Phylogenetic analysis and positive-selection site detecting of vascular endothelial growth factor family in vertebrates

Vascular endothelial growth factor (VEGF), known to play an important role in vascular homeostasis, vascular integrity and angiogenesis, is little known about the evolutionary relationship of its five members especially the role of gene duplication and natural selection in the evolution of the VEGF family. In this study, seventy-five full-length cDNA sequences from 33 vertebrate species were extracted from the NCBI's GenBank, UniProt protein database and the Ensembl database. By phylogenetic analyses, we investigated the origin, conservation, and evolution of the VEGFs. Five VEGF family members in vertebrates might be formed by gene duplication. The inferred evolutionary transitions that separate members which belong to different gene clusters correlated with changes in functional properties. Selection analysis and protein structure analysis were combined to explain the relationship of the site-specific evolution in the vertebrate VEGF family. Eleven positive selection sites, one transmembrane region and the active sites were detected in this process.     

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012. © 2012 Wiley Periodicals, Inc.     

Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts

Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.     

Cloning and Tissue Expressional Characterization of a Full-Length cDNA Encoding Human Neuronal Protein P17.3

A full-length cDNA of 595 bp was isolated froma human fetal brain cDNA library. It contains an openreading frame encoding 153 amino acids, with an 18-bp5UTR and a 118-bp 3UTR in which there isan atypicalpolyadenylation signal (ATTAAA). The calculatedmolecular weight of the deduced protein is 17.3 kU. Thepredicted isoelectric point is 4.89. On account of itshigh homology to mouse neuronal protein NP15.6(81.2% identity), the deduced protein was namedneuronal protein 17.3 (NP17.3). When its secondarystructure was examined by the GGBSM program of PCGENEsoftware, it was found that 32.6 and 15.0% of itsamino acids are involved in formingalpha-helices and beta-sheets, respectively. Examinedwith the PESTFIND program, a typical PEST region foundin rapidly degraded proteins was found between residue48and residue 68.     

Ultraviolet A regulates the stemness of human adipose tissue‐derived mesenchymal stem cells through downregulation of the HIF‐1α via activation of PGE2–cAMP signaling

Ultraviolet A (UVA) irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate the effects of UVA irradiation on the stemness properties of human adipose tissue‐derived mesenchymal stem cells (hAMSCs). Furthermore, we examined the UVA‐antagonizing effects of L ‐cysteine ethylester hydrochloride (ethylcysteine) and elucidated its action mechanisms. The results of this study showed that UVA reduced the proliferative potential and stemness of hAMSCs, as evidenced by reduced proliferative activity in the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and downregulation of OCT4, NANOG, and SOX2, stemness‐related genes. The mRNA level of hypoxia‐inducible factor (HIF)‐1α, but not HIF‐2α was reduced by UVA. Moreover, the knockdown of HIF‐1α using small interfering RNA (siRNA) for HIF‐1α was found to downregulate stemness genes, suggesting that UVA reduces the stemness through downregulation of HIF‐1α. In addition, we examined the mechanisms underlying the UVA‐mediated effects and found that UVA induced production of prostaglandin (PG) E2 and 3′‐5′‐cyclic adenosine monophosphate (cAMP), and that this effect was mediated through activation of activating protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB). The UVA effects were antagonized by ethylcysteine, and the effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show for the first time that UVA regulates the stemness of hAMSCs and its effects are mediated by downregulation of HIF‐1α via the activation of PGE₂–cAMP signaling. In addition, ethylcysteine may be used as an antagonizing agent to mitigate the effects of UVA. J. Cell. Biochem. 113: 3681–3691, 2012. © 2012 Wiley Periodicals, Inc.