Background Granulomatosis with polyangiitis (GPA) is a rare granulomatous vasculitis affecting small- and medium-sized blood vessels. In optimally treated patients with long-standing disease, the common cause of death is atherosclerosis even in the absence of typical risk factors. Objective To evaluate endothelial dysfunction in GPA patients. Methods 44 patients (21 men and 23 women) diagnosed with GPA and 53 controls matched for age, sex, BMI and typical risk factors for cardiovascular diseases (22 men and 31 women) were enrolled in the study. We measured each participant’s serum levels of vascular cell adhesion molecule-1 (VCAM-1), interleukin 6 (IL-6), and thrombomodulin. We also studied flow-mediated dilatation (FMD) of the brachial artery, intima-media thickness (IMT) of the common carotid artery and aortic stiffness using echocardiography. Results Patients with GPA showed a 15.9% increase in serum levels of VCAM-1 (p = 0.01), 66% of IL-6 (p < 0.001) and 50.9% of thrombomodulin (p < 0.001) compared to controls. FMD% was 48.9% lower in patients with GPA in comparison to controls (p < 0.001), after adjustment for potential confounders, with no differences regarding IMT or aortic stiffness. FMD% was negatively associated with duration of the disease (β = − 0.18 [95% CI: − 0.32 to − 0.04]), C-reactive protein (β = − 0.17 [95% CI: − 0.27 to − 0.07]), IL-6 (β = − 0.29 [95% CI: − 0.39 to − 0.19]), blood creatinine level (β = − 0.2 [95% CI: − 0.3 to − 0.1]), and IMT (β = − 0.14 (− 0.24 to − 0.04). In a multiple linear regression model, kidney function, IMT, pack-years of smoking, diabetes and level of VCAM-1 were independent predictors of lower FMD%. Conclusion GPA is characterized by endothelial dysfunction. FMD is a useful tool for the detection of endothelial injury. Keywords Endothelium · Atherosclerosis · Systemic vasculitis · Ultrasonography Introduction Endothelium plays a key role in vascular homeostasis. It Electronic supplementary material The online version of this acts as a barrier between tissues and circulating blood and article (https ://doi.org/10.1007/s0029 6-018-4061-x) contains supplementary material, which is available to authorized users. as a signal transducer that regulates vasomotor activity . Activation of endothelial cells leads to upregulation of adhe- * Jerzy Dropiński sion molecules, such as P-selectin, E-selectin, intercellular email@example.com adhesion molecule-1, and vascular cell adhesion molecule-1 Department of Anatomy, Jagiellonian University, Medical (VCAM-1), resulting in attachment and migration of cir- College, Cracow, Poland culating leukocytes. Differentiation of migrated monocytes Centre of Oncology, Maria Sklodowska-Curie Memorial into macrophages and the subsequent uptake of lipids by Institute, Cracow Branch, Cracow, Poland these cells results in foam cell generation and fatty streak 2nd Department of Internal Medicine, Jagiellonian formation. Further recruitment of inflammatory cells and University, Medical College, ul.Skawińska 8, 31-066 Cracow, proliferation of smooth muscle cells leads to the develop- Poland ment of atherosclerotic plaque . Endothelial function can Faculty of Mathematics and Natural Sciences, University be determined with a noninvasive ultrasound measurement of Rzeszow, Rzeszow, Poland Vol.:(0123456789) 1 3 1522 Rheumatology International (2018) 38:1521–1530 of flow-mediated dilatation (FMD) of a brachial artery. enrolled from the hospital personnel and relatives. They This is the direct measurement of the arterial endothelium’s were selected according to matching criteria. All partici- response to hyperemia (shear stress) that leads to the nitric pants received a detailed brief of the methodology and safety oxide release and vasodilatation. protocols for the study and provided written consent for their Granulomatosis with polyangiitis (GPA, formerly Wegen- participation. er’s granulomatosis) is the common vasculitis with a preva- lence of 3 per 100, 000 and peak incidence at the age of Study groups 50–60. Anti-neutrophil cytoplasmic antibodies (ANCA) are considered as the marker of the disease and are tar- The case group constituted 44 patients with GPA—21 men geted against proteinase 3 (PR3). Its most common clinical and 23 women. features are granulomatous lesions of the upper and lower The control group consist of 53 individuals, 22 men and airways accompanied by the kidney failure [3, 4]. Cardiac 31 women, matched to GPA patients by gender, age, body involvement is infrequent in GPA, but coronary heart dis- mass index (BMI) and smoking habit, as well as comor- ease, arrhythmias, pericarditis, and nonbacterial thrombotic bidities, including hypertension, hypercholesterolemia, and endocarditis can be