Development of a Novel Apigenin Dosage form as a Substitute for the Modern Triple Antithrombotic Regimen.

The simultaneous administration of three antiplatelet agents has been proposed as an efficient strategy for the secondary prevention of atherothrombotic events and is included in the European guidelines. However, this strategy presented an increased risk of bleeding; therefore, the identification of new antiplatelet agents, with improved efficacy and diminished side effects, is of great importance. In silico studies, UPLC/MS Q-TOF plasma stability, in vitro platelet aggregation experiments, and pharmacokinetic studies were exploited. In the present study, it has been predicted that the flavonoid apigenin could target different platelet activation pathways, including P2Y12, protease-activated receptor-1 (PAR-1), and cyclooxygenase 1 (COX-1). To enhance apigenin's potency, hybridization with docosahexaenoic acid (DHA) was performed, as fatty acids have illustrated potent efficacy against cardiovascular diseases (CVDs). The new molecular hybrid, termed 4'-DHA-apigenin, demonstrated enhanced inhibitory activity against platelet aggregation induced by thrombin receptor activator peptide-6 (TRAP-6), adenosine diphosphate (ADP), and arachidonic acid (AA), with respect to the parent apigenin. The 4'-DHA-apigenin hybrid illustrated an almost 2-fold enhanced inhibitory activity, with respect to apigenin, and an almost 3-fold enhanced inhibitory activity, with respect to DHA, for the ADP-induced platelet aggregation. Additionally, the hybrid presented a more than 12-fold enhanced inhibitory activity with respect to DHA for the TRAP-6 induced platelet aggregation. Furthermore, a 2-fold enhanced inhibitory activity was recorded for the 4'-DHA-apigenin hybrid for the AA-induced platelet aggregation with respect to apigenin. To surmount the reduced LC-MS based plasma stability, a novel dosage form in olive oil has been developed. The 4'-DHA-apigenin olive oil-based formulation presented an enhanced antiplatelet inhibitory effect in three activation pathways. To further explore the pharmacokinetic profile of 4'-DHA-apigenin in olive oil formulations, a UPLC/MS Q-TOF protocol has been established to quantify the serum levels of apigenin after oral administration to C57BL/6J wild type mice. The olive oil-based formulation of 4'-DHA-apigenin demonstrated an increase in apigenin bioavailability of 262 %. This study may offer a new therapeutic strategy tailored to improve the treatment of CVDs.

PCSK9 is minimally associated with HDL but impairs the anti-atherosclerotic HDL effects on endothelial cell activation.

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) regulates the cell-surface localization of LDL receptors in hepatocytes and is associated with LDL and lipoprotein(a) [Lp(a)] uptake, reducing blood concentrations. However, the connection between PCSK9 and HDL is unclear. Here, we investigated the association of plasma PCSK9 with HDL subpopulations and examined the effects of PCSK9 on the atheroprotective function of HDL. We examined the association of PCSK9 with HDL in apoB-depleted plasma by ELISA, native PAGE, and immunoblotting. Our analyses showed that upon apoB-depletion, total circulating PCSK9 levels were 32% of those observed in normolipidemic plasma, and only 6% of PCSK9 in the apoB-depleted plasma, including both the mature and furin-cleaved forms, was associated with HDL. We also show human recombinant PCSK9 abolished the capacity of reconstituted HDL to reduce the formation of ROS in endothelial cells, while a PCSK9-blocking antibody enhanced the capacity of human HDL (in apoB-depleted plasma) to reduce ROS formation in endothelial cells and promote endothelial cell migration. Overall, our findings suggest that PCSK9 is only minimally associated with HDL particles, but PCSK9 in apoB-depleted plasma can affect the atheroprotective properties of HDL related to preservation of endothelial function. This study contributes to the elucidation of the pathophysiological role of plasma PCSK9 and highlights further the anti-atherosclerotic effect of PCSK9 inhibition.

Pleiotropic effects of apolipoprotein C3 on HDL functionality and adipose tissue metabolic activity.

