Imbalance in the production between vascular endothelial growth factor and endostatin in Kawasaki disease

Abstract

To investigate whether an imbalance exists in the production between angiogenic and antiangiogenic growth factors in patients with Kawasaki disease (KD), we measured the serum levels of vascular endothelial growth factor (VEGF) and endostatin (ES) in 35 patients with KD, 15 patients with acute febrile diseases (disease controls) and 15 healthy children. KD patients had significantly higher VEGF levels and lower ES levels (P < 0·01) in the acute and subacute phases than the disease control and healthy children. KD patients with coronary artery lesions (CAL, n = 10) had significantly higher VEGF levels and lower ES levels (P < 0·05) in the subacute and convalescent phases than those without CAL (n = 25). The ratios of VEGF/ES in sera of KD patients with CAL were significantly higher (P < 0·05) in the acute and convalescent phases compared to those without CAL. Furthermore, the occurrence of CAL significantly correlated with the VEGF/ES ratio above 10 × 10⁻³ in the subacute phase of KD (Odds ratio 17·25, P = 0·005). The findings in the present study indicate that an imbalance exists in the production between VEGF and ES in patients with KD while also suggesting that KD patients with a high VEGF/ES ratio have a significantly greater risk of CAL involvement.

Introduction

Endothelial cells (ECs) play an important role in the physiological and pathological processes of haemostasis, inflammation and angiogenesis [1]. ECs consist of a huge population of cells and respond to several stimulators including infectious agents, proinflammatory cytokines, growth factors and oxidative stress [1]. Vascular endothelial growth factor (VEGF), which is expressed in normal tissues (lung, kidney, adrenal gland, heart, liver and stomach), cells (macrophages, neutrophils and smooth muscle cells) and tumours, is a potent angiogenic factor that is known to stimulate EC proliferation [2,3]. Endostatin (ES) is a C-terminal of collagen type XVIII which specifically inhibits VEGF-induced EC migration and tumour growth [4,5]. As a result, ES directly antagonizes the biological effects of VEGF. The serum levels of both VEGF and ES have been reported to increase in several types of malignant diseases including soft tissue sarcoma [6], renal cell carcinoma [7], breast cancer [8] and non-Hodgkin lymphoma [9]. On the other hand, patients with rheumatoid arthritis have been reported to have a high level of VEGF with a normal level of ES in both serum and joint fluid samples, thus suggesting an imbalance in the production between VEGF and ES [10].

Kawasaki disease (KD) is an acute febrile illness that predominantly affects infants and children [11]. This disease is characterized as one type of systemic vasculitis which may have coronary artery lesions (CAL) [12]. The immune system shows a marked activation during the acute phase of KD, thus suggesting that activated effector cells induce an increased production of cytokines, including IFN-γ, IL-1, IL-6 and TNF-α, which can thus cause endothelial cell activation and damage [13]. Recently, it has been noted that growth factors and proteolytic enzymes are also involved in the pathogenesis of CAL in KD. Yasukawa et al. [14] reported that CAL in acute KD had abundant expression of VEGF and its receptors. Matrix matalloproteinases (MMP)-2 and -9 were expressed at the CAL in KD, thus suggesting that angiogenesis occurs in the remodeling process of the arterial walls in acute KD vasculitis [15]. Furthermore, an immunohistochemical study demonstrated that intimal proliferation and neoangiogenesis are accompanied by an over-expression of VEGF at the CAL in the late phase of KD [16]. Although the serum levels of VEGF have been reported to increase in the acute phase of KD [17,18], there has so far been no report on the serum levels of ES in this disease. In the present study, we measured the serum levels of both VEGF and ES from the acute through the convalescent phases of KD and investigated the kinetics of both angiogenic and antiangiogenic factors.

Materials and methods

Subjects

We studied 35 patients with KD (aged 7 months to 7 years; median 25 months; male/female = 21/14), 15 patients with acute febrile diseases (disease control, DC; aged 10 months to 9 years; median 29 months; male/female 8/7) and 15 healthy children (HC, aged 9 months to 6 years; median 24 months; male/female 9/6). The DC group included six patients with pneumonia, three with infectious mononucleosis, three with enterocolitis, two with measles and one with lymphadenitis). HC were recruited as follows: when parents required an examination to determine blood type (ABO) or a parent/child DNA-matching test for their children in our out-patient clinic, we asked the parents to provide an additional blood sample (2–3 ml) from their children. Informed consent was obtained from the parents of all patients and all HC. The present study was approved by the institutional review board. All KD patients were hospitalized at the National Defence Medical College Hospital between May 1997 and July 2002. The KD patients were enrolled within 7 days of the onset of illness, with day 1 defined as the first day of fever symptoms. All KD patients met the diagnostic criteria for KD established by the Japanese Kawasaki Disease Research Committee and were typical cases which fulfilled the requirement of the criteria. All patients were administered both aspirin (30 mg/kg/day) and single dose of intravenous immunoglobulin (IVIG, 1–2 g/kg: 2 g/kg for 32 KD patients and 1 g/kg for 3 KD patients).

