Liver Transplantation The Role of Arterial Stiffness in the Estimation of Cardiovascular Risk in Liver Transplant Recipients Lydia Sastre, MD,1,2 Raquel García, RN,1 Julián-Gonzalo Gándara, MD,1 Patricia Fernández-Llama, MD,3 Antonio J. Amor, MD,4 Cristina Sierra, MD,5 Laia Escudé, MD,1 Pablo Ruiz, MD,1 Jordi Colmenero, MD,1 Emilio Ortega, MD,4 Miquel Navasa, MD,1 and Gonzalo Crespo, MD1 Background. Long-term cardiovascular (CV) events are a frequent cause of death and disability after liver transplant (LT). Although a more in-depth, risk-adapted control of CV risk factors may result in improved post-LT CV outcomes, an accurate strati- fication of the CV risk of LT recipients to better implement preventive strategies is lacking. Aortic pulse wave velocity (aPWV) is a surrogate of arterial stiffness that has been suggested as a biomarker of CV risk; it has never been evaluated in adult LT recipients. Methods. In a single-center prospective study, we included 122 LT recipients at 12 (n = 39), 60 (n = 45), or 120 (n = 38) mo after LT. aPWV estimation by oscillometry, clinical assessment of CV risk factors, and CV risk estimation by standard clinical scores (systematic coronary risk evaluation and pooled cohort equation) were performed. The incidence of CV events during prospective follow-up was registered. Results. aPWV was independently associated with age and the grade of control of blood pressure. After a median follow-up of 35 mo, 15 patients (12%) presented a CV event. Higher aPWV, diabetes, past or present smoking habit, previous CV events, lower eGFR, being in systematic coronary risk evaluation or pooled cohort equation high-risk groups, and higher levels of total cholesterol, LDL-cholesterol, creatinine, and triglycerides were associated with the incidence of CV events at univariate analysis; aPWV, past or present smoking habit, and triglycerides were independent predictors of CV events. Conclusions. According to our results, aPWV mirrors CV risk in LT recipients and thus may be a useful CV risk biomarker in this population. Considering these preliminary results, its accuracy in stratifying risk requires confirmation in further studies. (Transplantation Direct 2022;8: e1272; doi: 10.1097/TXD.0000000000001272). INTRODUCTION (SCORE)7,8 to identify LT recipients at highest risk of CV Cardiovascular (CV) events are frequent causes of long- events is not perfect; thus, a simple, accurate, and objective term morbimortality in liver transplant (LT) recipients.1,2 system to identify patients at higher risk of long-term CV Although many clinical variables have been associated with events is lacking.6 the risk of post-LT CV events,3-6 the accuracy of such vari- The estimation of CV risk is clinically important, as it permits ables, or that of clinical risk scores like the Framingham to match the efforts of preventive interventions with the risk of Risk Score, the PROspective CArdiovascular Munster each individual. Although clinical risk scores are used to assess (PROCAM), or the Systematic COronary Risk Evaluation long-term CV risk and define the targets to consider controlled Received 26 September 2021. Revision received 1 November 2021. R.G. and J.G.G. performed research, critical review of article. P.F.L.L., A.J.A., C.S., Accepted 10 November 2021. L.E., P.R., J.C., and E.O. did critical review of article. M.N. designed study, critical 1 Liver Transplant Unit, Liver Unit, IDIBAPS, CIBERehd, Hospital Clínic, University review of article. G.C. designed study, performed research, wrote the article. of Barcelona, Barcelona, Spain. Supplemental digital content (SDC) is available for this article. Direct URL citations 2 Gastroenterology and Hepatology Department, Hospital Universitari Son appear in the printed text, and links to the digital files are provided in the HTML Espases, Palma de Mallorca, Spain. text of this article on the journal’s Web site (www.transplantationdirect.com). 3 Renal and Hypertension Units, Fundació Puigvert, IIB Sant Pau, Universitat Correspondence: Gonzalo Crespo, MD, Liver Transplant Unit, Liver Unit, Hospital Autónoma de Barcelona, Barcelona, Spain. Clínic, IDIBAPS, CIBERehd, University of Barcelona, Stair 7-9, 3rd Floor, Hospital 4 Department of Endocrinology and Nutrition, IDIBAPS, CIBERobn, Hospital Clínic, Villarroel 170 08036 Barcelona, Spain. (gcrespo@clinic.cat). Clínic, Barcelona, Spain. Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters 5 Hypertension and Vascular Risk Unit, Department of Internal Medicine, Kluwer Health, Inc. This is an open-access article distributed under the terms of IDIBAPS, Hospital Clínic, University of Barcelona, Barcelona, Spain. the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 The authors declare no conflicts of interest. (CCBY-NC-ND), where it is permissible to download and share the work provided This work was supported by the 2018 Research Grant from Fundación de la it is properly cited. The work cannot be changed in any way or used commercially Sociedad Española de Trasplante Hepático (FSETH) (Gonzalo Crespo). L.S. without permission from the journal. was the recipient of the 2017 Astellas Research Grant from the Fundació de la Societat Catalana de Trasplantament. ISSN: 2373-8731 L.S. designed study, performed research, and did critical review of article. DOI: 10.1097/TXD.0000000000001272 Transplantation DIRECT 2022 www.transplantationdirect.com 1 2 Transplantation DIRECT ■ 2022 www.transplantationdirect.com each CV condition, they are limited by their moderate accu- consumption. A complete physical examination was per- racy. Thus, novel ways to characterize CV risk are continu- formed including waist circumference measurement, and body ously being evaluated.9,10 Among these, estimation of arterial mass index was calculated. Metabolic syndrome was defined stiffness by measuring aortic pulse wave velocity (aPWV) has when at least 3 of the following 5 risk factors were met20: (1) been