An evaluation of biomarkers indicating endothelial cell damage, inflammation and coagulation in children with Henoch-Schönlein purpura

Introduction

Henoch-Schönlein purpura (HSP) is one of the most common vasculitic syndromes. The clinical manifestation is variable and characterized by non-thrombocytopenic cutaneous palpable purpura, joint manifestations, gastrointestinal (GI) findings, and hematuria/proteinuria [1]. The disease is usually self-limiting, but can rarely cause severe renal disease. Although HSP has been known for many years, the etiopathogenesis is not still clear. However, it has been proposed that vascular inflammation and endothelial dysfunction may play an important role in the pathogenesis [2], [3], [4].

HSP is an immune-mediated inflammatory disease. IgA deposition in vessel walls leads to endothelial cell damage and cytokines secretion [1]. The coagulation system and fibrinolysis are thus activated. Several studies have reported biochemical markers indicating endothelial cell damage or hemostatic parameters in HSP patients [5], [6], [7], [8], [9], [10].

Endocan is excreted from vascular endothelial cells, renal distal tubule cells, and bronchi [11], [12], [13]. Endocan has recently been investigated in various pathological processes, such as cancer, infections and rheumatological diseases [11], [12], [13], [14], [15]. Tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF) stimulate endocan expression and secretion from endothelial cells [13]. Increased levels of both have been observed in patients with HSP [10], [16]. We therefore presumed that endocan may be a vascular endothelium damage biomarker for HSP.

Our aim in this study was to investigate various inflammation and coagulation parameters and endocan as endothelial dysfunction markers in children with HSP and to compare these with healthy children. We also examined the relationship between endocan and clinical findings and laboratory tests in patients with HSP.

Materials and methods

Results

Laboratory tests

Following laboratory evaluation, significant mean erythrocyte sedimentation rate (ESR) (p=0.030), CRP (p=0.001), white blood cell (WBC) (p=0.025), NLR (p=0.02), D-dimer (p<0.001), triglyceride (p=0.043), globulin (p=0.014), IgA (p=0.001), and C₃ (p<0.001) value elevations were determined in the patients compared to the healthy controls. Mean HDL and albumin values were significantly lower in the patients than in the healthy controls. Patients’ mean endocan levels were 809.4±430.4 pg/mL, and were similar to those of the control group (775.6±374.1 pg/mL; p=0.884). Differences in other parameters between the groups were not significant. The results of these comparisons are given in Table 2.

Table 2:

Comparison of laboratory parameters between patients and controls.

	Patients (n=26)	Controls (n=26)	p-Value
Endocan (pg/mL) median (min–max)	698 (118–1867)	717 (236–1722)	0.884a
CRP (mg/L) mean±SD	8.5 (0.0–34.2)	0.7 (0.1–19.3)	0.001
ESR (mm/h) mean±SD	27±13	20±8	0.030
Leucocyte (/mm3) median (min–max)	10,435±3410	8430±2745	0.025a
Neutrophil/lymph, median (min–max)	1.88 (0.59–6.96)	1.01 (0.35–4.21)	0.002a
HCT (%) mean±SD	35.5±2.7	36.8±2.4	0.067
HB (g/L) mean±SD	12.1±1.1	12.6±0.7	0.069
PLT (/mm3) mean±SD	344,384±64,670	307,961±82,944	0.084
MPV (fL) mean±SD	9.6±0.8	10.0±0.9	0.121
PDW (%) mean±SD	10.9±1.6	11.8±1.7	0.076
PTT (s) mean±SD	11.8±1.0	11.7±0.7	0.837
INR (s) mean±SD	0.98±0.08	0.99±0.06	0.790
APTT (s) mean±SD	25.1±3.3	26.3±2.8	0.150
Fibrinogen (mg/dL) mean±SD	329±68	307±69	0.258
D-dimer (ng/mL) median (min–max)	1540 (324–6960)	280 (92–635)	<0.001a
Urea (mg/dL) median (min–max)	24 (12–47)	21 (11–37)	0.134a
Creatinine (mg/dL) mean±SD	0.38±0.12	0.44±0.17	0.336
Cholesterol (mg/dL) mean±SD	150±23	159±30	0.282
Triglyceride (mg/dL) median (min–max)	94 (37–413)	71 (36–173)	0.043a
LDL (mg/dL) mean±SD	84±18	89±24	0.377
HDL (mg/dL) mean±SD	44±9	53±13	0.008
Albumin (g/dL) mean±SD	3.97±0.33	4.41±0.31	<0.001
Globulin (g/dL) mean±SD	2.94±0.43	2.64±0.42	0.014
IgG (mg/dL) median (min–max)	1002 (545–1859)	943 (431–1564)	0.142a
IgE (IU/mL) median (min–max)	64 (7–1133)	65 (4–862)	0.585a
IgM (mg/dL) median (min–max)	89 (40–176)	109 (52–249)	0.407a
IgA (mg/dL) mean±SD	169±59	112±50	0.001
C3 (mg/dL) mean±SD	148±15	123±15	<0.001
C4 (mg/dL) median (min–max)	31 (8–234)	29 (10–44)	0.095a

1. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HCT, hematocrit; HB, hemoglobin; PLT, platelet; MPV, mean platelet volume; PDW, platelet distribution width; PTT, prothrombin time; INR, international normalized ratio; APTT, activated partial thromboplastin time; LDL, low-density lipoprotein; HDL, high-density lipoprotein; Ig, Immunoglobulin; C, complement. All variables are showed as mean±SD (Standard Deviation) or median (minimum–maximum) for continuous data with or without a normal distribution, respectively. ^aMann-Whitney U test. All other Student t test. Bold values indicate statistical significance at the p<0.05 level.

The receiver operating characteristic (ROC) curves for biochemical parameters are shown in Figure 1. The areas under the curve of D-dimer, NLR, IgA and C3 were 0.94, 0.74, 0.74 and 0.89, respectively. Our multivariate analysis, increased IgA, NLR, CRP, C3, D-dimer levels, and decreased albumin level were not related to HSP (Table 3). Serum endocan concentrations were inversely correlated with activated partial thromboplastin time (APTT) (r=−0.485, p=0.012), but did not correlate with other parameters.

Figure 1:

Receiver–operating characteristic (ROC) curve of the RDW for predicting HSP Henoch-Schonlein purpura. Areas under the curve of D-dimer, NLR, IgA and C3 are indicated by bold values.

Table 3:

Logistic regression analysis of some biomarkers.

Risk factor	p-Value	OR	95% CI
IgA increase	0.422	1.097	0.875–1.375
NLR increase	0.364	56.539	0.009–341257.247
CRP increase	0.517	1.167	0.731–1.863
C3 increase	0.583	0.925	0.702–1.220
D dimer increase	0.393	1.041	0.950–1.141
Albumin decrease	0.544	0.045	0.000–990.097

1. CI, Confidence interval; CRP, C-reactive protein; NLR, neutrophil/lenfocyte rate; OR, odds ratio.

No relation was determined between endocan and clinical findings in patients with HSP (Table 4).

Table 4:

Relationships between endocan and clinical findings in patients.

	n	Endocan (pg/mL)	p-Value
Gender
Male	14	959±491	0.046
Female	12	634±271
Season
Spring	3	814±206	0.733a
Summer	7	810±616
Autumn	9	709±265
Winter	7	934±500
Clinical findings rash
Present	26	807±405
Arthralgia/arthritis
Absent	17	813±500	0.975
Present	9	690±518
Abdominal pain
Absent	5	812±445	0.649a
Abdominal pain without GI bleeding
Present	12	856±412
Abdominal pain with GI bleeding
Present	9	752±368
Renal
Absent	20	1000±595	0.223
Present	6	765±409
Treatment NSAİ
Absent	6	629±380	0.251
Present	20	863±438
Steroid
Absent	15	738±362	0.339
Present	11	905±511
Urine analysis
Normal	20	753±339	0.710
>5 Erythrocyte hematuria
Present	6	808±525
Fetal occult blood
Negative	17	807±436	0.979
Positive	9	812±445

1. GI, Gastrointestinal; NSAID, nonsteroidal anti-inflammatory drug. All variables are showed as mean±SD. ^aKruskal-Wallis test – all other Student t test. Bold value indicate statistical significance at the p<0.05 level.

Discussion

HSP is a small vessel vasculitis drew on by IgA-immune deposits, complement proteins and neutrophil infiltration [2]. IgA is the most important component of the immune deposits in HSP. IgA is able to activate complements by inducing the alternative complement pathways and mannan-binding lectin. It is also capable of binding to some receptors, the most important of which is FcαRI, which is capable of inducing pro-inflammatory reactions such as phagocytosis, neutrophil migration, formation of reactive oxygen species (ROS), cytokine secretion and the release of neutrophil extracellular traps [2]. Proinflammatory factors, such as TNF-α, interleukin-1, interleukin-6, transforming growth factor (TGF)-b, VEGF, and chemokines are secreted as a result of inflammation and endothelial cell damage in HSP [8], [10], [16], [18].

