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CLINICAL STUDY: ATHEROSCLEROSIS

Association between the –514 ct polymorphism of the hepatic lipase gene promoter and unstable carotid plaque in patients with severe carotid artery stenosis

Elisabetta Faggin, PhD^*, Alberto Zambon, MD, PhD, Massimo Puato, MD^*, Samir S. Deeb, PhD, Sandra Bertocco, MD, Saverio Sartore, PhD, Gaetano Crepaldi, MD, Achille C. Pessina, MD, PhD^* and Paolo Pauletto, MD^*,*

^* Dipartimento di Medicina Clinica e Sperimentale, Università di Padova, Italy
Dipartimento di Scienze Mediche e Chirurgiche, Università di Padova, Italy
Department of Medical Genetics, University of Washington, Seattle, Washington, USA
Dipartimento di Scienze Biomediche e Sperimentali, Università di Padova, Italy

Manuscript received December 12, 2001; revised manuscript received June 5, 2002, accepted June 12, 2002.

^* Reprint requests and correspondence: Prof. Paolo Pauletto, Università degli Studi di Padova, Dipartimento di Medicina Clinica e Sperimentale, Via Giustiniani 2, 35128 Padova, Italy.
paolo.pauletto{at}unipd.it

	Abstract

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OBJECTIVES: We investigated the potential association between –514 CT polymorphism in the promoter of the hepatic lipase gene (LIPC) and the prevalence of inflammatory cells in the plaque of patients with severe carotid artery stenosis.

BACKGROUND: This common LIPC polymorphism has been related to the presence of an atherogenic lipoprotein pattern.

METHODS: We studied 68 consecutive patients undergoing carotid endarterectomy. The LIPC genotype was determined by polymerase chain reaction. Endarterectomy specimens were examined by immunocytochemistry using monoclonal antibodies for smooth muscle cells, macrophages, or lymphocytes.

RESULTS: In 50 of 68 patients who had evidence of previous ipsilateral ischemic events, 36 (72%) were carriers of the CC genotype, whereas only 14 (28%) were carriers of the CT/TT genotype (p = 0.002). Among the 18 patients without evidence of events, the two genotypes were equally distributed (9 vs. 9). The low-density lipoprotein (LDL) particles were denser in CC than in CT/TT genotype carriers (flotation rate: 0.315 ± 0.025 vs. 0.356 ± 0.019, p < 0.0005). The CC genotype was associated with an abundance of macrophages (6.7 ± 3.5 vs. 2.1 ± 2.1 cells/area unit in the CT/TT group, p < 0.0005) and a reduced number of smooth muscle cells (6.9 ± 6.2 vs. 14.5 ± 6.4 in the CT/TT group, p < 0.0005) in the plaque. An inverse relationship was found between LDL buoyancy and the number of macrophages in the plaque (r = –0.639, p < 0.0005).

CONCLUSIONS: We provide evidence, for the first time, that LIPC promoter –514 CT polymorphism, by modulating LDL density, significantly affects the number of macrophages in the plaque and possibly affects the occurrence of cerebrovascular events in patients with carotid artery stenosis.

Abbreviations and Acronyms   ANOVA   analysis of variance   apo   apolipoprotein   CARS   Carotid Atherosclerosis and Restenosis Study   CETP   cholesteryl-ester transfer protein   HDL   high-density lipoprotein   HL   hepatic lipase   LDL   low-density lipoprotein   LIPC   hepatic lipase gene   PCR   polymerase chain reaction   SMC   smooth muscle cells

Although infection represents a recognized factor in the pathogenesis of unstable plaque (4), the potential role of genetic factors is poorly defined. Recent studies have shown that the common –514 CT variant in the LIPC promoter is associated with different levels of hepatic lipase (HL) activity, low-density lipoprotein (LDL) buoyancy, and high-density lipoprotein-2 (HDL₂) cholesterol (7). In particular, compared with the CT and TT genotypes, the CC genotype is associated with higher HL activity and smaller, denser, more atherogenic LDL particles along with lower HDL cholesterol levels (7). Moreover, in the Familial Atherosclerosis Treatment Study (FATS), coronary angiography after intensive lipid-lowering therapy showed a significantly greater improvement in the atherosclerotic lesions of the CC than the CT and TT genotype carriers (8,9).