APOC3 is produced mainly by the liver and intestine and approximately half of plasma APOC3 associates with HDL. Though it was believed that APOC3 associates with HDL by simple binding to preexisting particles, recent data support that biogenesis of APOC3-containing HDL (APOC3-HDL) requires Abca1. Moreover, APOC3-HDL contributes to plasma triglyceride homeostasis by preventing APOC3 association with triglyceride-rich lipoproteins. Interestingly, APOC3-HDL also shows positive correlation with the morbidly obese phenotype. However, the roles of APOC3 in HDL functionality and adipose tissue metabolic activity remain unknown. Therefore, here we investigated the direct effects of APOC3 expression on HDL structure and function, as well as white adipose tissue (WAT) and brown adipose tissue (BAT) metabolic activity. C57BL/6 mice were infected with an adenovirus expressing human APOC3 or a recombinant attenuated control adenovirus expressing green fluorescent protein and blood and tissue samples were collected at 5 days postinfection. HDL was then analyzed for its apolipoprotein and lipid composition and particle functionality. Additionally, purified mitochondria from BAT and WAT were analyzed for uncoupling protein 1, cytochrome c (Cytc), and Cytc oxidase subunit 4 protein levels as an indirect measure of their metabolic activity. Serum metabolomic analysis was performed by NMR. Combined, our data show that APOC3 modulates HDL structure and function, while it selectively promotes BAT metabolic activation.

Oxidative stress in multi-port and single-port cholecystectomy.

BACKGROUND: This study was designed to analyze and compare plasma levels of 8-isoprostane (8-epiPGF2α), a biomarker of lipid peroxidation, and uric acid (UA), a marker of the antioxidant status, in standard laparoscopic (LC) and laparoendoscopic single-site cholecystectomy (LSSC). MATERIALS AND METHODS: Forty patients with noncomplicated cholelithiasis were randomized to undergo either LSSC (n = 20) or LC (n = 20). The patients had body mass index <30, American Society of Anesthesiologists score I or II, and no previous upper gastrointestinal surgery. Blood samples were taken preoperatively and 6 h and 24 h postoperatively. Levels of 8-epiPGF2α were determined using enzyme-linked immunosorbent assay, whereas levels of UA were calculated using automated analyzer. RESULTS: No significant differences were observed in operative data among the groups. Levels of 8-epiPGF2α were significantly higher in LSSC compared with LC at 6 h (P = 0.003) and 24 h (P < 0.001). 8-epiPGF2α levels showed significant changes over time in LC (LSSC: P = 0.720, LC: P < 0.001). UA levels were significantly higher in LC compared with LSSC, 24 h postoperatively (P = 0.021). No significant changes over time in the UA levels in both groups (LSSC: P = 0.056, LC: P = 0.205). CONCLUSIONS: LSSC is associated with increased oxidative stress compared with LC. Further studies are needed to confirm these results.

Differential membrane expression of Toll-like receptors and intracellular cytokine induction in peripheral blood monocytes of patients with chronic kidney disease and diabetic nephropathy.

BACKGROUND: Toll-like receptors (TLRs) are key players in the innate immune system whose activation leads to an inflammatory response. Inflammation plays an important role in the pathogenesis of chronic kidney disease (CKD) and diabetes mellitus. The aim of our study was to assess the proinflammatory state of nondialysis CKD patients by evaluating the membrane expression of TLR2 and TLR4 and the intracellular IL-1ß and IL-6 production in response to the ligand Pam3Cys-Ser-(Lys)4 (Pam3CSK4). METHODS: 85 nondialysis CKD patients [mean estimated glomerular filtration rate: 34 (17-90) ml/min/1.73 m(2)] were divided in 2 groups: 55 nondiabetic CKD patients (CKD group) and 30 patients with diabetic nephropathy (DN group). The two groups were compared with 36 healthy subjects (control group). TLR2 and TLR4 membrane expression in monocytes and Pam3CSK4-induced intracellular production of IL-1ß and IL-6 were assessed by flow cytometry. RESULTS: Both patient groups showed increased TLR2 membrane expression compared with the control group, both at baseline (p < 0.05 for both) and after Pam3CSK4 stimulation (p < 0.05 for both). The DN group exhibited significantly higher TLR4 expression at baseline compared to the CKD and control groups (p < 0.04 and p < 0.02, respectively). Intracellular IL-1ß and IL-6 levels at baseline were significantly lower in CKD patients compared to the DN and control groups. After Pam3CSK4 stimulation, intracellular IL-1ß and IL-6 increased in all groups, but were lower in the CKD group versus the control group or DN group, which exhibited higher levels than the controls. CONCLUSIONS: Nondialysis CKD patients showed significant alterations in TLR2 and TLR4 membrane expression, and impaired Pam3CSK4-induced cytokine production in monocytes, a phenomenon that is markedly influenced by the presence of diabetes.

