the human body, and it is possible that such interference will cause unexpected adverse reactions. (4) It has not been established how to introduce miRNAs into target cells safely and effectively, and the cost of this treatment will probably be high. Thus, miRNAs are still insufficient to be used as therapeutic targets in the clinical scene. Nevertheless, miRNAs may be important tools as IPF biomarkers. To use miRNAs clinically, further studies are necessary to

**Figure 4.** *The role of miRNAs in macrophages for IPF.*

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

creating a positive feedback loop [99]. In both BALF and the peripheral blood of IPF patients, the number of CD4 + CD25 + FOXP3+ regulatory T-cells (Tregs) is decreased compared with healthy people. Thus, Tregs' numerical and functional deficiency may play a central role in the initial phases of IPF pathogenesis [55].

In CD4+ T cells, the level of miR-155 expression is positively associated with the expression of the Th2 cytokines, IL-5 and IL-13, and miR-155 suppresses inhibitors of cytokine production including SOCS1 [100]. However, Foxp3 inhibition significantly decreased miR-146a expression in Tregs. The target gene of miR-146a is TNF receptor-associated factor 6 (TRAF6). Foxp3 and miR-146a have been suggested to regulate colonic inflammation and fibrosis by negatively regulating TRAF6 [101].

#### **4. Conclusions**

*Interstitial Lung Diseases*

activated by Th2 signals [89] [90]. Th2 cytokines, IL-4 and IL-13 (IL-4/IL-13), contribute to extensive tissue fibrosis in mouse models [91], and IL-4/IL-13-induced macrophages activate fibroblasts by secreting pro-fibrogenic cytokines. IL-4/IL-13 induced CCL18 in lung macrophages of IPF patients, and secreted CCL18 increased the production of collagen from fibroblasts [92]. Many transcriptional factors, such as STAT (signal transducers and activator of transcription) family, regulate macrophage polarization. M1 macrophage polarization is controlled via STAT1, and M2 macrophages activation is mediated by STAT6 [93]. To maintain STAT6 phosphorylation, M2 macrophages overcome strong expression of suppressor of cytokine signaling 1 (SOCS1), resulting in the prolongation of fibrogenesis (**Figure 4**).

IL-4/IL-13 induced miR-142-5p expression and suppressed miR-130a-3p expression in macrophages [94]. Transfection of macrophages with either miR-142-5p anti-sense oligonucleotides or miR-130-3p mimics significantly reduced the expression of M2 markers induced by IL-4 and regulated macrophage pro-fibrogenesis [94]. SOCS1 3′UTR has seven conserved sites complementary to miR-142-5p RNA, and PPARγ 3′UTR has seven conserved sites complementary to miR-130a-3p RNA. The expression of SOCS1 is reduced in IPF lungs, and bleomycin-induced lung fibrosis and inflammation were enhanced in SOCS1-deficient mice compared with WT mice [95]. Thus, miR-142-5p and miR-130a-3p regulate pulmonary

Downregulation of miR-185 has been demonstrated in IPF lung tissue and alveolar macrophages [29, 96]. MiR-185 inhibited cell growth and proliferation by directly targeting AKT1 [97]. Downregulation of miR-185 resulted in AKT activation via increased DNMT1 expression, leading to promoter hypermethylation and silencing of *PTEN* [77]. Moreover, miR-185 directly bound and degraded the 3′UTR of *AKT1* mRNA [98]. Finally, TGF-β1 inhibited the expression of miR-185 in macrophages, and thus TGF-β1 signaling has been suggested to induce proliferation and activation of macrophages through inhibition of miR-185 expression [98]. Some lymphocytic cytokines are considered profibrotic with direct effects on fibroblast and myofibroblast activity in IPF. Th1, Th2, and Th17 have been linked to IPF pathogenesis. The Th1 subset produces IL-1α, TNF-α, PDGF, and TGF-β1. The Th2 subset typical interleukins are IL-4 and IL-13, which are directly involved in fibroblast activation and ECM production. The Th17 subset indirectly promotes fibrosis by increasing TGF-β1 levels, and it is positively regulated by TGF-β1,

fibrosis through SOCS1 and PPARγ in macrophages.

