Ramon Cueto

Regulatory T cells and Atherosclerosis.

Atherosclerosis is a chronic autoimmune inflammatory disease. The involvement of both innate and adaptive immune responses in the pathogenesis of the disease has been well recognized. Tregs are an essential part of the immune system and have indispensable functions in maintaining immune system homeostasis, mediating peripheral tolerance, preventing autoimmune diseases, and suppressing inflammatory and proatherogenic immune response. Tregs carry out their immunosuppressive functions via several mechansims. One of the well-documented suppressive mechanisms of Tregs is the secretion of anti-inflammatory cytokines including IL-10, TGF-β, and IL-35. Studies have found that IL-10 and TGF-β have atheroprotective properties. In addition, Tregs can suppress the activity of proatherogenic effector T cells, suggesting an atheroprotective role. In fact, fewer Tregs are found in atherogenic ApoE-/- mice comparing to wild-type mice, suggesting an uncontrolled balance between weakened Tregs and effector T cells in atherogenesis. Some clinical studies of autoimmune diseases also suggest that decreased Tregs numbers are associated with increased disease activity. The importance of Tregs in many autoimmune diseases and experimental atherosclerosis has been established in in vivo and in vitro studies. However, the roles of Tregs in atherosclerosis in the clinical setting remains to be further characterized.

Inhibition of Caspase-1 Activation in Endothelial Cells Improves Angiogenesis: A NOVEL THERAPEUTIC POTENTIAL FOR ISCHEMIA.

Background: The interplay between dyslipidemia-induced inflammation and angiogenesis remains poorly understood. Results: Inhibition of caspase-1 improves VEGFR-2 signaling, tube formation, and blood perfusion in ischemic tissues. Conclusion: The suppression of caspase-1 improves angiogenesis and ischemia prognosis. Significance: Caspase-1 suppression is a novel therapeutic target for improvement of angiogenesis and ischemia under inflammatory environments. Deficient angiogenesis may contribute to worsen the prognosis of myocardial ischemia, peripheral arterial disease, ischemic stroke, etc. Dyslipidemic and inflammatory environments attenuate endothelial cell (EC) proliferation and angiogenesis, worsening the prognosis of ischemia. Under these dyslipidemic and inflammatory environments, EC-caspase-1 becomes activated and induces inflammatory cell death that is defined as pyroptosis. However, the underlying mechanism that correlates caspase-1 activation with angiogenic impairment and the prognosis of ischemia remains poorly defined. By using flow cytometric analysis, enzyme and receptor inhibitors, and hind limb ischemia model in caspase-1 knock-out (KO) mice, we examined our novel hypothesis, i.e. inhibition of caspase-1 in ECs under dyslipidemic and inflammatory environments attenuates EC pyroptosis, improves EC survival mediated by vascular endothelial growth factor receptor 2 (VEGFR-2), angiogenesis, and the prognosis of ischemia. We have made the following findings. Proatherogenic lipids induce higher caspase-1 activation in larger sizes of human aortic endothelial cells (HAECs) than in smaller sizes of HAECs. Proatherogenic lipids increase pyroptosis significantly more in smaller sizes of HAECs than in larger sizes of the cells. VEGFR-2 inhibition increases caspase-1 activation in HAECs induced by lysophosphatidylcholine treatment. Caspase-1 activation inhibits VEGFR-2 expression. Caspase-1 inhibition improves the tube formation of lysophosphatidylcholine-treated HAECs. Finally, caspase-1 depletion improves angiogenesis and blood flow in mouse hind limb ischemic tissues. Our results have demonstrated for the first time that inhibition of proatherogenic caspase-1 activation in ECs improves angiogenesis and the prognosis of ischemia.

Homocysteine induces inflammatory transcriptional signaling in monocytes.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Here, we studied transcriptional regulation in homocysteine (Hcy)-induced gene expression in monocytes (MC). We identified 11 Hcy-induced genes, 17 anti-inflammatory cytokine interleukin 10-induced, 8 pro-inflammatory cytokine interferon gamma (IFN gamma)-induced and 8 pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha)-induced genes through literature search. Binding frequency of 36 transcription factors (TFs) implicated in inflammation and MC differentiation were analyzed within core promoter regions of identified genes, and classified into 3 classes based on the significant binding frequency to the promoter of Hcy-induced genes. Class 1 TFs exert high significant binding frequency in Hcy-induced genes. Class 2 and 3 TFs have low and no significant binding frequency, respectively. Class 1 TF binding occurrence in Hcy-induced genes is similar to that in IFN gamma -induced genes, but not that in TNF alpha -induced. We conclude that Hcy is a pro-inflammatory amino acid and induces inflammatory transcriptional signal pathways mediated by class 1 TF. We term class 1 TF as putative Hcy-responsive TFs.