present in these patients [5–8]. diabetes mellitus. The increased morbidity from ischemic heart disease in GPA suggests that not only small vessels but also big ones Patients are affected [9 ]. Atherosclerosis and its complications are one of the lead- Each patient had a current or previous diagnosis of GPA ing cause of death even in properly treated patients with based on the criteria of the American College of Rheumatol- long-standing ANCA-associated vasculitis (AAVs) [10, ogy . We analyze only patients with the disease flare or 11]. However, the mechanism by which atherosclerosis is those, who were symptomatic and diagnosed with persistent promoted in these diseases is not explained by the classical disease. Disease activity was measured using the Birming- atherosclerotic risk factors and remains under investigation. ham Vasculitis Activity Score (BVAS) . Disease flare The relationship between inflammation, vascular dys- was defined as the presence of new symptoms (major or function and atherosclerosis is well-established. Premature minor item in BVAS). Persistent disease was defined as the atherosclerosis has been observed in patients with chronic presence of one or more persistent symptoms attributed to inflammatory diseases such as systemic lupus erythematosus active disease for more than 1 month but less than 3 months. [12, 13], systemic sclerosis [14, 15] and antiphospholipid For symptoms which occurred in patients since the onset syndrome [16, 17]. There is currently a lack of reliable data of GPA and were present for more than 3 months, we used on endothelial injury and development of premature athero- Vascular Damage Index (VDI) . Patients with congestive sclerosis in the setting of vasculitis. heart failure, coronary heart disease, uncontrolled hyperten- Previously, a few studies have analyzed the endothelial sion, liver failure, and cancer were excluded from the study damage and progression of atherosclerosis in patients with (for details see supplementary material). systemic vasculitis [18–21]. Their results, however, were inconsistent and limited in significance due to the small Main outcome variable number of subjects studied. For this reason, we sought to evaluate ultrasonographic and laboratory markers of In this case–control study, we analyzed whether GPA is endothelial injury in patients with GPA, which might be associated with vascular endothelial damage. We measured related to the premature and accelerated atherosclerosis and flow-mediated dilatation of the brachial artery, intima-media increased risk of cardiovascular events. thickness of the common carotid artery, aortic stiffness as well as evaluated serum levels of thrombomodulin and VCAM-1 in GPA patients and matched control subjects. Methods Procedures A case–control observational, retrospective study was car- ried out with approval of the Bioethics Committee of XXXX Laboratory analysis University Medical College (9th May 2013, number of pro- tocol: KBET/79/B/2013). The patients were recruited from Fasting blood samples were drawn in the morning from the the population of patients at the Department of Allergy antecubital vein using minimal stasis. Lipid profile, glucose, and Clinical Immunology at the University Hospital in liver enzymes, urine, creatinine with eGFR, complete blood XXXXX in the period between 2014 and 2017 who were cell and platelet count were analyzed by routine laboratory in the disease flare or symptomatic. The control group was techniques. C-reactive protein (CRP) was measured using 1 3 Rheumatology International (2018) 38:1521–1530 1523 analyzer VITROS 250 Johnson & Johnson. Blood samples by a forward stepwise selection procedure, verified by F Sne- were drawn into serum separation tubes, centrifuged at decore’s statistics, with F > 1. The R was used as a measure 2000×g for 20 min at room temperature, within 2 h from of the variance. To calculate odds ratios (ORs) with 95% sampling. The supernatant was frozen in aliquots and stored confidence intervals (CIs), unconditional multivariate logis- at -70 °C until analysis. Interleukin-6 (IL-6), VCAM-1, and tic regression was performed. The cut-off values for IL-6, soluble thrombomodulin were measured using standard- VCAM-1, FMD%, and thrombomodulin were determined ized ELISA method (all, R&D Systems, Minneapolis, MN, based on receiver operating characteristic (ROC) curves. USA). Anti-PR3 IgG was measured in all GPA subjects Results were considered statistically significant when the p using ELISA assay (EUROIMMUN, Lübeck, Germany). value was less than 0.05. Ultrasound examinations Results Ultrasound examinations were performed in a darkened, quiet, room, after at least 10 min rest in a supine position, Characteristics of patients