MRI of atherosclerosis and fatty liver disease in cholesterol fed rabbits

One in four Americans, including an increasing number of young adults, develops non-alcoholic fatty liver (NAFL) [1]. After viral hepatitis, NAFL ranks among the most common liver diseases in the USA and worldwide [1, 2]. The liver is a central organ of lipid metabolism and plasma lipoprotein synthesis. Genetic, environmental, and dietary factors can contribute to lipid accumulation in the liver, referred to as hepatic steatosis [2, 3]. In Western countries, fat- and carbohydrate-rich diets are associated with the development of obesity, insulin resistance, hypertension, and atherosclerosis [3]. An imbalance in hepatic lipid metabolism, deposition, or de novo synthesis promotes lipid buildup in hepatocytes, the initial stage of NAFL. While the course of simple steatosis is reversible and in most cases asymptomatic, the chronic excess of intracellular lipids induces lipotoxicity [4] and hepatocyte injury which in some individuals can progress to fibrosis, cirrhosis, [5] and finally hepatocellular carcinoma [2, 6]. Damaged hepatocytes release intracellular transaminases, plasma biomarkers of liver injury, and may undergo cell death. These inflammatory and pro-fibrotic processes in response to injury advance liver disease to non-alcoholic steatohepatitis (NASH). Limited effective pharmacological therapies exist currently, and thus NASH is considered as an irreversible stage of NAFL [5].

Histological grading of NAFL patient liver biopsies showed that NASH occurs in 10–30% of patients [1, 7]. While knowledge has grown regarding lifestyle and dietary impacts of liver disease from cohort studies [7, 8], the role of vascular inflammation and its potential systemic effects on liver disease has been challenging to establish mechanistically. In this study, we further explore our ongoing hypothesis that unresolved local inflammation can produce a chronic systemic inflammatory assault that affects other tissues and organs [9, 10]. Previously, we showed that oral inflammation with Porphyromonas gingivalis exposure greatly exacerbates aortic atherosclerosis in cholesterol fed rabbits. Here, we test the hypothesis that progressive atherosclerotic plaque inflammation can promote progression of NASH into a pro-inflammatory phenotype with fibrosis. NASH is associated with increased risk of thrombus formation and pro-coagulation factors as demonstrated by recent clinical cohort [11] and epidemiologic studies [12]. Inflamed vulnerable plaques prone to thrombosis release pro-coagulation factors and could also advance liver pathologies. A lack of comprehensive multi-organ studies has hindered the development of effective therapeutics for the liver, while advancing liver diseases may tie together cardiometabolic abnormalities and vascular disease. For example, general anti-inflammatory therapies may help to reduce chronic systemic inflammation. This observation is further substantiated by a recent trial in patients with a prior heart attack who were treated with a drug canakinumab (a monoclonal antibody that neutralized IL-1-beta) that led to a reduction in the risk of a second heart attack [13].

Diets may contain either pro or anti-inflammatory components that modulate the systemic inflammatory state [14, 15]. In particular, specific lipid species may exhibit pro-inflammatory [2, 4], neutral [16, 17], beneficial [10, 18], or resolving effects on the vasculature, liver, and other organs. Excessive cholesterol (CHOL) accumulation in the liver promotes CHOL crystal formation which may play a direct role in hepatic lipotoxicity [4, 19] and inflammation [20‐22]. Lipidomic analyses of human NASH livers showed an increase in CHOL levels, but not in simple steatosis, while levels of free fatty acids were unchanged [23‐25]. Several representative epidemiological studies demonstrated the association of total CHOL intake with an increased risk and severity of NAFL [15], cirrhosis, or liver cancer [26, 27].

