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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:354-364

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Articles

Lipoprotein Lipase Correlates Positively and Hepatic Lipase Inversely With Calcific Atherosclerosis in Homozygous Familial Hypercholesterolemia

Klaus A. Dugi; Irwin M. Feuerstein; Suvimol Hill; Joanna Shih; Silvia Santamarina-Fojo; H. Bryan Brewer Jr; Jeffrey M. Hoeg

the Department of Radiology (I.M.F., S.H.) of the Warren G. Magnuson Clinical Center of the National Institutes of Health and the Molecular Disease Branch (K.A.D., S.S.-F., H.B.B., J.M.H.) and the Office of Biostatistics Research (J.S.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.

Correspondence to Jeffrey M. Hoeg, Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bldg 10, Rm 7N115, 10 Center Dr, MSC 1666, Bethesda, MD 20892-1666. E-mail jeff@mdb.nhlbi.nih.gov.


*    Abstract
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Homozygous familial hypercholesterolemia (FH) is a rare genetic disorder that leads to premature atherosclerosis due to a defective LDL receptor. There is, however, a large degree of phenotypic heterogeneity at the level of atherosclerosis even in patients with identical mutations of the LDL receptor protein. Lipoprotein lipase (LPL) and hepatic lipase (HL) are crucial enzymes in lipoprotein metabolism, and both have been proposed as having proatherogenic as well as antiatherogenic effects. To evaluate a potential role for these enzymes in the severity of atherosclerosis, we correlated postheparin LPL mass and activity as well as HL activity with the volume of total calcific atherosclerosis (heart and thoracic aorta), coronary artery calcific atherosclerosis, and Achilles tendon width as measured by computed tomography in 15 FH homozygotes. LPL dimer and total mass were positively correlated with all three parameters (r=.65 to .87, P<.01) as was LPL activity (r=.52 to .63, P<.05). HL activity was negatively correlated with total and coronary artery calcified lesion volume (r=-.55 to .57, P<.05). In a multiple regression model of the coronary artery lesion volume, LPL dimer mass and HL activity together accounted for 84% of the variability (r=.92, P<.0001). In a multiple regression model of the total calcified lesion volume, HL activity, total cholesterol, age, and LPL dimer mass together accounted for 85% of the variability (r=.92, P=.0005). These data demonstrate a significant correlation of LPL mass and activity with the extent of calcific atherosclerosis in homozygous FH. It is not clear whether LPL is the cause or consequence of the observed correlation, but if the association between LPL and coronary artery lesions is also present in patients with other genetic dyslipoproteinemias, LPL could constitute a new risk factor for cardiovascular disease.


Key Words: atherosclerosis • coronary disease • macrophages • computed tomography x-ray • xanthomatosis


*    Introduction
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 *Introduction
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The genetic disease FH is caused by the absence of functional LDL receptors. While the heterozygous form of FH is fairly common, with a frequency of about 1:500, homozygous FH is extremely rare and estimated to occur in about 1 of 1 million US births. Homozygous FH is characterized by marked elevations of LDL-C, cutaneous as well as tendon xanthomas, arcus corneae, and generalized atherosclerosis developing during childhood. If untreated, death from myocardial infarction or sudden cardiac death typically occurs before age 30.1

Despite its rarity, homozygous FH has been a paradigm of human lipoprotein metabolism. This condition established the role of LDLs in human cardiovascular disease, the first known genetic basis for atherosclerosis, and played a key role in developing new therapeutic approaches for the prevention of atherosclerosis. In addition, FH represents an example of the polygenic nature of certain disease processes, such as atherosclerosis.2 There is a large degree of heterogeneity in the phenotypic expression of homozygous FH. Part of this heterogeneity may be due to distinct mutations in the LDL receptor gene that lead to different LDL receptor activities and LDL plasma levels.1 3 The protective influence of female gender, which is seen in normal and FH heterozygote probands, seems to be diminished4 or even absent5 in homozygous FH. Nonetheless, there is a large variability in the clinical course even in patients with identical mutations in the LDL receptor gene. For instance, one of two related FH patients with the same French Canadian mutation died of cardiovascular disease at the age of three years, while the other did not develop symptoms until the age of 19.6

