Research Article
Cell Biology International 10.1002/cbin.10999
Endothelial progenitor cells from aged subjects display decreased expression of Sirtuin 1, angiogenic functions and increased senescence† Running Title: Sirtuin 1 and age-related EPC senescence Impreet Kaur1, Preety Rawal1, Sumati Rohilla1, Mohsin Hassan Bhat2, Priyanka Sharma1, Hamda Siddiqui1, Savneet Kaur1,2* 1
School of Biotechnology, Gautam Buddha University, Greater Noida, India
2
Institute of Liver and Biliary Sciences, New Delhi, India
*Corresponding author: Dr. Savneet Kaur Past address Assistant Professor, School of Biotechnology, Gautam Buddha University, Greater Noida, (UP) 201310, India Current address Assistant Professor, Department of Molecular and Cellular Medicine Institute of Liver and Biliary Sciences D1, Vasant Kunj, New Delhi 110070 Email:
[email protected] Keywords: Aging; Angiogenesis; Endothelial progenitor cells (EPCs); p53, Senescence; Sirtuin 1 (SIRT1)
†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10999]
This article is protected by copyright. All rights reserved Received: 20 October 2017; Revised: 14 May 2018; Accepted: 25 May 2018
1
List of Abbreviations acLDL: acetylated low density lipoprotein CFU: Colony forming units EGF: Epidermal growth factor EPC: Endothelial progenitor cell FITC: Fluorescein isothiocyanate PBMC: Peripheral blood mononuclear cells PFA: Paraformaldehyde SA-b-Gal: Senescence-associated beta- galactosidase SIRT1: Sirtuin 1 VEGF: Vascular endothelial growth factor Vegfr2: Vascular endothelial growth factor receptor 2 UEA1: Ulex europaeus agglutinin 1 Abstract Studies have demonstrated that aging is associated with a substantial decline in numbers and angiogenic activity of endothelial progenitor cells (EPCs). In view of senescence being an important regulator of age-related cell survival and function, in the current study, we correlated EPCs numbers and functions with their senescence status and mechanisms in young and elderly subjects. Healthy young subjects (n=30, below 60 y) and old subjects (n=30, equal to or above 60 y) participated in the study. Subjects had no significant disease or risk factors of disease and aging was the only risk factor in the aged subjects. Enumeration of CD34-vegfr2 dual positive EPCs was performed. The ex vivo culture of EPCs was done to study colony formation, migration and senescence-associated beta-galactosidase activity. The expression of cell cycle and senescence regulatory proteins including, p53, p21 and sirtuin 1 (SIRT1), a deacetylase protein was studied in cultured EPCs by RT-PCR and immunofluorescence staining. In vivo proliferation, ex vivo colonies, migration and secretory ability of EPCs was significantly higher in young subjects than that in elderly subjects. EPCs in old subjects showed enhanced senescence and decreased expression of SIRT1 in comparison to that observed in young subjects. 2
An inhibition of SIRT1 in EPCs of young subjects led to significant increase in senescence and reduction of cell differentiation. The study suggests that EPCs have decreased proliferation and functions in aged subjects due to increased senescence which may be attributable to decreased expression of SIRT1.
1.
Introduction
Studies have demonstrated an important role of bone marrow-derived endothelial progenitor cells (EPCs) in endogenous repair mechanism of the endothelial monolayer (Chong et al., 2016; Asahara et al., 2011). EPCs represent a population of pluripotent cells that are mobilized into the peripheral blood from the bone marrow in response to stimulating signals emanating from tissue ischemia or traumatic injury. They integrate into the endothelial layer of blood vessels, contribute to postnatal angiogenesis and augment neo-vascularization of ischemic tissues (Eguchi et al., 2007; Nishimura and Asahara, 2005). Several studies report that aged individuals have a substantial decline in the numbers and angiogenic activity of EPCs, thereby, having impaired endogenous repair. (Heiss et al., 2005; Dimmeler and Vasa-Nicotera, 2003; Thijssen et al., 2006; Hoetzer et al., 2007). This decline of EPC function is attributed to telomere reduction in aged subjects, which results in increased senescence, altered cellular function and thereby reduced survival (Shawi and Autexier, 2008; Yang et al., 2008). However, besides telomere shortening, the effect of several other cell-intrinsic cell cycle regulators on EPC functions remains largely elusive. 3
In the current study, we investigated the numbers, proliferation, senescence status, colonyformation potential and angiogenic functions of EPCs in young and old subjects. Also, to gain deeper insights into senescence-associated mechanisms in EPCs, we analyzed the expression of senescence–associated cell cycle genes including p53, p21 and sirtuin 1 (SIRT1), a protein deactylase in these cells in both young and aged subjects. The role of SIRT1 in cell senescence was further addressed by transient inhibition of SIRT1 in EPC cultures from young subjects.
