High-Density Lipoproteins and Endothelial Function

Nitric Oxide

NO, originally called endothelium-derived relaxing factor, is produced by a constitutive endothelial NO synthase (eNOS) in response to various stimuli, including fluid shear stress and exposure to the neurohumoral factors acetylcholine, bradykinin, serotonin, and substance P. Although short-lived, NO is a potent vasodilator that induces smooth muscle relaxation through the activation of smooth muscle guanylate cyclase. NO also inhibits platelet aggregation and leukocyte adhesion to the endothelium. Inhibition of endothelium-dependent relaxation due to the decrease in the bioavailability of NO is the most prominent feature of endothelial dysfunction. An association between atherogenic lipids, such as LDL, and a decrease in acetylcholine-induced endothelium-dependent relaxation or even a paradoxical vasoconstrictive response is well documented.³ High HDL cholesterol in humans, however, is associated with normal endothelium-dependent relaxation in response to acetylcholine.⁴

Incubating isolated rabbit aorta with HDL reverses the decrease in endothelium-dependent relaxation caused by oxidized LDL at physiological HDL concentrations.⁵ Exactly how HDL achieves this effect is not entirely clear. Matsuda et al⁵ suggested that HDL acts by preventing the transfer of lysophosphatidylcholine (LPC) from oxidized LDL to endothelial cells. LPC is a major lipid component of oxidized LDL that can mimic many of the atherogenic effects of oxidized LDL, including the decrease in endothelium-dependent vasodilatation.⁶ Recently, Uittenbogaard et al⁷ showed that incubation of endothelial cells with oxidized LDL depletes plasma membrane caveolae of cholesterol and translocates eNOS from caveolae to an internal membrane compartment, making eNOS insensitive to stimulation by acetylcholine. HDL opposes this phenomenon by donating cholesteryl esters to endothelial cell caveolae and preventing the defective localization of eNOS.⁸ eNOS-receptor uncoupling is believed to be important in the early progression of endothelial dysfunction, but in the long term, it is thought that decreases in NO bioavailability are due to an increase in the generation of superoxide anion. Superoxide anion reacts with NO to form peroxynitrate, a less effective vasodilator than NO. HDL is an important antioxidant and may improve the bioavailability of NO by bolstering the antioxidant status of endothelial cells and decreasing the formation of superoxide anion. This concept was recently strengthened by Cominacini et al,⁹ who showed that the effect of oxidized LDL is predominantly due to the generation of superoxide anion and not impaired activation of eNOS. The physiological relevance of the effects of HDL on caveola structures and protection against oxidized LDL remains uncertain. Nevertheless, these data suggest that oxidized LDL and HDL have opposing effects on NO bioavailability in endothelial cells.

Prostaglandins

Prostacyclin (PGI₂) is a prostaglandin produced by endothelial cells that acts on smooth muscle prostacyclin receptors to activate adenylate cyclase and inhibit contraction. Like NO, prostacyclin inhibits platelet aggregation. Prostacyclin increases the synthesis of NO by endothelial cells, and in turn, NO increases the activity of prostacyclin on smooth muscle relaxation. Incubation of HDL with endothelial cells promotes prostacyclin synthesis¹⁰; HDL also prolongs the half-life of prostacyclin in the blood.¹¹ As with NO, the mechanism by which HDL increases prostacyclin synthesis is not entirely clear. Pomerantz et al¹² showed that HDL provides endothelial cells with arachidonic acid in the form of cholesteryl arachidonate. The provision of endothelial cells with arachidonate is important, because endothelial cells lack the enzyme required to synthesize arachidonic acid. Spector et al¹³ downplay the contribution of HDL, arguing that arachidonic acid bound to albumin in plasma is more likely to be the source of arachidonic acid for the endothelium in vivo; endothelial cells also use endoperoxides released by platelets for prostaglandin synthesis. Cells prelabeled with radioactive arachidonate and exposed to HDL will generate radiolabeled prostacyclin, suggesting that HDL stimulates the calcium-sensitive phospholipase A₂.¹² Tamagaki et al¹⁴ showed that IP₃ generation and an increase in intracellular calcium accompany prostacyclin synthesis in endothelial cells incubated with HDL. Furthermore, incubating endothelial cells with HDL and a calcium chelator eliminates the effect of HDL on prostacyclin synthesis,¹⁴ indicating that the effect of HDL is calcium-sensitive and again implicating activation of phospholipase A₂. Cyclooxygenase-2 expression in human umbilical vein endothelial cells is modestly increased by treatment with HDL, and HDL can act synergistically with the inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β to produce a large increase in cyclooxygenase-2 expression.¹⁵ Although the origin of arachidonate as a substrate for prostacyclin synthesis is debated, the data show that HDL mediates production of prostacyclin in vitro.

