At least 468 individual genes have been manipulated by molecular methods to study their effects on the initiation, promotion, and progression of atherosclerosis. Most clinicians and many investigators, even in related disciplines, find many of these genes and the related pathways entirely foreign. Medical schools generally do not attempt to incorporate the relevant molecular biology into their curriculum. A number of key signaling pathways are highly relevant to atherogenesis and are presented to provide a context for the gene manipulations summarized herein. The pathways include the following: the insulin receptor (and other receptor tyrosine kinases); Ras and MAPK activation; TNF-α and related family members leading to activation of NF-κB; effects of reactive oxygen species (ROS) on signaling; endothelial adaptations to flow including G protein-coupled receptor (GPCR) and integrin-related signaling; activation of endothelial and other cells by modified lipoproteins; purinergic signaling; control of leukocyte adhesion to endothelium, migration, and further activation; foam cell formation; and macrophage and vascular smooth muscle cell signaling related to proliferation, efferocytosis, and apoptosis. This review is intended primarily as an introduction to these key signaling pathways. They have become the focus of modern atherosclerosis research and will undoubtedly provide a rich resource for future innovation toward intervention and prevention of the number one cause of death in the modern world.

Molecular Biology of Atherosclerosis

Cytokines in atherosclerosis: Key players in all stages of disease and promising therapeutic targets.

Purpose: The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation, and tumor initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells. Experimental Design: We investigated the role of Notch activity in lung adenocarcinoma using a Notch GFP reporter construct and a γ-secretase inhibitor (GSI), which inhibits Notch pathway activity. Results: Transduction of lung cancer cells with Notch GFP reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 443 patients with lung adenocarcinoma. This correlation was further confirmed in an independent group of 89 patients with adenocarcinoma in which Hes-1 overexpression correlated with poor overall survival. Conclusions: Notch activity can identify lung cancer stem cell–like population and its inhibition may be an appropriate target for treating lung adenocarcinoma. Clin Cancer Res; 19(8); 1972–80. ©2013 AACR .

https://clincancerres.aacrjournals.org/content/clincanres/19/8/1972.full.pdf

Notch Pathway Activity Identifies Cells with Cancer Stem Cell–like Properties and Correlates with Worse Survival in Lung Adenocarcinoma

Beyond adiponectin and leptin: adipose tissue-derived mediators of inter-organ communication

Under various pathological conditions, including thrombosis and inflammation, extracellular nucleotide levels may increase because of both active release and passive leakage from damaged or dying cells. Once in the extracellular compartment, nucleotides interact with plasma membrane receptors belonging to the P2 purinergic family, which are expressed by virtually all circulating blood cells and in most blood vessels. In this review, we focus on the specific role of the 3 platelet P2 receptors P2Y1, P2Y12, and P2X1 in hemostasis and arterial thrombosis. Beyond platelets, these 3 receptors, along with the P2Y2, P2Y6, and P2X7 receptors, constitute the main P2 receptors mediating the proinflammatory effects of nucleotides, which play important roles in various functions of circulating blood cells and cells of the vessel wall. Each of these P2 receptor subtypes specifically contributes to chronic or acute vascular inflammation and related diseases, such as atherosclerosis, restenosis, endotoxemia, and sepsis. The potential for therapeutic targeting of these P2 receptor subtypes is also discussed.

Purinergic Receptors in Thrombosis and Inflammation

TRAIL attenuates the development of atherosclerosis in apolipoprotein E deficient mice

Aims Platelets have a fundamental role in atherothrombosis, but their role in early atherogenesis is unclear. The P2Y12 receptor is responsible for amplifying and sustaining platelet activation and P2Y12 inhibition is crucial in modulating the vessel wall response to injury. We therefore examined the role of platelet vs. vessel wall P2Y12 in early atherogenesis and considered the use of P2Y12 antagonists ticagrelor and clopidogrel in modulating this process.

Methods and results ApoE−/− and ApoE−/−P2Y12−/− male mice underwent bone marrow transplantation and were fed a western diet for 4 weeks before assessing atherosclerotic burden. Compared with ApoE−/− controls, platelet P2Y12 deficiency profoundly reduced platelet reactivity but had no effect on atheroma formation, whereas vessel wall P2Y12 deficiency significantly attenuated atheroma in the aortic sinus and brachiocephalic artery (both P < 0.001). ApoE−/− and ApoE−/−P2Y12−/− male mice fed western diet plus either twice-daily doses of ticagrelor (100 mg/kg) or daily clopidogrel (20 mg/kg) for 4 weeks exhibited no significant reduction in atheroma compared with control mice fed mannitol. Attenuated P-selectin expression confirmed platelet P2Y12 inhibition in drug-treated mice.

Conclusions Despite its major contribution to platelet reactivity, platelet P2Y12 has no effect on early atheroma formation, whereas vessel wall P2Y12 is important in this process. Ticagrelor and clopidogrel effectively reduced platelet reactivity but were unable to inhibit early atherogenesis, demonstrating that these P2Y12 inhibitors may not be effective in preventing early disease.

