Acidification of the intimal fluid: the perfect storm for atherogenesis

This article is available online at http://www.jlr.org Journal of Lipid Research Volume 56, 2015 203

Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

accumulation of lipoprotein-derived lipids into the intimal layer where they become modifi ed and, as a result, trigger a maladaptive immune response characterized by infi ltration of monocyte-derived macrophages into the arterial wall. Mature atherosclerotic lesions are gradually formed via chronic infl ammation, tissue remodeling, smooth muscle cell proliferation, fi brosis, and calcifi cation.

Some areas of the arterial tree are more prone to athero-sclerosis than others, due to their relatively thick intima ( 1–3 ). One major factor explaining the relationship be-tween intimal thickness and susceptibility to atherosclerosis is the absence of capillaries and lymphatics ( 4 ), which re-stricts the supply of oxygen and nutrition of intimal cells ( 5 ). Thus, hypoxia develops easily in the deep intima, and is further exacerbated by the additional increase in intimal thickness that occurs during atherogenesis ( 6, 7 ). Due to hypoxia, intimal cells become more dependent on glycoly-sis for energy production. Importantly, activated immune cells, including macrophages, favor energy production by glycolysis even in a normoxic environment ( 8 ). Similarly, proliferation of smooth muscle cells has been shown to in-volve stimulation of glycolysis ( 9 ). As cells with a glycolytic phenotype produce and secrete more protons than cells us-ing oxidative phosphorylation, both aerobic and anaerobic glycolysis cause acidifi cation of the extracellular fl uid.

Indeed, acidic pH is found in various infl ammatory sites ( 10–13 ), where local acidosis can affect the ongoing im-mune response ( 14, 15 ). The extrusion of intracellular pro-tons is important for the activity of immune cells, because, by extruding excess intracellular acid, the cells not only protect themselves from intracellular acidifi cation, but also deliver protons to the extracellular milieu to facilitate vari-ous cellular functions in a paracrine or autocrine fashion. For example, extrusion of intracellular protons allows sus-tained activity of NADPH oxidase, an enzyme present on the plasma membrane of phagocytes involved in mounting

Abstract Atherosclerotic lesions are often hypoxic and ex-hibit elevated lactate concentrations and local acidifi cation of the extracellular fl uids. The acidifi cation may be a conse-quence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycol-ysis for ATP synthesis even under normoxic conditions, re-sulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current under-standing of the effects of acidic extracellular pH on three key players in atherogenesis: macrophages, apoB-containing lipoproteins, and HDL particles. Acidic extracellular pH enhances receptor-mediated phagocytosis and antigen pre-sentation by macrophages and, importantly, triggers the se-cretion of proinfl ammatory cytokines from macrophages through activation of the infl ammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifi ca-tions of LDL and other apoB-containing lipoproteins, and strongly increases their affi nity for proteoglycans, and may thus have major effects on their retention and the ensuing cellular responses in the arterial intima. Finally, the decrease in the expression of ABCA1 at acidic pH may compromise cholesterol clearance from atherosclerotic lesions. Taken together, acidic extracellular pH amplifi es the proathero-genic and proinfl ammatory processes involved in atherogen-esis. —Öörni, K., K. Rajamäki, S. D. Nguyen, K. Lähdesmäki, R. Plihtari, M. Lee-Rueckert, and P. T. Kovanen. Acidifi ca-tion of the intimal fl uid: the perfect storm for atherogenesis. J. Lipid Res. 2015. 56: 203–214.

Supplementary key words apolipoproteins • high density lipopro-tein • infl ammation • low density lipoprotein • lipids/effl ux • lipopro-teins • macrophages/monocytes • phospholipases • proteoglycans • infl ammasome

Atherosclerosis is a chronic disease of the inner layer of the arterial wall, the intima. The disease involves slow

This study was supported by grants from the Academy of Finland, Finnish Foun-dation for Cardiovascular Research, the Magnus Ehrnrooth Foundation, the Sigrid Juselius Foundation, the Paulo Foundation, the Finnish Cultural Foun-dation, the Oskar Öfl und Foundation, the Finnish-Norwegian Medical Founda-tion, and Biomedicum Helsinki Foundation. Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation.

Manuscript received 15 April 2014 and in revised form 25 November 2014.

Published, JLR Papers in Press, November 25, 2014 DOI 10.1194/jlr.R050252

Acidifi cation of the intimal fl uid: the perfect storm for atherogenesis

Katariina Öörni , 1 Kristiina Rajamäki , Su Duy Nguyen , Katariina Lähdesmäki , Riia Plihtari , Miriam Lee-Rueckert , and Petri T. Kovanen

Wihuri Research Institute , Helsinki, Finland

Abbreviations: Fc � R, Fc � receptor; HIF-1 � , hypoxia-inducible transcription factor 1 � ; IL, interleukin; LAL, lysosomal acid lipase; LPS, lipopolysaccharide; NF, nuclear factor; PLA 2 , phospholipase A 2 ; RCT, reverse cholesterol transport; ROS, reactive oxygen species .

