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Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival in vitro. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation.

Induced Pluripotent Stem Cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Fibroblast (FB) is the adult somatic cell most commonly used for reprogramming and is defined by spindle-shape morphology, plastic-adherence and expression of several markers. Nonetheless, such properties also define Mesenchymal Stem Cells (MSCs), that also share with dermal FB the ability to differentiate into osteoblasts, adipocytes and chondroblasts. Moving from the hypothesis that the most striking difference between FBs and MSCs is in the name, we isolated and cultured human dermal and visceral FBs from tissue waste fragments of patients undergoing surgical procedures, and evaluated their morphology and expression of markers typical of MSCs, at gene and protein level. Additionally, we analyzed synthesis and release of specific growth factors in culture medium and, finally, we tested FB ability to differentiate into osteoblasts, adipocytes and chondroblasts. Interestingly, all FBs in culture adhered to plastic culture dishes, expressed markers typical of MSCs, like CD90, CD105, CD146,SSEA-4, ECM2, ID-1, and were morphologically undistinguishable. As reported for MSCs, FBs released EGF, FGF-4, GDNF, HGF, IGF, TGF-b and VEGF, and were capable of differentiating into cells of mesodermal origin. Our findings raise the question of whether FBs and MSCs are not admittedly the same population.We might infer that previously described differences are due to different stages of differentiation, which also account for reports on high variability in both expression of mesenchymal markers by MSCs and efficiency of FB reprogramming to iPSCs.

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to 40x28x6mm, to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation.

The essential requirement for a successful myocardial tissue engineering is that scaffolds and tissue constructs exhibit biomimetic activity toward cells. Our aim was to investigate the significance of cell-matrix interactions in cardiac tissue microenvironment in vitro. Cardiac fibroblasts from normal (CF-N) and pathological (CF-P) hearts with ischemic cardiopathy deposited extracellular matrix (Mx) and conditioned medium (Cm), which were then characterized by immunohistology, immunoblotting, ELISA-based protein array or mass spectrometry. Normal and pathological cardiac primitive cells CPC-N and -P) were cultured on CF-Mx-N and -P or in CF-Cm-N and -P and their proliferation, apoptosis, migration and maturation were evaluated by BrdU, TdT, scratch wound assays or RT-PCR, respectively. Laminin-1 and tenascin-X were detected in CF-Mx-P. The expression of IGFBP1, Il-6, EGF was higher in CF-Cm-P, while that of SCF, TGF-?3, BMP4 was higher in CF-Cm-N. Migration speed of CPC-P peaked at 21.3 ?m/h on CF-Mx-N. Proliferation of CPCs increased significantly on CF-Mx-P, while apoptosis diminished on both types of matrix. In CPC-N, maturation toward cardiomyocytes was activated by CF Mx-N and toward endothelial cells by CF-Mx-P; in CPC-P the trend toward smooth muscle cell-lineage was evident on both substrates. CF-Cm-N reduced apoptosis of CPC-N (2-fold), while CF-Cm-P reduced apoptosis of CPC-P (3.5-fold) and increased proliferation of CPC-N (1.6-fold, n=3, p

The genetic variation at the Apolipoprotein E locus (APOE) is an important determinant of plasma lipids and has been implicated in various human pathological conditions. The objective of the present study was to estimate the distribution of APOE alleles in five Venezuelan communities: two Amerindian tribes (Bari and Yucpa), one Negroid population from Curiepe, one Caucasoid population from Colonia Tovar and the mestizo urban population living in Caracas. The APOE*3 allele was the most common allele in all populations studied. However, a significant increase in the APOE*2 allele frequency in the Mestizo (18.96%) and Negroid (16.25%) populations was found. Similar to results reported in other Native American populations we have found that the APOE*2 allele is completely absent in the Bari and Yucpa Amerindians. Frequencies found in the Colonia Tovar population are in agreement with those reported in the population of Germany, indicating a high degree of relatedness. The results support the notion that the distribution of the APOE alleles shows ethnic variability.

none 9 none Guerra G., Zuccolo E., Tafuri D., Paccone M., Riboni L., De Luca A., Montagnani S., D’Angelo E., Moccia F. Guerra, G.; Zuccolo, E.; Tafuri, D.; Paccone, M.; Riboni, L.; De Luca, A.; Montagnani, S.; D’Angelo, E.; Moccia, F.

