- Email: [email protected]
Differential responsiveness of early- and late-passage endothelial cells to shear stress
The American Journal of Surgery 190 (2005) 763–769
Paper
Differential responsiveness of early- and late-passage endothelial cells to shear stress Fabio A. Kudo, M.D., Ph.D.a,b, Bohdan Warycha, M.D.a,b, Peter J. Juran, B.A.a,b, Hidenori Asada, M.D.a,b, Desarom Teso, M.D.a,b, Faisal Aziz, M.D.a,b, Jared Frattini, M.D.a,b, Bauer E. Sumpio, M.D., Ph.D.a,b, Toshiya Nishibe, M.D., Ph.D.c, Charles Cha, M.D.a,b, Alan Dardik, M.D., Ph.D.a,b,* a
Department of Surgery, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave., New Haven, CT 06519, USA b Department of Surgery, Veterans Administration Connecticut Healthcare Systems, 950 Campbell Avenue, West Haven, CT 06516, USA c Department of Cardiovascular Surgery, Fujita Health University School of Medicine, 1-98 kutsu kake-cho, Nagoya, Aichi 470-1192, Japan Manuscript received June 23, 2005; revised manuscript July 15, 2005 Presented at the 29th Annual Surgical Symposium of the Association of VA Surgeons, Salt Lake City, Utah, March 11–13, 2005
Abstract Background: The incidence of vascular disease increases with age. Because atherosclerosis and neointimal hyperplasia colocalize in areas of disturbed shear stress, the effects of orbital shear stress (SS) on endothelial cell proliferation, protein kinase B (Akt) activation, and functional activity were analyzed using a senescence model. Methods: Early- (p3 to 7) and late- (p28 to 32) passage bovine aortic endothelial cells were exposed to orbital SS (210 rpm) or static conditions (0 to 5 days). Cell proliferation was directly counted and confirmed with proliferating cell nuclear antigen reactivity. Phosphorylated and total Akt were assessed with Western blotting. Endothelial cell–induced smooth muscle cell migration was assessed with a Boyden chamber. Results: Late-passage endothelial cells demonstrated no increase in orbital SS stimulated proliferation compared with early-passage cells (P ⫽ .42). Late-passage endothelial cells demonstrated decreased Akt phosphorylation in response to SS compared with early passage cells (n ⫽ 6, P ⫽ .01). Late-passage cells induced 26% less smooth muscle cell migration than early-passage cells (n ⫽ 3, P ⫽ .03). Conclusions: Late-passage endothelial cells demonstrate decreased proliferation, Akt phosphorylation, and secretion of smooth muscle cell chemoattractants in response to orbital SS compared with early passage cells. These results suggest that late-passage endothelial cells respond to SS differently than early-passage cells and confirm the utility of the in vitro senescence model. © 2005 Excerpta Medica Inc. All rights reserved. Keywords: Aging; Apoptosis; Endothelium; Protein kinase B; Senescence; Shear stress
The incidence of cardiovascular disease increases with age; rates of hypertension, stroke, and acute coronary syndrome all increase dramatically in both elderly men and women [1]. Age-associated changes in vasculature, including intimal-medial thickening and increased arterial stiffening, may play a key role in promoting atherosclerotic disease and hypertension [1]. The worsened clinical outcomes of myocardial infarction, stroke, and several vascular procedures in * Corresponding author. Tel.: ⫹1-203-737-2213; fax: ⫹1-203-7372290. E-mail address: [email protected]
elderly patients, compared with younger patients, remains an important clinical problem. For example, in 1 report, octogenarians undergoing carotid endarterectomy had higher mortality, illness severity, and length of stay compared with younger patients [2]. Vascular endothelial cells are exposed to hemodynamic forces such as cyclic strain and hydrostatic pressure; however, unlike the rest of the vessel wall, they are uniquely exposed to shear stress (SS), the lateral frictional force of the blood flow over the luminal endothelial surface [3]. It is thought that arterial levels of laminar SS are responsible for maintaining the normally quiescent phenotype of vascular
0002-9610/05/$ – see front matter © 2005 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2005.07.017
764
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
endothelium, promoting cell survival and inhibiting cellular proliferation [4 –7]. Unlike these atheroprotective effects of laminar SS, the presence of disturbed or oscillatory SS, such as at the carotid bifurcation, is believed to have an atherogenic effect on the vasculature, promoting plaque formation and neointimal hyperplasia [8 –11]. We previously demonstrated that orbital SS stimulates endothelial cell proliferation and secretion of smooth muscle cell chemoattractants and may be an in vitro model of complex flow in vivo such as found at the human carotid bulb [12,13]. Endothelial cell exposure to laminar SS stimulates phosphorylation of the intracellular kinase protein kinase B (Akt), which inhibits apoptosis through phosphorylation and inactivation of the proapoptotic factors Bad and procaspase-9, thereby promoting endothelial cell survival [14 –16]. Although laminar SS stimulates Akt phosphorylation, nonlaminar flow does not, suggesting that the Akt pathway may play a role in the differential endothelial response to laminar or turbulent shear stress in vivo [12]. Although the effects of laminar SS on endothelial cell activation have been reported, the effects of aging on endothelial cell function or activation have not been explored as comprehensively. In addition, the Akt pathway has also been implicated in aging and thus may link the effects of SS and aging. Downregulation of the insulin and insulin-like growth factor–1/Akt pathway by way of caloric restriction has been demonstrated to increase longevity in numerous species, suggesting a relationship between aging and Akt activity [17]. Aged endothelial cells may have high basal rates of apoptosis secondary to downregulation of endothelial nitric oxide synthase, decreasing caspase inactivation by way of inhibition of S-nitrosylation of critical cysteine residues. Aged endothelial cells also may not demonstrate increased eNOS activity in response to laminar SS as do younger cells, consistent with resistance to laminar SS, which usually promotes survival and inhibits apoptosis [18]. To examine the effects of senescence on the endothelial cell response to SS, we compared early- with late-passage endothelial cell proliferation, apoptosis, and secretion of smooth muscle cell chemoattractants in response to orbital SS. In addition, because Akt phosphorylation is stimulated by SS, and alterations of Akt activation are associated with aging, we compared early-with late-passage endothelial cell Akt phosphorylation in response to orbital SS.
