HDL and plaque stability

What are High-Density Lipoproteins: HDLs are a broad group of plasma lipoprotein particles which may be isolated by density gradient centrifugation between densities of 1.063-1.21 g/ml.Particles vary in size, density and composition of lipids, proteins and apolipoproteins, and are present in normal serum at concentrations between 0.5 — 1.5 mg/ml (apo A-I). The major apolipoprotein of HDL is apo A-I, which is present in all HDL particles.Nascent particles, synthesised mainly in the liver, are discoidal in shape.Currently there are two distinct HDL-like preparations under investigation.(1) ETC-216 is made as a proteoliposome particle consisting of Apo A-I Milano, made as a bacterially expressed recombinant protein, and carried in a phosphatidylcholine liposome in a ratio of protein to lipid of 1:1.These particles need to be stored frozen until just prior to use and are not as stable as the reconstituted HDL (rHDL).(2) rHDL is more like nascent discoidal HDLs and are made using plasma purified human Apo A-I and soyabean phosphatidylcholine in a ratio of protein to lipid of 1:150 [1].This preparation is stored lyophilised and is stable for one month at 4^oC following reconstitution and will be used in this proposal.

Chronic Effects of HDLs: The plasma concentration of HDLs has been shown to inversely correlate with the incidence of cardiovascular disease [2,3].Lipoprotein levels have been associated with the extent and severity of cerebrovascular atherosclerosis [4], and a recent study revealed an independent negative association between HDL cholesterol and ischaemic stroke [5].In transgenic mouse models it has been demonstrated that elevation of plasma HDLs significantly reduced the extent of plaque formation [6-9]. ETC-216 given as a weekly infusion for five weeks to patients with acute coronary syndrome, reduced the volume of coronary plaques by 4%, as measured by intravascular ultrasound (IVUS) [10].The mechanism of this effect is unclear.

Acute effects of HDLs:One of the earliest cellular events in atherogenesis is the binding of leukocytes to the vessel wall. We showed that a four hour treatment with HDLs ablated cytokine-induced adhesion molecule expression in vitro and in vivo [11,12].In addition to an ameliorative effect on induction of adhesion molecules, Folgelman and colleagues were able to show that HDLs also inhibit the monocyte chemokine, MCP-1 following an overnight treatment with the lipoprotein [13]. In an experimental model of endothelial activation, four hours following an infusion of HDLs we observed a significant reduction of tissue damage [14,15], and inflammation [16] after oxidative insult. Figure 1 summarises our in vivo data showing the anti-inflammatory effect of rHDLs.Similar studies, using short peptide analogues of apo A-I demonstrated anti-inflammatory effects of HDL’s.As these peptides lack the ability to support reverse cholesterol transfer, these data support the idea that the anti-inflammatory effect of HDLs is independent of their ability to remove cholesterol from peripheral tissues [17,18].

The ability of HDLs to act as a mitogen is well documented [19] and recent data suggests that the lipoproteins may also influence cell migration in an FGF-dependent fashion [20].In addition, HDLs have been shown to inhibit apoptosis in both SMC and EC [21,22].The ability of HDLs to inhibit the apoptosis of EC may be of fundamental importance in reducing plaque erosion [23].Taken together these data support the idea that HDLs could influence plaque stability through acute modulation of cellularity.

HDLs have been shown to possess strong anti-oxidant properties, related largely to the function of paraoxonase, an HDL-associated enzyme [24,25]. In vitro studies have shown that ox-LDL induction of MMP 1 from primary human monocytes can be ablated by the addition of HDL [26], providing a complex mechanism for plaque stabilisation.In addition to the ability of HDL to prevent lipid oxidation, it has been suggested that HDL can modulate the deleterious effect of oxLDL through competition for the scavenger receptor CD36 on the macrophage.Interestingly, the expression of CD36 has been shown to be regulated through the degree of oxidation of LDL [27], and thus the ability of HDL to reduce oxidation of LDL would further augment the protective effect of this lipoprotein.

The numerous citations to clinical and pre-clinical data which clearly demonstrate HDL mediatesmeasurable effects on a broad spectrum of molecules observed in minutes and hours, lends support to our proposed study in which patients elected to undergoe carotid endarterectomy will be given an infusion of wither rHDLs or placebo 24 hours prior to removal of the atherosclerotic plaque.We will then analyse the cellular and molecular composition of the plaque to evaluate whether HDLs have an acute ability in vivo to stabilise the plaque.We are likely to see the molecular phenotype of the atherosclerotic plaque significantly changed following an infusion of rHDL compared to those patients who will receive carrier alone.

Bibliography of relevant references:

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2.Gordon T, Castelli WP, Hjortland MC et al.,(1977) High density lipoproteins as a protective factor against coronary heart disease: the Framingham study. Am J Med.62:707-714.