Effects of Serum Estradiol on Proprotein Convertase Subtilisin/Kexin Type 9 Levels and Lipid Profiles in Women Undergoing In Vitro Fertilization.

BACKGROUND: The mechanisms underlying the impact of estradiol (E2) on low-density lipoprotein cholesterol (LDL-C) levels are not completely understood, although a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) has been proposed. We aimed to investigate the association between levels of E2, PCSK9, and lipid parameters in premenopausal women undergoing in vitro fertilization (IVF). METHODS: Healthy women undergoing IVF in the Department of Obstetrics and Gynecology of the University General Hospital of Ioannina were recruited. Their levels of E2, PCSK9, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), LDL-C, and triglycerides (TGs) were measured 10 days after ovarian depression (E2min) and 7 days after ovarian stimulation (E2max). RESULTS: We included 34 consecutive women of median age 38 (interquartile range 26-46) years who underwent a full IVF cycle. As expected, E2 levels increased by 329.6% from E2min to E2max (108 [47-346] to 464 [241-2471] pg/mL, p < 0.05). During the same time, serum PCSK9 levels decreased by 30.8% (245 ± 80 to 170 ± 64 ng/mL, p < 0.05). TC, LDL-C, and TGs decreased by 0.4%, 3.8%, and 2.2%, respectively, while HDL-C levels increased by 5.3% (all p = NS). CONCLUSIONS: The rise in endogenous E2 during an IVF cycle was related with a significant decline in serum PCSK9 levels, but no significant change in plasma lipids during a 7-day period.

The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor of cardiovascular disease. Plasma Lp-PLA2 is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA2. We determined the plasma levels of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA2 concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA2 [apoB/Lp-PLA2⁻] was calculated by subtracting the apoB/Lp-PLA2 from total apoB. The apoB/Lp-PLA2 levels were 3.6-fold higher, while apoB/Lp-PLA2⁻ were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA2 and apoB/Lp-PLA2⁻ levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA2, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA2⁻. Considering that Lp-PLA2 is proatherogenic, the predominance of apoB/Lp-PLA2 particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease.

Oxidative stress markers in laparoscopic versus open colectomy for cancer: a double-blind randomized study.

BACKGROUND: Colorectal cancer as well as colorectal surgery is associated with increased oxidative stress through different mechanisms. In this study the levels of different oxidative stress markers were comparatively assessed in patients who underwent laparoscopic or conventional resection for colorectal cancer. METHODS: Sixty patients with colorectal cancer were randomly assigned to undergo laparoscopic (LS) or open surgery (OS). Lipid, protein, RNA, and nitrogen damage was investigated by measuring serum 8-isoprostanes (8-epiPGF2α), protein carbonyls (PC), 8-hydroxyguanosine (8-OHG), and 3-nitrotyrosine (3-NT), respectively. The primary end point of the study was to analyze and compare serum levels of the oxidative stress markers between the groups. RESULTS: Postoperative serum levels of 8-epiPGF2α, 3-NT, and 8-OHG were significantly lower in the LS group at 24 h after surgery (p < 0.05). At 6 h postoperatively, the levels of 8-epiPGF2α and 3-NT were significantly lower in the LS group (p < 0.05). No difference in the levels of PC was found between the two groups at any time point. In the OS group, postoperative levels of 8-epiPGF2α were significantly lower than the preoperative values (p < 0.01). In the LS group, the postoperative values of 8-epiPGF2α, 3-NT, and 8-OHG were significantly lower than the preoperative values (p < 0.05). CONCLUSION: Laparoscopic surgery for colorectal cancer is associated with lower oxidative stress compared to open surgery. 8-epiPGF2α was the most suitable marker for readily defining the oxidative status in patients who underwent surgery for colorectal cancer.

Levels of Circulating IgM and IgY Natural Antibodies in Broiler Chicks: Association with Genotype and Farming Systems.