4
2. Materials and Methods 2.1 Study Groups: A total of 60 healthy subjects, including, young (n=30, below 60 y, mean age= 32.9 + 11, Male: Female= 2:1) and old (n=30, equal to or above 60 y, mean age= 68.6 + 6.7, Male: Female= 4:1) subjects were recruited for the study group. Subjects with any form of cancer, renal disease, rheumatoid arthritis, pneumonia, nephropathy and cardio-vascular disease were excluded. 15 ml venous blood sample was collected in EDTA tubes. 2-3 ml blood was used for the isolation of PBMCs, which were used for EPC enumeration by flow cytometry. Rest of the blood was used for EPC isolation and culture. The study was approved by Human Ethics Committee of Gautam Buddha University and informed consent was obtained from all subjects prior to the study. 2.2 Enumeration of Circulating EPCs: EPCs in the peripheral blood were enumerated both in young and the old subjects by fluorescent-activated cell sorting (FACS). 2-3 ml of whole blood was used for the isolation of PBMCs by Ficoll (HiSep, Himedia) method using density centrifugation. After purification, about 6 x106 cells were taken in M-199 and stained with the antibodies, anti-human CD34 (FITC-conjugated, Santa Cruz Biotechnology) and anti-human vegfr2/Flk-1 (APC-conjugated, BD Biosciences). Of all the markers, we used CD34 and Flk-1 to characterize the circulating EPCs (Kaur et al., 2007). Unconjugated CD34 and vegfr2 antibodies (Santa Cruz Biotechnology) without flourophores were used as controls to assess nonspecific binding. The results were analyzed by BD FACS Aria III (BD Biosciences and DIVA software). For each sample, a minimum of 100,000 events were acquired. 2.3 EPC isolation and in vitro expansion: EPCs were cultured as previously described (Kaur et al., 2007). Briefly, mononuclear cells (MNCs) were isolated from a 10-12 ml blood sample by Ficoll method using density centrifugation. After purification with 2 washing steps 5
and RBC lysis, MNCs (1 x 106 cells/cm2) were plated onto fibronectin coated, 6-well plates with M-199 and 20% FBS. For ex-vivo expansion, the non-adherent cells were collected after 48 h, washed and replated onto 6-well plates with M-199, 20% FBS and growth factors including vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). Medium was changed every three days and all the subsequent assays were performed using cells harvested on day 7-8 with PBS-0.05% trypsin-0.02% EDTA. 2.3.1 Characterization of EPCs in culture: Functionality and viability of EPCs was assessed by ac-LDL uptake and lectin staining. After 7 days in culture, attached EPC-CFUs were stained for the uptake of DiI-labeled acetylated low-density lipoprotein (acLDL, Invitrogen) and concurrent binding of FITC-conjugated Ulex europaeus agglutinin I (UEA-1, Sigma) as earlier described (Kaur et al., 2007). The stained cells were visualized under an inverted fluorescent microscope. 2.3.2 Colony forming units of EPCs: Culture-enriched EPC colonies/CFUs on fibronectin-coated plates in both young and aged subjects were counted in 10 random microscopic fields under an inverted phase-contrast microscope and the average CFU counts per field were calculated. 2.4 Senescence-associated beta-galactosidase activity of EPCs: Senescence-associated beta- galactosidase (SA-b-Gal) activity was measured in the cell lysates using beta-galactosidase staining kit (Thermo Fisher Scientific, India) following the manufacturer’s protocol. Briefly, 50 µl of EPC lysates were collected after two weeks of culture and added onto 96 well plates. Then, 100 μL of β-Galactosidase assay reagent was added to each well and the plate was incubated for 30 min at 370C. Thereafter, absorbance was measured at 405 nm.