as well as from freshly harvested animals. Endothelial cells were seeded on 6-well plates and exposed to either orbital SS or static conditions as previously described [12]. Senescence The senescence-associated -galactosidase (SA-gal) staining kit (Cell Signaling Technology, Beverly, MA) was used to confirm cellular senescence. Briefly, early- and late-passage endothelial cells were grown to confluence and washed with phosphate-buffered saline before fixation at room temperature for 10 to 15 minutes in 1x fixative solution (2% formaldehyde and 0.2% glutaraldehyde). Cells were then washed twice with phosphate-buffered saline and incubated overnight at 37°C in SA-gal staining solution: 930 L 1x staining solution (40 mM citric acid and sodium phosphate at pH 6.0, 150 mmol/L NaCl, and 2 mmol/L MgCl2); 10 L staining supplement A (500 mmol/L potassium ferrocyanide); 10 L staining supplement B (500 mmol/L potassium ferricyanide); and 50 L 20 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside powder) in dimethylformamide. Cells were observed for blue staining under a microscope with 200x magnification. Apoptosis The in-situ death kit was employed to determine apoptosis (Roche Molecular Biochemicals, Indianapolis, Indiana) according to the manufacturer’s instructions as previously described [12]. Cell counting Early- and late-passage cells were detached with trypsin and cell numbers directly counted with an automatic counter (model ZM; Coulter Electronics, Hialeah, Florida) before application of SS and after 1, 3, or 5 days of exposure to SS as previously described [20]. PCNA Proliferation was measured by assessing staining with anti-proliferating cell nuclear antigen (PCNA) antibody (Clone PC10; Sigma), as previously described [12]. Cell staining
Methods Endothelial and smooth muscle cell culture and SS application Bovine aortic endothelial cells and smooth muscle cells were isolated, cultured in vitro, and identified as previously described [19]. Endothelial cells were passaged in a 1:4 ratio for 28 to 32 passages in 10% serum. Control cells were early passage cells (p3 to 7) harvested from the same animal
Endothelial cell morphology was evaluated with crystal violet staining after exposure to SS as previously described [12]. Smooth muscle cell migration assay Endothelial cells were exposed to orbital SS or static conditions for 16 hours. Conditioned medium was removed and placed into the lower chamber of a modified Boyden
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
765
microchemotaxis chamber (NeuroProbe Inc, Gaithersburg, Maryland), and smooth muscle cell migration was directly counted after 4 hours as previously described [12]. Immunoblot technique Akt and p53 expression was evaluated with Western blotting using anti-Akt antibody, antiphospho-specific Akt antibody, or anti-p53 antibody (Cell Signaling, Beverly, Massechussetts), as previously described [20]. Specificity of Akt activation was determined with the upstream phosphatidylinositol-3-kinase (PI3K) inhibitor wortmannin (25 nM), which inhibits Akt activation in several cell types [20]. Data analysis Data are depicted as means ⫾ SEMs. Data were compared using analysis of variance (ANOVA) and analyzed with StatView 5.0.1 software (SAS, Cary, North Carolina). P ⱕ .05 was considered significant.
Results To examine the effects of SS on endothelial cells using a model of cell senescence, endothelial cells were passaged in vitro for 28 to 32 passages. Late-passage endothelial cells were identified as senescent by reactivity with SA-gal and expression of p53 (Fig. 1A). Because the literature has conflicting reports regarding the rate of apoptosis in senescent endothelial cells [18,21], we determined the rate of apoptosis in late-passage endothelial cells using terminal deoxynucleotidyl transferase–mediated nick-end labeling staining. Late-passage cells did not demonstrate a markedly high rate of apoptosis, but the percentage of cells was slightly higher than that observed in early-passage cells (n ⫽ 3, 3.7% vs. 1.5%, P ⬍ .0001, ANOVA, Fig. 1B). Orbital SS stimulates endothelial cell proliferation in vitro and may be a model for disturbed or turbulent flow [12]. As expected, early-passage endothelial cells demonstrated increased proliferation in response to orbital SS compared with static conditions (n ⫽ 3, 36% increase, day 5, P ⫽ .03, Fig. 2A). Late-passage cells had no statistically significant increase in proliferation in response to orbital SS (n ⫽ 3, 24% increase, P ⫽ .42, Fig. 2A). Because the increase in proliferation in the orbital SS model is confined to the center of the culture well [12], we confirmed the lack of late-passage cells’ response to orbital SS with proliferating cell nuclear antigen staining. Examination of the center and periphery of the culture wells demonstrated that the increased proliferation of early-passage endothelial cells exposed to SS for 24 hours was confined to the center of the culture well (n ⫽ 3, P ⬍ .0001, Fig. 2B). Late-passage cells exposed to orbital SS demonstrated no increase in proliferation either in the center or in the periphery of the culture well (Fig. 2B).
Fig. 1. (A) Identification of late-passage endothelial cells as senescent. Representative photomicrograph of senescence-associated -galactosidase staining of confluent early- (P4) and late-passage (P28) endothelial cells; magnification, 200x (n ⫽ 3). Representative photomicrograph of Western blot demonstrated increased p53 expression in late-passage endothelial cells; beta-tubulin bands demonstrate equivalent loading. (B) Basal levels of apoptosis were checked in early- and late-passage endothelial cells by terminal deoxynucleotidyl transferase–mediated nick-end labeling staining. Late passage endothelial cells demonstrated 2.5-fold increased levels of apoptosis compared with early-passage endothelial cells (P ⬍ .0001). EC ⫽ endothelial cells.
Because endothelial cells normally align and elongate in the direction of blood flow, the alignment of late-passage endothelial cells in response to orbital SS was compared with the response of early-passage cells. Early-passage cells elongated and aligned in the direction of flow in the periphery but not in the center of the culture well as previously reported (Fig. 3) [12]. Although late-passage endothelial cells in the periphery of the culture well were smaller in size compared with the cells cultured in the center of the well, cells in the periphery of the well did not align in the direction of flow; endothelial cells in the periphery of the well were randomly aligned and polygonal in shape (Fig. 3). Because orbital shear stress stimulates endothelial cells to secrete proteins that stimulate smooth muscle cell chemotaxis, we compared the ability of early- and late-passage cells to induce smooth muscle cell migration. Early- and late-passage endothelial cells had similar basal secretion of smooth muscle cell chemoattractants under static conditions. However, late-passage endothelial cells secreted fewer smooth muscle cell chemoattractants than early-passage endothelial cells in response to orbital SS (n ⫽ 3, P ⫽ .03, Fig. 4). Because changes in Akt activity are associated with effects of aging [22], and late-passage cells have slightly more apoptosis than early-passage cells (Fig. 1), we determined whether late-passage cells have decreased rates of
766
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
Fig. 2. (A) Endothelial cell number, direct counts. (B) Endothelial proliferation, PCNA staining. PCNA staining was assessed both in the center and in the periphery of the culture well. PCNA ⫽ proliferating cell nuclear antigen, SS ⫽ shear stress.
Akt phosphorylation compared with early-passage cells. Consistent with previous reports, early-passage cells exposed to SS demonstrated increased Akt phosphorylation compared with cells exposed to static conditions, whereas late-passage cells did not show such a significant increase (n ⫽ 6, P ⫽ .01, Fig. 5). Both early- and late-passage endothelial cells expressed significantly lower levels of Akt phosphorylation in the presence of wortmannin, confirming the specificity of Akt activation in both early- and latepassage endothelial cells through a phosphatidylinosital3-kinase–mediated pathway (data not shown).