Naturally occurring antibodies (NAbs), which are major components of innate immunity, exist in circulation under healthy conditions without prior antigenic stimulation and are able to recognize both self- and non-self-constituents. The present study aimed at identifying potential immunological differences between commercial fast- and slow-growth broilers (n = 555) raised in conventional and free-range systems, respectively, through the use of the specificity, isotypes and levels of circulating NAbs. The possible beneficial effect of oregano-based dietary supplementation was also evaluated. To this end, serum IgM and IgY NAbs against self- (actin and DNA) and non-self- antigens (trinitrophenol and lipopolysaccharide) were measured by ELISA and further correlated with genotype, season and performance. Significantly higher levels of IgM NAbs against all antigens were found in slow-growth compared to fast-growth broilers. IgM NAb levels were also significantly increased in dietarily supplemented slow-growth broilers versus those consuming standard feed. Moreover, significantly elevated levels of anti-DNA IgY NAbs were found in fast-growth compared to slow-growth broilers, whereas the opposite was observed for anti-LPS IgY NAbs. Multivariate linear regression analysis confirmed multiple interactions between NAb levels, genotype, season and performance. Overall, serum NAbs have proven to be valuable innovative immunotools in the poultry industry, efficiently differentiating fast-growing versus slow-growing broilers, and dietary supplementation of plant extracts can enhance natural immunity.

Plasma Lp-PLA(2) mass and apoB-lipoproteins that carry Lp-PLA(2) decrease after sodium.

BACKGROUND: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) ) is a novel cardiovascular risk marker, which is predominantly complexed to apolipoprotein (apo) B-containing lipoproteins in human plasma. As increasing dietary sodium intake may decrease plasma apoB-containing lipoproteins, we tested whether a sodium challenge lowers plasma Lp-PLA(2) mass, as well as the levels of apoB-containing lipoprotein particles carrying Lp-PLA(2) (apoB-Lp-PLA(2) ), employing a newly developed enzyme-linked immunosorbent assay. MATERIALS AND METHODS: In 45 women and 31 men (mean age 44 ± 14 years), plasma Lp-PLA(2) mass (turbidimetric immunoassay), the level of apoB-Lp-PLA(2) , expressed in apoB concentration and lipoproteins were measured in response to a 3-day challenge with 9 g sodium chloride tablets daily. RESULTS: Urinary sodium excretion increased from 165 ± 60 to 321 ± 70 mmol/24 h (P<0.001) after salt loading. Plasma Lp-PLA(2) mass decreased from 618 (493-719) to 588 (465-698) µg/L (P<0.001), and apoB-Lp-PLA(2) decreased from 0.276 (0.200-0.351) to 0.256 (0.189-0.328) g LDL protein/L (P=0.004) in response to the sodium challenge together with decreases in plasma total cholesterol, nonhigh-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B and the total cholesterol/HDL cholesterol ratio (P<0.01 for all). Changes in plasma Lp-PLA(2) mass were correlated positively with changes in total cholesterol, LDL cholesterol and non-HDL cholesterol (r=0.260-0.276, P<0.05 to P<0.02), whereas changes in apoB-Lp-PLA(2) were correlated positively with changes in non-HDL cholesterol and in the total cholesterol/HDL cholesterol ratio (r=0.232-0.385, P<0.05-0.01). CONCLUSION: Both plasma Lp-PLA(2) mass levels and apoB-Lp-PLA(2) decrease in response to a short-term oral sodium challenge.

α-Defensins and hsCRP levels in inflammatory response of standard and laparoendoscopic single-site cholecystectomy.

