What Is Known/What Is New
What Is Known
- The microvascular endothelium has a role in normal mucosal immunity, mediating, and aggravating uncontrolled chronic inflammation.
- Endothelial dysfunction promotes endothelial cells activation with the recruitment of proinflammatory cytokines and leukocytes.
What Is New
- In children with inflammatory bowel diseases there is an endothelial dysfunction at diagnosis.
- Endothelial dysfunction persists at follow-up, sustained by chronic inflammation of the endothelial cells, despite anti-inflammatory therapy.
- Endothelial dysfunction correlates with fecal calprotectin, the more meaningful marker of intestinal inflammation.
Inflammatory bowel diseases (IBD), including 2 major entities, Crohn disease (CD) and ulcerative colitis (UC), are intestinal autoimmune disorders, characterized by chronic and intermittent inflammation of the gastrointestinal tract (1). The inflammatory process in IBD involves both immune and nonimmune cellular systems, including intestinal microvascular endothelial cells. Several studies have suggested a critical role of the microvascular endothelium in normal mucosal immunity, mediating and aggravating uncontrolled chronic inflammation so that the dysfunction of the endothelium has also been considered a pathogenic factor in IBD development (2,3). Endothelial dysfunction (ED) is defined as a condition in which the imbalance between vasodilatating and vasoconstricting substances induces both functional and structural alterations in vascular endothelium and promotes endothelial cells activation with the recruitment of proinflammatory cytokines and leukocytes, causing a gradual arterial thickening and stiffening in response to the inflammation process (4). However, it is still not clear if ED could be considered itself a starting mechanism or a consequence of the chronic inflammation which may affect arterial properties. Moreover, the finding of an increased risk of cardiovascular events and atherosclerosis in adult patients with IBD compared to the general population (5,6), underlines the importance of a better knowledge of ED as part of the complex IBD scenario. This is the first study which evaluates the presence of ED in a pediatric IBD population both at diagnosis and at follow-up. For this purpose we used the flow-mediated vasodilatation (FMD) method and the endothelial pulse amplitude test (EndoPAT), comparing the ED parameters obtained to a group of healthy controls (HC).
The study included all children referred to the Gastroenterology Pediatric Unit at the Department of Translational Medical Science of University of Naples “Federico II” of Naples, Italy, between January 2015 and October 2016. Patients were stratified in 2 main groups, newly diagnosed patients with IBD (T0) treatment naïve, both UC and CD, and HC. The HC were patients diagnosed with functional gastrointestinal disorders and they were not performing any kind of medical therapies. They were matched for sex and age to patients with IBD.
The diagnosis of IBD was based on clinical, radiological, endoscopic, and histological data according to the revised Porto criteria published in 2014 (7). At diagnosis, all patients underwent colonoscopy with mucosal biopsies performed by 2 expert endoscopists (E.M., C.S.), and small bowel imaging, including abdominal ultrasound and magnetic resonance enterography or small bowel follow through. Disease location and phenotype were defined on the basis of Paris classification (8). Mayo endoscopic subscore and the simplified endoscopic score for CD (SES-CD) were used to assess endoscopic activity in UC and CD, respectively (9). For each patient with IBD, clinical activity of disease was evaluated using the Pediatric Ulcerative Colitis (PUCAI) and the Pediatric Crohn Disease (PCDAI) Activity Index scores of disease (10,11). A PUCAI/PCDAI ≥ 10 was indicative for active disease. At the time of enrollment blood samples were taken from each patient to detect the main serum inflammation parameters including erythrocyte sedimentation rate (mm/h), C-reactive protein (CRP, mg/dL), hemoglobin concentration (g/dL), platelets count (×103/μL) and feces samples for fecal calprotectin (μg/g of stool) determination were also collected. For IBD patients these data were all collected at diagnosis (T0) and after 12 months (T1). Patients with a history of other chronic conditions like metabolic diseases, other inflammatory diseases, neoplasm, and cardiovascular events were excluded from the study.
Assessment of Endothelial Function
Patients’ endothelial functionality was assessed at the same time by 2 different not invasive techniques performed by the same operator: FMD and the EndoPAT. These data were collected from IBD patients’ treatment naïve at diagnosis (T0) and under therapy after 12 months (T1), and from HC. Before performing the endothelial evaluation the heart rate (beats per minute) and blood pressure (mmHg) of each patient were also collected.
