Aging Impairs the Beneficial Effect of Granulocyte Colony-Stimulating Factor and Stem Cell Factor on Post-Myocardial Infarction Re
http://www.100md.com
Stephanie Lehrke, Ramesh Mazhari, Daniel
参见附件。
the Department of Medicine, Division of Cardiology (S.L., R.M., D.J.D., M.Z., J.M.Z., K.H.S., A.S.C., J.M.H.)
The Institute for Cell Engineering (ICE) (S.L., R.M., D.J.D., M.Z., J.M.Z., K.H.S., A.S.C., J.M.H.)
the Department of Comparative Medicine (D.B., J.M.H.), Johns Hopkins University School of Medicine, Baltimore, Md.
Abstract
Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) are potential new therapies to ameliorate post-myocardial infarction (post-MI) remodeling, as they enhance endogenous cardiac repair mechanisms and decrease cardiomyocyte apoptosis. Because both of these pathways undergo alterations with increasing age, we hypothesized that therapeutic efficacy of G-CSF and SCF is impaired in old versus young adult rats. MI was induced in 6- and 20-month-old rats by permanent ligation of the left coronary artery. In young animals, G-CSF/SCF therapy stabilized and reversed a decline in cardiac function, attenuated left ventricular dilation, decreased infarct size, and reduced cardiomyocyte hypertrophy. Remarkably, these effects on cardiac structure and function were absent in aged rodents. This could not be attributed to ineffective mobilization of bone marrow cells or decreased quantity of c-Kit+ cells within the myocardium with aging. However, whereas the G-CSF/SCF cocktail reduced cardiac myocyte apoptosis in old as well as in young hearts, the degree of reduction was substantially less with age and the rate of cardiomyocyte apoptosis in old animals remained high despite cytokine treatment. These findings demonstrate that G-CSF/SCF lacks therapeutic efficacy in old animals by failing to offset periinfarct apoptosis and therefore raise important concerns regarding the efficacy of novel cytokine therapies in elderly individuals at greatest risk for adverse consequences of MI.
Key Words: aging myocardial infarction remodeling growth substances
Introduction
The administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) is proposed as a potential new therapy for cardiovascular regenerative medicine. Animal studies demonstrate that G-CSF alone or in combination with SCF ameliorate post-myocardial infarction (post-MI) remodeling.1–4 Early clinical trials appeared to support these findings,5–7 and initial concerns of stimulating restenosis in the infarct-related artery8 have not been substantiated.9,10 However, a recent randomized controlled trial in patients with a mean age of 60 years of age failed to demonstrate benefit from G-CSF administration following MI, despite ample CD34+ cell mobilization.11
One of the main mechanisms by which G-CSF and SCF exert favorable effects on cardiac remodeling is enhancement of endogenous cardiac repair mechanisms that include both bone marrow stem cell mobilization, engraftment, and differentiation12–15 as well as proliferation of cardiomyocytes.16 In addition, G-CSF inhibits cardiomyocyte apoptosis17 and accelerates healing by stimulating absorption of necrotic tissue and reducing granulation and scar tissue via expression of matrix metalloproteinases.3
It is important to address whether potentially new cardiac regenerative therapies will benefit elderly patients who, despite reperfusion therapy, have increased MI-related mortality and morbidity.18,19 This higher vulnerability can only partially be attributed to comorbidities, and there is increasing evidence for impaired endogenous cardiac repair mechanisms with aging. Increased age is associated with reduced angiogenic capacity20 and diminished cell cycling of cardiac stem cells.21,22 In addition, there is increased cardiomyocyte apoptosis in the aged heart both at baseline23 and after ischemia, which could contribute to the adverse prognosis of elderly individuals.24,25
Given emerging evidence of impaired regenerative capacity of the aging heart coupled with its higher propensity for apoptotic cell death, the key question arises of whether these defects, which are the target mechanisms for the cardioprotective effect of G-CSF/SCF, will attenuate its efficacy. Here using an established rodent model of cardiovascular aging we tested the hypothesis that G-CSF/SCF would have reduced efficacy in old compared with young adult animals because of impaired target mechanisms.
Materials and Methods
All procedures in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and were approved by the Johns Hopkins University Animal Care and Use Committee.
Rat MI Model
MI was induced by permanent left coronary artery ligation in 6- and 20-month-old F344 rats purchased from the National Institute on Aging. Twenty-month-old Fisher rats are shown to have physiological signs of cardiovascular aging and correspond approximately to humans of 50 to 60 years of age.26,27 Rats were intubated endotracheally and ventilated with 2% isoflurane in oxygen using a rodent respirator (Model 683, Harvard Apparatus Inc). The heart was exposed via a left lateral thoracotomy, and the left anterior descending coronary artery was ligated 3 mm below its origin with a 6-0 silk suture.
Four hours post-MI, rats were randomly assigned to receive a first subcutaneous injection of recombinant human G-CSF (100 μg/kg) and rat-SCF (200 μg/kg; both Amgen, Thousand Oaks, Calif) followed by daily injections for 5 days. Placebo animals received equal volumes of isotonic saline. Rats (n=29, young; n=25, old) were followed by serial echocardiography for the development of cardiac function and geometry. The echocardiographer was fully blinded to the treatment group. The animals were allocated into treatment (n=15, young; n=12, old) and placebo (n=14, young; n=13, old) groups. A second subset of animals was euthanized 7 days after MI for the assessment of circulating c-Kit+ and CD34+ cells and myocardial apoptosis and were allocated into treatment (n=5, young; n=5, old) and placebo (n=5, young; n=6, old).
Mortality within 48 hours of MI was higher in 20-month-old compared with 6-month-old (27/63 [42.9%] versus 7/46 [15.2%]; P=0.003) rats without an effect of cytokine treatment. Only rats surviving beyond 48 hours were included in the primary long-term analyses.
Echocardiography
Transthoracic echocardiograms were obtained using a Sonos 5500 (Agilent, Hewlett Packard, Palo Alto, Calif) equipped with a 15-MHz linear transducer. For additional details, see the online data supplement, available at http://circres.ahajournals.org.
Complete Blood Count/Flow Cytometric Analysis of CD34+ and c-Kit+ Cells
Complete white blood counts and numbers of circulating CD34+ and c-Kit+ cells were determined at baseline and at day 7 after MI and cytokine administration by FACS analysis (Becton Dickinson). For additional details, see the online data supplement.
Histology
Infarct size and myocyte width in noninfarcted remote myocardium were determined from explanted hearts. Immunohistochemistry for c-Kit (DAKO rabbit anti-human CD117 antibody; A 4502) and mast cells were performed using deparaffinized and rehydrated tissue sections. Apoptosis was quantified by TUNEL analysis. For additional details, see the online data supplement.
Western Blot Analysis
Western blots were performed on heart extracts as described by Raju et al28 using polyclonal anti-phospho-STAT3 antibody (1:1000, Cell Signaling Technology), polyclonal anti-phospho-ERK antibodies (1:1000, Cell Signaling Technology Inc), and anti-phospho-caspase-3 (SC-7148, Santa Cruz Biotechnology). Immunoblots were detected using enhanced chemiluminescent Kits (SuperSignal; Pierce) and analyzed with a densitometer (Bio-Rad). Membranes were stripped and reprobed with polyclonal anti-STAT3 antibody (1:1000, Cell Signaling Technology), polyclonal anti-ERK antibodies (1:1000, Cell Signaling Technology Inc), or monoclonal anti-GAPDH antibodies (1:10 000, Research Diagnostics Inc).
Results
Cardiac Function and Structure
Echocardiographic parameters at baseline and at 2 days after MI were not different between age and treatment groups (Table 1). Importantly, at day 2 after MI, 2D ejection fraction (EF) showed a similar decline in all 4 groups, suggesting a comparable degree of initial myocardial injury (Table 1 and Figure 1A).
