Increased expression of basic fibroblast growth factor in skin of patients with systemic sclerosis
Published Web Locationhttps://doi.org/10.5070/D36s6582vr
Increased expression of basic fibroblast growth factor in skin of patients with systemic sclerosisDepartment of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow -226 014 India. firstname.lastname@example.org
A Lawrence, D Khanna, R Misra, and A Aggarwal
Dermatology Online Journal 12 (1): 2
Increased collagen deposition is a hallmark of systemic sclerosis (SSc). Several fibrogenic cytokines play a role in this sclerosis. The role of basic fibroblast growth factor (bFGF), the most potent fibrogenic cytokine, is poorly understood in SSc. Skin biopsies from forearm of 13 patients with SSc and 3 normal individuals were analyzed by immunohistochemistry using avidin biotin-system to amplify the signal. In addition serum levels of bFGF were also measured in 30 patients including these 13 and 23 healthy controls. Thirteen patients with SSc were all females and had a median age of 26.5 years, median disease duration of 2.25 years. Of these thirteen, seven had diffuse and six had limited disease. The skin biopsies from patients showed increased expression of bFGF in the basal layer of epidermis, dermis (periappendageal, perivascular, matrix tissue) as compared to normal tissues. The expression of bFGF did not correlate with duration of disease or skin score. In contrast, only low levels of bFGF were detectable in 4/30 sera from SSc patients as compared to 3/23 from healthy controls (p = ns). Overexpression of bFGF in skin of patients with SSc along with normal serum levels suggests that bFGF probably acts in an autocrine or paracrine manner in fibrogenesis.
Systemic sclerosis (SSc) is a non-organ-specific autoimmune disease that is characterized by fibrosis and excessive laying down of collagen in the skin, gastrointestinal tract, and the lungs. Based on the extent of sclerosis it is divided into two subtypes, namely limited and diffuse. The exact pathogenic mechanisms leading to sclerosis are still unclear, but microvascular injury, fibroblast dysfunction, and excessive release of fibrogenic cytokines are thought to play a major role . Compared to transient activation of fibroblasts in tissue repair, SSc fibroblasts continue to produce excessive amounts of collagen and extracellular matrix. Connective tissue growth factor (CTGF), transforming growth factor-β (TGF-β) and fibroblast growth factors (FGF) are important fibrogenic cytokines . Blockade of TGF-β with anti-TGF-β antibodies reduces cutaneous sclerosis and deposition of collagen in animal models of SSc .
Basic FGF (bFGF), also known as fibroblast growth factor 2 (FGF 2), is a potent mitogen for cells of mesodermal and neuroectodermal origin including fibroblasts and endothelial cells . Basic FGF lacks a classic leader sequence for secretion but is probably released by exocytosis. The released bFGF stays bound to the extra cellular matrix and its controlled release plays a vital role in regulation of fibrosis and angiogenesis . In addition to endothelial cells, T cells also secrete bFGF. Microvascular injury in SSc could lead to increased production of bFGF from endothelial cells, which can act synergistically with TGF-β in inducing fibrosis as has been shown in neonatal mice model of fibrosis .
Serum levels of bFGF were shown to be elevated in 31 of 74 patients with SSc and were found to correlate with presence of plasma endothelin levels and presence of anti-centromere antibodies . Because most bFGF has autocrine and paracrine function, its expression at the site of injury (i.e., skin in SSc) would be more relevant. Thus we studied the expression of basic FGF in the skin biopsies of patients with SSc and also measured its levels in the serum.
Expression of bFGF in skin biopsy
Biopsy of affected skin was obtained from the forearm of SSc patients fulfilling ACR criteria after obtaining informed consent, and transported to laboratory in saline. After snap freezing in liquid nitrogen the blocks were stored at -70°C. Later 5 µm thin cryostat sections were cut and air dried. The slides were fixed in acetone for 20 minutes, dried and stored at -40°C. To stain, the sections were rehydrated with phosphate-buffered saline (PBS 0.1M pH 7.4/7.2) for 10 minutes. After washing the sections were incubated with PBS containing 0.4 percent sodium azide for 15 minutes to block endogenous peroxidase. Following washing, the sections were blocked with PBS containing 10 percent bovine serum albumin for 1 hour and were overlaid with rabbit anti-human bFGF antibody (Sigma chemicals, USA) diluted 1:1000 in PBS containing 1 percent BSA and incubated in a moist chamber for 90 minutes at 37°C. After washing thrice with PBS, 1:15 diluted goat anti-rabbit Ig conjugated with biotin (Sigma, USA) in PBS-BSA was added and incubated in a moist chamber for 30 minutes and washed thrice in PBS. 1:15 extravidin-peroxidase conjugate (Sigma USA) was added and slides were incubated for 30 minutes. The slides were developed with AEC substrate kit (Sigma, USA) as per the manufacturer's instructions. After washing with double distilled water the slides were counterstained with Mayer's hematoxylin and viewed under the microscope. With every slide negative (without addition of basic FGF antibody but with the addition of rabbit immunoglobulin) and positive control (astrocytoma tissue sections) were also stained (Figs. 1A and 1B). The staining was scored on a scale of 0-3 depending on the intensity of stain. The negative control was taken as zero and the positive control as 3. The Epidermis, dermis and peri-vascular/peri-appendageal areas were scored separately. The study was approved by the institutional ethics committee.
