Elevated serum levels of proinflammatory cytokines and biomarkers of matrix remodeling in never-treated patients with familial hypercholesterolemia

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Elevated serum levels of proinflammatory cytokines and biomarkers of matrix remodeling in never-treated patients with

familial hypercholesterolemia

Mariame El Messal

^a

, Jean-Louis Beaudeux

^b,c,

*, Anas Drissi

^d

, Philippe Giral

^c,e

, Rachid Chater

^a,d

, Eric Bruckert

^c,e

, Ahmed Adlouni

^d

, M. John Chapman

^c

aFaculte´ des Sciences Aı¨n Chock, Universite´ Hassan II Aı¨n Chock, Casablanca, Morocco

bFe´de´ration de Biochimie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 47-83 boulevard de l’hoˆpital, F75651 Paris Cedex 13, France

cDyslipidemia and Atherosclerosis Research Unit, National Institute for Health and Medical Research (INSERM), Unit 551, Hoˆpital Pitie´-Salpeˆtrie`re, 75651 Paris Cedex 13, France

dFaculte´ des Sciences Ben M’Sik, Universite´ HassanII-Mohammedia, Casablanca, Morocco

eService d’Endocrinologie-Me´tabolisme, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 47-83 boulevard de l’hoˆpital, F75651 Paris Cedex 13, France Received 14 September 2005; received in revised form 30 September 2005; accepted 30 September 2005

Available online 8 November 2005

Abstract

Background: Familial hypercholesterolemia (FH) is a common inherited disorder of lipoprotein metabolism, whose origin involves mutations in the gene coding for the low-density lipoprotein receptor protein. Although FH is monogenic, wide variation occurs in the onset and severity of atherosclerosis in these patients.

Methods: Since data on levels of inflammatory proteins and/or active factors in FH patients who have never received lipid-lowering treatment are lacking, serum levels of MMP-3, active MMP-9 and TIMP-1 as well as pro-inflammatory cytokines (TNF-a, IL-18) were determined in never-treated homozygous FH Moroccan patients (n= 4) and compared to those of heterozygous FH subjects (n= 7) and of healthy control subjects (n= 5).

Results: When compared to controls, homozygous FH patients exhibited levels of active MMP-9 and TIMP-1 (p< 0.05), and of both high sensitive-CRP and IL-18 which were significantly elevated (p< 0.05 andp< 0.01, respectively). In heterozygous FH patients, intermediate values between FH homozygotes and healthy controls were observed for these markers, with the exception of MMP-9 activity whose levels were significantly elevated (p< 0.05). Multivariate analysis revealed a positive correlation between apolipoprotein B, TIMP-1 and IL-18 levels, and between hs-CRP and IL-18 (p< 0.01).

Conclusions: Although the sample size of this FH group was limited, our data suggest that nontreated homozygous FH patients, and to a lesser degree heterozygous FH patients, exhibit not only a markedly proinflammatory vascular state but also pronounced extracellular matrix remodeling, as reflected by elevated circulating levels of inflammatory cytokines and MMPs.

Keywords:Atherosclerosis; Inflammation; Metalloproteinase; Familial hypercholesterolemia; Matrix remodeling

1. Introduction

Familial hypercholesterolemia (FH) is a common autosomal dominantly inherited disorder of lipid/cholesterol metabolism whose origins involve either mutations in the low-density lipoprotein (LDL) receptor gene or in the gene encoding apolipoprotein B or the in PCSK9 gene[1 – 4]. As a result of the disruption of LDL receptor function, patients

doi:10.1016/j.cca.2005.09.027

Abbreviations:FH, familial hypercholesterolemia; MMP, matrix metalloproteinase; TIMP, tissue inhibitor metalloproteinase; TNF-a, tumor necrosis factor; IL, interleukine; Hs-CRP, high sensitive C-reactive protein;

CAD, coronary artery disease.

* Corresponding author. Fe´de´ration de Biochimie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 47-83 boulevard de l’hoˆpital, F75651 Paris Cedex 13, France. Tel.: +33 1 42 16 21 87; fax: +33 1 42 16 21 75.

E-mail address:jean-louis.beaudeux@psl.aphp.fr (J.-L. Beaudeux).

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with FH display markedly elevated circulating levels of LDL cholesterol from birth, ranging from 7 to 15 mmol/l in heterozygous patients to up to move than 25 mmol/l in the homozygous state. As high levels of LDL-cholesterol are a major risk factor for cardiovascular disease (CAD), FH subjects exhibit a significantly increased risk of premature CAD, due to the formation of atherosclerotic lesions in childhood[5,6].

