Car diac allograft vasculopathy (CAV) is a limiting fa ctor for the long-term survival of heart transplan t recipients1,2. CAV is characterized by the devel opment of diffuse concentric fibromuscular intimal hyperplasia in epicardial and smaller intram yocardial arteries along with focal, eccentric ath erosclerotic plaques in the larger epicardial arte ries3,4. The development of these lesions may lead to the progressive narrowing of the lumen5. According to the response to injury hypo thesis of CAV, these lesions are the res ult of cumulative endothelial injury induced¨ by alloimmune responses as well as non-immuno logical risk factors such as ischemia-reperfusion¨ injury, viral infections, and metabolic disor ders3,6.
Early diagnosis of CAV is essential ¨to implement appropriate prevention and treatment ¨measures. Clinical prediction models of CAV are currently not available and may be useful for non-invasive diagnostic and prognostic purpos es. The general aim of this doctoral thesis i s to develop diagnostic prediction models for ¨prevalent CAV. The specific centra l hypothesis of this doctoral thesis is¨ that biomarkers of endothelial homeostasis discrim inate between CAV-negative and CAV-positive heart¨ transplant recipients.
Endothelial home ostasis reflects the balance between endothel ial injury and endothelial repair. In chapter 1, we investigated whether biomarkers related to endothelial injury and endothelial repair dis criminate between CAV-negative and CAV-positi ve heart transplant recipients. Fifty-two patients ¨undergoing coronary angiography between 5 and 15¨ years after heart transplantation were recrui ted in this study. Flow cytometry was applied to q uantify endothelial progenitor cells (EPCs),¨ circulating endothelial cells (CECs), and circulat ing endothelial microparticles (CEMPs). Cell cultu re was used for quantification of circulating EPC¨ number and hematopoietic progenitor cell ¨(HPC) number and for analysis of EPC function. EPC number and EPC function did not differ betw een CAV-negative and CAV-positive patients. I n univariable models, age, creatinine, steroid dos e, granulocyte colony-forming units, apoptoti c CECs, and apoptotic CEMPs discriminated between CAV-positive and CAV-negative patients.¨ The logistic regression model containing apop totic CECs and apoptotic CEMPs as independent ¨predictors provided high discrimination between CAV- positive and CAV-negative patients ( c-statistic 0.812; 95% CI 0.692-0.932). In a logistic regression model with age and creati nine as covariates, apoptotic CECs (p=0.0112) and apoptotic CEMPs (p=0.0141) were independent p redictors (c-statistic 0.855; 95% CI 0.756-0.953). These two biomarkers remained independent pr edictors when steroid dose was introduced in the m odel. Taken together, the high discriminative ¨ability of apoptotic CECs and apoptotic CEMPs is¨ a solid foundation for the development of clinical ¨prediction models of CAV.
In chapter 2, pat ients with stable native coronary artery ¨disease (CAD) were compared with heart trans plant recipients with CAV. After all, CAV is a par ticular type of arteriosclerosis with many similar ities but also significant differences compar ed to native CAD. Atherosclerosis in patients with ¨stable native CAD is characterized by the pr esence of atheromata that contain a lipid cor e filled with extracellular cholesterol and cellul ar debris and are covered by a fibrous cap. I n contrast, fibromuscular intimal hyperplasia is t he most prominent lesion type of CAV and mainly consists of smooth muscle cells and extrac ellular matrix7. Endothelial injury is assume d to play a key role in the initiation and pr ogression of both native CAD and CAV2,8. In t he response-to-injury hypothesis of atherosclerosi s of Ross and Glomset, endothelial injury was orig inally defined as endothelial denudation resu lting from focal desquamation of endothelium9,10. Later versions of the response-to-injury hypo thesis emphasized endothelial dysfunction rather than denudation8,11. Cellular biomarkers of en dothelial injury (CEMPs and CECs) may discrim inate between endothelial activation and irreversi ble endothelial damage. The hypothesis that e ndothelial injury and circulating platelet micropa rticles (CPMPs) are distinct in both types of ¨arteriosclerosis was investigated.
The geometric mean of the concentration of CECs (CD45- CD31bright VEGFR-2+) was 2.90-fold (p<0. 001) and 2.34-fold (p<0.05) higher in pati ents with stable native CAD (n=80) and with C AV (n=30), respectively, compared to healthy contr ols (n=25). No significant difference in tota l, Annexin V negative, and Annexin V positive (apo ptotic) CECs was observed between patients with na tive CAD and with CAV. The concentration of Annexi n V negative CEMPs (CD144+ CD42a-) was 59.2% (p< ;0.01) higher in transplant recipients with CAV th an in native CAD patients but no difference i n Annexin V positive CEMPs was observed. The media n value of total CD61+ CPMPs in native CAD patient s was 69.4% (p<0.001) and 71.6% (p<0.00 1) lower compared to healthy controls and transpla nt recipients with CAV, respectively. These differ ences were even more pronounced when CD42a+CD31+ C PMPs were quantified. In conclusion, the selective increase of Annexin V negative CEMPs and the ¨absence of a difference in Annexin V positiv e CECs strongly suggest increased endothelial acti vation but not endothelial apoptosis in CAV-p ositive patients compared to stable CAD patie nts. Use of antiplatelet drugs likely underli es the strikingly lower levels of CPMPs in patient s with native CAD.
