Platelets exacerbate cardiovascular inflammation in a murine model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Increased platelet counts and activation are observed during the course of KD, and elevated platelet counts are associated with higher risks of developing intravenous immunoglobulin resistance and coronary artery aneurysms. However, the role of platelets in KD pathogenesis remains unclear. Here, we analyzed transcriptomics data generated from the whole blood of patients with KD and discovered changes in the expression of platelet-related genes during acute KD. In the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, LCWE injection increased platelet counts and the formation of monocyte-platelet aggregates (MPAs), upregulated the concentration of soluble P-selectin, and increased circulating thrombopoietin and interleukin 6 (IL-6). Furthermore, platelet counts correlated with the severity of cardiovascular inflammation. Genetic depletion of platelets (Mpl-/- mice) or treatment with an anti-CD42b antibody significantly reduced LCWE-induced cardiovascular lesions. Furthermore, in the mouse model, platelets promoted vascular inflammation via the formation of MPAs, which likely amplified IL-1B production. Altogether, our results indicate that platelet activation exacerbates the development of cardiovascular lesions in a murine model of KD vasculitis. These findings enhance our understanding of KD vasculitis pathogenesis and highlight MPAs, which are known to enhance IL-1B production, as a potential therapeutic target for this disorder.

Tumor-expressed factor VII is associated with survival and regulates tumor progression in breast cancer.

Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumor-expressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liver-derived FVII have opposing effects on EMT and metastasis.

In silico analysis reveals PRDX4 as a prognostic and oncogenic marker in renal papillary cell carcinoma.

BACKGROUND: Alterations in the tumor microenvironment leads to the accumulation of reactive oxygen species (ROS). When in low levels, ROS act as a signaling molecule and contribute to tumor cell proliferation whereas its elevation results in oxidative stress and eventually cell death. It is known that antioxidant systems regulate the ROS levels and thus cell fate. Among these systems, peroxiredoxins (PRDXs) were found to be upregulated in various cancers. However their exact contribution to carcinogenesis is not yet clear. AIM: Herein, the expression pattern and prognostic value of PRDXs were explored in cancer setting by using in silico analysis tools and publicly available datasets. RESULTS: Pan-cancer analysis revealed that PRDXs are differentially expressed in normal and tumor tissues. Further analysis showed that higher PRDX4 levels was associated with poor prognosis and clinicopathological and histological features associated with a more aggressive renal papillary cell carcinoma (KIRP) profile. Hypoxia, ER stress and protein folding were shown to be pathways positively correlated with PRDX4 levels. Furthermore, PRDX4 was found to be strong regulator of protein homeostasis. Kaplan-Meier analysis revealed that PRDX4 is a potent prognostic marker in Type 2 KIRP and this might be due to increased ER stress and oxidative stress levels in this subtype. CONCLUSIONS: The data suggest that PRDX4 can be used as a prognostic marker for KIRP patients. Its association with more aggressive tumor characteristics also underlines that it might be used for targeted therapy.

Identification of thioredoxin domain containing family members' expression pattern and prognostic value in diffuse gliomas via in silico analysis.

BACKGROUND: Gliomas are the most prevalent primary tumors of the central nervous system. Their aggressive nature and the obstacles arising during therapy highlights the importance of finding new prognostic markers and therapy targets for gliomas. TXNDC genes are members of the thioredoxin superfamily and were shown to play a role in redox homeostasis, protein folding, electron transfer and also acting as cellular adapters. The well known contribution of these processes in cancer progression prompted us to investigate if TXNDC family members may also play a role in carcinogenesis, in particular diffuse gliomas. METHODS: The present study used in silico analysis tools GEPIA, UCSC Xena, Gliovis, cBioPortal, and Ivy GAP to evaluate the expression pattern, prognostic value and clinical significance of TXNDC family members in diffuse gliomas. RESULTS: Our analysis showed that TXNDC family members' expression pattern differ between tumors and healthy tissues and among tumors with different grades. The detailed analysis of TXNDC5 in glioma pathogenesis revealed that TXNDC5 expression is associated with more aggressive clinical and molecular features and poor therapy success both in LGG and GBM samples. Kaplan-Meier survival curves represented a worse prognosis for patients with leveated TXNDC5 levels in LGG and all grade glioma patients. The levels of TXNDC5 was shown to be possibly regulated by hypoxia-ER stress axis and a potential mechanism for TXNDC5-driven glioma progression was found to be extracellular matrix (ECM) production which is known to promote tumor aggressiveness. CONCLUSIONS: Our results uncovered the previously unknown role of TXNDC family members in glioma pathogenesis and showed that TXNDC5 levels could serve as a predictor of clinical outcome and therapy success and may very well be used for targeted therapy.

Integrin regulation by tissue factor promotes cancer stemness and metastatic dissemination in breast cancer.

Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with ß1-integrin leading to inactivation of ß1-integrin. Inhibition of TF signaling induces a shift in TF-binding from α3ß1-integrin to α6ß4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin ß1 and ß4-dependent reduction in metastasic dissemination.

Targeting IRE1 endoribonuclease activity alleviates cardiovascular lesions in a murine model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of noncongenital heart disease in children. Studies in mice and humans propound the NLRP3/IL-1ß pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated to our knowledge. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of inositol-requiring enzyme 1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1ß secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis and highlight IRE1 RNase activity as a potential new therapeutic target.

Spondyloarthritides: Theories and beyond.