Atherosclerosis is recognized as a chronic inflammatory disease of the arterial wall in which an inflammatory response is a key event that leads to the formation of atheromatous lesions[7]. Fragilisation of the fibrous cap of atherosclerotic plaques is intimately related to the presence of an inflammatory state which plays a crucial role in plaque rupture, the major pathophysiological event underlying acute coronary syndromes[8 – 10]. Expression of a spectrum of inflammatory factors, such as C-reactive protein (CRP) and proinflammatory cytokines including TNF-aand IL-18, constitutes a strong link between inflammation and atherogenesis in coronary artery disease [11]. Proinflammatory cytokines are responsible for vascular endothelial dysfunc- tion and activation, leading for example to the overexpression of endothelial adhesion molecules [12], to enhanced uptake of oxidized LDL through increased expression of scavenger receptors in monocyte-derived macrophages [13], to plaque fragilisation [14] and to induction of cell proliferation in the arterial wall[15]. The matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) constitute an additional group of biomarkers and of actors in the inflammatory process, and play a central role in subendo- thelial vascular extracellular matrix remodeling [16 – 18].

Experimental data have established that MMPs and TIMPs are involved in the development of atherosclerotic lesions and their fragilisation [16 – 19]; moreover, we recently observed that asymptomatic hyperlipidemic subjects at high cardiovascular risk display elevated circulating levels of MMP-3, MMP-9 and TIMP-1[20].

There is a paucity of information on the inflammatory state in untreated FH subjects, and more specifically on circulating levels of cytokines, MMPs and TIMPs [21,22].

However, although the cause of FH is monogenic, wide variation exists in the onset and severity of atherosclerotic disease in these patients [23]. We hypothesized that comprehensive evaluation of the inflammatory state of never-treated FH patients might provide not only further insight into variation in disease severity but might also improve cardiovascular risk assessment. We therefore determined serum levels of several inflammatory markers, i.e. high sensitive CRP (hs-CRP), IL-18 and tumor necrosis factor (TNF)-a, and of matrix remodeling factors, i.e.

MMP-3, MMP-9 and TIMP-1, in homozygous FH patients who had never received either lipid-lowering or anti- inflammatory treatment. We equally evaluated the potential relationships between these inflammatory molecules and the plasma lipid profile.

2. Materials and methods 2.1. Patients

Three groups of Moroccan subjects were studied: 4 homozygous FH subjects (M/F ratio: 3/1; age: 16.2T8.0 years, meanTSD); 7 heterozygous FH subjects (M/F ratio:

5/2; age: 42.5T22.6 years), and 5 healthy control subjects (M/F ratio: 3/2; age: 24.2T7.2 years). The diagnosis of FH was established using Simon Broome’s criteria [24] and confirmed by the presence of LDL receptor gene mutation.

The control subjects and FH patients are issued from 3 unrelated families with distinct LDL receptor gene mutations. All patients included in the study had never been treated for hypercholesterolemia. Exclusion criteria for both groups were: hypothyroidism, renal deficiency, hepatic disease, diabetes mellitus, arthritis, cancer and treatment with anti-inflammatory drugs. All patients gave their informed consent prior to inclusion in the study. The mean body mass indices were 22.7T7.4, 23.4T5.9 and 22.1T7.2 kg/m² in homozygous, heterozygous and control groups, respectively. CAD had been documented by ECG and cardiac scan in 1 homozygous and 1 heterozygous FH patient. Control individuals had no past history or evidence of CAD, or hypertension.

2.2. Analysis of circulating lipids, lipoproteins and inflammatory markers

Venous blood samples were collected by venipuncture after a 12-h overnight fast. Serum total cholesterol (TC) and triglyceride levels (TG) were measured by routine enzy- matic methods (Boehringer Mannheim, FRG). Serum HDL cholesterol (HDL-C) was enzymatically determined by separating HDL from plasma by precipitation of the (LDL + VLDL) fraction with a phosphotungstic acid – magnesium chloride solution (Boehringer Mannheim, FRG). Serum LDL-C was calculated according to the Friedewald formula [25]. Apolipoproteins AI and B, lipoprotein (a) [Lp(a)], and hs-CRP levels were measured using immunonephelometric methods on a BNII analyzer (Dade-Behring, France) using the manufacturer’s reagents.

Detection limit for hs-CRP was 0.2 mg/l; intra- and interassay CV were < 5% [20].

Serum levels of MMP-3, active MMP-9 and TIMP-1 proteins were determined using commercially available enzyme-linked immunoassays (R&D Systems, France).

Intra-assay CVs were < 7%; between assay CVs were

< 9%. Sensitivities were 0.009, 0.08, and 0.005 ng protein/

ml for MMP-3, TIMP-1 and active MMP-9, respectively.

Serum TNF-aand IL-18 concentrations were measured using commercially available enzyme-linked immunoassays (Bender Med Systems GmbH and R&D Systems, France, respectively). Intra-assay CVs were < 10%; between assay CVs were < 15% (< 10% for IL-18). Detection limits were 5.8 and 12.5 pg protein/ml for TNF-aand IL-18, respectively.

