- Email: [email protected]
In vitro senescence occurring in normal human endothelial cells can be rescued by ectopic telomerase activity
In Vitro Senescence Occurring in Normal Human Endothelial Cells Can Be Rescued by Ectopic Telomerase Activity A.T.L. Young, J.R.T. Lakey, A.G. Murray, J.C. Mullen, and R.B. Moore ABSTRACT 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 (-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.
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N THE LAST TWO DECADES, organ transplantation has been the adopted treatment modality for end-stage human diseases. The universal application of this treatment protocol relies heavily on the availability of donor resources that have been in shortage as the demands for organ transplantation continue to rise, leading to the utilization of organs from older donors. This lengthening of the waiting lists is, in part, attributed to the general dysfunction of the transplanted organ as constitutive cells of an aging organ have a much reduced ability to thrive following transplantation. The limited longevity of aging allografts is associated with replicative senescence, a state of irreversible growth arrest. The onset of cellular replicative senescence can be explained by telomere shortening occurring in normal somatic cells. Telomeres consist of hexamer repeat sequences (TTAGGG) located at the distal extremities of eukaryotic chromosomes and whose function is to protect chromosomal ends from genetic mutation and instability. Virtually all somatic cells undergo telomere attrition as the cells divide until they reach the Hayflick limit, at which proliferation ceases and the cells become senescent with the exception of germ cells and cancer cells that possess
telomerase activity. Expression of telomerase, a reverse transcriptase that is normally absent in normal somatic cells, has been shown to renew replicative capacity by means of telomere maintainence via the de novo synthesis of telomeric DNA. Telomerase expression in transduced endothelial cells has been shown to extend replication in vitro and preserve a healthy phenotype.1 The therapeutic potential provided by telomerase hopefully will renew aging
From the Department of Surgery (A.T.L.Y., J.R.T.L., J.C.M., R.B.M.), Surgical-Medical Research Institute, and the Department of Medicine (A.G.M.), University of Alberta, Edmonton, Canada. Supported in part by the Edmonton Civic’s Employees Charitable Assistance Fund, S. Jean Milner Fund, AHFMR and JDF/CHIR granted to Drs. R.B. Moore and J.R.T Lakey who are AHFMR scholars. Address reprint requests to Dr Jonathan R.T. Lakey, Assistant Professor of Surgery, Director of Clinical Islet Laboratory, Surgical-Medical Research Institute, University of Alberta, 1074 Dentistry/Pharmacy Building, Edmonton, Alberta T6G 2N8 Canada. E-mail: [email protected]
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/03/$–see front matter doi:10.1016/j.transproceed.2003.08.032
Transplantation Proceedings, 35, 2483–2485 (2003)
2483
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cells’ divisional capacity in response to transplant injury and therefore, enhances long-term allograft survival. We are working toward improving the longevity of transplanted solid organs by extending cellular life span of human endothelial cells via the ectopic expression of human catalytic telomerase (hTERT) and examining the preclinical feasibility of using nonviral lipid transfection for gene delivery, which eliminates many of the biosafety concerns associated with the use of viral vectors. This study examined the natural senescence of human endothelial cells (HUVECs) in culture and the in vitro effects of telomerase expression following transfection. Our ultimate goal is to perform transfection on the endothelium, an immediate target of rejection, of organ transplants with the telomerase gene and monitor graft survival. MATERIALS AND METHODS Primary endothelial cells (ECs) were isolated from human umbilical cord vein (HUVECs) as previously described.2 Isolated ECs were cultured in complete M199 media supplemented with 20% fetal bovine serum, L-glutamine and antibiotics at 37 °C and 5% CO2. Endothelial cell growth supplement was also provided to the cell culture. ECs were plated onto a 6-well transwell plate for transfection with a mixture prepared from telomerase DNA and Effectene lipids. The transfection mixture was added dropwise to the cell monolayer with swirling and incubated under standard culture conditions for 2 days prior to cell collection for further assays. In vitro senescence was analyzed by cell staining that include the use of (1) senescence-associated -galactosidase (SA--gal) at pH 6,3 carboxyfluorescein diacetate succinimidyl-ester (CFDA-SE) dye,4 and propidium iodide (PI) for S-phase cycling cells. Following transfection, cells were collected at different time intervals (3h, 1-3d) and assessed for transfection efficiency. Control and transfected cell pellets were lysed to isolate the plasmid vector and ran on an agarose gel to determine transfection outcome. Furthermore, the effects of telomerase transfection in culture and on telomere length were assessed by (1) changes in cell morphology, (2) time of recovery from serum starvation, and (3) telomere length measurement by Southern blot and quantitative fluorescent in situ hybridization (q-FISH) using a telomere-specific fluorescent probe.
