Targeting the smooth muscle cell KEAP1-Nrf2-STING axis with pterostilbene attenuates abdominal aortic aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a life-threatening aortic disease, and to date, there are currently no effective pharmacological treatments to address this condition. Activation of cytosolic DNA sensing adaptor stimulator of interferon genes (STING) signaling is a crucial mechanism in AAA formation. PURPOSE: This study investigated pterostilbene (Pt), a naturally occurring polyphenol and resveratrol analogue, as a STING inhibitor for preventing AAA. METHODS: We evaluated the effect of Pt on AAA formation in angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE-/-) mice. We used histological analysis, MMP activity measurement, western blot, and immunohistochemistry to detect AAA formation and development. We applied RNA sequencing, molecular docking, cellular thermal shift assay (CETSA) and functional studies to dissect the molecular mechanism of Pt-regulating KEAP1-Nrf2-STING signaling. We conditionally knocked down Nrf2 in vascular smooth muscle cells (VSMCs) in vivo to investigate its role in Pt-mediated protective effects on AAA. RESULTS: Pt effectively blocked the formation of AAA in AngII-infused ApoE-/- mice. Whole transcriptome sequencing analysis revealed that nuclear factor erythroid 2-related factor 2 (Nrf2) and STING pathway in VSMCs were linked to the anti-AAA effects of pterostilbene. Mechanistically, Pt upregulated Nrf2 target genes (e.g., HO-1 and NQO1) through activation of the KEAP1/Nrf2 signaling, which restricted the immunostimulatory axis of mtDNA-STING-TBK1-NF-κB, thereby alleviating VSMC inflammation and preserving the VSMC contractile phenotype. Subsequently, molecular docking and CETSA revealed a binding mode between Pt and KEAP1/Nrf2. Intriguingly, the inhibitory effect of Pt on STING signaling and the protective role of Pt in AAA were largely abrogated by VSMC-specific Nrf2 knockdown in mice. CONCLUSION: Collectively, naturally derived Pt shows promising efficacy for the treatment of AAA by targeting the KEAP1-Nrf2-STING axis in VSMCs.

The natural polyphenol fisetin in atherosclerosis prevention: a mechanistic review.

The incidence and mortality rate of atherosclerotic cardiovascular disease (ASCVD) is increasing yearly worldwide. Recently, a growing body of evidence has unveiled the anti-atherosclerotic properties of fisetin, a natural polyphenol compound. In this article, we reviewed the pharmacologic actions of fisetin on experimental atherosclerosis and its protective effects on disease-relevant cell types such as endothelial cells, macrophages, vascular smooth muscle cells, and platelets. Based on its profound cardiovascular actions, fisetin holds potential for clinical translation and could be developed as a potential therapeutic option for atherosclerosis and its related complications. Large-scale randomized clinical trials are warranted to ascertain the safety and efficacy of fisetin in patients with or high risk for ASCVD.

A novel mouse model of familial combined hyperlipidemia and atherosclerosis.

Within the context of residual cardiovascular risk in post-statin era, emerging evidence from epidemiologic and human genetic studies have demonstrated that triglyceride (TG)-rich lipoproteins and their remnants are causally related to cardiovascular risk. While, carriers of loss-of-function mutations of ApoC3 have low TG levels and are protected from cardiovascular disease (CVD). Of translational significance, siRNAs/antisense oligonucleotide (ASO) targeting ApoC3 is beneficial for patients with atherosclerotic CVD. Therefore, animal models of atherosclerosis with both hypercholesterolemia and hypertriglyceridemia are important for the discovery of novel therapeutic strategies targeting TG-lowering on top of traditional cholesterol-lowering. In this study, we constructed a novel mouse model of familial combined hyperlipidemia through inserting a human ApoC3 transgene (hApoC3-Tg) into C57BL/6 J mice and injecting a gain-of-function variant of adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-D377Y concurrently with high cholesterol diet (HCD) feeding for 16 weeks. In the last 10 weeks, hApoC3-Tg mice were orally treated with a combination of atorvastatin (10 mg·kg-1·d-1) and fenofibrate (100 mg·kg-1·d-1). HCD-treated hApoC3-Tg mice demonstrated elevated levels of serum TG, total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Oral administration of atorvastatin and fenofibrate significantly decreased the plaque sizes of en face aorta, aortic sinus and innominate artery accompanied by improved lipid profile and distribution. In summary, this novel mouse model is of considerable clinical relevance for evaluation of anti-atherosclerotic drugs by targeting both hypercholesterolemia and hypertriglyceridemia.