and controls using high-quality ultrasonograph (Sequoia 512 with a 10 MHz linear array ultrasonic transducer, MountainView, Demographic, clinical and laboratory characteristics of the Ca, USA). Before examination, the subjects refrained from studied subjects, including basic laboratory tests, ultrasound eating for at least 10 h. Examinations were conducted by parameters, and cardiovascular risk factors were given in two independent ultrasonography experts and considered Table 1. Both groups were similar in age, sex, BMI as well as parameters constituted a mean of three subsequent meas- prevalence of comorbidities (hypercholesterolemia, hyper- urements. A complete transthoracic echocardiogram (TTE) tension, and diabetes mellitus), smoking habit, and family was performed in every participant with estimation of ejec- history of cardiovascular diseases. Parameters describing tion fraction of the left ventricle (EF) and systolic pulmo- GPA activity, as well as current and past therapy were given nary artery pressure in accordance to standard methods . in Table 2. The median duration of the disease was 4.5 years. Flow-mediated dilatation (FMD) of the brachial artery was More than half of the patients had active disease at the time measured in accordance to Celermayer’ method . Aortic of evaluation. All of them had detectable anti-PR3. Most of stiffness was expressed as a percentage of aortic systolic them were being treated with steroids currently or in the past diameter (ASD) and aortic diastolic diameter (ADD): aortic with other immunosuppressive agents, such as: azathioprine, stiffness % = [(ADD − ASD)/ASD] × 100%. The intima- cyclophosphamide, methotrexate, mycophenolate mofetil media thickness (IMT) of the carotid artery was also meas- and rituximab. Additionally, GPA patients were receiv- ured and in the further analysis we used a mean value of the ing statins, beta-blockers, angiotensin-converting enzyme IMT measured on the right and left common carotid artery inhibitors or angiotensin receptor antagonists, diuretics and (for details see supplementary material). calcium channel blockers. Lungs were the most commonly involved organs, followed by paranasal sinuses and kidneys. Statistical analysis Basic laboratory tests and basic transthoracic The results were compared between the case and control echocardiographic parameters groups using STATISTICA 12.5 Software. Continuous variables, all non-normal distributed values (verified by the As expected, GPA patients were characterized by higher Shapiro–Wilk test), were given as median and interquartile inflammatory markers, such as CRP, IL-6 (reference range: range and compared by the Mann–Whitney U test. Cate- 0.45–9.96 pg/ml) and white blood cells, as well as impaired gorical variables were presented as numbers (percentages) kidney function and lower hemoglobin level (Table 1). and compared by χ test. Potential confounders were identi- Moreover, there were characterized by higher triglycerides. fied as: age, BMI, sex, and comorbidities such as arterial In TTE GPA subjects had larger left and right ventricles hypertension, diabetes mellitus and hypercholesterolemia. and left atria, thicker posterior walls and interventricular To adjust for these, obtained results of FMD%, IMT, aortic septa, as well as lower ejection fraction and higher systolic stiffness%, IL-6, VCAM-1, and thrombomodulin were log- pulmonary artery pressure. transformed and a one way covariance analysis (ANCOVA) was performed, to achieve the overall p value. The univariate Laboratory markers of endothelial injury linear regression tests (with adjustment for aforementioned confounders) were used to analyze associations between two GPA patients had a 15.9% higher levels of VCAM-1 selected parameters. Independent determinants of FMD% (p = 0.01) and a 50.9% increased thrombomodulin con- were established in multiple linear regression model, built centrations (p < 0.001) in peripheral blood, comparing to 1 3 1524 Rheumatology International (2018) 38:1521–1530 Table 1 A summary of demographic, laboratory and echocardiographic parameters in patients with granulomatosis with polyangiitis and con- trols Patients, n* = 44 Controls, n = 53 p value Age (years) 59 (46–65) 48 (43–61) 0.07 Male gender, number (%) 21 (47.6) 22 (41.5) 0.67 Body mass index (kg/m2) 26.1 (24.1–29.6) 26.6 (23.9–29.1) 0.93 Basic laboratory tests Hemoglobin (g/dl) 12.25 (10.55–13.55) 13.7 (12.7–15) < 0.001 3 a Red blood cells (10 /ul) 4.13 (3.7–4.5) 4.5 (4.2–4.9) < 0.001 3 a White blood cells (10 /ul) 7.46 (5.76–10.06) 5.9 (5.03–6.96) < 0.001 Platelet count (10 /ul) 235.5 (171–287) 225 (200–275) 0.67 Total cholesterol (mmol/l) 4.7 (3.9–5.4) 4.9 (4.2–5.25) 0.58 Low-density lipoprotein (mmol/l) 2.4 (1.9–3.2) 3.1 (2.5–3.6) 0.003 Triglycerides (mmol/l) 1.7 (1.2–2.1) 1.1 (0.7–1.5) 0.002 Glucose (mmol/l) 5 (4.45–5.43) 4.95 (4.72–5.2) 0.95 Creatinine (mmol/l) 101.9 (72.5–240) 76.1 (68.3–90) 0.01 Urea (mmol/l) 7.55 (5.8–12.7) 4.56 (3.93–5.3) < 0.001 2 a Estimated glomerular filtration rate (ml/min/1.73 m ) 60 (26–67) 60 (60–80) 0.01 Alanine transaminase (U/l) 21.5 (15–32) 22.5 (14–28) 0.72 C-reactive protein (mg/dl) 7.6 (5–19.4) 1.2 (1–2.1) < 0.001 Interleukin-6 (pg/ml) 5.03 (3.02–10.5) 1.7 (1.08–2.16) < 0.001 Echocardiographic parameters Left ventricular diastolic diameter (cm) 4.8 (4.6–5.3) 4.7 (4.5–4.9) 0.29 Left ventricular systolic diameter (cm) 3 (3–3.4) 3 (2.9–3.1) 0.049 Right ventricular diameter (cm) 2.2 (2–2.3) 2.1 (1.9–2.3) 0.01 Left atrial diameter (cm) 3.9 (3.7–4.1) 3.7 (3.5–3.9) 0.004 Left ventricle posterior wall thickness (cm) 1.05 (0.9–1.2) 0.9 (0.8–1) < 0.001 Interventricular septum thickness (cm) 1.1 (1–1.2) 0.9 (0.8–1) < 0.001 Ejection fraction (%) 65 (60–68) 68 (68–70) < 0.001 Pulmonary artery pressure (mmHg) 32 (30–36) 32 (26–32) 0.01 Laboratory parameters of endothelial injury Vascular cell adhesion molecule-1 (ng/ml) 957 (749.1–1273.4) 804.6 (694.4–936.7) 0.01 Thrombomodulin (ng/ml) 8.9 (5.2–1.4) 4.3 (3.9–4.7) < 0.001 Ultrasound parameters of endothelial injury and atherosclerosis Relative increase of flow-mediated dilatation of a brachial artery 5.26 (4.08–8.01) 10.3 (8.89–12,5) < 0.001 Aortic stiffness (%) 7.14 (4–9.09) 7.4 (6.25–10.34) 0.27 Median value of intima-media thickness of a common carotid artery (cm) 0.07 (0.06–0.08) 0.07 (0.06–0.08) 0.20 Other cardiovascular risk factors Hypertension n(%) 21 (50) 16 (30.2) 0.08 Diabetes mellitus n(%) 9 (21.43) 6 (11.3) 0.2 Hypercholesterolemia n(%) 14 (33.33) 19 (35.8) 0.68 Smoking currently n(%) 3 (7.14) 4 (7.55) 0.9 In the past n(%) 13 (30.95) 15 (28.3) 0.33 Smoking (packs/years) 0 (0–15) 0 (0–3) 0.64 Positive family history of cardiovascular diseases n(%) 9 (21.42) 7 (13.2) 0.32 Categorical variables are presented as numbers (percentage), continuous variables as median and interquartile range. The results which are sta- tistically significant are marked healthy individuals. However, in ANCOVA analysis we confounders (age, sex, BMI, hypercholesterolemia, hyper- documented that only thrombomodulin levels remained tension, and diabetes mellitus, p < 0.001). The VCAM-1 was higher in GPA subjects after adjustment for potential similar in GPA and control groups in this analysis (p = 0.54). 1 3 Rheumatology International (2018) 38:1521–1530 1525 Table 2 Clinical characteristics of the patients (n = 44) with granulo- and β = 0.28 [95% CI: 0.27–0.29], VCAM-1 and thrombo- matosis with polyangiitis modulin, respectively) and IL-6 level (β = 0.27 [95% CI: 0.15–0.39], and β = 0.4 [95% CI: 0.27–0.53], VCAM-1 and Patients thrombomodulin, respectively). Moreover, we demonstrated Duration of the disease (years) 4.5 (1–9) strong positive association between white blood cells and Active disease n (%) 26 (59.1) thrombomodulin (β = 0.2 [95% CI: 0.11–0.29]). Table 3 BVAS in active disease 9 (8–10) demonstrates the most important associations of selected Persistent disease n(%) 16 (36.36) laboratory and echocardiographic parameters after adjust- BVAS in persistent disease 4 (3–5) ment for confounders with linear regression models. We Anti-proteinase 3 antibodies (IU/ml) 20.5 (5–65) documented positive associations between laboratory param- VDI score in eligible patients 3 (0–5) eters of endothelial damage and kidney function, anti-PR3 Organ involvement level as well as interventricular septum and posterior wall Cutaneous vasculitis n (%) 13 (30.95) thickness (see Table 3). Granulomatous lesions in ears/hearing disturbances 11 (26.19) GPA patients with hypertension had 30. 6% higher levels n (%) of VCAM-1 (1178.3 [815.2–1600.1] vs. 893.8 [635.4–959.8] Granulomatous lesions in larynx n (%) 6 (14.63) ng/ml, p = 0.02) and 41% higher thrombomodulin levels Paranasal sinuses inflammation n (%) 30 (71.42) (11.3 [7.8–17.6] vs. 5.9 [4.4–9.5] ng/ml, p = 0.01) than Bone destruction of paranasal sinuses n(%) 16 (38.1) the remaining GPA patients. Moreover, GPA patients with Chronic kidney disease n (%) 22 (52.38) chronic kidney disease were characterized by higher throm- Lungs n (%) 31 (73.81) bomodulin level (11.7 [9.4–17.6) vs. 5.4 [4.4–6.9] ng/ml, Peripheral nerves n (%) 10 (23.8) p < 0.001) and VCAM-1 level (1258.2 [893.2–1457.7] vs. Gastrointestinal system n (%) 1 (2.38) 747.4 [546.9–917.5] ng/ml, p < 0.001). Other comorbidities Heart n (%) 1 (2.38) had no impact on laboratory markers of endothelial damage. Treatment characteristic Patients taking statins and antihypertensive medications had Current steroids n (%) 37 (88.1) increased