1 To whom correspondence should be addressed. e-mail: [email protected]

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204 Journal of Lipid Research Volume 56, 2015

glycolysis, known as the Warburg effect, was fi rst described in cancer cells by the world-renowned biochemist Otto Warburg ( 28 ). Curiously, it seems that aerobic glycolysis is specifi cally upregulated by proinfl ammatory activation of immune cells, not by anti-infl ammatory activation of im-mune cells; classically activated proinfl ammatory M1 mac-rophages and T helper cells display a glycolytic phenotype, whereas alternatively activated M2 macrophages and regu-latory T cells with anti-infl ammatory properties are charac-terized by enhanced oxidative phosphorylation ( 27 ). Basal metabolism in resting mouse peritoneal macrophages is predominantly glycolytic though metabolic fl ux is slow, and the rate of glycolytic fl ux is greatly increased by classical M1 type activation (LPS + IFN- � ) and innate activation (LPS or other TLR agonists alone), but not by alternative M2 type activation [interleukin (IL)-4/IL-13; IL-10] ( 31 ). The im-portance of HIF-1 � and glycolysis in macrophage energy metabolism under normoxia is further highlighted by the drastic decrease in steady state ATP levels in HIF-1 � -defi cient macrophages ( 32 ) and by the marked increase in the expression of glycolytic enzymes during differentiation of human monocytes into macrophages ( 33 ). Similar to macrophages, proinfl ammatory activation of dendritic cells through toll-like receptors and of T lymphocytes through the T cell receptors triggers a HIF-1 � -dependent increase in glycolytic metabolism ( 34, 35 ). The rationale behind the strong induction of aerobic glycolysis in activated macro-phages and other immune cells most likely lies in the strong induction of various biosynthetic pathways and prolifera-tion in these cells; glycolysis is not only a rapid source of ATP, but a high glycolytic rate also promotes accumulation of glycolytic intermediates that are mainly fed into the pen-tose phosphate pathway for the production of amino acids, nucleotides, and NADPH ( 8 ). Another possible rationale for glycolytic energy production is the compensation of a shift in mitochondrial function from production of ATP to-ward production of mitochondrial reactive oxygen species (ROS), which was recently shown to have an important role in macrophage bactericidal activity ( 36 ).

Taken together, levels of both anaerobic and aerobic glycolysis are likely to increase in the intimal cells during lesion development, resulting in increased production of lactate and H + . The excess H + and lactate are secreted from the cells through the activity of several pumps, ex-changers, and transporters, which locally decrease the ex-tracellular pH ( Fig. 1 ). Indeed, both increased lactate concentrations ( 26 ) and extracellular acidifi cation ( 22 ) are observed in atherosclerotic lesions.

EFFECT OF LOCAL ACIDOSIS ON IMMUNE FUNCTIONS OF MACROPHAGES

It has been known for decades that macrophages are able to adapt to and survive the local acidosis that develops at acute infl ammatory sites ( 37 ). Thus, macrophages, the most abundant immune cells in atherosclerotic lesions, will remain viable despite the acidic microenvironment of the atherosclerotic intima and are subject to acidosis-induced

a key bactericidal response, the oxidative burst ( 16, 17 ). Aci-dosis greatly enhances the receptor-mediated uptake of op-sonized bacteria into macrophages thereby promoting effi cient clearance of an infection ( 18 ), and it also boosts antigen presentation by these cells via enhancing fl uid-phase endocytosis and increasing the expression of mole-cules involved in antigen presentation ( 19, 20 ). Extracellular acidity is also needed for the hydrolytic activity of lysosomal enzymes secreted by the phagocytes, and thereby tends to augment the extracellular destruction of bacteria. By allow-ing the secreted lysosomal cathepsins to retain their activity, extracellular acidity also facilitates the movement of im-mune cells into the site of action ( 21 ).

Similar to other infl ammatory sites, acidic extracellular pH is also found in atherosclerotic lesions. In the report by Naghavi et al. ( 22 ), pH values as low as 6.8 were measured using a microelectrode in the subendothelial areas of hu-man carotid plaques, and differences as great as 1.0 pH units were noted within most plaques. In addition, visual-ization of the plaques with two pH-sensitive fl uorescent dyes indicated that the pH may reach values even below 6. In this review, we describe results from various experimen-tal settings providing strong supportive evidence that an acidic microenvironment in the arterial intima could have a direct role in atherogenesis. Importantly, in the context of atherosclerosis, many of the acidity-induced physiologi-cal cellular functions that have evolved to aid, e.g., in the killing of bacteria, become maladaptive and actually may aggravate atherogenesis, particularly by affecting several key elements of the intimal lipoprotein metabolism in-volved in the progression of atherosclerosis.

MECHANISMS OF LOCAL ACIDIFICATION IN ATHEROSCLEROTIC PLAQUES

Tissue hypoxia and acidifi cation may be linked via en-hancement of glycolytic cellular metabolism at the hy-poxic areas. Hypoxic cells have been visualized in human carotid atherosclerotic lesions using the hypoxia marker pimonidazole that becomes reductively activated by intra-cellular redox enzymes at oxygen tensions � 10 mmHg and forms adducts with thiol groups of proteins ( 23, 24 ). Immunohistochemical staining of the pimonidazole ad-ducts showed strong hypoxia in macrophages near the deep intimal core regions of the lesions and, furthermore, the hypoxic macrophages colocalized with nuclear stain-ing of hypoxia-inducible transcription factor 1 � (HIF-1 � ), a major regulator of cellular response to hypoxia that in-duces, e.g., the metabolic switch to glycolysis ( 25 ). Consistent with the data from human lesions, hypoxia was detected in macrophages present in the lipid core region of advanced rabbit atherosclerotic plaques, and, moreover, high lac-tate levels were measured in the same areas indicating in-duction of glycolysis ( 26 ).