RESULTS In Vitro Senescence
The morphology of HUVECs changed as they aged in culture with increasing passage number (p). Aging cells became larger and multinucleated. When comparing normal ECs of passage 2, 7, and 13 we observed a gradual loss of the cobblestone appearance in the cell monolayer and late-passaged cell cultures required an extended period of culture time to reach confluence. Moreover, the fraction of SA--gal stained (blue) cells increased with subsequent passaging as the number of senescent cells increased. Immortalized HeLa cultures had about 7% blue-staining cells as opposed to the 35% of p2 HUVECs, which showed sign of senescence (stained blue) with an increase to 80% in cultures of p12 HUVECs. Young cell cultures (p2– 4, 19.00 ⫾ 1.00%) also showed a higher percentage of cycling cells
YOUNG, LAKEY, MURRAY ET AL
in S-phase compared to the intermediate passages (p5–7, 17.00 ⫾ 1.57%) or senescing cultures (p8 –10, 14.00 ⫾ 3.95%), whereas cancer cells (HeLa, 25%) showed minimal senescence. When cell injury was induced in the monolayer by scraping off some ECs in a marked area, cells of p7 recovered quicker than p13 cultures, whose rates of proliferation and migration into the wounded area were slow. In Vitro Duration of Lipid Transfection
Following transfection with a control (SV--gal) and a telomerase vector (Ni-Tel), both vectors were detected in transfected cells 3 hours posttransfection. Presence of the vectors was undetectable on day 1 and at subsequent time points of detection using ethidium bromide staining. Although the duration of the plasmid vector inside cells was very transient; nonetheless, Effectene provided an effective means of delivering the gene into human cells. In Vitro Effects of Telomerase in Culture
Differences in cell morphology were observed between untransfected and telomerase transfected ECs of p3 and p13 examined 2 weeks posttransfection. Transfected ECs appeared healthier and younger looking compared to untransfected controls. Furthermore, cells transfected with telomerase underwent faster recovery after serum replacement in culture media deprived of fresh serum for 2 weeks (Fig 1). Telomerase-positive ECs recovered from serum stress immediately 2 days following media replacement, whereas cells transfected with a control vector (SV-gal) did not show rapid recovery. The assessment of cell proliferation in normal passaged ECs using the CFDA-SE dye showed a declining rate in the proliferative index as cells aged. The proliferative indices of normal HUVECs of p2, p5, p9, and p11 assessed 3 days post-CFDA-SE staining were 2.60, 1.75, 1.35, and 0. Telomerase-transfected HUVECs at p3 were cultured to p6 prior to CFDA-SE staining to acquire enough cells for the assay. Telomerase-transfected cells and untransfected controls were stained at p6 and assessed for proliferation 3 days poststaining. The proliferative index of control cells was 1.50, whereas telomerase-positive cells demonstrated an index of 2.20 comparable to early passaged cells. Comparative Telomere Length
Using the q-FISH assay, telomere length was assessed in p2 and p7 HUVECs as well as in HeLa cells. Telomere signals (hybridized telomere DNA with Cy3-fluorescent–labeled telomere-specific probe) were stronger in early passaged HUVECs and weakest in cancer cells with shorter telomeres. The mean fluorescent telomere signal, in arbitrary units (AU), for p2 HUVECs was 367 ⫾ 132 AU (n ⫽ 4), p7 HUVECs was 230 ⫾ 41 AU (n ⫽ 7), and HeLa was 134 ⫾ 3 AU (n ⫽ 3).