Comments
Fig. 3. Representative photomicrographs of endothelial cells in the center and periphery of the culture well. Magnification 200x.
We demonstrated that late-passage endothelial cells had no increase in proliferation as well as decreased secretion of smooth muscle cell chemoattractants in response to SS compared with early-passage cells. Late-passage cells also had decreased Akt phosphorylation in response to SS compared with early passage cells. These results suggest that
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
Fig. 4. Bar graph depicts smooth muscle cell migration in response to exposure to conditioned medium from early or late passage endothelial cells under static or orbital shear stress conditions (n ⫽ 3). *P ⫽ .03. SMC ⫽ smooth muscle cells.
early- and late-passage endothelial cells differ in their responses to orbital SS. Turbulent SS stimulates cellular division and DNA replication, suggesting a mechanism for the localization of atherosclerosis to certain microenvironments such as the carotid bifurcation [23]. Although the effects of turbulent or disturbed SS have not been thoroughly studied in the context of aging, deficits in age-associated vascular function suggest that age-associated changes in vascular cells may play an important role in the development of clinically significant vascular disease. For instance, compared with younger rats, older rats have substantially increased neointimal growth and cellular proliferation in response to vessel injury [24]. Older rats also show increased levels of elastases and gelatinases such as type-2 metalloproteinase, increased levels of which may be responsible for defects in basement membrane integrity and concomitant hyperpermeability in smooth muscle cells and circulating inflammatory cells [25]. Endothelial dysfunction in aging rats—including increased expression of adhesion molecules, decreased endothelial nitric oxide synthase activity, and decreased vascular endothelial growth factor expression with accompanying impairments in angiogenesis—may predispose vessels to atherosclerotic damage [26 –28]. To mimic the in vivo process of aging in cells, we used an accepted in vitro model of cellular senescence. Cellular senescence is defined by the irreversible cessation of cellular and DNA replication with maintenance of cellular metabolic activity. The buildup with time of senescent cells in tissue is believed to influence aging, wound repair, tissue maintenance, and tumor growth [21]. Body tissue in elderly people is not devoid of proliferating or quiescent cells, and the majority of cells are not in a senescent state; however, it has been suggested that dysregulation of extracellular matrix components—including fibronectin, thrombospondin, osteonec-
767
tin, and procollagens derived from senescent cells—may disrupt overall tissue integrity and play a role in the aging process [21]. In addition, senescent cells display increased proteinase activity without corresponding increases in antiproteinase activity; this metabolic disproportion suggests the importance of senescence in stimulating tissue degeneration associated with aging [29]. Finally, the incidence of cellular senescence is proportional to both the replicative age of in vitro samples and the donor age of in vivo samples, confirming an association between senescence and aging [30,31]. We demonstrated decreased proliferation in late-passage endothelial cells in response to orbital SS (Figs. 2A and 2B). Early-passage cells demonstrated increased proliferation when exposed to orbital SS as previously described [12]. The lack of stimulation of proliferation of late-passage endothelial cells with orbital SS is consistent with previous studies of senescent cells. Compared with cells of early replicative age, senescent cells have alterations in the expression of growth regulatory genes—such as the inhibited expression of Id1 and Id2, c-fos, and mdm2—thus conferring resistance to mitogens and proliferative stimuli [21]. Senescent cells are further characterized by the upregulated expression of cyclin-dependent kinase (CDK) inhibitor p21 [32]. Overexpression of p21 inhibits several additional cellcycle regulatory elements, including cyclin-dependent kinases, Rb, and E2F [29]. These reports suggest several mechanisms by which late-passage endothelial cells are resistant to proliferative stimuli such as orbital SS. Unlike actively replicating cells, senescent cells have been shown to resist apoptosis through an increase in Bcl-2 expression [21]. Resistance to apoptosis is believed to contribute to long-term accumulation of senescent cells in aged tissue. We demonstrate that late-passage cells have slightly
Fig. 5. Bar graph represents the mean relative ratio of phosphorylated to total Akt both at 0 minutes and after 30 minutes of static conditions or orbital shear stress (n ⫽ 6). *P ⫽ .01. A representative Western Blot is shown; total Akt bands demonstrate equivalent loading. Akt ⫽ protein kinase B.
768
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
higher basal levels of apoptosis (Fig. 1B), similar to other reports [18]. The slightly higher rate of apoptosis in latepassage endothelial cells compared with early passage cells may reflect changes in either basal or inducible Akt activity. Exposure of early-passage endothelial cells to orbital SS has previously been shown to activate intracellular signal transduction cascades such as the PI3K-Akt pathway [6,12,33,34]. We demonstrated decreased Akt phosphorylation in response to orbital SS but similar basal phosphorylation levels as early passage cells (Fig. 5), suggesting that the basal PI3K-Akt axis is functional in late-passage endothelial cells, but it may be deficient in response to external stimuli such as SS. Akt activation has been shown to induce arrest of cellular division through a p53/p21– dependent pathway [35]. Consistent with this observation, late-passage cells demonstrated increased expression of p53 compared with early-passage cells (Fig. 1A). Akt also inhibits the mammalian forkhead transcription factor FOXO3a, an inhibitor of the p53/p21 pathway and a regulator of the expression of antioxidant genes that protect vascular endothelium from oxidative stress [35]. Vascular smooth muscle cell migration is a crucial step in the formation of atherosclerotic plaque and neointimal hyperplasia. When activated by cytokines or vessel injury, smooth muscle cells migrate into the intima and secrete extracellular matrix proteins, including proteoglycans, glycosaminoglycans, collagen, elastin, laminin, fibronectin, thrombospondin, and vitronectin [36]. Smooth muscle cells represent the largest cellular component of atherosclerotic plaque, and drugs such as sirolimus that inhibit smooth muscle cell proliferation and vascular inflammation also decrease restenosis [37]. We previously demonstrated that orbital SS stimulates early-passage endothelial cells to secrete smooth muscle cell chemoattractants such as PDGF-BB and IL-1␣, suggesting that orbital SS may be an in vitro model of the disturbed, or turbulent, shear forces that colocalize with plaque and neointima in vivo [13]. In this study, we demonstrate that late-passage endothelial cells secrete fewer smooth muscle cell chemoattractants than early-passage cells in response to orbital SS. Although these results suggest that senescent cells may not play a role in age-associated smooth muscle cell chemoattraction, these results may also reflect a limitation of our orbital SS model. However, senescent cells may be less responsive to therapeutic strategies optimized for younger cells and suggests decreased responsiveness to some therapies in elderly patients [38,39]. Although the senescence model affords a useful in vitro means to investigate potential links to aging, it is not a specific model of aging. To confirm the applicability of our senescence model to vascular pathology in older patients, results should be tested in a true aging model using cells harvested from young and old animals. In addition, results obtained using orbital SS should be confirmed using other models of turbulent flow, such as the cone and plate viscometer. Finally, midrange passages of cells could be tested
in this model to increase its applicability and potentially detect a dose response. We described decreased proliferation, Akt phosphorylation, and secretion of smooth muscle cell chemoattractants by late-passage endothelial cells in response to orbital SS. These results suggest that late-passage endothelial cells are senescent, with different properties than early passage cells, and provide rationale to study the effects of aging on vascular cells. Rational therapeutic strategies for vascular disease in elderly patients should therefore target pathologic changes associated with the aging process.
