Doctor of Medicine and Doctor of Philosophy (MDPhD)
The role of smooth muscle cell lysosomal acid lipase in atherosclerosis
Lipids are important players in the host response to sepsis. High-density lipoprotein (HDL) binds avidly to pathogen lipids, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), neutralizing their inflammatory effects. Pathogen lipids within HDL are transferred to low-density lipoprotein (LDL) and cleared from the circulation by the liver in a process mediated by the LDL receptor (LDLR). In recent decades, more patients are surviving sepsis and being discharged from hospital. Studies analyzing long-term outcomes of sepsis have demonstrated a greater risk of late death, re-infection(s), late organ dysfunction(s), and re-hospitalization(s) in sepsis survivors compared to subjects never exposed to sepsis. Inadequate clearance of pathogens at the acute phase of sepsis and persistent immune dysfunction are possible factors associated with increased risk of adverse long-term outcomes in sepsis survivors. In this work, we hypothesized that plasma HDL-cholesterol levels are positively associated with decreased risk of sepsis-associated acute kidney injury (AKI), late kidney impairment or death, and that genetic variants in genes known to regulate HDL-C would impact the risk of AKI during sepsis. Finally, the role of proprotein convertase subtilisin-kexin type 9 (PCSK9),a major regulator of LDL plasma levels and LDL receptor metabolism in the long-term outcomes of sepsis was evaluated through: i) analysis of the impact of PCSK9 loss-of-function genotype in a composite outcome composed by 1-year death or infection-related readmission (IRR), and ii) analysis of the effects of PCSK9 inhibitors on the long-term inflammation in mouse model of sepsis. This study demonstrated that low plasma levels of HDL-C measured at sepsis admission increased significantly the risk of AKI, kidney dysfunction and/or long-term death. Moreover, the HDL-related cholesteryl ester transfer protein (CETP) variant rs1800777 (allele A) was strongly associated with low levels of HDL-C and increased risk of AKI during sepsis. Last, we observed that the presence of multiple PCSK9 loss-of-function alleles decreased the risk of the death or IRR in sepsis-survivors.
Smooth muscle cells (SMCs) are the predominant cell type in the intima of human atherosclerosis-prone arteries and promote initial retention of atherogenic lipoproteins in the deep intima. We previously found that ≥50% of foam cells in intermediate coronary atheromas are of SMC origin and that intimal SMCs have reduced expression of the cholesterol exporter protein ATP-binding cassette transporter A1 (ABCA1). ABCA1 expression is dependent on the flux of cholesterol out of lysosomes, generated via the hydrolysis of lipoprotein-derived cholesteryl esters (CEs) to cholesterol by lysosomal acid lipase (LAL), and subsequent generation of oxysterols such as 27-hydroxycholesterol by CYP27A1 for promotion of gene transcription via the nuclear liver X receptor (LXR). In the present studies we tested the hypothesis that SMCs have reduced lysosomal function that contributes to foam cell formation. Chapter 2 investigates and defines the role of lysosomal function in cholesterol metabolism using a mouse LAL KO peritoneal macrophage model. Chapter 3 investigates lysosomal function and cholesterol metabolism differences between human monocyte-derived macrophages (HMMs) and arterial SMCs treated with aggregated LDL (agLDL). Unlike HMMs, lipid loading of SMCs did not significantly increase 27-hydroxycholesterol or ABCA1 levels and did not decrease new cholesterol synthesis. Microscopy revealed sequestration of CEs in lysosomes of SMCs, while HMMs displayed mostly cytosolic CE accumulations. We did not find evidence of a lysosomal functional defect from lipid induced loss of acidity or loss of lysosomal proteolytic function in SMCs. Instead, LAL levels were markedly higher in macrophages compared to SMCs (LAL activity 23.4-times higher in agLDL loaded HMMs compared to SMCs, p
