Manuela Martins-Green

Thirdhand Tobacco Smoke: Emerging Evidence and Arguments for a Multidisciplinary Research Agenda

Background: There is broad consensus regarding the health impact of tobacco use and secondhand smoke exposure, yet considerable ambiguity exists about the nature and consequences of thirdhand smoke (THS). Objectives: We introduce definitions of THS and THS exposure and review recent findings about constituents, indoor sorption–desorption dynamics, and transformations of THS; distribution and persistence of THS in residential settings; implications for pathways of exposure; potential clinical significance and health effects; and behavioral and policy issues that affect and are affected by THS. Discussion: Physical and chemical transformations of tobacco smoke pollutants take place over time scales ranging from seconds to months and include the creation of secondary pollutants that in some cases are more toxic (e.g., tobacco-specific nitrosamines). THS persists in real-world residential settings in the air, dust, and surfaces and is associated with elevated levels of nicotine on hands and cotinine in urine of nonsmokers residing in homes previously occupied by smokers. Much still needs to be learned about the chemistry, exposure, toxicology, health risks, and policy implications of THS. Conclusion: The existing evidence on THS provides strong support for pursuing a programmatic research agenda to close gaps in our current understanding of the chemistry, exposure, toxicology, and health effects of THS, as well as its behavioral, economic, and sociocultural considerations and consequences. Such a research agenda is necessary to illuminate the role of THS in existing and future tobacco control efforts to decrease smoking initiation and smoking levels, to increase cessation attempts and sustained cessation, and to reduce the cumulative effects of tobacco use on morbidity and mortality.

MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300.

CBP/p300 recruitment to enhancer‐bound complexes is a key determinant in promoter activation by many transcription factors. We present a novel mechanism of activating such complexes and show that pre‐assembled Elk‐1–p300 complexes become activated following Elk‐1 phosphorylation by changes in Elk‐1–p300 interactions rather than recruitment. It is known that Elk‐1 binds to promoter in the absence of stimuli. However, it is unclear how activation of Elk‐1 by mitogen‐acivated protein kinase (MAPK)‐mediated phosphorylation leads to targeted gene transactivation. We show that Elk‐1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk‐1 C‐terminus and the p300 N‐terminus. Phosphorylation on Ser383 and Ser389 of Elk‐1 by MAPK enhances this basal binding but, most importantly, Elk‐1 exhibits new interactions with p300. These interaction changes render a strong histone acetyltransferase activity in the Elk‐1‐associated complex that could play a critical role in chromatin remodeling and gene activation. The pre‐assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate‐early response genes, in particular those involved in stress responses.

Firsthand cigarette smoke alters fibroblast migration and survival: implications for impaired healing

Although it is known that high levels of cigarette smoke lead to cell death, little is known about the effects of low‐to‐moderate levels of smoke components that are found in vivo, such as those experienced by cells in tissues. Clinical studies and experimental data show that smokers heal poorly and are more prone to develop fibrotic diseases. Here we show the effects of first‐hand cigarette smoke on fibroblasts, cells that are critically involved in these processes. Using doses of smoke found in the tissues of smokers and a variety of cell and molecular approaches, we show that these doses of cigarette smoke do not cause cell death but rather stimulate fibroblasts to produce stress response and survival proteins such as interleukin‐8, PKB/Akt, p53, and p21 that in turn contribute to an increase in cell survival. In addition, smoke‐treated cells show a decrease in cell migration, which can be explained by the increased cell adhesion and alterations in cytoskeletal elements. We also show that these levels of smoke cause changes in mitochondrial morphology with a minimum loss of function and these changes are the result of exposure to reactive oxygen species. We conclude that the increase in cell survival may lead to a build‐up of connective tissue in the area of a wound, potentially leading to delayed healing and/or fibrosis and that the alterations in the cytoskeleton and in cell adhesion result in inhibition of cell migration, a process that could lead to nonclosure of the wound for lack of proper fibroblast migration to form the healing tissue.

The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure

Chemokines are small cytokines primarily known for their roles in inflammation. More recently, however, they have been implicated in processes involved in development of the granulation tissue of wounds, but little is known about their functions during this process. Fibroblasts play key roles in this phase of healing: some fibroblasts differentiate into myofibroblasts, α-smooth muscle actin (SMA)-producing cells that are important in wound closure and contraction. Here we show that the CXC chemokine chicken chemotactic and angiogenic factor (cCAF) stimulates fibroblasts to produce high levels of α-SMA and to contract collagen gels more effectively than do normal fibroblasts, both characteristic properties of myofibroblasts. Specific inhibition of α-SMA expression resulted in abrogation of cCAF-induced contraction. Furthermore, application of cCAF to wounds in vivo increases the number of myofibroblasts present in the granulation tissue and accelerates wound closure and contraction. We also show that these effects in culture and in vivo can be achieved by a peptide containing the NH2-terminal 15 amino acids of the cCAF protein and that inhibition of α-SMA expression also results in inhibition of N-peptide–induced collagen gel contraction. We propose that chemokines are major contributors for the differentiation of fibroblasts into myofibroblasts during formation of the repair tissue. Because myofibroblasts are important in many pathological conditions, and because chemokines and their receptors are amenable to pharmacological manipulations, chemokine stimulation of myofibroblast differentiation may have implications for modulation of functions of these cells in vivo.

