Effects of Early and Late Time-Restricted Feeding on Parameters of Metabolic Health: An Explorative Literature Assessment.

Chrono-nutrition (meal timing) aligns food consumption with one's circadian rhythm. The first meal (e.g., breakfast) likely promotes synchronization of peripheral circadian clocks, thereby supporting metabolic health. Time-restricted feeding (TRF) has been shown to reduce body weight (BW) and/or improve cardiovascular biomarkers. In this explorative literature assessment, 13 TRF randomized controlled trials (RCTs) were selected from PubMed and Scopus to evaluate the effects of early (eTRF: first meal before 10:30 a.m.) and late TRF (lTRF: first meal after 11:30 a.m.) on parameters of metabolic health. Although distinct variations in study design were evident between reports, TRF consistently decreased energy intake (EI) and BW, and improved insulin resistance as well as systolic blood pressure. eTRF seemed to have a greater beneficial effect than lTRF on insulin resistance (HOMA-IR). Importantly, most studies did not appear to consider chronotype in their evaluation, which may have underestimated TRF effects. TRF intervention may be a promising approach for risk reduction of human metabolic diseases. To conclusively determine benefits of TRF and identify clear differences between eTRF and lTRF, future studies should be longer-term (≥8 weeks) with well-defined (differences in) feeding windows, include participants chronotypically matching the intervention, and compare outcomes to those of control groups without any dietary limitations.

Geranylgeranyl transferase 1 modulates autophagy and apoptosis in human airway smooth muscle.

Geranylgeranyl transferase 1 (GGT1) is involved in the posttranslational prenylation of signaling proteins, such as small GTPases. We have shown that blocking the formation of isoprenoids with statins regulates survival of human lung mesenchymal cells; thus, we tested the hypothesis that GGT1 may specifically modulate programmed cell death pathways in these cells. To this end, human airway smooth muscle (HASM) cells were treated with the selective GGT1 inhibitor GGTi-298. Apoptosis was seen using assays for cellular DNA content and caspase activation. Induction of autophagy was observed using transmission electron microscopy, immunoblotting for LC3 lipidation and Atg5-12 complex content, and confocal microscopy to detect formation of lysosome-localized LC3 punctae. Notably, GGT1 inhibition induced expression of p53-dependent proteins, p53 upregulated modulator of apoptosis (Noxa), and damage-regulated autophagy modulator (DRAM), this was inhibited by the p53 transcriptional activation inhibitor cyclic-pifithrin-α. Inhibition of autophagy with bafilomycin-A1 or short-hairpin RNA silencing of Atg7 substantially augmented GGTi-298-induced apoptosis. Overall, we demonstrate for the first time that pharmacological inhibition of GGT1 induces simultaneous p53-dependent apoptosis and autophagy in HASM. Moreover, autophagy regulates apoptosis induction. Thus, our findings identify GGT1 as a key regulator of HASM cell viability.

Therapeutic potential of targeting regulatory mechanisms of hepatic stellate cell activation in liver fibrosis.

Hepatic fibrosis is a manifestation of different etiologies of liver disease with the involvement of multiple mediators in complex network interactions. Activated hepatic stellate cells (aHSCs) are the central driver of hepatic fibrosis, given their potential to induce connective tissue formation and extracellular matrix (ECM) protein accumulation. Therefore, identifying the cellular and molecular pathways involved in the activation of HSCs is crucial in gaining mechanistic and therapeutic perspectives to more effectively target the disease. In addition to a comprehensive summary of our current understanding of the role of HSCs in liver fibrosis, we also discuss here the proposed therapeutic strategies based on targeting HSCs.

The mevalonate cascade as a target to suppress extracellular matrix synthesis by human airway smooth muscle.

