Dysregulation of angiogenic homeostasis in ... - Wiley Online Library

International Journal of Rheumatic Diseases 2013; 16: 448–454

ORIGINAL ARTICLE

Dysregulation of angiogenic homeostasis in systemic sclerosis Hanan Mohamed FAROUK,1 Sherine Hosny HAMZA,1 Samah A. EL BAKRY,1 Sahar S. YOUSSEF,2 Iman Mohamed ALY,3 Afaf A. MOUSTAFA,3 Naglaa Youssef ASSAF4 and Al Hussein M. EL DAKRONY5 Departments of 1Internal Medicine and Rheumatology, 2Dermatology and Venereology, 3Clinical Pathology, 4Rheumatology and Rehabilitation, Faculty of Medicine, Ain Shams University, and 5Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Cairo, Egypt

Abstract Background: Systemic sclerosis (SSc) is a connective tissue disorder characterized by tissue hypoxia and excessive fibrosis of skin and internal organs. Objective: To evaluate the possible role of angiogenesis imbalance in the pathogenesis of SSc. Subjects and methods: Twenty-five SSc patients and 20 age- and sex-matched healthy controls were included. Assay of serum vascular endothelial growth factor (VEGF) and endostatin was done for all patients and controls using enzyme-linked immunosorbent assay. Patients were subjected to modified Rodnan skin score (mRss), pulmonary function tests (PFTS) and skin biopsies for histopathological skin thickness score assessment. Results: There was significant increase in the mean levels of serum VEGF and endostatin in SSc patients compared to controls (t = 4.07, P < 0.001). Mean values of serum endostatin was significantly increased in late compared to early stages of disease (t = 6.65, P < 0.01). A significant positive correlation was found between serum levels of endostatin, mRss and histopathological skin thickness score (r = 0.99, 0.94, respectively, P < 0.01). SSc patients with ischemic manifestations had significantly higher levels of serum endostatin compared to those without ischemic manifestations (t = 6.27, P < 0.001). SSc patients with restricted PFTS had significantly higher levels of serum endostatin compared to those without pulmonary manifestations (t = 4.3, P < 0.001). Conclusion: Angiogenic inhibitor (endostatin) is induced and outweighs angiogenic inducer (VEGF) in late stages of SSc. Increased serum endostatin is associated with skin sclerosis severity and pulmonary fibrosis and favors SSc disease progression. Key words: angiogenesis, endostatin, skin, systemic sclerosis, vascular endothelial growth factor.

INTRODUCTION Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by

Correspondence: Dr Samah Abdel Rahman El Bakry, Department of Internal Medicine and Rheumatology, Faculty of Medicine, Ain Shams University, Building 6 Makka St. El Sefarat district, Nasr City, Cairo, Eygpt. Email: [email protected]

pronounced fibroproliferative alterations in the microvasculature, and frequent cellular and humoral immunity abnormalities, culminating in a severe and often progressive fibrotic process.1 Angiogenesis is a complex multistep process that is under tight control of angiogenesis inducers and inhibitors. Under normal conditions the levels of angiogenesis inducers and inhibitors are balanced and angiogenesis does not occur in healthy tissues.2 Increasing evidence indicates that vascular damage is a primary

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Angiogenesis in systemic sclerosis