This study investigates whether local vascular inflammation in the lipid-rich atherosclerotic plaques of cholesterol fed rabbits promotes chronic systemic effects and accelerate liver disease from NAFL to NASH. For this, we used a well-established rabbit model of human atherosclerosis that replicates histological features of both, stable and vulnerable plaques [early (types II and III) and advanced (types IV, Va, Vc, VI)] at the end of the 3 months protocol [28]. In vivo MRI of the atherosclerotic aorta has provided quantitative imaging features that are characteristic of vulnerable plaques and predictive of thrombosis [29, 30]. Injury of the aortic endothelium in combination with 1% CHOL feeding has produced highly inflamed atherosclerotic plaques [28, 31]. Severe NASH developed only in rabbits receiving both 1% CHOL diet and injury, in comparison to rabbits receiving either 1% CHOL diet or normal diet with injury alone.

The focus of this study was to compare liver disease in 1% CHOL fed rabbits with and without aortic injury and to address whether high vascular inflammation affected the liver. As shown by our in vivo MRI, ex vivo MRI, and histology of aortic atherosclerotic plaques, rabbits without endothelial injury developed smaller plaques with low levels of inflammation, whereas rabbits with injury developed more advanced plaques with high inflammation and a much higher frequency of disruption. We hypothesized that comparison of these groups with the same high CHOL diet (1%) would provide a better understanding of the impact of atherosclerotic inflammation that adversely affects other tissues such as the liver. Moreover, rabbits fed normal diets with or without injury did not have signs of progressive disease (plaques) in the aorta and lacked signs of liver steatosis or portal fibrosis.

Our rabbit model with CHOL feeding and injury is an established model of human atherosclerosis resembling most of the stages categorized by the American Heart Association (as noted in the Introduction and in [28]). Rabbits with 1% CHOL + injury also have numerous vulnerable plaques that after pharmacological triggering disrupt and form large thrombi (Fig. 2). Our in vivo results demonstrated greater Gd uptake in the 1% CHOL + injury group compared to the 1% CHOL without injury, which is a marker of active inflammation and neovascularization occurring in the vessel wall [29, 39]. The 1% CHOL rabbits had a smaller number of thrombi as compared to the 1% CHOL + injury group (Table 2). Similar results were previously observed by Abela et al. [31] using 1% CHOL-fed rabbits with endothelial injury compared to 1% CHOL-fed rabbits without injury, or injured rabbits fed normal diets. In our study, the 1% CHOL, normal diet, or normal diet + injury groups had relatively thin vessel walls compared to the 1% CHOL + injury group. Moreover, histological inflammation, compositional heterogeneity, and lipid contents were substantial in the vessel walls of the 1% CHOL + injury group (Figs. 3 and 4).

In addition to representing the human plaques, rabbits have high plasma LDL, VLDL, and other important plasma components, such as CRP [40‐42]. Plasma free CHOL and triglycerides were elevated in 1% CHOL + injury rabbits (Table 3), as were all the lipoprotein subfractions HDL, LDL, VLDL (Additional file 1: Table S2). The AST to ALT ratio, heightened in the 1% CHOL + injury rabbits (Table 3), is a clinically valuable marker of liver dysfunction [37, 38]. An increase in the AST to ALT ratio was observed in the 1% CHOL fed rabbits without injury group (Additional file 1: Table S1), but to a lesser degree that did not reach statistical significance. A study of 70 patients with NASH revealed mean AST to ALT ratios of 0.7, 0.9, and 1.4 for subjects with no fibrosis, mild fibrosis, or cirrhosis, respectively [38]. In a larger clinical cohort, patients with no liver fibrosis and diabetes had a mean AST to ALT ratio of 0.78, and this was elevated to 0.98 (n = 204) in diabetic patients with advanced fibrosis (n = 142). Advanced fibrosis in the study was defined via histological diagnosis of either bridging fibrosis or cirrhosis [43]. GGT is also relevant to liver function, is responsible for the extracellular catabolism of the antioxidant glutathione, and is suspected to be important beyond liver damage in chronic subclinical inflammation and systemic oxidative demand [44‐46].