Induction of MI resulted in a substantial impairment of cardiac function, as left ventricular EF decreased significantly during the first 2 weeks in all groups (P<0.0001) without any effects of cytokine treatment or age (P=NS). Whereas ventricular function remained severely impaired during the 8-week follow-up period among young placebo animals, there was a dramatic improvement of EF from week 2 to 8 among young G-CSF/SCF-treated rats (P=0.0001; absolute increase, 22.5±3.8%), reversing the initial severe left ventricular dysfunction (Figure 1A) and representing an almost 50% recovery. Remarkably, this effect of cytokine treatment was completely absent in old animals, where EF in both treatment and placebo groups deteriorated similarly to young placebo animals and remained low until week 8 (Figure 1B). Similarly, whereas the G-CSF/SCF cocktail ameliorated left ventricular enlargement in young animals (P=0.003), this effect was completely absent in old animals (Figure 1C and 1D).
Effect of G-CSF/SCF on Infarct Size and Compensatory Cardiomyocyte Hypertrophy
Induction of MI resulted in the development of substantial transmural scarring of the left ventricle. Infarct size expressed as percentage of left ventricular perimeter was comparable between young placebo (26.1±3.1%), old G-CSF/SCF-treated (27±6%) and old placebo animals (28.1±4.9%) but was significantly reduced in young G-CSF/SCF-treated rats (14.7±3.5%; P=0.04 versus young placebo; Figure 2A).
Reactive hypertrophy of cardiomyocytes in areas remote from the scar region was evident in both age groups. Whereas the cytokine treatment completely prevented this compensatory myocyte hypertrophy in young animals (P<0.0001 versus young placebo; Figure 2B), it was without effect in the old rats.
White Blood Count/Circulating CD34+ and c-Kit+ Cells
Age-associated hematological changes at baseline are summarized in Table 2. These findings are in accordance with those reported previously.29 The percentage of circulating mononuclear cells expressing CD34 (0.45±0.04% young versus 0.55±0.07% old) and c-Kit (0.36±0.07% young versus 0.46±0.07% old) did not differ significantly between age groups at baseline. At 1 week post-MI, the neutrophil count was markedly elevated in G-CSF/SCF-treated groups in both young (2.17±0.25 103/μL versus 0.87±0.05 103/μL at baseline, P=0.008) and old (3.16±0.69 103/μL versus 1.19±0.28 103/μL at baseline, P=0.04) animals. Whereas the percentage of CD34+ cells was increased compared with baseline in all groups regardless of treatment, the percentage of circulating c-Kit+ cells remained relatively unchanged in all groups (Figure 3A and 3B). See Figure I in the online data supplement.
Interestingly, G-CSF/SCF treatment increased splenic weight among young (996±42 mg treated versus 828±25 mg placebo; P<0.01) but not among old rats (1570±157 mg treated versus 1345±124 mg placebo; P=NS). This difference remained significant after correction for body weight.
c-Kit+ Cells in the Peri-infarct Area
Next we examined the effect of G-CSF/SCF on the number of c-Kit+ cells in the infarct region. Cells expressing membrane c-Kit were detected in the peri-infarct zone at day 7 after MI and were 2-fold more abundant in young versus old animals, although this did not reach statistical significance. Cytokine treatment increased c-Kit+ cells in young (&14-fold; P<0.01 versus young placebo) and old (&17-fold; P<0.05 versus old placebo; Figure 4A) rats. Although in old rats, it did not reach a level exceeding that of young rats. Thus, the lack of response to G-CSF/SCF in old rats is not attributable to the absence of c-Kit+ cell mobilization within the heart. At 8 weeks after MI, cardiac c-Kit+ cells were essentially undetectable in all groups (data not shown).
Effect of G-CSF/SCF on Myocyte Apoptosis
As identified by TUNEL staining, cardiomyocyte apoptosis in the infarct border zone as well as in remote areas was significantly increased with old age in both placebo and treatment groups. G-CSF/SCF treatment reduced cardiomyocyte apoptosis in the infarct border zone in both young (28±1.7% versus 36.4±2.8% young placebo; P<0.05) and old (41.0±2.9% versus 51.3±2.5% old placebo; P<0.01; Figure 5A) animals. In areas remote from the infarct, cytokine treatment diminished cardiac myocyte apoptosis in young animals, from 37.8±3.5% to 25.7±1.4% (P<0.01), but did not reduce apoptosis in old animals (Figure 5B). Importantly, old G-CSF/SCF-treated rats had higher apoptotic indices than young placebo animals. Thus, the response to G-CSF/SCF was profoundly impaired in old versus young hearts.
Western Blot Analysis
Western blot analysis of the molecular pathways involved in apoptosis revealed increased activity of STAT3, a pathway implicated in G-CSF cardiac protection, following G-CSF/SCF treatment in young but not old infarcted rats (Figure 6A and 6B). On the other hand, activation of the proapoptotic caspase-3 pathway was greater in old versus young rats after MI (P<0.05 for old cytokines versus no cytokines). Importantly, G-CSF/SCF treatment did not reduce caspase-3 activation in old rats following MI (Figure 6C and 6D). Finally, the extracellular signal-regulated kinase (ERK) pathway was activated after MI in both old and young rats (P<0.01 in young and P<0.02 in old rats), and this was not affected by cytokine treatment (Figure 6E and 6F).
Discussion
The major new finding of this study is that the efficacy of G-CSF and SCF to attenuate post-MI remodeling is dramatically reduced with age. Whereas in young animals, the cytokine treatment markedly improved cardiac function, decreased infarct size, and ameliorated cardiomyocyte hypertrophy in residual myocytes, these effects were absent in post-MI aged rodents. Importantly, this was not attributable to reduced mobilization of stem cells in the circulation or heart. Rather, the degree of reduction in apoptosis was substantially less in old versus young hearts. In light of ongoing clinical trials, these findings have important implications for assessing novel therapies in elderly individuals at greatest risk for adverse consequences of MI.
Our findings in young animals agree with those of Orlic et al, who first demonstrated the potential of G-CSF/SCF to repair cardiac injury by mobilization of bone marrow stem cells.1 A number of subsequent animal studies confirmed the healing effect of G-CSF given alone or in combination with SCF and suggested additional mechanisms by which the cardioprotective effect is achieved, including antiapoptotic effects,17 enhanced cardiomyocyte proliferation,16 and side population cell mobilization.12 Dawn et al have recently shown that either SCF or Flt-3 ligand added to G-CSF are superior to G-CSF alone in reversing post-MI remodeling and causing bone marrow mobilization.15
This study offers important new insights with regard to age as a host determinant limiting responsiveness to G-CSF/SCF. MI increases in frequency as a function of age, and the elderly also experience higher mortality and morbidity.18,19,30 The aged heart shows marked physiological and structural alterations and varies significantly in its response to different stressors, including ischemia.31,32 Compared with young hearts, aged hearts exhibit an elevated rate of apoptotic cardiomyocytes both at baseline23 and after ischemia/reperfusion injury.24 A lower capacity for endogenous cardiac repair with aging is supported by reports of reduced ability for neoangiogenesis20 and cardiac stem cell senescence.21,22,33 G-CSF and SCF exert their effects by targeting these mechanisms that are altered in the aging heart.
The mobilization of bone marrow stem cells, their homing to the injured heart, and consequent cardiac regeneration are the main underpinnings of the beneficial effect of G-CSF/SCF on cardiac remodeling.1,4,13,15 Advanced age is a well-known limiting factor for the mobilization of bone marrow cells in donors being treated with G-CSF for leukopheresis of hematopoietic progenitor cells.34 We excluded poor bone marrow cell mobilization as the reason for the lack of effect seen in aged animals by demonstrating similar increases of neutrophils and circulating CD34+ and c-Kit+ cells after G-CSF/SCF in both age groups. Furthermore, the number of c-Kit+ cells in the peri-infarct area was similarly elevated by G-CSF/SCF treatment regardless of age. These cells have the potential to originate either from bone marrow1,35 or from the heart itself, where they represent endogenous cardiac stem cells.36 There is increasing support for the presence of an endogenous population of c-Kit+ cardiac stem cells that proliferate in response to injury and that may be targets for novel approaches of activation.37
There is also evidence that cardiac stem cells, depleted after MI, are replenished by both endogenous proliferation and mobilization of bone marrow-derived stem cells. In this regard, bone marrow-derived mesenchymal stem cells have lower colony formation activity in old compared with young rats.38,39 In addition to endogenous defects in either cardiac or bone marrow-derived cardiac precursor cells, reduced receptiveness to cellular regeneration may be another important mechanism for impaired cardiac repair in old adult animals. Support for this view is emerging, with data showing reduced responsiveness to cardiac regenerative therapy with intravenous MSC infusion in old versus young rats.40 Work is ongoing to explore these different potential mechanistic components.