Translation from the 5' untranslated region shapes the integrated stress response

How cells keep going in the face of adversity When cells experience stresses that affect their ability to process newly synthesized proteins, they turn down their rates of translation to help them survive the stress. They also turn on the translation of proteins that will help them cope with the misfolded proteins generated during stress. How do they turn down translation in general, but maintain or increase translation of specific proteins? Starck et al. developed an approach that allowed them to look at the translation of specific messenger RNAs that were not down-regulated by stress. They identified a motif that helped keep chaperone protein synthesis going. Science, this issue p. 10.1126/science.aad3867 Protein translation from open reading frames with alternative initiation codons occurs during induction of cellular stress responses. INTRODUCTION Protein synthesis is controlled by a plethora of developmental and environmental conditions. One intracellular signaling network, the integrated stress response (ISR), activates one of four kinases in response to a variety of distinct stress stimuli: the endoplasmic reticulum (ER)–resident kinase (PERK), the interferon-induced double-stranded RNA–dependent eIF2α kinase (PKR), the general control nonderepressible 2 (GCN2), or the heme-regulated inhibitor kinase (HRI). These four kinases recognize a central target and phosphorylate a single residue, Ser51, on the α subunit of the eukaryotic initiation factor 2 (eIF2α), which is a component of the trimeric initiation factor eIF2 that catalyzes translation initiation at AUG start codons. Phosphorylation of eIF2α down-regulates eIF2-dependent protein synthesis, which is important in development and immunity but also is implicated in neurodegeneration, cancer, and autoimmunity. However, protein synthesis does not cease on all mRNAs during the ISR. Rather, eIF2α phosphorylation is required for expression of select mRNAs, such as ATF4 and CHOP, that harbor small upstream open reading frames (uORFs) in their 5′ untranslated regions (5′ UTRs). Still other mRNAs sustain translation despite ISR activation. We developed tracing translation by T cells (3T) as an exquisitely sensitive technique to probe the translational dynamics of uORFs directly during the ISR. With 3T, we measured the peptide products of uORFs present in the 5′ UTR of the essential ER-resident chaperone, binding immunoglobulin protein (BiP), also known as heat shock protein family A member 5 (HSPA5), and characterized their requirement for BiP expression during the ISR. RATIONALE We repurposed the sensitivity and specificity of T cells to interrogate the translational capacity of RNA outside of annotated protein coding sequences (CDSs). 3T relies on insertion of a tracer peptide coding sequence into a candidate DNA sequence. The resulting mRNAs harboring the nested tracer peptide coding sequence are translated to produce tracer peptides. These translation products are processed and loaded onto major histocompatibility complex class I (MHC I) molecules in the ER and transit to the cell surface, where they can be detected by specific T cell hybridomas that are activated and quantified using a colorimetric reagent. 3T provides an approach to interrogate the thousands of predicted uORFs in mammalian genomes, characterize the importance of uORF biology for regulation, and generate fundamental insights into uORF mutation-based diseases. RESULTS 3T proved to be a sensitive and robust indicator of uORF expression. We measured uORF expression in the 5′ UTR of mRNAs at multiple distinct regions, while simultaneously detecting expression of the CDS. We directly measured uORF peptide expression from ATF4 mRNA and showed that its translation persisted during the ISR. We applied 3T to study BiP expression, an ER chaperone stably synthesized during the ISR. We showed that the BiP 5′ UTR harbors uORFs that are exclusively initiated by UUG and CUG start codons. BiP uORF expression bypassed a requirement for eIF2 and was dependent on the alternative initiation factor eIF2A. Both translation of the UUG-initiated uORF and eIF2A were necessary for BiP expression during the ISR. Unexpectedly, the products of uORF translation are predicted to generate MHC I peptides active in adaptive immunity. We propose that this phenomenon presents an extracellular signature during the ISR. CONCLUSION Our findings introduce the notion that cells harbor a distinct translation initiation pathway to respond to a variety of environmental conditions and cellular dysfunction. We showed that cells utilize a distinct, eIF2A-mediated initiation pathway, which includes uORF translation, to sustain expression of particular proteins during the ISR. 3T offers a valuable method to characterize the thousands of predicted translation events in 5′ UTRs and other noncoding RNAs and, expanded to a genome-wide scale, can complement ribosome profiling and mass spectrometry in uORF and short ORF discovery. Our observations underscore the importance of translation outside of annotated CDSs and challenge the very definition of the U in 5′ UTR. 3T reveals the translational landscape of the genome outside of annotated coding sequences. Tracer peptide coding sequences are inserted into regions outside the annotated CDS, such as uORFs. When translated, they generate peptides that are transported into the ER, loaded onto MHC I, and transit to the cell surface. T cell hybridomas that recognize the specific tracer peptide–MHC I complex become activated, which is detected using a colorimetric substrate. Translated regions distinct from annotated coding sequences have emerged as essential elements of the proteome. This includes upstream open reading frames (uORFs) present in mRNAs controlled by the integrated stress response (ISR) that show “privileged” translation despite inhibited eukaryotic initiation factor 2–guanosine triphosphate–initiator methionyl transfer RNA (eIF2·GTP·Met-tRNAiMet). We developed tracing translation by T cells to directly measure the translation products of uORFs during the ISR. We identified signature translation events from uORFs in the 5′ untranslated region of binding immunoglobulin protein (BiP) mRNA (also called heat shock 70-kilodalton protein 5 mRNA) that were not initiated at the start codon AUG. BiP expression during the ISR required both the alternative initiation factor eIF2A and non–AUG-initiated uORFs. We propose that persistent uORF translation, for a variety of chaperones, shelters select mRNAs from the ISR, while simultaneously generating peptides that could serve as major histocompatibility complex class I ligands, marking cells for recognition by the adaptive immune system.

