Hypertriglyceridemia is a sign of abnormal lipid metabolism, and an independent risk factor in atherosclerotic development[4]. APOC3 is a key regulator of TG metabolism, is a water-soluble low molecular weight lipoprotein that is present in the plasma along with HDLs, VLDLs, CM and LDLs[22]. Studies show that elevated levels of APOC3 inhibits the activity of LPL and HL, which delays triglyceride-rich lipoprotein clearance and increases its levels in the plasma, eventually leading to impaired TG metabolism[23]. Although the in vivo studies on APOC3 are mainly based on mouse models, the rabbit model has several advantages such as easier maintenance, suitable size of the aorta, high fecundity and short gestation periods[24], and similar lipid metabolism and cardiovascular pathophysiology as humans[25]. For instance, the hepatic LDL receptor is normally inactive in the rabbits as in humans, which makes it a highly suitable model for studying the mechanistic basis of AS, as well as the effects of lipid-lowering drugs[26]. Furthermore, rabbits have abundant plasma cholesteryl ester transfer protein, which can help develop strategies to raise plasma HDL-c levels[27]. Finally, both humans and rabbits are more sensitive to an HFD compared to mice[25, 28].
We designed one sgRNA targeting exon 2 of rabbit APOC3 and finally obtained three KO rabbits by CRISPR/Cas9 gene-editing. None of the three founder rabbits contained APOC3 in their livers and small intestine, and no APOC3 protein was detected in plasma. Under an NC diet, the plasma TG levels of the KO rabbits decreased, but the plasma TC and LDL-C levels had no significant difference compared with the control group. It is worth noting that after an HFD, in addition to plasma TG levels, plasma TC and LDL-C levels also began to differ from the control group, indicating that the lack of APOC3 affects cholesterol transport, and this change is aggravated by a high-fat intake. Interestingly, all lipid indices were higher in AC1 compared to AC2 and AC3, which could be due to sex differences.
The plasma lipoprotein profiles of the WT and APOC3 KO rabbits showed significant differences; while APOB and APOE were significantly reduced in the APOC3 KO rabbits, APOA1 was enriched and led to the high HDL levels. This also confirmed that APOC3 may increase plasma TG levels by hindering APOE-mediated uptake of CM and VLDL in the liver by competitively inhibiting APOE binding to its receptor. We then detected a significant increase in plasma LPL activity in three KO rabbits and HL activity in two of them (perhaps due to the insufficient sample size). LPL is present on the surface of extrahepatic capillary endothelial cells, and hydrolyzes CM and VLDLs[29, 30]. Its activity can be evaluated in terms of the amount of free fatty acids. Due to the role of HL in assisting the conversion of VLDL[31, 32], a large amount of VLDL accumulated in the plasma of the WT rabbits after 6 weeks of high-fat feeding, but few lipoproteins accumulated in the plasma of the KO rabbits. As the main regulator of HDL-C[33], the increase of HL activity also leads to an increase in HDL-C. Taken together, the absence of APOC3 significantly altered the lipoprotein profile and lipcatabolic related enzymes of the high-fat feeding rabbits, inhibited hypertriglyceridemia and improved their fat tolerance by enabling rapid clearance of the TGs.
Atherosclerosis is a chronic inflammatory disease, and the inflammatory process is important in both the initiation and progression of lesion development[34–36]. After detection, plasma IL-1β and TNF-a decreased significantly and the whole-blood monocyte, neutrophil, and platelet counts were significantly lower in KO rabbits than the WT rabbits. IL-1β and TNF-α can increase endothelial permeability and lipoprotein permeability[37, 38]. Extensive monocyte recruitment plays an important role in the development of early atherosclerotic lesions[34, 39, 40]. Neutrophils aggravate endothelial dysfunction, attract monocytes, enter atherosclerotic lesions, and accelerate the formation of foam cells[41]. Studies have confirmed that platelets contribute to the atherosclerotic process at both the early (endothelial disruption) and final stages (rupture of the vulnerable plaque), participating in the process by releasing chemokines, inflammatory mediators, and microparticles[42, 43]. Therefore, it can be boldly speculated that APOC3 is closely related to the formation and development of atherosclerosis, in which a series of inflammatory responses play an important role.
After 12 weeks of high-fat diet, the aortic tree lesion coverage rate of the WT rabbit was as high as 21%, while that in KO rabbits was much lower (only 3%). HE, MT, and immunohistochemical staining showed the WT rabbit had obvious atherosclerotic lesions and increased intima thickening and collagen content, accompanied by the accumulation of macrophages and the proliferation of smooth muscle cells, while KO rabbits only had slight early atherosclerotic lesions. However, there were no obvious pathological changes in the coronary arteries of the two groups. For the formation of coronary artery plaques in rabbits, higher blood lipids (such as seen in Watanabe heritable hyperlipidemic rabbits or LDLR KO rabbits) or other additional conditional interventions, such as hybridization or surgical ligation are usually required. Taken together, lack of APOC3 restrains atherogenesis, although the exact mechanistic basis needs to be elucidated.
Statins are often used to treat dyslipidemia, especially to control the elevation of LDL-C; although clinicians generally believe that the benefits of statins are exaggerated, while the potential side effects are underestimated[44]. In addition, other risk factors for atherosclerosis still exist, such as TG and TG-rich lipoproteins[45]. However, the efficacy of existing triglyceride-lowering drugs is still controversial. Therefore, using experimental animal models that are more similar to the characteristics of human lipid metabolism to study the association between genes and cardiovascular disease may be conducive to the development and application of new drugs.