The cytoplasmic sensor, the AIM2 inflammasome: A precise therapeutic target in vascular and metabolic diseases.

Cardio-cerebrovascular diseases encompass pathological changes in the heart, brain and vascular system, which pose a great threat to health and well-being worldwide. Moreover, metabolic diseases contribute to and exacerbate the impact of vascular diseases. Inflammation is a complex process that protects against noxious stimuli but is also dysregulated in numerous so-called inflammatory diseases, one of which is atherosclerosis. Inflammation involves multiple organ systems and a complex cascade of molecular and cellular events. Numerous studies have shown that inflammation plays a vital role in cardio-cerebrovascular diseases and metabolic diseases. The absent in melanoma 2 (AIM2) inflammasome detects and is subsequently activated by double-stranded DNA in damaged cells and pathogens. With the assistance of the mature effector molecule caspase-1, the AIM2 inflammasome performs crucial biological functions that underpin its involvement in cardio-cerebrovascular diseases and related metabolic diseases: The production of interleukin-1 beta (IL-1ß), interleukin-18 (IL-18) and N-terminal pore-forming Gasdermin D fragment (GSDMD-N) mediates a series of inflammatory responses and programmed cell death (pyroptosis and PANoptosis). Currently, several agents have been reported to inhibit the activity of the AIM2 inflammasome and have the potential to be evaluated for use in clinical settings. In this review, we systemically elucidate the assembly, biological functions, regulation and mechanisms of the AIM2 inflammasome in cardio-cerebrovascular diseases and related metabolic diseases and outline the inhibitory agents of the AIM2 inflammasome as potential therapeutic drugs.

A proteome-wide screen identifies the calcium binding proteins, S100A8/S100A9, as clinically relevant therapeutic targets in aortic dissection.

Aortic dissection (AD) is a fatal cardiovascular disease with limited pharmacotherapies. To discover novel therapeutic targets for AD, the present study was conducted on ascending aorta samples from AD patients versus those from control subjects using proteomic analysis. Integrated proteomic data analysis identified S100 calcium-binding proteins A8 and A9 (S100A8/A9) as new therapeutic targets for AD. As assessed by ELISA, the circulating levels of S100A8/A9 were elevated in AD patients. In addition, we validated the upregulation of S100A8/A9 in a mouse model of AD. In vitro and in vivo studies substantiated that S100A8/A9, as danger-associated molecular pattern molecules, promotes the smooth muscle cells phenotypic switch by inhibiting serum response factor (SRF) activity but elevating NF-κB dependent inflammatory response. Depletion of S100A8/A9 attenuates the occurrence and development of AD. As a proof of concept, we tested the safety and efficacy of pharmacological inhibition of S100A8/A9 by ABR-25757 (paquinimod) in a mouse model of AD. We observed that ABR-25757 ameliorated the incidence of rupture and improved elastin morphology associated with AD. Further single-cell RNA sequencing disclosed that the phenotypic switch of vascular smooth muscle cells (VSMCs) and inflammatory response pathways were responsible for ABR-25757-mediated protection against AD. Thus, this study reveals the regulatory mechanism of S100A8/A9 in AD and offers a potential therapeutic avenue to treat AD by targeting S100A8/A9.

Growth differentiation factor 15 is dispensable for acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced liver injury (AILI) has been recognized as a pivotal contributor to drug-induced liver failure in Western countries, but its molecular mechanism remains poorly understood. Growth differentiation factor 15 (GDF15) is a pleiotropic factor that alleviates non-alcoholic liver steatohepatitis, liver fibrosis and liver injury. The aim of the present study was to examine the possibility whether GDF15 confers protection against AILI. We found that the gene expression of Gdf15 was increased significantly after APAP overdose in mice. Next, the role of Gdf15 in AILI was evaluated by hepatic Gdf15 overexpression (using adeno-associated virus serotype 8), injection with recombinant human GDF15 (rhGDF15) and Gdf15 knockout mice after challenge with APAP. A marked elevation of Gdf15 was observed after AILI. However, there were no significant differences in AILI-related liver injury and JNK phosphorylation after Gdf15 overexpression, rhGDF15 injection or Gdf15 deficiency. Together, we conclude that, despite a noticeable elevation of Gdf15 level after AILI, Gdf15 is dispensable for APAP-induced AILI. Our study further suggests that genomic analysis of mRNA expression after APAP overdose is of limited relevance unless followed up by a functional analysis of candidate genes in vivo.

Endothelin-1 dependent expression of GAG genes involves NOX and p38 mediated Smad linker region phosphorylation.