As stated above, several types of activated immune cells, including the macrophages abundant in atheromas, favor energy production by glycolysis even in a normoxic environ-ment [reviewed in ( 8, 27 )]. This phenomenon of aerobic

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Acidifi cation of the intimal fl uid 205

One of the central features emerging from studies of macrophages in acidic environments is the modulation of various cellular uptake mechanims by pH. At pH 6.5, the avidity of heat-aggregated human IgG immune complexes to monocyte and macrophage Fc � receptors (Fc � Rs) was double that at pH 7.3 ( 39 ). Furthermore, preincubation of macrophages at pH 6.0 led to enhanced Fc � R-mediated phagocytosis of IgG-opsonized latex beads and bacteria at pH 7.4 without increasing the binding of particles to the cell surface ( 18 ). Thus, exposure to an acidic environment enhances Fc � R-mediated uptake both via increased bind-ing to Fc � R and via increased activity of the internalization machinery. In a recent study, expression of the phosphati-dylserine receptor, stabilin-1, on macrophages was shown to be increased at pH 6.8, resulting in enhanced clearance of apoptotic cells ( 40 ). Finally, extracellular acidosis enhanced fl uid phase endocytosis by macrophages and dendritic cells, accompanied by increased expression of molecules involved in antigen presentation, including ma-jor histocompatibility complex I and CD86 ( 19, 20 ).

Macrophage-generated ROS are an important compo-nent of the bactericidal activity of the cells; however, ex-cessive ROS production may cause oxidative damage to the producing cell and its surroundings . Macrophage su-peroxide production in response to phorbol myristate ac-etate was decreased both by extracellular and intracellular acidosis ( 41, 42 ). On the other hand, acidic pH increases the rate of superoxide dismutation into hydrogen perox-ide, and protonation of superoxide anions at acidic pH generates a species with increased reactivity ( 43 ). Regard-ing atherosclerosis, the net effect of the apparently two-way modulatory infl uences of extracellular acidosis on ROS production are refl ected by increased iron-catalyzed extracellular LDL oxidation by macrophages, which could be explained by enhancement of the hydrogen peroxide-dependent Fenton reaction producing hydroxyl radicals ( 44 ). In addition to promoting oxidative modifi cations,

modulation of their immune functions. Of note, extracel-lular acidity also decreases the intracellular pH of macro-phages ( 38 ), which greatly amplifi es the number of pathways potentially modulated by extracellular acidosis. Because macrophages have a central role in triggering and maintaining the infl ammatory reaction in the arterial wall throughout all stages of atherogenesis, any modifi cation in their function would profoundly affect several mecha-nisms at play in lesion progression. Here, we focus on the effects of acidic extracellular pH on cells of the monocyte-macrophage lineage (summarized in Fig. 2 ). For a review of the effects of pH on lymphocytes and neutrophils, we commend the excellent review by Lardner ( 14 ).

Fig. 1. Enhanced glycolysis leads to extracellular acidifi cation. After proinfl ammatory activation, macrophages produce energy predominantly through glycolysis in both hypoxic and normoxic conditions (the “Warburg effect”), which leads to the formation and secretion of lactate and H + via the activity of various pumps, exchangers, and transporters located in the plasma membrane.

Fig. 2. The effects of local extracellular acidity on macrophage immune functions. Acidic environment enhances fl uid phase endocytosis, Fc � -receptor-mediated phagocytosis, clearance of apoptotic cells, and antigen presentation. Extracellular acidity also activates the NLRP3 infl ammasome, which results in the se-cretion of two potent proinfl ammatory cytokines, IL-1 � and IL-18.

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triggers secretion of proinfl ammatory mediators and may enhance foam cell formation ( 55 ). Of note, acidosis also increases LDL oxidation by macrophages, potentially con-tributing to the pool of immunogenic modifi ed LDL spe-cies in the lesions. On the other hand, enhancement of apoptotic cell clearance by macrophages at low pH may be a more anti-atherogenic effect. Importantly, low pH triggers the secretion of the potent proinfl ammatory cytokines, IL-1 � and IL-18, in macrophages. For example, IL-1 � in-creases the expression of adhesion molecules on endothe-lial cells to attract more infl ammatory cells into the lesion and IL-18 contributes to induction of highly proathero-genic IFN- � in T cells ( 56 ). Low pH can also boost antigen presentation in macrophages and thus contribute to induc-tion of adaptive immune responses in the lesions. Thus, aci-dosis promotes many key immune functions of macrophages with a predominantly proinfl ammatory effect. Although these effects may be benefi cial for effi cient clearance of acute infl ammation, they seem maladaptive in the context of atherosclerosis. In atherosclerosis, the local infl amma-tory response develops mainly under sterile conditions and excessive unresolved infl ammation promotes lesion devel-opment and instability of the plaques.