In addition to ischemia induced necrosis, apoptosis contributes to cardiomyocyte loss after MI.23,41–43 Experimental studies show that apoptosis of cardiomyocytes remains elevated for months after the infarct and correlates with postinfarction left-ventricular remodeling.44 G-CSF is directly antiapoptotic in cardiomyocytes, endothelial cells, and myofibroblasts,17 activating its myocardial cell-surface receptor and leading, in turn, to JAK/Stat pathway activation and stimulation of the antiapoptotic protein bcl-2. In line with these findings, we demonstrated a reduction of apoptotic cells associated with Stat3 phosphorylation by G-CSF/SCF treatment in young animals. Whereas apoptosis of cardiomyocytes in infarct, border zone, and remote myocardium was reduced in young animals, only cardiomyocyte apoptosis in the infarct border zone was decreased in old animals. Additionally, G-CSF/SCF did not activate Stat3 or reduce phospho-caspase-3 in old hearts. Furthermore, apoptotic indices in old treated animals equaled or exceeded those of young control animals.
Whereas G-CSF/SCF increased c-Kit+ cells in the injured myocardium in old animals, they failed to reduce cardiomyocyte apoptosis to levels seen in young animals. In the context of a balance between the potential for new tissue formation through c-Kit+ cells and cardiomyocyte loss through apoptosis, the latter outweighs the former in aged animals, likely explaining the lack of cardioprotective effect in the old.
There is increasing evidence for reduced efficacy of other cytokine pathways with age, including reduced platelet-derived growth factor (PDGF)-AB-induced neoangiogenesis45 (which can be rescued by endothelial progenitor cells from young animals46) and reduced tumor necrosis factor (TNF)- cardioprotection attributable to downregulated TNF- receptors.46 Cytokine receptors are downregulated with aging in organs other than the heart,47 and a similar phenomenon could contribute to the present results.
Intriguingly, data from several human trials of G-CSF therapy following MI show a variance that may be related to the mean age of the study subjects. For example, 3 studies,5,9,11 each with subjects of a mean age of 60 years or more, found no statistically significant benefits in any objectively measured parameter. In these trials, the only significant clinical benefit was a reduction in subjective symptoms of myocardial ischemia in the 13 patients reported by Wang et al.5 In light of our results, it is interesting to note that the largest of these studies11 showed that older patients were capable of effectively mobilizing the CD34+ cells, suggesting that the reduced efficacy in this population can be attributed to a local defect in response to G-CSF therapy. Taken together, these reports contrast sharply with more encouraging data from 2 studies with subjects of mean ages of 507 and 586 years. The latter studies showed statistically significant benefits across a breadth of objective end points, including regional wall motion, myocardial perfusion, and EF, suggesting that younger patients may benefit substantially from these therapies.
In summary, we show that the beneficial effects of G-CSF/SCF on post-MI cardiac function and remodeling are dramatically diminished with age. Mobilization of circulating precursor cells and increased cardiac c-Kit+ cells were preserved in the older animals. This loss of responsiveness could be attributed, at least in part, to failure of G-CSF/SCF to adequately reduce apoptosis particularly in remote areas in rats of advanced age. At a molecular level, G-CSF failed to activate the anti-apoptotic pathways in older animals. Together, these findings have important mechanistic and therapeutic implications in an aging population at high risk for MI and its complications and need to be taken into consideration in future clinical trials.
Acknowledgments
The G-CSF and rat-SCF were kindly provided by Amgen (Thousand Oaks, Calif).
Sources of Funding
This work was supported by The Johns Hopkins University School of Medicine Institute for Cell Engineering (ICE), The Donald W. Reynolds Foundation, and NIH grants RO1 HL-65455, R21 HL-72185, and RO1 NIA AG078915.
Disclosures
None.
Footnotes
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received March 14, 2006; revision received July 14, 2006; accepted July 17, 2006.
References
Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001; 98: 10344–10349.
Iwanaga K, Takano H, Ohtsuka M, Hasegawa H, Zou Y, Qin Y, Odaka K, Hiroshima K, Tadokoro H, Komuro I. Effects of G-CSF on cardiac remodeling after acute myocardial infarction in swine. Biochem Biophys Res Commun. 2004; 325: 1353–1359. [Order article via Infotrieve]
Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai M, Misao Y, Lu C, Suzuki K, Goto K, Komada A, Takahashi T, Kosai K, Fujiwara T, Fujiwara H. Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac function and remodeling by postinfarction granulocyte colony-stimulating factor treatment. Circulation. 2004; 109: 2572–2580.
Norol F, Merlet P, Isnard R, Sebillon P, Bonnet N, Cailliot C, Carrion C, Ribeiro M, Charlotte F, Pradeau P, Mayol JF, Peinnequin A, Drouet M, Safsafi K, Vernant JP, Herodin F. Influence of mobilized stem cells on myocardial infarct repair in a nonhuman primate model. Blood. 2003; 102: 4361–4368.
Wang Y, Tagil K, Ripa RS, Nilsson JC, Carstensen S, Jorgensen E, Sondergaard L, Hesse B, Johnsen HE, Kastrup J. Effect of mobilization of bone marrow stem cells by granulocyte colony stimulating factor on clinical symptoms, left ventricular perfusion and function in patients with severe chronic ischemic heart disease. Int J Cardiol. 2005; 100: 477–483. [Order article via Infotrieve]
Kuethe F, Figulla HR, Herzau M, Voth M, Fritzenwanger M, Opfermann T, Pachmann K, Krack A, Sayer HG, Gottschild D, Werner GS. Treatment with granulocyte colony-stimulating factor for mobilization of bone marrow cells in patients with acute myocardial infarction. Am Heart J. 2005; 150: 115. [Order article via Infotrieve]
Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T, Schumichen C, Freund M, Nienaber CA. Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005; 112: 3097–3106.
Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet. 2004; 363: 751–756. [Order article via Infotrieve]
Valgimigli M, Rigolin GM, Cittanti C, Malagutti P, Curello S, Percoco G, Bugli AM, Della PM, Bragotti LZ, Ansani L, Mauro E, Lanfranchi A, Giganti M, Feggi L, Castoldi G, Ferrari R. Use of granulocyte-colony stimulating factor during acute myocardial infarction to enhance bone marrow stem cell mobilization in humans: clinical and angiographic safety profile. Eur Heart J. 2005; 26: 1838–1845.
Jorgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P, Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization by granulocyte-colony stimulating factor. Preliminary intracoronary ultrasound results from a double-blind randomized placebo-controlled study of patients treated with percutaneous coronary intervention for ST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. 2006; 111: 174–177. [Order article via Infotrieve]
Zohlnhofer D, Ott I, Mehilli J, Schomig K, Michalk F, Ibrahim T, Meisetschlager G, von Wedel J, Bollwein H, Seyfarth M, Dirschinger J, Schmitt C, Schwaiger M, Kastrati A, Schomig A. Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA. 2006; 295: 1003–1010.
Adachi Y, Imagawa J, Suzuki Y, Yogo K, Fukazawa M, Kuromaru O, Saito Y. G-CSF treatment increases side population cell infiltration after myocardial infarction in mice. J Mol Cell Cardiol. 2004; 36: 707–710. [Order article via Infotrieve]
Fukuhara S, Tomita S, Nakatani T, Ohtsu Y, Ishida M, Yutani C, Kitamura S. G-CSF promotes bone marrow cells to migrate into infarcted mice heart, and differentiate into cardiomyocytes. Cell Transplant. 2004; 13: 741–748. [Order article via Infotrieve]
Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H, Muguruma Y, Tsuboi K, Itabashi Y, Ikeda Y, Ogawa S, Okano H, Hotta T, Ando K, Fukuda K. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood. 2004; 104: 3581–3587.
