Pulmonary arterial remodelling and role of endothelial to mesenchymal transition (EndMT) in patients with idiopathic pulmonary fibrosis (IPF)
Creators: name: Gaikwad, Archana
Publisher: name: University of Tasmania
Year: 2024

description: <b>Background</b>: Idiopathic pulmonary fibrosis (IPF) is a highly progressive interstitial lung disease which causes irreversible damage to the lungs. Global estimates suggest that 10 in 100,000 people are affected with this condition. IPF patients suffer from high mortality, with survival rate of only 2-3 years from diagnosis. Older and male population is more vulnerable. The injury to the lung alveolar region and its impairment is central to IPF pathophysiology. The severe damage to the alveolar-capillary membrane in the alveoli causes tissue disruption, aberrant fibrosis, and excessive extracellular matrix (ECM) deposition. Structural loss or degeneration of vasculature occurs in IPF, leading to comorbidities such as pulmonary hypertension (PH), further impacting patient survival. One of the potential mechanisms contributing to vascular remodelling and fibrosis in IPF is the process of endothelial-mesenchymal transition (EndMT). In EndMT, endothelial cells gain a mesenchymal phenotype and become actively matrix secreting fibroblast like cells. An in-depth understanding of the molecular pathways associated with EndMT could potentially lead to a better therapeutic modality for IPF patients. My thesis was designed to provide vital clinical evidence for quantitative assessment of arterial remodelling changes observed across various arterial sizes from a well-characterized IPF cohort. I also investigated arterial EndMT and its drivers in the patient tissues. Furthermore, I accessed the critical association between EndMT, vascular remodelling and IPF lung physiology.<br><b>Method:</b> I made vital morphological assessments of pulmonary arteries from resected lung tissues from IPF patients and comparable healthy normal controls (NC). Using lower magnification (4x), I initially captured arterial images of NC and IPF and classified their pulmonary arteries into six groups based on their size measurements (100-1000µm, interspaced 100 or 200µm). Measurements were done using pre-calibrated measuring tools within the image ProPlus7.0 software. I measured the external length (one end to the other end of adventitia) and luminal length (one end to the other end of the intima layer margin). Further, based on arterial sizes, I used specific magnifications, for example, 63x for 100-199μm, 40x for 200-399μm and 400-599μm, and 20x for larger arteries 600-1000μm for measuring total arterial thickness and thickness of individual layers intima, media, and adventitia. I also measured elastin deposition in them. For layer measurements, I made area of interest (AOI) for each layer. Intima encompassed areas starting from the luminal border toward the inner elastin bordering the media. Likewise, the media layer had areas that contained the inner elastin border to their external elastin bordering the adventitia. While the adventitial layer consisted of the outer boundary of the external elastin layer to the outer end of the connective tissue.<br>Once arterial remodelling was established, I further assessed EndMT role in vascular modification using optimized immunohistopathology techniques. First, I quantified the presence of intimal VE-cadherin and N-cadherin in the endothelial cell junctional and cytoplasm. Further, total arterial and individual layers mesenchymal maker- N-cadherin, S100A4, and vimentin percent expression were measured. I correlated these EndMT marker expressions with vascular remodelling changes and percent DLCO. Finally, to further establish the EndMT pathway driving these vascular remodelling, I made a quantitative immunohistochemical assessment of drivers- TGF-β1, pSmad-2/3, Smad-7, and β-catenin. These EndMT drivers' expressions in total arterial and individual layers were measured. Moreover, I evaluated whether these drivers influenced mesenchymal expression and vascular remodelling.<br><b>Results:</b> I found significant vascular remodelling changes across pulmonary arterial sizes in IPF compared to NC, with a significant decrease in the number of large and medium-sized arteries. In comparison to arteries in healthy lungs, patients with IPF exhibited notable structural alterations such as endothelial proliferation into the lumen, muscular hypertrophy of the intima and medial layers, proliferative intima, and the presence of plexiform lesions. Additionally, excessive deposition of collagen and elastin in the adventitia was also observed. In certain cases, the arteries were completely remodelled and merged seamlessly with the surrounding tissues. I also noted that the increased deposition of elastin could potentially contribute to the arterial thickening observed in IPF lungs. The quantification of these remodelling results showed the most significant thickness was in intima in the arterial ranges of 200–399μm and 600– 1000μm, whereas medial and adventitial thicknesses were significant across the arterial range of 200–1000μm. IPF patients also exhibited increased total arterial elastin in all arterial ranges except smaller arteries compared to NC. Medial thickness and total elastin were associated with low carbon monoxide absorption.<br>The arterial cellular transition was indicated by the expression of active mesenchymal markers N-cadherin, vimentin and S100A4, and downregulation of junctional VE-cadherin expression. I also noted more cytoplasmic VE-cadherin expression in the endothelium throughout all arterial ranges in IPF than in NC. N-cadherin junctional and cytoplasmic expression in all arterial ranges was significant in IPF compared to NC. Consequently, the expression of N-cadherin, vimentin and S100A4 percentages was higher in the IPF arteries. However, S100A4 staining was lesser than vimentin and N-cadherin expression across all arterial sizes in IPF Furthermore, increased mesenchymal expression was associated with a detrimental impact on percent DLCO in IPF. I also discovered that increased vimentin and N-cadherin expression could influence vascular remodelling changes in IPF.<br>Among the EndMT driver expressions, I found a significant and marked increase in TGF-β1, pSmad-2/3, Smad-7, and β-catenin expression in the intimal, medial, and adventitia layers across all arterial sizes in IPF compared to NC. Interestingly, the expression of these markers was much higher in the intimal layer across the 100- 1000μm range, corresponding to the earlier upsurge in intimal mesenchymal marker expression observed in IPF. Intimal TGF-β1 and β-catenin expression levels were significantly associated with increased vimentin and N-cadherin expression in the intimal layer. Moreover, intimal TGF-β1 had shown a substantial correlation with enhanced intimal thickness and elastin disposition. Intimal β-catenin also demonstrated a significant association with increased intimal thickness. Moreover, total TGF-β1 significantly impacted percent DLCO.<br><b>Conclusion:</b> My thesis provides for the first time a novel size-based evaluation of arterial remodelling changes in well-classified tissues of patients suffering from IPF. I have critically assessed the role of EndMT in these remodelling changes, and experiments conducted during my thesis provide an in-depth insight into the factors and drivers that facilitate these drastic changes in IPF. Overall, I identified a reduction in the vasculature and an increase in arterial thickness and ECM deposition. All these intrinsic factors were associated with a severe decline in lung function in these patients. EndMT was identified to be highly active across arterial sizes and layers. Also, I identified that TGF-β/Smad-dependent and β-catenin-dependent Wnt signaling pathways could be crucial drivers of EndMT and vascular remodelling. My vital research findings present the need for better diagnostic and therapeutic options for vascular remodelling in IPF patients. Targeting EndMT-associated pathways could help develop a novel treatment that can prevent disease progression and improve the quality of life for IPF patients.