Hyperexpression of Low-Density Lipoprotein Receptors and Hydroxy-Methylglutaryl-Coenzyme A-Reductase in Human Pinguecula and Primary Pterygium

Abstract

purpose. There is now increasing evidence that pterygium and pinguecula are tumorlike tissues and that cell growth and DNA replication are closely linked to cholesterol metabolism. In this study, the expression of two main genes correlated to cholesterol metabolism—namely, the low-density lipoprotein receptor (LDL-R) gene and the hydroxy-methylglutaryl-coenzyme A-reductase (HMG-CoA-R) gene—were investigated in primary pterygium, pinguecula, and normal conjunctiva.

methods. Pterygium, pinguecula, and normal conjunctiva samples were obtained from 30 eyes (50% men) at the time of surgery. Total RNA extracted from the specimens was subjected to semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). Equal amounts of total RNA were reverse transcribed into cDNA. The cDNA was subsequently amplified by the PCR in the presence of specific primers for low-density lipoprotein receptor (LDL-R) and for hydroxy-methylglutaryl coenzyme A reductase (HMG-CoA-R).

results. Semiquantitative RT-PCR analysis revealed that the mRNA levels of LDL-R and HMG-CoA-R were increased in pterygia, compared with levels in both pingueculae and normal conjunctivae. Differences were statistically significant (P < 0.05), including pingueculae versus normal conjunctivae.

conclusions. This study indicates that pterygium and pinguecula have an altered metabolism of cholesterol—namely increased LDL-R and HMG-CoA-R mRNAs—as is characteristic of tumorlike tissues, and that the high expression of LDL receptors renders them amenable to be treated by photodynamic therapy with intravenously injected verteporfin.

Pinguecula and pterygium are both nonmalignant, slow-growing proliferations of conjunctival connective tissue in proximity to the limbus, generally arising in response to UVB radiation. ^{1 2} Pterygium can start out as pinguecula but can grow as an independent entity as well. Pterygium, but not pinguecula, results from the invasion of the cornea by hyperproliferating limbal epithelial cells, together with their supporting blood vessels, and may therefore stretch and distort the cornea and induce astigmatism and low vision. In the advanced stages, it can necessitate complex surgery for full visual rehabilitation. Surgical excision of pterygium, however, is often associated with high rates of recurrence, despite multiple variations in surgical techniques and adjunctive treatments. ³ Recurrent pterygium may sometimes become a serious clinical challenge, either because it is an aggressive form or because patients refuse further surgery. 

Pterygium shows features that suggest excessive or disordered growth. Tumorlike histologic characteristics, ranging from mild dysplasia to carcinoma in situ and local invasiveness, have been described. ^{4 5} Controversial data have been accumulated on the role of p53 oncogene: although Dushku and Reid, ⁶ Tan et al., ⁷ and Weinstein et al. ⁸ reported high expression of p53 in the epithelium overlying the pterygium and speculated on the existence of a p53 gene mutation, other investigators have failed to detect an increased p53 protein level and p53 mutation in pterygia. ^{9 10} Oncogenic human papillomaviruses (HPVs), particularly types 16 and 18, which have been found in pterygia and limbal tumors ^{11 12} (particularly in certain geographic areas, such as Sardinia in Italy ¹³ ) can disrupt the p53-dependent programmed cell death pathway ¹⁴ and assume a potential oncogenic role, ¹⁵ eventually interacting with ultraviolet (UV) irradiation. ¹⁶ Moreover, UV light has long been associated as the etiologic agent for cutaneous malignancies such as melanoma, basal cell carcinoma, and squamous cell carcinoma; and recently, heparin-binding epidermal growth factor (HB-EGF), a potent mitogen, has been localized in pterygium tissue and has been demonstrated to be significantly induced by UVB in pterygium-derived epithelial cells. ¹⁷ Furthermore, pterygial fibroblasts exhibit characteristics of the transformed phenotype ¹⁸ —microsatellite instability and loss of heterozygosity ¹⁹ —and high levels of mitotic activity, ²⁰ all reported to be common findings in neoplastic tissues, supporting the hypothesis that pterygium may be a benign neoplastic lesion. 