Dawn B, Guo YR, Rezazadeh A, Huang YM, Stein AB, Hunt G, Tiwari S, Varma J, Gu Y, Prabhu SD, Kajstura J, Anversa P, Ildstad ST, Bolli R. Postinfarct cytokine therapy regenerates cardiac tissue and improves left ventricular function. Cir Res. 2006; 98: 1098–1105.
Hamamoto M, Tomita S, Nakatani T, Yutani C, Yamashiro S, Sueda T, Yagihara T, Kitamura S. Granulocyte-colony stimulating factor directly enhances proliferation of human troponin I-positive cells derived from idiopathic dilated cardiomyopathy through specific receptors. J Heart Lung Transplant. 2004; 23: 1430–1437. [Order article via Infotrieve]
Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara K, Komuro I. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med. 2005; 11: 305–311. [Order article via Infotrieve]
Aguirre FV, McMahon RP, Mueller H, Kleiman NS, Kern MJ, Desvigne-Nickens P, Hamilton WP, Chaitman BR. Impact of age on clinical outcome and postlytic management strategies in patients treated with intravenous thrombolytic therapy. Results from the TIMI II Study. TIMI II Investigators. Circulation. 1994; 90: 78–86.
Thiemann DR, Coresh J, Schulman SP, Gerstenblith G, Oetgen WJ, Powe NR. Lack of benefit for intravenous thrombolysis in patients with myocardial infarction who are older than 75 years. Circulation. 2000; 101: 2239–2246.
Reed MJ, Edelberg JM. Impaired angiogenesis in the aged. Sci Aging Knowledge Environ. 2004; 2004: e7.
Chimenti C, Kajstura J, Torella D, Urbanek K, Heleniak H, Colussi C, Di Meglio F, Nadal-Ginard B, Frustaci A, Leri A, Maseri A, Anversa P. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res. 2003; 93: 604–613.
Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A, Sussman MA, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri A. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94: 514–524.
Kajstura J, Cheng W, Sarangarajan R, Li P, Li B, Nitahara JA, Chapnick S, Reiss K, Olivetti G, Anversa P. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol. 1996; 271: H1215–H1228. [Order article via Infotrieve]
Azhar G, Gao W, Liu L, Wei JY. Ischemia-reperfusion in the adult mouse heart influence of age. Exp Gerontol. 1999; 34: 699–714. [Order article via Infotrieve]
Liu L, Azhar G, Gao W, Zhang X, Wei JY. Bcl-2 and Bax expression in adult rat hearts after coronary occlusion: age-associated differences. Am J Physiol. 1998; 275: R315–R322. [Order article via Infotrieve]
Turturro A, Witt WW, Lewis S, Hass BS, Lipman RD, Hart RW. Growth curves and survival characteristics of the animals used in the Biomarkers of Aging Program. J Gerontol A Biol Sci Med Sci. 1999; 54: B492–B501.
Lakatta EG, Yin FC. Myocardial aging: functional alterations and related cellular mechanisms. Am J Physiol. 1982; 242: H927–H941. [Order article via Infotrieve]
Raju SVY, Zheng M, Schuleri KH, Phan AC, Bedja D, Saraiva RM, Yiginer O, Vandegaer K, Gabrielson KL, O’Donnell CP, Berkowitz DE, Barouch LA, Hare JM. Activation of the cardiac ciliary neurotrophic factor receptor reverses left ventricular hypertrophy in leptin-deficient and leptin-resistant obesity. Proc Natl Acad Sci U S A. 2006; 103: 4222–4227.
Ho AL, Gou YL, Rowlands DK, Chung YW, Chan HC. Effects of Bak Foong Pills and Menoease Pills on white blood cell distribution in old age female rats. Biol Pharm Bull. 2003; 26: 1748–1753. [Order article via Infotrieve]
Calle P, Jordaens L, De Buyzere M, Rubbens L, Lambrecht B, Clement DL. Age-related differences in presentation, treatment and outcome of acute myocardial infarction. Cardiology. 1994; 85: 111–120. [Order article via Infotrieve]
Loubani M, Ghosh S, Galinanes M. The aging human myocardium: tolerance to ischemia and responsiveness to ischemic preconditioning. J Thorac Cardiovasc Surg. 2003; 126: 143–147.
Schulman D, Latchman DS, Yellon DM. Effect of aging on the ability of preconditioning to protect rat hearts from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 2001; 281: H1630–H1636.
Sussman MA, Anversa P. Myocardial aging and senescence: where have the stem cells gone Annu Rev Physiol. 2004; 66: 29–48. [Order article via Infotrieve]
Anderlini P, Przepiorka D, Seong C, Smith TL, Huh YO, Lauppe J, Champlin R, Korbling M. Factors affecting mobilization of CD34+ cells in normal donors treated with filgrastim. Transfusion. 1997; 37: 507–512. [Order article via Infotrieve]
Fazel S, Cimini MC, Chen L, Li S, Angoulvant D, Fedak P, Verma S, Weisel RD, Keating A, Li RK. Activation of c-kit is necessary for mobilization of reparative bone marrow progenitor cells in response to ischemic cardiac injury. J Clin Invest. 2006; 116: 1865–1877.
Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114: 763–776. [Order article via Infotrieve]
Urbanek K, Torella D, Sheikh F, De Angelis A, Nurzynska D, Silvestri F, Beltrami CA, Bussani R, Beltrami AP, Quaini F, Bolli R, Leri A, Kajstura J, Anversa P. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proc Natl Acad Sci U S A. 2005; 102: 8692–8697.
Mouquet F, Pfister O, Jain M, Oikonomopoulos A, Ngoy S, Summer R, Fine A, Liao R. Restoration of cardiac progenitor cells after myocardial infarction by self-proliferation and selective homing of bone marrow-derived stem cells. Circ Res. 2005; 97: 1090–1092.
Zhang H, Fazel S, Tian H, Mickle DA, Weisel RD, Fujii T, Li RK. Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy. Am J Physiol Heart Circ Physiol. 2005; 289: H2089–H2096.
Lehrke S, Durand DJ, Mazhari R, Zimmet JM, Bedja D, Saraiva RM, Kuang JQ, Young R, Martin BJ, Gabrielson KL, Mills R, Hare JM. Effects of intravenous infusion of mesenchymal stem cells on post-myocardial infarction remodeling. J Am Coll Cardiol. 2006; 47: 192A Abstract.
Kajstura J, Liu Y, Baldini A, Li B, Olivetti G, Leri A, Anversa P. Coronary artery constriction in rats: necrotic and apoptotic myocyte death. Am J Cardiol. 1998; 82: 30K–41K. [Order article via Infotrieve]
Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G, Anversa P. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. 1996; 74: 86–107. [Order article via Infotrieve]
Cheng W, Reiss K, Li P, Chun MJ, Kajstura J, Olivetti G, Anversa P. Aging does not affect the activation of the myocyte insulin-like growth factor-1 autocrine system after infarction and ventricular failure in Fischer 344 rats. Circ Res. 1996; 78: 536–546.
Sam F, Sawyer DB, Chang DL, Eberli FR, Ngoy S, Jain M, Amin J, Apstein CS, Colucci WS. Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart. Am J Physiol Heart Circ Physiol. 2000; 279: H422–H428.
Xaymardan M, Zheng J, Duignan I, Chin A, Holm JM, Ballard VL, Edelberg JM. Senescent impairment in synergistic cytokine pathways that provide rapid cardioprotection in the rat heart. J Exp Med. 2004; 199: 797–804.
Cai D, Xaymardan M, Holm JM, Zheng J, Kizer JR, Edelberg JM. Age-associated impairment in TNF-alpha cardioprotection from myocardial infarction. Am J Physiol Heart Circ Physiol. 2003; 285: H463–H469.
Shiraha H, Gupta K, Drabik K, Wells A. Aging fibroblasts present reduced epidermal growth factor (EGF) responsiveness due to preferential loss of EGF receptors. J Biol Chem. 2000; 275: 19343–19351.
the Department of Medicine, Division of Cardiology (S.L., R.M., D.J.D., M.Z., J.M.Z., K.H.S., A.S.C., J.M.H.)