3.Assmann G, Schultze H. (1988) The Prospective Cardiovascular Munster (PROCAM) study: prevalence of hyperlipidaemia in persons with hypertension and/or diabetes mellitus and the relationship to coronary heart disease.Am Heart J.116:1713-1724.

4.Tell GS, Crouse JR, Furberg CD. (1988) Relation between blood lipids, lipoproteins and cerebrovascular atherosclerosis. Stroke.19:423-30.

5.Tanne D, Yaari S, Goldbourt U. (1997) High-density lipoprotein cholesterol and risk of ischaemic stroke mortality. Stroke.28:83-87.

6.Paterno R, Ruocco A, Postiglione A et al., Reconstituted high-density lipoprotein exhibits neuroprotection in two rat models of stroke.Cerebrovasc Dis.2004:17:204-211.

7.Rubin E, Krause RM, Spangler EA et al., (1991) Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.Nature;353:265-267.

8.Plump AS, Scott CJ, Breslow JL. (1994) Human apolipoprotein AI gene expression in the apolipoprtotein E-deficient mouse.Proc Natl Acad Sci.91:9607-9611.

9.Paszty C, Maeda N, Verstuyft J et al., Apolipoprotein AI transgene corrects apolipoprotein E-deficiency-induced atherosclerosis in mice.J Clin Invest.94;899-903.

10.Nissen SE, Tsunoda T, Tuzcu EM et al.,(2003) Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomised controlled trial.JAMA.290;2292-2300.

11.Cockerill GW, K-A Rye, JR Gamble et al., (1995). High density lipoproteins inhibit cytokine-induced expression of adhesion molecules on endothelial cells. Arteriosclerosis, Thromb Vasc Biol.15:1987-1994.

12.Cockerill GW, T Heuhns, C Stocker et al., Elevation of plasma HDL inhibits cytokine-induced induction of E-selectin in a porcine model of acute inflammation. Circulation. 103:108-112.

13.Navab M, Imes SS, Hama SY et al., (1991) Monocyte transmigration induced by a modification of low density lipoprotein in co-culture of human aortic wall cells is due to induction of monocyte chemotactic protein 1 and is abolished by high density lipoprotein.J Clin Invest.88;2039-2046.

14.Cockerill GW, MC McDonald, H Mota-Filipe et al., (2001) High-density lipoproteins reduce organ injury and organ dysfunction in hemorrhagic shock. FASEB J.15:1941-1952.

15.Theimerman C, Patel NS, Kvala EO, GW Cockerill et al.,(2003) High density lipoproteins (HDL) reduces renal ischaemia /reperfusion injury.J Am Soc Nephrol.14:1833-43.

16. Cussocrea S, Dugo L, Patel NS, Di Paola R, Cockerill GW, Genovese T, Thiemermann C. (2004) High-density lipoproteins reduce intestinal damage qassociated with ischemia/reperfusion and coliis. Shock.21:342-351.

17.Navab M, Anatharamaiah GM, Reddy ST et al.,(2003)Human apolipoprotein AI mimetic peptides for the treatment of atherosclerosis.Curr Oppin Investig Drugs.4:1100-1104.

18.Navab M, Anatharamaiah GM, Hama S et al., (2002) Oral administration of an Apo A-I mimetic peptide sytnthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol.Circulation.105;290-292.

19.Tauber JP, Cheng J, Massoglia S et al.,(1981) Effect of high and low density lipoproteins on proliferation of bovine vascular endothelial cells. In Vitro.17;519-530.

20.Murugesan G, Sa G, Fox PL (1994).High-density lipoprotein stimulates endothelial cell movement by aechanism distinct from basic fibroblast growth factor. Circ Res.74:1149-1156.

21. Sugano M, Tsuchida K, Makino N. High-density lipoproteins protect endothelial cells from tumour necrosis factor-alpha-induced apoptosis.(2000) Biochem Biophys Res Comm.272:872-876.

22.De Kroon RM, Mihovilovic M, Goodger ZV et al.,(2003) Apo E genotype-specific inhibition of apoptosis. J Lipid Res.44;1566-1573

23.Nofer JR, Levkau B, Wolinska I et al.,(2001) Suppression of endothelial cell apopotosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids.. J Biol Chem.276:34480-34485.

24.Mackness M, Abbot C, Arrol S et al.,(1993) Biochem J.294:829-834.

25.Van Lenten BJ, Wagner AC, Nayak DP et al.,(2001) Circulation.103:2283-2288.

26.Ardans JA, Economou AP, Martinson JM et al., (2002) J Leukoc Biol.71:1012-1018.

27.Kavanagh IC, Symes CE, Renaudin P et al., (2003) Degree of oxidation of low density lipoproteins affects expression of CD36 and PPAR gamma, but not cytokine production, by human monocyte-macrophages.Atherosclerosis.168:271-282