Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: A mechanism underlying RV fibrosis associated with cigarette smoke exposure

INTRODUCTION: Right ventricular dysfunction is associated with numerous smoking-related illnesses including chronic obstructive pulmonary disease (COPD) where it is present even in absence of pulmonary hypertension. It is unknown if exposure to cigarette smoke has direct effects on RV function and cardiac fibroblast proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to cigarette smoke (CS) and determined mechanisms of smoke-induced changes in cardiac fibroblast signaling and fibrosis. METHODS: AKR mice were exposed to cigarette smoke for six weeks followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat cardiac fibroblasts (CF) exposed to cigarette smoke extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. RESULTS: Mice exposed to CS had significantly decreased RV function as determined by TAPSE. There were no changes in LV parameters. RV collagen content was significantly elevated but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased cardiac fibroblast proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CONCLUSION: CS and nicotine have direct effects on cardiac fibroblasts to induce proliferation and fibrosis which may negatively affect right heart function

Platelet activation in experimental murine neonatal pulmonary hypertension

Serotonin (5-HT) contributes to the pathogenesis of experimental neonatal pulmonary hypertension (PH) associated with bronchopulmonary dysplasia (BPD). Platelets are the primary source of circulating 5-HT and is released upon platelet activation. Platelet transfusions are associated with neonatal mortality and increased rates of BPD. As BPD is often complicated by PH, we tested the hypothesis that circulating platelets are activated and also increased in the lungs of neonatal mice with bleomycin-induced PH associated with BPD. Newborn wild-type mice received intraperitoneal bleomycin (3 units/kg) three times weekly for 3 weeks. Platelets from mice with experimental PH exhibited increased adhesion to collagen under flow (at 300

Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro

Fibrotic disorders account for over one third of mortalities worldwide. Despite great efforts to study the cellular and molecular processes underlying fibrosis, there are currently few effective therapies. Dual-stage polymerization reactions are an innovative tool for recreating heterogeneous increases in extracellular matrix (ECM) modulus, a hallmark of fibrotic diseases in vivo. Here, we present a clickable decellularized ECM (dECM) crosslinker incorporated into a dynamically responsive poly(ethylene glycol)-α-methacrylate (PEGαMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol-ene Michael addition between PEGαMA (8-arm, 10 kg mol-1) and the clickable dECM resulted in hydrogels with an elastic modulus of E = 3.6 ± 0.24 kPa, approximating healthy lung tissue (1-5 kPa). Next, residual αMA groups were reacted via a photo-initiated homopolymerization to increase modulus values to fibrotic levels (E = 13.4 ± 0.82 kPa) in situ. Hydrogels with increased elastic moduli, mimicking fibrotic ECM, induced a significant increase in the expression of myofibroblast transgenes. The proportion of primary fibroblasts from dual-reporter mouse lungs expressing collagen 1a1 and alpha-smooth muscle actin increased by approximately 60% when cultured on stiff and dynamically stiffened hybrid-hydrogels compared to soft. Likewise, fibroblasts expressed significantly increased levels of the collagen 1a1 transgene on stiff regions of spatially patterned hybrid-hydrogels compared to the soft areas. Collectively, these results indicate that hybrid-hydrogels are a new tool that can be implemented to spatiotemporally induce a phenotypic transition in primary murine fibroblasts in vitro