Thromboxane A₂ (TXA₂) is another prostaglandin produced by endothelial cells, but unlike prostacyclin, TXA₂ is a potent vasoconstrictor and platelet activator. The ratio of prostacyclin to thromboxane is believed to be important for cardiovascular health. HDL can modify this balance. In particular, Oravec et al¹⁶ found that the HDL₂ subfraction was able to dose-dependently inhibit the formation of TXA₂ in cultured endothelial cells. HDL₃ stimulated TXA₂ formation, but the net effect was a favorable PGI₂:TXA₂ ratio at all but the highest concentrations of HDL used.

Platelet-Activating Factor

Platelet-activating factor (PAF) is a juxtacrine signaling phospholipid synthesized by activated endothelial cells. PAF stimulates platelet aggregation and smooth muscle contraction via a specific 7-transmembrane-domain PAF receptor. HDL inhibits the synthesis of PAF in activated cultured endothelial cells.¹⁷ This inhibitory activity is independent of the HDL PAF-acetyl hydrolase enzyme and is mediated by a decrease in the activity of cellular acetyl-coenzyme A:lyso-PAF acetyltransferase.¹⁷ Numerous signaling pathways regulate the acetyltransferase, but to date the mechanism used by HDL has not been defined. In human studies, HDL-cholesterol levels correlate inversely with platelet-thrombus formation assessed in a Badimon chamber, strengthening the concept that HDL particles modulate platelet function in vivo.¹⁸

Primary Hemostasis

NO inhibits platelet aggregation by increasing platelet cGMP generation. HDL restores impaired NO synthesis in endothelial cells that have been exposed to atherogenic lipids.⁸ HDL also acts directly on platelets to induce endogenous NO synthesis.¹⁹ Prostacyclin inhibits platelet aggregation by increasing platelet cAMP via a G-coupled protein. As noted before, prostacyclin synthesis in endothelial cells is stimulated by HDL, probably through a combination of substrate provision in the form of cholesteryl arachidonate¹² and calcium signaling.¹⁴ Conversely, HDL inhibits the synthesis of the prostaglandin platelet activator TXA₂.¹⁶ HDL further inhibits platelet activation by suppressing a PAF synthetic enzyme in activated endothelial cells.¹⁷ Endothelial cells express von Willebrand factor (vWF), a platelet ligand essential for platelet adhesion and aggregation in response to vascular injury. Circulating vWF in patients is inversely associated with HDL.²⁰ These various mechanisms may have an overall significance in vivo, because human data indicate an inverse association between HDL and platelet-dependent thrombus formation.¹⁸

Secondary Hemostasis

Tissue factor pathway inhibitor (TFPI) is secreted by both endothelial cells and the liver and opposes coagulation by acting on the extrinsic pathway. Epidemiological associations between circulating TFPI and HDL in humans are equivocal; one study has shown a negative association between HDL cholesterol and TFPI,²¹ whereas another showed a positive association between HDL cholesterol and TFPI and apoA-I and TFPI.²²

Thrombomodulin is an endothelial membrane protein that binds thrombin, permitting it to activate the anticoagulant serine protease protein C. Soluble thrombomodulin is often elevated in the plasma of patients with atherosclerosis, but to date no association between HDL and thrombomodulin has been identified. HDL does seem to have an endothelium-independent ability to activate proteins C and S.²³