**12**

**Figure 4.**

*The role of miRNAs in macrophages for IPF.*

IPF is a heterogeneous disease that is affected and developed by various genetic backgrounds and environments. The recent development and approval of antifibrotic agents such as nintedanib and pirfenidone, both of which reduced the lung function decline rate in IPF patients in clinical trials, offer hope that it may be possible to alter the increased mortality associated with IPF; however, these therapeutic effects are not sufficient for curing the disease. To reveal new therapeutic IPF targets, genetic screening of IPF patients, including miRNAs, is an effective option. Over the past decade, the role of miRNAs in both promoting and alleviating chronic respiratory disease has become increasingly apparent. In IPF, some miRNAs have been indicated to be associated with EMT, EndoMT, fibrocyte differentiation, or ECM deposition. Investigation of miRNAs and their target genes associated with IPF may lead to new therapy. However, there have been some problems with applying miRNAs in the clinical scene: (1) multiple groups performing studies with similar experimental conditions have reported different and often contrasting results. Hence, collection methods and specimen parts need to be standardized, and better assays are needed for high-confidence miRNA measurements. (2) It is not clear whether miRNA misregulation is a direct cause of disease or an indirect result of gene regulatory processes. Most studies did not attempt to establish the necessity and sufficiency of the miRNA under investigation. (3) It is still difficult to interfere with miRNA expression in the human body, and it is possible that such interference will cause unexpected adverse reactions. (4) It has not been established how to introduce miRNAs into target cells safely and effectively, and the cost of this treatment will probably be high. Thus, miRNAs are still insufficient to be used as therapeutic targets in the clinical scene. Nevertheless, miRNAs may be important tools as IPF biomarkers. To use miRNAs clinically, further studies are necessary to elucidate the association between miRNAs and IPF.

*Interstitial Lung Diseases*

#### **Author details**

Koichi Takagi1 , Munekazu Yamakuchi<sup>2</sup> \*, Teruto Hashiguchi2 and Hiromasa Inoue2

1 Department of Pulmonary Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

2 Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**15**

*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

> [9] Medina PP, Nolde M, Slack FJ. OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma. Nature. 2010;**467**(7311):86-

90. DOI: 10.1038/nature09284

10.1083/jcb.201608063

[11] Li XX, Liu YM, Li YJ, Xie N, Yan YF, Chi YL, et al. High glucose concentration induces endothelial cell proliferation by regulating cyclin-D2 related miR-98. Journal of Cellular and Molecular Medicine. 2016;**20**(6):1159- 1169. DOI: 10.1111/jcmm.12765

[12] Takagi K, Yamakuchi M, Matsuyama T, Kondo K, Uchida A, Misono S, et al. IL-13 enhances mesenchymal transition

of pulmonary artery endothelial cells via down-regulation of miR-424/503 in vitro. Cellular Signalling. 2018;**42**:270-280. DOI: 10.1016/j.

[13] Kim J, Hwangbo C, Hu X, Kang Y,

cellsig.2017.10.019

Papangeli I, Mehrotra D, et al. Restoration of impaired endothelial myocyte enhancer factor 2 function rescues pulmonary arterial hypertension. Circulation. 2015;**131**(2):190-199. DOI: 10.1161/

circulationaha.114.013339

[14] Lu TX, Munitz A, Rothenberg ME. MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression. Journal of Immunology. 2009;**182**(8):4994- 5002. DOI: 10.4049/jimmunol.0803560

[15] Mattes J, Collison A, Plank M, Phipps S, Foster PS. Antagonism

[10] Chowdhury SR, Reimer A, Sharan M, Kozjak-Pavlovic V, Eulalio A, Prusty BK, et al. Chlamydia preserves the mitochondrial network necessary for replication via microRNA-dependent inhibition of fission. The Journal of Cell Biology. 2017;**216**(4):1071-1089. DOI:

[1] Fernandez IE, Eickelberg O. New cellular and molecular mechanisms of lung injury and fibrosis in

idiopathic pulmonary fibrosis. Lancet. 2012;**380**(9842):680-688. DOI: 10.1016/

[2] Jin HL, Dong JC. Pathogenesis of idiopathic pulmonary fibrosis: From initial apoptosis of epithelial cells to lung remodeling. Chinese Medical Journal. 2011;**124**(24):4330-4338

[3] Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. The FASEB Journal. 2004;**18**(7):816-827.

[4] Raghu G, Remy-Jardin M, Myers JL, Richeldi L, Ryerson CJ, Lederer DJ, et al. Diagnosis of idiopathic pulmonary fibrosis: An official ATS/ERS/JRS/ALAT clinical practice guideline. American Journal of Respiratory and Critical Care Medicine. 2018;**198**(5):e44-e68. DOI:

DOI: 10.1096/fj.03-1273rev

10.1164/rccm.201807-1255ST

10.1038/ncb0309-228

[5] Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity: MicroRNA biogenesis pathways and their regulation. Nature Cell Biology. 2009;**11**(3):228-234. DOI:

[6] Bushati N, Cohen SM. MicroRNA

[7] Leung AK, Sharp PA. MicroRNA functions in stress responses. Molecular

Baltimore D. MicroRNA regulation

Cell. 2010;**40**(2):205-215. DOI: 10.1016/j.molcel.2010.09.027

[8] O'Connell RM, Rao DS,

of inflammatory responses. Annual Review of Immunology. 2012;**30**:295-312. DOI: 10.1146/ annurev-immunol-020711-075013

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*The Role of miRNAs in Idiopathic Pulmonary Fibrosis DOI: http://dx.doi.org/10.5772/intechopen.82771*

#### **References**

*Interstitial Lung Diseases*

**14**

**Author details**

Koichi Takagi1

provided the original work is properly cited.

