Using the Theory of Planned Behavior to Anticipate DIY E-juice Mixing among Young Adult International E-cigarette Users.

Background: Trends in young adult use of electronic nicotine delivery systems (ENDS) and experimentation with do-it-yourself (DIY) e-juice mixing are growing around the world. Theoretical frameworks for examining secondary behaviors (i.e., mixing) embedded within a primary behavior (i.e., vaping) are limited, leading to challenges in scholarly understanding of behavioral performance. This study explored the theoretically driven factors surrounding ENDS users' decision to mix DIY e-juice through a multiple behavior test of the theory of planned behavior (TPB). Methods: An international sample of young adult participants aged 18-19 (n=203) was recruited from Prolific for an online crosssectional survey. Path modeling tested four theoretically driven models to explore behavioral performance of mixing. Findings: The data supported TPB expectations and revealed new paths for secondary behavior. Primary perceptions of attitudes, norms, and intention were predictive of the same secondary perceptions. In addition, for both primary and secondary behaviors, perceived norms were a function of perceived attitudes. For the secondary behavior, normative influence was experienced indirectly through perceived attitudes. Conclusion: DIY e-juice mixing is a product of perceived attitudes and behavioral control surrounding mixing as well as perceived attitudes, norms, and intention surrounding general ENDS use. While unregulated DIY experimentation increases among youth, these findings provide a lens for public health efforts seeking to reach and reduce use. Understanding DIY e-juice behaviors is essential to anticipate stockpiling behaviors and negative outcomes from amateur experimentation.

Surface Lin28A expression consistent with cellular stress parallels indicators of senescence.

AIMS: Declining cellular functional capacity resulting from stress or ageing is a primary contributor to impairment of myocardial performance. Molecular pathway regulation of biological processes in cardiac interstitial cells (CICs) is pivotal in stress and ageing responses. Altered localization of the RNA-binding protein Lin28A has been reported in response to environmental stress, but the role of Lin28A in response to stress in CICs has not been explored. Surface Lin28A redistribution is indicative of stress response in CIC associated with ageing and senescence. METHODS AND RESULTS: Localization of Lin28A was assessed by multiple experimental analyses and treatment conditions and correlated to oxidative stress, senescence, and ploidy in adult murine CICs. Surface Lin28A expression is present on 5% of fresh CICs and maintained through Passage 2, increasing to 21% in hyperoxic conditions but lowered to 14% in physiologic normoxia. Surface Lin28A is coincident with elevated senescence marker p16 and beta-galactosidase (ß-gal) expression in CICs expanded in hyperoxia, and also increases with polyploidization and binucleation of CICs regardless of oxygen culture. Transcriptional profiling of CICs using single-cell RNA-Seq reveals up-regulation of pathways associated with oxidative stress in CICs exhibiting surface Lin28A. Induction of surface Lin28A by oxidative stress is blunted by treatment of cells with the antioxidant Trolox in a dose-dependent manner, with 300 µM Trolox exposure maintaining characteristics of freshly isolated CICs possessing low expression of surface Lin28A and ß-gal with predominantly diploid content. CONCLUSION: Surface Lin28A is a marker of environmental oxidative stress in CICs and antioxidant treatment antagonizes this phenotype. The biological significance of Lin28 surface expression and consequences for myocardial responses may provide important insights regarding mitigation of cardiac stress and ageing.

'Youthful' phenotype of c-Kit+ cardiac fibroblasts.

Cardiac fibroblast (CF) population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for CF biology and interventional strategies to influence injury response and aging-associated remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within CF population has not been addressed. An inducible reporter mouse detects c-Kit promoter activation with Enhanced Green Fluorescent Protein (EGFP) expression in cardiac cells. Coincidence of EGFP and c-Kit with the DDR2 fibroblast marker was confirmed using flow cytometry and immunohistochemistry. Subsequently, CFs expressing DDR2 with or without c-Kit was isolated and characterized. A subset of DDR2+ CFs also express c-Kit with coincidence in ~ 8% of total cardiac interstitial cells (CICs). Aging is associated with decreased number of c-Kit expressing DDR2+ CFs, whereas pathological injury induces c-Kit and DDR2 as well as the frequency of coincident expression in CICs. scRNA-Seq profiling reveals the transcriptome of c-Kit expressing CFs as cells with transitional phenotype. Cultured cardiac DDR2+ fibroblasts that are c-Kit+ exhibit morphological and functional characteristics consistent with youthful phenotypes compared to c-Kit- cells. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration, including phospho-ERK and pro-caspase 3. The phenotype of c-kit+ on DDR2+ CFs correlates with multiple characteristics of 'youthful' cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2+ CF population and provides a fundamental basis for future studies to influence myocardial biology, response to pathological injury and physiological aging.

Fundamentals of vaping-associated pulmonary injury leading to severe respiratory distress.

