Matthew W. Gorr

Chronic graft-versus-host disease (cGVHD) significantly contributes to late mortality after allogeneic stem cell transplantation, with bronchiolitis obliterans syndrome (BOS) being a particularly lethal and treatment-resistant complication despite available therapies. Bromodomain and extraterminal (BET) proteins are epigenetic readers driving inflammatory transcriptional programs across multiple cell types. We hypothesized that BET inhibition would suppress inflammatory T and B cells and decrease macrophage polarization to a profibrotic phenotype, alleviating disease. In an established BOS cGVHD model, BET inhibition reduced germinal center (GC) formation and responses through a reduction of the CXCL13:CXCR5 axis and inflammatory T follicular helper/GC B cells in the spleen, along with a reduction in plasma cell infiltration within the lung. Mice with cGVHD had elevated pathogenic immunoglobulin G1 (IgG1) and IgM levels, both in circulation and deposited on lung tissue, which were attenuated under BET inhibition. Single-cell RNA-sequencing analysis revealed distinct cell states in the BOS lung vs control. In cGVHD mice, gene set enrichment analysis revealed the upregulation of profibrotic Arginase1 and Tgfb1 expression in alveolar macrophages (AM) and interstitial macrophages (IM), which was significantly reduced with BET inhibition. Furthermore, BET inhibition targeted lung-infiltrating M2 macrophages through the selective depletion of CD206+FcγR+ IM and AM, ultimately resulting in reduced collagen deposition and improved lung function. Our findings reveal a previously unrecognized mechanistic axis of BET regulation during cGVHD fibrosis and highlight BET inhibition as a promising therapeutic strategy.

BACKGROUND: Mitochondrial dysfunction caused by abnormally high RyR2 (ryanodine receptor) activity is a common finding in cardiovascular diseases. Mechanisms linking RyR2 gain of function with mitochondrial remodeling remain elusive. We hypothesized that RyR2 hyperactivity in cardiac disease increases [Ca ²⁺ ] in the mitochondrial intermembrane space (IMS) and activates the Ca ²⁺ -sensitive protease calpain, driving remodeling of mitochondrial cristae architecture through cleavage of structural protein OPA1 (optic atrophy protein 1). METHODS: We generated a highly arrhythmogenic rat model of catecholaminergic polymorphic ventricular tachycardia, induced by RyR2 gain-of-function mutation S2236L(Ser2336Leu) ^{( +/-)} . We created a new biosensor to measure IMS-[Ca ²⁺ ] in adult cardiomyocytes with intact Ca ²⁺ cycling. We used ex vivo whole heart optical mapping, confocal and electron microscopy, as well as in vivo/in vitro gene editing techniques to test the effects of calpain in the IMS. RESULTS: We found altered mitochondrial cristae structure, increased IMS-[Ca ²⁺ ], reduced OPA1 expression, and augmented mito-reactive oxygen species emission in catecholaminergic polymorphic ventricular tachycardia myocytes. We show that calpain-mediated OPA1 cleavage led to disrupted cristae organization and, thereby, decreased electron transport chain supercomplex assembly, resulting in accelerated reactive oxygen species production. Genetic inhibition of calpain activity in IMS reversed mitochondria structural defects in catecholaminergic polymorphic ventricular tachycardia myocytes and reduced arrhythmic burden in ex vivo optically mapped hearts. CONCLUSIONS: Our data suggest that RyR2 hyperactivity contributes to mitochondrial structural damage by promoting an increase in IMS-[Ca ²⁺ ], sufficient to activate IMS-residing calpain. Calpain activation leads to proteolysis of OPA1 and cristae widening, thereby decreasing assembly of electron transport chain components into supercomplexes. Consequently, excessive mito-reactive oxygen species release critically contributes to RyR2 hyperactivation and ventricular tachyarrhythmia. Our new findings suggest that targeting IMS calpain may be beneficial in patients at risk for sudden cardiac death.

The popularity of electronic cigarettes (e-cigs) has risen in recent years, largely due to perceptions of reduced harm compared with traditional tobacco cigarettes. E-cig use has spread into vulnerable populations such as adolescents and individuals of reproductive age, yet their safety during pregnancy remains poorly understood. E-cigs differ from conventional cigarettes in composition, and their aerosolized components may uniquely affect the uterine environment and placental function. Proper placental development, maternal vascular remodeling, and tone are essential for healthy pregnancy outcomes, but it remains unclear how e-cig aerosol components affect these processes. To address this gap, we investigated how in utero exposure to sub-ohm e-cig aerosols, with and without nicotine, affects maternal and fetal cardiovascular function, uterine vascular function, and placental morphology. Pregnant female FVB/J mice were exposed to filtered air, vehicle (propylene glycol/vegetable glycerin; PG:VG or PV), or vehicle with nicotine (PV + Nicotine) from embryonic day (E) 0.5 to E17.5 (4 h/day, 5 days/week). Maternal and fetal cardiovascular parameters were assessed at E16.5 via echocardiography and ultrasound. At E17.5, maternal, fetal, and placental tissues were collected for histological and molecular analysis. E-cig exposure resulted in maternal diastolic dysfunction, reduced uterine artery blood flow, fetal bradycardia, structural alterations in the placenta, and reduced placental sufficiency. These effects were observed in both e-cig exposure groups, with some differences between the PV and PV + Nicotine groups, suggesting that exposure to aerosolized e-cig vehicle components alone can also contribute to gestational impairments. Our findings demonstrate that e-cig exposure during pregnancy disrupts cardiovascular adaptation and fetal development, highlighting the need for reevaluation of e-cig safety during pregnancy and guiding future mechanistic studies of its effects on placental and vascular dysfunction.

