Metaflammation, an atypical, metabolically induced, chronic lowgrade inflammation, plays an important role in the development of obesity, diabetes, and atherosclerosis. An important primer for metaflammation is the persistent metabolic overloading of the endoplasmic reticulum (ER), leading to its functional impairment. Activation of the unfolded protein response (UPR), a homeostatic regulatory network that responds to ER stress, is a hallmark of all stages of atherosclerotic plaque formation. The most conserved ERresident UPR regulator, the kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), is activated in lipid-laden macrophages that infiltrate the atherosclerotic lesions. Using RNA sequencing in macrophages, we discovered that IRE1 regulates the expression of many proatherogenic genes, including several important cytokines and chemokines. We show that IRE1 inhibitors uncouple lipid-induced ER stress from inflammasome activation in both mouse and human macrophages. In vivo, these IRE1 inhibitors led to a significant decrease in hyperlipidemia-induced IL-1β and IL-18 production, lowered T-helper type-1 immune responses, and reduced atherosclerotic plaque size without altering the plasma lipid profiles in apolipoprotein E-deficient mice. These results show that pharmacologic modulation of IRE1 counteracts metaflammation and alleviates atherosclerosis.endoplasmic reticulum stress | unfolded protein response | metaflammation | lipotoxicity | atherosclerosis C omplex molecular interactions between environment, diet, and genetics that take place at the metabolic and immune interface provoke a low-grade, chronic inflammatory responsemetaflammation-that engages cells of the immune system (macrophages, neutrophils, and lymphocytes) and metabolic tissues (adipocytes, hepatocytes, and pancreatic cells) (1). An important primer for metaflammation is chronic metabolic overloading of organelles, such as the endoplasmic reticulum (ER) and mitochondria, which results in impairment of their functions (2).The ER serves essential cellular functions that include the synthesis and folding of secreted and transmembrane proteins, calcium storage, and lipid synthesis for membrane biogenesis or energy storage. Disruption of any of these functions leads to ER stress and the subsequent activation of an elaborate network of adaptive responses, collectively known as the unfolded protein response (UPR) (3). The UPR reestablishes homeostasis through both transcriptional and translational layers of control. The UPR signals through three mechanistically distinct branches that are initiated by the ER-resident protein folding sensors inositolrequiring enzyme 1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) (3).IRE1 controls the phylogenetically most conserved branch of the UPR found from fungi to metazoans. It has an ER-lumenal sensor domain that recognizes unfolded peptides and cytosolic kinase and endoribonuclease (RNase) domains that relay the information to downstrea...