We sought to determine whether there is a relationship between the –514 CT LIPC promoter variant and the presence of unstable plaque in patients enrolled in the Carotid Atherosclerosis and Restenosis Study (CARS). This follow-up study of patients who had carotid endarterectomy was aimed at evaluating the molecular and cellular features of the primary lesions and their potential role in restenosis and cerebrovascular events (10). Our study provides evidence for the first time that the CC genotype of the common –514 CT variant in the LIPC promoter is associated with denser LDL, with an increased number of macrophages in the plaque, and with the rate of cerebrovascular events in patients with carotid artery stenosis.

	Methods

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Investigations of the LIPC promoter genotype, cell composition of endarterectomy specimens, and prevalence of cerebrovascular events, as well as analysis of the data, were carried out in blinded manner by different researchers. Blood samples were taken in ethylenediaminetetra-acetic acid from the antecubital vein before surgery. Samples were used for routine biochemical assays, and deoxyribonucleic acid (DNA) extraction for the LIPC promoter and cholesteryl-ester transfer protein (CETP) genotyping.

Patients.   Sixty-eight consecutive patients who had enrolled in CARS (10) and had no evidence of present or past atrial fibrillation were studied. Most of them were recognized as having had ipsilateral cerebrovascular events of ischemic origin, including transient ischemic attack, stroke, and minor stroke, before carotid endarterectomy was performed. This was established by the previous medical report and/or computed tomographic scan. All of the patients had significant carotid artery narrowing (70%) at angiography.

Lipid and lipoprotein determinations
Plasma, LDL, HDL cholesterol, triglycerides, apolipoprotein (apo) B, and apo A-I were measured as previously described (11). Lipoprotein (a) levels were determined with a monoclonal antibody-based immunonephelometric assay (12).

DNA extraction and analysis of LIPC promoter and CETP polymorphism
Deoxyribonucleic acid was obtained from whole blood stocked at –80°C, and analysis was performed using the Quantum Prep AquaPure Genomic DNA blood kit (BIO-RAD, Milan, Italy). As described previously (7), screening for the LIPC –514 CT polymorphism was carried out by polymerase chain reaction (PCR) amplification using the primer pair—forward: 5'-CCTACCCCGACCTTTGGCAG-3'; and reverse: 5'-GGGGTCCAGGCTTTCTTGG-3'.

Amplification was carried out in a 10-µl reaction with an initial denaturation at 94°C for 2 min, followed by 35 cycles of amplification at 92°C for 15 s, annealing at 64°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 7 min.

Five microliters of the PCR product was digested with the restriction enzyme DraI, followed by electrophoresis on 1% agarose gel.

Screening for the CETP gene TaqIB polymorphism was carried out by PCR amplification followed by agarose gel electrophoresis, as previously reported (13).

Density-gradient ultracentrifugation for apo B–containing lipoproteins
This technique, designed to optimize the resolution of apo B–containing lipoproteins, is a modification of a previous method (14). After creating a discontinuous salt density gradient in an ultracentrifuge tube, the samples were centrifuged at 65,000 rpm for 90 min ( ) at 10°C in a Sorvall TV-865B vertical rotor. Thirty-eight 0.45-ml fractions were then collected from the bottom of the centrifuge tube. Cholesterol was measured in each fraction. The relative flotation rate (Rf), which characterizes LDL peak buoyancy, was obtained by dividing the fraction number containing the LDL cholesterol peak by the total number of fractions collected.

Immunohistochemistry for cell composition of endarterectomy specimens
The endarterectomy specimens taken at surgery and suitable for analysis (absence of fragmentation, fissuring, or loss of structure) were investigated for cell composition, as previously described (10). In brief, the specimens were frozen in liquid nitrogen immediately after they were obtained and stored at –80°C until use. Eight-micrometer-thick seriate cryosections were processed for immunohistochemistry using the following monoclonal antibodies: 1) SM-E7, specific to smooth muscle myosin heavy chains, which recognizes SMCs independently from their phenotype; 2) HAM56, which recognizes human macrophages; and 3) CD45RO, which recognizes lymphocytes.

