Evolving evidence suggests nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness (AHR). We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (ET-1), and collagen (COL1A1, and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary lung fibroblasts (LFs) exposed to nicotine (50 µg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 µg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist, rosiglitazone. Compared to unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo.  Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, while treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3’UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

Extract of the Japanese apricot (JAE) has biological properties as an antioxidant and anti-inflammatory agent. We hypothesized that JAE might exert therapeutic effects on cigarette smoke (CS)-induced DNA damage and cytotoxicity. In this study, we found that concentrated JAE protects against cigarette smoke extract (CSE)-induced cytotoxicity and DNA damage accompanied by increased levels of aldehyde dehydrogenase (ALDH)2, 3A1, and Werner’s syndrome protein (WRN) in immortalized human bronchial epithelial cells (HBEC2) and normal human epidermal keratinocytes (NHEK). Using the centrifugal partition chromatography (CPC) method, we identified an undescribed compound, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal (which we named FA-1), responsible for the protective effects against CSE. This chemical structure has not been reported from a natural source to date. Protective effects of isolated FA-1 against CSE were observed in both HBEC2 and NHEK cells. The studies described herein suggest that FA-1 isolated from JAE protects against CSE-induced DNA damage and apoptosis by augmenting multiple isozymes of ALDH and DNA repair and reducing oxidative stress.

Sickle cell disease (SCD)-associated pulmonary hypertension (PH) causes significant morbidity and mortality. Here, we defined the role of endothelial specific peroxisome proliferator-activated receptor γ (PPARγ) function and novel PPARγ/HUWE1/miR-98 signaling pathways in the pathogenesis of SCD-PH. PH and right ventricular hypertrophy (RVH) were increased in chimeric Townes humanized sickle cell (SS) mice with endothelial-targeted PPARγ knockout (SSePPARγKO) compared with chimeric littermate control (SSLitCon). Lung levels of PPARγ, HUWE1, and miR-98 were reduced in SSePPARγKO mice compared with SSLitCon mice, whereas SSePPARγKO lungs were characterized by increased levels of p65, ET-1, and VCAM1. Collectively, these findings indicate that loss of endothelial PPARγ is sufficient to increase ET-1 and VCAM1 that contribute to endothelial dysfunction and SCD-PH pathogenesis. Levels of HUWE1 and miR-98 were decreased, and p65 levels were increased in the lungs of SS mice in vivo and in hemin-treated human pulmonary artery endothelial cells (HPAECs) in vitro. Although silencing of p65 does not regulate HUWE1 levels, the loss of HUWE1 increased p65 levels in HPAECs. Overexpression of PPARγ attenuated hemin-induced reductions of HUWE1 and miR-98 and increases in p65 and endothelial dysfunction. Similarly, PPARγ activation attenuated baseline PH and RVH and increased HUWE1 and miR-98 in SS lungs. In vitro, hemin treatment reduced PPARγ, HUWE1, and miR-98 levels and increased p65 expression, HPAEC monocyte adhesion, and proliferation. These derangements were attenuated by pharmacological PPARγ activation. Targeting these signaling pathways can favorably modulate a spectrum of pathobiological responses in SCD-PH pathogenesis, highlighting novel therapeutic targets in SCD pulmonary vascular dysfunction and PH.

Endothelial-to-mesenchymal transition (EndoMT) plays an important role in pulmonary hypertension (PH). Also, the molecular mechanisms regulating EndoMT in PH remain to be defined. In this study, we first showed that reduced expression of the transcription factors ETV2 (ETS variant 2) and PPARγ (Peroxisome Proliferator-Activated Receptor gamma) along with reduced endothelial markers and increased EndoMT markers were consistently observed in lungs and pulmonary artery endothelial cells (PAECs) of idiopathic pulmonary arterial hypertension (IPAH) patients, in hypoxia-exposed mouse lungs, human PAECs, and in induced EndoMT cells. Base on this observation, we aimed to investigate the function of ETV2 and PPARγ in EndoMT. We have explored the function of ETV2 and PPARγ and its mechanism in PH using in Etv2+/- mice or PPARγ KO mice. Etv2+/- mice spontaneously developed PH and right ventricular hypertrophy, associated with increased EndoMT markers and decreased EC markers. PPARγ transcriptionally activated the ETV2 promoter. Endothelial PPARγ expression in mice is positively correlated with ETV2 expression, but inversely with EndoMT markers.Overexpression of ETV2 in hypoxia-exposed rat pulmonary artery led to vascular relaxation. We conclude that PPARγ-ETV2 signaling can function as a novel pathway in PH pathogenesis by attenuating EndoMT.