BACKGROUND: Laparoendoscopic single-site (LESS) surgery is an evolution of laparoscopic surgery aiming at decreasing the patient's parietal trauma associated with abdominal operations. LESS has been found so far to be efficient and have the same good results as the standard four-port laparoscopic cholecystectomy. α-Defensins are antimicrobial peptides of the organism. They are the first cell components against pathogens. Cytokines are also mediators in the response to trauma. The aim of this study was to compare the inflammatory reaction in LESS and four-port laparoscopic cholecystectomy. METHODS: Forty patients with noncomplicated cholelithiasis were randomly assigned into one of two groups. Group A included the patients who would undergo four-port laparoscopic cholecystectomy and group B included the patients who would undergo LESS cholecystectomy. These patients had a BMI < 30, were ASA I or II, and had no previous upper-GI surgery. Blood was taken preoperatively and 6 and 24 h postoperatively. hsCRP (with automated analyzer) and α-defensins (using ELISA) were calculated for each sample. The same postoperative protocol was followed for both groups. Mann-Whitney U test was used to analyze the results. Pain was calculated with a visual analog scale (VAS) for shoulder and abdomen at 6 and 24 h. Hospital stay, nausea, and pain medication needed was noted. RESULTS: The α-defensins value was statistically significantly higher in the 24-h samples (P < 0.001) for LESS cholecystectomy. No statistically significant difference was shown for hsCRP, even though P = 0.05 for the 24-h samples with the values of LESS higher. No LESS was converted to a classical laparoscopic cholecystectomy, and none of the patients of either group needed conversion to open cholecystectomy. Pain was statistically significantly less for the LESS group at the 24-h interval (P < 0.0001). Less medication was needed for LESS patients after the 6th postoperative hour (P = 0.007). CONCLUSION: Higher inflammatory reaction in LESS cholecystectomy could be the result of greater tension on the tissues. More studies are needed to conclude if this has a significant clinical expression.

Toll-like receptors in the inflammatory response during open and laparoscopic colectomy for colorectal cancer.

BACKGROUND: Surgical interventions activate a cascade of reactions that result in an aseptic inflammatory reaction. This inflammatory response initiates the organism's innate immunity. Laparoscopic surgery reduces the trauma, and patients benefit from diminished surgical trauma and maintained immune function. Cytokine levels and C-reactive protein (CRP) are related to the magnitude of surgical trauma and surgical stress. Toll-like receptors (TLRs) 2 and 4 are the first sensor-recognition receptors of the invading pathogens for the innate immune response. This study aimed to compare the inflammatory response and then the stress response during laparoscopic and open colectomy for cancer by calculating TLR-2 and TLR-4 as the first sensor-recognition receptors together with interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and high-sensitivity CRP (hsCRP). METHODS: A total 40 patients with colorectal cancer were randomized in two groups: group A (open colectomy, n = 20) and group B (laparoscopic colectomy, n = 20). An epidural catheter was placed in all patients 1 h preoperatively. Rupivocaine was administered perioperatively and 48 h postoperatively. Blood samples were taken for calculation of IL-6, TNF-α, hsCRP, TLR-2, and TLR-4 preoperatively and 5 min after deflation of pneumoperitoneum (group B) or 5 min after division of the colon (group A), then 6 and 24 h postoperatively. RESULTS: The mean operative time was 115 for group A and 142 min for group B. The mean blood loss was respectively 240 and 105 ml (P < 0.001), and the mean hospital stay was respectively 8 and 5 days (P < 0.05). The IL-6 level was significant higher in group A than in group B at 6 and 24 h postoperatively (P < 0.0001), and the hsCRP level was significant higher in group A than in group B at 24 h postoperatively (P < 0.001). The TNF-α values did not differ between the two groups. The TLR-2 level was significantly higher in group A than in group B at 5 min (P = 0.013) and 24 h (P = 0.007) postoperatively. The TLR-4 level was significant higher in group A than in group B at 5 min postoperatively (P = 0.03). CONCLUSION: The inflammatory response and the resultant stress response are significantly less during laparoscopic colectomy than during open colectomy for colorectal cancer. This is an obvious short-term clinical benefit for the patient, providing tinder for further study to investigate the long-term results of laparoscopic colectomy versus open colectomy for colorectal cancer.

Oxidized phospholipids and lipoprotein-associated phospholipase A2 (Lp-PLA2 ) in atherosclerotic cardiovascular disease: An update.

Inflammation and oxidative stress conditions lead to a variety of oxidative modifications of lipoprotein phospholipids implicated in the occurrence and development of atherosclerotic lesions. Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is established as an independent risk biomarker of atherosclerosis-related cardiovascular disease (ASCVD) and mediates vascular inflammation through the regulation of lipid metabolism in the blood and in atherosclerotic lesions. Lp-PLA2 is associated with low- and high-density lipoproteins and Lipoprotein (a) in human plasma and specifically hydrolyzes oxidized phospholipids involved in oxidative stress modification. Several oxidized phospholipids (OxPLs) subspecies can be detoxified through enzymatic degradation by Lp-PLA2 activation, forming lysophospholipids and oxidized non-esterified fatty acids (OxNEFAs). Lysophospholipids promote the expression of adhesion molecules, stimulate cytokines production (TNF-α, IL-6), and attract macrophages to the arterial intima. The present review article discusses new data on the functional roles of OxPLs and Lp-PLA2 associated with lipoproteins.