thrombomodulin blood level (10.7 [7.5–15.2] vs. Current steroids dose (mg/day of prednisone) 8 (4–20) 5.6 [4.1–11.9] ng/ml, p = 0.03, and 11.1 [7.1–16.1] vs. 5.4 Systemic steroids therapy (years) 2 (0.5–5) [3.9–8.1] ng/ml, p = 0.01, respectively), Moreover, those Immunosuppressive treatment (currently or in the past) treated with antihypertensives had higher level of VCAM-1 Azathioprine n (%) 12 (28.57) (1089.7 [884.5–1293.1] vs. 803.4 [520.1–905.3] ng/ml, Cyclophosphamide n (%) 37 (88.1) p = 0.01). Total dose of cyclophosphamide (grams) 8.15 (3.9–19) In the subgroups analysis, patients in persistent disease or Methotrexate n (%) 5 (11.9) in active disease had similar results of laboratory parameters Mycophenolate mofetil n (%) 2 (5.26) of endothelial injury (thrombomodulin 12.63 [5.99–18.86] Rituximab n (%) 13 (30.95) vs. 10.18 [5.24–11.4] ng/ml, p = 0.19, VCAM-1 1099.05 Internal medicine medications [863.94–1263.64] vs. 1065.82 [745.64–1273.38] ng/ml, Angiotensin-converting enzyme inhibitors or angio- 12 (28.57) p = 0.58). tensin receptor antagonists n (%) Statins n (%) 21 (51.22) Ultrasound parameters of endothelial injury Beta-blockers n (%) 17 (40.48) Diuretics n (%) 12 (28.57) GPA patients had 48.9% decrease in FMD% compared to Calcium channel blockers n (%) 12 (28.57) controls (p < 0.001, also after adjustment for potential con- Categorical variables are presented as numbers (percentage), continu- founders: age, sex, BMI, hypercholesterolemia, hyperten- ous variables as median and interquartile range sion, diabetes mellitus p < 0.001), and markedly higher risk n number, BVAS Birmingham Vasculitis Activity Score, VDI vascular of diminished FMD% defined as values below the cut-off damage index point of 8.51 (OR 4.9 [95% CI: 2.88–8.23]). In Table 3 are given selected associations of FMD% with Moreover, patients with GPA had increased risk of elevated other laboratory and ultrasound parameters. As presented, VCAM-1 (OR 5.75 [95% CI: 2–16.38], reference range: FMD% was negatively associated with white blood cells (β 349–991 ng/ml), and thrombomodulin (OR 6.71 [95% CI: = − 0.24 [95% CI: − 0.32 to − 0.15]), CRP (β = − 0.17 [95% 3.37–13.3], reference range: 2.9–5.3 ng/ml) compared to the CI: − 0.27 to − 0.07]), IL-6 (β = − 0.29 [95% CI: − 0.39 healthy individuals (cut-off points: 1213.96 and 5.9 ng/ml, to − 0.19]) and the blood creatinine level (β = − 0.2 [95% respectively). As expected, both endothelial injury mark- CI:− 0.3 to − 0.1]) in univariate linear regression models. ers were related to the CRP (β = 0.18 [95% CI: 0.08–0.28], Interestingly, FMD% was also negatively related to smoking 1 3 1526 Rheumatology International (2018) 38:1521–1530 Table 3 Correlations of selected laboratory and echocardiographic parameters in GPA patients Flow-mediated dilata- Intima-media thick- Aortic stiffness%, β Vascular cell adhe- Trombomodulin (ng/ tion% β (95% CI) ness (cm) β (95% CI) (95% CI) sion molecule-1 (ng/ ml), β (95% CI) ml), β (95% CI) White blood cells − 0.24 (− 0.32 to − 0.07 (− 0.02 to 0.16) − 0.23 (− 0.36 to − 0.08 (− 0.02 to 0.18) 0.2 (0.11 to 0.29)* (10 /ul) 0.15)* 0.1)* C-reactive protein − 0.17 (− 0.27 to − − 0.03 (− 0.13 to − 0.19 (− 0.31 to − 0.18 (0.08 to 0.28)* 0.28 (0.27 to 0.29)* (mg/dl) 0.07)* 0.07) 0.7)* Interleukin-6 (pg/ml) − 0.29 (− 0.39 to − − 0.03 (− 0.13 to 0.08 (− 0.06 to 0.22) 0.24 (0.14 to 0.34)* 0.29 (0.2 to 0.38)* 0.19)* 0.07) Creatinine(mmol/l) − 0.2 (− 0.3 to − 0.18 (0.08 to 0.28)* 0.27 (0.15 to 0.39)* 0.39 (0.29 to 0.49)* 0.6 (0.53 to 0.67)* 0.1)* Urea(mmol/l) − 0.06 (− 0.16 to 0.11 (0 to 0.22) 0.11 (− 0.04 to 0.26) 0.27 (0.06 to 0.38)* 0.63 (0.55 to 0.71)* 0.04) Estimated glomerular 0.05 (− 0.05 to 0.15) − 0.12 (− 0.24 to 0) 0.00 (− 0.19 to 0.19) − 0.23 (− 0.36 to − − 0.48 (− 0.38 to − filtration rate (ml/ 0.1)* 0.58)* min/1.73 m ) Interventricular sep- − 0.23 (− 0.33 to − 0.12 (0.01 to 0.23)* 0.01 (− 0.13 to 0.15) 0.29 (0.17 to 0.41)* 0.43 (0.33 to 0.53)* tum thickness (cm) 0.13)* Posterior wall thick- − 0.29 (− 0.39 to − 0.16 (0.05 to 0.27)* 0.00 (− 0.14 to 0.14) 0.35 (0.24 to 0.46)* 0.5 (0.41 to 0.59)* ness (cm) 0.19)* Flow-mediated dilata- – − 0.12 (− 0.22 to − 0.25 (0.1 to 0.4)* 0.03 (− 0.09 to 0.15) − 0.07 (− 0.17 to 0.03) tion% 0.02)* Intima-media thick- − 0.14 (− 0.24 to − – 0.00 (− 0.18 to 0.18) − 0.09 (− 0.22 to 0.05 (− 0.05 to 0.15) ness (cm) 0.04)* 0.04) Aortic stiffness% 0.2 (− 0.07 to 0.43) 0.00 (− 0.12 to 0.12) – 0.31 (0.17 to 0.44)* 0.16 (0.03 to 0.29)* Vascular cell adhesion 0.02 (− 0.08 to 0.12) − 0.08 (− 0.18 to 0.3 (0.17 to 0.43)* – 0.57 (0.49 to 0.65)* molecule-1 (ng/ml) 0.02) Thrombomodulin (ng/ − 0.09 (− 0.19 to 0.05 (− 0.05 to 0.15) 0.19 (0.04 to 0.34)* 0.72 (0.62 to 0.82)* – ml) 0.01) Smoking (packs/years) − 0.33 (− 0.44 to − 0.04 (− 0.02 to 0.1) − 0.17 (− 0.47 to − 0.2 (− 0.47 to 0.07) − 0.04 (− 0.3 to 0.2) 0.12)* 0.13) Steroids time of treat- − 0.07 (− 0.23 to 0.1) 0.19 (0.03 to 0.35)* − 0.19 − 0.43 to 0.05 − 0.1 − 0.29 to 0.09 0.05 − 0.13 to 0.22 ment (years) Duration of the − 0.18 (− 0.32 to − 0.27 (0.14 to 0.41)* 0.13 (− 0.08 to 0.34) − 0.1 (− 0.28 to 0.08) 0.09 (− 0.06 to 0.24) disease 0.04)* Concentration of anti- 0.00 (− 0.14 to 0.14) − 0.08 (− 0.23 to − 0.04 (− 0.24 to 0.19 (0. 