Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques.

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffinembedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core

Thrombin generation in human coronary arteries after percutaneous transluminal balloon angioplasty

AbstractObjectives. The aim of this study was to investigate the relation between coronary atherosclerotic plaque injury and activation of the coagulation cascade.Background. Thrombus formation after atherosclerotic plaque disruption has been implicated in the pathogenesis of atherosclerosis, unstable angina and myocardial infarction.Methods. Biochemical markers of thrombin generation (prothrombin fragment F1+2) and thrombin activity (fibrinopeptide A) were measured in coronary blood before, during and immediately after percutaneous transluminal coronary angioplasty. After demonstrating that blood withdrawal through an angioplasty catheter does not artifactually elevate the plasma levels of these markers in patients after heparinization, coronary artery samples ware collected proximal and distal to the lesion before and distal to the lesion after baltoon inflation in 26 patients.Results. Plasma levels of F1+2measured proximal to the lesion before angioplasty (median 0.47 nmol/liter, 95% confidence interval [CI] 0.40 to 0.50) were significantly elevated after angioplasty (median 0.55 nmol/liter, 95% CI 0.46 to 0.72, p = 0.001). In contrast, plasma fibrinopeptide A levels measured proximal to the lesion before angioplasty (median 2.0 ng/ml, 95% CI 1.3 to 22) were similar to those measured after angioplasty (median 1.8 ng/ml, 95% CI 1.3 to 3.0, p = NS). After we defined a normal range of interassay variability on the basis of values obtained from samples drawn proximal and distal to the lesion before angioplasty, seven patients (27%) had a significant increase in F1+2plasma levels. A significant increase in plasma fibrinopeptide A occurred in five of these seven patients. Lesions with dissection, filling defects or haziness on postangioplasty angiography were associated with more thrombin generation than lesions without these features.Conclusions. Markers of thrombio generation and activity can be collected safely and assayed accurately in heparinized blood withdrawn through aa angioplasty catheter. Balloon dilation of coronary stenoses increases thrombin generation and activity within the coronary artery in a substantial subgroup of patients undergoing angioplasty

Transcript of The Dory Derby Accident

This story is an excerpt from a longer interview that was collected as part of the Launching through the Surf: The Dory Fleet of Pacific City project. In this story, Don Grotjohn recounts an accident that occurred during a Dory Derby competition

Functional and clinical implications of genetic structure in 1686 Italian exomes

To reconstruct the phenotypical and clinical implications of the Italian genetic structure, we thoroughly analyzed a whole-exome sequencing data set comprised of 1686 healthy Italian individuals. We found six previously unreported variants with remarkable frequency differences between Northern and Southern Italy in the HERC2, OR52R1, ADH1B, and THBS4 genes. We reported 36 clinically relevant variants (submitted as pathogenic, risk factors, or drug response in ClinVar) with significant frequency differences between Italy and Europe. We then explored putatively pathogenic variants in the Italian exome. On average, our Italian individuals carried 16.6 protein-truncating variants (PTVs), with 2.5% of the population having a PTV in one of the 59 American College of Medical Genetics (ACMG) actionable genes. Lastly, we looked for PTVs that are likely to cause Mendelian diseases. We found four heterozygous PTVs in haploinsufficient genes (KAT6A, PTCH1, and STXBP1) and three homozygous PTVs in genes causing recessive diseases (DPYD, FLG, and PYGM). Comparing frequencies from our data set to other public databases, like gnomAD, we showed the importance of population-specific databases for a more accurate assessment of variant pathogenicity. For this reason, we made aggregated frequencies from our data set publicly available as a tool for both clinicians and researchers (http://nigdb.cineca.it; NIG-ExIT)

Predictors, type, and impact of bleeding on the net clinical benefit of long-term ticagrelor in stable patients with prior myocardial infarction

