Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells

Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique.     

Persistence of high-replicative capacity in cultured fibroblasts from nonagenarians

Earlier studies on human fibroblast cultures have demonstrated an inverse relationship between the total number of population doublings (PDs) and donor age. As more recent studies were unable to replicate these findings, we set out to analyze growth characteristics of fibroblast cultures from nonagenarians who represent the extreme of human lifespan. Therefore, we obtained skin biopsies from 68 participants of the Leiden 85-plus Study, all aged 90 years. None of the 68 strains failed to proliferate and all were easily cultured under highly standardized conditions. Within a time window of 30 months, all strains displayed a high and reproducible replicative capacity that was maintained for at least 50 PDs. A decline in mitotic activity was observed between 26 and 81 PDs. Out of the 68 cell strains, 58 strains reached the post-mitotic phase with an onset between 51 and 108 PDs. The growth pattern of each senescent strain was fitted by a piecewise linear model, which allowed calculation of the transition towards the phase of decreased growth speed, as well as by a nonlinear continuous model; goodness-of-fit was high and not different between the models (both > 0.99). Growth characteristics were not associated with morbidity or mortality of the donors. We conclude that fibroblasts from nonagenarians maintain a high-replicative capacity despite a huge variability in the onset of senescence. These results cast further doubt on the association between in vitro growth characteristics of fibroblast cultures and the length of human lifespan.     

Enhanced control of proliferation in telomerized cells

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but “escape” to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Nondividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytize debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.     

Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization

Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.     

CCN2 is necessary for the function of mouse embryonic fibroblasts

CCN2 is expressed by mesenchymal cells undergoing active tissue remodeling, and is characteristically overexpressed in connective tissue pathologies such as fibrosis and cancer. However, the physiological roles and mechanism of action of CCN2 are largely unknown. Here, we probe the contribution of CCN2 to the biology of mouse embryonic fibroblasts (MEFs) using genome-wide mRNA expression profiling, proteomic and functional bioassay analyses. We show that ccn2-/- mouse embryonic fibroblasts (MEFs) have significantly reduced the expression of pro-adhesive, pro-inflammatory and pro-angiogenic genes such as interleukin-6 (IL-6), ceruloplasmin, thrombospondin-1, lipocalin-2 and syndecan 4. Anti-syndecan 4 antibody reduced ERK phosphorylation in ccn2+/+ MEFs. In ccn2+/+ MEFs, the MEK inhibitor U0126 and dominant negative ras reduced expression of IL-6 and lipocalin-2. Overexpressing syndecan 4 in ccn2-/- MEFs restored IL-6 and lipocalin-2 mRNA expression. Syndecan 4 has been shown to mediate cell migration. We found that ccn2+/+ MEFs migrated significantly faster than ccn2-/- MEFs; anti-syndecan 4 antibody and U0126 reduced the migration of ccn2+/+ MEFs to that of ccn2-/- MEFs. These results collectively support the notion that syndecan 4 acts downstream of CCN2 in MEFs, and that reduced syndecan 4 expression contributes to at least part of the ccn2-/- phenotype. Further, these results suggest that CCN2 is required for MEFs to contribute to aspects of tissue remodeling. Consistent with this notion, whereas ccn2+/+ MEFs displayed actin stress fibers and focal adhesions at the cell periphery consistent with a migratory phenotype, ccn2-/- MEFs displayed reduced focal adhesions and actin stress fibers, and a reduced ability to transduce forces across a collagen gel matrix. Collectively, these results suggest that CCN2 supplies essential, non-redundant functions required for fibroblasts to properly participate in features of embryogenesis, and further suggest that CCN2 may play essential roles in adult wound healing, tissue repair and fibrogenesis.     

A Cre-loxP based system for studying horizontal gene transfer

We have previously shown that DNA can be transferred to phagocytosing cells via the uptake of apoptotic cells. We report a model system that facilitates study of antigen presentation of genes transferred specifically via horizontal gene transfer. Constructs were generated encoding the LacZ gene or the influenza A nucleoprotein silenced by a STOP sequence flanked by two loxP sites. These reporter genes were demonstrated to be silent in donor cells and become activated after phagocytosis of Cre-expressing fibroblasts or macrophages. These results provide a model system for studying the influence of horizontally transferred antigens on activation of the immune system.     

Regulation of various proteolytic pathways by insulin and amino acids in human fibroblasts

Intracellular protein degradation is a regulated process with several proteolytic pathways. Although regulation of macroautophagy has been investigated in some detail in hepatocytes and in few other cells, less is known on this regulation in other cells and proteolytic pathways. We show that in human fibroblasts insulin and amino acids reduce protein degradation by different signalling pathways and that this inhibition proceeds in part via the mammalian target of rapamycin, especially with amino acids, which probably increase lysosomal pH. Moreover, the regulatory amino acids (Phe, Arg, Met, Tyr, Trp and Cys) are partially different from other cells. Finally, and in addition to macroautophagy, insulin and amino acids modify, to different extents and sometimes in opposite directions, the activities of other proteolytic pathways.     

Vasoactive intestinal peptide inhibits the activation of murine fibroblasts and expression of interleukin 17 receptor C

Acute respiratory distress syndrome (ARDS) is an acute, severe, and refractory pulmonary inflammation with high morbidity and mortality. Excessive activation of fibroblast during the fibroproliferative phase plays a pivotal role in the prognosis of ARDS. Our previous study demonstrated that the vasoactive intestinal peptide (VIP) is mediated by lentivirus attenuates lipopolysaccharide (LPS)‐induced ARDS in a murine model, and VIP inhibits the release of interleukin‐17A (IL‐17A) from activation macrophages. However, the effects of VIP on the activation of murine fibroblast and expression of IL‐17 receptor (IL‐17R) in ARDS remain unclear. Here, a mouse model of ARDS was established by an intratracheal injection of LPS. We found that the gene expression of col3a1 and hydroxyproline contents in the lungs were significantly increased 24 h after LPS injection. IL‐17RC rather than IL‐17RA was increased in the lungs of mice with ARDS. In vitro, LPS activated NIH3T3 cells, which was suppressed by VIP in a dose‐dependent manner. In detail, VIP reduced the hydroxyproline content and col3a1 messenger RNA induced by LPS in NIH3T3 cells, as well as the expression of α‐smooth muscle actin. Furthermore, we found that VIP inhibited the expression of IL‐17R in the lungs of mice with ARDS and NIH3T3 cells stimulated with LPS, which was partly inhibited by antagonists of protein kinase A and protein kinase C. Taken together, our results demonstrated that VIP inhibited the activation of fibroblast via downregulation of IL‐17RC, which may contribute to the protective effects of VIP against ARDS in mice.