02 to 0.36)* 0.163 (0.001 to 0.33)* proteinase 3 antibod- 0.07) 0.16) ies (IU/ml) The resulting regression coefficients (β) were given after adjustment for age, sex, BMI, and comorbidities (hypertension, diabetes mellitus and hypercholesterolemia) *The results which are statistically significant are marked (packs/years) (β = − 0.33 [95% CI: − 0.44 to − 0.12)], dura- Among GPA patients, lower FMD% was observed in tion of the disease (β = − 0.18 [95% CI: − 0.32 to − 0.04]), those with hypertension (5 [2.9–5.9] vs. 6.4 [4.7–8.9], as well as posterior wall and interventricular septum thick- p = 0.01), diabetes (3.4 [2.9–5] vs. 6 [4.3–8.4], p = 0.01) and ness (β = − 0.29 [95% CI: − 0.39 to − 0.19], β = − 0.23 smoking currently or in the past (4.3 [2.9–5] vs. 6.7 [5–0.3]). [95% CI: − 0.33 to − 0.13], respectively). Interestingly, only those treated with azathioprine had lower A multiple regression model showed that various factors FMD% (4.13 [2.9–5.13] vs. 6.7 [4.9–8.4], p = 0.01) with- independently determined FMD%, including presence of out influence of other immunosuppressive drugs. Patients diabetes mellitus (β = − 0.41 [95% CI: − 0.55 to − 0.27]), treated with statins were also characterized by decrease in pack-years of smoking (β = − 0.14 [95% CI: − 0.29 to FMD% (4.5 [2.9–5.3] vs. 6.8 [5.1–9.4], p = 0.01). − 0.01]), IMT (β = − 0.34 [95% CI: − 0.5 to − 0.18]), serum The values of aortic stiffness% and IMT were similar urea (β = − 0.41 [95% CI: − 0.61 to − 0.21]) or VCAM-1 (β in both studied groups. Aortic stiffness% and IMT did not = − 0.33 [95% CI: − 0.53 to − 0.13]), (Table 4). correlate with laboratory parameters of endothelial injury. 1 3 Rheumatology International (2018) 38:1521–1530 1527 Table 4 Multiple linear regression model for a relative increase of flow-mediated dilatation of a brachial artery comparing patients and control group Patients Control 2 2 β (95% CI) R β (95% CI) R Duration of the disease (years) 0.15 (− 0.08 to 0.38) 0.51 – 0.32 Total cholesterol level (mmol/l) − 0.06 (− 0.19 to 0.07) 0.07 (− 0.05 to 0.19) Urea (mmol/l) − 0.41 (− 0.61 to − 0.21)* − 0.22 (− 0.36 to − 0.08)* Posterior wall thickness (cm) 0.03 (− 0.15 to 0.21) − 0.41 (− 0.56 to − 0.26) * Intima-media thickness of a common carotid − 0.34 (− 0.50 to − 0.18)* − 0.20 (− 0.35 to − 0.05)* artery (cm) Total dose of cyclophosphamide (grams) − 0.19 (− 0.41 to 0.03) – Smoking (packs/years) − 0.14 (− 0.29 to − 0.01)* – Diabetes mellitus − 0.41 (− 0.55 to − 0.27)* – Vascular cell adhesion molecule-1 (ng/ml) − 0.33 (− 0.53 to − 0.13)* − 0.09 (− 0.22 to 0.04) Adjustment statistics F = 2.84, p = 0.01 F = 4.2, p < 0.001 The resulting standardized regression coefficient (β) with 95% confidence intervals (95% CI) for a factor (independent variable) indicates the increase/decrease in standard deviations (SDs) of dependent variable, when that particular factor increases with 1 SD and all other variables in the model are unchanged. The results which are statistically significant are marked * However, IMT was related to FMD% (β = − 0.12 [95% CI: laboratory markers of endothelial injury increase while − 0.22 to − 0.02]), blood creatinine level (β = 0.18 [95% FMD decreases in association with inflammatory markers, CI: 0.08 to 0.28]), duration of steroid treatment (β = 0.19 such as IL-6 and CRP. These observations suggest that the [95% CI: 0.03 to 0.35]) duration of the disease (β = 0.27 most important predictor of endothelial damage in GPA is [95% CI: 0.14 to 0.41]), posterior wall thickness (β = 0.16 a persistent systemic inflammatory state. However, multi- [95% CI:0.05 to 0.27]), and interventricular septum thick- ple regression analysis shows that impaired FMD was also ness (β = 0.12 [95% CI:0.01 to 0.23]) (Table 3). independently determined by other factors, including kidney In the subgroup of GPA patients with chronic kid- insufficiency, diabetes as well as smoking habit. ney disease IMT was higher (0.08 [0.06–0.09] vs. 0.065 The associations of laboratory and ultrasound param- [0.055–0.075], cm p = 0.01). GPA subjects treated with eters of endothelial damage with markers of inflammation statins and with hypertension had higher IMT than remain- may indicate that the process of endothelial injury is more ing GPA patients (0.08 [0.07–0.09] vs. 0.06 [0.05–0.07] prominent in active phase of the disease. However, compar- cm, p = 0.01, and 0.08 [0.07–0.09] vs. 0.06 [0.06–0.07] cm, ing the subgroups in persistent disease or in active disease p = 0.001, respectively). Only one patient had an atheroscle- these parameters did not differ regardless of phase of the rotic plaque in the left common carotid artery. disease. According to Tervaert  atherosclerosis is accel- Aortic stiffness% was negatively associated with blood erated in the active phase but endothelial function returns to leukocyte count (β = − 0.23 [95% CI: − 0.36 to − 0.1] and normal when inflammation is pharmacologically controlled. CRP level (β = − 0.19 [95% CI: − 0.31 to − 0.7]. Comor- Another study showed that successful immunosuppressive bidities and medication had no impact on aortic stiffness%. treatment improves endothelial function, reaffirming the key Ultrasound parameters of endothelial injury were com- role of inflammation in pathogenesis of atherosclerosis in parable between the patients in the disease flare and in this disease . Such thesis was also recognized in children the persistent disease (FMD% 5.92 [3.35–8.89] vs. 5.86 with primary systemic vasculitis without common cardiovas- [4.35–7.14], p = 0.76, IMT 0.07 [0.06–0.09] vs. 0.07 cular risk factors . Moreover, Nienhuis et al.  dem- [0.06–0.08] cm, p = 0.92, aortic stiffness% 7.93 [4.56–10.53] onstrated impaired endothelium-dependent vasodilatation in vs. 7.69 [3.85–8.82], p = 0.4). microcirculation of the hand vessels of GPA patients, even if they did not have clinical manifestations of atherosclerosis. In our study, we did not document any significant differ - Discussion ences in IMT between GPA patients and healthy controls. This observation stays in line with results published by De This study demonstrates that GPA patients suffer vascular Souza et al. , who suggested that premature atheroscle- endothelial damage that is manifested by increased serum rosis in GPA patients might be postponed by use of statins levels of thrombomodulin and VCAM-1, as well as lower or prednisolone. This contradicts other reports [21, 31] that flow-mediated dilatation of the brachial artery. Both the have shown that GPA patients are characterized by higher 1 3 1528 Rheumatology International (2018) 38:1521–1530 IMT, indicating accelerated atherosclerosis. Potential dis- and endothelial dysfunction. Additionally, uremia has been crepancy between these results may be due to the small sam- considered as a nontraditional cardiovascular risk factor ple size, different treatment mode or duration of the disease . However, this relationship in GPA patients has not in evaluated patients. Importantly in our study, we found that been described in previous reports . In our study, we IMT related positively to the time course of GPA. observed positive association of blood creatinine, as well The reason of endothelial dysfunction in GPA remains as urea level with FMD%, VCAM-1, and thrombomodu- unknown. However, it seems that it might be related to the lin. GPA patients with chronic kidney disease also had pathogenesis of the disease and eventually its therapeutic increased levels of VCAM-1, thrombomodulin and IMT. possibilities. In our study, the level of anti-PR3 antibodies This is a novel finding of our study. Urea level in GPA was related to markers of endothelial injury. As it has been patients was also independent predictors of lower FMD% shown, autoantibodies in AAVs activate neutrophils which in a multiple linear regression model. then adhere to the inner vessel wall causing endothelial The lower FMD in subjects with GPA documented in impairment by release of proteolytic enzymes and trigger- our study is possibly related not only to the endothelial dys- ing vasculopathic cascade [32, 33]. Moreover, pro-inflam- function, but also to the arteries wall remodeling. In the matory cytokines increased in active systemic vasculitis previous study the blood vessels of these patients showed an depress endothelium-dependent relaxation in vitro, as well increased level of matrix metalloproteinases (markers of vas- as in vivo , while reactive oxygen species lead to oxida- cular remodeling) . Moreover, we have found a negative tion of lipoproteins which are responsible for endothelial correlation between FMD and interventricular septum and cell injury . posterior wall thickness. We also demonstrated that patients In the study by Clarke et al.  the level of endothe- with GPA are characterized by larger left and right ventricu- lial damage biomarkers was predominantly affected by lar diameters and left atrial diameter, as well as increased disease activity rather than by treatment. However, it has left ventricle