shown to be a sensitive biomarker of CV risk.11 In a large waist circumference ≥ 102 cm for men and ≥88 cm for women; meta-analysis, after adjustment for conventional CV risk fac- (2) triglycerides ≥ 150 mg/dL; (3) high-density lipoprotein tors, aPWV improved risk prediction, particularly in certain cholesterol (HDL-C) < 40 mg/dL for men and <50 mg/dL for subgroups, such as younger individuals at intermediate risk.12 women; (4) systolic BP ≥ 130 mm Hg or diastolic BP ≥ 85 Additionally, aPWV is useful to identify high-risk individuals in mm Hg; and (5) fasting serum glucose ≥ 100 mg/dL. patients with type 1 diabetes.13 Although the gold standard to Ten-year CV risk assessment was done by calculating the estimate aPWV is the measurement of carotid-femoral PWV by pooled cohort equation (PCE)21 and the SCORE22 using the tonometry, oscillometric methods that measure brachial blood corresponding online calculators. According to the algorithms, pressure (BP) have been validated as surrogates of aPWV and SCORE is not calculated in patients with type 2 diabetes or have also shown to predict CV events.14,15 previous CV events; in this latter group PCE is neither calcu- In solid organ transplant recipients, arterial stiffness has lated as according to the guidelines those group of patients are been mainly studied after kidney transplant, where it was already considered to be at high/very high CV risk. shown to be associated with clinical risk factors and to inde- A detailed anamnesis on previous CV disease was made, pendently predict mortality, CV events, and loss of renal and a review of clinical history was also performed. We spe- function.16 In contrast, data regarding arterial stiffness in LT cifically registered the following: recipients are scarce and mainly derived from the pediatric LT population.17,18 Consequently, we designed this cross-sectional - Previous episodes of acute coronary syndrome (myocardial study with prospective follow-up to investigate the potential infarction with or without ST elevation or unstable angina), association between aPWV and CV risk in adult LT recipients. stable angina, or coronary artery disease diagnosed by any means. - Previous episodes of ischemic or hemorrhagic stroke or MATERIALS AND METHODS transient ischemic attack. This is a prospective study performed in the Liver Transplant - Previous episodes of heart failure, based on signs and symp- Unit of Hospital Clinic, Barcelona, including 2 different parts: toms along with echocardiographic evidence of ventricular a cross-sectional evaluation of aPWV and CV risk factors, fol- dysfunction. lowed by a prospective follow-up on CV events after cross- - Previous diagnosis of peripheral artery disease, clinically sectional evaluation. diagnosed and confirmed with lower limb ultrasonography or ankle-brachial index (ABI < 0.9). Cross-sectional Assessment of aPWV - Previous episodes of arrhythmia, including atrial or ventric- and Cardiovascular Risk Factors ular fibrillation and flutter, atrioventricular block, torsades Between March 2017 and July 2018, consecutive adult LT de pointes, and sinus bradycardia requiring treatment. recipients with a follow-up of 12 (±3), 60 (±3), and 120 (±3) mo after LT seen in the Outpatient Clinic were considered Immunosuppression to participate in the study, and the cross-sectional evaluation Dose and trough levels of tacrolimus, cyclosporine A, or was performed. Retransplanted patients, those with multiple everolimus were registered when appropriate. Additionally, organ transplantation and patients infected with HIV were we also registered the dose of prednisone or mycophenolate excluded. In addition, patients were only included if they were mophetil when applicable. in the outpatient setting, without evidence of graft rejection or dysfunction, technical complications, or active infections. Arterial Stiffness We evaluated arterial stiffness through the estimation of Assessment of Cardiovascular Risk Factors aPWV using the validated Mobilograph device (IEM, Stolberg, The presence of diabetes mellitus (DM), arterial hyperten- Germany). Assessment was performed in a quiet, temperature- sion (AHT), and dyslipidemia and their grade of control were controlled examination room, in supine position and using an prospectively assessed according to the following criteria19: adequately sized cuff. Patients were in fasting status, and the assessment took place before the intake of immunosuppres- - AHT: office BP ≥ 140/90 mm Hg in untreated individu- sants and antihypertensive drugs. Procedures were performed als, or use of BP-lowering medication. Controlled BP was in triplicate; mean values were used for analyses. First, bra- defined as an office BP < 140/90 mm Hg. chial systolic and diastolic BP are obtained. Then, the brachial - DM: anytime glycemia ≥ 200 mg/dL, or fasting glyce- cuff is inflated to the diastolic blood level and held for 10 s to mia ≥ 126 mg/dL at least twice, or glycated hemoglobin record pulse waves, and a proprietary mathematical model (HbA1c) ≥ 6.5%, or use of antidiabetic drugs. Controlled (ARCSolver, Austrian Institute of Technology, Vienna, Austria) DM was defined as HbA1c < 7%. that combines several parameters from pulse wave and wave - Dyslipidemia: total cholesterol >200 mg/dL, or low-density separation analysis estimates aPWV.14 An aPWV ≥ 10 m/s was lipoprotein (LDL) cholesterol (LDL-C) >130 mg/dL, or tri- considered as subclinical organ damage.23 glyceride levels >150 mg/dL, or use of lipid-lowering drugs. Controlled dyslipidemia was defined as an LDL-C < 100 mg/dL. Blood Tests The same day of arterial stiffness assessment, a blood sample In addition, we prospectively evaluated smoking habit was obtained in fasting status. Liver and kidney function tests, (classified as never, current, or former) and the total pack-year as well as a lipid profile including total, HDL- and LDL-C, © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. Sastre