Spondyloarthritides (SpA) are a family of interrelated rheumatic disorders with a typical disease onset ranging from childhood to middle age. If left untreated, they lead to a severe decrease in patients' quality of life. A succesfull treatment strategy starts with an accurate diagnosis which is achieved through careful analysis of medical symptoms. Classification criterias are used to this process and are updated on a regular basis. Although there is a lack of definite knowledge on the disease etiology of SpA, several studies have paved the way for understanding plausible risk factors and developing treatment strategies. The significant increase of HLA-B27 positivity in SpA patients makes it a strong candidate as a predisposing factor and several theories have been proposed to explain HLA-B27 driven disease progression. However, the presence of HLA-B27 negative patients underlines the presence of additional risk factors. The current treatment options for SpAs are Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), TNF inhibitors (TNFis), Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and physiotherapy yet there are ongoing clinical trials. Anti IL17 drugs and targeted synthetic DMARDs such as JAK inhibitors are also emerging as treatment alternatives. This review discusses the current diagnosis criteria, treatment options and gives an overview of the previous findings and theories to clarify the possible contributors to SpA pathogenesis with a focus on Ankylosing Spondylitis (AS) and enthesitis-related arthritis (ERA).

MicroRNA-223 Regulates the Development of Cardiovascular Lesions in LCWE-Induced Murine Kawasaki Disease Vasculitis by Repressing the NLRP3 Inflammasome.

Kawasaki disease (KD), an acute febrile childhood illness and systemic vasculitis of unknown etiology, is the leading cause of acquired heart disease among children. Experimental data from murine models of KD vasculitis and transcriptomics data generated from whole blood of KD patients indicate the involvement of the NLRP3 inflammasome and interleukin-1 (IL-1) signaling in KD pathogenesis. MicroRNA-223 (miR-223) is a negative regulator of NLRP3 activity and IL-1ß production, and its expression has been reported to be upregulated during acute human KD; however, the specific role of miR-223 during KD vasculitis remains unknown. Here, using the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, we demonstrate increased miR-223 expression in LCWE-induced cardiovascular lesions. Compared with control WT mice, LCWE-injected miR-223-deficient mice (miR223 -/y ) developed more severe coronary arteritis and aortitis, as well as more pronounced abdominal aorta aneurysms and dilations. The enhanced cardiovascular lesions and KD vasculitis observed in LCWE-injected miR223 -/y mice correlated with increased NLRP3 inflammasome activity and elevated IL-1ß production, indicating that miR-223 limits cardiovascular lesion development by downmodulating NLRP3 inflammasome activity. Collectively, our data reveal a previously unappreciated role of miR-223 in regulating innate immune responses and in limiting KD vasculitis and its cardiovascular lesions by constraining the NLRP3 inflammasome and the IL-1ß pathway. These data also suggest that miR-223 expression may be used as a marker for KD vasculitis pathogenesis and provide a novel therapeutic target.

Inositol-requiring enzyme-1 regulates phosphoinositide signaling lipids and macrophage growth.

The ER-bound kinase/endoribonuclease (RNase), inositol-requiring enzyme-1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1's one known, specific RNA target, X box-binding protein-1 (XBP1) or the RNA substrates of IRE1-dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide-derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross-talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1's RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P3 ) 5-phosphatase-2 (INPPL1) is a direct target of miR-2137, which controls PI(3,4,5)P3 levels in macrophages. The modulation of cellular PI(3,4,5)P3 /PIP2 ratio and anabolic mTOR signaling by the IRE1-induced miR-2137 demonstrates how the ER can provide a critical input into cell growth decisions.

Interleukin-1 Beta-Mediated Sex Differences in Kawasaki Disease Vasculitis Development and Response to Treatment.

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 caseicell wall extract, or a combination of Lactobacillus caseicell 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 caseicell wall extract-injected mice showed enhanced Il1b expression and IL-1 signaling genes in males. Although the more severe IL-1ß-mediated disease phenotype observed in male mice was ameliorated by Anakinra treatment, the milder disease phenotype in female mice failed to respond. CONCLUSIONS: IL-1ß may play a central role in mediating sex-based differences in KD, with important implications for the use of anti-IL-1ß therapies to treat male and female patients with KD.

Intercepting the Lipid-Induced Integrated Stress Response Reduces Atherosclerosis.

BACKGROUND: Eukaryotic 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. OBJECTIVES: Potent 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. METHODS: The 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. RESULTS: The 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-1ß secretion in macrophages. Furthermore, ISR inhibitors suppress hyperlipidemia-induced inflammasome activation and inflammation, and reduce atherosclerosis. CONCLUSIONS: These 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-1ß 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.

Targeting IRE1 with small molecules counteracts progression of atherosclerosis.

Metaflammation, an atypical, metabolically induced, chronic low-grade 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 ER-resident 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-1ß 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.

Prevention of atherosclerosis by bioactive palmitoleate through suppression of organelle stress and inflammasome activation.

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-1ß (IL-1ß) and IL-18 concentrations in vivo in hyperlipidemic mice. Moreover, PAO prevents macrophage ER stress and IL-1ß 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.

Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.

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.

Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma.

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 procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Alternatively spliced tissue factor promotes breast cancer growth in a ß1 integrin-dependent manner.

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 ß1 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.

Tissue factor isoforms in cancer and coagulation: may the best isoform win.

Tissue factor (TF), the trigger of blood coagulation, is a 47 kDa membrane protein that also impacts on non-hemostatic processes, such as atherosclerosis, primary tumor growth and metastasis. TF binding to its ligand FVIIa induces activation of protease-activated receptor-2 and this event is thought to considerably influence atherosclerosis and tumor angiogenesis. TF-dependent activation of the coagulation cascade, rather than PAR-2 activation, then leads to the potentiation of metastasis. Importantly, a soluble alternatively spliced isoform of TF (asTF) has been discovered, but the function of asTF in hemostatic and non-hemostatic events is poorly understood. In this review, we aim to present a side-by-side evaluation of normally-spliced, full length TF (flTF) and asTF with regard to coagulant function, atherosclerosis, tumor progression and malignancy-associated thrombosis.