, Munekazu Yamakuchi<sup>2</sup>

Medical and Dental Sciences, Kagoshima, Japan

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Pulmonary Medicine, Kagoshima University Graduate School of

2 Department of Laboratory and Vascular Medicine, Kagoshima University

Graduate School of Medical and Dental Sciences, Kagoshima, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
\*, Teruto Hashiguchi2

and Hiromasa Inoue2

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*Interstitial Lung Diseases*

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2011;**6**(6):e21253. DOI: 10.1371/journal.

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[24] Huang C, Xiao X, Yang Y, Mishra A, Liang Y, Zeng X, et al. MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation. The Journal of Biological Chemistry. 2017;**292**(40):16420-16439.

DOI: 10.1074/jbc.M117.805747

[25] Zhang S, Liu H, Liu Y, Zhang J, Li H, Liu W, et al. miR-30a as potential therapeutics by targeting TET1 through regulation of Drp-1 promoter hydroxymethylation in idiopathic pulmonary fibrosis. International Journal of Molecular Sciences.

2017;**18**(3). DOI: 10.3390/ijms18030633

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pone.0006718

pone.0021253

jcb.27185

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[17] Qin HB, Xu B, Mei JJ, Li D, Liu JJ, Zhao DY, et al. Inhibition of miRNA-221 suppresses the airway inflammation in asthma. Inflammation. 2012;**35**(4):1595- 1599. DOI: 10.1007/s10753-012-9474-1

[18] Pandit KV, Corcoran D, Yousef H, Yarlagadda M, Tzouvelekis A, Gibson KF, et al. Inhibition and role of let-7d in idiopathic pulmonary fibrosis. American Journal of

Respiratory and Critical Care Medicine. 2010;**182**(2):220-229. DOI: 10.1164/

[19] Liu G, Friggeri A, Yang Y, Milosevic J, Ding Q , Thannickal VJ, et al. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. The Journal of Experimental Medicine. 2010;**207**(8):1589-1597. DOI: 10.1084/

[20] Cushing L, Kuang PP, Qian J, Shao F, Wu J, Little F, et al. miR-29 is a major regulator of genes associated with pulmonary fibrosis. American Journal of Respiratory Cell and Molecular Biology. 2011;**45**(2):287-294. DOI: 10.1165/rcmb.2010-0323OC

[21] Pottier N, Maurin T, Chevalier B, Puissegur MP, Lebrigand K, Robbe-Sermesant K, et al. Identification of keratinocyte growth factor as a target of microRNA-155 in lung

10.1016/j.jaci.2011.04.005

rccm.200911-1698OC

jem.20100035

**16**

[28] Bibaki E, Tsitoura E, Vasarmidi E, Margaritopoulos G, Trachalaki A, Koutoulaki C, et al. miR-185 and miR-29a are similarly expressed in the bronchoalveolar lavage cells in IPF and lung cancer but common targets DNMT1 and COL1A1 show disease specific patterns. Molecular Medicine Reports. 2018;**17**(5):7105-7112. DOI: 10.3892/mmr.2018.8778

[29] Tsitoura E, Wells AU, Karagiannis K, Lasithiotaki I, Vasarmidi E, Bibaki E, et al. miR-185/AKT and miR-29a/ collagen 1a pathways are activated in IPF BAL cells. Oncotarget. 2016;**7**(46):74569-74581. DOI: 10.18632/ oncotarget.12740

[30] Dai WJ, Qiu J, Sun J, Ma CL, Huang N, Jiang Y, et al. Downregulation of microRNA-9 reduces inflammatory response and fibroblast proliferation in mice with idiopathic pulmonary fibrosis through the ANO1-mediated TGF-beta-Smad3 pathway. Journal of Cellular Physiology. 2019:**234**(3):2552-2565. DOI: 10.1002/jcp.26961

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[45] Hashimoto N, Phan SH, Imaizumi K, Matsuo M, Nakashima H, Kawabe T, et al. Endothelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis. American Journal of Respiratory Cell and Molecular Biology. 2010;**43**(2):161-172. DOI: 10.1165/rcmb.2009-0031OC

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s12916-015-0412-6

path.1700950212

japplphysiol.01104.2010

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Physiology. 2016;**311**(6):L1050-L1L61. DOI: 10.1152/ajplung.00252.2016

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