Evaluation of plasma PCSK9 concentrations, transcript of LDL receptor, as well as the total number of monocyte LDL receptors in acute coronary syndrome patients.

BACKGROUND: Before our study, there were no data concerning complex evaluation of: plasma PCSK9 concentrations, transcript LDL receptor (LDLR), as well as the total amount of monocytes' LDLR in acute coronary syndrome (ACS) patients. PCSK9 levels in a few cohort studies were found to correlate with the number of white blood cells (WBC) or platelets (PLT). The study aims to evaluate PCSK9-LDLR concentrations, as well as to find any association between PCSK9 and WBC or PLT. METHODS: The study group included 95 consecutive patients with acute myocardial infarction, in whom angiography/angioplasty of the culprit vessel was performed. The control group consisted of 10 healthy young volunteers. Thirty patients from the studied group were qualified for further percutaneous revascularization after 3 months. Laboratory tests were performed using commercially available kits. LDLR expression on monocyte surface was measured by flow cytometry, but the mRNA level for LDLR was established by real time polymerase chain reaction. The PCSK9 plasma concentration was measured by ELISA kits. RESULTS: Higher concentration of PCSK9 and amount of LDLR on monocytes surface were observed in patients with ACS compared with healthy young volunteers (number of LDLRs on monocytes [reaction units] 10.8 ± 9.6 vs. 41.8 ± 11.8, p < 0.001, PCSK9 [ng/mL] 295.4 ± 76.4 vs. 213 ± 63.2, p < 0.001). A similar relationship was observed after application of 3-month intensive lipid-lowering therapy in patients with ACS (n = 30, PCSK9 [ng/mL] 281.1 ± 59.5 vs. 358.5 ± 74.7, p < 0.001, LDLR transcript [reaction units] 0.6 ± 0.32 vs. 1.87 ± 0.24, p < 0.001, number of LDLRs on monocytes [reaction units] 5.9 ± 3.1 vs. 22.3 ± 3.8, p < 0.001). There were no significant differences in levels of PCSK9, LDLR between patients with ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI). There was no relation of the PCSK9 with WBC as well as with PLT. CONCLUSIONS: We observed significantly higher concentration of PCSK9, and significantly higher levels of mRNA LDLR transcript in patients with ACS compared with healthy young volunteers. A similar pattern was observed after 3 months of intensive statin therapy among patients with ACS. There were no differences in these parameters between patients with STEMI vs. NSTEMI. The results of the study require confirmation in a larger population of patients.

Highly upregulated expression of CD36 and MSR1 in circulating monocytes of patients with acute coronary syndromes.

Acute Coronary Syndromes (ACS) are a group of disorders caused by the significant reduction of circulation in coronary arteries. The most common reason of the dysfunction is a blood clot formed in place of plaque rupture. The role of scavenger receptors in development and progression of atherosclerosis has been confirmed in many animal experiments, however the knowledge about contribution of the receptors in the development of ACS symptoms in humans still remains insufficient. The aim of this work was to define the expression of two scavenger receptors: CD36 and MSR1 in monocytes of patients with ACS after the onset of symptoms and after the 6 months of treatment. The analysis of CD36 and MSR1 expression was carried out with the use of real-time PCR and flow cytometry. Analyses of lipid and glucose concentration in blood and the level of inflammatory markers in plasma were performed additionally for all ACS patients. All data obtained during the research were analyzed using statistical tests, such as Mann Whitney test, Wilcoxon test, or correlation. In all patients with symptoms of ACS the amount of CD36 and MSR1 mRNA in circulating monocytes, as well as the density of both receptors on the cells surface was significantly higher. Re-analysis of subjects after 6 months of treatment, showed a significant decrease in the CD36 and MSR1 expression in all patients who received atorvastatin. The results of presented studies demonstrate that both investigated receptors are involved in the development and/or progression of ACS.

Proprotein convertase subtilisin/kexin type 9: a new target molecule for gene therapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel target for controlling plasma levels of low-density lipoprotein cholesterol (LDL-C) and decreasing the risk of cardiovascular diseases. At present it is clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels and fail to protect a significant percentage of patients from cardiovascular events. Therefore development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through inhibition of PCSK9 expression and protease activity is a highly desirable approach to overcome hypercholesterolemia. Since there are several agents which are being evaluated in human preclinical and clinical trials, this review summarizes current therapeutic strategies targeting PCSK9, including specific antibodies, antisense oligonucleotides, small interfering RNAs (siRNAs) and other small-molecule inhibitors.

Expression of 5S rDNA in the oocytes of water frogs.