Primary antibodies (except for CD45RO) were applied to freshly cut unfixed cryosections (7 µm), as previously described (10). For the antilymphocyte antibody, cryosections were first fixed in acetone for 10 min at –20°C and then washed briefly in phosphate-buffered saline (PBS). The secondary antibody was anti-mouse immunoglobulin G (IgG), coupled with horseradish peroxidase diluted in PBS, bovine serum albumin (1%), and human serum (1%). The bound IgG was revealed by incubation in the amino-ethyl-carbazole (Sigma, St. Louis, Missouri) solution. Nuclei were demonstrated using the bis-benzimide stain (Hoechst 33258, Sigma). Sections were examined with a Zeiss Axioplan microscope equipped with a high-resolution telecamera (Hamamatsu CCD, Shizuoka, Japan), using an x40 Planapo objective lens. Assessment of the different cell types was carried out by the same two observers, who were unaware of the outcome of the ultrasound follow-up studies.

For cell counting, we considered three standard areas (7 x 10³ µm²) of the plaque: the basal, shoulder, and cap regions. We evaluated the size of each cell population by assessing, on seriate sections, the number of cells (per area unit) positive to SM-E7, HAM56, and CD45RO. Moreover, the relative prevalence of each cell type was calculated as the percentage of positive cells to the total number of nuclei per area.

To ascertain the reproducibility of the assessment of the various cell types, three standard sites were selected in eight randomly chosen endarterectomy specimens: 1) proximal (cranially); 2) intermediate; and 3) distal (caudally). The length of specimens ranged from 0.5 to 2.6 cm. In the three sites, seriate cryosections were cut at 8-µm intervals and processed (immunocytochemistry) for SMCs, macrophages, and lymphocytes. Variability in cell counts obtained from the three sites was assessed for each cell type by the use of analysis of variance (ANOVA) for repeated measurements within subjects (i.e., specimens) and the coefficient of variation of the mean difference of repeated measurements. No difference between repeated assessments of SMCs (F = 2.482, p = 0.120), macrophages (F = 0.225, p = 0.801), and lymphocytes (F = 1.298, p = 0.304) was found by ANOVA. The coefficients of variation were 5.3% for SMCs, 4.8% for macrophages, and 3.4% for lymphocyte measurements.

Statistical analysis
Continuous variables were average, expressed as the mean value ± SD and compared by ANOVA with the Bonferroni correction. The prevalence of categorical variables was evaluated by two-way contingency tables, expressed as the percent rate, and compared with the Pearson chi-square test. The occurrence of a cerebrovascular event as a categorical variable was placed into a logistic regression analysis. The number of macrophages per area unit was also played into a multiple regression analysis without forcing any item into the equation, obtaining one multiple correlation coefficient (r). The SYSTAT package (SPSS Inc., Chicago, Illinois) was used, in which ANOVA automatically adjusts the significance level according to the number of variables (i.e., different genotypes) tested.

	Results

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Among the 68 patients, 45 were carriers of the CC genotype, 22 of the CT genotype, and one of the TT genotype of the LIPC promoter (Table 1). The occurrence of a previous ipsilateral transient ischemic attack or minor stroke was documented in 50 patients. Among them, 36 (72%) were carriers of the CC genotype, whereas only 14 (28%) were carriers of the CT genotype (p = 0.002). Among the 18 patients without evidence of previous events, nine were carriers of the CC, eight of the CT, and one of the TT genotype. Values of blood pressure and plasma lipids and the prevalence of risk factors for atherosclerosis were similar between the two genotype groups, but the LDL buoyancy (Rf) was significantly lower in the CC group than in the CT/TT group (Table 1). The distribution of the different CETP genotypes was similar between the CC and CT/TT genotype groups (Table 1), as well as in patients who had events compared with those who did not (data not shown).

