Reverse transcription–polymerase chain reaction for messenger RNA (mRNA) for the pigment epithelium–derived factor gene (PEDF) in the nasal mucosa of healthy control subjects and patients with allergic rhinitis. The PEDF mRNA was expressed in both control and allergic rhinitis mucosa. Expression levels of PEDF mRNA were consistently increased in the tissue from patients with allergic rhinitis compared with the tissue from healthy controls. Bp indicates base pair; GAPDH, the gene for glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase; +, positive; −, negative.
Comparison of the ratio of messenger RNA (mRNA) for the pigment epithelium–derived factor gene (PEDF) to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) in the nasal mucosa of healthy control subjects and patients with allergic rhinitis. The expression level of PEDF mRNA was significantly increased in patients with allergic rhinitis compared with controls. Bars represent mean values; limit lines, standard deviations.
Immunolocalization of pigment epithelium–derived factor (PEDF) and vascular endothelial growth factor (VEGF) proteins. A, PEDF protein is expressed in the epithelium, submucosal glands, and a few scattered stromal cells. The expression of PEDF protein in allergic rhinitis mucosa was consistently stronger than in the control mucosa (original magnification ×200). B, VEGF protein is expressed in the epithelium, submucosal glandular epithelium, and a few scattered stromal cells (original magnification ×200).
Representative Western blots of pigment epithelium–derived factor (PEDF) protein expression in nasal mucosal samples from healthy control subjects and patients with allergic rhinitis. The number indicates the size of the immunodetected protein.
Kang HJ, Park H, Won Chae S, Hwang SJ, Lee SH, Lee SH, Lee H. Increased Expression of Pigment Epithelium–Derived Factor in Allergic Rhinitis. Arch Otolaryngol Head Neck Surg. 2008;134(10):1094-1098. doi:10.1001/archotol.134.10.1094
To investigate the expression of messenger RNA (mRNA) of the gene for pigment epithelium–derived factor (PEDF) (OMIM *172860) and PEDF protein and to localize the PEDF protein in the nasal mucosa of patients with allergic rhinitis and of control subjects.
Investigation of PEDF mRNA and PEDF protein expression in the nasal mucosa using reverse transcription–polymerase chain reaction, Western blotting, and immunohistochemical staining.
We used inferior turbinate mucosal samples from 10 patients with allergic rhinitis and 10 matched healthy control subjects.
We extracted PEDF mRNA from the inferior turbinate mucosa samples and performed reverse transcription–polymerase chain reaction analysis. We used Western blotting to analyze differences in expression levels of PEDF protein between patients with allergic rhinitis and healthy controls, and the PEDF protein was localized immunohistochemically.
The expression levels of PEDF mRNA and PEDF protein in the nasal mucosa were significantly increased in patients with allergic rhinitis compared with those in nonallergic controls. The PEDF protein was expressed in the epithelium and submucosal glands.
We found that PEDF protein is expressed in the human nasal mucosa, and its expression is increased in allergic rhinitis. These results suggest a possible contribution of PEDF to the chronic inflammation of the nasal mucosa in allergic rhinitis.
Allergic rhinitis is one of the most common chronic diseases in the industrialized world. It is histologically characterized by a marked infiltration of inflammatory cells, increased vascular permeability, plasma transudation, and vasodilation in the nasal mucosa. Increased angiogenesis in the nasal mucosa has also been reported.1
Angiogenesis is the growth of new blood vessels from existing ones. This process is important in the pathogenesis of many diseases such as cancer, diabetic retinopathy, and rheumatoid arthritis.2 Under physiologic conditions, angiogenesis depends on the balance of angiogenic and angiostatic mediators within the vascular microenvironment.3
Increased angiogenesis and increased expression of the angiogenic mediator vascular endothelial growth factor (VEGF) in the nasal mucosa of patients with allergic rhinitis has been documented.1,4 Although the importance of the angiostatic mediator pigment epithelium–derived factor (PEDF) in ocular and pulmonary diseases has been demonstrated in previous studies,5,6 data regarding PEDF has not been described in the nasal mucosa in the setting of allergic rhinitis. In this study, we investigated the expression and localization of PEDF in the nasal mucosa of patients with allergic rhinitis and compared them with the expression and localization of PEDF in healthy control subjects.