Acknowledgment This research was supported by the Dennis W. Jahnigen Career Development Scholarship Program, which is administered by the American Geriatrics Society, through an initiative funded by The John A. Hartford Foundation of New York City and The Atlantic Philanthropies (A. D., C. C.); an American College of Surgeons Faculty Research Fellowship (A. D., C. C.); and the Pacific Vascular Research Foundation (A. D.).
References [1] Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises. Part I: aging arteries: a “set up” for vascular disease. Circulation 2003;107:139 – 46. [2] Kazmers A, Perkins AJ, Huber TS, et al. Carotid surgery in octogenarians in Veterans Affairs medical centers. J Surg Res 1999;81:87–90. [3] Paszkowiak JJ, Dardik A. Arterial wall shear stress: observations from the bench to the bedside. Vasc Endovasc Surg 2003;37:47–57. [4] Dimmeler S, Haendeler J, Rippmann V, et al. Shear stress inhibits apoptosis of human endothelial cells. FEBS Lett 1996;399:71– 4. [5] Levesque MJ, Nerem RM, Sprague EA. Vascular endothelial cell proliferation in culture and the influence of flow. Biomaterials 1990; 11:702–7. [6] Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 1998;18:677– 85. [7] Lin K, Hsu PP, Chen BP, et al. Molecular mechanism of endothelial growth arrest by laminar shear stress. Proc Natl Acad Sci U S A 2000;97:9385–9. [8] Bakker SJ, Gans RO. About the role of shear stress in atherogenesis. Cardiovasc Res 2000;45:270 –2. [9] Ku DN, Giddens DP, Zarins CK, et al. Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis 1985;5:293–302. [10] Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA 1999;282:2035– 42. [11] Zarins CK, Giddens DP, Bharadvaj BK, et al. Carotid bifurcation atherosclerosis. Quantitative correlation of plaque localization with flow velocity profiles and wall shear stress. Circ Res 1983;53:502–14. [12] Dardik A, Chen L, Frattini J, et al. Differential effects of orbital and laminar shear stress on endothelial cells. J Vasc Surg 2005;41:869 – 80. [13] Dardik A, Yamashita A, Aziz F, et al. Shear stress-stimulated endothelial cells induce smooth muscle cell chemotaxis via platelet-derived growth factor-BB and interleukin-1a. J Vasc Surg 2005;41:321–31.
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769 [14] Dimmeler S, Assmus B, Hermann C, et al. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res 1998;83:334 – 41. [15] Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:231– 41. [16] Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318 –21. [17] Al-Regaiey KA, Masternak MM, Bonkowski M, et al. Long-lived growth hormone receptor knockout mice: interaction of reduced insulin-like growth factor i/insulin signaling and caloric restriction. Endocrinology 2005;146:851– 60. [18] Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res 2001;89:709 –15. [19] Haga M, Chen A, Gortler D, et al. Shear stress and cyclic strain may suppress apoptosis in endothelial cells by different pathways. Endothelium 2003;10:149 –57. [20] Haga M, Yamashita A, Paszkowiak J, et al. Oscillatory shear stress increases smooth muscle cell proliferation and Akt phosphorylation. J Vasc Surg 2003;37:1277– 84. [21] Raffetto JD, Leverkus M, Park HY, et al. Synopsis on cellular senescence and apoptosis. J Vasc Surg 2001;34:173–7. [22] Smith AR, Hagen TM. Vascular endothelial dysfunction in aging: loss of Akt-dependent endothelial nitric oxide synthase phosphorylation and partial restoration by (R)-alpha-lipoic acid. Biochem Soc Trans 2003;31(Pt 6):1447–9. [23] Helmlinger G, Geiger RV, Schreck S, et al. Effects of pulsatile flow on cultured vascular endothelial cell morphology. J Biomech Eng 1991;113:123–31. [24] Hariri RJ, Alonso DR, Hajjar DP, et al. Aging and arteriosclerosis. I. Development of myointimal hyperplasia after endothelial injury. J Exp Med 1986;164:1171– 8. [25] Cheng L, Mantile G, Pauly R, et al. Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo. Circulation 1998;98:2195–201. [26] Cernadas MR, Sanchez de Miguel L, Garcia-Duran M, et al. Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ Res 1998;83:279 – 86. [27] Rivard A, Fabre JE, Silver M, et al. Age-dependent impairment of angiogenesis. Circulation 1999;99:111–20.
769
[28] Rivard A, Berthou-Soulie L, Principe N, et al. Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity. J Biol Chem 2000;275: 29643–7. [29] Campisi J, Dimri GP, Nehlin JO, et al. Coming of age in culture. Exp Gerontol 1996;31:7–12. [30] Murano S, Thweatt R, Shmookler Reis RJ, et al. Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence. Mol Cell Biol 1991; 11:3905–14. [31] Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America 1995;92:9363–7. [32] Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 1994;211:90 – 8. [33] Go YM, Park H, Maland MC, et al. Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells. Am J Physiol 1998;275(5 Pt 2):H1898 –H1904. [34] Hu Y, Hochleitner BW, Wick G, et al. Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stressactivated protein kinases, in in vitro propagated endothelial cells. Exp Gerontol 1998;33:601–13. [35] Miyauchi H, Minamino T, Tateno K, et al. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway. Embo J 2004;23:212–20. [36] Raines EW. The extracellular matrix can regulate vascular cell migration, proliferation, and survival: relationships to vascular disease. Int J Exp Pathol 2000;81:173– 82. [37] Morice MC, Serruys PW, Sousa JE, et al. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 2002;346:1773– 80. [38] Fenton M, Barker S, Kurz DJ, et al. Cellular senescence after single and repeated balloon catheter denudations of rabbit carotid arteries. Arterioscler Thromb Vasc Biol 2001;21:220 – 6. [39] Fournet-Bourguignon MP, Castedo-Delrieu M, Bidouard JP, et al. Phenotypic and functional changes in regenerated porcine coronary endothelial cells: increased uptake of modified LDL and reduced production of NO. Circ Res 2000;86:854 – 61.