et al 3 triglycerides and HbA1C levels were determined. A serum to evaluate baseline variables associated with the incidence of sample was frozen at –80°C and stored for further studies. CV events during follow-up, considering the number of events only variables statistically significant in the univariate analy- Prospective Assessment of CV Events sis were included in multivariate models. Since SCORE and After the cross-sectional assessment, patients were prospec- PCE scoring are not performed in a significant proportion of tively followed until December 31, 2020, to evaluate the inci- patients as per definitions of the algorithms (SCORE is not dence of new-onset CV events. During this time, the incidence calculated in type 2 diabetic patients and patients with previ- of the following CV events was recorded: ous CV events, whereas PCE is not calculated in patients with previous CV events), for Cox-regression analysis purposes - Acute coronary syndrome (including myocardial infarction these variables were categorized. SCORE was categorized as with or without ST elevation or unstable angina). ≥15% (including diabetic patients and those with previous - Ischemic or hemorrhagic stroke, including transient CV events) versus <15%,22 and PCE was categorized as ≥20% ischemic attack. (including patients with previous CV events) versus <20%.21 - Peripheral artery disease based on the Fontaine classification A P < 0.05 was considered statistically significant for all tests. and confirmed with lower limb ultrasonography or ABI < 0.9. - Arrhythmias, that included atrial or ventricular fibrillation Ethical Issues and flutter, atrioventricular block, torsades de pointes, and The study was performed in accordance with the sinus bradycardia requiring treatment. Declaration of Helsinki, the Declaration of Istanbul, and - Heart failure, diagnosed on signs and symptoms of volume the E6 Good Clinical Practice Standards ICH. All the study, overload with echocardiographic evidence of ventricular data were treated anonymously with restricted access only by dysfunction. authorized personnel for the purposes of the study. The study was approved by the Clinical Research Ethical Committee of Statistical Analysis Hospital Clínic, Barcelona (approval number HCB/2016/490). Continuous variables are expressed as medians and inter- All patients were properly informed about the study and pro- quartile range, and categorical variables are expressed in vided written consent for inclusion. absolute numbers and percentages. Patients were grouped according to the time of post-LT follow-up: 12, 60, or 120 mo (Groups A, B, and C, respectively). Nonparametric tests RESULTS (Kruskal-Wallis, Fisher test) were used to study differences During the study period, 168 patients were considered between groups. The association between aPWV and the rest for the study, and finally 122 met inclusion criteria and were of the variables was studied with uni- and multivariate linear included. Among them, 39 formed group A (12 mo after LT), regression analysis and graphically represented by dot plots or 45 group B (60 mo), and 38 group C (120 mo). The flowchart bar graphs. Variables with a P <0.1 in the univariate analysis of the study is depicted in Figure 1, and the main character- were included in the multivariate analysis. Time-dependent istics of the whole cohort and according to the timing groups univariate and multivariate Cox-regression analysis were used are shown in Table 1. FIGURE 1. Flowchart of the study. LT, liver transplant. 4 Transplantation DIRECT ■ 2022 www.transplantationdirect.com TABLE 1. Baseline characteristics of the whole cohort and according to the timing group All Group A Group B Group C Variable n = 122 n = 39 n = 45 n = 38 P Sex (male) 87 (71) 27 (69) 30 (67) 30 (79) 0.351 Age (y) 61 (55–68) 58 (54–62) 62 (53–68) 67 (59–71) 0.001 Etiology of liver disease 0.132 HCV 44 (36) 13 (33) 18 (40) 13 (34) Alcohol 36 (29) 8 (20) 12 (27) 16 (42) NASH/cryptogenic 11 (9) 6 (16) 3 (7) 2 (5) Indication of LT (HCC) 47 (38) 22 (56) 18 (40) 7 (18) 0.006 Never smoker 49 (40) 12 (31) 22 (44) 17 (45) 0.211 Previous CV events 20 (16) 5 (13) 5 (11) 10 (26) 0.113 Arterial hypertension 81 (66) 20 (51) 30 (67) 31 (82) 0.005 Diabetes mellitus 61 (50) 22 (56) 21 (47) 18 (47) 0.427 Dyslipemia 58 (47) 18 (46) 19 (42) 21 (55) 0.430 SCORE (%) (n = 58) 4 (2–5) 3 (1–4) 3 (1–6) 5 (4–6) 0.01 PCE (%) (n = 102) 11.6 (6.4–21.3) 9 (3.2–21.8) 12.8 (6.2–20.4) 14.8 (9.4–23.8) 0.189 Metabolic syndrome 60 (50) 19 (50) 19 (42) 22 (58) 0.493 eGFR (mL/min/1.73m2) 69 (52–88) 71 (61–95) 73 (60–89) 62 (50–75) 0.051 Creatinine (mg/dL) 1.06 (0.85–1.33) 1.09 (0.82–1.29) 0.99 (0.82–1.22) 1.19 (0.96–1.41) 0.089 Total cholesterol (mg/dL) 175 (153–198) 177 (166–210) 172 (153–197) 176 (144–190) 0.298 LDL-cholesterol (mg/dL) 102 (85–120) 110 (88–124) 102 (86–122) 95 (84–111) 0.218 HDL-cholesterol (mg/dL) 46 (37–60) 52 (28–60) 46 (37–63) 45 (34–55) 0.320 Triglycerides (mg/dL) 111 (85–150) 111 (86–158) 99 (78–133) 124 (97–161) 0.086 HbA1c (%) 5.6 (5.1–6.3) 5.6 (5.1–6.3) 5.7 (5.1–6.2) 5.6 (5.2–6.5) 0.748 BMI (kg/m2) 27.1 (24.6–31.1) 26.9 (24.2–30.8) 26.8 (24.1–31.9) 27.8 (25.7–30) 0.677 CNI-free immunosuppression 21 (17) 2 (5) 4 (9) 15 (40) <0.001 aPWV (m/s) 8.8 (7.6–9.9) 8.1 (7.3–9.1) 9.1 (7.8–10.1) 9.5 (8.2–10.4) 0.002 aPWV >10 (m/s) 30 (25) 3 (8) 12 (27) 15 (40) 0.001 Data are given as absolute count (%) or median (IQR). Bold indicates variables with statistically significant differences between groups. aPWV, aortic pulse wave velocity; BMI, body mass index; CNI, calcineurin inhibitor; CV, cardiovascular; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDL, high-density lipoprotein; IQR, interqiartile range; LDL, low-density lipoprotein; LT, liver transplant; NASH, nonalcoholic steatohepatitis; PCE, pooled cohort equation; SCORE, systematic coronary risk evaluation. CV Risk Factors shows the correlation between aPWV and age, time