The Institute for Cell Engineering (ICE) (S.L., R.M., D.J.D., M.Z., J.M.Z., K.H.S., A.S.C., J.M.H.)
the Department of Comparative Medicine (D.B., J.M.H.), Johns Hopkins University School of Medicine, Baltimore, Md.
Abstract
Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) are potential new therapies to ameliorate post-myocardial infarction (post-MI) remodeling, as they enhance endogenous cardiac repair mechanisms and decrease cardiomyocyte apoptosis. Because both of these pathways undergo alterations with increasing age, we hypothesized that therapeutic efficacy of G-CSF and SCF is impaired in old versus young adult rats. MI was induced in 6- and 20-month-old rats by permanent ligation of the left coronary artery. In young animals, G-CSF/SCF therapy stabilized and reversed a decline in cardiac function, attenuated left ventricular dilation, decreased infarct size, and reduced cardiomyocyte hypertrophy. Remarkably, these effects on cardiac structure and function were absent in aged rodents. This could not be attributed to ineffective mobilization of bone marrow cells or decreased quantity of c-Kit+ cells within the myocardium with aging. However, whereas the G-CSF/SCF cocktail reduced cardiac myocyte apoptosis in old as well as in young hearts, the degree of reduction was substantially less with age and the rate of cardiomyocyte apoptosis in old animals remained high despite cytokine treatment. These findings demonstrate that G-CSF/SCF lacks therapeutic efficacy in old animals by failing to offset periinfarct apoptosis and therefore raise important concerns regarding the efficacy of novel cytokine therapies in elderly individuals at greatest risk for adverse consequences of MI.
Key Words: aging myocardial infarction remodeling growth substances
Introduction
The administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) is proposed as a potential new therapy for cardiovascular regenerative medicine. Animal studies demonstrate that G-CSF alone or in combination with SCF ameliorate post-myocardial infarction (post-MI) remodeling.1–4 Early clinical trials appeared to support these findings,5–7 and initial concerns of stimulating restenosis in the infarct-related artery8 have not been substantiated.9,10 However, a recent randomized controlled trial in patients with a mean age of 60 years of age failed to demonstrate benefit from G-CSF administration following MI, despite ample CD34+ cell mobilization.11
One of the main mechanisms by which G-CSF and SCF exert favorable effects on cardiac remodeling is enhancement of endogenous cardiac repair mechanisms that include both bone marrow stem cell mobilization, engraftment, and differentiation12–15 as well as proliferation of cardiomyocytes.16 In addition, G-CSF inhibits cardiomyocyte apoptosis17 and accelerates healing by stimulating absorption of necrotic tissue and reducing granulation and scar tissue via expression of matrix metalloproteinases.3
It is important to address whether potentially new cardiac regenerative therapies will benefit elderly patients who, despite reperfusion therapy, have increased MI-related mortality and morbidity.18,19 This higher vulnerability can only partially be attributed to comorbidities, and there is increasing evidence for impaired endogenous cardiac repair mechanisms with aging. Increased age is associated with reduced angiogenic capacity20 and diminished cell cycling of cardiac stem cells.21,22 In addition, there is increased cardiomyocyte apoptosis in the aged heart both at baseline23 and after ischemia, which could contribute to the adverse prognosis of elderly individuals.24,25
Given emerging evidence of impaired regenerative capacity of the aging heart coupled with its higher propensity for apoptotic cell death, the key question arises of whether these defects, which are the target mechanisms for the cardioprotective effect of G-CSF/SCF, will attenuate its efficacy. Here using an established rodent model of cardiovascular aging we tested the hypothesis that G-CSF/SCF would have reduced efficacy in old compared with young adult animals because of impaired target mechanisms.
Materials and Methods
All procedures in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and were approved by the Johns Hopkins University Animal Care and Use Committee.
Rat MI Model
MI was induced by permanent left coronary artery ligation in 6- and 20-month-old F344 rats purchased from the National Institute on Aging. Twenty-month-old Fisher rats are shown to have physiological signs of cardiovascular aging and correspond approximately to humans of 50 to 60 years of age.26,27 Rats were intubated endotracheally and ventilated with 2% isoflurane in oxygen using a rodent respirator (Model 683, Harvard Apparatus Inc). The heart was exposed via a left lateral thoracotomy, and the left anterior descending coronary artery was ligated 3 mm below its origin with a 6-0 silk suture.
Four hours post-MI, rats were randomly assigned to receive a first subcutaneous injection of recombinant human G-CSF (100 μg/kg) and rat-SCF (200 μg/kg; both Amgen, Thousand Oaks, Calif) followed by daily injections for 5 days. Placebo animals received equal volumes of isotonic saline. Rats (n=29, young; n=25, old) were followed by serial echocardiography for the development of cardiac function and geometry. The echocardiographer was fully blinded to the treatment group. The animals were allocated into treatment (n=15, young; n=12, old) and placebo (n=14, young; n=13, old) groups. A second subset of animals was euthanized 7 days after MI for the assessment of circulating c-Kit+ and CD34+ cells and myocardial apoptosis and were allocated into treatment (n=5, young; n=5, old) and placebo (n=5, young; n=6, old).
Mortality within 48 hours of MI was higher in 20-month-old compared with 6-month-old (27/63 [42.9%] versus 7/46 [15.2%]; P=0.003) rats without an effect of cytokine treatment. Only rats surviving beyond 48 hours were included in the primary long-term analyses.
Echocardiography
Transthoracic echocardiograms were obtained using a Sonos 5500 (Agilent, Hewlett Packard, Palo Alto, Calif) equipped with a 15-MHz linear transducer. For additional details, see the online data supplement, available at http://circres.ahajournals.org.
Complete Blood Count/Flow Cytometric Analysis of CD34+ and c-Kit+ Cells
Complete white blood counts and numbers of circulating CD34+ and c-Kit+ cells were determined at baseline and at day 7 after MI and cytokine administration by FACS analysis (Becton Dickinson). For additional details, see the online data supplement.
Histology
Infarct size and myocyte width in noninfarcted remote myocardium were determined from explanted hearts. Immunohistochemistry for c-Kit (DAKO rabbit anti-human CD117 antibody; A 4502) and mast cells were performed using deparaffinized and rehydrated tissue sections. Apoptosis was quantified by TUNEL analysis. For additional details, see the online data supplement.
Western Blot Analysis
Western blots were performed on heart extracts as described by Raju et al28 using polyclonal anti-phospho-STAT3 antibody (1:1000, Cell Signaling Technology), polyclonal anti-phospho-ERK antibodies (1:1000, Cell Signaling Technology Inc), and anti-phospho-caspase-3 (SC-7148, Santa Cruz Biotechnology). Immunoblots were detected using enhanced chemiluminescent Kits (SuperSignal; Pierce) and analyzed with a densitometer (Bio-Rad). Membranes were stripped and reprobed with polyclonal anti-STAT3 antibody (1:1000, Cell Signaling Technology), polyclonal anti-ERK antibodies (1:1000, Cell Signaling Technology Inc), or monoclonal anti-GAPDH antibodies (1:10 000, Research Diagnostics Inc).
Results
Cardiac Function and Structure
Echocardiographic parameters at baseline and at 2 days after MI were not different between age and treatment groups (Table 1). Importantly, at day 2 after MI, 2D ejection fraction (EF) showed a similar decline in all 4 groups, suggesting a comparable degree of initial myocardial injury (Table 1 and Figure 1A).
Induction of MI resulted in a substantial impairment of cardiac function, as left ventricular EF decreased significantly during the first 2 weeks in all groups (P<0.0001) without any effects of cytokine treatment or age (P=NS). Whereas ventricular function remained severely impaired during the 8-week follow-up period among young placebo animals, there was a dramatic improvement of EF from week 2 to 8 among young G-CSF/SCF-treated rats (P=0.0001; absolute increase, 22.5±3.8%), reversing the initial severe left ventricular dysfunction (Figure 1A) and representing an almost 50% recovery. Remarkably, this effect of cytokine treatment was completely absent in old animals, where EF in both treatment and placebo groups deteriorated similarly to young placebo animals and remained low until week 8 (Figure 1B). Similarly, whereas the G-CSF/SCF cocktail ameliorated left ventricular enlargement in young animals (P=0.003), this effect was completely absent in old animals (Figure 1C and 1D).