6
2.5 Chemotaxis/Migration of EPCs: EPCs were detached, harvested by centrifugation and resuspended in M199 (without serum) and then placed in the upper chamber of a modified Boyden chamber consisting of uncoated polycarbonate filter membranes of 8 µm pore size (Kaur et al., 2009). The chamber was placed in a 24-well culture dish containing M199 only, M199 with VEGF (20 ng/ml) and M199 with FBS (20%). After 24 h incubation at 370C, the lower side of the filter was washed with PBS and fixed with 4% paraformaldeyde for 2 min. Then cells were washed and permeabilized by 100% methanol for 20 min. For quantification, cell nuclei were stained with 0.5% crystal violet. The upper side of the filter containing the non-migrating cells was scraped with a cotton swab. Cells and those migrating into the lower chamber were counted manually at 40X objective in random microscopic fields. 2.6 Evaluation of conditioned medium from EPC cultures and ELISAs for secretory factors: EPCs from young and old subjects after 8-10 days of culture were washed four times with serum-free M199 media, and then incubated with media only at 37°C in a humidified 5% (v/v) CO2 incubator for 24h. The medium containing secreted proteins was collected, centrifuged (1000g, 10 min) at 4°C, and filtered with a 0.22-μm low protein binding membrane. The collected media (designated as the EPC secretome) was stored at −80°C and analyzed for angiogenic growth factors, PDGF-BB and VEGF by ELISA kits (Thermo Fisher Scientific, India) as per manufacturer’s instructions. 2.7 RT-PCR of cell cycle genes: Total RNA from cultured EPCs was isolated by using RNA sure kit (Genetix Biotech Asia). RNA (300 ng) from each sample was subjected to reverse transcription using the kit according to the manufacturer’s protocol (Genetix Biotech Asia). Taqman PCR master mix was used for all PCR experiments. About 10 pmol of gene-specific primer sets were used for p53 (sense: 5’-AGGTTGGCTCTGACTGTACC-3' and antisense: 5’7
AAAGCTGTTCCGTCCCAGTA-3’), p21 (sense: 5’-TGGACCTGTCACTGTCTTGT-3’ and antisense:
5’-GGCGTTTGGAGTGGTAGAAA-3’),
p16
(sense:
5’-
GTCGACCTGGCTGAGGAG-3’ and antisense: 5’-CTTTCAATCGGGGATGTCTG-3’) to look for their expression in the young and aged populations. Data was normalized by using 18S RNA (sense:
5’-GTAACCCGTTGAACCCCATT-3’
and
antisense:
5’-
CCATCCAATCGGTAGTAGCG-3’) 2.8 Protein Expression of Senescent markers in cultured EPC:
The protein
expression of p53, p21, SIRT1, acetylated-p53 (Lys382) was analyzed in the cultured EPCs by immunoflourescence staining in both the study groups. The cells were fixed with 4% PFA for 810 min at -200C. Then after a wash with PBS, the cells were permeabilized using 0.1% Triton X 100 for 10 min. For staining, the following antibodies, anti-human p53, p21 (Sanatcruz Biotechnology), SIRT1, acetylayed-p53 (Thermo Fisher Scientific) were added to about 2.5 x106 cells for 45 min at 4°C. After a wash with cold PBS, the cells were then incubated with FITCconjugated anti-mouse secondary IgG antibody for p53, p21 and acetylayed-p53 (Sanatcruz Biotechnology) or rhodamine-conjugated anti-mouse secondary IgG antibody for SIRT1 (Sanatcruz Biotechnology) for 45 min at 4°C in dark. The cells were further stained with DAPI for 5 min to stain the nucleus of these cells. 2.8.1 SIRT1 inhibition in EPCs from young subjects: 8-10 day EPC cultures (5 x 105 cells/well) in six well plates were transfected with 100nmole/l control siRNA or SIRT1 siRNA (Santa Cruz Biotechnology) using Lipofectamine. After 48 h of transfection, the number of differentiated EPCs was counted and cell lysates were collected for assay of SA-b-Gal activity. 2.9 Statistical Analysis: Results are expressed as mean ± standard deviation (SD). Normality of the data was checked by online Kolmogorov-Smirnov test. Differences between the 8
two groups were compared using unpaired student’s t test. A value of P< 0.05 was considered significant.