Smooth muscle cells promote fibroproliferative airway remodeling in asthma, and transforming growth factor ß1 (TGFß1) is a key inductive signal. Statins are widely used to treat hyperlipidemia. Growing evidence indicates they also exert a positive impact on lung health, but the underlying mechanisms are unclear. We assessed the effects of 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase inhibition with simvastatin on the fibrotic function of primary cultured human airway smooth muscle cells. Simvastatin blocked de novo cholesterol synthesis, but total myocyte cholesterol content was unaffected. Simvastatin also abrogated TGFß1-induced collagen I and fibronectin expression, and prevented collagen I secretion. The depletion of mevalonate cascade intermediates downstream from HMG-CoA underpinned the effects of simvastatin, because co-incubation with mevalonate, geranylgeranylpyrophosphate, or farnesylpyrophosphate prevented the inhibition of matrix protein expression. We also showed that human airway myocytes express both geranylgeranyl transferase 1 (GGT1) and farnesyltransferase (FT), and the inhibition of GGT1 (GGTI inhibitor-286, 10 µM), but not FT (FTI inhibitor-277, 10 µM), mirrored the suppressive effects of simvastatin on collagen I and fibronectin expression and collagen I secretion. Moreover, simvastatin and GGTI-286 both prevented TGFß1-induced membrane association of RhoA, a downstream target of GGT1. Our findings suggest that simvastatin and GGTI-286 inhibit synthesis and secretion of extracellular matrix proteins by human airway smooth muscle cells by suppressing GGT1-mediated posttranslational modification of signaling molecules such as RhoA. These findings reveal mechanisms related to evidence for the positive impact of statins on pulmonary health.

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells.

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-ß(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-ß(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-ß(1). The failure by TGF-ß(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-ß(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.

Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c.

Statins inhibit 3-hydroxy-3-methyl-glutarylcoenzyme CoA (HMG-CoA) reductase, the proximal enzyme for cholesterol biosynthesis. They exhibit pleiotropic effects and are linked to health benefits for diseases including cancer and lung disease. Understanding their mechanism of action could point to new therapies, thus we investigated the response of primary cultured human airway mesenchymal cells, which play an effector role in asthma and chronic obstructive lung disease (COPD), to simvastatin exposure. Simvastatin induced apoptosis involving caspase-9, -3 and -7, but not caspase-8 in airway smooth muscle cells and fibroblasts. HMG-CoA inhibition did not alter cellular cholesterol content but did abrogate de novo cholesterol synthesis. Pro-apoptotic effects were prevented by exogenous mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, downstream products of HMG-CoA. Simvastatin increased expression of Bax, oligomerization of Bax and Bak, and expression of BH3-only p53-dependent genes, PUMA and NOXA. Inhibition of p53 and silencing of p53 unregulated modulator of apoptosis (PUMA) expression partly counteracted simvastatin-induced cell death, suggesting a role for p53-independent mechanisms. Simvastatin did not induce mitochondrial release of cytochrome c, but did promote release of inhibitor of apoptosis (IAP) proteins, Smac and Omi. Simvastatin also inhibited mitochondrial fission with the loss of mitochondrial Drp1, an essential component of mitochondrial fission machinery. Thus, simvastatin activates novel apoptosis pathways in lung mesenchymal cells involving p53, IAP inhibitor release, and disruption of mitochondrial fission.

Simvastatin inhibits TGFß1-induced fibronectin in human airway fibroblasts.

BACKGROUND: Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-ß1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFß1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects. METHODS: We used simvastatin (1-15 µM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 µM) and farnesyl transferase (FT; FTI-277, 10 µM) were used to determine whether GGT1 and FT contribute to TGFß1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 µM) or farnesylpyrophosphate (30 µM). RESULTS: Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFß1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFß1-induced signaling. Asthmatic fibroblasts exhibited greater TGFß1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin. CONCLUSIONS: We conclude that TGFß1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

The role of osteoprotegerin (OPG) in fibrosis: its potential as a biomarker and/or biological target for the treatment of fibrotic diseases.

