Murine models do not recapitulate the pathophysiology of age-related venous thrombosis in humans

Abstract

Venous thromboembolism (VTE) encompasses both deep vein thrombosis (DVT) and pulmonary embolism (PE), a very serious and potentially lethal complication of DVT 1.The Surgeon General's Call to Action to Prevent Deep Vein Thrombosis and Pulmonary Embolism [Internet]. 2008. http://www.ncbi.nlm.nih.gov/books/NBK44178/. Accessed 20 April 2012.Google Scholar. The strongest but most unchangeable risk factor for VTE is age 2.Wong P. Baglin T. Epidemiology, risk factors and sequelae of venous thromboembolism.Phlebology. 2012; 27: 2-11Crossref PubMed Scopus (27) Google Scholar. After the age of 55, the risk of VTE goes up substantially and in those 75 years and older there is a ten-fold increase compared with the overall population 1.The Surgeon General's Call to Action to Prevent Deep Vein Thrombosis and Pulmonary Embolism [Internet]. 2008. http://www.ncbi.nlm.nih.gov/books/NBK44178/. Accessed 20 April 2012.Google Scholar, 3.Silverstein R.L. Bauer K.A. Cushman M. Esmon C.T. Ershler W.B. Tracy R.P. Venous thrombosis in the elderly: more questions than answers.Blood. 2007; 110: 3097-101Crossref PubMed Scopus (68) Google Scholar. The relationship between aging and VTE is not fully understood. Healthy adult wild-type mice have demonstrated outstanding utility in modeling both the natural history of DVT and human disorders associated with hemostasis and thrombosis without requiring genetic manipulation 4.Diaz J.A. Obi A.T. Myers D.D. Wrobleski S.K. Henke P.K. Mackman N. Wakefield T.W. Critical review of mouse models of venous thrombosis.Arterioscler Thromb Vasc Biol. 2012; 32: 556-62Crossref PubMed Scopus (180) Google Scholar. While several groups have described phenotypic changes occurring in genetic mouse models of premature aging 5.Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y.I. Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2806) Google Scholar, 6.Cardenas J.C. Owens A.P. Krishnamurthy J. Sharpless N.E. Whinna H.C. Church F.C. Overexpression of the cell cycle inhibitor p16INK4a promotes a prothrombotic phenotype following vascular injury in mice.Arterioscler Thromb Vasc Biol. 2011; 31: 827-33Crossref PubMed Scopus (20) Google Scholar, 7.Hemmeryckx B. van Hove C.E. Fransen P. Emmerechts J. Kauskot A. Bult H. Lijnen H.R. Hoylaerts M.F. Progression of the prothrombotic state in aging Bmal1-deficient mice.Arterioscler Thromb Vasc Biol. 2011; 31: 2552-9Crossref PubMed Scopus (26) Google Scholar, there is limited literature on mouse models of natural aging and its effects on venous thrombosis. Stasis is an established risk factor for venous thrombosis and stasis promoted by immobility is common in the elderly. Accordingly, aging in mice has been found to enhance stasis-induced thrombosis with marked increases in several endothelial and circulating factors 8.McDonald A.P. Meier T.R. Hawley A.E. Thibert J.N. Farris D.M. Wrobleski S.K. Henke P.K. Wakefield T.W. Myers D.D. Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis.Thromb Res. 2010; 125: 72-8Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar. Conversely, others found no increase in arterial thrombosis, changes in hemostatic factors or inflammatory mediator expression in aged mice 9.Stämpfli S.F. Akhmedov A. Gebhard C. Lohmann C. Holy E.W. Rozenberg I. Spescha R. Shi Y. Lüscher T.F. Tanner F.C. Camici G.G. Aging induces endothelial dysfunction while sparing arterial thrombosis.Arterioscler Thromb Vasc Biol. 2010; 30: 1960-7Crossref PubMed Scopus (27) Google Scholar. However, they did report impaired arterial endothelium vaso-relaxation in these mice 9.Stämpfli S.F. Akhmedov A. Gebhard C. Lohmann C. Holy E.W. Rozenberg I. Spescha R. Shi Y. Lüscher T.F. Tanner F.C. Camici G.G. Aging induces endothelial dysfunction while sparing arterial thrombosis.Arterioscler Thromb Vasc Biol. 2010; 30: 1960-7Crossref PubMed Scopus (27) Google Scholar. These studies collectively suggest that mouse models of aging and thrombosis do not fully recapitulate the multifactorial pathophysiology of thrombosis in humans. In this study, we characterize hemostatic parameters and two venous thrombosis models in aged C57BL/6 mice to understand the relationship between aging and thrombosis in mice and correlate this with VTE in elderly humans. Vascular occlusion times were measured in mice aged 2, 6 and 12 months after treatment with FeCl3 (7.5%) to the saphenous vein as previously described 6.Cardenas J.C. Owens A.P. Krishnamurthy J. Sharpless N.E. Whinna H.C. Church F.C. Overexpression of the cell cycle inhibitor p16INK4a promotes a prothrombotic phenotype following vascular injury in mice.Arterioscler Thromb Vasc Biol. 2011; 31: 827-33Crossref PubMed Scopus (20) Google Scholar, 10.Buyue Y. Whinna H.C. Sheehan J.P. The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis.Blood. 2008; 112: 3234-41Crossref PubMed Scopus (48) Google Scholar. Consistent with the observation that humans have an increased risk of venous thrombosis with age, a significantly shorter time to occlusion was seen in 12-month-old mice (6.9 ± 1.2 min) compared with 2-month-old mice (9.1 ± 1.5 min) (Fig. 1A). Mice aged 2 and 12 months were analysed in a model of inferior vena cava (IVC) stasis-induced thrombosis to compare thrombus formation and resolution over time, as previously described 8.McDonald A.P. Meier T.R. Hawley A.E. Thibert J.N. Farris D.M. Wrobleski S.K. Henke P.K. Wakefield T.W. Myers D.D. Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis.Thromb Res. 2010; 125: 72-8Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar. In contrast to our findings with the FeCl3 injury model, no significant difference between young and old mice was observed in thrombus formation or resolution following IVC ligation (Fig. 1B). Circulating thrombin/antithrombin (TAT) complex levels and thrombin generation were used as markers of baseline hypercoagulability in plasma from aged mice. In contrast to humans, who demonstrate age-related increases in markers of coagulation 11.Mari D. Mannucci P.M. Coppola R. Bottasso B. Bauer K.A. Rosenberg R.D. Hypercoagulability in centenarians: the paradox of successful aging.Blood. 1995; 85: 3144-9Crossref PubMed Google Scholar, 12.Haidl H. Cimenti C. Leschnik B. Zach D. Muntean W. Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT).Thromb Haemost. 2006; 95: 772-5Crossref PubMed Scopus (110) Google Scholar, mice at 2, 6 and 12 months of age displayed no difference in circulating TAT levels (Fig. 1C). Aged mice also demonstrated reduced peak thrombin generation and prolonged lagtime and time to peak in plasma samples assayed by calibrated automated thrombography (Fig. 1D), suggesting a difference in kinetics of thrombin generation between aged and young mice. Interestingly, several changes in hemostatic parameters observed in aged mice are discordant with historical data describing human aging. Aged C57BL/6 mice had increased platelet, lymphocyte and neutrophil counts compared with young mice (data not included), consistent with previous findings 13.Hemmeryckx B. Emmerechts J. Bovill E.G. Hoylaerts M.F. Lijnen H.R. Effect of ageing on the murine venous circulation.Histochem Cell Biol. 2012; 137: 537-46Crossref PubMed Scopus (19) Google Scholar. However, humans experience a decrease in platelet 14.Segal J.B. Moliterno A.R. Platelet counts differ by sex, ethnicity, and age in the United States.Ann Epidemiol. 2006; 16: 123-30Crossref PubMed Scopus (169) Google Scholar, lymphocyte 15.Sparrow D. Silbert J.E. Rowe J.W. The influence of age on peripheral lymphocyte count in men: a cross-sectional and longitudinal study.J Gerontol. 1980; 35: 163-6Crossref PubMed Scopus (17) Google Scholar, neutrophil and monocyte 16.McArthur W.P. Bloom K. Taylor M. Wheeler T. Smith J. Magnusson N.I. Peripheral blood leukocyte populations in the elderly with and without periodontal disease.J Clin Periodontol. 1996; 23: 846-52Crossref PubMed Scopus (7) Google Scholar counts with age, suggesting blood composition in mice and humans follows different trends during aging. Additionally, while aging in humans is associated with increases in prothrombotic biomarkers 11.Mari D. Mannucci P.M. Coppola R. Bottasso B. Bauer K.A. Rosenberg R.D. Hypercoagulability in centenarians: the paradox of successful aging.Blood. 1995; 85: 3144-9Crossref PubMed Google Scholar, 12.Haidl H. Cimenti C. Leschnik B. Zach D. Muntean W. Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT).Thromb Haemost. 2006; 95: 772-5Crossref PubMed Scopus (110) Google Scholar, we observed no difference in plasma TAT levels and reduced thrombin generation in aged mice. Mice are frequently used as models of both aging and thrombosis. We observed that the age-related susceptibility to venous thrombosis is dependent upon the thrombosis model used. In agreement with the notion that aging is associated with oxidative stress 17.Kregel K.C. Zhang H.J. An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations.Am J Physiol Regul Integr Comp Physiol. 2007; 292: R18-36Crossref PubMed Scopus (693) Google Scholar, we found that directly damaging the vessel wall with FeCl3 and inducing oxidative damage shortened the time to occlusion in aged mice. However, we observed no difference in stasis-induced thrombosis and resolution over time between young and old mice, suggesting that this model is a poor representation of the natural age-induced increase in venous thrombosis observed in humans. There are several limitations to this study, including the relatively small cohort of animals compared in our murine models, which reduced the maximum potential to detect differences. Additionally, this study is not a comprehensive analysis of thrombotic risk factors. There are other cellular and molecular mechanisms that contribute to the age-related risk of VTE aside from the parameters tested here. In conclusion, our data suggest that natural aging in mice does not accurately represent the age-related hypercoagulability and increase in venous thrombosis seen in humans. Although mice may display some age-related susceptibility to thrombosis following vascular injury, the pathophysiology driving this phenotype is likely to be unrelated to the cellular and molecular risk factors associated with human aging. J. C. Cardenas designed/performed experiments, analyzed data and wrote the manuscript; M. M. Aleman and J.-G. Wang performed experiments; A. S. Wolberg and H. C. Whinna analyzed data and reviewed the manuscript; F. C. Church designed and supervised the study, analyzed data and wrote the manuscript. Thrombosis [C14.907.355.830], Embolism and Thrombosis [C14.907.355], Age Factors [N06.850.490.250], Saphenous Vein [A07.231.908.819], Vena Cava, Inferior [A07.231.908.949.648]. The authors state that they have no conflict of interests. We thank C. Rein-Smith, D. Monroe and N. Mackman for providing critical feedback regarding data interpretation. This work was supported in part by the American Heart Association 11PRE76300005 to J. C. Cardenas and the National Institutes of Health T32 HL697668 to J. C. Cardenas, R21AG031068 to F. C. Church, HL094740 to A. S. Wolberg and HL112608 to M. M. Aleman.American Heart Association11PRE76300005National Institutes of HealthT32 HL697668R21AG031068HL094740HL112608