Endothelial function was measured by evaluating brachial artery responses to endothelium-dependent stimuli, using ultrasound. Subjects were fasting for at least 8 hours, in a quiet and comfortable dark room. Artery diameter was measured on B-mode ultrasound (Samsung RS80A machine). Images were captured at right brachial artery in a long axis projection between 2 and 8 cm above the elbow. A sphygmomanometer cuff was placed in the distal site to the artery. After image baseline acquisition, the artery was occluded by inflating the cuff to suprasystolic pressure for 5 minutes, creating distal ischemia. After dilation, reactive hyperaemia occurs, which means that flow in the brachial artery increases to accommodate the dilated resistance vessels. The brachial artery was imaged for the first 2 minutes of reactive hyperaemia. The vessel diameter was defined as the average of the 3 consecutive maximum diameter. FMD was defined as the maximal percentage change in brachial artery diameter after reactive hyperaemia compared with baseline. FMD was calculated as follows: (hyperemic diameter − baseline diameter)/baseline diameter × 100%.
Endothelial Pulse Amplitude Testing
The EndoPAT is a Food and Drug Administration–approved device consisting of a fingertip plethysmograph capable of sensing volume changes in the digit with each arterial pulsation. The EndoPAT testing protocol was described previously (12,13) (Itamar Medical Ltd, Caesarea, Israel). This is a noninvasive technique that records endothelium-mediated changes in the vascular tone with a pair of plethysmographic biosensors situated on the second fingertip of both hands. The endothelium-mediated changes in the vascular tone were obtained by creating a distal hyperemic response that was elicited by occluding flow through the brachial artery for 5 minutes by inflating a blood pressure cuff to 50 mmHg above systolic pressure. The EndoPAT data were analyzed with the proprietary software package, without any input from the examiner. The pulse wave amplitude – reactive hyperaemia index (RHI), a measure of arterial stiffness, was calculated as the ratio of the average pulse wave amplitude value, over a 1-minute epoch starting after cuff release, to the preocclusion baseline pulse wave.
A cut off value of 1.67 was used on the basis of adult data (14).
The results from the tested arm were correct for potential systemic changes, normalizing them with the pulse wave amplitude from the nontested arm.
An informed consent was filled by subjects’ parents and also by the patients if they were at least 13 years old after a careful explanation of the study procedures. The study protocol was approved by the Institutional Review Board “Carlo Romano” of the University of Naples “Federico II.”
Variables were screened for their distribution, and appropriate parametric or nonparametric tests were adopted as necessary. The Mann-Whitney test for continuous variables and χ2 test for categorical variables were used, where appropriate. Correlations between continuous variables were evaluated through linear regression and expressed by the Spearman correlation coefficient. Statistical significance was predetermined P < 0.05. Percentages were rounded to the nearest whole numbers. SPSS version 20 was used for all statistical analyses (SPSS Inc, Chicago, IL).
During the study period a total of 44 patients were enrolled. Among this, 26 (59%) were patients with IBD and 18 (41%) were HC. At T0 the mean age ± standard deviation (SD) of patients and HC was 13.3 ± 2.7 and 12.5 ± 3.8, respectively. Out of the 44 patients, 20 (45.5%) were boys. No significant differences were found among the 2 groups when considering demographic characteristics such as the sex and the median age of patients. In the IBD group, 11 (42%) were diagnosed with UC and 15 (58%) with CD. The mean disease activity Index score at diagnosis was 48.3 ± 17.3 for patients with UC and 41.8 ± 14.3 for CD. In the UC group 4 (15.4%) showed pancolitis (E4) at the endoscopy, whereas in the CD group 11 (42.3%) had an ileocolonic localization (L3) with nobody presenting with an upper-gastrointestinal localization (L4). The patients at T0 started induction therapies continued for 6 weeks, after induction therapy all the patients started maintenance therapy which remained stable until T1. Baseline characteristics and therapies of the patients with IBD enrolled are described in Table 1. Characteristics of the study population are described in Table 2.
Endothelial Function Assessment
The mean FMD ± SD at the time of IBD diagnosis was significantly lower in the cases group compared with controls (18.4 ± 9.1 vs 26.3 ± 16.5; P = 0.04) (Fig. 1). When considering the patients with IBD at follow-up after 12 months of disease the median FMD value was reduced compared to the diagnosis, although the difference was not statistically significant (18.4 ± 9.1 vs 16.7 ± 2.1; P = 0.3). However, when compared to the control group the FMD value at follow-up was even more significantly reduced (16.7 ± 2.1 vs 26.3 ± 16.5; P = 0.004). No differences were found when we evaluated RHI. The mean RHI between patients with IBD and HC at baseline (1.5 ± 0.3 vs 1.6 ± 0.5; P = 1.0) and at follow-up (1.5 ± 0.4 vs 1.6 ± 0.5; P = 0.9) did not show significant change as assessed by FMD. Data are shown in Table 3.