Nasal mucosal biopsy specimens of the inferior turbinate were obtained from 10 patients (5 men and 5 women; age range, 27-43 years; median age, 34 years) with allergic rhinitis at Korea University Hospital, Seoul, between September 1, 2005, and April 30, 2006. All biopsy specimens were obtained from the lower edge of the inferior turbinate, approximately 2 cm posterior to the anterior margin, with the use of Blakesley forceps (Karl Storz, Tuttlingen, Germany) by the same investigator (H.-M.L). The patients who were included in this study presented with a clinical diagnosis of allergic rhinitis (sneezing, rhinorrhea, and/or nasal congestion on most days) of at least 2 years' duration, had results of a nasal smear demonstrating eosinophilia, and had a documented positive reaction (≥3+) to 1 or more perennial allergens (ie, Dermatophagoides pteronyssinus, Dermatophagoides farinae, cockroach, molds, cat fur, and dog hair) on a commercially available skin prick test kit (Allergy Therapeutics, West Sussex, England) within the study period. All of the study subjects exhibited symptoms of allergic rhinitis at the time of the evaluation.
The patients discontinued treatment with cromolyn sodium (sodium cromoglicate), antihistamines, and corticosteroids at least 4 weeks before entering this study. No subject had an infection of the respiratory tract 4 weeks before the study began. Smokers and subjects who had bronchial asthma that required the regular use of inhaled corticosteroids or systemic corticosteroids were excluded from the study.
Normal inferior turbinate biopsy specimens from 10 matched patients (5 men and 5 women; age range, 24-42 years; median age, 33 years) who were undergoing augmentation rhinoplasty were used as controls (control mucosa). All of the control subjects had negative results of a skin prick test, no eosinophilia on the nasal smear finding, and no evidence of inflammation in the nasal cavity. Informed consent was obtained from all patients and controls, and the study protocols were approved by the institutional review board of Korea University College of Medicine.
Tissues were cut into 2 portions. One portion was immediately flash-frozen in liquid nitrogen and stored at −70°C for subsequent RNA processing and Western blot analysis. The other portion was fixed with 4% paraformaldehyde in 0.1M phosphate-buffered saline solution (pH, 7.4) overnight at 4°C and then embedded in paraffin for immunohistochemical staining.
Total RNA was extracted from the perennial allergic rhinitis mucosa specimens using a commercially available reagent (TRIzol; Gibco BRL, Grand Island, New York) according to the manufacturer's suggestions. Total RNA from each sample underwent reverse transcription (RT) in 20 μL of reaction mixture containing 2.5 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL) and 50pM random hexanucleotides at 42°C for 60 minutes. Oligonucleotide primers for polymerase chain reaction (PCR) analysis were commercially synthesized (Bioneer Co, Daejon, South Korea) on the basis of the published sequences. Oligonucleotide primers were designed according to the following published sequences for PEDF: sense primer 5′-CAG AAG AAC CTC AAG AGT GCC-3′ and antisense primer 5′-CTT CAT CCA AGT AGA AAT CC-3′. Amplification of the complementary DNA was performed using 30 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by a final extension cycle of 72°C for 7 minutes. Specificity of the 310-base pair (bp) PCR product was verified by predicted size, restriction digestion, and DNA sequencing. As negative controls, the PEDF primer or the reverse transcriptase was omitted from some RT-PCR reactions. The primers 5′-GTG GAT ATT GTT GCC ATC AAT GAC C-3′ and 5′-GCC CCA GCC TTC ATG GTG GT-3′ for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) (OMIM *138400) were run simultaneously as an internal control (the expected size of this PCR product was 270 bp). The PCR product was analyzed by means of gel electrophoresis. To analyze the RT-PCR results semiquantitatively, the gel images were scanned and the intensity of the PCR products was measured using a commercially available image software package (National Institutes of Health, Bethesda, Maryland). The relative intensities of individual bands on a gel image were determined as the ratio of the intensity of PEDF messenger RNA (mRNA) to that of GAPDH mRNA.
Slides were deparaffinized and then serially rehydrated in graded alcohol. For PEDF immunostaining, the rehydrated sections were soaked in citrate buffer and heated in a microwave oven for 15 minutes at 500 W. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 5 minutes. Nonspecific binding was blocked with 10% normal horse serum for 20 minutes. After a brief rinse, each section underwent immunoreaction for 1 hour with primary antibodies at room temperature. The primary antibodies consisted of monoclonal anti–PEDF antibody (Chemicon International Inc, Temecula, California) and monoclonal anti–VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, California). The sections were washed in phosphate-buffered saline solution, incubated for 1 hour with biotinylated anti–mouse IgG (Elite Kit; Vector Laboratories Inc, Burlingame, California), and then treated with avidin-biotin complex for 1 hour. Then, 3-3′-tetradiaminobenzidine (Vector Laboratories Inc) was applied as a chromogen. For the negative control, nonimmune serum IgG or phosphate-buffered saline solution was used instead of the primary antibody. Sections were visualized after counterstaining with Mayer hematoxylin.