BACKGROUND: We report the expression pattern of 5S rDNA in the eggs of water frogs Rana lessonae, Rana ridibunda and Rana esculenta using the quantitative real-time PCR. This kind of research had never been performed before. RESULTS: 5S rDNA relative expression of the Rana ridibunda oocytes is approximately six times higher in comparison to the Rana lessonae oocytes. The oocytes of the investigated Rana esculenta frogs, in respect of 5S rDNA relative expression ratio, were very similar to the Rana ridibunda oocytes. CONCLUSION: We suggest the possibility of using 5S rDNA as the internal control gene, in the studies of relative mRNA quantitative assays in water frog oocytes, because of its characteristic specific expression pattern in the Rana lessonae, Rana ridibunda and Rana esculenta oocytes.

The effect of atorvastatin treatment on lipid profile and adhesion molecule levels in hypercholesterolemic patients: relation to low-density lipoprotein receptor gene polymorphism.

Inflammation has been indicated to play a major role in the development of atherosclerosis. The beneficial effect of statins has been suggested to be related to their anti-inflammatory properties. We have studied plasma levels of soluble adhesion molecules in patients with hypercholesterolemia before and after 3 months of treatment with atorvastatin and evaluated possible relations to the mutations in low-density lipoprotein receptor (LDLR) gene. In patients with no LDLR gene polymorphism (group A), lower baseline levels of total cholesterol and LDL cholesterol were found than in patients with LDLR gene polymorphism (group B). The soluble adhesion molecules sICAM-1, sE-selectin and sP-selectin, but not sVCAM-1 and sL-selectin, were higher in group B than in group A. sICAM-1 levels decreased in group A by 7% (p = 0.007) and in group B by 21% (p = 0.039), whereas levels of sVCAM-1 decreased in group A by 12% (p = 0.001) and in group B patients by 19% (p = 0.039). Atorvastatin did not change sE-selectin nor sP-selectin levels in group A. However, in group B, the treatment reduced E-selectin and sP-selectin levels by 39% (p = 0.007) and 24% (p = 0.007), respectively. Atorvastatin attenuates the inflammatory reaction in hypercholesterolemic patients, but in patients with LDLR gene polymorphism, this effect is more profound.

The evaluation of ghrelin mRNA expression in human somatotroph adenomas.

Ghrelin is one of the peptides involved into GH-release, binding to specific GHS receptors on hypothalamus and pituitary. The ghrelin peptide and ghrelin mRNA have been detected in several regions of hypothalamus, in normal pituitary, as well as in various types of pituitary adenoma, with different levels of expression in different tumour types. We decided to determine the expression of ghrelin in somatotroph adenomas. Human pituitary somatotroph adenoma tissues were obtained at the time of transsphenoidal surgery from 3 acromegalic patients and studied for ghrelin mRNA expression. Before surgery each patient received a somatostatin analogue treatment at doses 20 mg, 30 mg, 30 mg at 30 days intervals. 20 mg of each tissue sample was used for the isolation of total cellular RNA. The reverse transcription and real-time PCR were performed according to Korbonits et al. method. The reverse transcription of total RNA to cDNA was performed using Super Script TM Rnase H RT kit according to manufacturer protocol. We wished to determine the number of copies of ghrelin gene within the single cell. We used the beta-actin, and the GAPDH genes as a reference molecules for standard curve calculation. Ghrelin mRNA was not detected in any examined tissues. We postulate that the absence of the ghrelin gene transcript is mainly due to the treatment with somatostatin analogues administered preoperatively, which could have suppressed the ghrelin gene transcription.

Monogenic hypercholesterolaemias--an evaluation of apolipoprotein B100 and LDL receptor gene polymorphisms.

INTRODUCTION: Normally the N-terminal domain of the LDL receptor (LDLR) binds to low-density lipoprotein through apolipoprotein B100. Mutations within the LDL receptor and/or apolipoprotein B-100 genes compromising this process may lead to congenital monogenic hypercholesterolaemias known as familial hypercholesterolaemia or familial defective apolipoprotein B-100. These diseases are inherited as autosomal dominant traits. AIM: To search for LDLR and apoB100 gene mutations in a Polish population of patients with hypercholesterolaemia and to determine their types and locations. An attempt was also made to evaluate the influence of identified gene mutations on the modification of protein product sequence and the severity of its functional impairment. METHODS: LDLR and apoB100 gene analyses using PCR, SSCP and automated sequencing techniques were performed in 190 hypercholesterolaemic patients. Flow cytometry was used to measure the influence of Cys152Trp mutation in the LDLR gene on ligand binding and internalisation. The OLA method was used for the preparation of adequate genetic markers allowing rapid detection of one of the most deleterious mutations of the apoB100 gene. RESULTS AND CONCLUSIONS: Three brand new mutations, not reported so far, have been detected--Pro2712Leu and Ile3532 in the apoB100 gene, and Cys347Ser in the LDLR gene--and numerous changes in the nucleotide sequence of the LDL receptor gene have been confirmed. The observations of functionality of the mutated receptor gene with flow cytometry suggested the dysfunction of LDLR due to Cys152Trp polymorphism reported in many studies.