Paper
Differential responsiveness of early- and late-passage endothelial cells to shear stress Fabio A. Kudo, M.D., Ph.D.a,b, Bohdan Warycha, M.D.a,b, Peter J. Juran, B.A.a,b, Hidenori Asada, M.D.a,b, Desarom Teso, M.D.a,b, Faisal Aziz, M.D.a,b, Jared Frattini, M.D.a,b, Bauer E. Sumpio, M.D., Ph.D.a,b, Toshiya Nishibe, M.D., Ph.D.c, Charles Cha, M.D.a,b, Alan Dardik, M.D., Ph.D.a,b,* a
Department of Surgery, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave., New Haven, CT 06519, USA b Department of Surgery, Veterans Administration Connecticut Healthcare Systems, 950 Campbell Avenue, West Haven, CT 06516, USA c Department of Cardiovascular Surgery, Fujita Health University School of Medicine, 1-98 kutsu kake-cho, Nagoya, Aichi 470-1192, Japan Manuscript received June 23, 2005; revised manuscript July 15, 2005 Presented at the 29th Annual Surgical Symposium of the Association of VA Surgeons, Salt Lake City, Utah, March 11–13, 2005
Abstract Background: The incidence of vascular disease increases with age. Because atherosclerosis and neointimal hyperplasia colocalize in areas of disturbed shear stress, the effects of orbital shear stress (SS) on endothelial cell proliferation, protein kinase B (Akt) activation, and functional activity were analyzed using a senescence model. Methods: Early- (p3 to 7) and late- (p28 to 32) passage bovine aortic endothelial cells were exposed to orbital SS (210 rpm) or static conditions (0 to 5 days). Cell proliferation was directly counted and confirmed with proliferating cell nuclear antigen reactivity. Phosphorylated and total Akt were assessed with Western blotting. Endothelial cell–induced smooth muscle cell migration was assessed with a Boyden chamber. Results: Late-passage endothelial cells demonstrated no increase in orbital SS stimulated proliferation compared with early-passage cells (P ⫽ .42). Late-passage endothelial cells demonstrated decreased Akt phosphorylation in response to SS compared with early passage cells (n ⫽ 6, P ⫽ .01). Late-passage cells induced 26% less smooth muscle cell migration than early-passage cells (n ⫽ 3, P ⫽ .03). Conclusions: Late-passage endothelial cells demonstrate decreased proliferation, Akt phosphorylation, and secretion of smooth muscle cell chemoattractants in response to orbital SS compared with early passage cells. These results suggest that late-passage endothelial cells respond to SS differently than early-passage cells and confirm the utility of the in vitro senescence model. © 2005 Excerpta Medica Inc. All rights reserved. Keywords: Aging; Apoptosis; Endothelium; Protein kinase B; Senescence; Shear stress
The incidence of cardiovascular disease increases with age; rates of hypertension, stroke, and acute coronary syndrome all increase dramatically in both elderly men and women [1]. Age-associated changes in vasculature, including intimal-medial thickening and increased arterial stiffening, may play a key role in promoting atherosclerotic disease and hypertension [1]. The worsened clinical outcomes of myocardial infarction, stroke, and several vascular procedures in * Corresponding author. Tel.: ⫹1-203-737-2213; fax: ⫹1-203-7372290. E-mail address: [email protected]
elderly patients, compared with younger patients, remains an important clinical problem. For example, in 1 report, octogenarians undergoing carotid endarterectomy had higher mortality, illness severity, and length of stay compared with younger patients [2]. Vascular endothelial cells are exposed to hemodynamic forces such as cyclic strain and hydrostatic pressure; however, unlike the rest of the vessel wall, they are uniquely exposed to shear stress (SS), the lateral frictional force of the blood flow over the luminal endothelial surface [3]. It is thought that arterial levels of laminar SS are responsible for maintaining the normally quiescent phenotype of vascular
0002-9610/05/$ – see front matter © 2005 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2005.07.017
764
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
endothelium, promoting cell survival and inhibiting cellular proliferation [4 –7]. Unlike these atheroprotective effects of laminar SS, the presence of disturbed or oscillatory SS, such as at the carotid bifurcation, is believed to have an atherogenic effect on the vasculature, promoting plaque formation and neointimal hyperplasia [8 –11]. We previously demonstrated that orbital SS stimulates endothelial cell proliferation and secretion of smooth muscle cell chemoattractants and may be an in vitro model of complex flow in vivo such as found at the human carotid bulb [12,13]. Endothelial cell exposure to laminar SS stimulates phosphorylation of the intracellular kinase protein kinase B (Akt), which inhibits apoptosis through phosphorylation and inactivation of the proapoptotic factors Bad and procaspase-9, thereby promoting endothelial cell survival [14 –16]. Although laminar SS stimulates Akt phosphorylation, nonlaminar flow does not, suggesting that the Akt pathway may play a role in the differential endothelial response to laminar or turbulent shear stress in vivo [12]. Although the effects of laminar SS on endothelial cell activation have been reported, the effects of aging on endothelial cell function or activation have not been explored as comprehensively. In addition, the Akt pathway has also been implicated in aging and thus may link the effects of SS and aging. Downregulation of the insulin and insulin-like growth factor–1/Akt pathway by way of caloric restriction has been demonstrated to increase longevity in numerous species, suggesting a relationship between aging and Akt activity [17]. Aged endothelial cells may have high basal rates of apoptosis secondary to downregulation of endothelial nitric oxide synthase, decreasing caspase inactivation by way of inhibition of S-nitrosylation of critical cysteine residues. Aged endothelial cells also may not demonstrate increased eNOS activity in response to laminar SS as do younger cells, consistent with resistance to laminar SS, which usually promotes survival and inhibits apoptosis [18]. To examine the effects of senescence on the endothelial cell response to SS, we compared early- with late-passage endothelial cell proliferation, apoptosis, and secretion of smooth muscle cell chemoattractants in response to orbital SS. In addition, because Akt phosphorylation is stimulated by SS, and alterations of Akt activation are associated with aging, we compared early-with late-passage endothelial cell Akt phosphorylation in response to orbital SS.