Lysophospholipids and their G protein-coupled receptors in atherosclerosis.

Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc.

Caspase-1 Plays a Critical Role in Accelerating Chronic Kidney Disease-Promoted Neointimal Hyperplasia in the Carotid Artery

To determine whether caspase-1 is critical in chronic kidney disease (CKD)-mediated arterial neointimal hyperplasia (NH), we utilized caspase−/− mice and induced NH in carotid artery in a CKD environment, and uremic sera-stimulated human vascular smooth muscle cells (VSMC). We made the following findings: (1) Caspase-1 inhibition corrected uremic sera-mediated downregulation of VSMC contractile markers, (2) CKD-promoted NH was attenuated in caspase−/− mice, (3) CKD-mediated downregulation of contractile markers was rescued in caspase null mice, and (4) expression of VSMC migration molecule αvβ3 integrin was reduced in caspase−/− tissues. Our results suggested that caspase-1 pathway senses CKD metabolic danger signals. Further, CKD-mediated increase of contractile markers in VSMC and increased expression of VSMC migration molecule αvβ3 integrin in NH formation were caspase-1 dependent. Therefore, caspase-1 is a novel therapeutic target for the suppression of CKD-promoted NH.

Lysophospholipid Receptors, as Novel Conditional Danger Receptors and Homeostatic Receptors Modulate Inflammation—Novel Paradigm and Therapeutic Potential

There are limitations in the current classification of danger-associated molecular patterns (DAMP) receptors. To overcome these limitations, we propose a new paradigm by using endogenous metabolites lysophospholipids (LPLs) as a prototype. By utilizing a data mining method we pioneered, we made the following findings: (1) endogenous metabolites such as LPLs at basal level have physiological functions; (2) under sterile inflammation, expression of some LPLs is elevated. These LPLs act as conditional DAMPs or anti-inflammatory homeostasis-associated molecular pattern molecules (HAMPs) for regulating the progression of inflammation or inhibition of inflammation, respectively; (3) receptors for conditional DAMPs and HAMPs are differentially expressed in human and mouse tissues; and (4) complex signaling mechanism exists between pro-inflammatory mediators and classical DAMPs that regulate the expression of conditional DAMPs and HAMPs. This novel insight will facilitate identification of novel conditional DAMPs and HAMPs, thus promote development of new therapeutic targets to treat inflammatory disorders.

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets—“Sand Out and Gold Stays”

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of “Sand out and Gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

Analyses of caspase-1-regulated transcriptomes in various tissues lead to identification of novel IL-1β-, IL-18- and sirtuin-1-independent pathways

BackgroundIt is well established that caspase-1 exerts its biological activities through its downstream targets such as IL-1β, IL-18, and Sirt-1. The microarray datasets derived from various caspase-1 knockout tissues indicated that caspase-1 can significantly impact the transcriptome. However, it is not known whether all the effects exerted by caspase-1 on transcriptome are mediated only by its well-known substrates. Therefore, we hypothesized that the effects of caspase-1 on transcriptome may be partially independent from IL-1β, IL-18, and Sirt-1.MethodsTo determine new global and tissue-specific gene regulatory effects of caspase-1, we took novel microarray data analysis approaches including Venn analysis, cooperation analysis, and meta-analysis methods. We used these statistical methods to integrate different microarray datasets conducted on different caspase-1 knockout tissues and datasets where caspase-1 downstream targets were manipulated.ResultsWe made the following important findings: (1) Caspase-1 exerts its regulatory effects on the majority of genes in a tissue-specific manner; (2) Caspase-1 regulatory genes partially cooperates with genes regulated by sirtuin-1 during organ injury and inflammation in adipose tissue but not in the liver; (3) Caspase-1 cooperates with IL-1β in regulating less than half of the genes involved in cardiovascular disease, organismal injury, and cancer in mouse liver; (4) The meta-analysis identifies 40 caspase-1 globally regulated genes across tissues, suggesting that caspase-1 globally regulates many novel pathways; and (5) The meta-analysis identified new cooperatively and non-cooperatively regulated genes in caspase-1, IL-1β, IL-18, and Sirt-1 pathways.ConclusionsOur findings suggest that caspase-1 regulates many new signaling pathways potentially via its known substrates and also via transcription factors and other proteins that are yet to be identified.

