Problem or question being addressed: Investigating the role of endothelial-epithelial crosstalk in the regulation of ATII cells differentiation and its possible therapeutic implications in idiopathic pulmonary fibrosis.Rationale for your approach: Pulmonary fibrosis (PF) is a progressive disease in which scarring of lung tissue leads to death from respiratory failure (1). Currently available drugs, pirfenidone and nintedanib, have only a modest effect on survival (2). The lack of effective therapies is likely due to a poor understanding of the pathogenetic mechanisms driving disease onset and progression, especially in its idiopathic form (Idiopathic Pulmonary Fibrosis, IPF) (3). While lung fibroblasts are clearly the ultimate effectors of fibrotic tissue deposition(4), emerging evidence indicates that IPF should be considered an epithelium-driven disease (5). The alveolar epithelium is composed of fully differentiated alveolar type I (ATI) cells, which perform gas exchange, and alveolar type II (ATII) cells, which produce surfactant proteins and act as progenitor cells, giving rise to new ATI cells upon injury. Loss of regenerative potential in ATII cells could represent a major driver of IPF (6). Recent evidence indicates that alveologenesis and ATII cell activation depend on close physical and functional interactions of ATII cells with lung endothelial cells (ECs) through the secretion of angiocrine signals (8), although, to our knowledge, the role of the endothelium in IPF has never been investigated. Among the regulators of alveolar epithelial function are microRNAs (miRNAs), small non-coding RNA molecules that function by repressing multiple target messenger RNAs (mRNAs) (9). Several miRNAs are involved in lung epithelial repair, epithelial-mesenchymal transition (EMT), and collagen production, all of which are relevant pathological mechanisms in IPF (9).Details of suggested approach: We recently demonstrated that ATII cells isolated from IPF patients fail to trans-differentiate to ATI cells (9) and undergo both EMT and senescence. Delivery of miR- 200c-3p to ATII cells from IPF patients rescued their trans-differentiation capacity, supporting the therapeutic potential of miR-200c in IPF (9). Whether miR-200c-3p can also act on other cell types of the lung remains elusive. Here, we will induce PF by intra-tracheal bleomycin administration, the most used protocol worldwide. We will isolate ATII cells from mouse lungs at 14 and 30 days after bleomycin administration and quantify their trans-differentiation to ATI cells. We expect control ATII cells to efficiently trans-differentiate to ATI cells. In contrast, ATII cells from lungs exposed to bleomycin for either 14 or 30 days are expected to show a drastic impairment in trans- differentiation. After confirming the suitability of the bleomycin model of PF in reproducing the trans-differentiation defect of ATII cells, we will assess the therapeutic potential of miR-200c-3p. Thus, we will deliver either miR-200c-3p or a control miRNA by aerosol immediately after bleomycin administration and assess the development of PF. The control miRNA should not affect the extent of PF. Conversely, mice that will receive miR-200c-3p should develop a milder disease with attenuated collagen deposition. Next, we aim to assess whether miR-200c-3p can revert established PF. Thus, we will deliver miR-200c-3p 14 days after bleomycin administration and sacrifice animals after an additional 16 days. We expect miR-200c-3p to significantly reduce PF, both in a preventing and therapeutic manner. To investigate additional mechanisms by which miR-200c-3p could prevent pulmonary fibrosis, we will interrogate the miRNA target prediction software miRtarbase to identify its direct targets in murine cells. Three targets have been experimentally validated by luciferase 3’UTR reporter assay: Flt1, Zeb1, and Zeb2. As these genes, especially Flt1, are mainly expressed by ECs, we hypothesized that these cells might be involved in the therapeutic effect exerted by miR- 200c-3p. To identify the main targets of miR-200c-3p in ECs, we will isolate lung ECs and transfect them with miR-200c-3p, a control miRNA, and siUBC (as an indicator of transfection efficiency). Next, to evaluate the role of Flt1 in the crosstalk between alveolar endothelial and epithelial cells, we will cross Cdh5-ERT2 mice with Flt1flox/flox mice to selectively knockout Flt1 in ECs upon Tamoxifen