In chapter 3, the relatio n between high density lipoproteins (HDL) and ¨CAV was investigated. The prevalence and the ¨incidence of CAV have been reported to be in creased in heart transplant recipients with decrea sed high density lipoprotein (HDL) cholesterol lev els12-15. The association between HDL cholesterol¨ and CAV may reflect causation but might also¨ be due to residual confounding. One such confoundi ng factor is insulin resistance, which is considered to play a role in the pathogenesis of CAV. ¨A triglyceride/HDL cholesterol ratio of greater t han 3 has been recognized as a marker of ¨insulin resistance in overweight subjects16¨ and constituted a risk factor for CAV and maj or adverse cardiac events in heart transplant reci pients17,18.
Remodelling of HDL in hear t transplant recipients is significantly affected¨ by a lower activity of cholesterol ester tran sfer protein, phospholipid transfer protein,¨ and hepatic lipase19,20. Consequently, these patie nts are characterized by an increased proportion o f large HDL particles and reduced pre-ß1-HDL ¨in the presence of normal or even elevated H DL cholesterol levels19,20. These alterations may be partially explained by corticosteroid use2 1 but may also be potentiated by statin intake22. The modified HDL metabolism and associated co mpositional changes of HDL particles may lead to a n impaired function of these lipoproteins. Reduced ¨HDL function may also occur as a result of o ngoing inflammation23.
We hypothesized that¨ HDL function may be impaired in these patients and ¨may discriminate between CAV-positive and CA V-negative patients. Cholesterol efflux capac ity of apolipoprotein B-depleted plasma was analys ed using a validated assay24. The vasculoprot ective function of HDL was studied by means o f an EPC migration assay. HDL cholesterol lev els were similar in heart transplant patients ¨compared to healthy controls. However, norma lized cholesterol efflux and vasculoprotective fun ction were reduced by 24.1% (p<0.001) and¨ by 27.0% (p<0.01), respectively, in heart transplant recipients compared to healthy con trols. HDL function was similar in patients w ith and without cardiac allograft vasculopathy (CA V) and was not related to C-reactive protein (CRP) levels. An interaction effect (p=0.0584) was observed between etiology of heart failure b efore transplantation and steroid use as fact ors of HDL cholesterol levels. Lower HDL chol esterol levels occurred in patients with prior ischemic cardiomyopathy not taking steroids. How ever, HDL function was independent of the etiology of heart failure before transplantation and¨ steroid use. The median C-reactive protein (CRP) l evel was 2.24-fold (p=0.082) higher in patien ts with CAV than in patients without CAV. In concl usion, HDL function is impaired in heart tran splant recipients but is unrelated to CAV-sta tus.
In chapter 4, the potential of endothel ium-enriched microRNAs (miRNAs) as putative b iomarkers for the prediction of CAV was inves tigated. MiRNAs are small, non-coding, single -stranded RNA sequences that regulate gene express ion at the post-transcriptional level. Because miR NAs circulate in remarkably stable forms in blood2 5,26, they have a significant potential as biomark ers. Several reports indicate that miRNAs may play a role in endothelial homeost asis27,28. In this study, a candidate-ba sed approach using circulating levels of endo thelium-enriched miRNAs (miR-21-5p, miR-92a-3 p, miR-92a-1-5p, miR-126-3p, miR-126-5p) to p redict CAV was evaluated. Circulating levels of endothelium-enriched miRNAs (miR-21-5p, miR-92a -3p, miR-92a-1-5p, miR-126-3p, miR-126-5p) were qu antified by real-time RT-PCR. The discriminative a bility of logistic regression models was quantifie d using the concordance statistic (c-statistic). P lasma levels of miR-21-5p, miR-92a-3p, miR-126-3p, and miR-126-5p were 1.86-fold (p=NS), 1 .91-fold (p<0.05), 1.74-fold (p=0.074), and 1.7 3-fold (p=0.060) higher, in patients with CAV ¨than in patients without CAV. Recipient age¨ (c-statistic 0.689 (95% CI 0.537-0.842)), serum cr eatinine (c-statistic 0.703 (95% CI 0.552-0.8 54)), levels of miR-92a-3p (c-statistic 0.682 ¨(95% CI 0.533-0.831)), and levels of miR-126 -5p (c-statistic 0.655 (95% CI 0.502-0.807)) predi cted CAV-status in univariable models. In a multiv ariable logistic regression model with recipient a ge and creatinine as covariates, miR-126-5p (chi-s quare=4.374; df=1; p=0.0365), miR-92a-3p (chi -square=6.007; df=1; p=0.0143), and the combination of miR-126-5p and miR-92a-3p (chi square=8. 162; df=2; p=0.0169) added significant inform ation. The model with age, creatinine, miR-12 6-5p and miR-92a-3p as covariables conferred¨ good discrimination between patients without CAV and patients with CAV (c-statistic 0.800 (95% CI 0.674-0.926)). In addition, miR-92a-3 p (chi-square=5.454; df=1; p=0.0195) and not miR-1 26-5p (chi-square=2.037; df=1; p=0.1535) ¨added value in a model with apoptotic CECs and ap optotic CEMPs as predictors (c- statistic0.847 (95 % CI 0.740-0.954)). In conclusion, endotheliu m-enriched miRNAs have predictive ability for ¨CAV beyond clinical predictors.