event in the pathogenesis of SSc that leads to vascular tone dysfunction and reduced capillary blood flow with consequent tissue ischemia and hypoxia that induces complex cellular and molecular mechanisms in the attempt to recover endothelial cell function and tissue perfusion. Nevertheless, in SSc patients there is no evidence of significant angiogenesis and the disease evolves toward chronic tissue ischemia with progressive and irreversible structural changes in multiple vascular beds, culminating in the loss of capillaries. An imbalance between angiogenic and angiostatic factors might explain the pathogenetic mechanisms of SSc vasculopathy.3 Numerous biomarkers have been described in SSc; however, aside from disease-specific autoantibodies, other biomarkers for pathogenesis and evaluation of organ involvement, disease activity and therapeutic response have not been thoroughly validated and require further studies.4 Vascular endothelial growth factor (VEGF) is a homodimeric heparin-binding glycoprotein that has been characterized as a key factor in the induction of angiogenesis, proliferation and migration of endothelial cells.5 Endostatin is a non-collagenic domain proteolytic fragment of collagen XVIII. It is an angiogenesis inhibitor that is endogenously produced from blood vessels in the skin and lung tissues frequently involved in SSc.6 There are several previous studies that measured circulating levels of these factors in SSc patients,7,8 but potential association of these levels with histopathological skin changes in these patients has not previously been examined. So, the aim of the present study was to evaluate the possible role of angiogenesis imbalance in the pathogenesis of SSc, by measuring the serum levels of the main angiogenic inducer (VEGF) and main angiogenic inhibitor (endostatin) and correlating them with various parameters of disease activity, severity and changes in skin biopsy.

PATIENTS AND METHODS Clinical evaluation The present case control study was carried out on 25 SSc patients who attended the rheumatology and dermatology outpatient clinics of Ain Shams and Cairo University Hospitals. Patients were recruited from April 2011 to February 2012. SSc was diagnosed according to the criteria of the American College of Rheumatology.9 They were classified as limited or diffuse cutaneous sub-


types according to the criteria of LeRoy.9 SSc patients were further classified according to disease duration into early and late sub-types; patients with disease duration less than 3 years were considered to have early disease and those with disease duration more than 3 years were considered to have late disease.10 Twenty healthy volunteers matching in age and sex served as a control group. Written consent was obtained from every patient and control. The study was approved by Ain Shams Medical Ethics Committee. Patients were subjected to the following; full medical history-taking with special emphasis on disease duration and thorough clinical examination with special emphasis on the presence of ischemic manifestations (digital ulcers, pitting scars, infarcts or gangrene). The severity and extent of skin sclerosis were assessed using the modified Rodnan skin score (mRss).11 Pulmonary function tests (PFTS) were performed on all patients and included forced vital capacity (%FVC) and diffusion capacity of carbon monoxide (%DLCO). The restrictive pattern of pulmonary function was based on a decreased percentage of FVC (%FVC < 80%) and a decreased percentage of DLCO (%DLCO < 75%) of the predicted values.

Laboratory assessment Venous blood (8 mL) was withdrawn from each subject and 5 mL was placed in ethylenediaminetetraacetic acid (EDTA) for the determination of erythrocyte sedimentation rate (ESR) by the Westergren method. A 3-mL aliquot was placed in a clean flat tube and serum was separated and kept at 20°C until used. Assays of serum endostatin and VEGF were performed using a Quantikine enzyme-linked immunosorbent assay (ELISA) kit for human endostatin immunoassay and human VEGF immunoassay, respectively (R&D Systems, Minneapolis, MN, USA: 751035.3). The duplicate readings for each standard, control and sample were subtracted from the average zero standard optical density. A standard curve was constructed using a log/log graph. Antibody profile was not performed as it is not routinely done for scleroderma patients.

Histopathological assessment Punch skin biopsies (4 mm in diameter) were taken from clinically affected areas of SSc patients. Sections were cut from paraffin embedded tissue and stained with hematoxylin and eosin (H&E, Fig. 1). Sections were analyzed using standard light microscopy and histopathological skin thickness score12 was applied.

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variance (ANOVA) test was used to compare more than two groups regarding numerical parameters. Correlation coefficient test was used to rank different variables positively or inversely against each other. Results were considered significant at P < 0.05.

RESULTS

Figure 1 Skin specimen of systemic sclerosis patient showing epidermal thinning, loss of rete ridges, vacuolar interface dermatitis (thin arrows), dermal sclerosis (arrow head), perivascular mononuclear inflammatory infiltrate (dashed arrow) and thick sclerosed collagen bundles (double-headed thick arrow) (H&E 9 200).

