The fate of an inhaled cigarette puff in the human respiratory tract.

OBJECTIVE: Cigarette smoking can lead to a host of adverse health effects such as lung and heart disease. Increased lung cancer risk is associated with inhalation of carcinogens present in a puff of smoke. These carcinogenic compounds deposit in the lung at different sites and trigger a cascade of events leading to adverse outcomes. Understanding the site-specific deposition of various smoke constituents will inform the study of respiratory diseases from cigarette smoking. We previously developed a deposition model for inhalation of aerosol from electronic nicotine delivery systems. In this study, the model was modified to simulate inhalation of cigarette smoke consisting of soluble and insoluble tar, nicotine, and cigarette-specific constituents that are known or possible human carcinogens. MATERIALS AND METHODS: The deposition model was further modified to account for nicotine protonation and other cigarette-specific physics-based mechanisms that affect smoke deposition. Model predictions showed a total respiratory tract uptake in the lung for formaldehyde (99%), nicotine (80%), and benzo[a]pyrene (60%). RESULTS: The site of deposition and uptake depended primarily on the constituent's saturation vapor pressure. High vapor pressure constituents such as formaldehyde were preferentially absorbed in the oral cavity and proximal lung regions, while low vapor pressure constituents such as benzo[a]pyrene were deposited in the deep lung regions. Model predictions of exhaled droplet size, droplet retention, nicotine retention, and uptake of aldehydes compared favorably with experimental data. CONCLUSION: The deposition model can be integrated into exposure assessments and other studies that evaluate potential adverse health effects from cigarette smoking.

KLF4 (Kruppel-Like Factor 4)-Dependent Perivascular Plasticity Contributes to Adipose Tissue inflammation.

OBJECTIVE: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-CreERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP+ cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP+; and unintended proinflammatory consequences. Using newly generated Myh11-DreERT2tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4Δ/Δ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1+ lymphatic endothelial cells in the epididymal AT. CONCLUSIONS: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Novel Autoimmune IgM Antibody Attenuates Atherosclerosis in IgM Deficient Low-Fat Diet-Fed, but Not Western Diet-Fed Apoe-/- Mice.

OBJECTIVE: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic Apoe-/- mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of Apoe-/- mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of Apoe-/- mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in Apoe-/- mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, sIgM-/-Apoe-/-, mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age. CONCLUSIONS: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.

Klf4 has an unexpected protective role in perivascular cells within the microvasculature.

Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.

Cardiovascular Outcomes among Combustible-Tobacco and Electronic Nicotine Delivery System (ENDS) Users in Waves 1 through 5 of the Population Assessment of Tobacco and Health (PATH) Study, 2013-2019.

BACKGROUND: Prior studies have not clearly established risk of cardiovascular disease (CVD) among smokers who switch to exclusive use of electronic nicotine delivery systems (ENDS). We compared cardiovascular disease incidence in combustible-tobacco users, those who transitioned to ENDS use, and those who quit tobacco with never tobacco users. METHODS: This prospective cohort study analyzes five waves of Population Assessment of Tobacco and Health (PATH) Study data, Wave 1 (2013-2014) through Wave 5 (2018-2019). Cardiovascular disease (CVD) incidence was captured over three intervals (Waves 1 to 3, Waves 2 to 4, and Waves 3 to 5). Participants were adults (40+ years old) without a history of CVD for the first two waves of any interval. Change in tobacco use status, from exclusive past 30 day use of any combustible-tobacco product to either exclusive past 30 day ENDS use, dual past 30 day use of ENDS and combustible-tobacco, or no past 30 day use of any tobacco, between the first two waves of an interval was used to predict onset of CVD between the second and third waves in the interval. CVD incidence was defined as a new self-report of being told by a health professional that they had congestive heart failure, stroke, or a myocardial infarction. Generalized estimating equation (GEE) analyses combined 10,548 observations across intervals from 7820 eligible respondents. RESULTS: Overall, there were 191 observations of CVD among 10,548 total observations (1.7%, standard error (SE) = 0.2), with 40 among 3014 never users of tobacco (1.5%, SE = 0.3). In multivariable models, CVD incidence was not significantly different for any tobacco user groups compared to never users. There were 126 observations of CVD among 6263 continuing exclusive combustible-tobacco users (adjusted odds ratio [AOR] = 1.44; 95% confidence interval (CI) 0.87-2.39), 15 observations of CVD among 565 who transitioned to dual use (AOR = 1.85; 0.78-4.37), and 10 observations of CVD among 654 who quit using tobacco (AOR = 1.18; 0.33-4.26). There were no observations of CVD among 53 who transitioned to exclusive ENDS use. CONCLUSIONS: This study found no difference in CVD incidence by tobacco status over three 3 year intervals, even for tobacco quitters. It is possible that additional waves of PATH Study data, combined with information from other large longitudinal cohorts with careful tracking of ENDS use patterns may help to further clarify this relationship.

BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma.

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma.

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-ß activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P < 0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P < 0.001) and 133.3-fold (P < 0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P < 0.001) and 2.7-fold reduction (P < 0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.

KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis.

Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using Myh11-CreER(T2) ROSA floxed STOP eYFP Apoe(-/-) mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Krüppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation-sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.