Full-length tissue factor (flTF), the coagulation initiator, is overexpressed in breast cancer (BrCa), but associations between flTF expression and clinical outcome remain controversial. It is currently not known whether the soluble alternatively spliced TF form (asTF) is expressed in BrCa or impacts BrCa progression. We are unique in reporting that asTF, but not flTF, strongly associates with both tumor size and grade, and induces BrCa cell proliferation by binding to β1 integrins. asTF promotes oncogenic gene expression, anchorage-independent growth, and strongly up-regulates tumor expansion in a luminal BrCa model. In basal BrCa cells that constitutively express both TF isoforms, asTF blockade reduces tumor growth and proliferation in vivo. We propose that asTF plays a major role in BrCa progression acting as an autocrine factor that promotes tumor progression. Targeting asTF may comprise a previously unexplored therapeutic strategy in BrCa that stems tumor growth, yet does not impair normal hemostasis.regulated pre-mRNA processing | outside-in signaling I n breast cancer (BrCa), proteins that modulate splicing events such as ASF/SF2 and SR(Serine/Arginine-rich)p55, are frequently up-regulated and contribute to cell transformation (1, 2). BrCa cells exhibit specific alternative splicing signatures that were proposed as potential prognostic factors in BrCa (3). Alternative splicing of proteins, such as spleen tyrosine kinase (Syk), p53, phosphatase and tensin homolog (PTEN), chemokine (C-X-C motif) receptor 3 (CXCR3), and ras-related C3 botulinum toxin substrate 1 (Rac1) impacts BrCa cell behavior and therefore, disease progression (4-8).Full-length tissue factor (flTF) is the initiator of blood coagulation (9). Following vascular damage, flTF binds its ligand FVII(a), which triggers clot formation. Aside from subendothelial tissues, flTF is also abundant on cancer cells (9) and fuels tumor progression by modulating integrin α3β1 function, cell migration (10), and FVIIa-dependent protease activated receptor (PAR)2 activation, but flTF-β1 integrin complexation enhances PAR2 activation (11). flTF-dependent PAR2 activation results in the production of VEGF, CXCL1, and IL-8, thus promoting the angiogenic switch and consequently, tumor growth in vivo (10, 11).Alternative splicing of TF pre-mRNA results in the deletion of exon 5 and thus a frameshift in exon 6, yielding a transmembrane domain-lacking isoform that can be secreted (12). Human and murine alternatively spliced TF (asTF) contain novel C termini with poor homology to one another or any other protein, and the murine asTF C terminus is longer than that of human asTF (12,13). High expression of asTF in tumor cell lines suggests a role in tumor progression (10,14). Subcutaneous growth of pancreatic cancer cells overexpressing asTF results in larger and more vascularized tumors (15). We recently discovered that asTF induces angiogenesis, independent of PAR2 activation, by acting as an integrin ligand (16). Thus, flTF and asTF facilitate cellular signaling via ...

De novo lipogenesis (DNL), the conversion of glucose and other substrates to lipids, is often associated with ectopic lipid accumulation, metabolic stress, and insulin resistance, especially in the liver. However, organ-specific DNL can also generate distinct lipids with beneficial metabolic bioactivity, prompting a great interest in their use for the treatment of metabolic diseases. Palmitoleate (PAO), one such bioactive lipid, regulates lipid metabolism in liver and improves glucose utilization in skeletal muscle when it is generated de novo from the obese adipose tissue. We show that PAO treatment evokes an overall lipidomic remodeling of the endoplasmic reticulum (ER) membranes in macrophages and mouse tissues, which is associated with resistance of the ER to hyperlipidemic stress. By preventing ER stress, PAO blocks lipid-induced inflammasome activation in mouse and human macrophages. Chronic PAO supplementation also lowers systemic interleukin-1β (IL-1β) and IL-18 concentrations in vivo in hyperlipidemic mice. Moreover, PAO prevents macrophage ER stress and IL-1β production in atherosclerotic plaques in vivo, resulting in a marked reduction in plaque macrophages and protection against atherosclerosis in mice. These findings demonstrate that oral supplementation with a product of DNL such as PAO can promote membrane remodeling associated with metabolic resilience of intracellular organelles to lipid stress and limit the progression of atherosclerosis. These findings support therapeutic PAO supplementation as a potential preventive approach against complex metabolic and inflammatory diseases such as atherosclerosis, which warrants further studies in humans.

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the pro-coagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