after LT, The overall prevalence of DM, AHT, and dyslipidemia was grade of control of BP, and eGFR. 50%, 66%, and 47%, respectively (Table 1). Twenty patients (16%) had had a previous CV event, either before or after LT. Arterial Hypertension and aPWV As expected, patients in group C were older and presented Overall, 64 patients (52%) were receiving BP-lowering more frequently AHT than the rest of the groups, whereas medication. Most patients (n = 35) were taking 1 single medi- there were no statistically significant differences in the rest of cation, although 20 and 9 patients were under 2 and 3 anti- clinical CV risk factors. hypertensive drugs, respectively. The most frequent group of antihypertensive drugs used were angiotensin II type 1 recep- Aortic Pulse Wave Velocity tor blockers (28 patients, 23%), followed by calcium channel Median aPWV was 8.8 m/s. Thirty patients (25%) presented blockers (n = 23, 18%) and angiotensin-converting-enzyme an aPWV >10 m/s (8% in group A, 27% in group B, and 40% inhibitors (n = 14, 11%). in group C, P = 0.001). Univariate and multivariate linear aPWV increased in parallel with the number of BP-lowering regression analyses of clinical variables associated with aPWV medications used. However, for each category of number of are shown in Table 2. In the univariate analysis, arterial stiff- medications used, aPWV tended to be lower if BP was within ness was significantly and directly associated with age, timing the predefined limits of control (<140/90 mm Hg) (Figure 3), group, hepatocellular carcinoma as the indication of LT, pres- with differences being statistically significant for the groups of ence of AHT, lack of AHT control, DM, metabolic syndrome, 0 and 3 BP-lowering medications. body mass index, waist circumference, PCE, SCORE, and his- In the group of patients on a single antihypertensive drug, tory of previous episodes of CV events, and arterial stiffness we did not find statistically significant differences in aPWV was inversely associated with estimated glomerular filtration according to the type of antihypertensive drug taken. Similarly, rate (eGFR). Additionally, it was significantly associated with in patients with 2 or 3 medications including calcium channel using a CNI-free immunosuppressive regimen, and, in the few blockers, angiotensin II type 1 receptor blockers, or angiotensin- patients with cyclosporine A, with lower trough levels of this convertingenzyme inhibitors as mainstay of antihypertensive immunosuppressant. In the multivariate analysis, the variables treatment together with any other drug (mainly betablockers independently associated with aPWV were age and AHT, either or diuretics), there were no statistically significant differences in as a diagnosis or when evaluated as controlled BP. Figure 2 aPWV depending on the main antihypertensive taken. © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. Sastre et al 5 TABLE 2. Uni- and multivariate lineal regression analysis of variables associated with aPWV Univariate Multivariate Variable B 95% CI P B 95% CI P Age (y) 0.134 0.122, 0.146 <0.001 0.130 0.083, 0.177 <0.001 Gender (male) –0.349 –1.00, 0.307 0.294 Etiology of liver disease (NASH) –0.433 –1.47, 0.604 0.363 Never smoker –0.257 –0.855, 0.341 0.396 Tobacco pack-y 0.009 –0.004, 0.021 0.187 Indication of LT (HCC) 0.777 0.178, 1.375 0.011 0.850 Timing (Group A/B/C) 0.661 0.306, 1.017 <0.001 0.195 SCORE (n = 58)a 0.314 0.107, 0.193 <0.001 PCE (n = 102)a 0.089 0.07, 0.108 <0.001 AHTa 1.691 1.143, 2.238 <0.001 Controlled BP –1.095 –1.674, –0.515 <0.001 –0.620 –0.813, –0.428 <0.001 Diabetes 0.652 0.068, 1.236 0.029 0.314 Controlled diabetes –0.360 –1.148, 0.427 0.367 Dyslipidemia 0.443 –0.148, 1.035 0.140 Body mass index 0.086 0.024, 0.147 0.006 0.533 Waist circumference (cm) 0.044 0.022, 0.066 0.002 0.165 Metabolic syndromea 1.003 0.445, 1.561 0.001 0.846 Creatinine(mg/dL) 0.254 –0.654, 1.162 0.581 eGFR (mL/min/1.73 m2) –0.028 –0.042, –0.013 <0.001 0.065 Total cholesterol 0.005 –0.002, 0.012 0.177 LDL-C 0.004 –0.006, 0.014 0.424 HDL-C 0.006 –0.011, 0.022 0.479 Tryglicerides 0.003 –0.001, 0.008 0.143 CNI-free IMS 0.864 0.105, 1.623 0.026 0.789 Cyclosporin A-based IMS 0.151 –0.116, 1.339 0.105 Tacrolimus trough levels (ng/mL) (n = 77) –0.147 –0.333, 0.040 0.121 Cyclosporin A trough levels (ng/mL) (n = 23)a –0.010 –0.018, –0.002 0.018 Previous CVE 1.016 0.248, 1.785 0.01 0.559 Bold indicates variables with statistically significant differences between groups. a Considering the low number of patients under immunosuppression with cyclosporine A, the variable “Cyclosplorin A trough levels” was not included in multivariate analysis. Additionally, cardiovascular risk scores (“SCORE” and “PCE”) and “Metabolic syndrome” were not included in multivariate analysis considering they include variables already studied in the model. Finally, the model was constructed including either “Controlled BP” (shown in the table) or “Arterial hypertension.” In this latter case, independent variables remained age and arterial hypertension. AHT, arterial hypertension; aPWV, aortic pulse wave velocity; BP, blood pressure; CNI, calcineurin inhibitor; CVE, cardiovascular events; eGFR, estimated glomerular filtration rate; HCC, hepatocellular carcinoma; HDL-C, high-density lipoprotein cholesterol; IMS, immunosuppression; LDL-C, low-density lipoprotein cholesterol; LT, liver transplantation; NASH, nonalcoholic steatohepatitis; PCE, pooled cohort equation; SCORE, systematic coronary risk evaluation. Prediction of Cardiovascular Events com/TXD/A396). In models with clinical variables, aPWV and During a median follow-up of 35 mo (31–38) after absence of smoking habit (inversely) were independently asso- assessment, 15 patients (12%) presented a new CV event. ciated with CV events; additionally, triglyceride levels were Characteristics of these patients and events are detailed in also independently associated with CV