It is not known whether the lipolytic enzymes LPL and HL influence the clinical course of homozygous FH. LPL is a crucial enzyme in lipid metabolism, hydrolyzing TGs in chylomicrons and VLDLs.7 LPL has been ascribed both proatherogenic and antiatherogenic roles8 ; its potential antiatherogenic role has been derived from several studies. LPL activity is positively correlated with HDL-C levels9 10 and increases the clearance of atherogenic TG-rich lipoproteins.11 Transgenic mice overexpressing LPL exhibit a reduction in VLDLs12 13 14 due to enhanced clearance12 and increased HDL2-C12 and blunted hypertriglyceridemia and hypercholesterolemia when fed diets high in carbohydrates or cholesterol.12 13 14 The long-term administration of compound NO-1886, which increases LPL activity, leads to elevated levels of HDL-C and an inhibition of CA atherosclerosis in rats.15

The notion that LPL may be proatherogenic16 17 is supported by the fact that LPL is expressed in atherosclerotic lesions,18 19 increases monocyte adhesion to aortic endothelial cells,20 traps lipoproteins in the subendothelial matrix,21 22 23 24 and mediates the formation of foam cells25 26 27 28 29 and cholesterol ester accumulation in smooth muscle cells.30

Important functions of HL in lipid metabolism include the hydrolysis of phospholipids and TGs in IDLs and HDLs.31 The potential proatherogenic role of HL stems from the fact that premenopausal women are partly protected from heart disease by having higher HDL levels than men and that these elevated HDL levels are due to a lower HL activity.32 In addition, increased HL activity is associated with the more atherogenic LDL subclass pattern B.33 Conversely, the presence of premature cardiovascular disease in at least some HL-deficient patients34 35 36 suggests an antiatherogenic role of HL. Moreover, overexpression of HL in mice led to a 42% reduction of aortic cholesterol compared with control mice.37

To ascertain whether LPL or HL could play a role in the phenotypic heterogeneity of homozygous FH, we measured LPL mass and activity as well as HL activity in the preheparin and postheparin plasma of 15 homozygous FH patients. The values were correlated with calcific atherosclerosis as measured by EBT. In this article we describe the significant positive correlation of LPL and the significant negative correlation of HL with the extent of calcific atherosclerosis in homozygous FH that indicate a potential role of these lipolytic enzymes in the phenotypic heterogeneity of FH.


*    Methods
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Patients
Fifteen consecutive homozygous FH patients were studied at the Clinical Center of the National Institutes of Health. These patients were originally diagnosed as having homozygous FH based on their plasma lipoprotein concentrations, family history, and the presence of tuberous and tendinous xanthomata. In the meantime, the diagnosis has been confirmed in 8 of the 15 patients by quantification of LDL receptor activity.3 All patients studied so far (patients 5 through 7, 9, 11, and 13 through 15) have LDL receptor activities <30% of normal, the value that differentiates between homozygous and heterozygous FH as defined by Goldstein et al.38 In addition, the presence of the apoB3500 mutation was ruled out in all patients by using a polymerase chain reaction–based analysis. Untreated lipoprotein concentrations and EBT results have been reported in some of these patients.39 Seven male and eight female patients were evaluated (average age at the time of the EBT, 26.4±12.6 years; range, 3 to 53 years). All patients received 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, and most were given bile acid–binding resins and probucol. None of the patients received fibrates. Six patients underwent biweekly LDL apheresis or plasma exchange and had not received treatment within 2 weeks of evaluation. One patient had undergone liver transplantation. The study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute. Each patient (or, for the minor patients, the legal guardian) gave informed consent for these studies.

EBT and Conventional CT
CT of the Achilles tendon was performed on a GE 9800 scanner (General Electric). Patients were scanned supine with their feet in the neutral position. The displayed field of view was 0.25 m. Contiguous 10-mm-thick transaxial sections were obtained through the Achilles tendons from the level of midcalcaneus until they merged into the gastrocnemius muscles. Tendon width was defined as the greatest distance between the most medial and lateral extent of the tendon. The values for Achilles tendon width in this study represent the means of both measured feet.

EBT was performed as described.39 In brief, EBT of the heart and thoracic aorta was performed by using an Imatron C-100XL CT scanner (Imatron Co) without intravenous contrast material. The calcified volume score, which represents the volume of calcified tissue in cubic millimeters, was calculated by multiplying the calcified area of a lesion by slice thickness in millimeters. The CA volume is the sum of calcified tissues of the left main, left anterior descending, left circumflex, right, and posterior descending CAs. The aortic root was defined as the section of ascending aorta within 20 mm of either coronary ostium. Lesions were defined as ostial if the calcified plaque was in direct contact with the coronary ostium. "Total" volume represents the sum of volumes from the CAs and the entire aorta.