The key regulator of initial HDL particle formation by cells is the ATP-binding cassette transporter A1 (ABCA1). ABCA1 expression is regulated primarily by oxysterol dependent activation of the liver X receptor (LXR). We investigated the role of lysosomal cholesterol on ABCA1 regulation by studying the lysosomal disorder Cholesteryl Ester Storage Disease (CESD). CESD is caused by genetic mutations in the LIP-A gene that result in only 5% of normal activity of lysosomal acid lipase (LAL), an enzyme that hydrolyzes cholesteryl esters (CE) and triglycerides on internalized lipoproteins specifically within the lysosome. We hypothesized that the flux of unesterified cholesterol out of the lysosomes from LAL-mediated hydrolysis of LDL cholesteryl esters is a key regulator of cellular ABCA1 expression, HDL formation and reverse cholesterol transport (RCT). We found that primary skin fibroblasts derived from individuals with CESD had impaired upregulation of ABCA1 in response to LDL loading, reduced phospholipid and cholesterol efflux to apoA-I, lower production of 27-hydroxycholesterol (27-OH) production in response to LDL loading and reduced α-HDL particle formation. This defect was recapitulated in normal fibroblasts following treatment with LAL inhibitors, whereas, treatment with conditioned medium from normal fibroblasts containing secreted LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation and production of 27-OH by CESD cells. We further investigated the role of LAL in RCT from macrophages specifically using an immortalized macrophage cell line created from LAL-deficient mouse peritoneal macrophages (LAL-/-). LAL-/- macrophages exhibited reduced basal and cholesterol-stimulated ABCA1 expression in culture, and reduced ability to support RCT in LAL-/- mice compared to wild-type (LAL⁺/⁺) macrophages injected into LAL⁺/⁺ mice. ABCA1 protein expression was reduced in LAL-/- mouse liver and mRNA expression of several LXR-dependent genes involved in reverse cholesterol transport (ABCG1, ABCG5, ABCG8, CYP7A1, SR-B1) were differentially modulated compared to LAL⁺/⁺ controls. These results indicate a critical role of LAL in promoting lysosomal flux of cholesterol for ABCA1 expression, cellular cholesterol efflux and RCT in vivo.
The removal of cholesterol from cells and formation of high-density lipoprotein (HDL) particles is critically dependent on the membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1). Previous studies from the Francis laboratory have determined that the regulation of ATP-binding cassette transporter A1 (ABCA1) expression is impaired in model intimal-type arterial smooth muscle cells (SMCs) and human intimal SMCs in atherosclerotic coronary arteries, providing a novel explanation for cholesterol accumulation and foam cell formation in human atheroma. Growing evidence appears to emphasize the importance of the lysosomal-mitochondrial oxysterol pathway in regulating ABCA1 expression. We hypothesized that the reduced expression of ABCA1 in model intimal-type arterial SMCs is due to impaired enzyme sterol 27-hydroxylase (CYP27A1) expression, resulting in impaired oxysterol production critical for the activation of ABCA1 gene expression via the Liver X receptor (LXR) pathway. Our results show that intimal-type arterial SMCs exhibit a reduced expression of CYP27A1 mRNA and protein. Exogenous treatment of these cells with LXR agonists increases ABCA1 expression, indicating an intact LXR-mediated activation of ABCA1 gene expression. Despite successful transfection of CYP27A1 in these SMCs, intimal-type arterial SMCs do not increase their expression of ABCA1. The expression of steroidogenic acute regulatory protein D1 (StARD1), responsible for the delivery of cholesterol to CYP27A1, is also reduced in intima-type SMCs. We also show preliminary results of reduced human arterial intimal SMC-specific CYP27A1 expression in coronary arteries with native atherosclerosis. The findings of this thesis provide a narrative highlighting the importance of the intracellular transport of cholesterol via StaRD1 to CYP27A1 for the activation of LXR-dependent ABCA1 expression in arterial SMCs, and provide further insight into the dysregulation of ABCA1 expression in human atherosclerotic arterial SMCs that may contribute to the foam cell population and subsequent plaque formation in human atherogenesis.