MicroRNA regulation in cardiovascular disease.

The molecular biology dogma that DNA replicates its genetic information within nucleotide sequences and transcribes it to RNA where it codes for the generation of mRNA, failed to consider a significant part of the genetic code. Although it has been generally assumed that most genetic information is executed by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is transcribed into non-coding RNA (ncRNA), many of which are alternatively spliced and/or processed into smaller functional RNA molecules. ncRNAs are predominantly involved in processes that require highly specific nucleic acid recognition, revealing a, so far hidden, layer of internal signals that control various levels of gene expression in developmental and (patho)physiological processes. MicroRNAs (miRNAs) are a large class of evolutionary conserved, small ncRNAs, typically 18 to 24 nucleotides in length, that primarily function at the posttranscriptional level by interacting with the 3' untranslated region (UTR) of specific target mRNAs in a sequence-specific manner. Despite the advances in miRNA discovery, the role of miRNAs in physiological and pathological processes is just rising, revealing their cellular functions in proliferation and differentiation, apoptosis, the stress response and tumorgenesis. MiRNA expression profiling and the manipulation of their expression in cardiac tissue has led to the discovery of regulatory roles for these small ncRNAs during cardiac development and disease, implicating them in regulation of cardiac gene expression. Here we review the basic mechanisms by which cardiovascular miRNAs are regulated in the larger context of cardiogenesis and in cardiovascular disease.

Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation.

OBJECTIVE: Adhesion of monocytes to endothelium can be supported by monocyte-monocyte interactions resulting in the formation of cell aggregates at the vessel wall (clusters). Since platelets that are bound to the injured vessel wall support monocyte adhesion and platelet activation in the circulation leads to formation of platelet-monocyte complexes (PMCs), we examined whether adhesion of PMCs to the vessel wall enhances monocyte clustering. METHODS AND RESULTS: The effect of PMC formation in monocyte adhesion and clustering on human umbilical vein endothelial cells (HUVECs) was studied in vitro with a perfusion system. In the presence of 10% to 20% PMCs, monocyte adhesion and cluster formation to stimulated HUVECs increased 2-fold above levels obtained with pure monocytes. While the observed effects increased with higher PMC levels, blocking-monoclonal antibodies directed against platelet-associated P-selectin or monocyte P-selectin glycoprotein ligand-1 (PSGL-1) reversed adhesion and clustering to control values. In the presence of PMCs, blocking L-selectin decreased adhesion by 25%. When PMCs were present, clustering was only supported by L-selectin at higher shear. These data indicate that monocyte adhesion to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering. These interactions are mainly mediated by P-selectin and PSGL-1. CONCLUSIONS: PMCs in the circulation might be proatherogenic, and prevention of their formation is a possible therapeutic goal.

Occludin expression in microvessels of neoplastic and non-neoplastic human brain.

The tight junction protein occludin 'glues' normal, adjacent brain microvessel endothelial cells together. Malignant brain tumours cause cerebral oedema because they have leaky endothelial tight junctions, which allow plasma fluid to enter the brain from the microvessel lumen. In order to identify molecular abnormalities in tumour endothelial tight junctions, we investigated occludin expression in microvessels from adult human non-neoplastic brain tissue using immunohistochemistry and immunoblotting. The proportions of microvessels immunolabelling for occludin were >2/3 in 5/5 non-neoplastic brain tissue samples, >1/3 in 5/5 low grade (Daumas-Duport I or II) astrocytomas and <1/3 in 5/5 high grade (III or IV) astrocytomas and 6/6 metastatic adenocarcinomas. Six non-neoplastic brain tissue immunoblots gave a 55-kDa occludin band, three low-grade astrocytomas gave 55-kDa and 60-kDa bands, 13 high-grade astrocytomas gave 60-kDa or no band and four adenocarcinomas did not give an occludin band. Expression of 55-kDa occludin inversely correlated with the presence of contrast enhancement on computed tomograms (P < 0.001). Electron microscopy showed open endothelial tight junctions in 0/2 non-neoplastic human brain specimens and 2/2 high-grade astrocytomas. We suggest that loss of 55-kDa occludin expression in human brain tumours may contribute to endothelial tight junction opening. Characterizing the molecular pathology of brain endothelial tight junctions may facilitate the design of novel drugs against cerebral oedema.

Saliva ELISA: a method for the diagnosis of chronic Chagas disease in endemic areas.

An indirect enzyme linked immunosorbent assay (ELISA) was applied to saliva to detect chronic infection by Trypanosoma cruzi in humans. Saliva samples from 114 Chagas' disease chronically infected individuals, characterized by three serological tests and clinical evaluation and from 100 healthy controls were tested for T. cruzi specific IgG antibodies. At dilution of 1 in 2, specific antibodies were detected in saliva samples from 103 of 114 samples from infected patients and 5 of 100 controls (sensitivity 90.4%, specificity 95%). There was no significant correlation between the antibody titre and cardiac or gastrointestinal tract disease. This assay possesses some advantages over other methods as saliva collection is non-invasive, requires no special equipment and whole saliva gave reproducible results. Although serology remains the gold standard for T. cruzi infection, these results suggest that T. cruzi specific salivary antibody detection may provide a screening diagnostic test and contribute to epidemiological studies of chronic trypanosomiasis infection in endemic areas.