HDL and macrophages: explaining the clinical failures and advancing HDL-based therapeutics in cardiovascular diseases?

KEYWORDS:

• High density lipoproteins
• macrophages
• inflammation
• atherosclerosis

Atherosclerosis, a chronic inflammatory disease affecting medium- and large-sized arteries, is the main underlying cause of cardiovascular syndromes such as myocardial infarction and stroke [1]. Atherosclerosis is commonly viewed as a chronic inflammatory disease, driven by (modified) lipids. This rendered lipid status a major target for intervention in or even reversal of this disease [2]. One of the most effective strategies appeared to be lowering of plasma cholesterol levels, in particular low-density lipoprotein (LDL) cholesterol levels by statins and more recently by pro-protein convertase subtilisin/kexin type 9 (PCSK9) inhibitors [2]. With the growing insight that high-density lipoproteins (HDL), unlike LDL, protects against cardiovascular diseases (CVDs), an increasing number of studies focus on elevating HDL as therapeutic target. The physiological role of HDL is to transport cholesterol from the periphery (e.g. the vessel wall) back to the liver for degradation. Therefore, elevated HDL levels should lead to attenuated atherosclerosis. However, as summarized in the following, the outcome of several clinical trial studies investigating the therapeutic effects of HDL-elevating drugs was disappointing which fostered a still ongoing lively debate. In this editorial we will focus on several recent publications which fuelled this debate regarding the potency of HDL as therapeutic target for atherosclerosis.

Already more than 50 years ago, the Framingham Heart Study was the first study to provide compelling evidence of an inverse relationship between HDL and the risk of a cardiovascular event [3]. This rooted the concept that HDL, ‘the good cholesterol,’ could potentially protect against atherosclerosis-related diseases (HDL hypothesis). During the 1990s this notion was confirmed in several animal studies. For example, Badimon et al. observed a regression of atherosclerotic lesions after infusion of cholesterol-fed rabbits with HDL isolated from human plasma [4]. Moreover, Rubin et al. could show that transgenic overexpression of human apoA-I in mice, resulting in high circulating HDL levels, inhibited early atherogenesis [5].

Based on the aforementioned epidemiological and experimental data, all pointing to beneficial effects of HDL in CVD, major efforts were made to develop HDL-elevating therapies. Besides the lifestyle interventions, like increased physical activity, a lot of pharmacological strategies, such as niacin, CETP inhibitors, PPAR agonists (fibrates), and direct HDL/apoA-I mimetics, have been developed and tested in human clinical trials. Surprisingly, several of these trials had to be discontinued due to unforeseen, detrimental side-effects or did not show the expected beneficial outcome on CVD risk. The HDL hypothesis recently suffered another blow with the study of Keene et al. [6]. Their meta-analysis of 39 clinical trials aiming to raise plasma HDL levels showed that niacin, fibrates, and CETP inhibitors fail to reduce all-cause mortality or even the incidence of cardiovascular events like myocardial infarction or stroke. Furthermore, recent Mendelian randomization analyses by Voight et al. showed that polymorphisms exclusively altering HDL levels actually did not affect the risk of myocardial infarctions [7].

After decades of research the originally described association of HDL and cardiovascular risk, the actual basis of the HDL hypothesis, thus seems to be not so straightforward as initially expected.

The recent discrepant findings of studies focusing on vascular cell-type-specific effects of HDL may offer some enlightenments. Clearly, HDL exerts anti-inflammatory effects on endothelial cells and smooth muscle cells (SMCs), evidenced by strongly reduced secretion of critical cytokines/chemokines [8,9]. Recent studies on the effects of HDL on macrophages reported rather conflicting results. However, the use of different cell states (naïve or lipid-loaded cells), HDL preparations and concentrations, or readout models may at least partly explain these observed discrepancies.

