Percentage Of Senescent Cells Research Articles

Influence of RNA interference-induced repression of p53 expression on senescence in human skin fibroblasts

Objective To establish a cell line with repressed expression of p53 by transfecting a plasmid construct expressing short hairpin RNA(shRNA)targeting p53 into human skin fibroblasts(HSFs),and to evaluate the effect of repression of p53 expression on the senescence in HSFs.Methods The eukaryotic expressing plasmid pGCsi-p53 containing shRNA targeting p53 gene was transfected into HSFs with lipofectamine.Subsequently,the cells were selected by G418,and resistant cell clones were chosen and expanded.Reverse transcription-PCR and real time fluorescence-based quanitative PCR were performed to determine the expression of p53 gene,and Western blot to detect the expression of p53 protein in HSFs.The senescence in HSFs was evaluated by SA β-gal staining,and cell proliferation by methyl thiazolyl tetrazolium(MTT)assay.Results A HSF clone with repressed expression of p53 was established successfully.The expressions of p53 mRNA and protein were downregulated in transfected HSFs compared with untransfected HSFs(0.09 ± 0.03 vs.0.32 ± 0.04,0.11 ± 0.04 vs.0.84 ± 0.05,both P ＜ 0.01).The percentage of senescent cells was 13.47％ ± 1.01％ in the transfected HSFs,significantly lower than that in untransfected HSFs(18.10％ ± 0.66％,P ＜ 0.05).As MTT assay showed,the proliferation was accelerated in transfected HSFs compared with untransfected HSFs(P ＜ 0.05).Conclusions The repression of p53 expression decelerates the senescence in HSFs,but promotes the proliferation of HSFs. Key words: Fibroblasts; Cell aging; Genes,p53; RNA interference

Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just as important for causing cellular senescence, cannot be measured properly using these methods. Stress-induced telomere shortening caused by, e.g. oxidative damage happens in a stochastic manner leaving just a few single telomeres critically short. It is now possible to visualize these few ultra-short telomeres due to the advantages of the newly developed Universal single telomere length assay (STELA), and we therefore believe that this method should be considered the method of choice when measuring the length of telomeres after exposure to oxidative stress. In order to test our hypothesis, cultured human mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells estimated by senescence-associated β-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage of senescent cells was absent. Based on the findings in the present study, it seems reasonable to conclude that Universal STELA is superior to TRF in detecting telomere damage caused by exposure to oxidative stress. The choice of method should therefore be considered carefully in studies examining stress-related telomere shortening as well as in the emerging field of lifestyle studies involving telomere length measurements.

Abstract 3059: CCL2 overexpression in an epithelial ovarian cancer cell line results in reduced tumorigenic potential

Abstract Chromosome 17 abnormalities are frequently observed in epithelial ovarian cancer (EOC), suggesting that one or more genes mapping to this chromosome contribute to EOC development and/or progression. We have previously performed a comparative transcriptome analysis of primary cultures of normal ovarian surface epithelial (NOSE) cells and malignant, serous subtype ovarian tumor (TOV) samples in order to identify differentially expressed chromosome 17 genes. CCL2, at 17q12, was significantly differentially underexpressed at least 3-fold in all TOV samples. We have confirmed underexpression in an independent series of 99 serous EOC tumor samples. Interestingly, CCL2 was identified as a significantly up-regulated gene occurring as a consequence of tumor suppression in the characterization of a novel EOC cell line model developed by transferring chromosome 3 fragments into the tumorigenic OV-90 EOC cell line. We have further characterized CCL2 expression in a series of EOC cell lines that differ in their tumorigenic potential, and have shown the lowest levels of gene expression occur in tumorigenic cell lines. DNA sequencing did not reveal any evidence of mutations. To determine if overexpression affects tumorigenicity or growth characteristics, we have generated stable overexpressing OV-90 clones using a commercially available pDream: CCL2 expression vector. Overexpression was verified by ELISA. In cell culture, the CCL2 overexpressing clones appeared less tightly packed and more elongated as compared with OV-90 cells. The cell viability of the CCL2 clones appears to be reduced when compared to the parental OV-90 cell line in XTT cell proliferation assays. The ability of the CCL2 clones to form colonies was also reduced as compared to OV-90 in a colony formation assay. There is also evidence of an increase in the percentage of senescent cells in the CCL2 clones based on a beta-galactosidase staining assay. Both CCL2 overexpressing clones exhibit latency in tumor formation in intraperitoneal tumorigenicity assays involving SCID mice as compared with the parental OV-90 cell line. This is the first report of stable CCL2 transfection into an EOC cell line. Our results suggest that CCL2 expression may reduce the tumorigenic potential of EOC cells, and draws attention to the role of the innate immune system and the tumor microenvironment in EOC development and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3059. doi:10.1158/1538-7445.AM2011-3059