Correlations Among Endothelial Function and Inflammation Markers
After correlating the endothelial function parameters with the serological markers of inflammation and with disease activity and disease location, data showed only a significant inverse correlation between FMD and fecal calprotectin (r2: 0.17; P = 0.04) at T0, as it is shown in Figure 1. This result was consistent with the hypothesis that higher inflammation assessed in this case by the fecal calprotectin was related to a minor dilatation in response to vasoconstriction. No other significant correlations were detected by linear regression analysis for the other clinical and serological inflammation markers such as PUCAI or PCDAI (P = 0.7), erythrocyte sedimentation rate (P = 0.5), C-reactive protein (P = 0.5), hemoglobin (P = 0.8), and fibrinogen (P = 0.3).
Comparison of the Characteristics of the Subjects With Ulcerative Colitis Versus Crohn Disease
When the IBD cohort was stratified by IBD subtypes, UC versus CD, there were no significant differences at baseline (T0) in age (P = 0.5), sex (P = 0.2), and disease duration as both groups were enrolled at the time of diagnosis. At T0, patients with CD showed a lower level of hemoglobin compared to UC (P = 0.01) and a tendency in higher fibrinogen level (P = 0.09). As it is shown in Supplementary Table 1A (Supplemental Digital Content 1, http://links.lww.com/MPG/B643), all the other inflammation markers and activity scores of disease were not significantly different between the 2 groups both at T0 and at T1. Moreover, at both time points, despite an evident reduction of CD FMD values no significant differences were detected when comparing to UC (P = 0.3 and P = 0.6, T0 and T1, respectively). Interestingly RHI showed a tendency to lower levels at T1 in CD compared to UC (P = 0.08). Cardiovascular characteristics and ED parameters of IBD subgroups are shown in Supplementary Table 1B (Supplemental Digital Content 1, http://links.lww.com/MPG/B643). No significant correlations were found when comparing serological and clinical markers of disease to ED parameters in both IBD subgroups.
There is evidence that several chronic inflammatory disorders are associated to ED (15–17). It is, however, still not clear if ED could be considered itself a pathogenic factor or a consequence of the chronic inflammation, which may affect arterial properties.
In this study we explored the ED in pediatric patients with IBD. Our findings show that in children with IBD there is an ED demonstrated from a decreased FMD significantly lower than that in HC. Strikingly we found that FMD further decreases at follow-up, suggesting that despite remission of the disease and the anti-inflammatory therapy, most probably ED and vascular disease persist sustained by chronic inflammation of the endothelial cells.
ED is diagnosed using physical and biochemical methods. Physical methods are based on assessing vasodilation in large arteries in response to increased flow and receptor stimulation. FMD is the most widely used and the most sensitive (18,19); moreover, it is a measure that seems to correlate significantly with invasive methods of testing coronary and brachial endothelial function (20–22).
We demonstrated that the FMD in patients with IBD is significantly lower than that in HC. There are 2 previous pediatric studies demonstrating impaired FMD in patients with IBD compared with controls in accordance with our results (23,24). Aloi et al found that FMD was significantly worse in patients with severe disease than in those in remission or with mild disease, whereas we found that the dysfunction of FMD was related to the duration of the disease and not to the severity of the disease. This discrepancy could be explained by the fact that the previous study is a cross-sectional study in which the FMD is measured after several years of disease and not at the diagnosis; therefore, this measurement could be influenced by different factors including therapies. Ozturk et al also found significantly decreased FMD levels in patients with IBD compared with controls. Consistent with our findings they also did not find any significant differences between IBD subgroups (patients with UC and CD).
Together with the FMD assessment, we also used the RHI analysis as a more objective instrument to evaluate ED. Unexpectedly, we found no significant difference in RHI between the 2 groups studied.
Nevertheless RHI analysis has been validated only in adult population (13) to assess microvascular function, and its use among children is scarce (25,26).
Only 2 pediatric studies used EndoPAT in pediatric patients with IBD (27,28). In contrast with our results, these studies showed that RHI values were significantly lower in patients with IBD than in controls.
This discrepancy could, however, be due to the fact that at the beginning of the disease like in our population the ED is not enough developed to be detected from RHI measurements. Indeed we analyzed patients at the diagnosis and after 1 year of follow-up, whereas both previous studies evaluated patients with a disease duration of approximately 3 to 5 years. Moreover it must be acknowledged that there are no specific pediatric probes. We used probes designed for adults, and these may be too large for children's fingers, therefore unable to detect volume changes in the fingertip and to record it as pulse amplitude.
