Among LDLR-independent pathways, suppressing apoB [94] production and inhibiting the function of apoC-III [95] and angiopoietin like-3 protein (ANGPTL-3) [52,55] have been studied in greater detail [96]. ApoB is a large 550 kDa protein and is essential for the assembly and secretion of VLDL by the liver [10,97]. VLDL undergoes lipolysis to form LDL [98,99]. ApoB also serves as a ligand for LDLR-mediated cellular uptake by the peripheral tissues and clearance from circulation via the liver [69]. While suppression of apoB synthesis in the liver leads to reductions in the apoB-containing atherogenic lipoproteins [47], it may cause fatty liver as a result of accumulation of fat in the liver, as seen in familial hypobetalipoproteinemia (FHBL) individuals with reduced levels of apoB [100,101], partly as a result of enhanced intracellular degradation of apoB [102]. FHBL individuals are also reported to have intestinal lipid accumulation and postprandial lipemia [103], which in some cases is associated with the risk of diabetes [104]. The hypolipidemic actions of both apoC-III and ANGPTL3 inhibitors are associated with lowering of atherogenic TLRs [59,105] via activation of lipoprotein lipase (LPL) [106,107,108,109]. LPL is an important player in the metabolism of TLRs [47], resulting in cardio-protection [110]. Human apoC-III is a small apoprotein with a molecular weight of 10 kDa, predominantly synthesized in the liver and intestine and primarily associated with chylomicrons, VLDL and HDL [111]. ApoC-III is an inhibitor of LPL that hydrolyzes TG-rich VLDL and impairs its clearance [108]. Thus, inactivation of apoC-III should prove beneficial to individuals with hyperlipidemia. Indeed, mice lacking apoC-III are shown to have increased LPL activity and enhanced lipolysis of TRLs and show lack of postprandial lipemia [112]. In rodents and non-human primates, apoC-III ASOs lowered plasma apoC-III and TG [95]. Silencing apoC-III activity via antisense therapy lowered TRLs in diabetic [113] and non-diabetic [114] individuals as a result of enhanced LPL activity [20]. Despite increased lipolysis of TRLs by LPL and reduced TG in apoC-III-deficient mice [112], no difference in atherosclerosis burden was noticed in the apoC-III-deficient mice compared to the control on LDLR-deficient background [115], although humans with apoC-III loss-of-function mutations showed apparent cardio-protection in carriers compared to non-carriers, based on subclinical atherosclerosis findings measured by calcium score [116]. Rare variants of apoC-III loss-of-function mutations in humans have also been reported to provide cardio-protection [117,118]. Inhibition of apoC-III accelerates its clearance of TRLs from circulation and reduces the risk of ASCVD. Lowering or loss of function of apoCIII results in the reduced secretion of chylomicron remnants and activation of LPL, leading to enhanced clearance of atherogenic lipoproteins [119]. Thus, apoCIII increases production of chylomicron remnants and also impedes clearance of TG-rich apoB lipoproteins. However, apoC-III antisense therapy is associated with increased risk of thrombocytopenia [120]. Newer apoC-III antagonists such as ASO olezarsen (formerly AKCEA-APOCIII-LRx) and short interfering RNA (siRNA) ARO-APOC3 appear to show efficacy with less risk of thrombocytopenia [120].

ANGPTL3 is a 460-amino-acid secretory glycoprotein expressed in the liver and implicated in the metabolism of lipids through modulation of LPL. ANGPTL3 inhibits lipoprotein lipase (LPL) [60] and endothelial lipase (EL) [121] in tissues, leading to formation of TRLs [122,123,124,125]. Thus, inactivation of ANGPTL3 leads to LPL activation and enhanced clearance of TRLs from the circulation [60]. ANGPTL3 inhibition also reduces hepatic VLDL secretion [126], resulting in the lowering of blood LDL and TG. Indeed, loss-of-function mutations affecting the gene encoding ANGPTL3 are linked with lower total cholesterol, LDL-C and TG levels and provide cardio-protection [22,55,127]. Consistent with the human data, ANGPTL3-null mice showed lower LDL and TG as a result of enhanced LPL activity and accelerated clearance of TRLs [106]. ANGPTL3 antibodies have been found effective in reducing LDL in humans lacking LDLR (HoFH), where LDLR-dependent therapies have been ineffective [128]. Thus, ANGPTL3 offers a unique opportunity to lower atherogenic lipoproteins in patients not able to benefit from LDLR-dependent therapies.