9
3. Results 3.1 Enumeration of EPCs: Results demonstrated that the percentage of CD34-vegfr2 dual positive EPCs in circulation was higher in the young subjects as compared to the aged subjects, however the difference was not statistically significant (Fig. 1 and Supplementary Fig.1). 3.2 Culture, Characterization and colony formation of EPCs: Circulating EPCs were cultured for about 7-10 days in the presence of growth factors and characterized by positive staining for DiacLDL uptake and FITC-UEA1 lectin binding assay (Fig. 2a-c). Since the formation of EPC clusters in culture is one of the measures of angiogenic ability of EPCs, we next examined the number of these clusters in the young and old subjects. More number of EPC clusters was observed in most of the young subjects in comparison to the aged subjects (21.1 + 5 versus 14.6 + 3.4, P< 0.05). There were, however, some aged subjects that showed good number of EPC colonies in culture and vice-versa. The cells in the clusters of the young subjects showed the presence of more numbers of spindle-shaped and well-differentiated cells than that observed in the old subjects (Fig. 2d-h). 3.3 Migratory, Secretory and Senescent Features of EPCs: EPCs migrate towards a chemotactic signal, like VEGF in culture. Our results showed that the number of migrated cells in the elderly subjects was significantly decreased as compared to the young subjects (P< 0.05, Fig. 3a). To assess the percentage of senescent cells, EPC cultures from the young and aged subjects were stained with SA-b-gal. A higher activity represents more number of senescent cells. EPCs from old subjects displayed increased b-gal activity compared to the young subjects (0.45 + 0.1 versus 0.21 + 0.3, P< 0.05, Fig. 3b).
10
The pro-angiogenic and secretory function of EPCs was determined in the study groups by ELISA assays of two important angiogenic factors, VEGF and PDGF-BB. Results showed that the levels of pro-angiogenic factors, VEGF and PDGF-BB was considerably enhanced in most of the EPCs of young subjects as compared to the old subjects, suggesting defects in the secretory functions of EPCs in the old group (P< 0.05 each, Fig. 3c and 3d). There was enough variability in VEGF levels in the young subjects with some showing low VEGF levels similar to that observed in the old subjects and some showing very high VEGF levels. In the old subjects, however, we observed consistently low levels of VEGF. 3.4 Expression of cell cycle genes/proteins: To elucidate the intrinsic mechanisms involved in the observed decline in the proliferation and functions of EPCs in the aged subjects, we next investigated the gene and protein expression of cell cycle genes including p53, p21 and p16 in the EPCs. The RT-PCR analysis revealed that the gene expression of p53 and p16 was slightly enhanced in the old subjects and the expression of p21 was significantly increased in the old subjects as compared to the young subjects (P< 0.05, Fig. 4a). The protein expression of p53 was very less and not well evident in immunoflourescence staining in the EPCs of young subjects. In the old subjects, some cells appeared to show p53 expression, although in these subjects too, a very meager and inadequate intensity of fluorescence was observed (Fig. 4b). The expression of p21 was increased in the EPCs of old subjects in comparison to that observed in the young subjects (Fig. 4c). 3.5 Expression of SIRT1: Since the gene and protein expression of p53 was not very different among the young and aged groups unlike the expression of p21, we hypothesized that p53 may be post-translationally regulated via SIRT1-mediated deacetylation. To this end, we first compared the protein expression of SIRT1 in the EPCs between the young and aged 11
subjects. Results showed decreased expression of SIRT1 in the old subjects as compared to the young subjects (Fig. 5a). To further validate, if p53 acetylation correlates with decreased activity of SIRT1, next, we compared the expression of acetylated-p53 in the EPCs of young and old subjects. It was observed that the expression of acetylayed-p53 was distinctly up-regulated in the old subjects in comparison to the young subjects (Fig. 5b). 3.6 Inhibition of SIRT1 in young EPCs: To validate the role of SIRT1 in EPC senescence, we next inhibited the expression of SIRT1 in cultured EPCs from young subjects. The SIRT1-siRNA transfection resulted in more than 60% reduction in SIRT1 gene expression (Fig.6a). Although some cell death was observed in both control and SIRT1-siRNA transfected cultures, as compared to the control-siRNA treated EPCs, the SIRT1-siRNA treated cells showed a significant increase in the beta-galactosidase activity (0.22 + 0.06 versus 0.62 + 0.06) and a substantial reduction in the number of differentiated spindle shaped EPCs (12.25 + 2 versus 5 + 2.1) after 48 h of transfection (Fig. 6b-6d).