as well as from freshly harvested animals. Endothelial cells were seeded on 6-well plates and exposed to either orbital SS or static conditions as previously described [12]. Senescence The senescence-associated -galactosidase (SA-gal) staining kit (Cell Signaling Technology, Beverly, MA) was used to confirm cellular senescence. Briefly, early- and late-passage endothelial cells were grown to confluence and washed with phosphate-buffered saline before fixation at room temperature for 10 to 15 minutes in 1x fixative solution (2% formaldehyde and 0.2% glutaraldehyde). Cells were then washed twice with phosphate-buffered saline and incubated overnight at 37°C in SA-gal staining solution: 930 L 1x staining solution (40 mM citric acid and sodium phosphate at pH 6.0, 150 mmol/L NaCl, and 2 mmol/L MgCl2); 10 L staining supplement A (500 mmol/L potassium ferrocyanide); 10 L staining supplement B (500 mmol/L potassium ferricyanide); and 50 L 20 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside powder) in dimethylformamide. Cells were observed for blue staining under a microscope with 200x magnification. Apoptosis The in-situ death kit was employed to determine apoptosis (Roche Molecular Biochemicals, Indianapolis, Indiana) according to the manufacturer’s instructions as previously described [12]. Cell counting Early- and late-passage cells were detached with trypsin and cell numbers directly counted with an automatic counter (model ZM; Coulter Electronics, Hialeah, Florida) before application of SS and after 1, 3, or 5 days of exposure to SS as previously described [20]. PCNA Proliferation was measured by assessing staining with anti-proliferating cell nuclear antigen (PCNA) antibody (Clone PC10; Sigma), as previously described [12]. Cell staining
Methods Endothelial and smooth muscle cell culture and SS application Bovine aortic endothelial cells and smooth muscle cells were isolated, cultured in vitro, and identified as previously described [19]. Endothelial cells were passaged in a 1:4 ratio for 28 to 32 passages in 10% serum. Control cells were early passage cells (p3 to 7) harvested from the same animal
Endothelial cell morphology was evaluated with crystal violet staining after exposure to SS as previously described [12]. Smooth muscle cell migration assay Endothelial cells were exposed to orbital SS or static conditions for 16 hours. Conditioned medium was removed and placed into the lower chamber of a modified Boyden
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
765
microchemotaxis chamber (NeuroProbe Inc, Gaithersburg, Maryland), and smooth muscle cell migration was directly counted after 4 hours as previously described [12]. Immunoblot technique Akt and p53 expression was evaluated with Western blotting using anti-Akt antibody, antiphospho-specific Akt antibody, or anti-p53 antibody (Cell Signaling, Beverly, Massechussetts), as previously described [20]. Specificity of Akt activation was determined with the upstream phosphatidylinositol-3-kinase (PI3K) inhibitor wortmannin (25 nM), which inhibits Akt activation in several cell types [20]. Data analysis Data are depicted as means ⫾ SEMs. Data were compared using analysis of variance (ANOVA) and analyzed with StatView 5.0.1 software (SAS, Cary, North Carolina). P ⱕ .05 was considered significant.
Results To examine the effects of SS on endothelial cells using a model of cell senescence, endothelial cells were passaged in vitro for 28 to 32 passages. Late-passage endothelial cells were identified as senescent by reactivity with SA-gal and expression of p53 (Fig. 1A). Because the literature has conflicting reports regarding the rate of apoptosis in senescent endothelial cells [18,21], we determined the rate of apoptosis in late-passage endothelial cells using terminal deoxynucleotidyl transferase–mediated nick-end labeling staining. Late-passage cells did not demonstrate a markedly high rate of apoptosis, but the percentage of cells was slightly higher than that observed in early-passage cells (n ⫽ 3, 3.7% vs. 1.5%, P ⬍ .0001, ANOVA, Fig. 1B). Orbital SS stimulates endothelial cell proliferation in vitro and may be a model for disturbed or turbulent flow [12]. As expected, early-passage endothelial cells demonstrated increased proliferation in response to orbital SS compared with static conditions (n ⫽ 3, 36% increase, day 5, P ⫽ .03, Fig. 2A). Late-passage cells had no statistically significant increase in proliferation in response to orbital SS (n ⫽ 3, 24% increase, P ⫽ .42, Fig. 2A). Because the increase in proliferation in the orbital SS model is confined to the center of the culture well [12], we confirmed the lack of late-passage cells’ response to orbital SS with proliferating cell nuclear antigen staining. Examination of the center and periphery of the culture wells demonstrated that the increased proliferation of early-passage endothelial cells exposed to SS for 24 hours was confined to the center of the culture well (n ⫽ 3, P ⬍ .0001, Fig. 2B). Late-passage cells exposed to orbital SS demonstrated no increase in proliferation either in the center or in the periphery of the culture well (Fig. 2B).
Fig. 1. (A) Identification of late-passage endothelial cells as senescent. Representative photomicrograph of senescence-associated -galactosidase staining of confluent early- (P4) and late-passage (P28) endothelial cells; magnification, 200x (n ⫽ 3). Representative photomicrograph of Western blot demonstrated increased p53 expression in late-passage endothelial cells; beta-tubulin bands demonstrate equivalent loading. (B) Basal levels of apoptosis were checked in early- and late-passage endothelial cells by terminal deoxynucleotidyl transferase–mediated nick-end labeling staining. Late passage endothelial cells demonstrated 2.5-fold increased levels of apoptosis compared with early-passage endothelial cells (P ⬍ .0001). EC ⫽ endothelial cells.
Because endothelial cells normally align and elongate in the direction of blood flow, the alignment of late-passage endothelial cells in response to orbital SS was compared with the response of early-passage cells. Early-passage cells elongated and aligned in the direction of flow in the periphery but not in the center of the culture well as previously reported (Fig. 3) [12]. Although late-passage endothelial cells in the periphery of the culture well were smaller in size compared with the cells cultured in the center of the well, cells in the periphery of the well did not align in the direction of flow; endothelial cells in the periphery of the well were randomly aligned and polygonal in shape (Fig. 3). Because orbital shear stress stimulates endothelial cells to secrete proteins that stimulate smooth muscle cell chemotaxis, we compared the ability of early- and late-passage cells to induce smooth muscle cell migration. Early- and late-passage endothelial cells had similar basal secretion of smooth muscle cell chemoattractants under static conditions. However, late-passage endothelial cells secreted fewer smooth muscle cell chemoattractants than early-passage endothelial cells in response to orbital SS (n ⫽ 3, P ⫽ .03, Fig. 4). Because changes in Akt activity are associated with effects of aging [22], and late-passage cells have slightly more apoptosis than early-passage cells (Fig. 1), we determined whether late-passage cells have decreased rates of
766
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
Fig. 2. (A) Endothelial cell number, direct counts. (B) Endothelial proliferation, PCNA staining. PCNA staining was assessed both in the center and in the periphery of the culture well. PCNA ⫽ proliferating cell nuclear antigen, SS ⫽ shear stress.
Akt phosphorylation compared with early-passage cells. Consistent with previous reports, early-passage cells exposed to SS demonstrated increased Akt phosphorylation compared with cells exposed to static conditions, whereas late-passage cells did not show such a significant increase (n ⫽ 6, P ⫽ .01, Fig. 5). Both early- and late-passage endothelial cells expressed significantly lower levels of Akt phosphorylation in the presence of wortmannin, confirming the specificity of Akt activation in both early- and latepassage endothelial cells through a phosphatidylinosital3-kinase–mediated pathway (data not shown).