The central ¨hypothesis at the start of this doctoral the sis was that biomarkers of endothelial homeostasis discriminate between CAV-negative and CAV-po sitive heart transplant recipients. The validity o f this hypothesis has been convincingly demonstrat ed. The refinement and validation of these models¨ in a larger follow-up study may lead to a clinically useful model that can be applied ¨for monitoring heart transplant recipients.
1. Stehlik, J., Edwards, L.B., Kuc heryavaya, A.Y., Benden, C., Christie, J .D., Dobbels, F., Kirk, R., Rahmel, A.O. ,Her tz, M.I. The Registry of the International So ciety for Heart and Lung Transplantation : Twenty-eighth Adult Heart Transplant Report--201 1. J Heart Lung Transplant 3 0, 1078-1094 (2011).
2. Schmauss, D . ,Weis, M. Cardiac allograft vasculopathy: recent ¨developments. Circulation 1 17, 2131-2141 (2008).
3. Vassa lli, G., Gallino, A., Weis, M., von Scheidt, W., Kappenberger, L., von Segesser, L.K. ¨,Goy, J.J. Alloimmunity and nonimmunologic risk f actors in cardiac allograft vasculopathy. Eur¨ Heart J 24, 1180-1188 ( 2003).
4. Rahmani, M., Cruz, R.P., Gran ville, D.J. ,McManus, B.M. Allograft vasculopathy¨ versus atherosclerosis. Circ Res 99, 801-815 (2006).
5. Kapadia, S.R ., Nissen, S.E. ,Tuzcu, E.M. Impact of intrav ascular ultrasound in understanding transplant cor onary artery disease. Curr Opin Cardiol 14, 140-150 (1999).
6. Ross, R. The pathogenesis of atherosclerosis- -an update. N Engl J Med 314, 488-500 (1986).
7. Lu, W.H., Palatnik, K., Fishbein, G.A., Lai, C., Levi, D.S. , Perens, G., Alejos, J., Kobashigawa, J. ,Fishbei n, M.C. Diverse morphologic manifestations of card iac allograft vasculopathy: a pathologic stud y of 64 allograft hearts. J Heart Lu ng Transplant 30, 1044- 1050 (2011).
8. Ross, R. Atherosclerosis--an ¨inflammatory disease. N Engl J Med 340, 115-126. (1999).
9. Ro ss, R. ,Glomset, J.A. Atherosclerosis and the arterial smooth muscle cell: Proliferation o f smooth muscle is a key event in the genesis ¨of the lesions of atherosclerosis. Science 180, 1332-1339 (1973).
1 0. Ross, R. ,Glomset, J.A. The pathogenesis of ath erosclerosis (first of two parts). N Engl ¨J Med 295, 369-377 (1976).11. Ross, R. The pathogenesis of athero sclerosis: a perspective for the 1990s. N ature 362, 801-809. (1993).12. Parameshwar, J., Foote, J., Sharples, L. , Wallwork, J., Large, S. ,Schofield, P. Lipi ds, lipoprotein (a) and coronary artery disea se in patients following cardiac transplantat ion. Transpl Int 9, 481- 485 (1996).
13. Valantine, H., Rickenbacker, ¨P., Kemna, M., Hunt, S., Chen, Y.D., Reaven, G. , Stinson, E.B. Metabolic abnormalities characterist ic of dysmetabolic syndrome predict the devel opment of transplant coronary artery disease: a prospective study. Circulation 103, 2144-2152 (2001).