Mesenchymal Stem Cells Derived from Rat Epicardial Versus Epididymal Adipose Tissue

Objective(s) Some investigation has indicated that adipose-derived stem cells possess different surface epitopes and differentiation potential according to the localization of fat pad from which the cells were derived. In the present study proliferation capacity and aging of such cells were explored. Materials and Methods Adherent cells were isolated from the collagenase digests of adipose tissues excised from rat epicardial and epididymal regions and propagated with several subcultures. The cells were then investigated whether or not they were able to differentiate into bone, cartilage and adipose cell lineages. Studied cells from two adipose tissues were also compared with respect to their in vitro proliferation capacity. The presence of senescent cells in the culture was determined and compared using senescence-associated (SA) s-galactosidase staining method. Results Successful differentiations of the cells were indicative of their mesenchymal stem cells (MSCs) identity. Epicardial adipose-derived cells tended to have a short population doubling time (45±9.6 hr) than the epididymal adipose-derived stem cells (69±16 hr, P< 0.05). Colonogenic activity and the growth curve characteristics were all better in the culture of stem cells derived from epicardial compared to epididymal adipose tissue. Comparatively more percentage of senescent cells was present at the cultures derived from epididymal adipose tissue (P< 0.05). Conclusion Our data emphasize on the differences existed between the stem cells derived from adipose depots of different anatomical sites in terms of their proliferative capacity and in vitro aging. Such data can help understand varying results reported by different laboratories involved in adipose stem cell investigations.

Birth weight and characteristics of endothelial and smooth muscle cell cultures from human umbilical cord vessels

BackgroundLow birth weight has been related to an increased risk for developing high blood pressure in adult life. The molecular and cellular analysis of umbilical cord artery and vein may provide information about the early vascular characteristics of an individual. We have assessed several phenotype characteristics of the four vascular cell types derived from human umbilical cords of newborns with different birth weight. Further follow-up studies could show the association of those vascular properties with infancy and adulthood blood pressure.MethodsEndothelial and smooth muscle cell cultures were obtained from umbilical cords from two groups of newborns of birth weight less than 2.8 kg or higher than 3.5 kg. The expression of specific endothelial cell markers (von Willebrand factor, CD31, and the binding and internalization of acetylated low-density lipoprotein) and the smooth muscle cell specific α-actin have been evaluated. Cell culture viability, proliferation kinetic, growth fraction (expression of Ki67) and percentage of senescent cells (detection of β-galactosidase activity at pH 6.0) have been determined. Endothelial cell projection area was determined by morphometric analysis of cell cultures after CD31 immunodetection.ResultsThe highest variation was found in cell density at the confluence of endothelial cell cultures derived from umbilical cord arteries (66,789 ± 5,093 cells/cm2 vs. 45,630 ± 11,927 cells/cm2, p < 0.05). Morphometric analysis indicated that the projection area of the artery endothelial cells (1,161 ± 198 and 1,544 ± 472 μm2, p < 0.05), but not those derived from the vein from individuals with a birth weight lower than 2.8 kg was lower than that of cells from individuals with a birth weight higher than 3.5 kg.ConclusionThe analysis of umbilical cord artery endothelial cells, which demonstrated differences in cell size related to birth weight, can provide hints about the cellular and molecular links between lower birth weight and increased adult high blood pressure risk.

Age-Related Differences Between Unrestricted Somatic Stem Cells from Cord Blood and Bone Marrow Derived Mesenchymal Stroma Cells.