Fibrosis is defined by excessive formation and accumulation of extracellular matrix proteins, produced by myofibroblasts, that supersedes normal wound healing responses to injury and results in progressive architectural remodelling. Fibrosis is often detected in advanced disease stages when an organ is already severely damaged and can no longer function properly. Therefore, there is an urgent need for reliable and easily detectable markers to identify and monitor fibrosis onset and progression as early as possible; this will greatly facilitate the development of novel therapeutic strategies. Osteoprotegerin (OPG), a well-known regulator of bone extracellular matrix and most studied for its role in regulating bone mass, is expressed in various organs and functions as a decoy for receptor activator of nuclear factor kappa-B ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Recently, OPG has been linked to fibrosis and fibrogenesis, and has been included in a panel of markers to diagnose liver fibrosis. Multiple studies now suggest that OPG may be a general biomarker suitable for detection of fibrosis and/or monitoring the impact of fibrosis treatment. This review summarizes our current understanding of the role of OPG in fibrosis and will discuss its potential as a biomarker and/or novel therapeutic target for fibrosis.

Important lessons learned from studies on the pharmacology of glucocorticoids in human airway smooth muscle cells: Too much of a good thing may be a problem.

Glucocorticoids (GCs) are the treatment of choice for chronic inflammatory diseases such as asthma. Despite proven effective anti-inflammatory and immunosuppressive effects, long-term and/or systemic use of GCs can potentially induce adverse effects. Strikingly, some recent experimental evidence suggests that GCs may even exacerbate some disease outcomes. In asthma, airway smooth muscle (ASM) cells are among the targets of GC therapy and have emerged as key contributors not only to bronchoconstriction, but also to airway inflammation and remodeling, as implied by experimental and clinical evidence. We here will review the beneficial effects of GCs on ASM cells, emphasizing the differential nature of GC effects on pro-inflammatory genes and on other features associated with asthma pathogenesis. We will also summarize evidence describing how GCs can potentially promote pro-inflammatory and remodeling features in asthma with a specific focus on ASM cells. Finally, some of the possible solutions to overcome these unanticipated effects of GCs will be discussed.

Pleiotropic effects of statins: A focus on cancer.

The statin drugs ('statins') potently inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase by competitively blocking the active site of the enzyme. Statins decrease de novo cholesterol biosynthesis and thereby reduce plasma cholesterol levels. Statins exhibit "pleiotropic" properties that are independent of their lipid-lowering effects. For example, preclinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. Furthermore, statins show chemo-sensitizing effects by impairing Ras family GTPase signaling. However, whether statins have clinically meaningful anti-cancer effects remains an area of active investigation. Both preclinical and clinical studies on the potential mechanisms of action of statins in several cancers have been reviewed in the literature. Considering the contradictory data on their efficacy, we present an up-to-date summary of the pleiotropic effects of statins in cancer therapy and review their impact on different malignancies. We also discuss the synergistic anti-cancer effects of statins when combined with other more conventional anti-cancer drugs to highlight areas of potential therapeutic development.

Insulin-induced laminin expression promotes a hypercontractile airway smooth muscle phenotype.

Airway smooth muscle (ASM) plays a key role in the development of airway hyperresponsiveness and remodeling in asthma, which may involve maturation of ASM cells to a hypercontractile phenotype. In vitro studies have indicated that long-term exposure of bovine tracheal smooth muscle (BTSM) to insulin induces a functional hypercontractile, hypoproliferative phenotype. Similarly, the extracellular matrix protein laminin has been found to be involved in both the induction and maintenance of a contractile ASM phenotype. Using BTSM, we now investigated the role of laminins in the insulin-induced hypercontractile, hypoproliferative ASM phenotype. The results demonstrate that insulin-induced hypercontractility after 8 days of tissue culture was fully prevented by combined treatment of BTSM-strips with the laminin competing peptides Tyr-Ile-Gly-Ser-Arg (YIGSR) and Arg-Gly-Asp-Ser (RGDS). YIGSR also prevented insulin-induced increases in sm-myosin expression and abrogated the suppressive effects of prolonged insulin treatment on platelet-derived growth factor-induced DNA synthesis in cultured cells. In addition, insulin time-dependently increased laminin alpha2, beta1, and gamma1 chain protein, but not mRNA abundance in BTSM strips. Moreover, as previously found for contractile protein accumulation, signaling through PI3-kinase- and Rho kinase-dependent pathways was required for the insulin-induced increase in laminin abundance and contractility. Collectively, our results indicate a critical role for beta1-containing laminins, likely laminin-211, in the induction of a hypercontractile, hypoproliferative ASM phenotype by prolonged insulin exposure. Increased laminin production by ASM could be involved in the increased ASM contractility and contractile protein expression in asthma. Moreover, the results may be of interest for the use of inhaled insulin administrations by diabetics.