Plasma VEGF and Leptin Values in Patients With Gastric Intestinal Metaplasia and Metabolic Syndrome.

Intestinal metaplasia of the stomach (IM) is considered a pre-cancerous lesion and is a potential precursor to adenocarcinoma. Metabolic syndrome (MetS) has been associated with lesions to the gastrointestinal tract such as the risk of developing Barett esophagus. Vascular endothelial growth factor and leptin have been associated with either gastrointestinal tract carcinogenesis or MetS. In this context, this study was designed to analyze plasma levels of VEGF and leptin in patients with IM and MetS. Four groups of 137 participants (a control group and three patient groups, IM, MetS and IM- MetS) were created. Inclusion criteria for the presence of IM were endoscopic findings and histological confirmation, while for MetS the ATP III and IDF guidelines. Levels of plasma vascular endothelial growth factor (VEGF) and leptin (Leptin) were determined. VEGF levels were increased in IM (IM vs Control, p=0,011) and IM-MetS groups (IM-MetS vs Control, p <0.001 and IM-MetS vs MetS, p=0.001). Leptin levels were found to be increased in the MetS group (MetS vs. Control, p <0.001 and MetS vs IM, p <0.001) and in IM-MetS (IM-MetS vs Control, p = 0.002, IM-MetS vs IM, p=0.033). Patients with intestinal metaplasia and metabolic syndrome (I M - Me t S g r o u p) have elevated levels of VEGF, while leptin levels were associated predominantly with MetS and not with IM.

Urine 8-Hydroxyguanine (8-OHG) in Patients Undergoing Surgery for Colorectal Cancer.

PURPOSE: Cellular RNA is less compact than DNA, more easily accessible to ROS and therefore could be more susceptible to oxidative damage. This study was conceived in order to analyze the RNA oxidative damage in the urine of patients undergoing operation for colorectal cancer (CRC), to compare with healthy controls, and correlate with the stage. MATERIALS AND METHODS: The study population was constituted by a group of 147 patients and a group of 128 healthy controls. Urine and blood samples were collected before the colonoscopy in all participants and 24 hours post-operatively for those who underwent surgery. Urine 8-hydroxyguanine (8-OHG) was determined as marker of RNA oxidation, and serum uric acid (UA) as antioxidant marker. RESULTS: Preoperatively, 8-OHG (ng/ml) values of CRC patients were found to be significantly higher than those of controls (p = 0.001). More specifically, stages II/III had significantly higher 8-OHG values (p < 0.001 and p = 0.007) than stages 0/I. Post-operatively, 8-OHG values were similar to controls (p = 0.053). Preoperatively, UA values (mg/dl) were significantly lower (p = 0.001), while postoperatively were similar to controls (p = 0.069). CONCLUSION: Oxidative RNA damage occurs in CRC patients. Stages II/III are associated with higher values of 8-OHG than stages 0/I. 8-OHG could act as a marker for the identification of patients with advanced disease.

Apolipoprotein A-I peptide models as probes to formulate potential inhibitors of the low-density lipoprotein oxidation.

Apolipoprotein A-I (apoA-I), which constitutes the principal protein component of high-density lipoprotein, is responsible for its major antiatherogenic functions. Aiming at contributing to the development of potent inhibitors of low-density lipoprotein (LDL) peptide models of helices 4,6 and 9,10 of apoA-I were designed and synthesized. Specific amino acid substitutions, resulting in transformation of the original helix class A and Y to G according to the Schiffer and Edmundson helical wheel representation, were introduced in order to validate the contribution of these modifications in the inhibitory activity of the synthesized peptide models against the LDL oxidation. The role of Met at positions 112 (helix 4) and 148 (helix 6) as oxidant scavenger was also investigated. The helical characteristics of all the peptide models were studied by CD in membrane-mimicking microenvironments and compared with the original helices.