Serum levels of bFGF
Blood was collected and serum separated and stored at -70°C until analysis. The assay was done using bFGF ELISA kit (R&D system, USA). The manufacturer's instructions were followed. It had a sensitivity of 10 pg/ml.
The proportion of patients and controls having detectable serum bFGF was compared by Fisher's exact test. P value less than 0.05 was considered significant.
The study included 30 serum samples from patients with SSc and 23 samples from healthy controls. Of these 30 patients all but 3 were females. Fifteen each had limited and diffuse disease. The median age and duration of disease were 26 years (range 14-50) and 3 years (range 1-10) respectively. Twenty had evidence of interstitial lung disease.
The levels of basic FGF levels were undetectable in the majority of patients. Only 4/30 (11-22 pg/ml) patients had values above detectable limit of 10 pg/ml as compared to 3/23 (14-32pg/ml) healthy controls (p = ns). Three of these four patients had limited disease.
Skin biopsy specimens were obtained from fifteen patients (13 included for analysis as in 2 the sections were not satisfactory) and three healthy controls undergoing biopsy for some other indication (such as removal of neurofibroma or lipoma).
All thirteen were females, of which six had Limited SSc (Table 1). The median age was 28 (range 15-50) and the median duration of disease was 2 years (range 1-8 years). Raynaud phenomenon was present in twelve patients whereas eight patients had symptomatic interstitial lung disease and eight had esophageal dysmotility.
The expression of bFGF was increased in all three areas in patients with SSc (Table 1). In the epidermis, maximum expression was seen in the basal layer (Figs. 1D and 1E). In dermis where the staining was maximal, it was present both extracellular in the loose connective tissue (Fig. 1F) as well as associated with cells. Appendageal cells showed an increase in bFGF staining. Perivascular cells also expressed bFGF. The intensity of expression was no different in limited versus diffuse disease and did not correlate with duration of disease or skin score.
|Figure 1A||Figure 1B|
|1. Astrocytoma sections stained with antibodies to basic FGF. a: positive control, b: isotype control|
|Figure 2A||Figure 2B|
Our data show that there is increased expression of bFGF in skin of patients with SSc. In contrast, serum bFGF was detectable in only a minority of patients.
Elevated serum bFGF has been shown in patients with SSc in only one previous study . Our prevalence of 13 percent is much lower than their prevalence of 42 percent; furthermore we did not find any difference between healthy controls and patients. This difference could be due to a smaller number of patients in our study, difference in assay (even though our assay had a high sensitivity), or a difference in patient population. They also found its prevalence higher in patients with limited disease. Our inability to detect basic FGF in serum could be related to the fact that, after secretion into the local milieu, bFGF remains sequestered in the extra-cellular matrix .
Immunohistochemically detectable bFGF was increased in all three areas of the skin in patients with SSc but more so in the dermis. However, the levels of immunoreactive bFGF varied considerably among various patients. This is analogous to variation in expression of TGF-β in scleroderma . Basic FGF has been shown to be present in basal keratinocytes, histiocytes, and endothelial cells in normal skin , similar to that observed by us even though the expression is low. Whereas in SSc, besides these cells, cells of skin appendages and dermal connective tissue also had increased expression of bFGF. This suggests that bFGF is secreted locally and sequestered in the dermal connective tissue and that release from there leads to fibrosis.
Several mechanisms for the role of basic fibroblast growth factor in fibrosis are given in the literature. Studies have shown that FGF increases the fibroblast proliferation whereas anti-Hu FGF antibodies can block this . Recent studies have highlighted the role of platelet-derived growth factor (PDGF) in animal models of fibrosis. Imatinib mesylate, which inhibits platelet derived growth factor receptor kinase in addition to Bcr-Abl kinase, has been shown to prevent fibrosis in both bleomycin-induced  as well as radiation-induced  lung fibrosis in animal models. Basic FGF modulates the receptor expression of platelet-derived growth factor (PDGF), which in turn regulates b-FGF receptor level [13, 14]. In addition, basic FGF also mediates mitogenic effect of PDGF on vascular smooth muscle cells . Therefore, basic FGF and PDGF can act synergistically in an autocrine and paracrine manner. . .TGF-β, another important cytokine in SSc, up-regulates the expression of PDGF and PDGF receptor on fibroblasts. Although TGF-β regulates the synthesis of collagen and extracellular matrix components in fibroblasts and can induce most of the features of transformed fibroblasts in vitro experiments, the sequence of events leading to the establishment of perpetuating fibrosis is not known. Mice experiments  suggest the potential role of basic FGF in inducing and sustaining the profibrotic phenotype in scleroderma fibroblasts. It is possible that basic FGF might be playing a role in maintaining the abnormal phenotype of systemic sclerosis fibroblasts by acting downstream of TGF-β and PDGF in an autocrine/paracrine manner.
We need to further study coexpression of bFGF and TGF-β to see that they are present together in milieu to have synergistic effect.
Acknowledgment: The study was funded by a grant from Sanjay Gandhi Postgraduate Institute to AA. Ethics approval: This study was approved by the institutional ethics committee.
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