ACIDIC EXTRACELLULAR pH INCREASES THE RETENTION OF ATHEROGENIC LIPOPROTEINS

Atherosclerosis is characterized by the extra- and intra-cellular accumulation of lipoprotein-derived lipids. Low extracellular pH affects many of the processes involved in lipid accumulation ( Fig. 3 ). Upon entering the subendo-thelial arterial intima, LDL particles encounter a dense extracellular matrix network rich in proteoglycans, collagen, and elastin, with which the LDL particles tend to interact. Especially important is the interaction with proteoglycans ( 57, 58 ), which initiates LDL retention in the intima ( 59, 60 ), particularly at the atherosclerosis-prone sites, where the proteoglycan composition favors the retention of apoB-containing lipoproteins ( 61, 62 ). In addition to LDL, other apoB-containing lipoproteins (chylomicron rem-nants, VLDL, and IDL) also bind to proteoglycans, albeit less tightly ( 63–65 ), and can therefore contribute to lipid accumulation in the intima ( 66 ). Recently, Mendelian ran-domization studies have provided strong supportive evidence for the causative roles of both LDL and triglyceride-rich lipoproteins in the development of cardiovascular disease ( 67 ).

The affi nity of lipoproteins for proteoglycans is quite low at neutral pH, but acidic pH signifi cantly enhances the binding of all three atherogenic apoB-100-containing lipoproteins (VLDL, IDL, and LDL) to human aortic pro-teoglycans ( 68, 69 ). The lipoprotein-proteoglycan interac-tion is mediated by certain positively charged sequences in apoB which contain lysine and arginine residues and nega-tively charged sulfate and carboxyl groups in the proteo-glycan glycosaminoglycan chains. At acidic pH, additional sequences of apoB are likely to be important for the inter-action: because the pK a of histidine side chains is � 6.0, the

acidic pH may also promote nitrosative damage, because an acidic environment induces the expression of the in-ducible nitric oxide synthase in macrophages, resulting in nitrite accumulation in the culture medium ( 45 ).

Activated macrophages secrete a plethora of proinfl am-matory cytokines and mediators that contribute to the in-fl ammatory reaction in the atherosclerotic intima. Bellocq et al. ( 45 ) have shown that 2 h culture in medium adjusted to pH 7.0 activates nuclear factor (NF)- � B, the key inducer of proinfl ammatory cytokine expression in rat and mouse macrophages via a positive feedback loop of TNF- � secre-tion and autocrine signaling. In contrast, other studies have shown that low pH (pH 7.0–5.5) inhibits LPS-induced TNF- � secretion (mediated by NF- � B) in mouse and rab-bit, but not in human macrophages ( 46–49 ). Accordingly, an inhibitory effect by low pH on LPS-induced NF- � B sig-naling was found in mouse, but not in human macro-phages ( 46, 49 ). Differences in the study setups and the pH range used likely explain the contrasting results obtained in mouse macrophages. Human macrophages, based on Gerry and Leake ( 49 ), seem less sensitive to modulation of NF- � B activity by low pH, but more data on NF- � B target binding at a wider range of acidic pH values is required before fi rm conclusions can be made.

Recently, we and others have shown that acidic pH stimu-lates the secretion of the key proinfl ammatory and proath-erogenic cytokine IL-1 � in primary human monocytes and monocyte-derived macrophages ( 38, 50 ). IL-1 � production is tightly regulated; the inactive procytokine, pro-IL-1 � , is only expressed following specifi c stimulation, and cleavage of the procytokine by caspase-1 protease is required for bio-logical activity. Monocytes constitutively express active caspase-1, and thus IL-1 � is proteolytically activated and secreted immediately after induction of pro-IL-1 � expres-sion ( 51 ), as exemplifi ed by acidic pH-induced pro-IL-1 � expression and secretion ( 50 ). In contrast, caspase-1 in macrophages is in the inactive pro-form and, therefore, pro-IL-1 � expression and caspase-1 activation are both required for secretion of mature IL-1 � ( 51 ). We showed that extra-cellular acidity has no effect on pro-IL-1 � expression in mac-rophages. However, when macrophages were stimulated with LPS to produce pro-IL-1 � , extracellular pH 6.0–7.0 triggered activation of caspase-1 via the NLRP3 infl amma-some and high-level secretion of mature IL-1 � , as well as that of IL-18, another caspase-1 target cytokine ( 38 ). We also found synergy between low pH and cholesterol crystals, another activator of the NLRP3 infl ammasome ( 52, 53 ), in induction of IL-1 � secretion ( 38 ). Confi rming the strong proinfl ammatory potential of acidic environment, a very recent microarray study compared macrophage gene ex-pression at pH 7.4 and 6.8 and found 353 differentially expressed genes that showed marked enrichment of path-ways related to infl ammation and immune responses ( 54 ).

How might these changes in macrophage immune func-tion relate to atherogenesis in the arterial wall? As discussed above, extracellular acidosis increases the Fc � R-mediated uptake of immune complexes. Immune complexes of mod-ifi ed LDL and corresponding antibodies have been found in atheromas, and their uptake to macrophages via Fc � Rs

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values ( 75 ) and LPL is able to bind to lipid droplets at least at pH values as low as pH 6.5 ( 76 ). Together, these pieces of information suggest that acidic pH would most likely also enhance this type of lipoprotein retention in the arte-rial intima.