Monomeric G-proteins as signal transducers in airway physiology and pathophysiology.

Monomeric G-proteins, also referred to as small GTPases, function as biological hubs being activated by extracellular stimuli and regulate downstream signalling events, which result in different cellular responses. The importance of these mechanisms is mirrored by the fact that several pathological conditions, including allergic asthma, are associated with derailed GTPases signalling. For this reason attention has been focused on the role of monomeric G-proteins and their effectors in airway (patho)physiology. In this article we review our current knowledge on the regulation and functions of Ras and Rho GTPase signalling under physiological and pathophysiological conditions in the pulmonary system. Based on recent findings concerning novel regulatory proteins for Ras family members, we further discuss potential future directions for therapeutical interventions in asthma.

Airway smooth muscle in asthma: phenotype plasticity and function.

Clinical asthma is characterized by reversible airway obstruction which is commonly due to an exaggerated airway narrowing referred to as airway hyperresponsiveness (AHR). Although debate exists on the complex etiology of AHR, it is clear that airway smooth muscle (ASM) mediated airway narrowing is a major contributor to airway dysfunction. More importantly, it is now appreciated that smooth muscle is far from being a simple cell with only contractile ability properties. Rather, it is more versatile with the capacity to exhibit numerous cellular functions as it adapts to the microenvironment to which it is exposed. The emerging ability of individual smooth muscle cells to undergo changes in their phenotype (phenotype plasticity) and function (functional plasticity) in response to physiological and pathological cues is an important and active area of research. This article provides a brief review of the current knowledge and emerging concepts in the field of ASM phenotype and function both under healthy and asthmatic conditions.

Non-genomic Effects of Glucocorticoids: An Updated View.

Glucocorticoid (GC) anti-inflammatory effects generally require a prolonged onset of action and involve genomic processes. Because of the rapidity of some of the GC effects, however, the concept that non-genomic actions may contribute to GC mechanisms of action has arisen. While the mechanisms have not been completely elucidated, the non-genomic effects may play a role in the management of inflammatory diseases. For instance, we recently reported that GCs 'rapidly' enhanced the effects of bronchodilators, agents used in the treatment of allergic asthma. In this review article, we discuss (i) the non-genomic effects of GCs on pathways relevant to the pathogenesis of inflammatory diseases and (ii) the putative role of the membrane GC receptor. Since GC side effects are often considered to be generated through its genomic actions, understanding GC non-genomic effects will help design GCs with a better therapeutic index.

Rho kinase inhibitors: a novel therapeutical intervention in asthma?

In asthma, inflammatory mediators that are released in the airways by recruited inflammatory cells and by resident structural cells result in airway hyperresponsiveness caused by increased bronchoconstriction. In addition, chronic inflammation appears to drive remodelling of the airways that contributes to the development of fixed airway obstruction and airway hyperresponsiveness in chronic asthma. Airway remodelling includes several key features such as excessive deposition of extracellular matrix proteins in the airway wall (fibrosis) and increased abundance of contractile airway smooth muscle encircling the airways. Current asthma therapy fails to inhibit these features satisfactorily. This review focuses on Rho kinase as a potential drug target in asthma, as compelling evidence from animal models and ex vivo studies suggests a central role for this enzyme and its associated signalling in acute and chronic airway hyperresponsiveness.

