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Background: Age-related arterial alterations affecting cells, matrix and biomolecules are the main culprit for initiation and progression of cardiovascular disease. The objective of this study is to gain further insights into the complex mechanism of elastic tissue ageing in human aortic blood vessels. Methods: One hundred and nineteen human aortic tissue samples were collected from adult patients (101 males, 18 females; age 40–86 years) undergoing coronary artery bypass grafting. Overall extracellular matrix architecture was examined by multiphoton laser scanning microscopy and histology. Matrix metalloproteinases 2 and 9, corresponding tissue inhibitors 1 and 2 as well as desmosine were determined. mRNA levels of tropoelastin were assessed by quantitative RT-PCR. Results: Age-related destruction of the vascular elastic laminas as well as a loss of interlamina cross-links were observed by laser scanning microscopy. These results were confirmed by histology indicating increasing interlamina gaps. There were no significant differences in matrix turnover or desmosine content. A steady decrease in tropoelastin mRNA by about 50% per 10 years of age increase was observed. Conclusions: Our findings indicate that ageing is accompanied by a destruction of the elastic vascular structure. However, tropoelastin expression analysis suggests that elastogenesis occurs throughout life with constantly decreasing levels.

(n ‫؍‬ 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/sodA gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus, 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus, 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius, 0/2; Streptococcus lutetiensis, 0/5; Leuconostoc mesenteroides, 4/0; and Lactococcus lactis, 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sodA gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus.

Human Wharton's jelly stem cells (HWJSC) emerged as a potential source of viable cells for use in tissue engineering. In this work, we have analyzed the transdifferentiation capabilities of HWJSC towards transdifferentiated endothelial-like cells (Tr-ELC) in order to establish the potential usefulness of these cells in vascular tissue engineering. Our results show that Tr-ELC became more polygonal and less proliferative than HWJSC, resembling the structure and proliferation rate of the endothelial cells. In addition, the markers of mesenchymal undifferentiation CD9, E-cad, PODXL, and SSEA-4 are downregulated in Tr-ELC, suggesting that these cells can be in the process of adult differentiation. Besides, RT-PCR and microarray analyses revealed that some genes with a role in defining the endothelial phenotype and structure are upregulated (VEGF-R1, EDF1, AAMP, CD31, CD34, CDH5, and ICAM2) or downregulated (VEGF) in Tr-ELC, although a number of genes related to relevant endothelial cell functions (CD36, ECE2, VWF, THBD, PGI2, ECE1, and ACE) did not change or were only partially induced. All this implies that HWJSC are able to efficiently transdifferentiate towards Tr-ELC at the phenotypical level following a hierarchical pattern of gene activation, with an earlier induction of morphological and phenotypical genes.

The development of lower extremity venous insufficiency (VI) during pregnancy has been associated with placental damage. VI is associated with increased oxidative stress in venous wall. We have investigated potential disturbance/dysregulation of the production of reactive oxygen species (ROS) in placenta and its eventual systemic effects through the measurement of malondialdehyde (MDA) plasma levels in women with VI. A total of 62 women with VI and 52 healthy controls (HCs) were studied.Levels of nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), 2 (NOX2), inducible nitric oxide synthase (iNOS), endothelial (eNOS), poly(ADP-ribose) polymerase PARP (PARP) and ERK were measured in placental tissue with immunohistochemistry and RT-qPCR. Plasma and placental levels of MDA were determined by colorimetry at the two study times of 32 weeks of gestation and post-partum. Protein and gene expression levels of NOX1, NOX2, iNOS, PARP and ERK were significantly increased in placentas of VI. eNOS activity was low in both study groups, and there were no significant differences in gene or protein expression levels. Women with VI showed a significant elevation of plasma MDA levels at 32 weeks of gestation, and these levels remained elevated at 32 weeks post-partum. The MDA levels were significantly higher in placentas of women with VI. Placental damage that was found in the women with VI was characterized by overexpression of oxidative stress markers NOX1, NOX2, and iNOS, as well as PARP and ERK. Pregnant women with VI showed