Comments
Fig. 3. Representative photomicrographs of endothelial cells in the center and periphery of the culture well. Magnification 200x.
We demonstrated that late-passage endothelial cells had no increase in proliferation as well as decreased secretion of smooth muscle cell chemoattractants in response to SS compared with early-passage cells. Late-passage cells also had decreased Akt phosphorylation in response to SS compared with early passage cells. These results suggest that
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
Fig. 4. Bar graph depicts smooth muscle cell migration in response to exposure to conditioned medium from early or late passage endothelial cells under static or orbital shear stress conditions (n ⫽ 3). *P ⫽ .03. SMC ⫽ smooth muscle cells.
early- and late-passage endothelial cells differ in their responses to orbital SS. Turbulent SS stimulates cellular division and DNA replication, suggesting a mechanism for the localization of atherosclerosis to certain microenvironments such as the carotid bifurcation [23]. Although the effects of turbulent or disturbed SS have not been thoroughly studied in the context of aging, deficits in age-associated vascular function suggest that age-associated changes in vascular cells may play an important role in the development of clinically significant vascular disease. For instance, compared with younger rats, older rats have substantially increased neointimal growth and cellular proliferation in response to vessel injury [24]. Older rats also show increased levels of elastases and gelatinases such as type-2 metalloproteinase, increased levels of which may be responsible for defects in basement membrane integrity and concomitant hyperpermeability in smooth muscle cells and circulating inflammatory cells [25]. Endothelial dysfunction in aging rats—including increased expression of adhesion molecules, decreased endothelial nitric oxide synthase activity, and decreased vascular endothelial growth factor expression with accompanying impairments in angiogenesis—may predispose vessels to atherosclerotic damage [26 –28]. To mimic the in vivo process of aging in cells, we used an accepted in vitro model of cellular senescence. Cellular senescence is defined by the irreversible cessation of cellular and DNA replication with maintenance of cellular metabolic activity. The buildup with time of senescent cells in tissue is believed to influence aging, wound repair, tissue maintenance, and tumor growth [21]. Body tissue in elderly people is not devoid of proliferating or quiescent cells, and the majority of cells are not in a senescent state; however, it has been suggested that dysregulation of extracellular matrix components—including fibronectin, thrombospondin, osteonec-
767
tin, and procollagens derived from senescent cells—may disrupt overall tissue integrity and play a role in the aging process [21]. In addition, senescent cells display increased proteinase activity without corresponding increases in antiproteinase activity; this metabolic disproportion suggests the importance of senescence in stimulating tissue degeneration associated with aging [29]. Finally, the incidence of cellular senescence is proportional to both the replicative age of in vitro samples and the donor age of in vivo samples, confirming an association between senescence and aging [30,31]. We demonstrated decreased proliferation in late-passage endothelial cells in response to orbital SS (Figs. 2A and 2B). Early-passage cells demonstrated increased proliferation when exposed to orbital SS as previously described [12]. The lack of stimulation of proliferation of late-passage endothelial cells with orbital SS is consistent with previous studies of senescent cells. Compared with cells of early replicative age, senescent cells have alterations in the expression of growth regulatory genes—such as the inhibited expression of Id1 and Id2, c-fos, and mdm2—thus conferring resistance to mitogens and proliferative stimuli [21]. Senescent cells are further characterized by the upregulated expression of cyclin-dependent kinase (CDK) inhibitor p21 [32]. Overexpression of p21 inhibits several additional cellcycle regulatory elements, including cyclin-dependent kinases, Rb, and E2F [29]. These reports suggest several mechanisms by which late-passage endothelial cells are resistant to proliferative stimuli such as orbital SS. Unlike actively replicating cells, senescent cells have been shown to resist apoptosis through an increase in Bcl-2 expression [21]. Resistance to apoptosis is believed to contribute to long-term accumulation of senescent cells in aged tissue. We demonstrate that late-passage cells have slightly
Fig. 5. Bar graph represents the mean relative ratio of phosphorylated to total Akt both at 0 minutes and after 30 minutes of static conditions or orbital shear stress (n ⫽ 6). *P ⫽ .01. A representative Western Blot is shown; total Akt bands demonstrate equivalent loading. Akt ⫽ protein kinase B.
768
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769
higher basal levels of apoptosis (Fig. 1B), similar to other reports [18]. The slightly higher rate of apoptosis in latepassage endothelial cells compared with early passage cells may reflect changes in either basal or inducible Akt activity. Exposure of early-passage endothelial cells to orbital SS has previously been shown to activate intracellular signal transduction cascades such as the PI3K-Akt pathway [6,12,33,34]. We demonstrated decreased Akt phosphorylation in response to orbital SS but similar basal phosphorylation levels as early passage cells (Fig. 5), suggesting that the basal PI3K-Akt axis is functional in late-passage endothelial cells, but it may be deficient in response to external stimuli such as SS. Akt activation has been shown to induce arrest of cellular division through a p53/p21– dependent pathway [35]. Consistent with this observation, late-passage cells demonstrated increased expression of p53 compared with early-passage cells (Fig. 1A). Akt also inhibits the mammalian forkhead transcription factor FOXO3a, an inhibitor of the p53/p21 pathway and a regulator of the expression of antioxidant genes that protect vascular endothelium from oxidative stress [35]. Vascular smooth muscle cell migration is a crucial step in the formation of atherosclerotic plaque and neointimal hyperplasia. When activated by cytokines or vessel injury, smooth muscle cells migrate into the intima and secrete extracellular matrix proteins, including proteoglycans, glycosaminoglycans, collagen, elastin, laminin, fibronectin, thrombospondin, and vitronectin [36]. Smooth muscle cells represent the largest cellular component of atherosclerotic plaque, and drugs such as sirolimus that inhibit smooth muscle cell proliferation and vascular inflammation also decrease restenosis [37]. We previously demonstrated that orbital SS stimulates early-passage endothelial cells to secrete smooth muscle cell chemoattractants such as PDGF-BB and IL-1␣, suggesting that orbital SS may be an in vitro model of the disturbed, or turbulent, shear forces that colocalize with plaque and neointima in vivo [13]. In this study, we demonstrate that late-passage endothelial cells secrete fewer smooth muscle cell chemoattractants than early-passage cells in response to orbital SS. Although these results suggest that senescent cells may not play a role in age-associated smooth muscle cell chemoattraction, these results may also reflect a limitation of our orbital SS model. However, senescent cells may be less responsive to therapeutic strategies optimized for younger cells and suggests decreased responsiveness to some therapies in elderly patients [38,39]. Although the senescence model affords a useful in vitro means to investigate potential links to aging, it is not a specific model of aging. To confirm the applicability of our senescence model to vascular pathology in older patients, results should be tested in a true aging model using cells harvested from young and old animals. In addition, results obtained using orbital SS should be confirmed using other models of turbulent flow, such as the cone and plate viscometer. Finally, midrange passages of cells could be tested
in this model to increase its applicability and potentially detect a dose response. We described decreased proliferation, Akt phosphorylation, and secretion of smooth muscle cell chemoattractants by late-passage endothelial cells in response to orbital SS. These results suggest that late-passage endothelial cells are senescent, with different properties than early passage cells, and provide rationale to study the effects of aging on vascular cells. Rational therapeutic strategies for vascular disease in elderly patients should therefore target pathologic changes associated with the aging process.