Our report is the first study to our knowledge tracking advanced stage plaques that are highly inflamed in relation to liver disease in rabbits. The 1% CHOL + injury rabbits had significantly increased triglyceride deposition in the liver as observed by in vivo MRS at 2 and 3 months compared to 1% CHOL non-injured rabbits at the same time points (Fig. 5). Note that both groups were fed a normal chow diet for the last month (our standard protocol [28, 29, 32] and shown in our timeline), and the liver triglyceride in each group did not increase between 2 and 3 months. There were insignificant gains in weight over the entire 3 month time period in each cholesterol fed group. Therefore, the accumulation of body fat is not a contributor to the differences in liver pathologies between the two cholesterol fed groups of rabbits. We also found that endothelial injury alone without cholesterol feeding does not induce visible histologic liver steatosis with inflammation (as shown in Fig. 6).

Taken together, above results strongly suggest that vascular inflammation from lipid-rich plaques in the setting of the high cholesterol diet is a major contributor to the liver pathology. After completion of the MRI and MRS studies, the color of the CHOL-fed rabbit’s livers changed from normal deep red to pale or yellowish, most notable for the 1% CHOL + injury group (Fig. 6a). The most remarkable and physiologically substantial differentiation of the livers was revealed by histology, which showed propagating fibrosis in the 1% CHOL + injury rabbits, as compared to the other study groups (Fig. 6b).

Some fibrosis was noted in the 1% CHOL-fed rabbits without injury. In rabbits fed 1% CHOL alone, a mean of 3.65% liver fibrosis by area was found compared to 8.06% liver fibrosis by area in the 1% CHOL + injury rabbits (Fig. 7a). The propagation to bridging fibrosis far beyond the near-boundaries of the liver portal triad (bile duct, hepatic portal vein, and hepatic artery) was observed only in the 1% CHOL + injury rabbits (Fig. 7b). These findings suggests that liver fibrosis is initiated around the blood vessels, and with a more severe disease, collagen fibrosis increased to propagate into the hepatic sinusoids. The three main structure of the liver, the portal vein, artery, and bile duct, are surrounded by loose myofibroblasts and the first layer of hepatic and non-parenchymal cells. These portal myofibroblasts (and not hepatic stellate cells) are hypothesized to be the point of portal fibrosis in the early stages of cholestatic fibrosis [47].

Several localized diseases that are characterized by unresolved inflammation have now been linked to cardiovascular disease (CVD). Previous studies in rabbits have demonstrated that periodontitis greatly promotes atherosclerotic plaque inflammatory processes [9]. High circulating inflammatory mediators have been postulated to contribute to “vulnerable blood”, a systemic characteristic of high-risk for cardiovascular events in humans [9, 10, and references therein]. One study has also linked periodontitis to liver inflammation [48]. Psoriasis is another example of a link between discrete sites of inflammatory pathology that promotes vascular inflammation and early atherosclerosis. Psoriasis becomes a systemic inflammatory disease because of the failure to resolve localized inflammation and the secretion of high levels of neutrophils and inflammatory mediators into the blood [49]. Our new evidence from CHOL-fed rabbits without periodontitis or psoriasis shows that discrete pathological regions of lipid-rich inflamed pro-thrombotic plaques contribute to the progression of liver disease to advanced stages with high inflammation and fibrosis.

This study has demonstrated that progressive atherosclerotic vascular inflammation is associated with worsening liver disease in rabbits. The liver disease appears to synergized between diet, aortic injury, and atherosclerotic inflammation, whereby the injury coupled with local plaque inflammation drives disease progression in both areas. These observations support the emerging broad hypothesis that unresolved inflammation may impart systemic effects. These findings provide a basis for detailed investigation of the mechanisms and molecules released from the plaques that can lead to inflammation and fibrosis in the liver. An important test of our overall hypothesis will be to assess the impact of pro-resolving mediators of inflammation, including lipoxins and resolvins, molecules that have been shown to be potent in treating or possibly preventing many inflammation-associated diseases [10].