Cigarette Smoke Toxins Deposited on Surfaces: Implications for Human Health

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.

Cell and molecular mechanisms of insulin-induced angiogenesis

Angiogenesis, the development of new blood vessel from pre‐existing vessels, is a key process in the formation of the granulation tissue during wound healing. The appropriate development of new blood vessels, along with their subsequent maturation and differentiation, establishes the foundation for functional wound neovasculature. We performed studies in vivo and used a variety of cellular and molecular approaches in vitro to show that insulin stimulates angiogenesis and to elucidate the signalling mechanisms by which this protein stimulates microvessel development. Mice skin injected with insulin shows longer vessels with more branches, along with increased numbers of associated α‐smooth muscle actin‐expressing cells, suggesting the appropriate differentiation and maturation of the new vessels. We also found that insulin stimulates human microvascular endothelial cell migration and tube formation, and that these effects occur independently of VEGF/VEGFR signalling, but are dependent upon the insulin receptor itself. Downstream signalling pathways involve PI3K, Akt, sterol regulatory element‐binding protein 1 (SREBP‐1) and Rac1; inhibition of these pathways results in elimination of endothelial cell migration and tube formation and significantly decreases the development of microvessels. Our findings strongly suggest that insulin is a good candidate for the treatment of ischaemic wounds and other conditions in which blood vessel development is impaired.

Vascular Endothelial Growth Factor Activation of Sterol Regulatory Element Binding Protein: A Potential Role in Angiogenesis

By stimulating the migration and proliferation of endothelial cells (ECs), vascular endothelial growth factor (VEGF) is a potent angiogenic factor. However, the molecular mechanism involved in the VEGF-induced angiogenesis remains elusive. We hypothesized that sterol regulatory element binding proteins (SREBPs), transcription factors governing cellular lipid homeostasis, play an important role in regulating angiogenesis in response to VEGF. VEGF activated SREBP1 and SREBP2 in ECs, as demonstrated by the increased SREBPs, their cleavage products, and the upregulation of the targeted genes. VEGF-induced SREBP activation depended on SREBP cleavage-activating protein (SCAP), because knocking down SCAP by RNA interference (RNAi) inhibited SREBP activation in response to VEGF. SREBP activation was also blocked by 25-hydroxycholesterol (25-HC). To verify the functional implication of SREBPs in VEGF-induced angiogenesis, we tested the role of SREBPs in EC migration and proliferation. SCAP RNAi or 25-HC inhibited VEGF-induced pseudopodia extension and migration of ECs. Both treatments inhibited VEGF-induced EC proliferation, with cell growth arrested at the G0/G1 phase and a concomitant decrease of the S phase. Blocking the PI3K-Akt pathway inhibited the VEGF-activated SREBPs, demonstrating that PI3K-Akt regulates SREBPs. Consistent with our in vitro data, SREBP1 was detected in newly developed microvasculatures in a rabbit skin partial-thickness wound-healing model. SREBP inhibition also markedly suppressed VEGF-induced angiogenesis in chick embryos. In summary, this study identifies SREBPs as the key molecules in regulating angiogenesis in response to VEGF.

A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity.

Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼

The chicken Chemotactic and Angiogenic Factor (cCAF), a CXC chemokine

25B/year in the US alone [1]. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.