In vivo proof-of-concept as well as pharmacology studies of therapeutic agents (new chemical entities (NCEs)/new biological entities (NBEs)), are first evaluated in relevant animal models since testing agents may show species [129,130,131] and strain [132] specificity, which can be examined using mammalian cells derived from animals. These studies are designed to determine various parameters for pharmacological effects, which assist in the selection of appropriate animal species for further in vivo pharmacology and toxicology studies. The combined results from in vitro and in vivo studies assist in the extrapolation of the findings to humans. In vivo studies to assess pharmacological activity, including defining mechanism(s) of action, are often used to support the rationale of the proposed use of the drug product in clinical studies. Several animal models have been described to assess atherosclerosis in hypercholesterolemic conditions [133,134]. Some examples of animal models that have been used to determine the association between LDL receptor activity and circulating LDL are described in pharmacology studies below.

In mice, a clear association between LDLR activity and circulating LDL levels was demonstrated by either overexpressing the LDL receptor or making LDL-receptor-deficient mice. Hofmann et al. [135] generated a transgenic mouse model expressing the human LDL receptor gene. These transgenic mice cleared intravenously injected ¹²⁵I-labeled LDL from blood eight to ten times more rapidly than normal mice. The plasma concentrations of apoproteins B-100 and E, the two ligands for the LDL receptor, declined by more than 90 percent, but the concentration of another apoprotein, apoA-I, was unaffected. These studies demonstrated that overexpression of the LDL receptor can dramatically lower the concentration of LDL in vivo. To further confirm that, indeed, an association exists between LDL receptor activity and circulating LDL concentration, the same group of researchers [13] generated a mouse model lacking the LDL receptor by homologous recombination. Total plasma cholesterol levels were two-fold higher in homozygous mice than those of wild-type littermates, owing to a seven- to nine-fold increase in atherogenic lipoproteins (IDL and LDL). No significant change in HDL was observed. Despite the mouse lipoprotein profile differing from that of humans, the LDLR-mediated regulation of circulating LDL appears to be similar between mice and humans in terms of LDLR-mediated clearance of LDL lipoproteins. The half-lives of intravenously administered ¹²⁵I-VLDL and ¹²⁵I-LDL were prolonged by 30-fold and 2.5-fold, respectively. Intravenous injection of a recombinant replication-defective adenovirus encoding the human LDL receptor restored expression of the LDL receptor protein in the liver and increased the clearance of ¹²⁵I-VLDL, further confirming the inverse association of LDL receptor activity and LDL level and the role of the LDL receptor in reversing the hypercholesterolemic effects of LDL receptor deficiency. An LDLR deficient mouse model has been extensively employed to ask important biologic questions, including the mechanism of atherosclerosis progression by nuclear hormone receptors [136,137,138], HDL receptors [139] and players in the cholesterol efflux pathway [140].

Both LDLR [13] as well as apoE knockout mice [141,142] develop hypercholesterolemia and atherosclerosis, the latter showing more severe symptoms than the former. Using these mice, mechanisms of atherogenesis have been studied extensively [143,144,145]. WT rats and mice develop very sparse atherosclerotic lesions, even on high-fat and high-cholesterol diets. Sithu et al. [146] developed an LDLR KO model in Sprague-Dawley rats and studied lipoprotein metabolism and atherosclerotic lesion formation. Lack of LDLR protein in rats caused a significant increase in plasma total cholesterol and triglycerides; the rats gained more weight and were more glucose intolerant than WT rats on normal rat chow diet. Feeding a Western diet resulted in increased obesity and age-dependent increases in glucose intolerance, as well as significant atherosclerotic lesions in the aortic arch as well as throughout the abdominal aorta in the LDLR KO rats. These findings demonstrated a tight association between LDLR and LDL.