1 4. Cooke, G.E., Eaton, G.M., Whitby, G., Kenn edy, R.A., Binkley, P.F., Moeschberger, M.L.¨ ,Leier, C.V. Plasma atherogenic markers in congestive heart failure and posttransplant (he art) patients. J Am Coll Cardiol 36, 509-516 (2000).
15. Sanchez-Gom ez, J.M., Martinez-Dolz, L., Sanchez-Lazaro, I., A lmenar, L., Sanchez-Lacuesta, E., Munoz-Giner , B., Portoles, M., Rivera, M., Valera-Roman, A., Gonzalez-Juanatey, J.R., Tejada-Ponce, D., Ag uero, J., Buendia, F. ,Salvador, A. Influence ¨of metabolic syndrome on development of cardiac a llograft vasculopathy in the transplanted heart. Transplantation 93, 106-111 (2012).
16. McLaughlin, T., Abbasi, F., Cheal, K., Chu, J., Lamendola, C. ,Reaven , G. Use of metabolic markers to identif y overweight individuals who are insulin resi stant. Ann Intern Med 139, 802-809 (2003).
17. Biadi, O., Potena, L. , Fearon, W.F., Luikart, H.I., Yeung, A., Ferrara, ¨R., Hunt, S.A., Mocarski, E.S. ,Valantine, H.A. I nterplay between systemic inflammation and markers ¨of insulin resistance in cardiovascular prognosis ¨after heart transplantation. J Heart Lung Tra nsplant 26, 324-330 (2007).18. Raichlin, E.R., McConnell, J.P., Le rman, A., Kremers, W.K., Edwards, B.S., Kushwaha, S.S., Clavell, A.L., Rodeheffer, R. J. ,Frantz, R.P. Systemic inflammation and metabol ic syndrome in cardiac allograft vasculopathy. J Heart Lung Transplant 26, 826-833 (2007).
19. Atger, V., Leclerc , T., Cambillau, M., Guillemain, R., Marti, C., Mo atti, N. ,Girard, A. Elevated high density li poprotein concentrations in heart transplant recip ients are related to impaired plasma cholesteryl e ster transfer and hepatic lipase activity. Ath erosclerosis 103, 29-41 (1993).
20. Sviridov, D., Chin-Dusting , J., Nestel, P., Kingwell, B., Hoang, A., Ol chawa, B., Starr, J. ,Dart, A. Elevated HDL c holesterol is functionally ineffective in car diac transplant recipients: evidence for impa ired reverse cholesterol transport. Transplant ation 81, 361-366 (2006).
21. Stamler, J.S., Vaughan, D.E. ,Loscalzo, J. ¨Immunosuppressive therapy and lipoprotein ab normalities after cardiac transplantation. Am¨ J Cardiol 68, 389- 391 (1991).
22. Yamashita, S., Tsubakio-Yama moto, K., Ohama, T., Nakagawa-Toyama, Y. ,Nis hida, M. Molecular mechanisms of HDL-choleste rol elevation by statins and its effects on H DL functions. J Atheroscler Thromb 17, 436-451 (2010).
23. Fish er, E.A., Feig, J.E., Hewing, B., Hazen, S.L. ,Smith, J.D. High-density lipoprotein functi on, dysfunction, and reverse cholesterol transport . Arterioscler Thromb Vasc Biol 32, 2813-2820 (2012).
24. Khera , A.V., Cuchel, M., de la Llera-Moya, M., Rodrigue s, A., Burke, M.F., Jafri, K., French, B.C., Phillips, J.A., Mucksavage, M.L., Wilensky, R .L., Mohler, E.R., Rothblat, G.H. ,Rader, D.J . Cholesterol efflux capacity, high-density l ipoprotein function, and atherosclerosis. N En gl J Med 364, 127-135 ( 2011).
25. Suarez, Y., Fernandez-Hernando, C ., Yu, J., Gerber, S.A., Harrison, K.D., Pobe r, J.S., Iruela-Arispe, M.L., Merkenschlager, M. ,Sessa, W.C. Dicer-dependent endothelial micro RNAs are necessary for postnatal angiogenesis. Proc Natl Acad Sci U S A 105, 14082-14087 (2008).
26. Kuehbac her, A., Urbich, C., Zeiher, A.M. ,Dimmeler, S. Ro le of Dicer and Drosha for endothelial microRNA ex pression and angiogenesis. Circ Res 101, 59-68 (2007).
27. Scott, E. , Loya, K., Mountford, J., Milligan, G. ,Baker, A. H. MicroRNA regulation of endothelial homeost asis and commitment-implications for vascular rege neration strategies using stem cell therapies . Free Radic Biol Med 64, 52-60 (2013).
28. Yamakuchi, M. Micro RNAs in Vascular Biology. Int J Vasc Med 2012, 794898 (2012 ).