The use of traditional nicotine delivery products such as tobacco has long been linked to detrimental health effects. However, little work to date has focused on the emerging market of aerosolized nicotine delivery known as electronic nicotine delivery systems (ENDS) or electronic cigarettes, and their potential for new effects on human health. Challenges studying these devices include heterogeneity in the formulation of the common components of most available ENDS, including nicotine and a carrier (commonly composed of propylene glycol and vegetable glycerin, or PG/VG). In the present study, we report on experiments interrogating the effects of major identified components in e‐cigarettes. Specifically, the potential concomitant effects of nicotine and common carrier ingredients in commercial “vape” products are explored in vitro to inform the potential health effects on the craniofacial skeleton through novel vectors as compared to traditional tobacco products. MC3T3‐E1 murine pre‐osteoblast cells were cultured in vitro with clinically relevant liquid concentrations of nicotine, propylene glycol (PG), vegetable glycerin (VG), Nicotine+PG/VG, and the vape liquid of a commercial product ( Juul ). Cells were treated acutely for 24 h and RNA‐Seq was utilized to determine segregating alteration in mRNA signaling. Influential gene targets identified with sparse partial least squares discriminant analysis (sPLS‐DA) implemented in mixOmics were assessed using the PANTHER Classification system for molecular functions, biological processes, cellular components, and pathways of effect. Additional endpoint functional analyses were used to confirm cell cycle changes. The initial excitatory concentration (EC50) studied defined a target concentration of carrier PG/VG liquid that altered the cell cycle of the calvarial cells. Initial sPLS‐DA analysis demonstrated the segregation of nicotine and non‐nicotine exposures utilized in our in vitro modeling. Pathway analysis suggests a strong influence of nicotine exposures on cellular processes including metabolic processes and response to stimuli including autophagic flux. Further interrogation of the individual treatment conditions demonstrated segregation by treatment modality (Control, Nicotine, Carrier (PG+VG), Nicotine+PG/VG) along three dimensions best characterized by: latent variable 1 (PLSDA‐1) showing strong segregation based on nicotine influence on cellular processes associated with cellular adhesion to collagen, osteoblast differentiation, and calcium binding and metabolism; latent variable 2 (PLSDA‐2) showing strong segregation of influence based on PG+VG and Control influence on cell migration, survival, and cycle regulation; and latent variable 3 (PLSDA‐3) showing strong segregation based on Nicotine and Control exposure influence on cell activity and growth and developmental processes. Further, gene co‐expression network analysis implicates targets of the major pathway genes associated with bone growth and development, particularly craniofacial (FGF, Notch, TGFβ, WNT) and analysis of active subnetwork pathways found these additionally overrepresented in the Juul exposure relative to Nicotine+PG/VG. Finally, experimentation confirmed alterations in cell count, and increased evidence of cell stress (markers of autophagy), but no alteration in apoptosis. These data suggest concomitant treatment with Nicotine+PG/VG drives alterations in pre‐osteoblast cell cycle signaling, specifically transcriptomic targets related to cell cycle and potentially cell stress. Although we suspected cell stress and well as cytotoxic effects of Nicotine+PG/VG, no great influence on apoptotic factors was observed. Further RNA‐Seq analysis allowed for the direct interrogation of molecular targets of major pathways involved in bone and craniofacial development, each demonstrating segregation (altered signaling) due to e‐cigarette‐type exposure. These data have implications directed toward ENDS formulation as synergistic effects of Nicotine+PG/VG are evidenced here. Thus, future research will continue to interrogate how varied formulation of Nicotine+PG/VG affects overall cell functions in multiple vital systems.

Respiratory immunity is dysregulated in preexisting asthma, and further perturbations by EC use could exacerbate asthma severity. However, the extent of their toxicity and the involved mechanisms contributing to the deleterious impact of EC exposure on patients with preexisting asthma have not been delineated. We found that EC has unique biological impacts in lungs and potential sex differences with loss of TFAM, a key mtDNA regulator, in lung epithelial region from our animal EC study.

The in utero environment is sensitive to toxicant exposure, altering the health and growth of the fetus, and thus sensitive to contaminant exposure. Though recent clinical data suggest that e-cigarette use does no further harm to birth outcomes than a nicotine patch, this does not account for the effects of vaping during pregnancy on the long-term health of offspring. Pregnant mice were exposed to: 1) e-cigarette vapor with nicotine (PV + Nic; 2% Nic in 50:50 propylene glycol: vegetable glycerin), 2) e-cigarette vapor without nicotine [PV; (50:50 propylene glycol:vegetable glycerin)], or 3) HEPA filtered air (FA). Dams were removed from exposure upon giving birth. At 5 mo of age, pulmonary function tests on the offspring revealed female and male mice from the PV group had greater lung stiffness (Ers) and alveolar stiffness (H) compared with the FA group. Furthermore, baseline compliance (Crs) was reduced in female mice from the PV group and in male mice from the PV and PV + Nic groups. Lastly, female mice had decreased forced expiratory volume (FEV0.1) in the PV group, but not in the male groups, compared with the FA group. Lung histology revealed increased collagen deposition around the vessels/airways and in alveolar tissue in PV and PV + Nic groups. Furthermore, goblet hyperplasia was observed in PV male and PV/PV + Nic female mice. Our work shows that in utero exposure to e-cigarette vapor, regardless of nicotine presence, causes lung dysfunction and structural impairments that persist in the offspring to adulthood.