In neoplastic tissue, modifications in cholesterol synthesis and metabolism are common findings. Within the normal cell, the distribution of cholesterol is highly compartmentalized, with most of the cellular cholesterol concentrated in the plasma membranes. Cholesterol is a major component of the cell membrane and is derived mainly from two sources: endogenously, by synthesis from acetyl-coenzyme-A (CoA) through mevalonate, with the pivotal role of HMG-CoA-reductase activity, the rate-limiting enzyme of cholesterol biosynthesis, and exogenously, by the internalization of cholesteryl-rich lipoproteins through the LDL receptor pathway. ²¹  

It has been suggested that an increased demand for free cholesterol derived from both exogenous and endogenous sources is part of the cellular response to a mitogenic stimulus, and it is satisfied by an increased amount of HMG-CoA-reductase activity and expression in LDL receptors per cell. ²² A correlation between alterations of cholesterol homeostasis and rate of cell growth has been found in our laboratory in different types of human tumors. ^{23 24} When cholesterol metabolism was evaluated in leukemic cells, we also found an increase in the mRNA levels of LDL receptor, HMG-CoA-reductase, and acyl-coenzyme A:cholesterol transferase (ACAT) activity. ²⁵  

Despite increasing evidence that pterygium is a disorder involving an abnormal growth, to the best of our knowledge, only one nearly 50-year-old study has been conducted to investigate lipid presence in pterygium. ²⁶ Moreover, this information is related to recent studies indicating that photodynamic therapy (PDT) with verteporfin may be useful for several neovascular conditions involving the anterior segment of the eye, such as corneal neovascularization ^{27 28} and pterygium. ²⁹ Because cellular uptake of verteporfin by means of LDL-R is necessary for the achievement of the cytotoxic response to PDT, it would be interesting to know the level of expression of LDL receptors in clinically advanced pterygia. 

In the present study, we examined the expression of two main genes correlated to cholesterol metabolism—namely, LDL-R and HMG-CoA-R mRNAs—in primary pterygium, pinguecula, and normal conjunctiva. 

Materials and Methods

Patients

Ten patients with primary pterygium (mean age, 63.0 ± 10.57 years [SD]) and 10 patients with pinguecula (mean age, 55.5 ± 12.09 years [SD]) were included in the study. All patients had blood cholesterol levels within normal limits. Diagnosis of pterygium and pinguecula was based on clinical history and evaluation of signs and symptoms. Patients with primary pterygium (five men and five women) had at least a 5-year history of a slow growing lesion, with a corneal extension of at least 4 mm, as measured with a caliper, from the limbus to the corneal vertex. Pterygia morphology was clinically graded according to the system of Tan et al., ³⁰ based on the assessment of pterygium translucency: atrophic (T1), intermediate (T2), or fleshy (T3) pterygium. All pterygia collected in this study were T3. Patients with pinguecula (five men and five women) had a 3-year history of a conjunctival globule on the sclera near the limbus. All patients with pinguecula and pterygium had never undergone surgery. The control group was age matched (mean age 62.5 ± 10.89 years) and included five men and five women undergoing surgery for retinal detachment. No subject in the control group had any inflammatory signs or symptoms. The research adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all the patients before tissues were collected. 

All tissue samples were obtained at the time of surgery from the inner canthus with microforceps. As a rule, within 1 hour of excision, samples were placed in sterile boxes containing a preservative solution (Eurocollins; Roche Biochemicals, Mannheim, Germany), stored at 4°C, and transported to the laboratory where they were cleaned, washed, weighed, and stored at −80°C in guanidine-thiocyanate buffer (4 M) until molecular analyses were performed. 

RNA Isolation and RT-PCR Analysis

The small amount of specimens led us to use RT-PCR in these studies. The specificity of RT-PCR has already been validated in a previous study. ³¹ Total RNA was isolated from samples by the guanidine isothiocyanate phenol-chloroform extraction method. ³² The integrity of RNA was evaluated by agarose gel electrophoresis, RNA yield was quantified spectrophotometrically at 260 nm, and A260/A280 ratios were determined to check for protein contamination. 