Studies have shown high D-dimer levels in patients with HSP [5], [9]. Hyperfibrinolysis secondary to endothelial damage has also been reported in patients with HSP [10]. Activation of inflammation causes the coagulation system to be activated. There are two suppositions on this subject; the one of them is that the circulating immune complexes act on the endothelial cells, and plasminogen proactivators are secreted. The other supposition is that proteolytic enzymes are secreted from inflammatory tissue cells, and these increase D-dimers [5], [19].

This study measured various biomarkers in patients with HSP and healthy controls – inflammatory factors as CRP, ESR, WBC, NLR, albumin and globulin, immunological markers including IgA, IgM, IgE and IgG, vascular permeability-related biomarkers such as C3 and C4, coagulation parameters including platelets, MPV, PDW, PT, APTT, D-dimer and fibrinogen, and endothelial dysfunction markers such as endocan and lipid profiles, and also performed other routine tests such as urea and Hb. CRP, ESR, WBC, NLR, globulin, C3 and IgA values were significantly higher, while albumin values were lower in patients with HSP than in the controls. CRP, ESR and WBC are non-specific markers of inflammatory activity. Previous studies have reported high levels of inflammatory parameters in patients with HSP [7], [20], [21], [22]. The NLR is a useful marker in inflammatory diseases. Various studies have reported that high blood NLR is associated with HSP [23], [24], [25]. Lymphocyte apoptosis lead to decreased lymphocytes and increased NLR in inflammation [25]. NLR may be useful in predicting organ involvement in HSP [23], [24]. However, contradictory have been reported on the role of the NLR in HSP [25]. Further investigations are therefore needed to assess its true value.

We also observed a significant increase in serum levels of D-dimer in HSP patients. However, platelets, PT and aPTT, standard coagulation tests, were within reference ranges. These results suggest that activated coagulation is secondary to the endothelial inflammation stimulated by IgA immune deposits in patients with HSP [10]. Wang et al. [19] reported raised D-dimer level as a risk factor for renal damage in patients with HSP. Our multivariate logistic regression analysis did not showed an association between raised D-dimer level and HSP. However, our patient group included all HSP patients with and without renal involvement. The number of patients with renal involvement was low and no multivariate logistic regression analysis could be performed in this group.

Recent studies have showed that IgA autoantibodies, which are cross-reactive with endothelial cells, are present in patients with HSP [7]. Only a few studies to date have investigated parameters that may show endothelial cell damage [3], [6], [10], [26]. These studies have frequently investigated Factor XIII and von Willebrand factor as markers of endothelial damage [3]. One previous study reported high von Willebrand antigen and low Factor XIII levels in patients with HSP [3], [6]. Söylemezoglu et al. [6] reported significantly increased von Willebrand factor during the acute phase of HSP and that this correlated well with CRP in the active phase, suggesting that this maybe a good indicator of vascular inflammation and endothelial damage.

The present study investigated endocan, a marker of epithelial cell damage, in patients with HSP. Endocan is associated with endothelial dysfunction [13]. Interaction between endocan and adhesion molecules causes transport of leukocytes to regions of inflammation and subsequent endothelial dysfunction [14]. High levels of endocan have been showed in the rheumatological diseases, including Behcet’s disease, systemic lupus, and systemic sclerosis [13], [14], [15]. Balta et al. [13] observed significantly higher serum endocan levels in patients with Behçet’s disease. CRP, ESR and disease activity correlated positively with endocan in these patients [13].

In our study, endocan levels were similar in the patients with HSP and the controls. We observed no correlation between endocan and other laboratory parameters. However, positive correlation was determined between APTT and endocan. No relation was determined between endocan and clinical findings.

HDL-C levels in our study were lower in patients with HSP than in the control group, but no correlation was determined with endocan. HDL exhibits anti-inflammatory, antithrombotic, and antioxidant effects. It also protects against atherosclerosis and endothelial dysfunction [14].

There are some limitations of our study. First, it is the small sample size. This may explain why no difference was determined between endocan levels in the two groups. Second, most of the HSP patients in our study were mild cases. Lastly, Factor XIII and von Willebrand Factor could not be determined due to our financial limitations.

In conclusion, endocan may be a useful predictor of endothelial dysfunction in patients with HSP. However, our results do not provide supportive evidence for an association between endocan and HSP. To the best of our knowledge, this is the first study to investigate endocan in children with HSP. Further studies of endocan involving a larger sample population are now needed. This will help clarify the possible role of endocan in the pathogenesis of HSP.