The tissue retrieved at the endarterectomy procedure was suitable for the blind analysis of the cell composition in 45 cases: twenty-seven of these had the CC, and 18 had the CT genotype (Table 2). The CC genotype was associated with an increased number of macrophages (6.7 ± 3.5 vs. 2.1 ± 2.1 cells per area unit in the CT/TT group, p < 0.0005) and a reduced number of SMCs (6.9 ± 6.2 vs. 14.5 ± 6.4 in the CT/TT group, p < 0.0005) in the plaque (Table 2, Fig. 1). The same pattern of cell composition was present in the cap region of the plaque (Table 2). A strong, inverse relationship was found between LDL buoyancy and the number of macrophages in the plaque (r = –0.639, p < 0.0005). After adjusting for LDL density, the difference between the HL genotype groups in the number of macrophages in the plaque was no longer significant. Moreover, it turned out that in patients with previous cerebrovascular events, the inverse relationship between LDL buoyancy and macrophages was present in the CC but not in the CT/TT genotype carriers (Fig. 2). The amount of lymphocytes was similar between the two genotype groups (Table 2). Medial layers underlying the plaque (Fig. 1) were present in 50% of specimens. Those specimens from patients carrying the CC genotype showed a slightly higher number of SMCs, as compared with specimens from patients with the CT/TT genotype (Table 2).

View larger version (116K): [in this window] [in a new window]   Figure 1 Representative micrographs of carotid endarterectomy specimens form CC (A and C) and CT (B and D) genotype carriers. A prevalence of macrophages (stained with HAM56) was found in specimens obtained from the CC carriers, whereas smooth muscle cells (SMCs) (stained with SM-E7) were more numerous in specimens obtained from the CT carriers. The insets in B and C display, at high magnification, the staining of plaque areas for macrophages and SMCs, respectively. The number of cells per area unit was assessed by computerized image analysis (see Methods and ref. 10). m = tunica media; pl = atherosclerotic plaque. Bar = 80 µm. Magnification x20 Planapo objective.

View larger version (21K): [in this window] [in a new window]   Figure 2 Relationship between low-density lipoprotein (LDL) density and number of macrophages in the atherosclerotic plaque. The analysis was carried out in patients from the two genotype groups who had cerebrovascular events. n/au = cells/area unit.

	Discussion

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This study provides novel evidence that an association exists between the CC genotype of the LIPC promoter and an abundance of macrophages in atherosclerotic lesions. This association is likely due to the presence of denser, more atherogenic LDL in carriers of the CC genotype, who also showed an increased susceptibility to cerebrovascular events. Previous studies in patients with coronary artery disease receiving intensive cholesterol-lowering therapy showed that LDL density is inversely related to the progression of coronary artery lesions (15), but information on the LIPC genotype and the cell composition of atherosclerotic lesions was not available.

Hepatic lipase polymorphism and plaque composition.   The –514 CT polymorphism in the promoter region of the HL gene is related to high HL activity, small and dense LDL particles, and low HDL₂ cholesterol (7,16). Subjects with small, dense LDL particles are at higher risk of coronary artery disease than subjects with large, buoyant LDL (17,18). Potential mechanisms underlying this increased atherogenicity of small LDL particles include an increased rate of LDL oxidation (19). This leads to a higher tendency of oxidized LDL to become entrapped in the subintima (20), where they increase the expression of chemoattractants (21), inflammatory (22), and adhesion molecules (23) by endothelial and SMCs. Moreover, these lipoproteins induce endothelial dysfunction independent of HDL and LDL cholesterol and triglyceride concentrations (24) and are associated with increased thickness of the carotid intima-media in healthy men (25).

Hepatic lipase activity is a key determinant of LDL size, accounting for 30% to 45% of LDL size and density variability (12,26). In addition, serum triglyceride levels and CETP activity are other regulatory factors (27). Triglyceride levels, however, were similar between the two groups in our series, and this is likely to have blunted the potential role of CETP in determining LDL particle size. The CETP genotypes were not associated with either the number of plaque macrophages or cerebrovascular risk in these patients, as confirmed by the results of the multivariate analyses. Other risk factors were similar between the two LIPC genotype groups (Table 1) and were not significantly associated with the occurrence of events and the cellular composition of the lesions (Tables 3 and 4).