A highly constrained cyclic (S,S)-CDC- peptide is a potent inhibitor of carotid artery thrombosis in rabbits.

Inhibition of platelet aggregation is indispensable for the treatment of acute arterial thrombotic episodes. We have previously reported the synthesis of a highly constrained cyclic peptide, that incorporates the -CDC- sequence, (S,S) PSRCDCR-NH(2), which potently inhibits aggregation and fibrinogen binding to human platelets in vitro. We have tested the safety and efficacy of the peptide on the electrically induced carotid artery thrombosis experimental rabbit model. The peptide's effects on carotid blood flow, thrombus weight, in vitro and ex vivo platelet aggregation, and bleeding and hemostatic parameters were evaluated. The peptide was administered via the femoral vein. Carotid blood flow was continuously monitored for 90 min after electrical thrombus formation. The peptide, at 12 mg/kg, prevented total artery occlusion and significantly preserved carotid artery's patency compared with placebo and eptifibatide. Furthermore, (S,S) PSRCDCR-NH(2) administration at 12 mg/kg reduced thrombus weight, whereas it inhibited ex vivo ADP, arachidonic acid (AA) and collagen-induced platelet aggregation. Moreover (S,S) PSRCDCR-NH(2) at 12 mg/kg presented significantly higher inhibitory effects on AA and collagen-induced ex vivo platelet aggregation compared to eptifibatide. The peptide at any dose did not affect the coagulation cascade, the bleeding times or the hemostatic response of the animals. Thus highly constrained cyclic peptides like (S,S) PSRCDCR-NH(2) that incorporate the -CDC- motif and fulfil certain conformational criteria represent novel compounds that potently inhibit thrombus formation, ex vivo platelet aggregation and carotid artery occlusion superiorly to other non-RGD peptides, such as YMESRADR, without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Association between PCSK9 Levels and Markers of Inflammation, Oxidative Stress, and Endothelial Dysfunction in a Population of Nondialysis Chronic Kidney Disease Patients.

Proprotein convertase subtilisin/kexin 9 (PCSK9) plays an important role in lipid metabolism while available literature regarding its involvement in the pathogenesis of atherosclerosis and in the expression of genes associated with apoptosis and inflammation is constantly increasing. Patients with chronic kidney disease (CKD) experience disproportionately increased cardiovascular morbidity and mortality due to dyslipidemia, accelerated atherosclerosis, inflammation, oxidative stress, and other risk factors. In the present cross-sectional study, we investigated the possible association of serum PCSK9 levels with markers of inflammation, oxidative stress, and endothelial damage in patients with CKD. Patients and Methods. Ninety-two patients with CKD stages II-ΙV (eGFR CKD-EPI 47.3 ± 25.7 ml/min/1.73 m2, mean age 66 years, 51 men) were included in the study. Plasma PCSK9 levels were correlated with comorbidities (arterial hypertension, diabetes mellitus, and history of cardiovascular disease), renal function indices (eGFR, proteinuria-UPR/24 h), lipid parameters (LDL-cholesterol, HDL-cholesterol, triglycerides, Lp(a), APO-A1, and APO-B), and soluble biomarkers of inflammation, oxidative stress, and endothelial damage (hs-CRP, fibrinogen, 8-epiPGF2a, ox-LDL, IL-6, TNF-α, sICAM-1, and sVCAM-1). Results. The mean plasma value of PCSK9 was 278.1 ng/ml. PCSK9 levels showed direct correlation with serum triglycerides (p = 0.03), Lp(a) (p = 0.01), and sICAM-1 levels (p = 0.03). There was no significant correlation between PCSK9 levels and indices of the renal function, other lipid profile parameters, inflammatory markers, or comorbidities. Multiple regression analysis showed a significant effect of Lp(a) on PCSK9 levels, and for each unit of higher Lp(a), an increase by 3.082 is expected (95% CI: 0.935-5.228, p = 0.006). At the same time, patients receiving statins are expected to have on average 63.8 ng/ml higher PCSK9 values compared to patients not receiving statins (95% CI: 14.6-113.5, p = 0.012). Conclusion. Plasma levels of PCSK9 in nondialysis CKD patients are correlated with endothelial dysfunction and lipid metabolism parameters. Statin intake increases PCSK9 levels significantly in this patient population. PCSK9 levels are not correlated with the severity of kidney disease. Major prospective studies are necessary to investigate the role of PCSK9 in the atherosclerotic cardiovascular outcome in CKD.