The PCR primers for HMG-CoA-R, LDL-R, and β-actin were designed from published human gene sequences (Table 1) . Reverse transcription–polymerase chain reaction (RT-PCR) was performed to evaluate the expression of these genes in the samples, with the gene β-actin as the internal control. In brief, equal amounts of total RNA (1 μg) were reverse transcribed into cDNA, using the random hexamer method. cDNA was subsequently amplified by PCR in a DNA thermal cycler (model 480; PerkinElmer, Boston, MA) in the presence of specific primers, according to the instructions provided by the manufacturer (GeneAmp RNA PCR Kit; PerkinElmer Cetus, Milan, Italy). PCR amplification was performed in a DNA thermal cycler (model 480; PerkinElmer) using the protocols shown in Table 1 . Preliminary experiments demonstrated that with these PCR conditions the product amplification-to-RNA relationship was linear for the cycle number used over the range of 500 to 1500 ng total RNA. Working in these conditions, PCR products separated on agarose and stained with ethidium bromide were characterized by a major band of the predicted size (Table 1) . 

Blot Analysis

During the PCR reaction the nonradioactive label digoxigenin-11-dUTP (DIG; Roche Biochemicals) was incorporated and immunodetected with anti-DIG Fab fragments conjugated to alkaline phosphatase and visualized with a chemiluminescence substrate (CSPD; Roche Biochemicals). Enzymatic dephosphorylation of CSPD (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1.3,7]decan}-4-yl)phenyl phosphate) by alkaline phosphatase led to light emission at a maximum wavelength of 477 nm, which was recorded on x-ray film. The DNA fragments were separated by electrophoresis on agarose and then blotted onto a nylon membrane for 16 hours in 10× SSC. The blot was exposed to x-ray film for 2 to 10 minutes. An image analysis system (Digital Science Band Scanner; Eastman Kodak, Rochester, NY, containing an HP ScanJet; Hewlett-Packard, Palo Alto, CA, with ID Image Analysis Software; Eastman Kodak) assessed the mass (in nanograms) of the bands in the autoradiograms. The overall procedure was standardized by expressing the amount of PCR product for each target mRNA relative to the amount of product formed for β-actin. Because a low yield of PCR products is often obtained when cDNA segments are coamplified with an internal standard gene in the same tube, the relative levels of gene expression were determined by comparing the PCR products of the target cDNA and β-actin gene processed in separate tubes. 

Statistical Analysis

Differences between LDL-R and HMG-CoA-R levels in pterygium, pinguecula, and normal conjunctiva were analyzed with Student’s t-test. Results are given as the mean ± SD. For all statistical analyses the level of significance was set at P ≤ 0.05. 

Results

Patients

The mean ages of patients with pterygium and pinguecula were similar. Of the 10 patients with pterygium, all were in the T3 stage of the disease. Four of these patients had a bilateral form. Of the 10 patients with pinguecula, all had the bilateral form. 

Blot Analysis

Figure 1 shows autoradiographs of PCR-amplified mRNAs of HMG-CoA-R, LDL-R, and β-actin genes representative of 10 normal conjunctivae (Con), 10 pterygia (Ptr), and 10 pingueculae (Ping). The specific product bands for the investigated genes and the β-actin gene are of the size indicated in Table 1 . The mRNAs of the HMG-CoA-R were expressed in all tissues studied, although in normal conjunctiva mRNA was barely detected (Fig. 1A) . The transcripts in pterygium and pinguecula were upregulated when compared with constantly expressed β-actin mRNA levels (Fig. 1C) , which served as an internal control. Similar results were obtained when mRNA levels of LDL-R were investigated (Fig. 1B) . The transcripts of HMG-CoA-R and LDL-R were more elevated in pterygia than in pingueculae or normal conjunctivae. 

Quantitative RT-PCR Analysis

We then assessed the mass (in nanograms) of the bands in the autoradiograms by determining the amount of PCR product for each target mRNA relative to the amount of product formed for β-actin, by using the image analysis system described in the Methods section. 

Quantitative densitometric determination of bands revealed a statistically significant (P < 0.05) increase of LDL-R mRNA levels: 5.49-fold in pterygia (19.05 ± 1.78 ng [SD]) and 3.03-fold in pingueculae (10.53 ± 2.19 ng), compared with the control (3.47 ± 0.52 ng; Fig. 2A ). The differences were statistically significant (P < 0.05), including those between pterygia and pingueculae. Also, mRNA levels of HMG-CoA-R were significantly (P < 0.05) increased in pterygia (18.15 ± 2.36 ng) and pingueculae (11.86 ± 2.13 ng) by 5.74- and 3.75-fold, respectively, compared with normal conjunctiva (3.16 ± 1.19 ng; Fig. 2B ). The significance of these quantitative differences resulted not only from the comparison of an entire set of 10 tissue specimens, but also from the comparison of each single specimen from the three tissue groups. 

