Eduardo Fuentes

Mechanisms of Chronic State of Inflammation as Mediators That Link Obese Adipose Tissue and Metabolic Syndrome

The metabolic syndrome is a cluster of cardiometabolic alterations that include the presence of arterial hypertension, insulin resistance, dyslipidemia, and abdominal obesity. Obesity is associated with a chronic inflammatory response, characterized by abnormal adipokine production, and the activation of proinflammatory signalling pathways resulting in the induction of several biological markers of inflammation. Macrophage and lymphocyte infiltration in adipose tissue may contribute to the pathogenesis of obesity-mediated metabolic disorders. Adiponectin can either act directly on macrophages to shift polarization and/or prime human monocytes into alternative M2-macrophages with anti-inflammatory properties. Meanwhile, the chronic inflammation in adipose tissue is regulated by a series of transcription factors, mainly PPARs and C/EBPs, that in conjunction regulate the expression of hundreds of proteins that participate in the metabolism and storage of lipids and, as such, the secretion by adipocytes. Therefore, the management of the metabolic syndrome requires the development of new therapeutic strategies aimed to alter the main genetic pathways involved in the regulation of adipose tissue metabolism.

Role of PPARs in inflammatory processes associated with metabolic syndrome (Review).

Metabolic syndrome (MS) includes the presence of arterial hypertension, insulin resistance, dyslipidemia, cardiovascular disease (CVD) and abdominal obesity, which is associated with a chronic inflammatory response, characterized by abnormal adipokine production, and the activation of certain pro-inflammatory signaling pathways. Furthermore, the changes presented by the adipose tissue in MS favors the secretion of several molecular mediators capable of activating or suppressing a number of transcription factors, such as the peroxisome proliferator-activated receptors (PPARs), whose main functions include storage regulation and fatty acid catabolization. When they are activated by their ligands (synthetic or endogenous), they control several genes involved in intermediate metabolism, which make them, together with the PPAR gamma coactivator-1-α (PGC-1) and the silent information regulator T1 (SIRT1), good targets for treating metabolic diseases and their cardiovascular complications.

Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway.

Strawberry extract presents antiplatelet activity by inhibition of inflammatory mediator of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1β) and thrombus formation

Abstract Cardiovascular disease prevention is of high priority in developed countries. Healthy eating habits including the regular intake of an antithrombotic diet (fruit and vegetables) may contribute to prevention. Platelet function is a critical factor in arterial thrombosis and the effect strawberries have is still unclear. Therefore, the aim of this study was to systematically examine the action of strawberries in preventing platelet activation and thrombus formation. Strawberry extract concentration-dependently (0.1–1u2009mg/ml) inhibited platelet aggregation induced by ADP and arachidonic acid. At the same concentrations as strawberry inhibits platelet aggregation, it significantly decreased sP-selectin, sCD40L, RANTES, and IL-1β levels. The strawberry may exert significant protective effects on thromboembolic-related disorders by inhibiting platelet aggregation. Also, this suggests that antithrombotic activity may have novel anti-inflammatory effects.

Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

Background The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. Objectives The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. Results Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [nu200a=u200a6, p<0.01] and 72±1.9% occlusion at 60 min, [nu200a=u200a6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, nu200a=u200a6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. Conclusion Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

Bioassay-Guided Isolation and HPLC Determination of Bioactive Compound That Relate to the Antiplatelet Activity (Adhesion, Secretion, and Aggregation) from Solanum lycopersicum

In seeking the functionality of foodstuff applicable to medicine, ripe tomato fruits were found to show an antiplatelet activity. Therefore, the bioactive compound was isolated, structurally identified, and studied for an inhibitory effects on platelet adhesion, secretion, and aggregation. The concentration of adenosine in ripe tomato fruits (pulp and skin extracts) and its processing by-products (paste and pomace) was determined by reversed-phase high-performance liquid chromatography (HPLC). According to platelet aggregation inhibition induced by ADP, the total extract residual was fractionated by liquid-liquid separation, obtaining aqueous, ethyl acetate and petroleum ether extracts. The aqueous extract was subjected to repeated permeation over sephadex LH-20 and semipreparative TLC. The isolate finally obtained was identified as adenosine on the basis of ESI-MS, 1H NMR, HPLC, and UV spectra. Adenosine concentration dependently (2.3–457u2009μM) platelet aggregation inhibited induced by ADP. Also, adenosine present inhibition of platelet secretion and thrombus formation under flow conditions. The quantitative HPLC analysis revealed significant amounts of adenosine in ripe tomato fruits and its processing by-products. From these results, extracts/fractions of ripe tomato fruits and their processing by-products may be referred to as functional food and functional ingredients containing a compound that inhibits platelet function with a potent preventive effect on thrombus formation, as those that occur in stroke.