Heparin-like proteoglycans on the endothelial cell surface further inhibit coagulation by activating antithrombin III. The apoE apolipoprotein moiety on HDL increases the sulfation of these endothelial proteoglycans.²⁴ Other apoE-containing lipoproteins (eg, VLDL) do not behave the same way, for reasons that are not understood.²⁴ Once again, in vivo evidence from humans supports a role for HDL as a thrombin inhibitor, because HDL inversely associates with a marker of activated thrombin.²⁵

Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1

Endothelial cells regulate thrombolysis as well as coagulation. The endothelium releases tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), which together control the activity of plasmin, an enzyme that cleaves cross-linked fibrin found in thrombi. In cultured endothelial cells, LDL and glycated LDL decrease the generation of tPA and increase the generation of PAI-1.²⁶ HDL reverses this effect, restoring the normal release of tPA and PAI-1.²⁶ In humans, HDL has not been directly correlated with PAI-1 in the blood,^22,27 although one study found an inverse relationship between PAI-1 and the ratio of HDL cholesterol to total cholesterol.²⁷

Apoptosis

Apoptosis of endothelial cells has been demonstrated in many cardiovascular diseases, including atherosclerosis. Some atherogenic lipids and inflammatory cytokines are potent apoptotic stimuli for endothelial cells. Defects in the endothelium due to endothelial cell apoptosis would be consistent with the response-to-injury model of atherogenesis; furthermore, an intact endothelium is essential to mask the thrombogenic molecules that lie underneath. HDL exerts a protective effect by interfering with the apoptotic stimuli to which endothelial cells are exposed. Oxidized LDL induces endothelial cell apoptosis in culture by producing a large and sustained increase in intracellular calcium, an increase that HDL opposes.³⁴ The ability of HDL to counteract oxidized LDL involves direct HDL-endothelial cell signaling, because preincubation of oxidized LDL with HDL has no effect on the apoptotic potency of oxidized LDL, and the protective effect of HDL is independent of the HDL antioxidant enzyme paraoxanase.³⁴ Furthermore, HDL does not affect oxidized LDL uptake by endothelial cells, and cells that are incubated with HDL for >5 hours, washed, and exposed to oxidized LDL continue to be protected.³⁴ ApoA-I mimics HDL in these respects but is less effective, suggesting that the lipid component of HDL may have some intrinsic antiapoptotic activity.³⁴ HDL also suppresses the caspases required for apoptosis that are activated by TNF-α, although the mechanism for this has not been defined.³⁵

Proliferation and Migration

If focal endothelial defects play a part in atherogenesis, then repair of these defects also merits consideration. HDL has long been known to be an important endothelial mitogen. The proliferative effect of HDL on endothelial cells seems to require extracellular calcium and involves changes in intracellular pH¹⁴; HDL also seems to induce protein kinase C-dependent phosphorylation of a 27-kDa heat-shock protein in proliferating endothelial cells,³⁶ although the significance of this is not known. Endothelial migration is also important in vessel repair, which HDL promotes through an unknown mechanism, although it is separate from that of basic fibroblast growth factor.³⁷ Recently, Nofer et al³⁸ reported that HDL-derived lysosulfatide and sphingosylphosphorylcholine activate phosphoinositol-specific phospholipase C in fibroblasts and promote cell proliferation. These results have not been reproduced with endothelial cells, yet this suggests that HDL-derived lipids or their metabolites have important physiological actions.

Conclusions

Although it is now possible to conclude that HDL improves endothelial function (see Figure), fundamental questions regarding the relationship between HDL and endothelial cells remain unanswered. How HDL mediates changes in endothelial phenotype is not thoroughly understood. Investigation of the effects of HDL is strongly warranted, because endothelial dysfunction occurs very early in and is required for atherogenesis. Furthermore, endothelial dysfunction is reversible. Pharmacological modalities, including lipid-lowering agents and the antioxidant probucol, have been shown to improve endothelial function in humans.¹ Knowledge gained from the study of the effects of HDL on endothelial cells may promote the development of new treatments for endothelial dysfunction.

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Effects of HDL on endothelium.