BYPASS SENESCENCE WITH TELOMERASE
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Fig 1. Stress-induced proliferative recovery. Serum starvation of both telomerase-transfected (C and D) and SV--gal-transfected (A and B) HUVECs. ECs were starved for 2 weeks in the absence of media change (A and C). (D) Telomerase-transfected ECs recovered immediately 2 days following media replacement whereas cells transfected with the irrelevant (B) -gal vector did not show immediate recovery in 2 days.
DISCUSSION
The findings from this study showed that normal ECs underwent cell senescence with subculturing and telomerase transfection demonstrated positive effects on cell survival in culture. Aging ECs were slow to respond to stress compared to younger cells. Senescent cultures also demonstrated slower rates of proliferation. However, when transfected with telomerase, these cells responded like young cells having a high proliferative index comparable to early passaged HUVECs. Our laboratory is currently working on standardizing the FISH assay for routine telomere measurement. The advantages of flow-FISH (FISH accompanied by flow cytometry detection) over Southern blot include (1) simplicity, (2) time requirement, (3) sample size, and (4) the use of a fluorescent-labeled probe specific to the telomeric region only.5,6 Telomere length in telomerase-transfected cells and controls will be compared. As well, these groups will be compared to telomeres of normal passaged ECs to determine whether telomere length can be maintained at an early passage following transfection with telomerase, and
the effects of telomere length maintainence will be assessed subsequently in an animal model. Further examination of the role of telomerase in bypassing senescence and extending replicative life span is underway in an animal model. Using aortic interposition grafts, we will monitor graft survival following transplantation of an aortic graft genetically modified ex vivo with the telomerase gene. Hopefully, long-term graft survival could be achieved by applying this gene strategy to create more robust grafts for transplantation. REFERENCES 1. Yang J, Chang E, Cherry AM, et al: J Biol Chem 274:26141, 1999 2. Gimbrone MA: Prog Hemostas Thromb 3:1, 1976 3. Dimri GP, Lee X, Basile G, et al: Proc Natl Acad Sci USA 92:9363, 1995 4. Lyons AB, Hasbold J, Hodgkin PD: Methods Cell Biol 63:375, 2001 5. Hultdin M, Gronlund E, Norrback KF, et al: Nucleic Acids Res 26:3651, 1998 6. Baerlocher GM, Mak J, Tien T, et al: Cytometry 47:89, 2002
I
N THE LAST TWO DECADES, organ transplantation has been the adopted treatment modality for end-stage human diseases. The universal application of this treatment protocol relies heavily on the availability of donor resources that have been in shortage as the demands for organ transplantation continue to rise, leading to the utilization of organs from older donors. This lengthening of the waiting lists is, in part, attributed to the general dysfunction of the transplanted organ as constitutive cells of an aging organ have a much reduced ability to thrive following transplantation. The limited longevity of aging allografts is associated with replicative senescence, a state of irreversible growth arrest. The onset of cellular replicative senescence can be explained by telomere shortening occurring in normal somatic cells. Telomeres consist of hexamer repeat sequences (TTAGGG) located at the distal extremities of eukaryotic chromosomes and whose function is to protect chromosomal ends from genetic mutation and instability. Virtually all somatic cells undergo telomere attrition as the cells divide until they reach the Hayflick limit, at which proliferation ceases and the cells become senescent with the exception of germ cells and cancer cells that possess
telomerase activity. Expression of telomerase, a reverse transcriptase that is normally absent in normal somatic cells, has been shown to renew replicative capacity by means of telomere maintainence via the de novo synthesis of telomeric DNA. Telomerase expression in transduced endothelial cells has been shown to extend replication in vitro and preserve a healthy phenotype.1 The therapeutic potential provided by telomerase hopefully will renew aging