ACIDITY ENHANCES MODIFICATION OF apoB-CONTAINING LIPOPROTEINS

LDL particles isolated from atherosclerotic lesions show signs of various modifi cations, such as oxidation, proteoly-sis, and lipolysis, which are indicative of oxidative, proteo-lytic, and lipolytic enzyme activities in the lesions ( 77 ). Such modifi ed LDL particles are often aggregated and/or fused into large lipid droplets. Acidic extracellular pH may increase lipoprotein modifi cation ( 71, 78 ), which may in turn further decrease the extracellular pH (see below). Acidic proteases, such as cathepsins, are found extracellu-larly in normal and atherosclerotic intima, and they effi -ciently proteolyze apoB-100 leading to LDL particle fusion ( 79–81 ). Moreover, proteolysis of apoB-100 sensitizes the LDL particles to lipases, thereby promoting their lipolytic modification ( 82, 83 ). Interestingly, activated macro-phages secrete cathepsins together with H + ions and so, by acidifying their local microenvironment, provide optimal conditions for activity of these secreted proteases ( 84 ).

positive charge of the histidine residues increases as the pH decreases around this pH, histidine residues of apoB-100 may also be involved in the interaction with proteogly-cans. In contrast, the negative charge of the sulfate and carboxyl groups in the proteoglycans are unlikely to be affected by the degree of acidifi cation found in the arterial intima, because they have a pK a of <2.0 and 3.02–4.37, respectively ( 70 ). Thus, the intimal acidity, even if reach-ing a pH value of 6, would not result in protonation of the sulfate and carboxyl groups, and the net negative charge of the proteoglycans will be preserved, thereby allowing interactions with positively charged lipoproteins and other molecules. Interestingly, proteoglycans may contribute to extracellular acidifi cation, mediated by the attraction of H + to their negatively charged sulfate and carboxyl groups ( 71 ). Although the negatively charged groups attract H + ions and other cations, they do not bind the ions at the slightly acidic pH values present in the arterial intima. This can cause differences in the distribution of the ions and lower the pH in the vicinity of the proteoglycans.

Lipoproteins may be retained in the arterial intima also via bridging molecules, such as LPL ( 72–74 ). Thus, LPL binds to proteoglycans via ionic interactions of high affi n-ity and to lipoproteins via hydrophobic interactions. There are no studies in which the effect of acidic pH has been studied on this bridging property of LPL. However, the binding of LPL to heparin is enhanced at mildly acidic pH

Fig. 3. The effects of extracellular acidity on extracellular and intracellular cholesterol accumulation. Ex-tracellular acidity enhances retention, modifi cation, and aggregation of LDL, and so promotes both extra- and intracellular cholesterol accumulation. Extracellular acidity also remodels HDL particles with generation of pre � -HDL. However, in acidic environments, pre � -HDL is prone to degradation by acidic proteases. Moreover, acidity decreases the expression of the ABCA1 transporter and the secretion of apoE by macro-phage foam cells, so decreasing cholesterol effl ux from these cells.

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molecules is determined by interaction of the phosphate and amine groups with counterions, such as protons and hydroxide ions ( 109 ). At neutral pH, the lipid head groups associated with hydroxide ions are the predominant form, meaning a net negative charge, but the increased proton concentration of acidic pH tends to enhance the associa-tion of the protons with the head groups and so enhance their charge-neutral character ( 109, 110 ). This may en-hance the interaction between LDL and PLA 2 -V and so explain the observed acidity-increased activity of PLA 2 to-ward LDL ( 69 ). Acidity also affects the fate of the products of PLA 2 activity. At neutral pH and physiological albumin concentration, most of the FFAs and lysophospholipids generated through PLA 2 -dependent hydrolysis of LDL particles are readily scavenged by albumin; however, at acidic pH the ability of albumin to bind the lipolytic prod-ucts is decreased, and more of the lipolytic products re-main in the LDL particles ( 111 ). This likely results from the property of albumin to preferentially bind the FFAs in their anionic form ( 112, 113 ); at acidic pH the FFAs are largely uncharged.

SMase hydrolyzes sphingomyelin on the surface of lipo-protein particles into ceramide and phosphocholine. Se-cretory SMase, a product of the acid SMase gene, is able, at neutral pH, to hydrolyze PLA 2 -digested and proteolyzed LDL or apoC-III-enriched LDL, as well as LDL extracted from human atheromata ( 82, 114 ). Digestion induced by secretory SMase is promoted at acidic pH ( 82, 114, 115 ). In vitro, SMase-modifi ed LDL particles promptly aggregate, the aggregate size increasing by synergy with chondroitin-sulfate-rich proteoglycans and LPL ( 72 ). Aggregate size after SMase digestion also increases as the pH drops, and these aggregates at pH 5.5 can ultimately span several micrometers ( 116 ). Consistent with these in vitro results, some of the ceramide-containing LDL particles in human atherosclerotic lesions become large micron-sized aggre-gates ( 117 ). Ceramide efficiently displaces cholesterol from lipid bilayers into the crystalline phase, thus promot-ing cholesterol crystal nucleation ( 118, 119 ). Therefore, the combined actions of SMase and cholesterol esterase on LDL may be important for cholesterol crystallization ( 87, 94 ), particularly in acidic environments, where these enzymes are most active.