The golden Syrian (GS) hamster has been used with increasing frequency to study lipoprotein metabolism [147,148] and atherosclerosis [149,150] and to evaluate hypolipidemic agents [9,150] because of the hamster’s lipid metabolism being comparable to that of humans. The VLDL particles synthesized and secreted by hamster liver are similar to human liver with apoB-100 [97,151], but different than rats and mice that secrete both apoB-100 and B-48 [152]. As in humans, hamsters also show CETP activity in plasma [151]. These characteristics of hamsters make them a suitable animal model for evaluating lipid-modulating agents in preclinical studies. Hamsters also respond to high-cholesterol and high-fat diets similarly to humans [153] and develop more atherosclerosis compared to rats and mice [154]. These features of hamsters make them very useful to test hypolipidemic agents. Because of the similarity of hamster and human lipoprotein metabolism, an LDL-receptor-deficient hamster model was developed by Guo et al. [155] using CRISPR-Cas9 technology. Homozygous LDLR KO hamsters on a chow diet developed hypercholesterolemia with LDL as the dominant lipoprotein and spontaneous atherosclerosis. On a high-cholesterol, high-fat (HCHF) diet, these animals exhibited severe hyperlipidemia and atherosclerotic lesions in the aorta and coronary arteries. Moreover, the heterozygous LDLR KO hamsters on a short-term HCHF diet also had apparent hypercholesterolemia, which could be effectively ameliorated with several lipid-lowering drugs. Importantly, heterozygotes fed HCHF diets for 3-months developed accelerated lesions in their aortas and coronary arteries [156], whereas only mild aortic lesions were observed in WT hamsters. Thus, unlike other rodent animals, the levels of plasma cholesterol in hamsters can be significantly modulated by the intervention of dietary cholesterol, levels which were closely associated with severity of atherosclerosis in LDLR^+/− hamsters. Further validation of the heterozygous hamster (LDLR^+/−) model for testing therapeutic agents was shown in a study where hyperlipidemic heterozygous hamsters were treated with the PCSK9 monoclonal antibody [157].

Familial hypercholesterolemia (FH) is caused primarily by loss-of-function mutations in the LDLR gene [79,80], leading to elevated concentrations of LDL in heterozygous and, most notably, homozygous patients [14,55,158]. FH is also associated with significantly reduced concentrations of both high-density lipoprotein cholesterol (HDL-C) and its principal protein component, apolipoprotein (apo) A-I [159], as a result of increased apoA-I catabolism and decreased apoA-I synthesis [160]. A rabbit model for FH, the Watanabe heritable hyperlipidemic (WHHL) rabbit, was discovered and reported in 1980 [161,162]. These rabbits have an LDLR gene that encodes a four-amino-acid deletion in the cysteine-rich ligand-binding domain of the protein that severely disrupts LDLR function [163]. Homozygous WHHL rabbits are markedly hypercholesterolemic from birth and suffer from tendon xanthomas and atherosclerosis, both of which exhibit remarkable pathological resemblance to those observed in human HoFH. Also similar to the human condition [159], plasma HDL-C and apoA-I levels are abnormally low in these animals [163]. Overexpression of human lecithin: cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLR defective rabbits increased HDL-C and apoA-I levels [160]. Similar to humans [82], treatment with statins induced LDL receptor expression in WHHL heterozygotes [164], suggesting the WHHL rabbit as a useful LDLR-deficient model that mimics human HoFH.

Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics and size of the atherosclerotic plaques. The pig LDL receptor gene was inactivated to drive hypercholesterolemia and atherosclerosis by two groups of researchers using separate technologies [165,166]. These mini pigs with an ablated LDLR gene showed diet-induced exacerbation of FH phenotypes. Hypercholesterolemic heterozygotes (LDLR^+/−) showed similar characteristics as human heterozygotes (LDLR^+/−) and a similar response when treated with statins (atorvastatin, 3 mg/kg/day). Homozygous (LDLR^−/−) pigs also showed human-like advanced plaques and responded to statin (pitavastatin) treatment [167]. In the homozygous (LDLR^−/−) and heterozygotes (LDLR^+/−) Yucatan miniature pigs, another class of drug candidate, bempedoic acid, which inhibits ATP citrate lyase, showed a reduction in LDL and lesion formation [168]. The ablation of LDLR caused elevation of LDL in homozygous and heterozygous miniature pigs, suggesting an association between LDL receptor function and LDL concentration.