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2.3. Statistical analysis

Statistical analysis was performed with JMP 5.0 software (SAS, Cary, NY). All values are shown as the meanTSD. A p value of < 0.05 was considered statistically significant.

The ANOVA test was used for comparison between groups.

The Dunnett test was used for comparison between controls and heterozygous patients, and between controls and homozygous patients.

3. Results

Serum lipid and lipoprotein levels are summarized in Table 1. As expected, TC and TG were significantly elevated in both homozygous and heterozygous FH patients as compared to non-FH controls of the same ethnicity, but to a greater extent in the homozygous than in the heterozygous FH patient group (p< 0.001 and p< 0.05 for TC and TG, respectively). Levels of HDL-C were markedly decreased In homozygous patients (p< 0.05), while LDL-C and Apo B levels were markedly increased (p< 0.001). Finally, Lp(a) levels did not differ between the three groups. Furthermore, levels of active MMP-9 and TIMP-1 were significantly elevated in homozygous FH patients; elevation of MMP-3 levels was observed in homozygous FH patients as compared to controls, but was not significant. As shown inTable 2, levels of hs-CRP and IL-18, but not of TNF-a, were significantly higher in the homozygous FH group relative to the control group (p< 0.05 and p< 0.001, respectively).

Comparison of homozygous and heterozygous FH groups with controls revealed that heterozygous FH patients exhibited mean levels of TIMP-1, hs-CRP, IL-18 and TNF- a which were intermediate between those of homozygous FH subjects and controls, with the exception of active MMP-9 which was significantly elevated when compared to the two other groups (p< 0.05). Finally, multivariate analysis revealed that hs-CRP levels positively correlated with TC, Apo B and IL-18 (pairwise correlations) in

homozygous FH patients. A positive correlation was also observed between Apo B, IL-18 and TIMP-1 (p< 0.01).

4. Discussion

In the present study, we demonstrate for the first time that never-treated homozygous FH patients exhibited elevated circulating levels of the proinflammatory cytokine IL-18, the inflammatory marker and factor hs-CRP, and the matrix remodeling mediators MMP-9 and TIMP-1, as compared to normolipidemic control subjects of the same ethnicity.

Atherosclerosis is characterized by chronic inflammation of the arterial wall at lesion sites, as well as by activation of vascular endothelial cells, smooth muscle cells and resident macrophages. Activation of nuclear transcription factors such as NF-nB results in the production of several proinflammatory mediators by macrophages [26] that in turn activate hepatic expression and systemic release of inflammatory acute phase proteins such as hs-CRP. Periph- eral blood levels of CRP and of proinflammatory cytokines which are elevated in hypercholesterolemia have therefore been used to predict cardiovascular disease risk [27 – 29].

Our results confirm that severe hypercholesterolemia, as occurs in homozygous FH, is associated with fivefold increase in hs-CRP levels and thus with a chronic inflammatory state. We also showed that never-treated heterozygous patients exhibited twofold higher hs-CRP levels than controls. Therefore, as originally proposed [30,31], hs-CRP may serve to evaluate the severity of the inflammatory state involved in atherosclerotic disease in FH patients. Equally, circulating levels of the proinflammatory cytokine IL-18 (> 2-fold) were also significantly elevated in homozygous FH patients. Experimental studies have shown that mononuclear cells from FH patients produce proinflammatory cytokines to a greater extent than control cells

Table 1

Plasma lipid, lipoprotein and apolipoprotein levels in FH patients and normolipidemic subjects

Control (n= 5)

HMZ (n= 4)

HTZ (n= 7)

ANOVA test CT (mg/dl) 139T019 713T120*** 302T99*** 0.0001

TG (mg/dl) 54T21 133T70* 91T18 0.02

HDL-C (mg/dl) 42T5 21T7* 37T9 0.01

LDL-C (mg/dl) 89T17 665T117*** 246T98 0.0001

Apo AI (mg/dl) 132T41 80T24 112T21 0.17

Apo B (mg/dl) 69T23 353T53*** 147T47*** 0.0001

Lp(a) (mg/dl) 17T11 26T2 27T10 0.27

Control: normolipidemic, HTZ: heterozygous FH patients, HMZ: homozygous FH patients.

HDL-C: HDL cholesterol, LDL-C: LDL cholesterol.

ANOVA test. *p< 0.05, ***p< 0.001 vs. control group (Dunnett test).