Vessel wall, not platelet, P2Y12 potentiates early atherogenesis

Genetic, dietary and immune factors contribute to the pathogenesis of atherosclerosis in humans and mice. Complement activation is an integral part of the innate immune defence but also shapes cellular responses and influences directly triglyceride synthesis. Deficiency of Factor B of the alternative pathway (AP) of complement is beneficial in LDLR−/− mice fed a high fat diet. The serum glycoprotein properdin is a key positive regulator of the AP but has not been studied in experimental atherosclerosis. Atherosclerosis was assessed after feeding low fat (LFD) or high fat (HFD) Western type diets to newly generated LDLR−/− ProperdinKO (LDLR−/−PKO) and LDLR−/−PWT mice. Lipids, lymphocytes and monocytes were similar among genotypes, genders and diets. Complement C3, but not C3adesarg, levels were enhanced in LDLR−/−PKO mice regardless of diet type or gender. Non-esterified fatty acids (NEFA) were decreased in male LDLR−/−PKO fed a HFD compared with controls. All mice showed significant atherosclerotic burden in aortae and at aortic roots but male LDLR−/− mice fed a LFD were affected to the greatest extent by the absence of properdin. The protective effect of properdin expression was overwhelmed in both genders of LDLR−/−mice when fed a HFD. We conclude that properdin plays an unexpectedly beneficial role in the development and progression of early atherosclerotic lesions.

https://lra.le.ac.uk/bitstream/2381/32731/2/Protective%20role%20for%20properdin%20in%20progression%20of%20experimental%20murine%20atherosclerosis..pdf

Protective Role for Properdin in Progression of Experimental Murine Atherosclerosis

Atherosclerosis is a chronic progressive inflammatory disease which manifests in the arterial vascular tree. It is a major cause of cardiovascular morbidity and contributes significantly to mortality in the developed world. Triggers for this inflammatory process are elevated levels of cholesterol, bacterial infection and obesity. The immune response in atherosclerosis is essentially pro-atherogenic, leading to lipid accumulation and cellular changes within the arterial wall. Small-animal models of atherosclerosis are used to study the relevance of candidate factors (cells, genes, diets) in the development and progression of lesions. From a multidisciplinary viewpoint, there are challenges and limitations to this approach. Activation of complement determines or modifies the outcome of acute and chronic inflammation. This review dissects the role of complement in the early development as well as the progressive manifestation of murine atherosclerosis and the advances in knowledge provided by the use of specific mouse models. It gives a critical overview of existing models, analyses seemingly conflicting results obtained with complement-deficient mouse models, highlights the importance of interrelationships between pro-coagulpant activity, adipose tissue, macrophages and complement, and uncovers exciting avenues of topical research.

/pdf/the-role-of-complement-in-the-development-and-manifestation-496wttuw1w.pdf

The role of complement in the development and manifestation of murine atherogenic inflammation: novel avenues.

Abstract Background Inflammatory bowel disease (IBD) affects an estimated 270,000 people in Canada and is rapidly increasing in prevalence. All patients have relapsing disease, and a subset of individuals do not respond to current treatments. Further, there are no approved treatment options in Canada that reverse IBD-induced intestinal fibrosis. We have previously shown type 1 regulatory cells (Tr1s) are capable of suppressing inflammatory macrophages, promote barrier function of human intestinal epithelial cells, and induce differentiation of mucin-producing goblet cells. We hypothesize that Tr1 cells can prevent inflammatory damage and fibrosis in an mouse model of acute gut damage. Purpose We hypothesize that Tr1 cells can prevent inflammatory damage and fibrosis in an mouse model of gut damage. Here we evalute the therapeutic potential of Tr1 cells in an model of acute intestinal epithelial damage. Method Tr1 cells were isolated and expanded from CD4+ CD44high FOXP3- cells. Their phenotype was characterized by flow cytometry and cytokine secretion was measured via ELISA. WT B6 mice were given 2% DSS in H2O for 7 days, followed by H2O alone for 7 days. Prior to DSS treatment, mice were sub-lethally irradiated to facilitate engraftment, and given I.P. injections of PBS or 0.5 – 2 x 106 Tr1 cells. Mice weights and health scores were recorded daily. At the endpoint, blood, spleen, and mesenteric lymph nodes were analyzed for Tr1 cell engraftment (or lack thereof) for each mouse. Complete white blood counts were performed for each mouse. Additionally, proximal, medial, and distal portions of the ileum were processed for histologic scoring. Result(s) Tr1 cells isolated from CD4+ CD44high FOXP3- cells produce high levels of IL-10 following stimulation (>35,000 pg/ml/1 x 105 cells). Additionally, these cells express high levels of Tr1 markers CD49b and Lag-3. Optimization experiments indicated no significant differences between mice irradiated and given DSS and mice only given DSS (no irradiation). Our results suggest no significant differences in inflammatory cell infiltrate scores between control and Tr1 treated mice. However, gut architecture scores appeared to improve with increasing Tr1 doses. Further, weight change improved with Tr1 treatment, as compared to PBS controls. Interestingly, Tr1 treatment appeared to decrease total eosinophil and neutrophil counts from peripheral blood. Conclusion(s) Our initial findings indicate Tr1 adoptive transfer prior to acute damage via DSS improves gut damage and weight loss. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

/pdf/a36-investigating-type-1-regulatory-t-cells-as-a-therapy-for-14lrjc2e.pdf

Tanja Steiner

Papers

TRAIL attenuates the development of atherosclerosis in apolipoprotein E deficient mice

Vessel wall, not platelet, P2Y12 potentiates early atherogenesis

Protective Role for Properdin in Progression of Experimental Murine Atherosclerosis

The role of complement in the development and manifestation of murine atherogenic inflammation: novel avenues.

A36 investigating type 1 regulatory t cells as a therapy for inflammatory bowel disease using a mouse model of acute intestinal epithelial damage