Pharmacology of airway smooth muscle proliferation.

Airway smooth muscle thickening is a pathological feature that contributes significantly to airflow limitation and airway hyperresponsiveness in asthma. Ongoing research efforts aimed at identifying the mechanisms responsible for the increased airway smooth muscle mass have indicated that hyperplasia of airway smooth muscle, due in part to airway myocyte proliferation, is likely a major factor. Airway smooth muscle proliferation has been studied extensively in culture and in animal models of asthma, and these studies have revealed that a variety of receptors and mediators contributes to this response. This review aims to provide an overview of the receptors and mediators that control airway smooth muscle cell proliferation, with emphasis on the intracellular signalling mechanisms involved.

Myocardial Cell Signaling During the Transition to Heart Failure: Cellular Signaling and Therapeutic Approaches.

Cardiovascular disease leading to heart failure (HF) remains a leading cause of morbidity and mortality worldwide. Improved pharmacological and interventional coronary procedures have led to improved outcomes following acute myocardial infarction. This success has translated into an unforeseen increased incidence in HF. This review summarizes the signaling pathways implicated in the transition to HF following cardiac injury. In addition, we provide an update on cell death signaling and discuss recent advances in cardiac fibrosis as an independent event leading to HF. Finally, we discuss cell-based therapies and their possible use to avert the deteriorating nature of HF. © 2019 American Physiological Society. Compr Physiol 9:75-125, 2019.

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction.

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.

Inhalation of the Rho-kinase inhibitor Y-27632 reverses allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction.

BACKGROUND: In guinea pigs, we have previously demonstrated that the contribution of Rho-kinase to airway responsiveness in vivo and ex vivo is enhanced after active sensitization with ovalbumin (OA). Using conscious, unrestrained OA-sensitized guina pigs, we now investigated the role of Rho-kinase in the development of airway hyperresponsiveness (AHR) after the allergen-induced early (EAR) and late asthmatic reaction (LAR) in vivo. METHODS: Histamine and PGF2alpha PC100-values (provocation concentrations causing 100% increase in pleural pressure) were assessed before OA-challenge (basal airway responsiveness) and after the OA-induced EAR (5 h after challenge) and LAR (23 h after challenge). Thirty minutes later, saline or the specific Rho-kinase inhibitor Y-27632 (5 mM, nebulizer concentration) were nebulized, after which PC100-values were reassessed. RESULTS: In contrast to saline, Y-27632 inhalation significantly decreased the basal responsiveness toward histamine and PGF2alpha before OA-challenge, as indicated by increased PC100 -values. Both after the allergen-induced EAR and LAR, AHR to histamine and PGF2alpha was present, which was reversed by Y-27632 inhalation. Moreover, there was an increased effectiveness of Y-27632 to reduce airway responsiveness to histamine and PGF2alpha after the EAR and LAR as compared to pre-challenge conditions. Saline inhalations did not affect histamine or PGF2alpha PC100-values at all. Interestingly, under all conditions Y-27632 was significantly more effective in reducing airway responsiveness to PGF2alpha as compared to histamine. Also, there was a clear tendency (P = 0.08) to a more pronounced degree of AHR after the EAR for PGF2alpha than for histamine. CONCLUSION: The results indicate that inhalation of the Rho-kinase inhibitor Y-27632 causes a considerable bronchoprotection to both histamine and PGF2alpha. Moreover, the results are indicative of a differential involvement of Rho-kinase in the agonist-induced airway obstruction in vivo. Increased Rho-kinase activity contributes to the allergen-induced AHR to histamine and PGF2alpha after both the EAR and the LAR, which is effectively reversed by inhalation of Y-27632. Therefore, Rho-kinase can be considered as a potential pharmacotherapeutical target in allergic asthma.