events when included Table 3. In univariate Cox-regression analysis (Table 4), the in the model. In contrast, total cholesterol, LDL-cholesterol, variables associated with the incidence of new-onset CV diabetes, previous CV events, and creatinine/eGFR were not events were higher aPWV, the presence of DM, the absence independently associated with the incidence of CV events in of present or past smoking habit (inversely), having presented any model. Importantly, these results did not change when previous CV events, lower eGFR, and higher serum levels of age or the grade of control of BP were forced into the mod- creatinine, total cholesterol, LDL-cholesterol, triglycerides, and els. In the models including clinical algorithms, aPWV was being in the high-risk groups in SCORE or PCE algorithms. independently associated with CV events when confronted Considering the number of events, the number of variables sta- with SCORE, which was not an independent predictor of CV tistically significant in the univariate analyses, and the fact that events. In the model including aPWV and PCE, none of the clinical algorithms include variables individually associated variables were independently associated with CV events. with CV events in the analysis, we approached multivariate analyses by performing different models. First, we performed multivariate analyses including aPWV and individual clinical DISCUSSION variables (without risk algorithms). Smoking habit, previous In this study, we show for the first time that the assessment CV events, aPWV, and diabetes were included in all these mod- of arterial stiffness by estimating aPWV correlates with sev- els with triglycerides, LDL-cholesterol, or total cholesterol and eral CV risk factors in LT recipients, and more importantly, either eGFR or creatinine; second, we performed the multi- it seems to be associated with the risk of presenting new CV variate analyses including aPWV and either SCORE or PCE, events during follow-up. Thus, it may be an accurate predic- finally resulting in 8 models (Table S1, SDC, http://links.lww. tor of CV events in this population, showing the particular 6 Transplantation DIRECT ■ 2022 www.transplantationdirect.com FIGURE 2. Dot plots or graph bars showing the association between aPWV and age (A), timing group (B), grade of control of blood pressure (C), and eGFR (D). aPWV, aortic pulse wave velocity; eGFR, estimated glomerular filtration rate. FIGURE 3. Box-plots showing aPWV in patients under 0, 1, 2, or 3 antihypertensive drugs according to the grade of control of blood pressure. aPWV, aortic pulse wave velocity; BP, blood pressure. advantage of being a quantifiable, continuous parameter. other classic estimators of CV risk, should be the focus of Considering the potential association between aPWV and larger, probably multicenter, studies. CV risk after LT suggested by our data, investigating its inde- The relevance of long-term CV events in LT recipients is pendent role in the prediction of CV events, as compared with expected to increase in the near future. LT candidates are © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. Sastre et al 7 TABLE 3. Description of the patients who presented a CV event during follow-up Age Months at Previous between Previous aPWV liver Type of CV aPWV aPWV Smoking SCORE PCE Controlled HbA1C CV Sex (y) disease event and event (m/s) habit Group (%) (%) eGFR AHT BP DM (%) Dyslipemia events 1 Male 62 HCV Peripheral 32 8.5 Past A ≥15 23.8 94.8 Yes Yes Yes 5.5 Yes No artery disease 2 Male 72 Alcohol Heart failure 22 10.1 Past C N/A 21.3 49.8 Yes Yes No 6 No No 3 Male 60 HBV Ischemic 26 9.3 Past B ≥15 ≥20 34.2 Yes No Yes 8.7 Yes Yes stroke 4 Male 58 HCV Peripheral 19 9.6 Past A ≥15 27.5 67 Yes No Yes 7.6 Yes No artery disease 5 Female 65 Alcohol Heart failure 26 11.3 Never A ≥15 21.3 86.4 Yes No Yes 5.6 Yes No 6 Male 64 NASH Heart failure 13 9 Past A ≥15 ≥20 31.4 Yes Yes Yes 9.8 Yes Yes 7 Male 69 HCV Heart failure 17 10.1 Past B 7 19.2 58.4 Yes No No 5.5 Yes No 8 Male 56 Alcohol Acute 3 8.8 Past A ≥15 7.5 42 Yes No Yes 4.7 Yes No coronary syndrome 9 Male 70 Alcohol Peripheral 7 10.9 Active C N/A 54.7 44.3 Yes No Yes 5.1 No No artery disease 10 Female 61 Alcohol Arrythmia 11 9.5 Past A ≥15 10.8 98.9 Yes No Yes 5.9 No No 11 Male 66 Alcohol Ischemic 20 9.1 Past B ≥15 20.7 52 No Yes Yes 6.3 Yes No stroke 12 Male 69 Alcohol Acute 33 10.2 Active C ≥15 ≥20 53.2 Yes No Yes 5.9 No Yes coronary syndrome 13 Female 57 NASH Arrythmia 29 7.5 Active A ≥15 ≥20 35.4 No Yes Yes 5.1 No Yes 14 Male 77 HCV Acute 27 11.9 Never C N/A ≥20 50.3 Yes No Yes 7.2 Yes Yes coronary syndrome 15 Female 68 HCV Arrtyhmia 11 9.8 Past B ≥15 ≥20 69 Yes Yes No 5.1 No Yes AHT, arterial hypertension; aPWV, aortic pulse wave velocity; BP, blood pressure; CV, cardiovascular; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate: HbA1c, glycated hemoglobin; HBV, hepatitis B virus; HCV, hepatitis C virus; N/A, not applicable; NASH, nonalcoholic steatohepatitis; PCE, pooled cohort equation; SCORE, systematic coronary risk evaluation. increasingly older and present more metabolic comorbidities contributing to a better identification of populations that might and renal impairment,24-26 suggesting that the clinical impact benefit from more aggressive management of CV risk factors. of long-term CV events will be even more meaningful in the In our cohort, aPWV correlated with several characteris- next few years. Importantly, a number of studies have shown tics that have been shown to be associated with CV events that the grade of control of CV risk factors such as DM, AHT, in the general population and in LT recipients, and it was or dyslipemia27-29 is suboptimal in this particular population. independently associated with age and BP level. These results In contrast, a recent study demonstrated that, if achieved, an are supported by the physiopathological mechanisms under- optimal BP control is associated with a