Discussion

In the present study the hyperexpression of two main genes associated with cholesterol metabolism—namely LDL-R and HMG-CoA-R mRNA—were found in primary pterygia and, to a lesser extent, in pingueculae, in comparison with normal conjunctivae. Such hyperexpression of LDL-R, which regulates the internalization of cholesteryl-rich lipoproteins, and of HMG-CoA-reductase (HMG-CoA-R), which represents the rate-limiting step of endogenous cholesterol biosynthesis, reflects the increased requirement for cholesterol by the fibroblasts and/or the accompanying neovascular endothelial cells. The increase in cholesterol synthesis serves at least two essential functions: It provides free cholesterol for the biogenesis of new membranes, and it promotes protein prenylation, which is essential for signal transduction leading to DNA replication. Therefore, our results confirm the hypothesis that pterygium may be the result of an abnormal proliferative process, probably triggered by UV light exposure, since its prevalence rates correlate positively with latitude. ³³ It is interesting to note that pinguecula, traditionally considered a trivial lesion, also showed features of increased cholesterol metabolism, probably related to its neovascular component. This finding is in agreement with the hypothesis that pinguecula is a potentially proliferative tissue. ³⁴  

Moreover, recent studies indicate that PDT with verteporfin may be useful for several neovascular conditions involving the anterior segment of the eye, such as corneal neovascularization ^{27 28} and pterygium. ²⁹ Cellular uptake of verteporfin by means of LDL-R is necessary for the cytotoxic response to PDT. After intravenous injection, liposomally formulated verteporfin strongly binds to endogenous LDLs and is then incorporated by the rapidly growing endothelial cells, which expresses LDL-R at high levels through receptor-mediated endocytosis. In experimental corneal neovascularization, it has been shown that rapidly growing new vessels highly express LDL-R ³⁵ and that the receptors regulate the uptake of liposomal benzoporphyrin derivative, when it is injected into the bloodstream for PDT. The results of the present study, demonstrating hyperexpression of LDL-R in pinguecula and primary pterygium, support PDT with verteporfin as an alternative method of treating such lesions, as has been preliminarily suggested in clinical work on T2 pterygia. ²⁹  

In conclusion, the present study indicates that pterygium and pinguecula have an increased metabolism of cholesterol, as is characteristic of most tumorlike tissues, and that the high expression of LDL-R render the receptors amenable to treatment with PDT with verteporfin. 

 Supported in part by a grant obtained by EP from “Fondazione Banco di Sardegna,” Sardinia, Italy.

 Submitted for publication February 19, 2004; revised July 3, 2004; accepted July 8, 2004.

 Disclosure: E. Peiretti, None; S. Dessì, None; M. Putzolu, None. M. Fossarello, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primer Sequences and Size of PCR Products

Target mRNA	Primers	PCR Protocol	Expected Size of PCR Products (bp)
HMG-CoA reductase 31	5′-TACCATGTCAGGGGTACGTC-3′	95°C for 60 s, 60°C for 45 s, and 72°C for 45 s,	246
	5′-CAAGCCTAGAGACATAATCATC-3′	for 35 cycles
LDL-receptor 31	5′-CAATGTCTCACCAAGCTCTG-3′	96°C for 60 s, 60°C for 45 s, and 72°C for 45 s,	258
	5′-TCTGTCTCGAGGGGTACCTG-3′	for 35 cycles
β-Actin	5′-AGGGGCCGGACTCGTCATACT-3′	96°C for 30 s, 60°C for 59 s, and 72°C for 45 s,	202
	5′-GGCGGCACCACCATGTACCCT-3′	for 20 cycles

Figure 1.

 

Representative autoradiograms (two patients) of (A) HMG-CoA-R, (B) LDL-R, and (C) β-actin mRNA levels determined by RT-PCR analysis. C, normal conjunctiva; Ptr, pterygium; Ping, pinguecula.

Figure 2.

 

The concentration in 10 samples from each group (mean ± SD) of (A) LDL-R and (B) HMG-CoA-R mRNAs determined by comparing the PCR products of the target DNA and the β-actin gene. *P < 0.05 versus control.