Endothelin-1 (ET-1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET-1-mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho-Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST-1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). ET-1 treatment of human VSMCs resulted in an increase in phospho-Smad2L level. The TGF-ß receptor antagonist, SB431542 and the mixed ETA and ETB receptor antagonist bosentan, inhibited ET-1-mediated phospho-Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET-1-mediated phospho-Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post-ET-1 treatment were increased compared to untreated controls (p < 0.01). The ET-mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N-acetyl-L-cysteine (NAC). ET-1-mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation-dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation.

Endothelin-1 mediated glycosaminoglycan synthesizing gene expression involves NOX-dependent transactivation of the transforming growth factor-ß receptor.

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor (TGF) ß receptor (TGFBR1) stimulates glycosaminoglycan (GAG) elongation on proteoglycans. GPCR agonists thrombin and lysophosphatidic acid (LPA) via respective receptors transactivate the TGFBR1 via Rho/ROCK dependent pathways however mechanistic insight for ET-1 transactivation of the TGFBR1 remains unknown. NADPH oxidase (NOX) generates reactive oxygen species (ROS) and is a signalling entity implicated in the pathogenesis of many diseases including atherosclerosis. If implicated in this pathway, NOX/ROS would be a potential therapeutic target. In this study, we investigated the involvement of NOX in ET-1/ET receptor-mediated transactivation of TGFBR1 to stimulate mRNA expression of GAG chain synthesizing enzymes chondroitin 4-O-sulfotransferase 1 (C4ST-1) and chondroitin sulfate synthase 1 (ChSy-1). The invitro model used vascular smooth muscle cells that were treated with pharmacological antagonists in the presence and absence of ET-1 or TGF-ß. Proteins and phosphoproteins isolated from treated cells were quantified by western blotting and quantitative real-time PCR was used to assess mRNA expression of GAG synthesizing enzymes. In the presence of diphenyliodonium (DPI) (NOX inhibitor), ET-1 stimulated phospho-Smad2C levels were inhibited. ET-1 mediated mRNA expression of GAG synthesizing enzymes C4ST-1 and ChSy-1 was also blocked by TGBFR1 antagonists, SB431542, broad spectrum ET receptor antagonist bosentan, DPI and ROS scavenger N-acetyl-L-cysteine. This work shows that NOX and ROS play an important role in ET-1 mediated transactivation of the TGFBR1 and downstream gene targets associated with GAG chain elongation. As ROS is involved in GPCR to protein tyrosine kinase receptor transactivation, the NOX/ROS axis presents as the first common biochemical target in all GPCR to kinase receptor transactivation signalling.

Akt acts as a switch for GPCR transactivation of the TGF-ß receptor type 1.

Transforming growth factor (TGF)-ß signalling commences with the engagement of TGF-ß ligand to cell surface TGF-ß receptors (TGFBR) stimulating Smad2 carboxyl-terminal phosphorylation (phospho-Smad2C) and downstream biological responses. In several cell models, G protein-coupled receptors (GPCRs) transactivate the TGF-ß receptors type-1 (TGFBR1) leading to phospho-Smad2C, however, we have recently published that in keratinocytes thrombin did not transactivate the TGFBR1. The bulk of TGFBRs reside in the cytosol and in response to protein kinase B (Akt phosphorylation) can translocate to the cell surface increasing the cell's responsiveness to TGF-ß. In this study, we investigate the role of Akt in GPCR transactivation of the TGFBR1. We demonstrate that angiotensin II and thrombin do not phosphorylate Smad2C in human vascular smooth muscle cells and in keratinocytes respectively. We used Akt agonist, SC79 to sensitise the cells to Akt and observed that Ang II and thrombin phosphorylate Smad2C via Akt/AS160-dependent pathways. We show that SC79 rapidly translocates TGFBRs to the cell surface thus increasing the cell's response to the GPCR agonist. These findings highlight novel mechanistic insight for the role of Akt in GPCR transactivation of the TGFBR1.

Metformin in cardiovascular diabetology: a focused review of its impact on endothelial function.