Echocardiographic examinations

We evaluated the presence of CAL using two-dimensional echocardiographic examinations which were performed at 2–3 day intervals in the acute and subacute phases during hospitalization and at 2–3 month intervals in the convalescent phase at our out-patient clinic of the National Defence Medical College Hospital. The maximum internal diameters of both the right and left coronary arteries were determined. The arteries, which measured greater than 2·5 mm in patients aged <24 months and greater than 3·0 mm in patients aged ≥24 months, were considered to demonstrate dilation. When the dilated arteries regressed to the normal range within 6 months after the onset of KD, they were considered to demonstrate transient dilation. The arteries, which measured greater than 5·0 mm in all-aged patients, were considered to demonstrate aneurysms.

Study design

Serial blood samples were obtained from all KD patients in the acute phase (before IVIG therapy on days 4–6), in the afebrile subacute phase (after IVIG therapy on days 9–16), and in the convalescent phase, when C-reactive protein (CRP) was negative, on days 22–39. All serum samples were stored at −80°C until analysed. The serum levels of VEGF and ES were measured using an ELISA kit (VEGF, BioSource International, Inc. Camarillo, CA, USA; ES, GT (Techne Corporation), Minneapolis, MN, USA).

Statistical analysis

All data are expressed as the mean ± S.E. Any differences among the acute, subacute and convalescent phases in the same group were assessed by the Wilcoxon signed-rank test. Intergroup differences were analysed with the Mann–Whitney test. Odds ratios (OR) and the 95% confidence intervals (CI) were calculated to assess any associations between the CAL and the levels of VEGF and ES. The correlation of ES or VEGF level and laboratory data were studied by Spearman's rank correlation coefficients. A P-value <0·05 was considered to be significant.

Results

Three KD patients, who received 1 g/kg infusion, became afebrile immediately. Twelve of the 32 KD patients, who received 2 g/kg IVIG infusion, did not respond to the first course of IVIG and thus were re-treated with a second course of IVIG (1 g/kg). Furthermore, one of 12 KD patients received a third course of IVIG (1 g/kg). As a result, 10 of the 35 KD patients had CAL during hospitalization. Seven patients had very mild dilation (diameter 3·0–3·5 mm), and two patients also had mild moderate dilation (diameter 3·5–4·0 mm). These dilated arteries in 9 patients with KD patients were later proven to be transient during the follow-up at the out-patient clinic. One patient, who received IVIG infusion three times, had bilateral aneurysms in the left and right coronaries (max diameter 6·0 mm), which regressed to a diameter of 3·2 mm by 6 months after the onset of KD.

The mean VEGF levels in all KD patients with and without CAL were significantly higher (P < 0·01) than those of DC and HC (acute KD, 1107·8 ± 78·5 pg/ml; subacute KD, 1318·5 ± 94·1 pg/ml; convalescent KD, 616·3 ± 80·5 pg/ml; DC, 288·3 ± 34·3 pg/ml; HC, 243·4 ± 22·3 pg/ml). The KD patients with CAL had significantly higher VEGF levels in the subacute and convalescent phases (subacute, P < 0·01; convalescent, P < 0·05), but not in the acute phase, than the KD patients without CAL (Fig. 1). In KD patients with CAL, the mean VEGF levels in the subacute phase were significantly higher (P < 0·01) than the levels in the acute and convalescent phases.

Fig. 1.

The serum VEGF levels in HC, DC and KD patients with CAL (•) and without CAL (○). *P < 0·01, **P < 0·05.

The mean ES levels in all KD patients with and without CAL were significantly lower (P < 0·01) in the acute and subacute phases, but not in the convalescent phase, than those of DC and HC (acute KD, 161·7 ± 5·3 ng/ml; subacute KD, 164·8 ± 6·0 ng/ml; convalescent KD, 174·0 ± 6·2 ng/ml; HC, 188·6 ± 6·4 pg/ml; HC, 196·2 ± 6·0 ng/ml). The KD patients with CAL had significantly lower ES levels in the acute, subacute and convalescent phases than the KD patients without CAL (acute, P < 0·05; subacute and convalescent, P < 0·01) (Fig. 2). The ES level in the convalescent phase of the KD patients without CAL recovered to the same levels as observed in HC, but the ES level in the convalescent phase of the KD patients with CAL remained low.

Fig. 2.

The serum ES levels in HC, DC and KD patients with CAL (•) and without CAL (○). *P < 0·01, **P < 0·05.

We next evaluated the ratio of VEGF/ES in HC, DC and the KD patients. The mean ratios of VEGF/ES in all phases of the KD patients with and without CAL were significantly higher (P < 0·01) than those of DC and HC (acute KD, 7·2 ± 0·65 × 10⁻³; subacute KD, 8·99 ± 1·08 × 10⁻³; convalescent KD, 4·08 ± 0·73 × 10⁻³; DC, 1·53 ± 0·18 × 10⁻³; HC, 1·24 ± 0·10 × 10⁻³). The KD patients with CAL had a higher ratio in all phases than the KD patients without CAL (acute and convalescent, P < 0·05; subacute, P < 0·01) (Fig. 3). In the KD patients with CAL, the VEGF/ES ratio in the subacute phase were significantly higher (P < 0·01) than the ratios in the acute and convalescent phases. A logistic regression analysis was performed to evaluate the association between the occurrence of CA and a VEGF/ES ratio above 10 × 10⁻³. Although the OR was not significant in the acute phase of KD (OR 2·25, 95%CI 1·53–58·78, P = 0·641), the OR was statistically significant in the subacute phase of KD (OR 17·25, 95%CI 2·53–117·78, P = 0·005).