Effect of G-CSF/SCF on Infarct Size and Compensatory Cardiomyocyte Hypertrophy
Induction of MI resulted in the development of substantial transmural scarring of the left ventricle. Infarct size expressed as percentage of left ventricular perimeter was comparable between young placebo (26.1±3.1%), old G-CSF/SCF-treated (27±6%) and old placebo animals (28.1±4.9%) but was significantly reduced in young G-CSF/SCF-treated rats (14.7±3.5%; P=0.04 versus young placebo; Figure 2A).
Reactive hypertrophy of cardiomyocytes in areas remote from the scar region was evident in both age groups. Whereas the cytokine treatment completely prevented this compensatory myocyte hypertrophy in young animals (P<0.0001 versus young placebo; Figure 2B), it was without effect in the old rats.
White Blood Count/Circulating CD34+ and c-Kit+ Cells
Age-associated hematological changes at baseline are summarized in Table 2. These findings are in accordance with those reported previously.29 The percentage of circulating mononuclear cells expressing CD34 (0.45±0.04% young versus 0.55±0.07% old) and c-Kit (0.36±0.07% young versus 0.46±0.07% old) did not differ significantly between age groups at baseline. At 1 week post-MI, the neutrophil count was markedly elevated in G-CSF/SCF-treated groups in both young (2.17±0.25 103/μL versus 0.87±0.05 103/μL at baseline, P=0.008) and old (3.16±0.69 103/μL versus 1.19±0.28 103/μL at baseline, P=0.04) animals. Whereas the percentage of CD34+ cells was increased compared with baseline in all groups regardless of treatment, the percentage of circulating c-Kit+ cells remained relatively unchanged in all groups (Figure 3A and 3B). See Figure I in the online data supplement.
Interestingly, G-CSF/SCF treatment increased splenic weight among young (996±42 mg treated versus 828±25 mg placebo; P<0.01) but not among old rats (1570±157 mg treated versus 1345±124 mg placebo; P=NS). This difference remained significant after correction for body weight.
c-Kit+ Cells in the Peri-infarct Area
Next we examined the effect of G-CSF/SCF on the number of c-Kit+ cells in the infarct region. Cells expressing membrane c-Kit were detected in the peri-infarct zone at day 7 after MI and were 2-fold more abundant in young versus old animals, although this did not reach statistical significance. Cytokine treatment increased c-Kit+ cells in young (&14-fold; P<0.01 versus young placebo) and old (&17-fold; P<0.05 versus old placebo; Figure 4A) rats. Although in old rats, it did not reach a level exceeding that of young rats. Thus, the lack of response to G-CSF/SCF in old rats is not attributable to the absence of c-Kit+ cell mobilization within the heart. At 8 weeks after MI, cardiac c-Kit+ cells were essentially undetectable in all groups (data not shown).
Effect of G-CSF/SCF on Myocyte Apoptosis
As identified by TUNEL staining, cardiomyocyte apoptosis in the infarct border zone as well as in remote areas was significantly increased with old age in both placebo and treatment groups. G-CSF/SCF treatment reduced cardiomyocyte apoptosis in the infarct border zone in both young (28±1.7% versus 36.4±2.8% young placebo; P<0.05) and old (41.0±2.9% versus 51.3±2.5% old placebo; P<0.01; Figure 5A) animals. In areas remote from the infarct, cytokine treatment diminished cardiac myocyte apoptosis in young animals, from 37.8±3.5% to 25.7±1.4% (P<0.01), but did not reduce apoptosis in old animals (Figure 5B). Importantly, old G-CSF/SCF-treated rats had higher apoptotic indices than young placebo animals. Thus, the response to G-CSF/SCF was profoundly impaired in old versus young hearts.
Western Blot Analysis
Western blot analysis of the molecular pathways involved in apoptosis revealed increased activity of STAT3, a pathway implicated in G-CSF cardiac protection, following G-CSF/SCF treatment in young but not old infarcted rats (Figure 6A and 6B). On the other hand, activation of the proapoptotic caspase-3 pathway was greater in old versus young rats after MI (P<0.05 for old cytokines versus no cytokines). Importantly, G-CSF/SCF treatment did not reduce caspase-3 activation in old rats following MI (Figure 6C and 6D). Finally, the extracellular signal-regulated kinase (ERK) pathway was activated after MI in both old and young rats (P<0.01 in young and P<0.02 in old rats), and this was not affected by cytokine treatment (Figure 6E and 6F).
Discussion
The major new finding of this study is that the efficacy of G-CSF and SCF to attenuate post-MI remodeling is dramatically reduced with age. Whereas in young animals, the cytokine treatment markedly improved cardiac function, decreased infarct size, and ameliorated cardiomyocyte hypertrophy in residual myocytes, these effects were absent in post-MI aged rodents. Importantly, this was not attributable to reduced mobilization of stem cells in the circulation or heart. Rather, the degree of reduction in apoptosis was substantially less in old versus young hearts. In light of ongoing clinical trials, these findings have important implications for assessing novel therapies in elderly individuals at greatest risk for adverse consequences of MI.
Our findings in young animals agree with those of Orlic et al, who first demonstrated the potential of G-CSF/SCF to repair cardiac injury by mobilization of bone marrow stem cells.1 A number of subsequent animal studies confirmed the healing effect of G-CSF given alone or in combination with SCF and suggested additional mechanisms by which the cardioprotective effect is achieved, including antiapoptotic effects,17 enhanced cardiomyocyte proliferation,16 and side population cell mobilization.12 Dawn et al have recently shown that either SCF or Flt-3 ligand added to G-CSF are superior to G-CSF alone in reversing post-MI remodeling and causing bone marrow mobilization.15
This study offers important new insights with regard to age as a host determinant limiting responsiveness to G-CSF/SCF. MI increases in frequency as a function of age, and the elderly also experience higher mortality and morbidity.18,19,30 The aged heart shows marked physiological and structural alterations and varies significantly in its response to different stressors, including ischemia.31,32 Compared with young hearts, aged hearts exhibit an elevated rate of apoptotic cardiomyocytes both at baseline23 and after ischemia/reperfusion injury.24 A lower capacity for endogenous cardiac repair with aging is supported by reports of reduced ability for neoangiogenesis20 and cardiac stem cell senescence.21,22,33 G-CSF and SCF exert their effects by targeting these mechanisms that are altered in the aging heart.
The mobilization of bone marrow stem cells, their homing to the injured heart, and consequent cardiac regeneration are the main underpinnings of the beneficial effect of G-CSF/SCF on cardiac remodeling.1,4,13,15 Advanced age is a well-known limiting factor for the mobilization of bone marrow cells in donors being treated with G-CSF for leukopheresis of hematopoietic progenitor cells.34 We excluded poor bone marrow cell mobilization as the reason for the lack of effect seen in aged animals by demonstrating similar increases of neutrophils and circulating CD34+ and c-Kit+ cells after G-CSF/SCF in both age groups. Furthermore, the number of c-Kit+ cells in the peri-infarct area was similarly elevated by G-CSF/SCF treatment regardless of age. These cells have the potential to originate either from bone marrow1,35 or from the heart itself, where they represent endogenous cardiac stem cells.36 There is increasing support for the presence of an endogenous population of c-Kit+ cardiac stem cells that proliferate in response to injury and that may be targets for novel approaches of activation.37
There is also evidence that cardiac stem cells, depleted after MI, are replenished by both endogenous proliferation and mobilization of bone marrow-derived stem cells. In this regard, bone marrow-derived mesenchymal stem cells have lower colony formation activity in old compared with young rats.38,39 In addition to endogenous defects in either cardiac or bone marrow-derived cardiac precursor cells, reduced receptiveness to cellular regeneration may be another important mechanism for impaired cardiac repair in old adult animals. Support for this view is emerging, with data showing reduced responsiveness to cardiac regenerative therapy with intravenous MSC infusion in old versus young rats.40 Work is ongoing to explore these different potential mechanistic components.