Table 2

Plasma levels of inflammatory markers and matrix remodeling factors in FH patients and normolipidemic subjects

Control (n= 5)

HMZ (n= 4)

HTZ (n= 7)

ANOVA test CRPus

(mg/l)

1.02T0.82 5.67T2.96* 2.27T1.73 0.01 IL-18

(pg/ml)

315T128 792T235** 459T118 0.001 TNF-a

(pg/ml)

2.42T0.76 3.06T1.09 2.79T1.40 0.71 MMP-3

(ng/ml)

9.38T4.87 16.23T3.23 15.81T9.17 0.25 MMP-9

activity (ng/ml)

167T86 266T91* 529T236* 0.01

TIMP-1 (ng/ml)

109T24 197T80* 138T31 0.04

Control: normolipidemic, HTZ: heterozygous patients, HMZ: homozygous patients.

*p< 0.05, **p< 0.01 vs. control group (Dunnett test).

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[32]. Therefore elevated circulating levels of IL-18 probably reflect overexpression of this cytokine in macrophages of atherosclerotic tissue.

The substantive interest in MMPs, initially identified with respect to their matrix-degrading capacity during tissue invasion by transformed cells, was prompted by the observation that cell movement, as well as structural tissue rearrangement, requires degradation of the matrix scaffold.

Numerous in vitro and in situ reports indicate that vascular and inflammatory cells produce MMPs, but that the spectrum of such enzymes secreted basally or in response to stimuli is rather distinct [19]. As indicated above for circulating IL-18 levels, it is presumed that alteration of circulating metalloproteinase levels in atherosclerosis may reflect metalloproteinase activity within the arterial wall, and could therefore provide information on the remodeling activity within atheromatous lesions [20,33]. The present data showing significantly elevated active MMP-9 and TIMP-1 levels (and of MMP-3, although not significant) in never-treated homozygous patients are consistent with our earlier report [20], in which we documented significant elevation of serum levels of MMP-3, MMP-9 and TIMP-1 in asymptomatic hyperlipidemic subjects at high cardiovascular risk. This latter study equally revealed that MMP-3 and TIMP-1 are strongly positively associated with the presence of carotid lesions suggesting that these factors might be used as pertinent biological markers of atherogenesis in FH patients. It has been reported that peripheral blood levels of MMP-9 in patients with CAD, and especially in acute coronary syndromes, are significantly elevated as compared to control subjects [34]. Therefore, our data appear relevant to the prediction of the occurrence of cardiovascular events in homozygous FH patients although a study including a larger cohort of patients will be required to confirm the present results. Recently, MMP-3 levels were found to be elevated in FH heterozygous patients during lipid-lowering therapy, while MMP-9 and TIMP-1 did not change [22]. It is noteworthy that our FH patients (both homozygous and heterozygous) were free of any pharmacological treatment, and thus the changes we observed in systemic biological markers were not related to lipid-lowering nor to anti-inflammatory drug treatment or to LDL-apheresis.

Finally, our heterozygous patients exhibited significantly higher active MMP-9 levels as compared to the homozygous group. This unexpected finding could be pathophysio- logically relevant, as TIMP-1 levels were not increased to the same level in these patients, thereby suggesting marked disequilibrium between MMP and TIMP activity. We can therefore hypothesize that the resulting metalloproteinase activity might be greater in heterozygous than in homozygous never-treated patients. Additionally, this hypothesis could be supported by the large variance of MMP-9 activity values than a single uncorrected high value causing an unexpected elevated value of this parameter in the FH homozygous group. A further study, including the determi-

nation of MMP and TIMP activity within atheromatous tissue, is required to confirm this hypothesis.

Despite the limitation of the present study due to the small number of patients studied, originality of this investigation was to explore inflammatory and matrix remodeling biomarkers in a unique model of nontreated chronic hypercholesterolemia. It should be emphasized that homozygous FH patients and especially those free of any hypolipidemic treatment, are very rare. Moreover, although only a few subjects were included, differences in systemic values of inflammatory markers and MMP/TIMP were statistically significant, thus indicating the strength of our conclusions. It is equally relevant that mean age differed between groups and was much higher in the heterozygous group than in the homozygous group and in controls. However, this component did not skew our conclusions since homozygous patients exhibited values of inflammatory and matrix remodeling systemic markers which were elevated in relation to those in heterozygous subjects and controls in spite of a lower age, thus in agreement with the constantly observed premature atherosclerosis in FH subjects.

In summary, this study has demonstrated that both never- treated homozygous and heterozygous FH patients display elevated risk of coronary heart diseases which may progress to clinical and vascular lesions in direct relation to levels of MMP and cytokine expression. Further research is war- ranted in order to elucidate the pathophysiological link between proinflammatory/anti-inflammatory cytokine bal- ance and circulating MMPs and TIMPs in untreated FH patients.

Acknowledgements

This work was supported by the Coope´ration Maroco- Franc¸aise en Recherche Me´dicale (Accord CNCPRST- INSERM), by INSERM and Assistance Publique-Hoˆpitaux de Paris (contrat Interface).

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