As a first-line treatment for diabetes, the insulin-sensitizing biguanide, metformin, regulates glucose levels and positively affects cardiovascular function in patients with diabetes and cardiovascular complications. Endothelial dysfunction (ED) represents the primary pathological change of multiple vascular diseases, because it causes decreased arterial plasticity, increased vascular resistance, reduced tissue perfusion and atherosclerosis. Caused by "biochemical injury", ED is also an independent predictor of cardiovascular events. Accumulating evidence shows that metformin improves ED through liver kinase B1 (LKB1)/5'-adenosine monophosphat-activated protein kinase (AMPK) and AMPK-independent targets, including nuclear factor-kappa B (NF-κB), phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), forkhead box O1 (FOXO1), krüppel-like factor 4 (KLF4) and krüppel-like factor 2 (KLF2). Evaluating the effects of metformin on endothelial cell functions would facilitate our understanding of the therapeutic potential of metformin in cardiovascular diabetology (including diabetes and its cardiovascular complications). This article reviews the physiological and pathological functions of endothelial cells and the intact endothelium, reviews the latest research of metformin in the treatment of diabetes and related cardiovascular complications, and focuses on the mechanism of action of metformin in regulating endothelial cell functions.

The zinc finger transcription factor, KLF2, protects against COVID-19 associated endothelial dysfunction.

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1ß and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.

Metformin, Macrophage Dysfunction and Atherosclerosis.

Metformin is one of the most widely prescribed hypoglycemic drugs and has the potential to treat many diseases. More and more evidence shows that metformin can regulate the function of macrophages in atherosclerosis, including reducing the differentiation of monocytes and inhibiting the inflammation, oxidative stress, polarization, foam cell formation and apoptosis of macrophages. The mechanisms by which metformin regulates the function of macrophages include AMPK, AMPK independent targets, NF-κB, ABCG5/8, Sirt1, FOXO1/FABP4 and HMGB1. On the basis of summarizing these studies, we further discussed the future research directions of metformin: single-cell RNA sequencing, neutrophil extracellular traps (NETs), epigenetic modification, and metformin-based combination drugs. In short, macrophages play an important role in a variety of diseases, and improving macrophage dysfunction may be an important mechanism for metformin to expand its pleiotropic pharmacological profile. In addition, the combination of metformin with other drugs that improve the function of macrophages (such as SGLT2 inhibitors, statins and IL-1ß inhibitors/monoclonal antibodies) may further enhance the pleiotropic therapeutic potential of metformin in conditions such as atherosclerosis, obesity, cancer, dementia and aging.

Poly(aspartic acid) in Biomedical Applications: From Polymerization, Modification, Properties, Degradation, and Biocompatibility to Applications.

Poly(aspartic acid) (PASP) is an anionic polypeptide that is a highly versatile, biocompatible, and biodegradable polymer that fulfils key requirements for use in a wide variety of biomedical applications. The derivatives of PASP can be readily tailored via the amine-reactive precursor, poly(succinimide) (PSI), which opens up a large window of opportunity for the design and development of novel biomaterials. PASP also has a strong affinity with calcium ions, resulting in complexation, which has been exploited for bone targeting and biomineralization. In addition, recent studies have further verified the biocompatibility and biodegradability of PASP-based polymers, which is attributed to their protein-like structure. In light of growing interest in PASP and its derivatives, this paper presents a comprehensive review on their synthesis, characterization, modification, biodegradation, biocompatibility, and applications in biomedical areas.

Emodin in atherosclerosis prevention: Pharmacological actions and therapeutic potential.

Atherosclerotic plaque formation, destabilization and eventual rupture leads to the acute cardiovascular events including myocardial infarction and stroke. Emodin (PubChem CID#3220), (1,3,8-trihydroxy-6-methylanthracene-9,10-dione) is a pharmacologically bioactive constituent isolated from the traditional Chinese medicinal herb Radix rhizoma Rhei. This molecule has anti-oxidant, anti-inflammatory, anti-proliferative, anti-apoptotic and lipid-modulating effects. Experimental studies have demonstrated that emodin attenuates and stabilizes atherosclerotic plaques. In this mini-review, we provide a summary of the pharmacological actions of emodin in regulating vascular function and atherosclerosis, highlighting the therapeutic potential of this phytochemical in patients with cardiovascular disease.

Targeted Molecular Imaging of Cardiovascular Diseases by Iron Oxide Nanoparticles.

Cardiovascular disease is one of the major contributors to global disease burden. Atherosclerosis is an inflammatory process that involves the accumulation of lipids and fibrous elements in the large arteries, forming an atherosclerotic plaque. Rupture of unstable plaques leads to thrombosis that triggers life-threatening complications such as myocardial infarction. Current diagnostic methods are invasive as they require insertion of a catheter into the coronary artery. Molecular imaging techniques, such as magnetic resonance imaging, have been developed to image atherosclerotic plaques and thrombosis due to its high spatial resolution and safety. The sensitivity of magnetic resonance imaging can be improved with contrast agents, such as iron oxide nanoparticles. This review presents the most recent advances in atherosclerosis, thrombosis, and myocardial infarction molecular imaging using iron oxide-based nanoparticles. While some studies have shown their effectiveness, many are yet to undertake comprehensive testing of biocompatibility. There are still potential hazards to address and complications to diagnosis, therefore strategies for overcoming these challenges are required.