Plasma levels of lipoprotein-associated phospholipase A(2) are increased in patients with ß-thalassemia.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an independent cardiovascular risk factor. We investigated the plasma levels of Lp-PLA(2) activity and mass as a function of plasma lipid levels, LDL subclass profile, and oxidative stress in patients with ß-thalassemia. Thirty-five patients with ß-thalassemia major (ß-TM) and 25 patients with ß-thalassemia intermedia (ß-TI) participated in the study. Lp-PLA(2) activity and mass were measured in total plasma, in apolipoprotein (apo)B-depleted plasma (HDL-Lp-PLA(2)), and in LDL subclasses. Lp-PLA(2) activity produced and secreted from peripheral blood monocytes in culture was also determined. Patients with ß-thalassemia are characterized by a predominance of small-dense LDL particles, increased oxidative stress, and very high plasma levels of Lp-PLA(2) mass and activity, despite low LDL-cholesterol levels. A significant positive correlation between plasma Lp-PLA(2) activity or mass and 8-isoprostane (8-epiPGF2a) and ferritin levels as well as intima-media thickness (IMT) values was observed. An increase in secreted and cell-associated Lp-PLA(2) activity from monocytes in culture was observed in both patient groups. The HDL-Lp-PLA(2) activity and mass as well as the ratio of HDL-Lp-PLA(2)/plasma Lp-PLA(2) were significantly higher in both patient groups compared with the control group. In conclusion, patients with ß-thalassemia exhibit high plasma Lp-PLA(2) levels, attributed to increased enzyme secretion from monocytes/macrophages and to the predominance of sdLDL particles in plasma. Plasma Lp-PLA(2) is correlated with carotid IMT, suggesting that this enzyme may be implicated in premature carotid atherosclerosis observed in ß-thalassemia.

The role of lipoprotein-associated phospholipase A2 in atherosclerosis may depend on its lipoprotein carrier in plasma.

Platelet-activating factor (PAF) acetylhydrolase exhibits a Ca(2+)-independent phospholipase A2 activity and degrades PAFas well as oxidized phospholipids (oxPL). Such phospholipids are accumulated in the artery wall and may play key roles in vascular inflammation and atherosclerosis. PAF-acetylhydrolase in plasma is complexed to lipoproteins; thus it is also referred to as lipoprotein-associated phospholipase A2 (Lp-PLA2). Lp-PLA2 is primarily associated with low-density lipoprotein (LDL), whereas a small proportion of circulating enzyme activity is also associated with high-density lipoprotein (HDL). The majority of the LDL-associated Lp-PLA2 (LDL-Lp-PLA2) activity is bound to atherogenic small-dense LDL particles and it is a potential marker of these particles in plasma. The distribution of Lp-PLA2 between LDL and HDL is altered in various types of dyslipidemias. It can be also influenced by the presence of lipoprotein (a) [Lp(a)] when plasma levels of this lipoprotein exceed 30 mg/dl. Several lines of evidence suggest that the role of plasma Lp-PLA2 in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated. In this regard, data from large Caucasian population studies have shown an independent association between the plasma Lp-PLA2 levels (which are mainly influenced by the levels of LDL-Lp-PLA2) and the risk of future cardiovascular events. On the contrary, several lines of evidence suggest that HDL-associated Lp-PLA2 may substantially contribute to the HDL antiatherogenic activities. Recent studies have provided evidence that oxPL are preferentially sequestered on Lp(a) thus subjected to degradation by the Lp(a)-associated Lp-PLA2. These data suggest that Lp(a) may be a potential scavenger of oxPL and provide new insights into the functional role of Lp(a) and the Lp(a)-associated Lp-PLA2 in normal physiology as well as in inflammation and atherosclerosis. The present review is focused on recent advances concerning the Lp-PLA2 structural characteristics, the molecular basis of the enzyme association with distinct lipoprotein subspecies, as well as the potential role of Lp-PLA2 associated with different lipoprotein classes in atherosclerosis and cardiovascular disease.