Suzuki et al. [10] reported inhibitory (anti-inflammatory) effects of HDL on the type I interferon response in acetylated LDL-loaded foam cells, whereas all other studies used nonloaded macrophages. In support, anti-inflammatory effects were also observed by De Nardo et al. [11]. However, in their study a soybean phospholipid reconstituted HDL compound (CSL-111) was used at supraphysiological concentrations, two- to threefold higher than in humans. As several soybean components have already been shown to be anti-inflammatory in macrophages, conclusions on specific HDL effects should be taken with caution. In addition, this study reported anti-inflammatory effects of native HDL, although these experiments were performed in TLR4 mutant C3H/HeJ mice, which display a defective response to bacterial endotoxins like LPS. In contrast, we recently showed clear pro-inflammatory effects of HDL on macrophages using physiological concentrations of both native HDL and 1-palmitoyl-2-linoleoyl-phosphatidylcholine apoA-I reconstituted HDL in (macrophages from) C57Bl/6 mice [12]. Other studies supported such pro-inflammatory effects on macrophages, for example, the study by Smoak et al. [13] using lipid-free apoA-I (the main protein constituent of native HDL). However, it should be noted that in humans the bulk of apoA-I is contained in lipidated HDL, not in a lipid-free pre-HDL state. The critical importance of HDL subtype for its effects on macrophages was underpinned by the study of Sanson et al. [14], showing that especially the HDL₃ subtype induced M2 macrophage markers (anti-inflammatory phenotype) and reduced Il6 and Nos2 expression. A recent review by Rye and Barter [15] indeed highlighted that different HDL subfractions may have divergent physiological effects. To date, however, the majority of the observed HDL effects involve total HDL pools and cannot be attributed to a particular HDL subtype or constituent, which should be a major focus on future HDL research.

A still outstanding question is how these apparent discrepancies and cell-specific effects translate into the patient. It is virtually impossible to extrapolate the integrated range of cell-type- and subtype-specific effects of HDL for a complex disease pathology like atherosclerosis, throughout disease progression. Current therapeutic strategies targeting HDL could well lack precision with respect to HDL subtypes and targeted cell types, therefore resulting in mutually counteracting systemic effects, leading to an apparent ineffectiveness of the HDL-based therapy in reducing cardiovascular risk/mortality. Such (cell- or disease-) targeted therapy should possess an improved efficacy profile, not only for the treatment of atherosclerosis but also other pathologies. For example, HDL has been shown to reduce SMC proliferation [9], which might be detrimental in the context of atherosclerosis (resulting in plaque instability), but could be beneficial for neointimal hyperplasia as this is mainly caused by hyperproliferation of SMCs. Although the effect of HDL on plaque macrophages remains to be determined, any pro-inflammatory effect could have a negative impact on atherosclerosis, especially at late stages of the diseases when more inflammation leads to plaque destabilization; however they might be beneficial at earlier stages when stimulation of macrophage functions can improve clearance of modified lipids and cellular debris. Furthermore, enhanced innate immune responses could be valuable in patients with compromised immunity or for treatment of inflammatory diseases like rheumatoid arthritis or bacterial infections.

In conclusion, we believe that in order to push forward the development of effective HDL-based therapies for atherosclerosis, but also other pathologies, it will be crucial to further elucidate the effects of specific HDL subtypes and HDL constituents as this clearly can have a great influence on the outcome and therefore therapeutic efficacy. It should be noted that a large body of in vitro and ex vivo obtained evidence regarding HDL functionality and in particular its cholesterol efflux capacity has not yet been clearly validated in humans. Furthermore, special attention is required to also include and further specify the cell-type-specific effects of the various compounds. These investigations will be essential to continue developing more (cell type and disease stage) specific and targeted HDL-based approaches aiming at a reduction of cardiovascular risk.

Declaration of interest

E.P.C. van der Vorst is supported by the Deutsche Forschungsgemeinschaft (SFB 1123-A1) and the Alexander von Humboldt Foundation. K. Theodorou and M.M.P.C. Donners are supported by the Dutch Heart Foundation (Dr. E. Dekker grant nr. 2012T079). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Funding

This paper was not funded.