Oxidative modifi cation of lipoprotein particles leads to formation of lipid peroxides, which further decompose into aldehydes that react with the protein components of the particle. Acidic pH enhances oxidation of LDL by iron, nitric oxide, and myeloperoxidase [reviewed by Leake ( 71 )]. Interestingly, acidic pH induces the aggrega-tion of oxidized LDL ( 120 ), but, unexpectedly, LDL oxi-dized at acidic pH is less cytotoxic than LDL oxidized at neutral pH ( 121 ). In contrast to the proteolytic and lipolytic modifi cations that enhance LDL-proteoglycan binding, oxidation decreases the binding of LDL to proteoglycans due to the neutralization of positively charged lysine resi-dues in apoB-100 ( 122 ). However, oxidized LDL particles can bind to human aortic proteoglycans under acidic con-ditions despite the oxidation-induced decrease in their affi nity for proteoglycans ( 68 ).

Finally, when macrophages latch onto large aggregates of LDL, they create partially sealed compartments on the sur-face of these aggregates, drop the pH, and secrete lyso-somal acid lipase (LAL). This enzyme then hydrolyzes the cholesteryl esters of the aggregated lipoproteins into un-esterifi ed cholesterol and fatty acids. Unesterifi ed cho-lesterol can enter the cells and the fatty acids can further acidify the microenvironment ( 85, 86 ). This process is analogous to the way osteoclasts, macrophage-like cells, normally latch onto bone and degrade it through the for-mation of sealed compartments at low pH ( 85 ).

The free cholesterol generated from lipoproteins or other extracellular lipid deposits by the secreted acidic LAL may contribute to the nucleation and growth of cho-lesterol monohydrate crystals in the lesions. Large choles-terol crystals are easily visible by microscopic examination of atherosclerotic arteries, and they are a hallmark of ad-vanced atheromas; however, smaller crystals are also found in about one third of intermediate lesions that lack ne-crotic cores ( 87, 88 ). Using a new microscopic technique, cholesterol microcrystals were detected in the aortic wall of apoE-defi cient mice after just 2 weeks of high choles-terol diet, coinciding with the fi rst appearance of macro-phages ( 52 ). As discussed above, cholesterol crystals were recently shown to elicit an infl ammatory response via the NLRP3 infl ammasome, but the mechanisms of crystal nu-cleation in atherosclerotic lesions in vivo remain elusive. Electron microscopy of human atherosclerotic lesions has shown that cholesterol crystal growth occurs predomi-nantly in the matrix-embedded extracellular lipid deposits of the deep arterial intima, whereas most macrophage foam cells reside in the more superfi cial intimal layer ( 89 ). Although less frequent, cholesterol crystals were also found inside macrophage foam cells in human lesions ( 89 ), and thus, the original site of crystal nucleation could not be defi ned with certainty. However, more recent in vitro stud-ies have shown that cholesterol crystal nucleation can oc-cur both within macrophage foam cells ( 87, 90–93 ) and on the surface of enzyme-modifi ed LDL particles ( 94 ), of which the latter mechanism may indeed be amplifi ed by secreted LAL in an acidic environment. Acidity may also directly affect cholesterol crystallization and the interac-tion of cholesterol crystals with each other, lipoproteins, or cell membranes ( 95–97 ).

Phospholipase A 2 (PLA 2 ) enzymes hydrolyze sn-2 ester bonds in glycerophospholipids, yielding lysophospholip-ids and FFAs, which have been shown to have proinfl am-matory effects ( 98–102 ). In addition, as noted above, the FFAs may contribute to extracellular acidifi cation. Several secreted PLA 2 enzymes with different substrate specifi ci-ties and lipolytic activities are present in human athero-sclerotic arteries ( 98, 103, 104 ). PLA 2 -V is one of the enzymes that may modify lipoproteins in the intima ( 105, 106 ), and at acidic pH it is more active against lipoproteins ( 69 ). The activity of the PLA 2 s is largely determined by their ability to bind to substrate membranes ( 107, 108 ) and it is sensitive to changes in the lipid membrane induced, e.g., by acidic pH. The electric charge distribution at the membrane interface of zwitterionic phosphatidylcholine

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( 132 ). HDLs are also capable of mediating intercellular communication among different types of cells by a mecha-nism that involves the transfer of endogenous miRNAs among the body compartments, which may partly explain the high versatility of HDL function ( 133 ).

Cholesterol effl ux induced by HDL initiates reverse cholesterol transport (RCT), which transfers peripheral cholesterol to the liver for ultimate excretion in the gut ( 134 ). Because reduction of the cholesterol pool in arte-rial macrophages can prevent progression of atherosclero-sis or even induce its regression, the particular path of RCT that initiates in the macrophage foam cells (macro-phage-RCT) is the most relevant fraction of the total body RCT regarding atherosclerosis ( 135 ). In human macro-phage foam cells, enhanced cholesterol effl ux in response to LXR activation appears to be entirely dependent upon the lipid transporter ABCA1 ( 136 ), which promotes cholesterol effl ux to lipid-free apoA-I and to the nascent lipid-poor pre � -migrating HDL subpopulation (pre � -HDL) ( 137 ). Importantly, cholesterol loading induces a compen-satory response in macrophages by upregulating ABCA1 mRNA and protein expression ( 138 ).