decreased incidence of lying arterial stiffness, which is intimately related with the CV events and better survival of LT recipients, suggesting that degenerative changes that take place in the arteries as the there is a clear role for intensive monitoring and treatment consequence of aging and increased BP.11,31 Indeed, previous of these risk factors.30 However, the target levels (and thus studies in the general (nontransplant) population, using either the intensity of interventions) of HbA1c, LDL-cholesterol, or tonometry or the same oscillometric method that we used to BP differ according to baseline CV risk.19 CV risk is usually evaluate arterial stiffness, have also shown a close association estimated through clinical scores, whose usefulness in the LT between aPWV and age and BP.32,33 It remains to be investi- setting has not been clearly demonstrated. In this regard, it gated whether the increase of aPWV with age is steeper in seems clear that improving risk stratification in this popula- transplant recipients, hypothesizing a direct role of immuno- tion can be clinically relevant. suppression in a potentially higher increase of aPWV in these Pulse wave velocity is the most widely used noninvasive patients. This would be in line with the accelerated progres- method to estimate arterial stiffness. Arterial walls are thickened sion of atherosclerosis that possibly takes place in transplant and stiffened as a result of the loss of elastic fibers and increased recipients. In this regard, in our cross-sectional study, aPWV fibrosis, consequence of age and repetitive stress.31 aPWV was associated with using CNI-free immunosuppression and directly correlates with arterial stiffness or arteriosclerosis, and with lower levels of cyclosporine A in patients using this drug its measurement has been shown to improve the predictive abil- in the univariate analysis. These results are probably related ity of standard risk factors in the general population, potentially with the fact that those patients with a baseline higher CV (or 8 Transplantation DIRECT ■ 2022 www.transplantationdirect.com TABLE 4. with longer follow-up to prove whether aPWV independently Univariate Cox-regression analysis of variables associated adds to current clinical calculators, would favor including with the incidence of cardiovascular events during the evaluation of arterial stiffness in the investigation of CV follow-up risk of LT recipients. Additionally, it may be worthwhile to investigate the role of aPWV estimated before LT in the pre- Variable HR 95% CI P diction of long-term CV events. Considering the increasingly Age (y) 1.060 0.998-1.126 0.060 worsening CV profile of LT candidates,37 having a new tool Gender (male) 0.863 0.275-2.713 0.801 to stratify risk before LT could be of great interest. Similarly, Etiology of liver disease (NASH) 1.567 0.354-6.944 0.554 whether changes in aPWV according to the grade of control Never smoker 0.206 0.046-0.917 0.038 of BP improve the predictive ability of long-term CV events aPWV (m/s) 1.595 1.119-2.273 0.010 with respect to baseline measurements may also be clinically SCORE, ≥15%a 6.323 1.426-28.03 0.015 important, as well as to investigate the association of aPWV PCE, ≥20%a 4.182 1.33-13.153 0.014 with post-LT kidney dysfunction. AHT 3.424 0.773-15.176 0.105 We also examined the association between BP treatment and Controlled BP 0.371 0.132-1.102 0.059 aPWV in this population. Although results are difficult to inter- Diabetes 4.703 1.327-16.670 0.016 pret, we did not find any obvious association between the type HbA1C (mg/dL) 1.727 0.895-1.823 0.178 of antihypertensive used and aPWV. There is some evidence that BMI (kg/m2) 1.077 0.970-1.195 0.163 drugs antagonizing the renin-angiotensin system may be more Waist circumference (cm) 1.049 0.999-1.101 0.053 effective in decreasing arterial stiffness, probably because of a Metabolic syndrome 2.171 0.742-6.352 0.157 specific antifibrotic or anti-inflammatory effect on the matrix of Creatinine (mg/dL) 9.825 2.286-42.234 0.002 the arterial wall38,39; thus, it may be possible that the sample size eGFR (mL/min/1.73 m2) 0.959 0.931-0.989 0.008 and the difficulties in analyzing the different treatment regimens Total cholesterol (mg/dL) 1.012 1.001-1.024 0.032 including combinations of different drugs have hampered our LDL-cholesterol (mg/dL) 1.020 1.003-1.037 0.023 analysis. Triglycerides (mg/dL) 1.008 1.003-1.014 0.004 There are limitations to our study. Particularly, it is a sin- Previous CVE 3.651 1.297-10.278 0.014 gle-center study and the sample size is limited and includes Use of ASA 0.551 0.113-2.221 0.363 patients with 3 different post-LT follow-up timings; however, Bold indicates variables that showed statistical significance in univariate analysis. this has also permitted us to study (and discard) the poten- a Patients in whom SCORE and PCE are not calculated (those with diabetes/previous CV events for SCORE; previous CV events for PCE) are included in the very high-risk groups for each tial impact of time after LT in arterial stiffness. Additionally, variable (≥15% for SCORE, ≥20% for PCE). there were relatively few CV events in the prospective follow- AHT, arterial hypertension; aPWV, aortic pulse wave velocity; ASA, acetyl salicylic acid; BMI, up. For this reason, confirmation of our results in larger stud- body mass index; BP, blood pressure; CI, confidence interval; CVE, cardiovascular events; eGFR, estimated glomerular filtration rate; HbA1C, glycosylated hemoglobin; HR, hazard ratio; ies is warranted before recommending to implement them in LDL, low-density lipoprotein; NASH, nonalcoholic steatohepatitis; PCE, pooled cohort equation; clinical practice. Finally, although we used a device that has SCORE, systematic coronary risk evaluation. been tested against intra-arterial measurements,14 the estima- tion of aPWV was done by oscillometry instead of using the renal) risk are also those