12
4. Discussion The current study demonstrates decreased number of endogenous CD34-vegfr2-positive circulating EPCs in the elderly subjects in comparison to the young subjects. Also, EPCs cultured from these subjects had impaired colony forming potential and pro-angiogenic functions including migration towards a chemotactic signal and secretion of angiogenic factors but increased senescence as compared to that observed in the young group. These results are in concordance to the findings of Heiss et al., who observed that early EPCs from the old subjects showed significantly attenuated proliferation, migration, and survival (Heiss et al., 2005). To gain insights into the molecular mechanisms of reduced EPC proliferation and functions and enhanced senescence in the aged subjects, we next investigated the gene and protein expression of p53 and p21 in the EPCs. Depending on the severity of damage to the genome, p53 can activate genetic programs that halt cell proliferation transiently (G1 and G2 cell cycle arrest), permanently (senescence), or eliminate the cell altogether (apoptosis) (Murray-Zmijewski et al., 2008). It has been reported that p21 induction by p53 leads to inhibition of CDKs, which in turn results in hypophosphorylation of pRb, which very likely mediates cell cycle arrest during senescence (Bringold and Serrano, 2000). Our results showed that both the mRNA and protein expression of p53 was not significantly different between the two study groups, albeit the expression of p21 was substantially increased in the EPCs of aged subjects as compared to the young group. Since, the expression of p53 was not different in the young and aged subjects, we hypothesized that p53 may be epigenetically modified via deacetylases such as SIRT1. It has been earlier demonstrated that under conditions of oxidative stress, SIRT1, the most studied mammalian homologue of the Sirtuin family, is one of the crucial factors that regulate cell senescence through deacetylation of p53 (Vaziri et al., 2001). SIRT1 attenuates p53-mediated 13
functions through deacetylation of p53 at its C-terminal Lys382 residue (Luo et al., 2001). Studies have indicated that acetylated-p53 is more stable and has a higher half life than the un-acetylated p53 (Li et al., 2002). Our results revealed that the expression of SIRT1 was very much decreased in the EPCs of aged subjects in comparison to the young subjects. This is in accordance with earlier studies showing that the protein expression and transcription levels of SIRT1 decline in aged animals and human tissues, including lung, fat, heart and blood vessels (Zu et al., 2010). The decreased levels of SIRT1 in the aged subjects in our study also correlated with an increased expression of acetylated-p53 in EPCs of these subjects, indicating that a decrease in the levels of SIRT1 may result in the stabilization of p53 via an increase in the expression of acetylated-p53. Further, a transient inhibition of SIRT1 gene in EPC cultures from young subjects resulted in significant decrease in the number of differentiated spindle-shaped EPCs and enhancement of cell senescence, thus validating our results that a decrease in SIRT1 induces senescence in these vascular progenitors. SIRT1 over-expression is well known to exert protective effects against endothelial dysfunction by preventing stress-induced premature senescence in endothelial cells (Ota et al., 2007; Erusalimsky and Skene, 2009). SIRT1 has also been shown to play a key role in angiogenic response following ischemic injury and oxidative stress (Potente et al., 2007; Salminen et al., 2013; Zhang et al., 2016). A recent study by Vasasllo et al., 2014 have reported that decreased expression of SIRT1 leads to accelerated senescence and angiogenic defect of endothelial colony forming cells in preterm subjects (Vasasllo et al., 2014). 5. Conclusions We conclude that EPCs in aged adults display reduced SIRT1 expression than that observed in the younger adults. Decreased SIRT1 expression leads to senescence and reduced proliferative 14
capabilities of EPCs. This may be attributed to the accumulation of more stable acetylated-p53 and transcription of p53 target genes such as p21. An increased senescence may also result in decline of other angiogenic EPC functions including migration and secretion of pro-angiogenic factors in the aged subjects. Our study corroborates the role of SIRT1 as one of the important anti-senescence mediators in human vascular aging.
6. Acknowledgements We are extremely grateful to Dr. Ashok Mukopadhayay and Dr. Nirupama Trehanpati, National Institute of Immunology and Institute of Liver and Biliary Sciences (ILBS), Delhi for their support in conducting flow cytometry experiments.