Vaping of flavored liquids has been touted as safe alternative to traditional cigarette smoking with decreased health risks. The popularity of vaping has dramatically increased over the last decade, particularly among teenagers who incorporate vaping into their daily life as a social activity. Despite widespread and increasing adoption of vaping among young adults, there is little information on long-term consequences of vaping and potential health risks. This study demonstrates vaping-induced pulmonary injury using commercial JUUL pens with flavored vape juice using an inhalation exposure murine model. Profound pathological changes to upper airway, lung tissue architecture, and cellular structure are evident within 9 wk of exposure. Marked histologic changes include increased parenchyma tissue density, cellular infiltrates proximal to airway passages, alveolar rarefaction, increased collagen deposition, and bronchial thickening with elastin fiber disruption. Transcriptional reprogramming includes significant changes to gene families coding for xenobiotic response, glycerolipid metabolic processes, and oxidative stress. Cardiac systemic output is moderately but significantly impaired with pulmonary side ventricular chamber enlargement. This vaping-induced pulmonary injury model demonstrates mechanistic underpinnings of vaping-related pathologic injury.

VAPIng into ARDS: Acute Respiratory Distress Syndrome and Cardiopulmonary Failure.

"Modern" vaping involving battery-operated electronic devices began approximately one dozen years and has quickly evolved into a multibillion dollar industry providing products to an estimated 50 million users worldwide. Originally developed as an alternative to traditional cigarette smoking, vaping now appeals to a diverse demographic including substantial involvement of young people who often have never used cigarettes. The rapid rise of vaping fueled by multiple factors has understandably outpaced understanding of biological effects, made even more challenging due to wide ranging individual user habits and preferences. Consequently while vaping-related research gathers momentum, vaping-associated pathological injury (VAPI) has been established by clinical case reports with severe cases manifesting as acute respiratory distress syndrome (ARDS) with examples of right ventricular cardiac failure. Therefore, basic scientific studies are desperately needed to understand the impact of vaping upon the lungs as well as cardiopulmonary structure and function. Experimental models that capture fundamental characteristics of vaping-induced ARDS are essential to study pathogenesis and formulate recommendations to mitigate harmful effects attributable to ingredients or equipment. So too, treatment strategies to promote recovery from vaping-associated damage require development and testing at the preclinical level. This review summarizes the back story of vaping leading to present day conundrums with particular emphasis upon VAPI-associated ARDS and prioritization of research goals.

Regeneration of infarcted mouse hearts by cardiovascular tissue formed via the direct reprogramming of mouse fibroblasts.

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.

Pim1 maintains telomere length in mouse cardiomyocytes by inhibiting TGFß signalling.

AIMS: Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFß) signalling pathway where inhibiting TGFß signalling maintains telomere length. The relationship between Pim1 and TGFß has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFß signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. METHODS AND RESULTS: Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFß signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFß signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFß pathway genes. CONCLUSION: Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFß pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

Duchenne muscular dystrophy (DMD) cardiomyocyte-secreted exosomes promote the pathogenesis of DMD-associated cardiomyopathy.

Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.

PIM1 Promotes Survival of Cardiomyocytes by Upregulating c-Kit Protein Expression.

Enhancing cardiomyocyte survival is crucial to blunt deterioration of myocardial structure and function following pathological damage. PIM1 (Proviral Insertion site in Murine leukemia virus (PIM) kinase 1) is a cardioprotective serine threonine kinase that promotes cardiomyocyte survival and antagonizes senescence through multiple concurrent molecular signaling cascades. In hematopoietic stem cells, PIM1 interacts with the receptor tyrosine kinase c-Kit upstream of the ERK (Extracellular signal-Regulated Kinase) and Akt signaling pathways involved in cell proliferation and survival. The relationship between PIM1 and c-Kit activity has not been explored in the myocardial context. This study delineates the interaction between PIM1 and c-Kit leading to enhanced protection of cardiomyocytes from stress. Elevated c-Kit expression is induced in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1. Co-immunoprecipitation and proximity ligation assay reveal protein-protein interaction between PIM1 and c-Kit. Following treatment with Stem Cell Factor, PIM1-overexpressing cardiomyocytes display elevated ERK activity consistent with c-Kit receptor activation. Functionally, elevated c-Kit expression confers enhanced protection against oxidative stress in vitro. This study identifies the mechanistic relationship between PIM1 and c-Kit in cardiomyocytes, demonstrating another facet of cardioprotection regulated by PIM1 kinase.

Enhancing myocardial repair with CardioClusters.

Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such 'next generation' implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit+ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage.

Human CardioChimeras: Creation of a Novel "Next-Generation" Cardiac Cell.