posterior wall thickness and interventricular been shown that cyclophosphamide, a cytotoxic drug used septum thickness. In our opinion, these findings are most to induce remission in AAVs patients, directly injures likely related to the inflammatory process of the kidneys and endothelial cells leading to subsequent leakage of plasma to lungs, the organs, most often affected by GPA. Kidney dam- the extravascular space . Colleoni et al.  observed a age results in overload of the circulatory system, whereas the significant drop in the level of vascular endothelial growth inflammatory process in the lungs leads to the pulmonary factor in breast cancer patients after oral administration of hypertension. Both of these processes lead to the secondary cyclophosphamide in small doses, which also suggests its hypertrophy of the heart cavities, which we have found in anti-angiogenic effect. This finding was also confirmed by our patients. Observed differences might be also related to Folkman et al. , who found that systemic administration the cardiovascular system involvement in course of GPA and of cyclophosphamide, anthracyclines or paclitaxel, inhibits vessel wall remodeling, leading to the increased stiffness of neovascularization in the mouse cornea. In cancer treatment, the arteries and higher afterload, as well as heart hypertro- cyclophosphamide is used in higher doses than in AAVs, phy. These findings, however, require further investigation. however, it should be considered as an additional factor Smoking currently or in the past also influenced lower of potential endothelial damage. In our study, we did not FMD in GPA patients and it was an independent predictor observe differences in parameters of endothelial injury in of lower FMD. those patients treated vs. those not treated with cyclophos- Described changes may lead to the increased risk of car- phamide or other immunosuppressive drugs, such as meth- diovascular events, what has been previously demonstrated otrexate, mycophenolate mofetil, and rituximab (data not by Faurschou et al. . In his study, patients with GPA had shown). Only in those treated with azathioprine the FMD% 1.9 (95% CI: 1.4–2.4) higher risk of cardiovascular disease. value was lower. There are just a few reports describing Based on this result, we may speculate that early detec- potential role of azathioprine in endothelial cell injury [39, tion of endothelial dysfunction in GPA patients may help 40]. However, the majority of the GPA patients were also in selecting the most suitable preventive strategy. Tervaert treated with steroids. Prolonged steroid therapy is associated  suggested that patients with large-vessels vasculitis with hypertension, diabetes mellitus and change in the lipid should be treated with aspirin (75–125 mg/day) to prevent profile, all of which influence the risk of atherosclerosis and ischemic complications. Statins should also be advised in cardiovascular events . most patients with GPA for endothelial protection. Obvi- It has been demonstrated that renal insufficiency might ously, patients benefit from optimal immunosuppressive be related to the endothelial dysfunction in other autoim- treatment that controls inflammation and prevents from mune diseases  as well as in peritoneal dialysis . consequences of GPA. However, it is necessary to keep in Hypertension, one of the complications of kidney failure, mind that immunosuppressive treatment directly leads to is known to be implicated in increased arterial stiffness endothelial cell injury. 1 3 Rheumatology International (2018) 38:1521–1530 1529 Open Access This article is distributed under the terms of the Crea- Study limitation tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- The limited number of GPA patients decreases the power tion, and reproduction in any medium, provided you give appropriate of our findings. However, our study group is one of the big- credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. gest evaluated in the literature so far in terms of endothe- lial dysfunction. Moreover, the GPA is a rare disease and in our opinion every report is valuable. Patients with GPA References had some comorbidities (diabetes mellitus, hypertension or kidney insufficiency), which in the majority of analyzed 1. Deanfield JE, Halcox JP, Rabelink TJ (2007) Endothelial func- subjects were related to the systemic complications of vas- tion and dysfunction: Testing and clinical relevance. Circula- culitis and might be considered as a consequence of GPA. tion 115:1285–1295. h t t p s : / / d o i . o r g / 1 0 . 1 1 6 1 / C I RC U L AT I O We attempted to eliminate these confounding variables by NAHA.106.65285 9 2. 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