The role of hepatic SURF4 in the lipid metabolism and the development of atherosclerosis

Abstract

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of morbidity and mortality worldwide. Long-term elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) are the major risk factors for developing atherosclerotic lesions, which can eventually lead to myocardial infarction and stroke. Lowering plasma LDL-C levels remains the main way to prevent CVD. LDL is removed from the circulation mainly by the hepatic LDL receptor. Mutations in this receptor cause familial hypercholesterolemia, which is characterized by elevated plasma levels of LDL-C and increased risk of CVD. Statins, the most-prescribed drug lowering plasma cholesterol levels, increase the expression of LDL receptor and reduce cardiovascular events by 20% to 40%. However, about 15% of people treated with statins show statin intolerance and require alternative therapies to lower LDL-C levels. Another option is Proprotein convertase subtilisin/kexin type 9 (PCSK9), which promotes degradation of LDL receptors. Recently approved PCSK9 inhibitors can effectively reduce plasma LDL-C levels, but the treatment is expensive. PCSK9 siRNA therapy may be more affordable, but it is still too expensive for all eligible patients to use for primary prevention. Furthermore, the lipid-lowering effects of both statins and PCSK9 inhibitors depends mainly on enhancing clearance of LDL through increasing expression of LDL receptor. LDL is produced through catabolism of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein that is made exclusively by the liver. Triglycerides (TG) on circulating VLDL are hydrolyzed by lipoprotein lipase (LPL), leading to formation of intermediate-density lipoprotein that can then be converted to LDL. The drugs Mipomersen (inhibitor of apolipoprotein B100 (apoB100)) and Lomitapide (inhibitor of microsomal triglyceride transfer protein (MTP)) can reduce VLDL production and secretion, lowering plasma levels of LDL-C. However, both drugs have severe adverse effects such as hepatic lipid accumulation, liver damage and diarrhea. Thus, the need to identify novel therapeutic targets to reduce plasma cholesterol levels is urgent. This thesis aims to investigate the role of hepatic Surfeit locus 4 (Surf4), a cargo receptor located in the endoplasmic reticulum (ER) membrane, in regulating plasma lipid levels and the development of atherosclerosis by mediating PCSK9 and VLDL secretion from hepatocytes. In chapter 3, I investigated the role of Surf4 in PCSK9 secretion in vitro and in vivo. It has been reported that Surf4 mediates overexpressed PCSK9 secretion in HEK293T cells, which were derived from human embryonic kidney cells. Considering endogenous PCSK9 is mainly expressed and secreted by the liver in normal physiological condition, I determined the role of Surf4 in mediating PCSK9 secretion in human hepatoma derived cell lines, Huh7 and HepG2 cells. Interestingly, I found that knockdown of SURF4 did not inhibit endogenous PCSK9 secretion; instead, deficiency of SURF upregulated PCSK9 expression in both Huh7 and HepG2 cells. On the other hand, knockdown of hepatic Surf4 in wild type mice (C57BL6/J) has no effects on PCSK9 expression and secretion. Although the results from in vitro and in vivo studies were completely consistent, these findings indicate a negligible role of hepatic SURF4 in endogenous PCSK9 secretion from the liver and cultured hepatocytes. In chapter 4, we generated Surf4 hepatocyte-specific knockout (Surf4LKO) mice and found that plasma levels of total cholesterol (TC), TG, non-high-density lipoprotein cholesterol (HDL-C), and apoB were significantly reduced in Surf4LKO mice compared to Surf4Flox mice. I also found that Surf4 coimmunoprecipitated and colocalized with apoB100, and SURF4 silencing reduced secretion of apoB100 in cultured human hepatoma cells. Furthermore, knockdown of hepatic Surf4 in LDL receptor knockout (Ldlr-/-) mice significantly reduced TG secretion, plasma levels of TC, non-HDL-C, and apoB, and ameliorated the development of atherosclerosis. However, both Surf4LKO mice and Ldlr-/- mice with hepatic Surf4 knockdown displayed similar levels of liver lipids and plasma alanine aminotransferase (ALT) activity as their control mice, indicating that inhibition of hepatic Surf4 does not cause notable liver damage. Expression of stearoyl-CoA desaturase-1 (SCD1) was also reduced in the liver of Surf4 knockdown mice, suggesting a reduction in de novo lipogenesis. These findings suggest that deficiency of hepatic Surf4 reduced VLDL secretion, plasma lipid levels, and the risk of atherosclerosis without causing significant hepatic lipid accumulation or liver damage. In chapter 5, I investigated the role of hepatic SURF4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE-/-) mice. I found that, in apoE-/- mice fed a regular chow diet, knockdown of hepatic Surf4 expression significantly reduced TG secretion and plasma levels of non-HDL-C and TG without causing obvious hepatic lipid accumulation or liver damage. When hepatic Surf4 was knocked down in apoE-/- mice fed the Western-type diet (WTD), I observed a significant reduction in plasma levels of non-HDL-C, but not TG. Knockdown of hepatic Surf4 did not increase hepatic TC and TG levels or cause liver damage, but significantly diminished atherosclerotic lesions in apoE-/- mice fed with WTD. Therefore, these findings, together with the results from Ldlr-/- mice, indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce plasma lipid levels and the risk of ASCVD. In summary, the work performed during this Ph.D. thesis will shed light on the novel target of lowering plasma levels of cholesterol and TG and reducing the risk of atherosclerosis without causing obvious hepatic lipid accumulation.