Characterization of fibroblast population in healthy and diseased lung

Pulmonary hypertension (PH) is a vascular pathology characterized by alterations of the vascular wall leading to increased pulmonary arterial pressure and right ventricular failure. Many conditions are associated with the development of PH, such as ageing and metabolic diseases, such as diabetes mellitus. The sympathetic nervous system (SNS) plays an important role in the development and worsening of PH. SNS signalling, mediated by the secretion of norepinephrine (NE), promotes contraction of airway smooth muscle and vasoconstriction of the pulmonary vasculature. PH patients are characterized by increased muscle sympathetic nervous system activity in the lungs, which could worsen the progression of the disease by contributing to sustained pulmonary vasoconstriction. It has been previously shown that SLC1A3+ perivascular fibroblasts in other vasculatures express the norepinephrine transporter SLC6A2, uptaking NE and decreasing its bioavailability, therefore promoting vascular relaxation and increased blood flow. The aim of our work was to investigate the presence and the number of SLC6A2+ lung fibroblasts in conditions that predispose to the development of PH, such as aging and diabetes mellitus. We first identified the presence of SLC6A2+ fibroblasts in human lung samples through immunohistochemistry (IHC) staining of the NE transporter and PDGFRα, a fibroblast marker. We then proceeded to use a Slc1a3-CreERT2-Rosa26-tdTomato transgenic mouse line for further IHC analysis, which permitted us to lineage trace Slc1a3+ fibroblasts in the mouse lung and differentiate the fibroblasts in peribronchial, adventitial and alveolar regions based on their anatomical location. We established that 37% of peribronchial, 29% of adventitial and 28% alveolar fibroblasts express Slc6a2. Analysing aged murine samples, we observed a marked decrease in the number of tdTomato+ adventitial and alveolar fibroblasts, combined with a fibroblast-specific reduction in Slc6a2 expression in the adventitial region, which could lead to a reduced NE uptake in the pulmonary vessels of aged mice. PH onset is also associated with diabetes, therefore we next used streptozotocin (STZ) model of type 1 diabetes. STZ injected transgenic mice did not show a significant variation in the relative amount of tdTomato+ perivascular fibroblasts in the bronchial, adventitial or alveolar region. Mice exposed to a high fat diet, a model for diabetes type 2, did not show an increased number of Slc1a3+ cells in bronchial and adventitial region; while the alveolar region presented areas characterized by a significant increase in fibroblast density. Overall, our results highlight an alteration in the number of perivascular fibroblasts in PH-associated conditions, including an age-induced reduction of Slc6a2 expression that could impact disease onset and progression.