Impact of biliary cholesterol secretion on atherosclerosis development

Excessive accumulation of cholesterol in the arteries drives atherosclerosis development.  Reverse cholesterol transport (RCT) is believed to regress or stabilize atherosclerotic lesions.  During RCT, cholesterol from atherosclerotic lesions is effluxed to high density lipoproteins (HDL), delivered to the liver, secreted into the bile, and excreted in feces.  We hypothesized that reduced biliary cholesterol secretion would increase the development of atherosclerosis due to a reduction in RCT.  Decreased biliary cholesterol secretion was achieved by hepatic overexpression of human NPC1L1 (L1Tg) and/or knockdown of hepatic ABCG5/G8 function using an ABCG8 antisense oligonucleotide (ASO). LDLR-/- and LDLR-/-/L1Tg mice received either control or ABCG8 ASO and were fed a high fat/low cholesterol diet for 20 weeks.  L1Tg mice and mice with hepatic ABCG8 knockdown had an >70% reduction in biliary cholesterol. The dramatic decrease in biliary cholesterol did not increase plasma cholesterol, and in fact mice with hepatic ABCG8 knockdown had reduced VLDL cholesterol and increased HDL cholesterol. Even more surprising, aortic atherosclerosis was significantly decreased in mice with compromised biliary cholesterol secretion. LDLR-/-/L1Tg treated with ABCG8 ASO had a >90% reduction in biliary cholesterol yet had ~70% less atherosclerosis compared to LDLR-/- controls.  Reducing biliary cholesterol did not result in major changes in macrophage RCT, hepatic cholesterol content, bile acid composition, and fecal excretion of neutral sterol.  Although the hepatic expression of FMO3 was significantly decreased in mice with reduced biliary cholesterol secretion, there was not a corresponding decrease in plasma TMAO, which is produced by FMO3 and has been linked to increased CVD risk.  In conclusion, reducing biliary cholesterol secretion paradoxically reduced atherosclerosis development through a mechanism that we are currently working to define.  Studies are also underway to determine whether reduced biliary cholesterol secretion limits atherosclerosis development in mice fed a high cholesterol diet.

Targeting microRNA-33 to reduce intracranial atherosclerosis and other neurovascular hallmarks of vascular cognitive impairment and dementia      

Intracranial atherosclerosis (ICAS) is a public health concern for both its role in stroke and as a contributing factor to vascular cognitive impairment and dementia (VCID). It is becoming widely accepted that poor vascular health facilitates poor brain health and that changes are needed to delay or prevent onset of VCID. Atherosclerotic vascular disease (AVD) is a chronic, maladaptive inflammatory disease that can affect extra- and intracranial arteries. The combination of lipoprotein retention, endothelial cell inflammation, myeloid infiltration, intracellular cholesterol accumulation, impaired apoptotic cell clearance, and extracellular matrix degradation leads to formation of advanced, unstable atherosclerotic plaques that can limit or occlude blood flow to tissues causing acute or chronic tissue damage.  ICAS often plays a causative role in ischemic stroke and subsequent cognitive decline.  ICAS has also been linked to both clinical signs of cognitive decline and Alzheimer’s disease pathology. Intracranial compared to extracranial atherosclerosis has a delayed onset of ~20 years but increases in prevalence and severity in individuals 60 years or older.  With the steady rise in the percentage of US citizens above the age of 60, ICAS will play an ever-growing role in the morbidity and mortality caused by VCID. Reducing low-density lipoprotein (LDL) concentration with statins is a primary therapeutic approach to stabilize AVD and attenuate ischemic event risk.  However, statins only reduce stroke risk by ~20% and do not appear to reduce VCID suggesting that treating hypercholesterolemia alone is not an ideal approach for decreasing VCID.  The obvious need for additional therapies that regress or stabilize ICAS has been hampered by the paucity of suitable animal models.  During an R01-funded study to determine the impact of microRNA-33 (miR-33) antagonism on cardiovascular AVD, we fortuitously discovered that our NHP model had ICAS and other neurovascular hallmarks of VCID.  We believe analysis of intracranial arteries and brains from our NHPs could have a high impact on the field of VCID research because of the potential discovery of a new therapy and animal model for VCID.  

Therapeutic targeting of metabolic microRNAs as a new treatment paradigm for NASH

Nonalcoholic steatohepatitis (NASH) is a progressive subtype of nonalcoholic fatty liver disease (NAFLD) characterized by the presence of steatosis, inflammation and fibrosis. NASH is strongly associated with obesity and the metabolic syndrome and is rapidly becoming the leading cause of end-stage liver disease, liver transplantation and hepatocellular carcinoma, underscoring the unmet medical need for effective and safe therapies for treatment of NASH. We have discovered two microRNAs (miRNAs) that function as key regulators of metabolic homeostasis. By employing GWAS meta-analysis we uncovered miR-128-1 as linked to circulating cholesterol and lipid abnormalities as well as to obesity and type 2 diabetes. Importantly, our data imply that miR-128-1 could be a promising therapeutic target for NASH, as pharmacologic inhibition of its function in high fat diet-induced obese (DIO) mice almost completely prevents hepatic lipid accumulation and inflammation. We have also identified miR-22 as another major regulator of metabolic homeostasis, which functions by orchestrating multiple pro-lipogenic programs that promote obesity. Notably, inhibition of miR-22 in DIO mice leads to loss of body weight and a decrease in hepatosteatosis and inflammation. Our goal is to identify antimiR compounds for effective and safe inhibition of miR-128-1 and miR-22 and assess their therapeutic potential to treat NASH in highly relevant mouse and non-human primate NASH models. Furthermore, we will evaluate hepatic and circulating miR-128-1 and miR-22 as biomarkers for NASH. We envision that the development of combined diagnostics and miRNA-targeted therapeutics based on dysregulation of miR-128-1 and miR-22 has high therapeutic potential, and if successfully translated to the clinic, will significantly impact public health worldwide.