Various conditions present in the atherosclerotic in-tima, such as hypoxia ( 139 ), infl ammation ( 140 ), and oxi-dative stress ( 141 ), reduce the ABCA1-mediated cholesterol effl ux from macrophage foam cells. We found that acidic pH also reduces ABCA1-mediated effl ux of cholesterol from cultured human macrophage foam cells to apoA-I ( 142 ). Because the � -helical content and secondary struc-ture of lipid-free apoA-I are not affected by low pH ( 109 ), our fi nding strongly suggests that acidity impairs the func-tion of ABCA1 rather than the function of apoA-I. Con-sistent with this speculation, we found that impaired cholesterol effl ux from macrophages cultured in medium with a pH value of pH 5.5 or 6.5 is accompanied by pro-gressive reduction in the levels of the ABCA1 protein. In accord with this in vitro fi nding, the ABCA1 protein level is signifi cantly reduced in whole extracts of human carotid atheromas ( 143, 144 ), and, moreover, ABCA1 mRNA was found not to be expressed in the foam cells within the ne-crotic core of advanced plaques in human atherosclerotic aortas ( 145 ), where the intimal fl uid most likely has an acidic pH. It is plausible to assume that acidifi cation of the extracellular fl uid has additional effects on the activity of ABCA1 in cholesterol effl ux; this may be through altera-tions in the physical properties of ABCA1 and the spatial geometry of the plasma membrane, which is thought to be the main location of ABCA1-mediated apoA-I lipidation ( 146 ), or through effects on the electrostatic interaction between ABCA1 and apoA-I ( 147 ). Moreover, secretion of apoE, which also stimulates ABCA1-mediated lipid effl ux ( 148 ), is reduced in macrophages incubated at acidic pH ( 142 ). Taken together, these fi ndings support the notion that the cholesterol effl ux-mediating activity of ABCA1 in macrophages is reduced by the low extracellular pH found in advanced atherosclerotic lesions ( 9 ). Because the inter-action of ABCA1 with apoA-I inhibits the expression of in-fl ammatory cytokines in macrophages ( 149 ), the lack or low activity of ABCA1 in acidic microenvironments may

ACIDITY INCREASES LDL UPTAKE BY MACROPHAGES

The appearance of macrophage-derived foam cells in the intima is the hallmark of developing atherosclerotic lesions. Foam cells are formed when macrophages take up modifi ed apoB-containing lipoproteins via various mecha-nisms, including scavenger receptor-mediated uptake and phagocytosis ( 124 ). Also, aggregated LDL bound to the components of the extracellular matrix produced by smooth muscle cells are readily taken up by macrophages ( 72 ). Extracellular acidosis enhances the uptake of native and modifi ed LDL particles by macrophages through in-creases in the levels of cell surface proteoglycans, and of LDL-proteoglycan binding ( 69, 125 ). These proteoglycans are most likely heparan-sulfate-rich proteoglycans of the syndecan family ( 126, 127 ). Extracellular acidosis may also accelerate foam cell formation by enhancing lipoprotein modifi cations that promote lipoprotein uptake by macro-phages ( 69, 79, 116, 120 ). In addition, Howard Kruth has proposed a model of foam cell formation that does not involve LDL modifi cation or macrophage receptors. Thus, when macrophages are incubated with high LDL concen-trations comparable to those found the intimal interstitial fl uid ( 128 ), foam cells are formed as a result of fl uid phase pinocytosis of unmodifi ed LDL particles ( 129 ). Fluid phase pinocytosis by macrophages has been reported to be increased at acidic pH ( 19, 20 ). Thus, most of the pro-posed mechanisms involved in the uptake of lipoproteins by macrophages and foam cell formation are augmented at acidic extracellular pH ( Fig. 3 )

ACIDITY DECREASES CHOLESTEROL EFFLUX FROM MACROPHAGE FOAM CELLS AND INDUCES

HDL REMODELING

By promoting cholesterol effl ux from macrophage foam cells and by inducing anti-infl ammatory effects in macrophages and endothelial cells, HDL particles are also thought to possess strong atheroprotective functions in vivo ( 130 ). Although an inverse relation between plasma HDL-cholesterol levels and the rate of atherosclerosis pro-gression has been documented in experimental animal models and in human population studies by aid of imag-ing of atherosclerotic lesions, recent clinical and genetic studies have failed to confi rm the hypothesis of plasma HDL-cholesterol level being per se a determinant of, at least, the fi nal atherothrombotic events in humans ( 131 ). Such failures to therapeutically modify atherogenesis in humans have actually led to a conservative skepticism regarding the benefi ts of HDL-oriented therapies and may have their root cause in our incomplete comprehension of the high complexity of the HDL particles. Indeed, it cur-rently appears that the capability of HDL to prevent ath-erosclerosis depends on both quantitative and qualitative features of their proteome and lipidome, which ultimately translates itself into functional differences not detected by simply measuring the plasma levels of HDL-cholesterol

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regions of the arterial tree. In contrast to the rather straightforward scenario of a proatherogenic role of acid-ity on intimal accumulation of cholesterol derived from apoB- 100-containing lipoproteins, HDL metabolism in the acidic intimal fl uid appears to be complex and unpre-dictable in its potential outcomes. Such entanglement of HDL metabolism in advanced human atherosclerotic le-sions, if present, could be one of the root causes of the failure in designing a clinically relevant HDL-based strat-egy for anti-atherogenic therapies.