7. Funding The study was financially supported by Department of Science and Technology, India (SR/FT/LS-150/2010).
8. Disclosure of Interests The authors declare that they have no competing interests.
15
9. References Asahara T, Kawamoto A, Masuda H (2011) Circulating endothelial progenitor cells for vascular medicine. Stem Cells;29:1650-5 Bringold F, Serrano M (2003) Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol;35:317-29. Chong MS, Ng WK, Chan JK (2016) Endothelial Progenitor Cells in Regenerative Medicine: Applications and Challenges. Stem Cells Transl Med;5:530-8. Dimmeler S, Vasa-Nicotera M (2003) Aging of progenitor cells: limitation for regenerative capacity? J Am Coll Cardiol;42:2081-2. Eguchi M, Masuda H, Asahara T (2007) Endothelial progenitor cells for postnatal vasculogenesis. Clin Exp Nephrol;11:18-25. Erusalimsky JD and Skene C (2009) Mechanisms of endothelial senescence. Experimental Physio; 94:299–304. Heiss C, Keymel S, Niesler U, Ziemann J, Kelm M, Kalka C (2005) Impaired progenitor cell activity in age-related endothelial dysfunction. J Am Coll Cardiol;45:1441-8. Hoetzer GL, Van Guilder GP, Irmiger HM, Keith RS, Stauffer BL, DeSouza CA (2007) Aging, exercise, and endothelial progenitor cell clonogenic and migratory capacity in men. J Appl Physiol;102:847-52. Kaur S, Jayakumar K, Kartha CC (2007) The potential of circulating endothelial progenitor cells to form colonies is inversely proportional to total vascular risk score in patients with coronary artery disease. Indian Heart J;59:475-81. Kaur S, Santhosh Kumar T R, Uruno A, Sugawara A, Jayakumar K, Kartha CC (2009) Genetic engineering with endothelial nitric oxide synthase improves functional properties 16
of endothelial progenitor cells from patients with coronary artery disease: an in vitro study. Basic Res Cardiol;104:739-49. Li M, Luo J, Brooks CL, Gu W (2002) Acetylation of p53 inhibits its ubiquitination by Mdm2. J Biol Chem;277:50607-11. Luo J, Nikolaev AY, Imai S, Chen D, Su F, Shiloh A, Guarente L, Gu W (2001) Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell;107:137-48. Murray-Zmijewski F, Slee EA, Lu X (2008) A complex barcode underlies the heterogeneous response of p53 to stress. Nat Rev Mol Cell Biol;9:702-12. Nishimura H, Asahara T (2005) Bone marrow-derived endothelial progenitor cells for neovascular formation. EXS;94:147-54. Ota H, Akishita M, Eto M, Iijima K, Kaneki M, Ouchi Y (2007) Sirt1 modulates premature senescence-like phenotype in human endothelial cells. J Mol Cell Cardiol;43:571-9. Potente M, Ghaeni L, Baldessari D, Mostoslavsky R, Rossig L, Dequiedt F, Haendeler J, Mione M, Dejana E, Alt FW, Zeiher AM (2007) SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev;21: 2644–2658. Roy S, Javed S, Jain SK, Majumdar SS, Mukhopadhyay A (2012) Donor hematopoietic stem cells confer long-term marrow reconstitution by self-renewal divisions exceeding to that of host cells. PLoS One;7:e50693. Salminen A, Kaarniranta K, Kauppinen A (2013) Crosstalk between Oxidative Stress and SIRT1: Impact on the Aging Process. Int J Mol Sci;14: 3834–3859. Shawi M, Autexier C (2008) Telomerase, senescence and ageing. Mechanisms of Ageing and Development;129:3–10.
17
Thijssen DH, Vos JB, Verseyden C, van Zonneveld AJ, Smits P, Sweep FC, Hopman MT, de Boer HC (2006) Haematopoietic stem cells and endothelial progenitor cells in healthy men: effect of aging and training. Aging Cell;5:495-503. Vassallo PF, Simoncini S, Ligi I, Chateau AL, Bachelier R, Robert S, Morere J,Fernandez S, Guillet B, Marcelli M, Tellier E, Pascal A, Simeoni U, Anfosso F,Magdinier F, DignatGeorge F, Sabatier F (2014) Accelerated senescence of cord blood endothelial progenitor cells in premature neonates is driven by SIRT1 decreased expression. Blood;123:2116-26. Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, Guarente L,Weinberg RA (2001) hSIR2 (SIRT1) functions as an NAD-dependent p53 deacetylase. Cell;107:14959. Yang DG, Liu L, Zheng XY (2008) Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence. Ageing Res Rev;7:137-46. Zhang L, Kondo H, Kamikubo H, Kataoka M, Sakamoto W (2016) VIPP1 has a disordered C-terminal tail necessary for protecting photosynthetic membranes against stress in Arabidopsis. Plant Physiol;171:1983–1995. Breitenstein A, Wyss CA, Spescha RD, Franzeck FC, Hof D, et al. (2013) Peripheral blood monocyte Sirt1 expression is reduced in patients with coronary artery disease. PLoS One;8: e53106 Zu Y, Liu L, Lee MY, Xu C, Liang Y, Man RY, Vanhoutte PM, Wang Y (2010) SIRT1 promotes proliferation and prevents senescence through targeting LKB1 in primary porcine aortic endothelial cells. Circ Res;106:1384-93.