The anomalies of the basilar artery are rare when compared with those ones pertaining to the circle of Willis vessels. Partial duplication or fenestration is rather common (0.6% to 1.8% in the angiographic descriptions); on the other hand, other anomalies, including complete duplication, hypoplasia and aplasia are exceptional. A rare case of segmental hypoplasia of the basilar artery in a 49-year-old man with transient vertebrobasilar (VB) ischemia, explored by magnetic resonance imaging (MRI) and digital angiography (DA), is reported. The embryology, the clinical relevance and the magnetic resonance findings of this arterial anomaly are discussed, with a review of other six reported cases. The appearance of a segmental aplasia was suggested in our case by MRI, and successively confirmed not only by time-of-flight MR-angiography (TOF-MRA) but also by DA. Only ultrathin-slice T2-weighted (w) images revealed the real finding of basilar artery (BA) hypoplasia; this sequence, not employed in previously reported cases, is mandatory to allowing a clear differential diagnosis between BA aplasia and hypoplasia. In conclusion, segmental hypo-aplasia of the BA is an exceptional embryological anomaly. This anomaly may be of clinical importance, and it should be considered among the potential causes of vertebrobasilar insufficiency in young adults. These cases should be investigated by MR and MRA; we stress the importance of ultrathin slice T2-weighted sequences in order to discriminate between aplasia and hypoplasia., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Summary Background Genes linked to the major histocompatibility complex (MHC), have been implicated in atopic asthma. Asthma is highly prevalent in the Venezuelan population (estimated at 20%) and genetic markers are needed to identify populations at risk and plan intervention strategies. Objective To study the influence of the MHC class I and class II genes in the susceptibility to atopic asthma. Methods MHC-class I HLA-A, -C, -B and MHC-class II HLA-DR, -DQ, -DP gene haplotype frequencies were determined in 135 Venezuelan mestizos, 71 belong to 20 atopic asthmatic families and 64 unrelated controls. The index cases were 20 atopic asthmatics with positive skin-prick tests and specific serum immunoglobulin E (IgE) for Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f). To ascertain the genes associated with susceptibility to atopy and/or asthma, two control groups were studied, 41 non-atopic subjects with skin-prick negative test, and undetectable levels of specific IgE and 23 non-asthmatic atopic subjects with detectable specific IgE to Der p and Der f. A linkage analysis was performed in those families with two or more atopic siblings (with or without asthma). Results MHC-class I genes analysis showed that HLA-Cw7 was absent in the asthmatic patients studied, whereas the frequency of this allele was 14.3% in non-atopic controls (P o 0.017, PCo 0.19) and 20.8% in the atopic controls (P = 0.0066, PCo 0.07). MHCclass II gene analysis showed a significant increase of the HLA-DRB1*11 in the asthmatic patients compared with non-atopic controls (allele frequencies of 25.6 vs 4.4% P o 0.0017, PCo 0.02). There were no significant differences among asthmatic and atopic controls in the frequency of HLA-DRB1*11 (25.6 vs 17.4%). In contrast, the HLA-DRB1*1101˛ haplotypes were significantly higher in asthmatics compared with atopic and non-atopic controls (19.6% vs 2.2% vs 2.3%, PC< 0.05). The HLA-DRB1*1101, DQA1*0501, DQB1*0301 haplotype was found significantly increased in the patients vs non-atopic controls (15.4 vs 1.1%, PC< 0.01). The serum levels of specific IgE were detectable in both atopic asthmatics and atopic controls; however, it was higher in atopic asthmatics vs atopic controls Der p (median, 58.7 vs 2.7 kU/L, P< 0.001) and Der f (median, 46.9 vs 2.7 kU/L

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According to recent findings, pathological processes may deplete adult heart of stem cells. The scope of the present study was to assess proliferation, apoptosis and commitment of cardiac stem cells (CSCs) resident in normal (n=11) and pathological hearts with ischemic cardiomyopathy (n=20). Immunofluorescence and immunoblotting of CSCs isolated from normal and pathological hearts revealed higher expression of cardiac-specific markers in the latter. Proliferation of CSCs isolated from pathological hearts was 3.2±0.7-fold higher, while apoptosis induced by oxidative stress was 1.8±0.2-fold higher than in the normal cells. Asymmetric division, with unequal Notch distribution between daughter cells, occurred frequently in CSCs from ischemic hearts, while symmetric division was typical of normal cells. Gene expression was examined by stem cells specific PCR-based microarray. We confined our analysis to genes with at least 1.7-fold differential expression. Genes downregulated in pathological hearts belonged to the early stages of developmental process (snail1 homolog, jagged-1, ephrin b1), cell cycle control (mdm2, p15 and p16), neurogenesis, skeletal development, bone remodeling and cartilage development functional classes. Upregulated genes, among which OTF2, endothelin receptor, CD105 and MRG1, were involved in developmental maturation, mesenchymal cell differentiation, heart development and circulatory system processes. Major finding emerging from the analysis was the activation of the epithelial-mesenchymal transition and the upregulation of its inducer, TGFβ. The expression of genes related to TGFβ BMP-mediated signal transduction pathway was examined by PCR Array. The analysis confirmed the upregulation of genes as TGFβ-1 and -3, Nodal, ID1 and c-Myc, suggesting the activation of mesenchyme formation and CSCs differentiation towards cardiac cell lineages. In conclusion, CSCs in the normal and in the pathological heart differ in several respects and the differences reflect the activation of cardiac stem cells pool in the chronic pathological conditions. Given the apparent failure of intrinsic heart regeneration, further studies are warranted to optimize the strategies for CSCs application in regenerative medicine.