BackgroundEukaryotic cells can respond to diverse stimuli by converging at serine-51 phosphorylation on eukaryotic initiation factor 2 alpha (eIF2α) and activate the integrated stress response (ISR). This is a key step in translational control and must be tightly regulated; however, persistent eIF2α phosphorylation is observed in mouse and human atheroma.ObjectivesPotent ISR inhibitors that modulate neurodegenerative disorders have been identified. Here, the authors evaluated the potential benefits of intercepting ISR in a chronic metabolic and inflammatory disease, atherosclerosis.MethodsThe authors investigated ISR’s role in lipid-induced inflammasome activation and atherogenesis by taking advantage of 3 different small molecules and the ATP-analog sensitive kinase allele technology to intercept ISR at multiple molecular nodes.ResultsThe results show lipid-activated eIF2α signaling induces a mitochondrial protease, Lon protease 1 (LONP1), that degrades phosphatase and tensin-induced putative kinase 1 and blocks Parkin-mediated mitophagy, resulting in greater mitochondrial oxidative stress, inflammasome activation, and interleukin-1β secretion in macrophages. Furthermore, ISR inhibitors suppress hyperlipidemia-induced inflammasome activation and inflammation, and reduce atherosclerosis.ConclusionsThese results reveal endoplasmic reticulum controls mitochondrial clearance by activating eIF2α-LONP1 signaling, contributing to an amplified oxidative stress response that triggers robust inflammasome activation and interleukin-1β secretion by dietary fats. These findings underscore the intricate exchange of information and coordination of both organelles’ responses to lipids is important for metabolic health. Modulation of ISR to alleviate organelle stress can prevent inflammasome activation by dietary fats and may be a strategy to reduce lipid-induced inflammation and atherosclerosis.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow. Video LinkThe video component of this article can be found at

Objective: Kawasaki disease (KD) is the leading cause of acute vasculitis and acquired heart disease in children in developed countries. Notably, KD is more prevalent in males than females. We previously established a key role for IL (interleukin)-1 signaling in KD pathogenesis, but whether this pathway underlies the sex-based difference in susceptibility is unknown. Approach and Results: The role of IL-1 signaling was investigated in the Lactobacillus casei cell wall extract-induced experimental mouse model of KD vasculitis. Five-week-old male and female mice were injected intraperitoneally with PBS, Lactobacillus casei cell wall extract, or a combination of Lactobacillus casei cell wall extract and the IL-1 receptor antagonist Anakinra. Aortitis, coronary arteritis inflammation score and abdominal aorta dilatation, and aneurysm development were assessed. mRNA-seq (messenger RNA sequencing) analysis was performed on abdominal aorta tissue. Publicly available human transcriptomics data from patients with KD was analyzed to identify sex differences and disease-associated genes. Male mice displayed enhanced aortitis and coronary arteritis as well as increased incidence and severity of abdominal aorta dilatation and aneurysm, recapitulating the increased incidence in males that is observed in human KD. Gene expression data from patients with KD and abdominal aorta tissue of Lactobacillus casei cell wall extract-injected mice showed enhanced Il1b expression and IL-1 signaling genes in males. Although the more severe IL-1β–mediated disease phenotype observed in male mice was ameliorated by Anakinra treatment, the milder disease phenotype in female mice failed to respond. Conclusions: IL-1β may play a central role in mediating sex-based differences in KD, with important implications for the use of anti–IL-1β therapies to treat male and female patients with KD.

To cite this article: Kocat€ urk B, Versteeg HH. Tissue factor-integrin interactions in cancer and thrombosis: every Jack has his Jill. J Thromb Haemost 2013; 11 (Suppl. 1): 285-93.Summary. Tissue factor (TF) is a 47 kDa membrane protein that initiates coagulation by binding to FVII(a) and FX(a) and is a risk factor for thrombosis in many disease states. In addition to its coagulant activity, TF also influences cancer progression by triggering signaling effects via a group of G-protein coupled receptors named protease-activated receptors (PARs). TF localizes to cytoskeletal structures in migrating cells, influences cytoskeleton reorganization and promotes migration. Recently, integrins, important mediators of cell motility, have emerged as important binding partners for TF and influence both TF coagulant and PAR-2-dependent signaling functions. Direct binding of TF to integrins also impacts processes such as cell migration and signaling independent of PAR-2. A recently discovered alternatively spliced, soluble TF isoform also ligates integrins to augment angiogenesis, thus fuelling cancer progression. To date, the literature describes a complex interplay between different integrin subunits and distinct TF isoforms, but our understanding of TF-integrin bidirectional regulation remains clouded. In this review, we aim to summarize the existing knowledge on integrin-TF interaction and speculate on its relevance to physiology and pathology.