Acknowledgment This research was supported by the Dennis W. Jahnigen Career Development Scholarship Program, which is administered by the American Geriatrics Society, through an initiative funded by The John A. Hartford Foundation of New York City and The Atlantic Philanthropies (A. D., C. C.); an American College of Surgeons Faculty Research Fellowship (A. D., C. C.); and the Pacific Vascular Research Foundation (A. D.).
References [1] Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises. Part I: aging arteries: a “set up” for vascular disease. Circulation 2003;107:139 – 46. [2] Kazmers A, Perkins AJ, Huber TS, et al. Carotid surgery in octogenarians in Veterans Affairs medical centers. J Surg Res 1999;81:87–90. [3] Paszkowiak JJ, Dardik A. Arterial wall shear stress: observations from the bench to the bedside. Vasc Endovasc Surg 2003;37:47–57. [4] Dimmeler S, Haendeler J, Rippmann V, et al. Shear stress inhibits apoptosis of human endothelial cells. FEBS Lett 1996;399:71– 4. [5] Levesque MJ, Nerem RM, Sprague EA. Vascular endothelial cell proliferation in culture and the influence of flow. Biomaterials 1990; 11:702–7. [6] Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 1998;18:677– 85. [7] Lin K, Hsu PP, Chen BP, et al. Molecular mechanism of endothelial growth arrest by laminar shear stress. Proc Natl Acad Sci U S A 2000;97:9385–9. [8] Bakker SJ, Gans RO. About the role of shear stress in atherogenesis. Cardiovasc Res 2000;45:270 –2. [9] Ku DN, Giddens DP, Zarins CK, et al. Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis 1985;5:293–302. [10] Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA 1999;282:2035– 42. [11] Zarins CK, Giddens DP, Bharadvaj BK, et al. Carotid bifurcation atherosclerosis. Quantitative correlation of plaque localization with flow velocity profiles and wall shear stress. Circ Res 1983;53:502–14. [12] Dardik A, Chen L, Frattini J, et al. Differential effects of orbital and laminar shear stress on endothelial cells. J Vasc Surg 2005;41:869 – 80. [13] Dardik A, Yamashita A, Aziz F, et al. Shear stress-stimulated endothelial cells induce smooth muscle cell chemotaxis via platelet-derived growth factor-BB and interleukin-1a. J Vasc Surg 2005;41:321–31.
F.A. Kudo et al. / The American Journal of Surgery 190 (2005) 763–769 [14] Dimmeler S, Assmus B, Hermann C, et al. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res 1998;83:334 – 41. [15] Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:231– 41. [16] Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318 –21. [17] Al-Regaiey KA, Masternak MM, Bonkowski M, et al. Long-lived growth hormone receptor knockout mice: interaction of reduced insulin-like growth factor i/insulin signaling and caloric restriction. Endocrinology 2005;146:851– 60. [18] Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res 2001;89:709 –15. [19] Haga M, Chen A, Gortler D, et al. Shear stress and cyclic strain may suppress apoptosis in endothelial cells by different pathways. Endothelium 2003;10:149 –57. [20] Haga M, Yamashita A, Paszkowiak J, et al. Oscillatory shear stress increases smooth muscle cell proliferation and Akt phosphorylation. J Vasc Surg 2003;37:1277– 84. [21] Raffetto JD, Leverkus M, Park HY, et al. Synopsis on cellular senescence and apoptosis. J Vasc Surg 2001;34:173–7. [22] Smith AR, Hagen TM. Vascular endothelial dysfunction in aging: loss of Akt-dependent endothelial nitric oxide synthase phosphorylation and partial restoration by (R)-alpha-lipoic acid. Biochem Soc Trans 2003;31(Pt 6):1447–9. [23] Helmlinger G, Geiger RV, Schreck S, et al. Effects of pulsatile flow on cultured vascular endothelial cell morphology. J Biomech Eng 1991;113:123–31. [24] Hariri RJ, Alonso DR, Hajjar DP, et al. Aging and arteriosclerosis. I. Development of myointimal hyperplasia after endothelial injury. J Exp Med 1986;164:1171– 8. [25] Cheng L, Mantile G, Pauly R, et al. Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo. Circulation 1998;98:2195–201. [26] Cernadas MR, Sanchez de Miguel L, Garcia-Duran M, et al. Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ Res 1998;83:279 – 86. [27] Rivard A, Fabre JE, Silver M, et al. Age-dependent impairment of angiogenesis. Circulation 1999;99:111–20.
769
[28] Rivard A, Berthou-Soulie L, Principe N, et al. Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity. J Biol Chem 2000;275: 29643–7. [29] Campisi J, Dimri GP, Nehlin JO, et al. Coming of age in culture. Exp Gerontol 1996;31:7–12. [30] Murano S, Thweatt R, Shmookler Reis RJ, et al. Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence. Mol Cell Biol 1991; 11:3905–14. [31] Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America 1995;92:9363–7. [32] Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 1994;211:90 – 8. [33] Go YM, Park H, Maland MC, et al. Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells. Am J Physiol 1998;275(5 Pt 2):H1898 –H1904. [34] Hu Y, Hochleitner BW, Wick G, et al. Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stressactivated protein kinases, in in vitro propagated endothelial cells. Exp Gerontol 1998;33:601–13. [35] Miyauchi H, Minamino T, Tateno K, et al. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway. Embo J 2004;23:212–20. [36] Raines EW. The extracellular matrix can regulate vascular cell migration, proliferation, and survival: relationships to vascular disease. Int J Exp Pathol 2000;81:173– 82. [37] Morice MC, Serruys PW, Sousa JE, et al. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 2002;346:1773– 80. [38] Fenton M, Barker S, Kurz DJ, et al. Cellular senescence after single and repeated balloon catheter denudations of rabbit carotid arteries. Arterioscler Thromb Vasc Biol 2001;21:220 – 6. [39] Fournet-Bourguignon MP, Castedo-Delrieu M, Bidouard JP, et al. Phenotypic and functional changes in regenerated porcine coronary endothelial cells: increased uptake of modified LDL and reduced production of NO. Circ Res 2000;86:854 – 61.