Plasma Lipids and Lipoproteins
The fasting plasma concentrations of TGs, TC, LDL-C, and HDL-C were determined by using enzymatic assays.40 Measurements were obtained at the initial diagnosis and at the time of the lipase studies. Since some of the initial LDL levels were obtained from outside our laboratory, all LDL levels at diagnosis are reported as calculated by the Friedewald formula. All subsequent LDL measurements in our laboratory were directly quantified by using ultracentrifugation.40 Plasma for the determination of Lp(a) levels at diagnosis was not available from all patients. Lp(a) plasma concentrations were quantified by using a noncommercial sandwich enzyme-linked immunosorbent assay with a monoclonal anti-apo(a) antibody for capture and a polyclonal anti-apoB antibody for detection.41

Measurement of LPL Mass and LPL and HL Activities
After patients had fasted overnight, venous blood samples were drawn into 10-mL EDTA-containing tubes before and 10 minutes after a bolus injection of heparin (60 IU IV). The samples were immediately chilled to 4°C, centrifuged, divided into aliquots, flash-frozen on dry ice, and stored at -70°C until assayed. Preheparin and postheparin LPL and HL activities were quantified by using a triolein-phosphatidylcholine emulsion.42 Selective measurement of HL was based on the inactivation of LPL by 1.0 mol/L NaCl. LPL activity was calculated as the difference between total postheparin lipase activity and selective HL activity. The samples were quantified in quadruplicate, and postheparin plasma from pooled normal control subjects as well as an LPL-deficient patient were used to correct for interassay variation. Lipase activity is expressed as nanomoles of free fatty acids released per milliliter per minute. Total postheparin LPL mass was quantified six times by using an enzyme-linked immunosorbent assay with the 5D2 monoclonal antibody for capture and a polyclonal LPL antibody from chicken (kindly donated by Dr I.J. Goldberg, Columbia University, New York, NY) for measurement.43 This assay quantifies total LPL mass, including LPL dimers and monomers. To confirm our results and to enable comparison of the values of the FH homozygous patients with a large collective of control subjects, we also measured the LPL mass with a sandwich enzyme-linked immunosorbent assay using the 5D2 monoclonal antibody.44 Because the same epitope must be present twice for detection, this assay preferentially measures the LPL dimer.45 The LPL dimer mass, which represents incremental mass, is determined by subtracting the preheparin immunoreactivity from the postheparin mass. In patients undergoing LDL apheresis or plasma exchange, the heparin study was performed 2 weeks after the last treatment. The 30 male and 40 female normal control subjects were the first 70 volunteers responding to an advertisement.

Statistical Analysis
Statistical analyses were performed by using SPSS for Windows, release 5.0 (SPSS Inc). For linear statistical analysis the values of the calcified lesion volume were transformed by taking the cube root. Two-tailed bivariate correlations were determined by the Pearson coefficient. Parameters with a correlation coefficient >=.35 were entered into a multiple regression model by using stepwise multiple regression analysis. Comparisons between FH homozygotes and normal control subjects were performed by using an unpaired Student's t test for independent variables and according to Levene's test for equality of variances.


*    Results
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 *Results
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Table 1Down lists the age, gender, TC, LDL-C, HDL-C, Lp(a), and TG plasma concentrations of the 15 FH homozygotes. As expected, their TC (range, 12.7 to 33.2 mmol/L [488 to 1277 mg/dL]; mean, 20.6±6.0 mmol/L [792±232 mg/dL]) and LDL-C (range, 11.6 to 29.9 mmol/L [447 to 1153 mg/dL]; mean, 17.7±5.7 mmol/L [683±220 mg/dL]) untreated levels were grossly elevated compared with those of normal control subjects. HDL-C concentrations (range, 0.44 to 1.45 mmol/L [17 to 56 mg/dL]; mean, 0.83±0.23 mmol/L [32±9 mg/dL]) were lower than in control subjects. With treatment, the TC (mean, 11.3±4.4 mmol/L [434±168 mg/dL]) and LDL-C (mean, 9.5±4.5 mmol/L [366±173 mg/dL]) levels were greatly reduced but were still significantly above normal. Lp(a) plasma concentrations (range, 0 to 3.4 mmol/L [0 to 132 mg/dL]; mean, 1.1±1.1 mmol/L [42±44 mg/dL]) were elevated compared with those of control subjects, whereas the fasting TG concentrations (range, 0.61 to 2.2 mmol/L [54 to 198 mg/dL]; mean, 1.2±0.4 mmol/L [103±39 mg/dL]) were in the upper range of normal.


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  		Table 1. Lipoprotein Profiles of Patients Homozygous for FH

Table 2Down outlines the age, calcified lesion volume as measured by EBT, and Achilles tendon thickness as determined by conventional CT for each patient. The calcified lesion volume is listed separately for the CAs, aortic root, coronary ostia, and total volume, which is the sum of lesion volumes from the CAs and the entire thoracic aorta. Calcified lesions were detectable in all but the two youngest patients. The grossly elevated calcified lesion scores compared with those of control subjects <40 years of age39 illustrate the extreme acceleration of atherosclerosis in homozygous FH subjects. The mean lesion volume in the aortic root was almost twice that in the CAs. These data are in agreement with earlier observations demonstrating that the atherosclerosis in homozygous FH is unusually prevalent in the aortic root.4 46 With the exception of the 3-year-old boy (patient 1), all patients had an Achilles tendon width >3 SDs higher than that of normal control subjects.47


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  		Table 2. Calcified Lesion Volume and Tendon Thickness of Patients Homozygous for FH

Table 3Down lists the postheparin plasma values for LPL dimer and total mass as well as postheparin LPL and HL activities for the 15 FH homozygotes and mean±SD values for 70 normal volunteers seen at the University of Washington, Seattle. LPL dimer mass was quantified to confirm the total LPL mass values and correlations and to allow for comparison with a large collective of control subjects. Postheparin LPL dimer mass (456±351 versus 236±136 µg/L, P<.05) and LPL activity (245±74 versus 182±72 nmol·mL-1·min-1, P<.01) were significantly higher in FH homozygotes than in the control subjects, whereas postheparin HL activity was within the normal range when analyzed for all patients together (185±75 versus 210±98 nmol·mL-1·min-1, P=.37) or each gender separately (men, 215±67 versus 265±87 nmol·mL-1·min-1, P=.17; women, 160±76 versus 171±87 nmol·mL-1·min-1, P=.74). Like LPL dimer mass, total LPL mass was higher in plasma from FH homozygotes than in postheparin plasma pooled from eight normal control subjects (761±198 versus 395 µg/L).


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  		Table 3. Lipase Values of Patients Homozygous for FH

Fig 1Down shows the Pearson correlation coefficients of various study parameters with the calcified lesion volume of the 15 FH homozygous patients. Postheparin LPL dimer (r=.73, P<.01) and total mass (r=.65, P<.01) had a higher positive correlation coefficient than did TC or LDL-C at diagnosis (Fig 1ADown). Postheparin HL activity was negatively correlated with total calcified lesion volume (r=-.55, P<.05). Fig 1BDown shows the correlation of study parameters with the extent of calcific atherosclerosis in the CAs. LPL dimer mass showed the highest correlation with the CA lesion volume (r=.87, P<.01). Interestingly, TC and LDL-C were not significantly correlated with CA lesions. Only age, TC, and LPL mass reached significance when correlated with calcified lesions in the aortic root (Fig 1CDown). Fig 1DDown depicts the correlation of study parameters with tendon thickness as determined by CT. Age was very highly correlated with tendon width (r=.80, P<.001), whereas TC, LDL-C, and HL activity were not. Surprisingly, LPL mass (r=.68, P<.01) and activity (r=.58, P<.05) were correlated even with lipid deposition in this poorly vascularized tissue. Fig 1Down illustrates that HDL-C plasma concentrations were not correlated with calcified atherosclerosis in FH homozygotes. Similarly, fasting TG concentrations and Lp(a) were not correlated with lesion volume or other study parameters (data not shown). In contrast to postheparin lipase mass and activity, preheparin LPL immunoreactivity did not show any significant correlations with the extent of calcific atherosclerosis or other study parameters (data not shown). In addition, the extent of ostial atherosclerosis was not significantly correlated with any other variables.



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  		Figure 1. Bar graphs show correlations of various study parameters with the extent of calcific atherosclerosis in 15 FH homozygotes. Age indicates age at time of CT; LDL and HDL indicate LDL-C and HDL-C, respectively. Correlations are based on postheparin lipase values and off-treatment cholesterol values. Pearson correlation coefficients are given for (A) total calcific lesion volume as determined by EBT, (B) CA calcified lesion volume as determined by EBT, (C) aortic root lesion volume, and (D) tendon thickness as determined by conventional CT. *P<.05, **P<.01, ***P<.001.

Postheparin LPL activity and LPL dimer mass are not shown separately because they correlated very well with total postheparin LPL mass (total LPL mass and activity, r=.82, P<.001; LPL total and dimer mass, r=.82, P<.001; LPL dimer mass and LPL activity, r=.85, P<.001). Total postheparin LPL mass correlated best with CA calcific atherosclerosis (r=.72, P<.01; Fig 2BDown) but was also correlated with total calcified lesion volume (r=.65, P<.01; Fig 2ADown), aortic lesion volume (r=.60, P<.05; Fig 2CDown), and tendon thickness (r=.68, P<.01; Fig 2DDown). Postheparin HL activity was inversely correlated with the extent of calcific atherosclerosis in homozygous FH patients, while there was no significant association of HL with tendon thickness (Fig 3Down). As with LPL, the highest correlation was found with CA lesions (r=-.57, P<.05; Fig 3BDown). HL activity was also correlated with the total calcified lesion volume (r=-.55, P<.05; Fig 3ADown), but the correlation with aortic lesions (r=-.45, P=.09; Fig 3CDown) did not reach significance.



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  		Figure 2. Scatterplots show correlations of total postheparin LPL mass with extent of calcific atherosclerosis and tendon thickness in 15 FH homozygotes. Pearson correlation coefficients and statistical significance were determined by using the SPSS statistical program, which was also used to generate the plots. Shown are correlations with (A) total calcified lesion volume of aorta and CAs as determined by EBT, (B) CA lesion volume alone, (C) aortic root lesion volume alone, and (D) tendon thickness as determined by conventional CT.



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  		Figure 3. Scatterplots show correlations of postheparin HL activity with extent of calcific atherosclerosis and tendon thickness in 15 FH homozygotes. Pearson correlation coefficients and statistical significance were determined by using the SPSS statistical program, which was also used to generate the plots. Shown are correlations with (A) total calcified lesion volume of aorta and CAs as determined by EBT, (B) CA lesion volume alone, (C) aortic root lesion volume alone, and (D) tendon thickness as determined by conventional CT.

The results of the stepwise multiple regression analysis of those variables that had a linear correlation coefficient of >=.35 are summarized in Table 4Down. LPL and/or HL were part of every multiple regression model of total and CA lesion volumes. HL activity and LPL dimer mass alone accounted for 84% of the variability of CA calcific atherosclerosis (P<.0001). HL activity, age, TC at diagnosis, and LPL dimer mass accounted for 85% of the variability (P=.0005) for total atherosclerosis. Aortic lesion volume, which is a hallmark of the somewhat unusual distribution pattern of atherosclerosis in homozygous FH, was mostly accounted for by TC concentrations and age (r2=.72, P=.0005). Age alone accounted for 64% of the variability of tendon width (P=.0004).


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  		Table 4. Multiple Regression Analysis of the Correlation of Study Parameters With Calcified Lesion Volume and Tendon Thickness

Because both age and LPL mass were highly correlated with calcific atherosclerosis and tendon xanthoma, we analyzed the relation of total LPL mass, LPL activity, and HL activity with age. Total postheparin LPL mass (Fig 4ADown) and activity (Fig 4BDown) showed a high positive correlation with age (r=.79, P<.001 and r=.67, P<.01, respectively), whereas postheparin HL activity (Fig 4CDown) had only a slight negative correlation with age (r=-.29, P=.29).



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  		Figure 4. Scatterplots show correlations of postheparin lipases with age in 15 FH homozygotes. Age indicates age at time of CT. Pearson correlation coefficients and statistical significance were determined by using the SPSS statistical program, which was also used to generate the plots. Shown are correlations of age with postheparin (A) total LPL mass, (B) LPL activity, and (C) HL activity.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In order to investigate a potential role for the plasma lipases in the phenotypic heterogeneity of homozygous FH, we measured postheparin LPL and HL in 15 FH homozygotes and correlated the values with the extent of calcific atherosclerosis as measured by EBT. We chose EBT because it is noninvasive and can predict CA disease.48 Although EBT cannot detect uncalcified atherosclerotic lesions,49 it generally correlates well with histopathologic findings.50 51 In addition to measuring calcific deposits in the CAs, EBT enables the quantification of calcific atherosclerosis in the thoracic aorta. The data shown in Table 2Up confirm histopathologic studies4 46 that indicate that FH homozygous patients have a somewhat unusual distribution of atherosclerosis, with the majority of lesions found in the aortic root. To be able to investigate the more common site of atherosclerosis we also quantified the calcific lesions in the CAs alone.

There has been some discussion about the reproducibility of EBT measurements.49 52 While low reliability would impede longitudinal studies of individual patients, it would not diminish the correlation of EBT results with other parameters. In fact, highly significant correlations in the context of unreliable EBT measurements would make the associations even more noteworthy.

Since homozygous FH is a disease leading to greatly diminished life expectancy, various treatments have been tried to increase survival. The patients in the present study were under different forms of treatment that theoretically could have influenced their lipase levels. The patients undergoing LDL apheresis or plasma exchange regimens were analyzed 2 weeks after their last treatment. Chevreuil et al53 have demonstrated that LPL levels return to normal a maximum of 24 hours after the administration of high doses of heparin. It is conceivable that the periodic infusion of heparin in the patients undergoing pheresis treatment could induce LPL synthesis. Patients receiving periodic heparin infusions in the course of hemodialysis, however, do not show such an induced expression of LPL.54 55 Although stores of HL are not renewed as quickly as those of LPL,56 it is reasonable to assume that 14 days is sufficient to yield postheparin lipase activities comparable with those of patients not undergoing LDL apheresis. To ensure that the correlations found in this study were not caused by an artifact due to the pheresis treatment of some of our patients, we repeated the calculations after excluding the six patients (patients 3, 9, and 11 through 14) undergoing such treatments. Multiple regression analysis after exclusion of these patients was virtually unchanged. Although the statistical significance was reduced due to the smaller sample size, the same parameters were included in the statistical models of lesion volume and tendon width as when all patients were used (data not shown), indicating that the observed correlations were not caused by the pheresis treatment.

Patient 5 had undergone liver transplantation. Since LPL is solely synthesized in extrahepatic tissues and the LDL receptor does not seem to be involved in the clearance of LPL (see below), LPL levels should not be influenced by liver transplantation. Since HL is synthesized in the liver, however, a different level of expression due to genetic differences in the transplanted liver cannot be excluded. All patients were taking 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Studies of the influence of this class of lipid-lowering medication on lipase levels report no change in HL activity57 58 59 60 and either no change59 or a slight decrease60 or elevation57 58 in LPL activity. Since all patients received 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and their lipase mass and activities differed considerably, the influence of this drug on lipase levels in the present study is probably negligible. It has also been reported that probucol has no significant effect on LPL and HL levels in humans.61 62 63 To our knowledge, no data are available on the influence of bile acid–binding resins on lipase levels in humans. None of the patients received fibrates, which are known to increase LPL activity and mass.64 Thus, because the heparin studies were performed 2 weeks after the last plasmapheresis treatment, because none of the patients received fibrates, and because lipase mass and activity varied by 2.5-fold to sixfold between minimum and maximum levels, we believe that the various treatments that the patients received do not invalidate the observed significant correlations between lipase levels and calcific atherosclerosis.

Among the factors suggested as influencing the clinical outcome of FH are age, gender, and plasma levels of Lp(a) and LDL.1 Investigations of FH families in Puerto Rico65 and China66 suggest the presence of genetic and/or environmental factors that can normalize LDL-C levels in heterozygous FH and thus influence phenotypic heterogeneity. Such factors are difficult to evaluate in a small heterogeneous patient series like ours. Gender is not believed to play a significant role in the heterogeneity of homozygous FH,5 and the number of patients in our study was not large enough to analyze the influence of gender. Fig 1Up illustrates, not surprisingly, that age correlated very well with the extent of calcific atherosclerosis and tendon width in the FH homozygotes, and clinical outcome in FH is associated with age.1 Our study also confirmed that levels of Lp(a) are elevated in FH67 68 but failed to show a significant correlation with calcific atherosclerosis or tendon xanthoma. Results conflict with regard to a potential role of Lp(a) levels in modulating the clinical course of FH heterozygotes.68 69 70 Lp(a) levels did not significantly contribute to the phenotypic heterogeneity in the 15 patients of the present study.

TC and LDL-C concentrations at diagnosis were also positively correlated with calcific atherosclerosis and xanthomatosis. Interestingly, however, the correlation for LDL reached significance only for total lesion volume, although elevated LDL concentrations are the cause of atherosclerosis in FH. The total calcified lesion volume mainly represents the atherosclerosis in the thoracic aorta. We analyzed the thoracic aorta because atherosclerosis of the aortic arch is associated with mortality and morbidity in the general population71 and because atherosclerosis in homozygous FH patients shows an unusual predilection for this location.46 Since elevated LDL levels are the cause of atherosclerosis in FH and the thoracic aorta is the preferred location, it is not surprising that LDL is highly correlated with total calcific atherosclerosis and that TC and age are the only variables in a model of calcific atherosclerosis in the aorta (Table 4Up). Clearly, however, factors other than LDL plasma concentrations contributed to the phenotypic expression of calcific atherosclerotic disease in the CAs and to the development of tendon xanthomas in the 15 FH homozygotes.