Immunoreactivity for PEDF and VEGF proteins was evaluated for the intensity of staining. The results were scored for the absence of positivity or the presence of weakly positive, definitely positive, or strongly positive intensity. The degree and distribution of immunostaining were assessed under light microscopy by 2 independent observers who were blinded to the sample groups.
Tissues were homogenized, and cells were lysed in protein-extract buffer (30mM TRIS hydrochloride [pH, 8.0], 10mM edetic acid, 1% Triton X-100, 100mM sodium chloride, and 1mM phenylmethylsulfonyl fluoride) and stored at −70°C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were then blocked for 30 minutes in a buffer consisting of 20mM TRIS hydrochloride (pH, 7.6), 135mM sodium chloride, and 1% Tween 20 (Sigma-Aldrich Inc, St Louis, Missouri), with 5% nonfat dry milk and incubated at 4°C overnight with monoclonal anti–PEDF antibody at a dilution of 1:200. After incubation, the membrane was washed 3 times for 5 minutes in the TRIS-buffered saline–polysorbate 20 buffer and then treated with peroxidase-conjugated anti–mouse IgG antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, Pennsylvania) for 1 hour at room temperature. The membrane was washed, and substrate was added from an enhanced chemiluminescence reagent kit (ECL; Amersham Corp, Arlington Heights, Illinois) and exposed to x-ray film for 10 seconds. As an internal control, β-actin expression was analyzed using the β-actin antibody (Sigma-Aldrich Inc).
We used the paired t test to compare the PEDF mRNA expression levels between the allergic rhinitis and control groups. Statistical analysis was performed using a commercially available statistical software package (SPSS, version 8.0; SPSS Inc, Chicago, Illinois). The results were expressed as mean (SE). Significance of a difference in the PEDF:GAPDH mRNA ratio between the 2 groups was accepted at the 5% level of confidence.
We performed RT-PCR to detect the presence of PEDF mRNA in the human nasal mucosa. The PCR products of the expected size corresponding to 310 bp for PEDF mRNA were detected in the control and allergic rhinitis mucosal samples and showed 100% sequence homology with the published PEDF mRNA sequence. These results indicated that the human nasal mucosa expresses PEDF mRNA. On RT-PCR screening results, PEDF mRNAs were weakly amplified in control nasal mucosa. However, PEDF mRNAs were prominently amplified in allergic rhinitis mucosa (Figure 1); PEDF mRNA was not expressed in the negative control. As a control for RNA integrity and the RT-PCR procedure, the same RNA samples were also analyzed for GAPDH gene expression. The GAPDH primer produced a PCR product (270 bp) from each of the tissue samples. All samples were normalized with respect to GAPDH. Semiquantitative analysis of the PCR samples showed a statistically significant difference in the PEDF:GAPDH mRNA ratios between the allergic rhinitis and control mucosal samples (P < .03) (Figure 2).
Immunohistochemical staining was performed to localize the PEDF and VEGF proteins in the nasal mucosa. Positively immunostained cells appeared brown. The PEDF protein was expressed in the epithelium, submucosal glands, and a few scattered stromal cells in the allergic rhinitis mucosa but was barely detectable in the control mucosa (Figure 3A). We observed VEGF protein expression in the epithelium, submucosal glandular epithelium, and a few scattered stromal cells (Figure 3B). The PEDF and VEGF immunostained areas showed a similar distribution in the allergic rhinitis nasal mucosa. The distribution of PEDF and VEGF immunopositive cells are shown in the Table.
Western blot analysis showed a prominent immunoreactive band with an apparent molecular mass of approximately 50 kDa reacting with anti–PEDF antibody in allergic rhinitis mucosa (Figure 4). A less prominent band of the same molecular mass was detected in the control mucosa.
In this study, we have shown for the first time, to our knowledge, the presence of the potent angiostatic mediator PEDF in the human nasal mucosa; that it is localized to the epithelium, submucosal glands, and a few scattered stromal cells. Our results demonstrate that its expression is increased in the setting of allergic rhinitis.
Angiogenesis is the process by which new vessels grow from existing ones. It is increased in numerous diseases such as cancer and diabetic retinopathy, and in chronic inflammatory diseases such as rheumatoid arthritis and asthma.2,7,8 Angiogenesis is thought to result from a dynamic balance of modulators that can stimulate or inhibit new vessel growth in a given tissue.2 In the eye, an increased expression of the angiogenesis stimulator VEGF relative to the angiogenesis inhibitor PEDF resulted in retinal neovascularization, whereas increased PEDF levels were correlated with regression of retinal neovascularization.9,10
There is considerable evidence to suggest that angiogenesis and chronic inflammation are codependent.11 In an inflammatory tissue environment, VEGF is secreted, promoting angiogensis. Angiogenesis in turn contributes to perpetuation of chronic inflammation by promoting the migration of inflammatory cells to the site of inflammation and by supplying nutrients and oxygen to the proliferating inflamed tissue. Therefore, targeting angiogenesis to treat chronic inflammation and targeting chronic inflammation to affect angiogenesis may potentially be beneficial in the treatment of chronic inflammatory diseases.