BACKGROUND: Ticagrelor reduces ischemic risk but increases bleeding in patients with prior myocardial infarction. Identification of patients at lower bleeding risk is important in selecting patients who are likely to derive more favorable outcomes versus risk from this strategy. METHODS AND RESULTS: PEGASUS-TIMI 54 (Prevention of Cardiovascular Events in Patients With Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin—Thrombolysis in Myocardial Infarction 54) randomized 21 162 patients with prior myocardial infarction in a 1:1:1 fashion to ticagrelor 60 mg or 90 mg twice daily or placebo, with ticagrelor 60 mg approved for long-term use. TIMI major or minor bleeding was the primary end point for this analysis. Causes of bleeding were categorized by site and etiology, and independent predictors were identified. At 3 years, ticagrelor 60 mg increased the rate of TIMI major or minor bleeding by 2.0% versus placebo (1.4% placebo versus 3.4% ticagrelor). The bleeding excess was driven primarily by spontaneous gastrointestinal bleeds. A history of spontaneous bleeding requiring hospitalization and the presence of anemia were independent predictors of bleeding but not of ischemic risk. Patients with at least 1 risk predictor had 3-fold higher rates of bleeding with ticagre-lor 60 mg versus those who had neither (absolute risk increase, 4.4% versus 1.5%; P=0.01). Patients with neither predictor had a more favorable benefit profile with ticagrelor 60 mg versus placebo including lower mortality (hazard ratio, 0.79; 95% CI, 0.65–0.96; P interaction = 0.03). CONCLUSIONS: In patients with prior myocardial infarction, bleeding with ticagrelor 60 mg twice daily is predominantly spontaneous gastrointestinal. A history of spontaneous bleeding requiring hospitalization or the presence of anemia identifies patients at higher risk of bleeding, and the absence of either identifies patients likely to have a more favorable net benefit with ticagrelor

Bardet-Biedl syndrome with end-stage kidney disease in a four-year-old Romanian boy: a case report

<p>Abstract</p> <p>Background</p> <p>Bardet-Biedl syndrome is a significant genetic cause of chronic kidney disease in children. Kidney abnormalities are a major cause of morbidity and mortality in Bardet-Biedl syndrome, but the onset of end-stage renal disease at an early age and continuous ambulatory peritoneal dialysis, however, are not commonly mentioned in the literature.</p> <p>Case presentation</p> <p>We present the case of a four-year-old Romanian boy who presented to our department with 'febrile seizures'. After an initial evaluation, we diagnosed our patient as having hypertension, severe anemia and end-stage renal disease. He met the major and minor criteria for the diagnosis of Bardet-Biedl syndrome and underwent continuous ambulatory peritoneal dialysis.</p> <p>Conclusions</p> <p>Close follow-up for renal involvement in patients with Bardet-Biedl syndrome and Alström syndrome from an early age is highly recommended to prevent end-stage renal disease and so renal replacement therapy can be started immediately.</p

Microvascular complications identify a specific coronary atherosclerotic phenotype in patients with type 2 diabetes mellitus

Background: Patients with type 2 diabetes mellitus (T2DM) are considered as a homogeneous cohort of patients. However, the specific role of diabetic microvascular complications (DMC), in determining the features of coronary plaques is poorly known. We investigated whether the presence of DMC may identify a different phenotype of patients associated to specific clinical, angiographic, optical coherence tomography (OCT) features and different prognosis. Methods: We prospectively enrolled consecutive T2DM patients with obstructive coronary artery disease (CAD) at their first coronary event. Patients were stratified according to the presence or absence of DMC, including diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. OCT assessment of the culprit vessel was performed in a subgroup of patients. The incidence of major adverse cardiac events (MACEs) was assessed at follow-up. Results: We enrolled 320 T2DM patients (mean age 70.3 ± 8.8&nbsp;years; 234 [73.1%] men, 40% acute coronary syndrome, 60% chronic coronary syndrome). Patients with DMC (172 [53.75%]) presented a different clinical and biochemical profile and, of importance, a higher prevalence of multivessel CAD (109 [63.4%] vs. 68 [45.9%], p = 0.002). At OCT analysis, DMC was associated to a higher prevalence of large calcifications and healed plaques and to a lower prevalence of lipid plaques. Finally, MACEs rate was significantly higher (25 [14.5%] vs. 12 [8.1%], p = 0.007) in DMC patients, mainly driven by a higher rate of planned revascularizations, and DMC predicted the occurrence of MACEs (mean follow-up 33.4 ± 15.6&nbsp;months). Conclusions: The presence of DMC identifies a distinct diabetic population with more severe CAD but with a more stable pattern of coronary atherosclerosis

Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial Nitric Oxide Synthase

Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs) and impaired bioavailabilty of nitric oxide (NO) are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5' nuclease assays (TaqMan™ PCRs) to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA) and 15 with acute coronary syndromes (ACS) without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS) gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001) in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype