Proteolytically modified high density lipoproteins (HDL): Insights into HDL function in reverse cholesterol transport (RCT)

Thursday, 27 May 1999 Poster session: lntracellular trqfficking and assembly o f lipoproteins/reverse cholesterol transport

40

primary lipid abnormality is hypertriglyceridemia. In this 6 week (w), multicenter, double-blind, parallel study, 196 patients with triglycerides (TG) of 300 mg/dL (3.41 mmol/L) to 900 mg/dL (10.22 mmol/L) and LDL-C > 75 mg/dL ( 1.94 mmol/L) were randomized to placebo, S 20, 40 or 80 mg/day for 6 w. The baseline (BL, mg/dL) and mean % change (CH; median for TG and VLDL-C) from BL to w 6 for the major lipids and lipoproteins were:

TG BL

LDL-C % CH

VLDL-C

BL

** C H

BL

HDL-C

% CH

BL

*,* C14

placebo

387

-8.0

128

-I.0

82

-2.9

38

1.6

S 20 mg

402

-19

124

-25

86

-22

38

7.4

S 40 mg

411

-22

118

-27

92

-33

37

II

S 80 m g

408

-31

120

-36

97

~.13

35

12

S produced significant (p < 0.001) beneficial effects on all major lipid parameters compared to placebo and was well tolerated. S is an effective treatment for patients with hypertriglyceridemia. EFFICACY AND SAFETY OF MICRONISED FENOFIBRATE AND SIMVASTATIN IN PATIENTS WITH TYPE IIA OR IIB DYSLIPIDAEMIA G. Crepaldi 1, A. Barbato2, D. Delaval 2. On behalf of the Italian Stud), Group." JDirettore Istituto di Medicina Interna Policlinico Uniuersitario. Padova. Italy; :Gr. Fournier. Country missing! This multicentre, randomised, parallel groups trial compared micronised fenofibrate (MF) and simvastatin (SV) on total (TC), LDL and HDL cholesterol, Triglyceride (TG), fibrinogen and uric acid in Type lla or lib dyslipidaemia. After 3 months on diet alone, 206 patients (101 men, 105 women, mean age 54+11 yrs) were randomised to 6 months on MF 200 mg OD (n = 105) or SV 10 mg OD (n = I01). I77 patients were evaIuated for efficacy. Baseline value~ In mmol/15:SD and % change from baseline (n at last visit} Fenofibrate LDL

HDL

Ila (n = 60}

lib (n = 31}

all (n = 911

5.3 4- 0 7 3

5.2 4- 0.76

5.3 ± 0.75

- 2 6 . 6 ± 15.2 (n = 54)

- 1 3 . 0 4- 21.6 m = 301

- 2 1 . 7 ± 18.8 tn = 841

1.4 5: 0.31

I I 4- 0.25

1.3 4- 0,32

3 0 0 4- 26.9 ~

2 1 . 0 4 - 2 6 . 1 " (n = 8 4 )

15.9+24.5(n

=54)

Poster session: Intracellular trafficking and assembly of lipoproteins/reverse cholesterol transport PROTEOLYTICALLY MODIFIED HIGH DENSITY LIPOPROTEINS (HDL): INSIGHTS INTO HDL FUNCTION IN REVERSE CHOLESTEROL TRANSPORT (RCT) M. Lee 1, A. von Eckardstein2. A. Catapano 3, ET. Kovanen 1, t IIqhuri Researt'h Institute. Helsinki, Finland; "Instttute of Clinical Chemistt 3" and Laborato O, Medicine. Westphalian Wilhelms-Universi~., Get'mare': 3Institute of Pharmacological Sciences. Uniuersi O, of Milan, Italy High proportions of lipid-poor HDL are present in the arterial intimal fluid, where they are thought to actively promote cellular cholesterol elflux. A small discoidal pre[~,-migrating HDL subpopulation, containing only apolipoprotein (apo)A-I, acts as the earliest acceptor of cholesterol. Although apoA-I is the best activator of lecithin cholesterol acyltransferase (LCAT) in plasma, the apoA-l-containing lipoproteins found in interstitial fluid are poor substrates for LCAT. Here we studied, using the mast cell serine protease chymase as a model, how the two key biological functions of HDL in RCT, namely cholesterol effiux-inducing and LCAT-activating abilities, are affected by loss of integrity of the protein moiety of HDL. Chymase effectively degraded apoA-I without causing aggregation or fusion of the proteolytically modified HDL, or causing its uptake by macrophage foam cell cultures. However, the proteolyzed HDL induced cholesterol effiux from macrophage foam cells with lower affinity due to specific depletion of their minor lipid-poor subpopulations, prel~qLpA-I and LpA-IV, as demonstrated by 2D-PAGGE. Monoclonal antibodies against apoA-I disclosed that its central region, which is associated with cholesterol efflux and activation of LCAT, was highly susceptible to proteolytic modification by chymase. The two epitopes most rapidly modified by chymase were specific for prej'~l- and pre[~2-HDL. Interestingly, even extensive proteolytic degradation of several apoA-I epitopes failed to abolish the ability of HDL to activate LCAT. Thus, of the two HDL functions studied, only the ability to promote highaffinity efflux o f cholesterol from macrophage foam cells was sensitive Io proteolysis, These studies suggest that proteolytic modification o f epitopes on apoA-I and apoA-IV with depletion of the lipid-poor particles involved in the induction of high-affinity efflux of cholesterol from foam cells may lead to local inhibition of the initial step of RCT in the arterial intima. CHOLESTEROL EFFLUX FROM J774 RAT MACROPHAGES MEDIATED BY THE SERUM OF MICE EXPRESSING HUMAN APO AIV

tn = 301

7.5 5 : 0 . 7 4

TC

-20.1 4- 12.7 tn = 54) TG

7.6 ± 0.86

7.5 4- 0.83

13.0 4- 15.5 (n = 3(1t - 1 7 . 5 ± 14.0 (n = 84)

1.6 4- 0.43

2.9 4- 0.53

2.0 4- 0.83

- 3 1 . 8 4- 37.8"

~15.0 4- 1 7 . 7 " "

- 3 6 . 5 4- 32.2"

(11 = 54)

¢n = 301

(n = a4)

Slmvaatatln

Ila (n = 49)

lib (n = 37)

all (n = 86)

LDL

5.7 5= 0.78

5 2 5: 0.71

5.5 5: 0.78

- 3 0 . 5 4- 18.9 In = 46) HDL

TC

TG

24.7 4- 14.3"

- 2 8 . 2 :t: 1 7 . 3 " "

(n ~ 31)

(n = 77)

1.3 5 : 0 . 3 2

I.I ± 0,26

1,2 4- 0.31

13.8 4- 46.9 (n = 47)

12.4 5 : 2 0 . 2 (n = 34)

13.2 + 37.9 In = 81)

7.6 5 : 0 , 7

7.7 4- 076

7.6 4- 0.73

-21.85:14.5(n=47)

-18.7±9.6(n=34)

-20.54-12.7(n=811

1.5 :t: 0 . M

2.9 + 0.51

2.1 4- 0.83

1.8 ± 47.8 (n = 47)

-16.1 5 : 3 2 . 6 tn = 34)

- 5 , 7 ::1:42.8 (n = 8 l )

Difference bet~een treatmentx f o r % change: mp < 0,05, n p = O 00L *p = 0,002. " p = 0,0002. "'p

= 0.0001

Fibrinogen rose by 13.6% after SV (-3.3% with MF). Uric acid feli by 23.6% and 3.3% after MF and SV. Eleven patients experienced treatmentrelated adverse events (MF 8: abdominal pain [2], nausea Ill, diarrhoea Ill, biliary pain [l], CPK increase [2], SGPT increase Ill. SV 3: headache Ill, abdominal pain [I], enzyme abnormality [I]). MF was superior to SV in lla and Ilb Types, for TG and HDL. In Type ila the treatments did not differ significantly for TC and LDL cholesterol.

N. Fournier, V. Atger, M. Sturm, J.L. Paul, G. Rothblat, N. Moatti. Hrpital Broussais. AP-HP Paris; Laboratoire de Biochimie. Facult~ des Sciences Pharmaceutiques. Chdtenay-Malabrs; France; Allegheny Uniuersi O" of the Health Sciences BiochemistO, Department. Philadelphia. USA The role of apolipoprotein AIV (apo AIV) in lipoprotein metabolism has not been established. Some in vitro studies suggest that apo AIV plays a role in reverse cholesterol transport (RCT). The goal of these studies was to investigate the role of apo AIV in RCT by comparing cellular cholesterol effiux to serum from control mice and mice expressing human apo AIV. Maintened on a chow diet, the lipid profiles were similar in transgenic and control mice; in transgenic mice serum human apo AIV averaged 7500 I.tg/ml. The cholesterol donor cells were J774+pretreatment with cAMP (0.2 raM). Exposure of these cells to cAMP had previously been shown to upregulate cholesterol effiux to lipid-free apolipoproteins. The difference in effiux between control and cAMP J774 cells reflected the contribution of free apolipoprotein-mediated efflux. The cells were exposed to whole serum, lipoprotein depleted serum (LPDS) or isolated HDL from control or transgenic mice. Using control J774 cells not pretreated with cAME the effiux values were similar for whole serum or LPDS from either control or transgenic mice. Cyclic AMP treatment induced an increase in effiux to whole serum and LPDS from transgenic mice, without having any effect on serum or LPDS from control mice. With transgenic mice, the cAMP stimulation of effiux was inversely related to the serum concentration. Thus, the greatest cAMP stimulation of effiux (80%) was observed using 0.1% serum, corresponding to 8 p.g/ml human apo AIV. Cyclic AMP produced a dramatic increase in cholesterol efflux to the LPDS, with the greatest stimulation (200%) when LPDS was added to provide approximately 10 [Ltg/ml of human apo AIV. In contrast to the stimulation observed with whole serum or LPDS, the effiux induced by isolated HDL from control or transgenic mice was not enhanced by cAMP treatment of J774. Our

71st EAS £7ongress and Satellite Symposia