Role of Platelet-Derived Microvesicles As Crosstalk Mediators in Atherothrombosis and Future Pharmacology Targets: A Link between Inflammation, Atherosclerosis, and Thrombosis

Reports in the last decade have suggested that the role of platelets in atherosclerosis and its thrombotic complications may be mediated, in part, by local secretion of platelet-derived microvesicles (pMVs), small cell blebs released during the platelet activation process. MVs are the most abundant cell-derived microvesicle subtype in the circulation. High concentrations of circulating MVs have been reported in patients with atherosclerosis, acute vascular syndromes, and/or diabetes mellitus, suggesting a potential correlation between the quantity of microvesicles and the clinical severity of the atherosclerotic disease. pMVs are considered to be biomarkers of disease but new information indicates that pMVs are also involved in signaling functions. pMVs evoke or promote haemostatic and inflammatory responses, neovascularization, cell survival, and apoptosis, processes involved in the pathophysiology of cardiovascular disease. This review is focused on the complex cross-talk between platelet-derived microvesicles, inflammatory cells and vascular elements and their relevance in the development of the atherosclerotic disease and its clinical outcomes, providing an updated state-of-the art of pMV involvement in atherothrombosis and pMV potential use as therapeutic agent influencing cardiovascular biomedicine in the future.

PAMAM dendrimer derivatives as a potential drug for antithrombotic therapy.

Platelets are anucleated blood cells that play an important role both in the pathogenesis of atherosclerosis and subsequent thrombosis. Dendrimers have attracted great interest in biomedical applications. However, their interactions with cell compounds and compartments are nonselective, thus causing cytotoxicity and hemotoxicity. We derivatized PAMAM G4 and G5 dendrimers to evaluate their interactions with serum metabolites, their effects on the viability of red blood cells, and their antithrombotic properties. PAMAM G4 and G5 derivatives showed better hemocompatibility than the PAMAM G4 and G5 dendrimers without any derivatization (NH₂). PAMAM G4-Arginine-Tos and G4-Lysine-Cbz act as potent inhibitors of platelet aggregation induced by ADP. PAMAM G4-Arginine-Tos also showed inhibition of platelet aggregation induced by collagen, TRAP-6 and arachidonic acid. Moreover, G4-Arginine-Tos present inhibition of platelet secretion and thrombus formation under flow conditions. Based on our study, the PAMAM G4-Arginine-Tos derivative is hemocompatible and produces desirable antiplatelet and antithrombotic effects. Thus, this compound has potential applications as an antithrombotic drug or a drug delivery vehicle.

Mechanisms of endothelial cell protection by hydroxycinnamic acids.

An endothelial dysfunction generates a proatherogenic environment characterized by stimulating thrombus formation. Epidemiological studies have provided evidence of a protective role of healthy diets in the prevention of cardiovascular diseases. Hydroxycinnamic acids constitute abundant polyphenols in our diets as they are present in high levels in many widely consumed foods, such as fruit, vegetables and beverages. Therefore, it can be established that due to the hydroxycinnamic acid content (caffeic, chlorogenic, feluric and p-coumaric acids), fruit, vegetables and beverages contribute to endothelial protection (attenuates oxidative stress, improved nitric oxide bioavailability and decreased E-selectin, ICAM-1 and VCAM-1 expression, among others). In this article, we systematically examine the mechanisms of endothelium protection of hydroxycinnamic acids.

Protective mechanisms of adenosine 5'-monophosphate in platelet activation and thrombus formation.

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.