in which these regimens without CNI gold standard noninvasive method (applanation tonometry). or with less exposure to these drugs are more frequently used A recent large study has suggested that oscillometric methods to try to mitigate that increased risk. Longitudinal evaluation may slightly overestimate aPWV in elderly and hypertensive of arterial stiffness in transplant recipients will be useful to patients with respect to tonometry, although these differences evaluate the actual role of immunosuppression in the progress do not seem to be clinically relevant.40 Additionally, there is of aPWV. increasing evidence of the usefulness of oscillometry in clini- Notably, aPWV seemed to be able to identify patients with cal practice,41 and, from a clinical point of view, the key issue a higher risk of presenting new CV events after a median would be the ability of any tool to provide an accurate esti- follow-up of 35 mo. Although the number of events must mation of future risk. In this regard, oscillometric-based 24-h be taken into account when interpreting the results, we per- aPWV estimation has been shown to be a strong predictor formed several multivariate models with clinical variables, of cardiovascular events in hemodyalisis patients.42 Not less all of which showed that higher aPWV and past or present importantly, oscillometric methods are less time-consuming smoking habit are independently associated with the inci- and more user-friendly than tonometry, making their use dence of CV events, with triglycerides serum levels also being easier in clinical practice. Indeed, the device used works as independently associated with CV events if included in the a standard sphygmomanometer and permits, in the same analysis. As stated above, multivariate analysis must be inter- examination, to obtain BP measurement (including ambula- preted with caution; however, our results are coherent and tory 24-h measurements) and aPWV. Then, the results are supported by the fact that smoking habit and serum triglycer- downloaded and analyzed by a proprietary software. All ides, well known CV risk factors,34-36 were consistently found these procedures can be easily performed in a post-LT medi- as independent predictors of CV events. Additionally, when cal or nursing clinic. tested against a standard clinical algorithm like SCORE, Although we recognize and have tried to overcome these aPWV maintained its independent predictive value, whereas limitations, it is clear that larger studies are needed to confirm neither PCE nor aPWV retained statistical significance when our results, and indeed our work must be seen as a first step tested together. However, it must be stressed that such algo- on the road. Indeed, even considering the pilot frame of the rithms are intended to estimate long-term (10 y) risk, and our design, our results are consistent and encouraging, and may patients were followed for a median time of 35 mo. Thus, pave the path to evaluate these different forms of evaluation our results, although requiring confirmation in larger studies of CV risk in LT recipients. © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. Sastre et al 9 In conclusion, we provide the first data showing that Heart, Lung, and Blood Institute; American Heart Association; the estimation of arterial stiffness mirrors CV risk after LT. World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation. Further studies will be necessary as to investigate whether it 2009;120:1640–1645. can be implemented as a screening tool or used as a surrogate 21. Goff DC Jr, Lloyd-Jones DM, Bennett G, et al; American College of CV risk in studies evaluating long-term outcomes. of Cardiology/American Heart Association Task Force on Practice Guidelines. 2013 ACC/AHA guideline on the assessment of cardio- vascular risk: a report of the American College of Cardiology/American REFERENCES Heart Association Task Force on Practice Guidelines. Circulation. 1. Watt KDS, Pedersen RA, Kremers WK, et al. Evolution of causes and 2014;129:S49–S73. risk factors for mortality post-liver transplant: results of the NIDDK 22. Conroy RM, Pyörälä K, Fitzgerald AP, et al; SCORE Project Group. long-term follow-up study. Am J Transplant. 2010;10:1420–1427. Estimation of ten-year risk of fatal cardiovascular disease in Europe: 2. Åberg F, Gissler M, Karlsen TH, et al. Differences in long-term sur- the SCORE project. Eur Heart J. 2003;24:987–1003. vival among liver transplant recipients and the general population: a 23. Reference Values for Arterial Stiffness, Collaboration. Determinants of population-based Nordic study. Hepatology. 2015;61:668–677. pulse wave velocity in healthy people and in the presence of cardio- 3. Sastre L, García R, Gándara J-G, et al. Incidence, predictors, and vascular risk factors: ‘establishing normal and reference values’ Eur impact on survival of long-term cardiovascular events after liver trans- Heart J. 2010;31:2338–2350. plantation. Transplantation. 2020;104:317–325. 24. Kwong A, Kim WR, Lake JR, et al. OPTN/SRTR 2018 annual data 4. Thoefner LB, Rostved AA, Pommergaard H-C, et al. Risk factors for report: liver. Am J Transplant. 2020;20(Suppl S1):193–299. metabolic syndrome after liver transplantation: a systematic review 25. Giusto M, Berenguer M, Merkel C, et al. Chronic kidney disease after and meta-analysis. Transplant Rev (Orlando). 2018;32:69–77. liver transplantation: pretransplantation risk factors and predictors 5. VanWagner LB, Montag S, Zhao L, et al. Cardiovascular disease out- during follow-up. Transplantation. 2013;95:1148–1153. comes related to early stage renal impairment after liver transplanta- 26. De Luca L, Kalafateli M, Bianchi S, et al. Cardiovascular morbidity and tion. Transplantation. 