From the Department of Surgery (A.T.L.Y., J.R.T.L., J.C.M., R.B.M.), Surgical-Medical Research Institute, and the Department of Medicine (A.G.M.), University of Alberta, Edmonton, Canada. Supported in part by the Edmonton Civic’s Employees Charitable Assistance Fund, S. Jean Milner Fund, AHFMR and JDF/CHIR granted to Drs. R.B. Moore and J.R.T Lakey who are AHFMR scholars. Address reprint requests to Dr Jonathan R.T. Lakey, Assistant Professor of Surgery, Director of Clinical Islet Laboratory, Surgical-Medical Research Institute, University of Alberta, 1074 Dentistry/Pharmacy Building, Edmonton, Alberta T6G 2N8 Canada. E-mail: [email protected]
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/03/$–see front matter doi:10.1016/j.transproceed.2003.08.032
Transplantation Proceedings, 35, 2483–2485 (2003)
2483
2484
cells’ divisional capacity in response to transplant injury and therefore, enhances long-term allograft survival. We are working toward improving the longevity of transplanted solid organs by extending cellular life span of human endothelial cells via the ectopic expression of human catalytic telomerase (hTERT) and examining the preclinical feasibility of using nonviral lipid transfection for gene delivery, which eliminates many of the biosafety concerns associated with the use of viral vectors. This study examined the natural senescence of human endothelial cells (HUVECs) in culture and the in vitro effects of telomerase expression following transfection. Our ultimate goal is to perform transfection on the endothelium, an immediate target of rejection, of organ transplants with the telomerase gene and monitor graft survival. MATERIALS AND METHODS Primary endothelial cells (ECs) were isolated from human umbilical cord vein (HUVECs) as previously described.2 Isolated ECs were cultured in complete M199 media supplemented with 20% fetal bovine serum, L-glutamine and antibiotics at 37 °C and 5% CO2. Endothelial cell growth supplement was also provided to the cell culture. ECs were plated onto a 6-well transwell plate for transfection with a mixture prepared from telomerase DNA and Effectene lipids. The transfection mixture was added dropwise to the cell monolayer with swirling and incubated under standard culture conditions for 2 days prior to cell collection for further assays. In vitro senescence was analyzed by cell staining that include the use of (1) senescence-associated -galactosidase (SA--gal) at pH 6,3 carboxyfluorescein diacetate succinimidyl-ester (CFDA-SE) dye,4 and propidium iodide (PI) for S-phase cycling cells. Following transfection, cells were collected at different time intervals (3h, 1-3d) and assessed for transfection efficiency. Control and transfected cell pellets were lysed to isolate the plasmid vector and ran on an agarose gel to determine transfection outcome. Furthermore, the effects of telomerase transfection in culture and on telomere length were assessed by (1) changes in cell morphology, (2) time of recovery from serum starvation, and (3) telomere length measurement by Southern blot and quantitative fluorescent in situ hybridization (q-FISH) using a telomere-specific fluorescent probe.
RESULTS In Vitro Senescence
The morphology of HUVECs changed as they aged in culture with increasing passage number (p). Aging cells became larger and multinucleated. When comparing normal ECs of passage 2, 7, and 13 we observed a gradual loss of the cobblestone appearance in the cell monolayer and late-passaged cell cultures required an extended period of culture time to reach confluence. Moreover, the fraction of SA--gal stained (blue) cells increased with subsequent passaging as the number of senescent cells increased. Immortalized HeLa cultures had about 7% blue-staining cells as opposed to the 35% of p2 HUVECs, which showed sign of senescence (stained blue) with an increase to 80% in cultures of p12 HUVECs. Young cell cultures (p2– 4, 19.00 ⫾ 1.00%) also showed a higher percentage of cycling cells
YOUNG, LAKEY, MURRAY ET AL
in S-phase compared to the intermediate passages (p5–7, 17.00 ⫾ 1.57%) or senescing cultures (p8 –10, 14.00 ⫾ 3.95%), whereas cancer cells (HeLa, 25%) showed minimal senescence. When cell injury was induced in the monolayer by scraping off some ECs in a marked area, cells of p7 recovered quicker than p13 cultures, whose rates of proliferation and migration into the wounded area were slow. In Vitro Duration of Lipid Transfection
Following transfection with a control (SV--gal) and a telomerase vector (Ni-Tel), both vectors were detected in transfected cells 3 hours posttransfection. Presence of the vectors was undetectable on day 1 and at subsequent time points of detection using ethidium bromide staining. Although the duration of the plasmid vector inside cells was very transient; nonetheless, Effectene provided an effective means of delivering the gene into human cells. In Vitro Effects of Telomerase in Culture
