Acidic extracellular pH promotes accumulation of free cholesterol in human monocyte-derived macrophages via inhibition of ACAT1 activity.

BACKGROUND AND AIMS: In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. METHODS: - Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [3H]oleate-BSA complexes, and the formation of [3H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. RESULTS: - In acidic media, ACAT1-dependent cholesteryl [3H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. CONCLUSIONS: Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding.

BACKGROUND AND AIMS: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. METHODS: Mature human MCs were differentiated from human peripheral blood-derived CD34+ progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. RESULTS: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. CONCLUSIONS: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention.

Acidification of the intimal fluid: the perfect storm for atherogenesis.

Atherosclerotic lesions are often hypoxic and exhibit elevated lactate concentrations and local acidification of the extracellular fluids. The acidification may be a consequence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycolysis for ATP synthesis even under normoxic conditions, resulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current understanding 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 presentation by macrophages and, importantly, triggers the secretion of proinflammatory cytokines from macrophages through activation of the inflammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifications of LDL and other apoB-containing lipoproteins, and strongly increases their affinity 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 amplifies the proatherogenic and proinflammatory processes involved in atherogenesis.

Acidity increases the uptake of native LDL by human monocyte-derived macrophages.

The extracellular pH is locally decreased in advanced atherosclerotic lesions, particularly in lipid-rich areas of the lesions. Since accumulation of LDL-derived cholesterol and formation of foam cells are key processes in atherogenesis, we tested here the effects of acidic pH on the uptake of native LDL. First, human monocytes were differentiated into macrophages in the presence of granulocyte-monocyte-colony stimulating factor (GM-CSF) after which native LDL was applied to the monocyte-derived macrophages at pH 5.5, 6.5, or 7.5 and the binding and uptake of LDL by macrophages were determined. The lower the pH was, the higher was the binding and uptake of LDL by macrophages. Also, acidic pH was found to increase the production of cell surface proteoglycans by macrophages and binding of LDL to the glycosaminoglycan chains of the proteoglycans. The acidity-induced increase in the uptake of LDL by macrophages could be inhibited by pretreating the cells with heparinase and chondroitinase as well as by inhibiting the production of proteoglycans with NaClO(3). Thus, the observed increase in the uptake of native LDL to macrophages appears to depend on the increased ability of LDL to bind to cell surface proteoglycans at acidic pH. Taken together, our present results indicate that acidity increases the effective concentration of LDL on macrophage surfaces by increasing the amount of cell surface proteoglycans and by enhancing the binding of LDL to them and so promotes LDL uptake with ensuing foam cell formation.

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase.

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A2 (sPLA2-IIa and sPLA2-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA2-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA2-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA2-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.

Phospholipase A(2)-modified LDL particles retain the generated hydrolytic products and are more atherogenic at acidic pH.

OBJECTIVE: Hydrolysis of LDL by phospholipase A(2) (PLA(2)) generates free fatty acids (FFAs) and lysophospholipids (lysoPCs). Binding of the PLA(2)-modified LDL to proteoglycans, and their uptake by macrophages are increased. Since the extracellular pH is locally decreased in advanced atherosclerotic plaques, we examined the effects of acidic pH on PLA(2)-induced LDL modification and its proatherogenic consequences. RESULTS: LDL particles were avidly hydrolyzed by sPLA(2)-V at pH range 7.5-5.5. With decreasing pH, the ability of albumin to sequester the formed FFAs and lysoPCs from the sPLA(2)-V-modified LDL particles decreased, and, as a consequence, more of the hydrolytic products accumulated in the particles. At acidic pH, the sPLA(2)-V-modified LDL particles had higher binding strength to human aortic proteoglycans, and their uptake by human monocyte-derived macrophages and ensuing foam cell formation were enhanced. CONCLUSIONS: The present data show that the proatherogenic effects exerted by sPLA(2)-V-induced lipolysis of LDL are enhanced with decreasing pH and suggest that sPLA(2)-V is particularly atherogenic in advanced atherosclerotic lesions, in which local acidic conditions prevail.