During the past years haematopoietic stem cells from unrelated umbilical cord blood have been increasingly used for treatment of leukaemia and genetic diseases. Human cord blood also contains a non-haematopoietic, adherently growing, CD45 negative, Oct4, nanog and Sox2 negative cell population with intrinsic multipotent differentiation potential, described as Unrestricted Somatic Stem Cells (USSC) (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Liedtke et al. 2007, Liedtke et al.2008, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). USSC can be cultivated GMP-grade reaching 1×109 cells in Passage 4–5 (correlating to 26,5 – 28 cumulative population doublings) without losing multipotency. To better understand and characterise USSC, we carried out in vitro studies concerning age-related changes in USSC-lines from human cord blood and in mesenchymal stroma cells derived from human bone marrow (BM-MSC). Telomerase activity and telomeres are involved in cell proliferation as well as the regulation of cell senescence (Lansdorp 2008). In this study 7 USSC-lines and 9 BM-MSC were analyzed for proliferation and senescence at different population doublings (PD). In vitro USSC have a higher proliferation capacity and accordingly a comparably more extended lifespan than BM-MSC. They undergo about 35 to 45 CPD whereas mean level of CPD from BM-MSC reaches 25. Telomere length of 12 USSC, 5 BM-MSC and 5 clonal populations of USSC were calculated after several PD and telomerase activity was measured. Mean terminal restriction fragment's (TRF's) length calculated from USSC after 31 CPD averages 9.7 kbp whereas mean telomere length from BM-MSC decreases already after 20 CPD to 7.9 kbp. After 47 CPD in clonal USSC populations mean TRF's length is 6.1 kbp. Telomerase activity was analysed with RT-PCR and real time PCR. In contrast to previous publications telomerase activity was detected neither in BM-MSC (Parsch et al.) 2003 nor in USSCs (Manca et al. 2008). The percentage of senescent cells after the same number of CPD is significantly higher in BM-MSC than in USSC. About 80% of senescence was observed in BM-MSC but only 10% senescence in USSCs after 26 CPD. Wnt signalling is mandatory for self-renewal, cell proliferation and differentiation of haematopoietic stem cells (Reya et al. 2003). In primitive MSC populations Wnt signalling regulates mesenchymal lineage specification (Etheridge et al. 2004). For analysing the role of Wnt pathway in USSC development quantitative PCR Arrays have been carried out profiling the expression of 84 genes related to Wnt-mediated signal transduction. 8 different USSC populations have been analysed. In all USSCs factors essential for canonical and also non-canonical Wnt-signalling are present. Poor proliferating USSC with high adipogenic differentiation potential show a stronger expression of Wnt-signalling inhibitors like SFRP1, DKK1 and CXXC4. Based on age-related characteristics, USSC from cord blood are a much better source for regeneration compared to their adult MSC counterpart from bone marrow.

In vitro senescence occurring in normal human endothelial cells can be rescued by ectopic telomerase activity

Telomerase activation is a means to delay in vitro replicative senescence in human cells via telomere maintainence; however, this enzymatic activity is virtually absent in almost all normal somatic cells. As a result, cell senesce, leading to an eventual loss of graft function. Aging allografts, either due to cell injury related to transplantation and/or the use of organs from older donors, pose a threat to the long-term survival of a graft as constitutive cells of an aging organ have a much reduced ability to thrive after transplantation. In our study, human endothelial cells were found to undergo replicative senescence in culture with an increase in the percentage of senescent cells (β-gal staining at pH 6) and a decrease in both the fraction of S-phase cycling cells and the proliferative index measured using CFDA-SE dye. Aging endothelial cells also demonstrated slow rates of proliferation and migration compared to younger cells. Unlike control cells that were transfected with an irrelevant gene vector, telomerase-transfected endothelial cells recovered rapidly after media replacement in cultures that had been serum starved for 2 weeks. Telomerase-transfected cells also retained a high proliferative index comparable to young cells as opposed to untransfected control cells. This young phenotype provided by telomerase expression through restoration of the telomeres may help to increase the longevity of organ transplants.

2P-0473 Effect of anthocyanins from chokeberry (Aronia melanocarpa) on blood pressure, inflammatory mediators and cell adhesion molecules in patients with a history of myocardial infarction (MI)

Endothelial progenitor cells (EPC) may provide protection against atherosclerosis and plaque rupture by their innate ability to replace dysfunctional or damaged endothelial cells in plaque microvessels. There is evidence that angiotensin II may impair the angiogenic functions of EPCs in the atherosclerotic plaque by accelerating senescence and inhibiting their proliferation through oxidative stress induction.In this study, we examined whether chokeberry (Aronia melanocarpa) fruit extract, containing mainly anthocyanins with potent antioxidative properties, could protect EPCs against angiotensin-induced oxidative stress.EPCs were isolated from peripheral blood of young healthy volunteers and cultivated on fibronectin-coated plates in the presence or absence of angiotensin II (1 µM) and chokeberry extract (1–25 µg/ml).EPCs exposed to chokeberry extract prior to angiotensin II showed a significant increase of proliferation and telomerase activity, and a decrease in the percentage of senescent cells and intracellular ROS formation in comparison to angiotensin II treated cells. Furthermore, extract increased migration ability, adhesion to fibronectin and the angiogenic potential of EPC in vitro diminished by angiotensin II in a concentration-dependent manner. That effect was related to the activation of the Nrf2 transcription factor and the increase of HO-1 expression.Our results suggested that chokeberry extract may protect EPCs against angiotensin II-induced dysfunction and could play a potential role in the prevention of coronary artery disease.