Fig. 3.

The serum VEGF/ES levels in HC, DC and KD patients with CAL (•) and without CAL (○). *P < 0·01, **P < 0·05.

Individual differences in the values of VEGF and ES were large in the KD patients, and an overlap in the data was seen among each group of KD. One KD patient with bilateral aneurysms had the highest VEGF level but the second lowest ES level from the acute thorough the convalescent phases of KD. There were no significant correlations among VEGF, ES, VEGF/ES and laboratory data (C reactive protein, white blood cell counts, neutrophil counts, and platelet counts).

Discussion

In the present study, KD patients had both higher levels of VEGF and lower levels of ES from the acute through the subacute phases than DC and HC. Furthermore, KD patients with CAL had both higher levels of VEGF and lower levels of ES from the subacute through the convalescent phases than those without CAL. The VEGF/ES ratio in patients with CAL was significantly higher than in those without CAL from the acute through the convalescent phases of KD, and a significant correlation was found between the occurrence of CA and VEGF/ES ratio above 10 × 10⁻³ in the subacute phase.

VEGF is an angiogenic growth factor that has been implicated in both physiological and pathological angiogenesis [19]. Several investigators have reported the increased levels of angiogenic factors such as VEGF and hepatocyte growth factor (HGF) in KD, thus suggesting VEGF to be a marker of the hyperpermeability of local blood vessels [17] or a predictive indicator for the occurrence of CAL in acute KD [18,20]. As a result, the increased production of VEGF may promote coronary vascular injury by inducing the migration of ECs and enhancing vascular permeability. In fact, pathological studies have revealed that CAL in KD are characterized by subendothelial oedema, vascular EC damage and the infiltration of inflammatory cells [21,22]. On the other hand, an active remodeling process has been reported to occur in the CAL of acute KD [14,15] which thereafter continues several years after the onset of KD [16]. VEGF can recruit endothelial progenitor cells from the bone marrow into the peripheral circulation [23]. In addition, we recently reported that the number of endothelial progenitor cells increases from the subacute through the convalescent phases of KD, especially in patients with CAL [24]. Therefore, we hypothesize that the up-regulation of VEGF may also be involved in both the repair of EC injury and/or potential microneovascularization via a mobilization of endothelial progenitor cells from the bone marrow in KD.

ES is a potent inhibitor of angiogenesis and exhibits antiangiogenic activity by inhibiting the proliferation and migration of ECs in addition to inducing EC apoptosis [4, 5, 25]. ES is best known for its antitumour activity in animal models [4,26]. Several authors have reported that the ES levels in the serum have been reported to increase, concomitantly with the elevated levels of VEGF, in patients with many forms of cancers [6–9]. However, there have been only a few reports noting the serum levels of ES to decrease under certain conditions. The serum ES levels decreased significantly after reperfusion in patients with acute myocardial infarction [27], and the serum ES levels were lower in chronic lymphocytic leukaemia patients with progressive disease than in those with stable disease [28]. On the other hand, recent studies have also revealed the decreased levels of ES in local lesions. Eyes with a high vitreous fluid level of VEGF and a low ES level had a significantly greater risk of proliferative diabetic retinopathy progressing than did eyes with low VEGF and high ES levels [29]. In addition, reduced ES levels in pericardial fluid have also been reported to be associated with collateral development in patients with ischemic heart diseases [30]. Therefore, these findings suggest that the down-regulation of ES is involved in the process of neovascularization similar to the up-regulation of VEGF. The present study is the first to report that the serum ES levels are decreased in KD. The down-regulation of ES might facilitate EC migration, proliferation, and neovascularization by VEGF in KD vasculitis.

Angiogenesis is believed to be mediated by both pro-angiogenic and antiangiogenic factors in the human body. Although the balance of such types of production is maintained under physiological angiogenesis, it may be lost under certain types of pathological angiogenesis. The present study demonstrated an imbalance to exist in the production between VEGF and ES in KD vasculitis. Furthermore, the occurrence of CAL significantly correlated with the high VEGF/ES ratios in the subacute phase of KD, when CAL usually begins to be observed on echocardiographic examinations. Therefore, the high levels of VEGF/ES ratio may reflect the degree of severity of vasculitis in this disease, thus suggesting the high VEGF/ES ratio to be a risk factor for the occurrence of CAL. On the other hand, we cannot rule out that high VEGF/ES levels in KD may reflect the active remodeling and/or the repair of injured ECs by the host. To elucidate the significance of VEGF and ES in the pathogenesis of KD vasculitis and to investigate whether a correction of a VEGF/ES imbalance might be a new target for KD therapy, further studies are called for in the future.