A comprehensive data mining study shows that most nuclear receptors act as newly proposed homeostasis-associated molecular pattern receptors

BackgroundNuclear receptors (NRs) can regulate gene expression; therefore, they are classified as transcription factors. Despite the extensive research carried out on NRs, still several issues including (1) the expression profile of NRs in human tissues, (2) how the NR expression is modulated during atherosclerosis and metabolic diseases, and (3) the overview of the role of NRs in inflammatory conditions are not fully understood.MethodsTo determine whether and how the expression of NRs are regulated in physiological/pathological conditions, we took an experimental database analysis to determine expression of all 48 known NRs in 21 human and 17 murine tissues as well as in pathological conditions.ResultsWe made the following significant findings: (1) NRs are differentially expressed in tissues, which may be under regulation by oxygen sensors, angiogenesis pathway, stem cell master regulators, inflammasomes, and tissue hypo-/hypermethylation indexes; (2) NR sequence mutations are associated with increased risks for development of cancers and metabolic, cardiovascular, and autoimmune diseases; (3) NRs have less tendency to be upregulated than downregulated in cancers, and autoimmune and metabolic diseases, which may be regulated by inflammation pathways and mitochondrial energy enzymes; and (4) the innate immune sensor inflammasome/caspase-1 pathway regulates the expression of most NRs.ConclusionsBased on our findings, we propose a new paradigm that most nuclear receptors are anti-inflammatory homeostasis-associated molecular pattern receptors (HAMPRs). Our results have provided a novel insight on NRs as therapeutic targets in metabolic diseases, inflammations, and malignancies.

Identification of homocysteine-suppressive mitochondrial ETC complex genes and tissue expression profile – Novel hypothesis establishment

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD) which has been implicated in matochondrial (Mt) function impairment. In this study, we characterized Hcy metabolism in mouse tissues by using LC-ESI-MS/MS analysis, established tissue expression profiles for 84 nuclear-encoded Mt electron transport chain complex (nMt-ETC-Com) genes in 20 human and 19 mouse tissues by database mining, and modeled the effect of HHcy on Mt-ETC function. Hcy levels were high in mouse kidney/lung/spleen/liver (24–14 nmol/g tissue) but low in brain/heart (~5 nmol/g). S-adenosylhomocysteine (SAH) levels were high in the liver/kidney (59–33 nmol/g), moderate in lung/heart/brain (7–4 nmol/g) and low in spleen (1 nmol/g). S-adenosylmethionine (SAM) was comparable in all tissues (42–18 nmol/g). SAM/SAH ratio was as high as 25.6 in the spleen but much lower in the heart/lung/brain/kidney/liver (7–0.6). The nMt-ETC-Com genes were highly expressed in muscle/pituitary gland/heart/BM in humans and in lymph node/heart/pancreas/brain in mice. We identified 15 Hcy-suppressive nMt-ETC-Com genes whose mRNA levels were negatively correlated with tissue Hcy levels, including 11 complex-I, one complex-IV and two complex-V genes. Among the 11 Hcy-suppressive complex-I genes, 4 are complex-I core subunits. Based on the pattern of tissue expression of these genes, we classified tissues into three tiers (high/mid/low-Hcy responsive), and defined heart/eye/pancreas/brain/kidney/liver/testis/embryonic tissues as tier 1 (high-Hcy responsive) tissues in both human and mice. Furthermore, through extensive literature mining, we found that most of the Hcy-suppressive nMt-ETC-Com genes were suppressed in HHcy conditions and related with Mt complex assembly/activity impairment in human disease and experimental models. We hypothesize that HHcy inhibits Mt complex I gene expression leading to Mt dysfunction.