Artemisinin inhibits glycosaminoglycan chain synthesizing gene expression but not proliferation of human vascular smooth muscle cells.

Pleotropic growth factor, transforming growth factor (TGF)-ß drives the modification and elongation of glycosaminoglycan (GAG) chains on proteoglycans. Hyperelongated GAG chains bind and trap lipoproteins in the intima leading to the formation of atherosclerotic plaques. We have identified that phosphorylation of Smad2 linker region drives GAG chain modification. The identification of an inhibitor of Smad2 linker region phosphorylation and GAG chain modification signifies a potential therapeutic for cardiovascular diseases. Artemisinin renowned for its potent anti-malarial effects possesses a broad range of biological effects. Our aim was to characterise the anti-atherogenic role of artemisinin in vascular smooth muscle cells (VSMCs). We demonstrate that TGF-ß mediated Smad2 linker region phosphorylation and GAG chain elongation was attenuated by artemisinin; however, we observed no effect on VSMC proliferation. Our data demonstrates the potential for artemisinin to be developed as a therapy to inhibit the development of atherosclerosis by prevention of lipid deposition in the vessel wall without affecting the proliferation of VSMCs.

The Role of Toll-like Receptors in Atherothrombotic Cardiovascular Disease.

Toll-like receptors (TLRs) are dominant components of the innate immune system. Activated by both pathogen-associated molecular patterns and damage-associated molecular patterns, TLRs underpin the pathology of numerous inflammation related diseases that include not only immune diseases, but also cardiovascular disease (CVD), diabetes, obesity, and cancers. Growing evidence has demonstrated that TLRs are involved in multiple cardiovascular pathophysiologies, such as atherosclerosis and hypertension. Specifically, a trial called the Canakinumab Anti-inflammatory Thrombosis Outcomes Study showed the use of an antibody that neutralizes interleukin-1ß, reduces the recurrence of cardiovascular events, demonstrating inflammation as a therapeutic target and also the research value of targeting the TLR system in CVD. In this review, we provide an update of the interplay between TLR signaling, inflammatory mediators, and atherothrombosis, with an aim to identify new therapeutic targets for atherothrombotic CVD.

Smad2 linker region phosphorylation is an autonomous cell signalling pathway: Implications for multiple disease pathologies.

This review will cover the signalling pathways leading to the phosphorylation of the Smad linker region independent of Smad carboxy terminal phosphorylation. Characterising Smad linker region as a signalling pathway in its own right will encourage comprehensive signalling studies to provide solutions for successful discovery and exploitation of drug targets. The review describes Smad transcription factor signalling distinct from Transforming Growth Factor (TGF)-ß signalling. Novel signalling pathways represent new drug targets where these pathways are known to be involved in fibrosis, cancer and cardiovascular disease.

Vernolide-A and Vernodaline: Sesquiterpene Lactones with Cytotoxicity against Cancer.

Vernolide-A and vernodaline are sesquiterpene lactones isolated from genera of Vernonia. Vernolide-A and vernodaline have shown promising therapeutic properties, including antibacterial, antihelminth, and antioxidant activities. Recently, the anticancer properties of these sesquiterpene lactones have been investigated with the elucidation of effects on cell proliferation, metastasis, angiogenesis, and apoptosis. The antiproliferation and antimetastatic activities arise from targeting extracellular signal-regulated kinase 1 (ERK-1), extracellular signal-regulated kinase 2 (ERK-2), nuclear factor-κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase 2 (MMP-2), and matrix metalloproteinase 9 (MMP9). The induction of apoptosis is due to the enhancement of caspase 9, caspase 3, while inhibition of Bcl-2 and Bcl-xL results in the release of cytochrome c into the cytosol. The activity of vernolide-A and vernodaline is hypothesized to be due to thiol reactivity through the α-methylene-γ-lactone group of sesquiterpene lactones. This review will give a brief summary of the anticancer activity of vernolide-A and vernodaline and provide information on the underlying molecular mechanisms.

Smad linker region phosphorylation is a signalling pathway in its own right and not only a modulator of canonical TGF-ß signalling.

Transforming growth factor (TGF)-ß signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-ß signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-ß receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-ß can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-ß signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-ß signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.