2018;102:1096–1107. mortality is increased post-liver transplantation even in recipients with 6. Konerman MA, Fritze D, Weinberg RL, et al. Incidence of and risk no pre-existing risk factors. Liver Int. 2019;39:1557–1565. assessment for adverse cardiovascular outcomes after liver transplan- 27. Martínez-Saldivar B, Prieto J, Berenguer M, et al. Control of tation: a systematic review. Transplantation. 2017;101:1645–1657. blood pressure in liver transplant recipients. Transplantation. 7. Di Maira T, Rubin A, Puchades L, et al. Framingham score, renal 2012;93:1031–1037. dysfunction, and cardiovascular risk in liver transplant patients. Liver 28. Alvarez-Sotomayor D, Satorres C, Rodríguez-Medina B, et al. Transpl. 2015;21:812–822. Controlling diabetes after liver transplantation: room for improvement. 8. Guckelberger O, Mutzke F, Glanemann M, et al. Validation of cardi- Transplantation. 2016;100:e66–e73. ovascular risk scores in a liver transplant population. Liver Transpl. 29. Patel SS, Rodriguez VA, Siddiqui MB, et al. The impact of coronary 2006;12:394–401. artery disease and statins on survival after liver transplantation. Liver 9. Tibaut M, Caprnda M, Kubatka P, et al. Markers of atherosclerosis: Transpl. 2019;25:1514–1523. part 1 - serological markers. Heart Lung Circ. 2019;28:667–677. 30. VanWagner LB, Holl JL, Montag S, et al. Blood pressure control 10. Maki KC, Dicklin MR. Assessing cardiovascular disease risk and according to clinical practice guidelines is associated with decreased responses to preventive therapies in clinical practice. Curr Atheroscler mortality and cardiovascular events among liver transplant recipients. Rep. 2018;20:23. Am J Transplant. 2020;20:797–807. 11. Kim H-L, Kim S-H. Pulse wave velocity in atherosclerosis. Front 31. Cavalcante JL, Lima JAC, Redheuil A, et al. Aortic stiffness: cur- Cardiovasc Med. 2019;6:41. rent understanding and future directions. J Am Coll Cardiol. 12. Ben-Shlomo Y, Spears M, Boustred C, et al. Aortic pulse wave veloc- 2011;57:1511–1522. ity improves cardiovascular event prediction: an individual participant 32. Sánchez-Martínez M, Cruz JJ, Graciani A, et al. Pulse wave velocity meta-analysis of prospective observational data from 17,635 sub- and central blood pressure: normal and reference values in older peo- jects. J Am Coll Cardiol. 2014;63:636–646. ple in Spain. Rev Esp Cardiol (Engl Ed). 2018;71:1084–1086. 13. Tynjälä A, Forsblom C, Harjutsalo V, et al; FinnDiane Study Group. 33. Gómez-Sánchez M, Patino-Alonso MC, Gómez-Sánchez L, et al; Arterial stiffness predicts mortality in individuals with type 1 diabetes. EVA Group. Reference values of arterial stiffness parameters and their Diabetes Care. 2020;43:2266–2271. association with cardiovascular risk factors in the Spanish population. 14. Hametner B, Wassertheurer S, Kropf J, et al. Oscillometric estimation The EVA Study. Rev Esp Cardiol (Engl Ed). 2020;73:43–52. of aortic pulse wave velocity: comparison with intra-aortic catheter 34. Nordestgaard BG, Varbo A. Triglycerides and cardiovascular disease. measurements. Blood Press Monit. 2013;18:173–176. Lancet. 2014;384:626–635. 15. Omboni S, Posokhov IN, Kotovskaya YV, et al. Twenty-four-hour 35. Klein LW. Systemic and coronary hemodynamic effects of tobacco ambulatory pulse wave analysis in hypertension management: current products on the cardiovascular system and potential pathophysiologic evidence and perspectives. Curr Hypertens Rep. 2016;18:72. mechanisms. Cardio Rev. [Epub ahead of print. May 12, 2021]. doi: 16. Korogiannou M, Xagas E, Marinaki S, et al. Arterial stiffness in patients 10.1097/CRD.0000000000000395 with renal transplantation; associations with co-morbid conditions, 36. Hagström H, Nasr P, Ekstedt M, et al. Cardiovascular risk factors in evolution, and prognostic importance for cardiovascular and renal non-alcoholic fatty liver disease. Liver Int. 2019;39:197–204. outcomes. Front Cardiovasc Med. 2019;6:67. 37. Aghaulor B, VanWagner LB. Cardiac and pulmonary risk stratification 17. Dégi AA, Bárczi A, Szabó D, et al. Cardiovascular risk assessment in liver transplantation. Clin Liv Dis. 2021;25:157–177. in pediatric liver transplant patients. J Pediatr Gastroenterol Nutr. 38. Boutouyrie P, Lacolley P, Briet M, et al. Pharmacological modulation of 2019;68:377–383. arterial stiffness. Drugs. 2011;71:1689–1701. 18. Al Nasser Y, Moura MC, Mertens L, et al. Subclinical cardiovascular 39. Boutouyrie P, Chowienczyk P, Humphrey JD, et al. Arterial stiffness changes in pediatric solid organ transplant recipients: a systematic and cardiovascular risk in hypertension. Circ Res. 2021;128:864–886. review and meta-analysis. Pediatr Transplant. 2016;20:530–539. 40. Del Giorno R, Troiani C, Gabutti S, et al. Comparing oscillometric and 19. Piepoli MF, Hoes AW, Agewall S, et al; ESC Scientific Document tonometric methods to assess pulse wave velocity: a population- Group. 2016 European guidelines on cardiovascular disease preven- based study. Ann Med. 2021;53:1–16. tion in clinical practice: The Sixth Joint Task Force of the European 41. Omboni S, Posokhov I, Parati G, et al. Ambulatory blood pressure Society of Cardiology and Other Societies on Cardiovascular Disease and arterial stiffness web-based telemonitoring in patients at car- Prevention in Clinical Practice (constituted by representatives of 10 diovascular risk. First results of the VASOTENS (Vascular health societies and by invited experts) developed with the special contri- ASessment Of The hypertENsive patients) Registry. J Clin Hypertens. bution of the European Association for Cardiovascular Prevention & 2019;21:1155–1168. Rehabilitation (EACPR). Eur Heart J. 2016;37:2315–2381. 42. Sarafidis PA, Loutradis C, Karpetas A, et al. Ambulatory pulse wave 20. Alberti KGMM, Eckel RH, Grundy SM, et al. Harmonizing the meta- velocity is a stronger predictor of cardiovascular events and all-cause bolic syndrome: a joint interim statement of the International Diabetes mortality than office and ambulatory blood pressure in hemodialysis Federation Task Force on Epidemiology and Prevention; National patients. Hypertension. 2017;70:148–157.