Chronic venous insufficiency (CVI) is a multifactorial disease, commonly caused by valvular incompetence (clinically diagnosed by venous reflux) and venous hypertension. The incidence of these factors clearly increases with patient age, and aging is one of the risk factors involved. The activity of the PI3K/Akt/mTOR pathway is considered fundamental in vascular pathologies, and understanding its involvement would help in the development of possible therapeutic targets. This is an observational, analytical, and prospective cohort study that reviewed 110 patients with CVI scheduled to undergo stratified saphenectomy. They were distributed according to the presence (R = 81) or absence (NR = 29) of valvular incompetence (venous reflux) diagnosed clinically. Each of the groups was further divided according to age, with a cutoff point of 50 years (NR < 50 = 13, NR ≥ 50 = 16, R < 50 = 32, and R ≥ 50 = 49). The involvement of the PI3K/Akt/mTOR pathway, as well as that of HIF-1α and HIF-2α and of CD4+, CD8+, and CD19+ cells and mastocytes, was assessed. Saphenous vein tissue samples obtained during surgery were processed for RT-qPCR and immunohistochemistry. Patients with venous reflux showed a significant increase in mRNA and protein expression levels for PI3K/mTOR and HIF-1α/HIF-2α. The number of mast cells was significantly elevated in the R group. In distribution by age, PI3K/Akt/mTOR and HIF-1α were significantly higher in R < 50 patients. Furthermore, these patients had a significant increase in the number of CD4+, CD8+, and CD19+ cells and mastocytes in the saphenous vein wall. These findings provide a basis for the possible existence of changes in PI3K/Akt/mTOR pathway expression in young patients, with potential accelerated asynchronous aging that is enhanced by CVI.

Lower extremity venous insufficiency (VI) is a complication of pregnancy. The potential association of this venous disease with structural damage of the placenta has not been described. We analyzed the pattern of histopathologic lesions and the gene and protein expression of HIF1-α and apoptosis regulatory proteins. A prospective study was carried out on placenta samples from 43 women with pregnancy-associated VI and 24 age-matched pregnant healthy controls (HCs). Women with VI showed a significant increase in the number of villi (150.77 ± 42.55 VI versus 122.13 ± 27.74 HC) and in syncytial knots compared with those found in the placentas from HCs (67.15 ± 31.08 VI versus 42.49 ± 17.36 HC), and an increase in the number of bridges (32.40 ± 2.67 VI versus 22.73 ± 2.37 HC; P < .05). The mean number of syncytial nodes per villus is 1.37 ± 0.90 in the VI group and 0.49 ± 0.58 in the HC group (P < .001). Significant increases in the expression of Bax and caspase-3 and caspase-9 in the placentas from women with VI were observed compared with those found in HC. The expression of HIF-1α at both the messenger RNA and protein levels was also significantly increased in the placentas from women with VI. Our study demonstrates that placentas from women with pregnancy-associated VI show structural remodeling, with an increase in the number of villi and syncytial knots and enhanced apoptotic cellular death. Interestingly, this placental damage is associated with an increased expression of hypoxia-triggered molecular pathways, such as HIF-1α.

A competitive double antibody radioimmunoassay (RIA) for rat metallothionein (MT) has been developed that has a detection limit of 100 pg and a range of 100 to 100000 pg. The antibody was raised in rabbits against rat MT-2 but it crossreacts equally with MT-1 and MT-2. However, when the assay is done in the presence of 2-mercaptoethanol the antibody is more specific for MT-2. Zn- and Cd- saturated MTs have similar responses in the assay. Addition of Cu(II) to Zn-MT (more than 6 mol Cu/mol MT) in non-reducing conditions modifies the response of the antibody, probably because of Cu(II) oxidation and later MT polymerization. Standard curves developed in the presence of cytosols from brain cortex, hypothalamus or liver did not differ from the standard curve, indicating the absence of interfering substances in the assay. Furthermore, serial dilutions of those cytosols paralleled the response of the standard curve, indicating that the response of the antibody was specific. For comparison, MT levels in some brain areas measured with the present RIA were compared with those measured with an established RIA. In addition, the expected effect of dexamethasone and stress on liver MT levels was clearly identified by this RIA. The results suggest that the present RIA can be used for quantitation of metallothionein.