The ED represents the first step toward structural alterations in the arterial vessel wall, followed by atherosclerotic plaque formation (29). The link between atherosclerosis and IBD has been researched mainly in adult. Several studies have shown increased carotid intima thickness in patients with IBD (30–32) and a higher rate of cardiovascular diseases (33). Recently, Aloi et al (23) showed an intima media thickness significantly higher in pediatric patients with IBD than controls, confirming data reported in previous pediatric studies (34,35). Nevertheless, although an increased risk of developing atherosclerosis, the most common atherosclerosis-related risk factors such as total cholesterol and low-density lipoprotein cholesterol were significantly lower in patients with IBD than in control groups (36–38). In accordance with these results, we analyzed the major cardiovascular risk factors as body mass index, blood pressure, and lipid profile and we did not find significant differences. These results induce us to hypothesize that pediatric IBD can be considered as an independent atherosclerosis risk factor. Therefore ED could start early during the childhood period, underlying the importance of starting monitoring these patients precociously.
In our patients, we also correlated the endothelial function with inflammatory markers of disease. We found that at diagnosis FMD was inversely correlated with fecal calprotectin, which is the more meaningful marker of intestinal inflammation. Indeed it has been demonstrated that calprotectin with its proinflammatory function has a role in the development and progression of atherosclerosis. Both in vitro and in vivo studies suggested a proatherogenic role for calprotectin (39–42). Indeed, in human studies, the 2 calprotectin subunits S100A8 and S100A9 were detected in atherosclerotic plaques and elevated in serum of patients suffering from peripheral artery disease (43,44). Dorosz et al (45) recently suggested that, in the context of the IL27-induced vascular inflammation, calprotectin may be a novel attractive candidate as a regulator of monocyte recruitment to early atherosclerotic lesions, hence preventing the progression of inflammation and atherosclerosis. So, it could be supposed that those patients with an increased level of calprotectin may have an ED and an increased cardiovascular risk with a major risk of atherosclerosis, in the absence of the classic cardiovascular risk factors.
In clinical practice, there are increasing evidences that also prolonged corticosteroids therapy enhances the development of atherosclerosis even if this effect has not been definitively proven. Recently, a nation-wide Danish study has shown that adult patients with IBD undergoing steroids therapy had a higher risk to develop atherosclerosis and ischemic heart disease compared to patients performing 5-aminosalicylic acid or anti-tumor necrosis factorα therapies whose risk was lower (46). The correlation between corticosteroids long-term treatment and heart disease onset, however, seems to be a consequence of the exacerbation of coronary risk factors, including hypertension, hypercholesterolemia, hypertriglyceridemia, and impairment of glucose tolerance (47). In our cohort, 23% (6/26) patients with IBD performed steroid therapy during the induction period for a maximum of 2 weeks high-dosage therapy, which cannot really be considered as a prolonged steroids therapy. Indeed, in the Danish study, patients with higher risk were undergoing prolonged and recurrent therapy due to the severe clinical phenotype. Nevertheless, in our study we did not find differences in the cardiovascular risk factors analyzed (blood pressure, total cholesterol, low-density lipoprotein, and high-density lipoprotein) between cases and HC, and between patients performing steroids or undergoing enteral nutrition, suggesting that the short duration of the steroid therapy performed was not responsible for the ED found in our IBD cohort.
This study has some limitations. Firstly due to the small sample size it was not possible to find differences when stratifying patients according to the disease subtype. It also must, however, be taken into account that it is a monocentric study and that these are pediatric patients prospectively enrolled at the diagnosis; therefore, the number of patients enrolled was limited.
Another important limit was the lack of HC data at follow-up (T1). Anyway, according to several studies (48,49) the FMD starts to show significant age-related differences around 40s in men and 50s in women. Particularly in children there are evidences that between 5 and 15 years FMD changes are age independent (50). This is the reason why, even after 12 months, we considered HC data as a control reference for patients with IBD at both time points (T0 and T1), also considering the fact that the groups age (IBD and HC) were still not significantly different at T1.
Despite these limitations, an important novel finding of this study is that in pediatric patients with IBD already at diagnosis there is a decrease of FMD, which is a marker of ED. Consistently, FMD is inversely correlated with calprotectin, a marker of intestinal inflammation. Interestingly FMD further decreases at follow-up, probably sustained by chronic inflammation of the endothelial cells.
Nevertheless, additional multicenter studies in larger cohort of patients are required to confirm these results and to further clarify the role of IBD inflammation on ED, and to differentiate between IBD subtype.
Supplemental Figure 1, Supplemental Digital Content 2, http://links.lww.com/MPG/B644.
The authors would like to thank all of the young subject participants in the study.
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