The abundance of macrophages and the relative scarcity of SMCs in the plaque and fibrous cap of patients with the CC genotype versus the CT/TT genotype (Table 2) represent a novel finding. Carriers of the CC genotype had significantly denser LDL particles than did patients with the CT/TT genotype, confirming previous observations (7,28) where HL gene polymorphism accounted for 20% to 32% of the variance in HL levels. In the CC carriers, LDL density was strongly associated with increased plaque macrophages (Fig. 2), providing a potential pathophysiologic explanation for the association between HL genotype and plaque cellular composition—namely, macrophage abundance (Table 3). Although our study does not provide direct evidence of a causative role of LDL density in determining plaque cellular composition, available evidence supports a pathophysiologic link between small, dense LDL and plaque cellular composition. In particular, dense LDL particles can more easily permeate the endothelium, reaching the subendothelial space (29), where they are more susceptible to oxidation, as demonstrated by in vitro studies (30). Oxidized LDL affects the structure of proteoglycans produced by SMCs, increasing their affinity for both native and oxidized LDL (20). In addition, oxidized LDL increases the expression of chemoattractants such as interleukin-8 (21), inflammatory molecules such as matrix metalloproteinase-1 (22), and adhesion molecules such as monocyte hemoattractant protein-1, by endothelial and SMCs (23). In agreement with these observations, in a subset of our specimens, in subjects with the CC genotype, we found significantly increased expression of acidic fibroblast growth factor, a macrophage-related factor that is induced by oxidized LDL as well (31) (unpublished data). By these mechanisms, small, dense, oxidized LDL may play a role in plaque cellular composition, matrix remodeling, plaque destabilization, and clinical events.

Accordingly, when LDL density was included in the multivariate analysis, the LIPC polymorphism was no longer a predictor of the number of macrophages in the plaque. Thus, the association between LIPC polymorphism and the number of macrophages appears to be, at least partly, accounted for by the effect of the former on LDL density.

Although total HDL cholesterol was not associated with the abundance of macrophages (Table 3) in the plaque, we cannot rule out a possible additional effect of HDL subpopulations on plaque composition, specifically HDL₂ particles, which may be clinically relevant, because the HL polymorphism also significantly affects HDL₂ cholesterol levels.

Cerebrovascular events and plaque composition
The number of macrophages in the plaque predicted cerebrovascular events in our study group (Table 4). It is worthwhile to note that the LIPC polymorphism remained associated with cerebrovascular events even after adding LDL density to the model. This finding suggests that HL, which is significantly controlled by LIPC polymorphism (7,9), affects both plaque composition (Table 3, model 6) and cerebrovascular events (Table 4, models 6 and 7) by additional mechanism(s). This hypothesis is supported by recent evidence of a role of HL in atherogenic lipoprotein metabolism, independent of its lipolytic activity (32).

Lymphocytes were equally distributed in the lesions from the two main genotype groups, and this may indicate that LIPC polymorphism exerts a selective action on macrophages. In the CC genotype carriers, a reduced number of SMCs was found in the atherosclerotic lesions. Instead, the SMCs were abundant in the media underlying the lesions from patients of this group. On the whole, this outlines a typical feature of carotid lesions found in CARS subjects (10), whose lipid-laden lesions with a predominant inflammatory component showed a well-preserved tunica media, as compared with subjects whose lesions were mainly composed of SMCs and whose media was relatively poor of cells. This is probably part of a complex process that includes the activation of adventitial fibroblasts to myofibroblasts and subsequent translocation of these cells to the media, perhaps to replace medial SMCs that have migrated to form the intimal lesion (33). Unfortunately, information on parallel changes in cell composition of the adventitial layer was not available under these experimental conditions.

Another relative limitation of this study is related to the sample size of our study group. The size appears to be appropriate for the detailed investigation needed to define the pathophysiologic mechanism(s) accounting for genetic modulation of unstable carotid plaque. However, it may not be suitable for a compelling study of genetic association.

Conclusions and clinical implications
We have provided some novel pathophysiologic steps of a complex mechanism linking LIPC polymorphism, carotid plaque composition, and cerebrovascular events in patients with severe carotid artery stenosis. These findings may have potentially relevant clinical implications by disclosing the value of future, population-based studies of genetic association, which are needed to confirm the relevance of LIPC polymorphism as a marker to detect subjects at high risk of cerebrovascular events who are potential candidates for intensive primary care.

	Acknowledgments

 
The technical assistance of Daniela Vianello, BSc, was greatly appreciated.

	Footnotes

 
This work was supported by MURST project no. 9905157119, the Fondazione Cassa di Risparmio of Padova, the Biomedical Association for Vascular Research (Padova, Italy), and the National Institutes of Health grant no. HL64322 to Dr. Deeb.

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