In addition to ischemia induced necrosis, apoptosis contributes to cardiomyocyte loss after MI.23,41–43 Experimental studies show that apoptosis of cardiomyocytes remains elevated for months after the infarct and correlates with postinfarction left-ventricular remodeling.44 G-CSF is directly antiapoptotic in cardiomyocytes, endothelial cells, and myofibroblasts,17 activating its myocardial cell-surface receptor and leading, in turn, to JAK/Stat pathway activation and stimulation of the antiapoptotic protein bcl-2. In line with these findings, we demonstrated a reduction of apoptotic cells associated with Stat3 phosphorylation by G-CSF/SCF treatment in young animals. Whereas apoptosis of cardiomyocytes in infarct, border zone, and remote myocardium was reduced in young animals, only cardiomyocyte apoptosis in the infarct border zone was decreased in old animals. Additionally, G-CSF/SCF did not activate Stat3 or reduce phospho-caspase-3 in old hearts. Furthermore, apoptotic indices in old treated animals equaled or exceeded those of young control animals.
Whereas G-CSF/SCF increased c-Kit+ cells in the injured myocardium in old animals, they failed to reduce cardiomyocyte apoptosis to levels seen in young animals. In the context of a balance between the potential for new tissue formation through c-Kit+ cells and cardiomyocyte loss through apoptosis, the latter outweighs the former in aged animals, likely explaining the lack of cardioprotective effect in the old.
There is increasing evidence for reduced efficacy of other cytokine pathways with age, including reduced platelet-derived growth factor (PDGF)-AB-induced neoangiogenesis45 (which can be rescued by endothelial progenitor cells from young animals46) and reduced tumor necrosis factor (TNF)- cardioprotection attributable to downregulated TNF- receptors.46 Cytokine receptors are downregulated with aging in organs other than the heart,47 and a similar phenomenon could contribute to the present results.
Intriguingly, data from several human trials of G-CSF therapy following MI show a variance that may be related to the mean age of the study subjects. For example, 3 studies,5,9,11 each with subjects of a mean age of 60 years or more, found no statistically significant benefits in any objectively measured parameter. In these trials, the only significant clinical benefit was a reduction in subjective symptoms of myocardial ischemia in the 13 patients reported by Wang et al.5 In light of our results, it is interesting to note that the largest of these studies11 showed that older patients were capable of effectively mobilizing the CD34+ cells, suggesting that the reduced efficacy in this population can be attributed to a local defect in response to G-CSF therapy. Taken together, these reports contrast sharply with more encouraging data from 2 studies with subjects of mean ages of 507 and 586 years. The latter studies showed statistically significant benefits across a breadth of objective end points, including regional wall motion, myocardial perfusion, and EF, suggesting that younger patients may benefit substantially from these therapies.
In summary, we show that the beneficial effects of G-CSF/SCF on post-MI cardiac function and remodeling are dramatically diminished with age. Mobilization of circulating precursor cells and increased cardiac c-Kit+ cells were preserved in the older animals. This loss of responsiveness could be attributed, at least in part, to failure of G-CSF/SCF to adequately reduce apoptosis particularly in remote areas in rats of advanced age. At a molecular level, G-CSF failed to activate the anti-apoptotic pathways in older animals. Together, these findings have important mechanistic and therapeutic implications in an aging population at high risk for MI and its complications and need to be taken into consideration in future clinical trials.
Acknowledgments
The G-CSF and rat-SCF were kindly provided by Amgen (Thousand Oaks, Calif).
Sources of Funding
This work was supported by The Johns Hopkins University School of Medicine Institute for Cell Engineering (ICE), The Donald W. Reynolds Foundation, and NIH grants RO1 HL-65455, R21 HL-72185, and RO1 NIA AG078915.
Disclosures
None.
Footnotes
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received March 14, 2006; revision received July 14, 2006; accepted July 17, 2006.
References
Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001; 98: 10344–10349.
Iwanaga K, Takano H, Ohtsuka M, Hasegawa H, Zou Y, Qin Y, Odaka K, Hiroshima K, Tadokoro H, Komuro I. Effects of G-CSF on cardiac remodeling after acute myocardial infarction in swine. Biochem Biophys Res Commun. 2004; 325: 1353–1359. [Order article via Infotrieve]
Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai M, Misao Y, Lu C, Suzuki K, Goto K, Komada A, Takahashi T, Kosai K, Fujiwara T, Fujiwara H. Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac function and remodeling by postinfarction granulocyte colony-stimulating factor treatment. Circulation. 2004; 109: 2572–2580.
Norol F, Merlet P, Isnard R, Sebillon P, Bonnet N, Cailliot C, Carrion C, Ribeiro M, Charlotte F, Pradeau P, Mayol JF, Peinnequin A, Drouet M, Safsafi K, Vernant JP, Herodin F. Influence of mobilized stem cells on myocardial infarct repair in a nonhuman primate model. Blood. 2003; 102: 4361–4368.
Wang Y, Tagil K, Ripa RS, Nilsson JC, Carstensen S, Jorgensen E, Sondergaard L, Hesse B, Johnsen HE, Kastrup J. Effect of mobilization of bone marrow stem cells by granulocyte colony stimulating factor on clinical symptoms, left ventricular perfusion and function in patients with severe chronic ischemic heart disease. Int J Cardiol. 2005; 100: 477–483. [Order article via Infotrieve]
Kuethe F, Figulla HR, Herzau M, Voth M, Fritzenwanger M, Opfermann T, Pachmann K, Krack A, Sayer HG, Gottschild D, Werner GS. Treatment with granulocyte colony-stimulating factor for mobilization of bone marrow cells in patients with acute myocardial infarction. Am Heart J. 2005; 150: 115. [Order article via Infotrieve]
Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Korber T, Schumichen C, Freund M, Nienaber CA. Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI). Circulation. 2005; 112: 3097–3106.
Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet. 2004; 363: 751–756. [Order article via Infotrieve]
Valgimigli M, Rigolin GM, Cittanti C, Malagutti P, Curello S, Percoco G, Bugli AM, Della PM, Bragotti LZ, Ansani L, Mauro E, Lanfranchi A, Giganti M, Feggi L, Castoldi G, Ferrari R. Use of granulocyte-colony stimulating factor during acute myocardial infarction to enhance bone marrow stem cell mobilization in humans: clinical and angiographic safety profile. Eur Heart J. 2005; 26: 1838–1845.
Jorgensen E, Ripa RS, Helqvist S, Wang Y, Johnsen HE, Grande P, Kastrup J. In-stent neo-intimal hyperplasia after stem cell mobilization by granulocyte-colony stimulating factor. Preliminary intracoronary ultrasound results from a double-blind randomized placebo-controlled study of patients treated with percutaneous coronary intervention for ST-elevation myocardial infarction (STEMMI Trial). Int J Cardiol. 2006; 111: 174–177. [Order article via Infotrieve]
Zohlnhofer D, Ott I, Mehilli J, Schomig K, Michalk F, Ibrahim T, Meisetschlager G, von Wedel J, Bollwein H, Seyfarth M, Dirschinger J, Schmitt C, Schwaiger M, Kastrati A, Schomig A. Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA. 2006; 295: 1003–1010.
Adachi Y, Imagawa J, Suzuki Y, Yogo K, Fukazawa M, Kuromaru O, Saito Y. G-CSF treatment increases side population cell infiltration after myocardial infarction in mice. J Mol Cell Cardiol. 2004; 36: 707–710. [Order article via Infotrieve]
Fukuhara S, Tomita S, Nakatani T, Ohtsu Y, Ishida M, Yutani C, Kitamura S. G-CSF promotes bone marrow cells to migrate into infarcted mice heart, and differentiate into cardiomyocytes. Cell Transplant. 2004; 13: 741–748. [Order article via Infotrieve]
Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H, Muguruma Y, Tsuboi K, Itabashi Y, Ikeda Y, Ogawa S, Okano H, Hotta T, Ando K, Fukuda K. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood. 2004; 104: 3581–3587.