Differences in cell morphology were observed between untransfected and telomerase transfected ECs of p3 and p13 examined 2 weeks posttransfection. Transfected ECs appeared healthier and younger looking compared to untransfected controls. Furthermore, cells transfected with telomerase underwent faster recovery after serum replacement in culture media deprived of fresh serum for 2 weeks (Fig 1). Telomerase-positive ECs recovered from serum stress immediately 2 days following media replacement, whereas cells transfected with a control vector (SV-gal) did not show rapid recovery. The assessment of cell proliferation in normal passaged ECs using the CFDA-SE dye showed a declining rate in the proliferative index as cells aged. The proliferative indices of normal HUVECs of p2, p5, p9, and p11 assessed 3 days post-CFDA-SE staining were 2.60, 1.75, 1.35, and 0. Telomerase-transfected HUVECs at p3 were cultured to p6 prior to CFDA-SE staining to acquire enough cells for the assay. Telomerase-transfected cells and untransfected controls were stained at p6 and assessed for proliferation 3 days poststaining. The proliferative index of control cells was 1.50, whereas telomerase-positive cells demonstrated an index of 2.20 comparable to early passaged cells. Comparative Telomere Length
Using the q-FISH assay, telomere length was assessed in p2 and p7 HUVECs as well as in HeLa cells. Telomere signals (hybridized telomere DNA with Cy3-fluorescent–labeled telomere-specific probe) were stronger in early passaged HUVECs and weakest in cancer cells with shorter telomeres. The mean fluorescent telomere signal, in arbitrary units (AU), for p2 HUVECs was 367 ⫾ 132 AU (n ⫽ 4), p7 HUVECs was 230 ⫾ 41 AU (n ⫽ 7), and HeLa was 134 ⫾ 3 AU (n ⫽ 3).
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2485
Fig 1. Stress-induced proliferative recovery. Serum starvation of both telomerase-transfected (C and D) and SV--gal-transfected (A and B) HUVECs. ECs were starved for 2 weeks in the absence of media change (A and C). (D) Telomerase-transfected ECs recovered immediately 2 days following media replacement whereas cells transfected with the irrelevant (B) -gal vector did not show immediate recovery in 2 days.
DISCUSSION
The findings from this study showed that normal ECs underwent cell senescence with subculturing and telomerase transfection demonstrated positive effects on cell survival in culture. Aging ECs were slow to respond to stress compared to younger cells. Senescent cultures also demonstrated slower rates of proliferation. However, when transfected with telomerase, these cells responded like young cells having a high proliferative index comparable to early passaged HUVECs. Our laboratory is currently working on standardizing the FISH assay for routine telomere measurement. The advantages of flow-FISH (FISH accompanied by flow cytometry detection) over Southern blot include (1) simplicity, (2) time requirement, (3) sample size, and (4) the use of a fluorescent-labeled probe specific to the telomeric region only.5,6 Telomere length in telomerase-transfected cells and controls will be compared. As well, these groups will be compared to telomeres of normal passaged ECs to determine whether telomere length can be maintained at an early passage following transfection with telomerase, and
the effects of telomere length maintainence will be assessed subsequently in an animal model. Further examination of the role of telomerase in bypassing senescence and extending replicative life span is underway in an animal model. Using aortic interposition grafts, we will monitor graft survival following transplantation of an aortic graft genetically modified ex vivo with the telomerase gene. Hopefully, long-term graft survival could be achieved by applying this gene strategy to create more robust grafts for transplantation. REFERENCES 1. Yang J, Chang E, Cherry AM, et al: J Biol Chem 274:26141, 1999 2. Gimbrone MA: Prog Hemostas Thromb 3:1, 1976 3. Dimri GP, Lee X, Basile G, et al: Proc Natl Acad Sci USA 92:9363, 1995 4. Lyons AB, Hasbold J, Hodgkin PD: Methods Cell Biol 63:375, 2001 5. Hultdin M, Gronlund E, Norrback KF, et al: Nucleic Acids Res 26:3651, 1998 6. Baerlocher GM, Mak J, Tien T, et al: Cytometry 47:89, 2002