Background CardioChimeras produced by fusion of murine c-kit+ cardiac interstitial cells with mesenchymal stem cells promote superior structural and functional recovery in a mouse model of myocardial infarction compared with either precursor cell alone or in combination. Creation of human CardioChimeras (hCCs) represents the next step in translational development of this novel cell type, but new challenges arise when working with c-kit+ cardiac interstitial cells isolated and expanded from human heart tissue samples. The objective of the study was to establish a reliable cell fusion protocol for consistent optimized creation of hCCs and characterize fundamental hCC properties. Methods and Results Cell fusion was induced by incubating human c-kit+ cardiac interstitial cells and mesenchymal stem cells at a 2:1 ratio with inactivated Sendai virus. Hybrid cells were sorted into 96-well microplates for clonal expansion to derive unique cloned hCCs, which were then characterized for various cellular and molecular properties. hCCs exhibited enhanced survival relative to the parent cells and promoted cardiomyocyte survival in response to serum deprivation in vitro. Conclusions The generation of hCC is demonstrated and validated in this study, representing the next step toward implementation of a novel cell product for therapeutic development. Feasibility of creating human hybrid cells prompts consideration of multiple possibilities to create novel chimeric cells derived from cells with desirable traits to promote healing in pathologically damaged myocardium.

Adaptation within embryonic and neonatal heart environment reveals alternative fates for adult c-kit+ cardiac interstitial cells.

Cardiac interstitial cells (CICs) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c-Kit+ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast-like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast-like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra-embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra-embryonic tissue formation.

Cardiac tissue engineering therapeutic products to enhance myocardial contractility.

Researchers continue to develop therapeutic products for the repair and replacement of myocardial tissue that demonstrates contractility equivalent to normal physiologic states. As clinical trials focused on pure adult stem cell populations undergo meta-analysis for preclinical through clinical design, the field of tissue engineering is emerging as a new clinical frontier to repair the myocardium and improve cardiac output. This review will first discuss the three primary tissue engineering product themes that are advancing in preclinical to clinical models: (1) cell-free scaffolds, (2) scaffold-free cellular, and (3) hybrid cell and scaffold products. The review will then focus on the products that have advanced from preclinical models to clinical trials. In advancing the cardiac regenerative medicine field, long-term gains towards discovering an optimal product to generate functional myocardial tissue and eliminate heart failure may be achieved.

Cardiac regenerative therapy: Many paths to repair.

Cardiovascular disease remains the primary cause of death in the United States and in most nations worldwide, despite ongoing intensive efforts to promote cardiac health and treat heart failure. Replacing damaged myocardium represents perhaps the most promising treatment strategy, but also the most challenging given that the adult mammalian heart is notoriously resistant to endogenous repair. Cardiac regeneration following pathologic challenge would require proliferation of surviving tissue, expansion and differentiation of resident progenitors, or transdifferentiation of exogenously applied progenitor cells into functioning myocardium. Adult cardiomyocyte proliferation has been the focus of investigation for decades, recently enjoying a renaissance of interest as a therapeutic strategy for reversing cardiomyocyte loss due in large part to ongoing controversies and frustrations with myocardial cell therapy outcomes. The promise of cardiac cell therapy originated with reports of resident adult cardiac stem cells that could be isolated, expanded and reintroduced into damaged myocardium, producing beneficial effects in preclinical animal models. Despite modest functional improvements, Phase I clinical trials using autologous cardiac derived cells have proven safe and effective, setting the stage for an ongoing multi-center Phase II trial combining autologous cardiac stem cell types to enhance beneficial effects. This overview will examine the history of these two approaches for promoting cardiac repair and attempt to provide context for current and future directions in cardiac regenerative research.

Adult human cardiac stem cell supplementation effectively increases contractile function and maturation in human engineered cardiac tissues.

BACKGROUND: Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS: Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS: The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS: For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Transcribing the heart: faithfully interpreting cardiac transcriptional insights.

Transcriptional profiling continues to produce phenotypical data essential for understanding of basic cardiac biology and required to improve efficiency of cardiac regenerative and therapeutic approaches after injury. Accurate interpretation of cardiac transcriptional data comes with the unique challenges of heart biology including cardiomyocyte morphology, cryopreservation of limited samples and adequate selection of transcriptional platform at a single-cell resolution. Consequently, development and implementation of novel transcriptional platforms and creative bioinformatic analysis are essential to resolve standing questions in the field of cardiac regenerative medicine. Targeted bioinformatic approaches, advancing technological access, increase technical availability and fostering communication between interdisciplinary groups is critical to improve therapeutic approaches and to overcome challenges inherent to transcriptomic cardiac research.

Adult Cardiomyocyte Cell Cycle Detour: Off-ramp to Quiescent Destinations.

Ability to promote completion of mitotic cycling of adult mammalian cardiomyocytes remains an intractable and vexing challenge, despite being one of the most sought after 'holy grails' of cardiovascular research. While some of the struggle is attributable to adult cardiomyocytes themselves that are notoriously post-mitotic, another contributory factor rests with difficulty in definitive tracking of adult cardiomyocyte cell cycle and lack of rigorous measures to track proliferation in situ. This review summarizes past, present, and future directions to promote adult mammalian cardiomyocyte cell cycle progression, proliferation, and renewal. Establishing relationship(s) between cardiomyocyte cell cycle progression and cellular biological properties is sorely needed to understand the mechanistic basis for cardiomyocyte cell cycle withdrawal to enhance cardiomyocyte cell cycle progression and mitosis.