Dawn B, Guo YR, Rezazadeh A, Huang YM, Stein AB, Hunt G, Tiwari S, Varma J, Gu Y, Prabhu SD, Kajstura J, Anversa P, Ildstad ST, Bolli R. Postinfarct cytokine therapy regenerates cardiac tissue and improves left ventricular function. Cir Res. 2006; 98: 1098–1105.
Hamamoto M, Tomita S, Nakatani T, Yutani C, Yamashiro S, Sueda T, Yagihara T, Kitamura S. Granulocyte-colony stimulating factor directly enhances proliferation of human troponin I-positive cells derived from idiopathic dilated cardiomyopathy through specific receptors. J Heart Lung Transplant. 2004; 23: 1430–1437. [Order article via Infotrieve]
Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara K, Komuro I. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med. 2005; 11: 305–311. [Order article via Infotrieve]
Aguirre FV, McMahon RP, Mueller H, Kleiman NS, Kern MJ, Desvigne-Nickens P, Hamilton WP, Chaitman BR. Impact of age on clinical outcome and postlytic management strategies in patients treated with intravenous thrombolytic therapy. Results from the TIMI II Study. TIMI II Investigators. Circulation. 1994; 90: 78–86.
Thiemann DR, Coresh J, Schulman SP, Gerstenblith G, Oetgen WJ, Powe NR. Lack of benefit for intravenous thrombolysis in patients with myocardial infarction who are older than 75 years. Circulation. 2000; 101: 2239–2246.
Reed MJ, Edelberg JM. Impaired angiogenesis in the aged. Sci Aging Knowledge Environ. 2004; 2004: e7.
Chimenti C, Kajstura J, Torella D, Urbanek K, Heleniak H, Colussi C, Di Meglio F, Nadal-Ginard B, Frustaci A, Leri A, Maseri A, Anversa P. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res. 2003; 93: 604–613.
Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A, Sussman MA, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri A. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94: 514–524.
Kajstura J, Cheng W, Sarangarajan R, Li P, Li B, Nitahara JA, Chapnick S, Reiss K, Olivetti G, Anversa P. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol. 1996; 271: H1215–H1228. [Order article via Infotrieve]
Azhar G, Gao W, Liu L, Wei JY. Ischemia-reperfusion in the adult mouse heart influence of age. Exp Gerontol. 1999; 34: 699–714. [Order article via Infotrieve]
Liu L, Azhar G, Gao W, Zhang X, Wei JY. Bcl-2 and Bax expression in adult rat hearts after coronary occlusion: age-associated differences. Am J Physiol. 1998; 275: R315–R322. [Order article via Infotrieve]
Turturro A, Witt WW, Lewis S, Hass BS, Lipman RD, Hart RW. Growth curves and survival characteristics of the animals used in the Biomarkers of Aging Program. J Gerontol A Biol Sci Med Sci. 1999; 54: B492–B501.
Lakatta EG, Yin FC. Myocardial aging: functional alterations and related cellular mechanisms. Am J Physiol. 1982; 242: H927–H941. [Order article via Infotrieve]
Raju SVY, Zheng M, Schuleri KH, Phan AC, Bedja D, Saraiva RM, Yiginer O, Vandegaer K, Gabrielson KL, O’Donnell CP, Berkowitz DE, Barouch LA, Hare JM. Activation of the cardiac ciliary neurotrophic factor receptor reverses left ventricular hypertrophy in leptin-deficient and leptin-resistant obesity. Proc Natl Acad Sci U S A. 2006; 103: 4222–4227.
Ho AL, Gou YL, Rowlands DK, Chung YW, Chan HC. Effects of Bak Foong Pills and Menoease Pills on white blood cell distribution in old age female rats. Biol Pharm Bull. 2003; 26: 1748–1753. [Order article via Infotrieve]
Calle P, Jordaens L, De Buyzere M, Rubbens L, Lambrecht B, Clement DL. Age-related differences in presentation, treatment and outcome of acute myocardial infarction. Cardiology. 1994; 85: 111–120. [Order article via Infotrieve]
Loubani M, Ghosh S, Galinanes M. The aging human myocardium: tolerance to ischemia and responsiveness to ischemic preconditioning. J Thorac Cardiovasc Surg. 2003; 126: 143–147.
Schulman D, Latchman DS, Yellon DM. Effect of aging on the ability of preconditioning to protect rat hearts from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 2001; 281: H1630–H1636.
Sussman MA, Anversa P. Myocardial aging and senescence: where have the stem cells gone Annu Rev Physiol. 2004; 66: 29–48. [Order article via Infotrieve]
Anderlini P, Przepiorka D, Seong C, Smith TL, Huh YO, Lauppe J, Champlin R, Korbling M. Factors affecting mobilization of CD34+ cells in normal donors treated with filgrastim. Transfusion. 1997; 37: 507–512. [Order article via Infotrieve]
Fazel S, Cimini MC, Chen L, Li S, Angoulvant D, Fedak P, Verma S, Weisel RD, Keating A, Li RK. Activation of c-kit is necessary for mobilization of reparative bone marrow progenitor cells in response to ischemic cardiac injury. J Clin Invest. 2006; 116: 1865–1877.
Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114: 763–776. [Order article via Infotrieve]
Urbanek K, Torella D, Sheikh F, De Angelis A, Nurzynska D, Silvestri F, Beltrami CA, Bussani R, Beltrami AP, Quaini F, Bolli R, Leri A, Kajstura J, Anversa P. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proc Natl Acad Sci U S A. 2005; 102: 8692–8697.
Mouquet F, Pfister O, Jain M, Oikonomopoulos A, Ngoy S, Summer R, Fine A, Liao R. Restoration of cardiac progenitor cells after myocardial infarction by self-proliferation and selective homing of bone marrow-derived stem cells. Circ Res. 2005; 97: 1090–1092.
Zhang H, Fazel S, Tian H, Mickle DA, Weisel RD, Fujii T, Li RK. Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy. Am J Physiol Heart Circ Physiol. 2005; 289: H2089–H2096.
Lehrke S, Durand DJ, Mazhari R, Zimmet JM, Bedja D, Saraiva RM, Kuang JQ, Young R, Martin BJ, Gabrielson KL, Mills R, Hare JM. Effects of intravenous infusion of mesenchymal stem cells on post-myocardial infarction remodeling. J Am Coll Cardiol. 2006; 47: 192A Abstract.
Kajstura J, Liu Y, Baldini A, Li B, Olivetti G, Leri A, Anversa P. Coronary artery constriction in rats: necrotic and apoptotic myocyte death. Am J Cardiol. 1998; 82: 30K–41K. [Order article via Infotrieve]
Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G, Anversa P. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. 1996; 74: 86–107. [Order article via Infotrieve]
Cheng W, Reiss K, Li P, Chun MJ, Kajstura J, Olivetti G, Anversa P. Aging does not affect the activation of the myocyte insulin-like growth factor-1 autocrine system after infarction and ventricular failure in Fischer 344 rats. Circ Res. 1996; 78: 536–546.
Sam F, Sawyer DB, Chang DL, Eberli FR, Ngoy S, Jain M, Amin J, Apstein CS, Colucci WS. Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart. Am J Physiol Heart Circ Physiol. 2000; 279: H422–H428.
Xaymardan M, Zheng J, Duignan I, Chin A, Holm JM, Ballard VL, Edelberg JM. Senescent impairment in synergistic cytokine pathways that provide rapid cardioprotection in the rat heart. J Exp Med. 2004; 199: 797–804.
Cai D, Xaymardan M, Holm JM, Zheng J, Kizer JR, Edelberg JM. Age-associated impairment in TNF-alpha cardioprotection from myocardial infarction. Am J Physiol Heart Circ Physiol. 2003; 285: H463–H469.
Shiraha H, Gupta K, Drabik K, Wells A. Aging fibroblasts present reduced epidermal growth factor (EGF) responsiveness due to preferential loss of EGF receptors. J Biol Chem. 2000; 275: 19343–19351.
您现在查看是摘要介绍页，详见ORG附件。