administration (Flt1 KOiEC). Then, we will isolate ECs from either wild type (wt) or Flt1 KOiEC mice and co-culture them with ATII cells, aiming to understand if ECs can activate the trans-differentiation of impaired ATII cells. To discriminate whether this effect requires cell-to-cell contact or is mediated by secreted factors, we will collect the conditioned medium of alveolar lung ECs purified from either wt or Flt1 KOiEC mice. In parallel, we will purify ATII cells from both controland bleomycin-treated mice and will expose them to the collected EC supernatants. Next, we will evaluate whether endothelial Flt1 has an impact on PF development in vivo. For this, Flt1 KOiEC and control mice will be treated intratracheally with either bleomycin or PBS (control). We expect Flt1 KOiEC mice to develop minimal fibrosis with preserved lung structure in comparison with wt mice. Finally, to shed light on the molecular mechanism by which Flt1 KO ECs promote ATII trans-differentiation to ATI cells, thus preventing fibrosis, we will interrogate the secretome of these cells by mass spectrometry analysis. In particular, we will purify ECs from wt and Flt1 KOiEC mice and culture them in serum-free medium for three days. Supernatants will be collected, and purified proteins will be analyzed by mass spectrometry (LC-MS/MS). Last, we will validate the functional effect of the identified proteins.How it will affect the broader field: The role of endothelial-epithelial crosstalk in IPF pathogenesis is still unknown. It has been described that lung ECs drive the activation of ATII cells in a mouse model of lung regeneration (9). The existence of a complex endothelial-epithelial paracrine crosstalk in vitro and in vivo could place lung ECs as a relevant therapeutic target in the fight against IPF. This project is essential to study the cell-cell communication to better understand the pivotal role of alveolar endothelial cells in activating the alveolar epithelial cells, that in turn, in pathological conditions, activate the transition of fibroblast in myofibroblast, leading to extracellular matrix deposition and pulmonary fibrosis. Finally, we will describe a novel mechanism by which miR-200c- 3p protects from and reverts PF, identifying its targets and demonstrating a complex endothelial- epithelial paracrine crosstalk.References
Collaborators GBDCRD. Prevalence and attributable health burden of chronic respiratory diseases, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Respir Med. 2020;8(6):585-96.
Kang J, Han M, Song JW. Antifibrotic treatment improves clinical outcomes in patients with idiopathic pulmonary fibrosis: a propensity score matching analysis. Sci Rep. 2020;10(1):15620.
Fernandez Fabrellas E, Peris Sanchez R, Sabater Abad C, Juan Samper G. Prognosis and Follow-Up of Idiopathic Pulmonary Fibrosis. Med Sci (Basel). 2018;6(2).
Rehman M, Vodret S, Braga L, Guarnaccia C, Celsi F, Rossetti G, et al. High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation. JCI Insight. 2019;4(8).
Naikawadi RP, Disayabutr S, Mallavia B, Donne ML, Green G, La JL, et al. Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis. JCI Insight. 2016;1(14):e86704.
Parimon T, Yao C, Stripp BR, Noble PW, Chen P. Alveolar Epithelial Type II Cells as Drivers of Lung Fibrosis in Idiopathic Pulmonary Fibrosis. Int J Mol Sci. 2020;21(7).
Ding BS, Nolan DJ, Guo P, Babazadeh AO, Cao Z, Rosenwaks Z, et al. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell. 2011;147(3):539-53.
9. Moimas S, Salton F, Kosmider B, Ring N, Volpe MC, Bahmed K, et al. miR-200 family members reduce senescence and restore idiopathic pulmonary fibrosis type II alveolar epithelial cell transdifferentiation. ERJ Open Res. 2019;5(4).