Atherosclerotic lesions contain a heterogeneous mix-ture of macrophage phenotypes, and some of them may confer more resistance to acidity and hypoxia than others. Recent fi ndings indicate that macrophage proliferation within the plaque plays an important role in the regula-tion of the size of the macrophage population in athero-sclerotic lesions ( 156 ). This raises the intriguing possibility that the macrophage population in the plaque evolves over time through selection of acid-resistant macrophages, analogous to the evolution of an acid-resistant cell popula-tion in solid tumors ( 157 ). There may be an additional level of selection if smooth muscle cells in the atheroscle-rotic lesion are sensitive to acid-induced cellular toxicity. Indeed, extracellular acidosis inhibits proliferation and migration of vascular smooth muscle cells , and also increases their susceptibility to apoptosis ( 158 ). Thus, in response to the selection pressure from the microenvironmental pH, the lesion could gradually become enriched with acid-resistant macrophages and depleted of smooth muscle cells, a population imbalance that is found in the rupture-prone subset of atherosclerotic lesions ( 159 ). Such a sce-nario invites us to envision that acidic pH in atherosclerotic lesions not only promotes atherogenesis, but may also contribute to the often lethal atherothrombotic complica-tions of the disease.

After having defi ned the acidic intimal environment as a perfect soil for atherogenesis, we need to ask: how can it be changed back to neutral? Obviously, we do not know the answer, but are compelled to fi nd it. It is of great inter-est to note that recent developments in nanoparticle-based therapy of cancer are exploiting the acidic extracellular environment of a tumor for targeted drug delivery to can-cer cells ( 160 ). Importantly, recent understanding of the similarities between cancer cells and infl ammatory cells has unraveled the role of AMP-activated protein kinase as an inhibitor of glycolysis that boosts oxidative phosphory-lation ( 8, 27, 161 ). The AMP-activated protein kinase is activated by certain drugs and xenobiotics, most notably by the type 2 diabetes drug, metformin, and by the classic anti-infl ammatory drug, salicylate ( 8, 27, 161 ). This new information, coupled with the developing technologies for acid-dependent drug delivery, might guide us when searching for new therapeutics and anti-atherogenic strat-egies. The prospects of a successful novel proton-lowering strategy are increased when considering that attenuation of the rate of glycolysis in macrophages may be associated with a phenotypic shift from a proinfl ammatory into an anti-infl ammatory macrophage ( 8 ).

also exacerbate atherogenesis by leading to enhanced pro-infl ammatory responses in the lesional macrophages.

A small labile pool of apoA-I constantly recycles on and off HDL particles during metabolic remodeling in vivo ( 150, 151 ), thereby exchanging apoA-I molecules with the pre � -HDL pool. In this regard, we have shown that an acidic pH in vitro promotes remodeling of the mature HDL particles resulting in formation of pre � -HDL and fu-sion of the � -migrating HDL ( 152 ). Such remodeling was initiated by unfolding of the apolipoproteins on the sur-face of HDL particles, which was followed by the release of apoA-I from the particles, resulting in the generation of unstable apoA-I-defi cient HDL particles that then fuse. However, it is important to note that because lipid-poor apoA-I is extremely sensitive to proteolysis, the proteases known to be secreted by intimal cells will easily render it nonfunctional ( 153 ). Indeed, we have found that various acidic cathepsins found in atherosclerotic lesions ( 81, 79 ) also effectively degrade apoA-I both in lipid-free and lipid-poor forms with loss of their cholesterol effl ux-inducing activity ( 155 ). Thus, the production of HDL-derived lipid-free or lipid-poor apoA-I in atherosclerotic plaques may increase as a result of acidifi cation of the intimal fl uid, but such generated apoA-I species may also be degraded when acidic proteases gain in function in the low pH microenvi-ronment ( Fig. 3 ).

Based on the above-described fragmentary and appar-ently two-way processes regarding the effects of acidity on HDL-dependent mechanisms which regulate cholesterol effl ux from macrophage foam cells, the envisioned sce-nario of such acidity-dependent effects on atherogenesis remains undefi ned. Thus, while acidity induces remodel-ing of HDL and ensuing generation of lipid-poor apoA-I species, the lipid-poor species of apoA-I may easily be lost due to extracellular degradation by acidic proteases. Moreover, the expression of ABCA1 in macrophage foam cells at acidic pH is low or absent, and so may compromise cholesterol clearance from foam cells in an arterial seg-ment with an acidic extracellular pH ( 142 ). Because the fi rst of the three acidity-induced processes tends to in-crease, and the two latter ones tend to decrease choles-terol effl ux, it is impossible even to predict the net effect of acidity on cholesterol effl ux from macrophage foam cells in an acidic environment in vivo.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

By virtue of its ability to enhance extracellular and intra-cellular lipid accumulation and to promote proinfl amma-tory processes in macrophages, extracellular acidity has emerged as a novel and potentially crucial element of ath-erogenesis. Extracellular acidity modifi es various proin-fl ammatory functions of macrophages, e.g., by triggering the secretion of potent proinfl ammatory cytokines by mac-rophages via activation of the infl ammasome pathway ( 38 ). Acidity also aggravates extra- and intracellular ac-cumulation of cholesterol in the atherosclerosis-prone

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Acidification of the intimal fluid: the perfect storm for atherogenesis

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