The presence and distribution of sclerosis was assessed within the various layers of the dermis: papillary dermis (PD), superficial reticular dermis (SRD) median reticular dermis (MRD) and deep reticular dermis (DRD). Within each of these four dermal layers, the extent of fibrous thickening was assessed semi-quantitatively as follows: 0 = no fibrosis, 1 = slight fibrosis, 2 = moderate fibrosis, 3 = extensive fibrosis. 1 Grade 1 weak fibrosis, defined by the presence of: no fibrosis in the PD = 0, and slight fibrosis in SRD ≤ 1 or in the MRD ≤ + or in the DRD ≤ +. 2 Grade 2: moderate fibrosis, defined by all the cases which do not belong to grade 1 or 3. 3 Grade 3: severe fibrosis, defined by the presence of severe fibrosis in the DRD and in the MRD irrespective of the degree of fibrosis in the SRD and in the PD or severe fibrosis in DRD plus moderate fibrosis in the MRD and in the SRD as well as in the PD.

Statistical methodology Analysis of data was done by IBM computer using Statistical Program for Social Sciences, ver.12 (SPSS Inc. Chicago, IL, USA).13 Mean and standard deviations were used to describe quantitative variables. Numbers and percentage were used for qualitative variables. Unpaired t-test was used to compare quantitative variables in parametric data (SD < 50% mean). Analysis of

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This case control study included 25 SSc patients, 21 females (84%) and four males (16%). Their ages ranged 33–51 years with a mean of 40.3 5.86 years. Another 20 healthy individuals matching in age and sex severed as the control group. They were 17 females (85%), three males (15%). Their ages ranged 30–50 years with a mean of 38.9 3.8 years. SSc patients were further classified according to disease duration into early and late sub-types. Disease duration in patients with early disease (limited and diffuse) were 17.167 10.32 and 21.333 10.78 months, respectively, while it was 80.0 27.386 and 105.0 26.46 months in late disease, limited and diffuse respectively. There was a statistically significant difference between SSc patients and controls as regards mean serum levels of VEGF (106.48 50.2 and 38.6 14.57 ng/mL respectively, t = 4.8, P < 0.001), and mean serum levels of endostatin (194.9 130.6 and 55.7 20.2 ng/mL, respectively, t = 4.07, P < 0.001). There was a statistically significant difference between limited and diffuse cutaneous SSc patients as regards mRss (7.49 4.6 and 20.59 6.63, respectively; P < 0.001) and ESR (37.41 7.93 and 58.13 7.83 mm/h respectively; P < 0.001), while no significant difference was found between them regarding mean serum levels of VEGF (109.2 57.30 and 99.80 48.32 ng/mL respectively; P > 0.05) and endostatin (196.9 150.80 and 191 120.30 ng/mL, respectively; P > 0.05). There was statistically significant increase in the mean values of serum VEGF in early compared to late SSc patients (P < 0.001), while the mean values of serum endostatin was significantly higher in late compared to early SSc patients (P < 0.001; Table 1). The mean value of serum VEGF was significantly higher in SSc patients without digital ischemic manifestations (132 49.6) compared to those with ischemic manifestations (76.73 40.2; t = 2.9, P < 0.01), while SSc patients with ischemic manifestations (16 patients) had statistically significant increase in the mean values of serum enodstatin (302.3 112.7) compared to those without ischemic manifestations (nine patients; 94.73 20.1; t = 6.27, P < 0.001).


Angiogenesis in systemic sclerosis

Table 1 Comparison between early and late systemic sclerosis patients as regards various laboratory data Variable (Mean SD)

Serum VEGF (ng/mL) Serum edostatin (ng/mL)

Limited SSc Early (n = 6)

Late (n = 9)

163.4 24.1 96 17.01

74.7 4.38 260.1 150.3

t

4.38 2.65

P