To evaluate whether the plasma lipases might play a role in the heterogeneity of clinical outcome in homozygous FH, we correlated LPL and HL levels with the extent of calcific atherosclerosis. Fig 3A and 3BUpUp illustrates that HL activity had an inverse correlation with the extent of total and CA atherosclerosis in the 15 patients; this finding was supported by the multiple regression analysis (Table 4Up). Compared with the mean HL activity in 70 normal volunteers, the HL activity in the 15 patients was within the normal range (Table 3Up). HL is predominantly cleared by the LDL receptor–related protein,72 and it is therefore unlikely that the defect in the LDL receptor system would directly alter HL levels. Contradictory results exist about HL levels in FH. One study shows decreased HL activity in patients with type II hyperlipoproteinemia,73 while another demonstrates elevated HL activity in FH heterozygotes.74

The correlation data do not allow us to differentiate between HL as cause or consequence of the atherosclerosis. It is conceivable, however, that interindividual differences of HL levels influence the extent of vascular atherosclerosis. Overexpression of HL in transgenic rabbits lowers plasma levels of potentially atherogenic TG-rich remnant lipoproteins,75 possibly by increasing their binding to cell-surface glycosaminoglycans76 and the LDL receptor–related protein.77 In transgenic mice, overexpression of HL reduces cholesterol deposition in the aorta by 42%.37 HL can generate pre-ß1 HDL particles in vitro78 and in vivo (K.A. Dugi, unpublished data, 1996). Therefore, a potential mechanism by which HL might mediate a decrease in vascular lipid deposition could relate to reverse cholesterol transport.

While HL activity levels in the studied patients were within the normal range, LPL mass and activity levels were significantly elevated (Table 3Up). Raised levels of postheparin plasma LPL in FH might be expected if LPL were cleared by the LDL receptor. Recent studies, however, indicate that the liver removes LPL from the circulation by a combination of heparan sulfate glycosaminoglycans and the LDL receptor–related protein and that the LDL receptor does not play an important role in this process.79 An increase in LPL mass could be due to elevated levels of the LPL monomer and/or dimer. The correlation of LPL activity with total LPL mass in our study was highly significant (r=.82, P<.001) and similar to results44 in which the correlation was .83. This strong correlation indicates that there was little variability in specific LPL activity and thus might indicate a homogeneous relation of LPL monomer to dimer mass. To further elucidate this question, we measured the LPL mass with two different assays, one that preferentially quantifies the LPL dimer and another that recognizes total mass. Both total and dimer mass were elevated compared with those of normal subjects (Table 3Up). Together with the significant increase in LPL activity, these results suggest that the amount of active LPL dimer is elevated in the FH homozygous patients. This finding is supported by an observation in which three patients with heterozygous FH and CA disease were reported to have elevated LPL activity and LPL dimer mass.80

The correlation coefficients of age and LPL mass with calcific atherosclerosis and xanthoma are clearly similar (Fig 1Up). This could mean that the strong correlation of LPL with atherosclerosis is secondary to its correlation with age; Fig 4A and 4BUpUp reveals that both total postheparin LPL mass and activity are highly correlated with age. Previous studies have shown, however, that in normal and hypertriglyceridemic probands >20 years of age, LPL activity decreases with age.81 82 Unfortunately, no information is available about lipase levels in childhood. To exclude the possibility that reduced muscle and adipose tissue mass and hence possibly reduced LPL levels in our patients <20 years of age led to the observed positive association of LPL with age, we repeated the calculations after exclusion of these individuals. The correlations of HL and LPL with age were virtually unchanged after exclusion of the five individuals <20 years old (data not shown), indicating that the association is inherent to our patient group. Since total (r=.82, P<.001) and dimer (r=.85, P<.001) mass were highly correlated with LPL activity in our study as well as others,44 LPL mass is likely to show the same association with age. Taken together with the results from the multiple regression analysis (Table 4Up), the correlation of LPL with age in homozygous FH indicates that there is a pathophysiological reason for the observed relation or that LPL is linked to atherosclerosis, which also increases with age.

Fig 5Down illustrates several potential explanations for the correlation of LPL with calcific atherosclerosis in homozygous FH. First (Fig 5Down, I), the elevated levels of apoB and LDL in FH could lead to elevated levels of LPL. LPL can bind to an amino-terminal fragment of apoB83 and associates with large LDL particles in plasma.84 85 LPL mass and activity, however, did not show any positive correlation with either apoB or LDL, making this explanation less likely. A second possible explanation for the correlation of LPL with atherosclerosis (Fig 5Down, II) is based on the proposed proatherogenic actions of LPL. LPL, which is expressed in the atherosclerotic lesion,18 19 can trap lipoproteins in the subendothelial matrix, making them more susceptible to oxidation and subsequent uptake by macrophages and smooth muscle cells.21 22 23 24 In fact, LPL increases the adhesion of monocytes to aortic endothelial cells20 and through several actions mediates the formation of foam cells26 27 28 29 and cholesterol ester accumulation in smooth muscle cells.30 These LPL actions are independent of the LDL receptor. In fact, in LDL receptor–negative fibroblasts LPL increases surface-bound LDL over 140-fold.86 Since these LPL-mediated processes play a crucial role in atherogenesis, an increase of LPL in certain patients could lead to accelerated atherosclerosis and explain the correlation of LPL with calcific lesions. Finally (Fig 5Down, III), the extent of atherosclerosis in homozygous FH is likely to increase the amount of macrophages. Since macrophages18 25 and macrophage-derived foam cells19 synthesize LPL, the elevated levels of LPL in homozygous FH could be a consequence of severe atherosclerotic disease rather than a cause. The fact that the FH patients had elevated LPL levels compared with control subjects suggests that the association of atherosclerosis with LPL is, at least in part, caused by the atherosclerotic process. Higher LPL levels in the vascular lesion, on the other hand, may then further accelerate the buildup of plaques.



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  		Figure 5. Diagram shows possible explanations for the correlations of postheparin LPL mass and activity with the extent of calcific atherosclerosis in 15 FH homozygotes. I., LPL ({blacktriangleup}) binds to an amino-terminal fragment of apoB83 (B). The increase of apoB-containing LDL particles in homozygous FH together with the fact that LDL concentrations correlate with atherosclerosis could therefore account for the positive correlation of LPL with calcific atherosclerosis. II., LPL concentrations differ in FH homozygous patients. Since LPL can trap lipoproteins in the subendothelial matrix and increase the formation of foam cells and lipid accumulation in smooth muscle cells, higher LPL levels could accelerate atherosclerosis. III., Because of the extent of atherosclerosis found in FH homozygotes, a large number of macrophages is likely to be found in atherosclerotic lesions. Since macrophages are known to secrete LPL, the high levels of LPL mass and activity in patients with severe disease could be the consequence of atherosclerosis rather than its cause.

Although several parameters have been linked to the presence of CA disease, this is, to our knowledge, the first report of a significant linear association of the extent of atherosclerosis with the plasma levels of a certain protein or enzyme. The data do not allow us, however, to ascertain a causal role of LPL or HL in the phenotypic heterogeneity of homozygous FH. An intriguing study from Renier et al87 shows that macrophages from atherosclerosis-susceptible mouse strains constitutively express higher levels of LPL than those of resistant strains. This would suggest that high LPL expression by macrophages constitutes one of the inherited components of atherosclerosis. In humans, different levels of constitutive LPL expression could contribute to the phenotypic heterogeneity in homozygous FH. Prospective studies in newly diagnosed FH patients will provide further insight into the correlation of LPL with atherosclerosis. If the association of LPL with atherosclerosis is also found in patients with other genetic dyslipoproteinemias, LPL mass and activity levels may evolve as new risk factors for atherosclerosis.


*    Selected Abbreviations and Acronyms
 
CA = coronary artery
CT = computed tomography
EBT = electron beam tomography
FH = familial hypercholesterolemia
HDL-C = HDL cholesterol
HL = hepatic lipase
LDL-C = LDL cholesterol
Lp(a) = lipoprotein(a)
LPL = lipoprotein lipase
TC = total cholesterol


*    Acknowledgments
 
The authors thank Dr John Brunzell for the measurement of LPL dimer mass, provision of lipase data from normal subjects, and valuable discussion of the manuscript. We thank Steve Hashimoto for excellent technical assistance, Carlo Gabelli for the screening of patients for the apoB3500 mutation, and Betty Kuzmik for her invaluable help in organizing the various procedures performed on the FH patients.


*    Footnotes
 
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 17, 1994.

Received February 13, 1996; revision received June 4, 1996;
*    References
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