18
Figure Legends Figure 1: Dot plot showing the percentage of CD34-vegfr2 dual positive circulating EPCs in the young and old subjects (n= 8 each) as assessed by FACS analysis.
Figure 2: Characterization of EPCs on day 7-8 of culture by fluorescence microscopy . (a) DiIacLDL uptake (b) FITC-UEA lectin binding (c) overlay of (a) and (b). Scale bar represents 15µm. Phase contrast pictures of colony-forming units of EPCs in (d and e) young subjects (f and g) old subjects after 7–8 days of culture. Scale bar represents 15µm. (h) Dot plot depicting average number of EPC colonies per field in young and old subjects (n= 10 each).
Figure 3: Box plots showing (a) Average number of migrated EPCs towards absence and presence of 20 ng/ml VEGF in a modified boyden chamber counted using 40X objective in young and aged subjects (n= 6 each). (b) Senescence-associated beta-galactosidase activity (SAbgal) in the two groups (n= 5 each) (c) Levels of PDGF-BB (pg/ml) from EPC-conditioned media obtained from young and aged subjects (n= 8 each). (d) Levels of VEGF (pg/ml) from EPC-conditioned media obtained from young and aged subjects (n= 8 each).
Figure 4: (a) Relative mRNA expression of cell cycle and senescence-associated genes, p53, p21 and p16 in young and aged subjects. Results are expressed as mean + SD (n= 5 each) (b) Representative immunoflourescence images of protein expression of p53 in young (p53y) and old (p53o) subjects. Scale bar represents 15µm. (c) Representative immunoflourescence images depicting protein expression of p21 in young (p21y) and old (p21o) subjects. Scale bar represents
19
30µm.The expression of p53 and p21 expression was detected using FITC-labeled secondary antibody. DAPI was used for staining of DNA.
Figure 5: (a) Representative images showing immunoflourescence expression of SIRT1 protein in young (SIRT1y) and old (SIRT1o) subjects. Scale bar represents 15µm. (b) Representative images depicting protein expression of acetylated-p53 in young (acp53y) and old subjects (acp53o). Scale bar represents 15µm. The expression of SIRT1 and acetylated-p53 was detected by immunoflourescence using rhodamine-labeled and FITC-labeled secondary antibodies respectively. DAPI was used for staining of DNA.
Figure 6: SIRT1 inhibition in cultured EPCs of young subjects. (a) Relative mRNA expression of SIRT1 gene in control-siRNA and SIRT1-siRNA treated EPCs from young subjects. Results are expressed as mean + SD (n= 3 each). (b) Box plot showing senescenceassociated beta-galactosidase activity (SA-bgal) in control-siRNA and SIRT1-siRNA treated EPCs (n= 5 each). (c) Phase contrast pictures of control-siRNA and SIRT1-siRNA treated EPCs colonies. Scale bar represents 15µm. (d) Bar Diagram showing average number of spindleshaped EPCs per field in control-siRNA and SIRT1-siRNA treated cultures. Results are expressed as mean ± SD (n= 5 each).
Supplementary Figure 1: Enumeration of circulating EPCs in blood was done by measuring the percentage of CD34 and vegfr2 dual positive cells in the lymphocyte and monocyte gated PBMCs. Representative images depicting percentages of single stained and dual stained cells in
20
the controls and only dual stained cells in the young and old subjects are shown. Unconjugated antibodies (Abs) without flourophores were used as controls.
Figure 1
Figure 2
21
Figure 3
Figure 4
22
Figure 5
Figure 6
23