Increased angiogenesis in the nasal mucosa of patients with allergic rhinitis was demonstrated in a previous study.1 Vascular endothelial growth factor has been cited as a mediator that contributes to the inflammatory pathogenesis of allergic rhinitis by promoting angiogenesis. Thus, it has been proposed that the upregulation of VEGF in the nasal mucosa plays an important role in the angiogenesis of allergic rhinitis.4
Pigment epithelium–derived factor is a 50-kDa protein belonging to the serpin family that was first described in retinal pigmented epithelial cells.12 It is one of the most potent endogenous inhibitors of angiogenesis and exhibits its angiostatic activity selectively against newly forming vessels while sparing existing ones.13,14 Pigment epithelium–derived factor significantly suppresses endothelial cell migration and proliferation, thus playing an essential role in the control of angiogenesis.5
There appears to be a regulatory interaction between PEDF and VEGF. In retinal pigmented epithelial cells, VEGF directly upregulated PEDF expression, whereas, in another study, PEDF significantly suppressed VEGF-induced proliferation and migration of vascular endothelial cells.5,15 Although the exact pathogenic mechanisms are uncertain, these findings suggest that a relative balance in the expression levels of PEDF and VEGF may be important in maintaining homeostasis of regional vasculature.
The cellular sources of PEDF in the nasal mucosa of patients with allergic rhinitis may be a subject of interest. The epithelial cells and submucosal glands are possible candidates because they are immunopositive for PEDF. For the immunopositive cells in the stroma, we did not determine specifically what cells these were in the present study. A search of the English literature yielded no published reports that investigated the expression of PEDF in the nasal mucosa. In a study of lung tissue, fibroblasts and epithelial cells have been suggested as sources of PEDF.6 Because the nasal and bronchial mucosa present with numerous similarities, fibroblasts and epithelial cells might also express PEDF in the nasal mucosa. Additional studies that can elucidate the cellular sources of PEDF are necessary.
Considering the previously reported upregulation of VEGF and the increased expression of PEDF in allergic rhinitis demonstrated in the present study, there may also be a regulatory interaction between PEDF and VEGF in the human nasal mucosa. The increased expression of VEGF and concomitantly elevated level of PEDF in the allergic rhinitis nasal mucosa demonstrated in the present study support this hypothesis. The increased expression of PEDF in the nasal mucosa in the setting of allergic rhinitis, along with its selective angiostatic activity against new vessel formation, may contribute to a protective mechanism against neovascularization and subsequent perpetuation of chronic inflammation in allergic rhinitis. Pharmacological control of angiogenesis may be a novel therapy for the alleviation of chronic inflammatory states. Further studies to investigate how PEDF expression is coordinated with VEGF expression in the allergic rhinitis nasal mucosa are needed in the future.
In conclusion, the angiostatic mediator PEDF mRNA and PEDF protein are expressed in the human nasal mucosa, and their expression is increased in allergic rhinitis. We suggest a possible contribution of PEDF to the pathogenic mechanism in the chronic inflammation of the nasal mucosa in allergic rhinitis.
Correspondence: Heung-Man Lee, MD, PhD, Department of Otorhinolaryngology–Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, Seoul, Korea 152-703 (email@example.com).
Submitted for Publication: September 20, 2007; final revision received January 11, 2008; accepted January 30, 2008.
Author Contributions: Dr H.-M. Lee had full access to all the data in the study and takes full responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Kang and H.-M. Lee. Acquisition of data: Kang, Park, and H.-M. Lee. Analysis and interpretation of data: Kang, Chae, Hwang, Sang H. Lee, Seung H. Lee, and H.-M. Lee. Drafting of the manuscript: Kang, Park, and H.-M. Lee. Critical revision of the manuscript for important intellectual content: Kang, Chae, Hwang, Sang H. Lee, Seung H. Lee, and H.-M. Lee. Administrative, technical, and material support: Chae, Hwang, Seung H. Lee, and H.-M. Lee. Study supervision: Sang H. Lee and H.-M. Lee.
Financial Disclosure: None reported.
Funding/Support: This study was supported by a grant from the Korea University College of Medicine (Dr H.-M. Lee).