Clinical Burden and Therapeutic Limitations

Hyperlipidemia is a core component of metabolic syndrome and frequently coexists with obesity, insulin resistance, and hypertension. Its global prevalence continues to rise, driven by sedentary lifestyles, high-fat dietary patterns, and an aging population 4. While statins remain the cornerstone of pharmacological therapy, their clinical utility is occasionally constrained by adverse effects, residual cardiovascular risk, interindividual response variability, and long-term adherence challenges 5. These limitations have stimulated the development of alternative lipid-lowering strategies, such as PCSK9 inhibitors, cholesterol absorption inhibitors, CETP inhibitors, and nutraceutical-based interventions. Concurrently, the advent of precision medicine has amplified the demand for preclinical models that more accurately recapitulate human lipid metabolism and disease heterogeneity 6. Historically, traditional drug discovery pipelines have been hindered by poor translation from animal studies to clinical outcomes. This translational gap primarily stems from species-specific divergences in lipoprotein metabolism, inflammatory responses, and plaque biology 7,8.

Role of Animal Models in Hyperlipidemia Research

Animal models provide a critical foundation for investigating lipid metabolism, identifying therapeutic targets, and evaluating the safety and efficacy of candidate antihyperlipidemic agents prior to human trials 8. These models are broadly classified into three categories. First, induced models utilize dietary, chemical, or hormonal interventions to generate hyperlipidemia. Second, genetically modified models—encompassing knockout, knock-in, or transgenic animals—are designed to mimic inherited dyslipidemias, such as familial hypercholesterolemia. Third, spontaneous models, exemplified by the Watanabe heritable hyperlipidemic (WHHL) rabbit, naturally develop hyperlipidemia and atherosclerosis over time. Each model presents distinct advantages for mechanistic and translational research. However, species-specific limitations persist, notably differences in cholesteryl ester transfer protein (CETP) activity, apolipoprotein expression, and LDL receptor regulation. These factors demand careful consideration when interpreting experimental outcomes and extrapolating preclinical findings to human physiology 8,9,10.

Importance of Model Selection and Standardization

Appropriate model selection is paramount for the meaningful preclinical evaluation of antihyperlipidemic therapies. A mismatch between a compound's pharmacodynamic properties and the metabolic background of the selected model can yield misleading efficacy or safety conclusions. For instance, wild-type mice lack CETP activity, inherently limiting their suitability for evaluating HDL-targeted therapies. In contrast, rabbits and non-human primates express CETP, thereby more closely mirroring human lipoprotein dynamics 9,10,11. Beyond model selection, inadequate standardization of induction protocols, dietary compositions, outcome measures, and reporting practices continues to be a major source of irreproducibility in preclinical research. International guidelines and regulatory frameworks—including those established by the OECD, ICH, and the ARRIVE consortium—strongly emphasize the necessity of ethical conduct, methodological rigor, and transparent reporting in animal studies 12.

Emerging Trends in Model Development

Recent technological advancements have significantly expanded the scope and sophistication of animal models utilized in lipid research. CRISPR/Cas9-mediated genome editing has facilitated the generation of humanized models that express key human apolipoproteins, LDL receptors, or PCSK9 variants. These innovations have markedly enhanced the biological relevance of preclinical models for investigating inherited and complex lipid disorders 13. Furthermore, multi-omics approaches—encompassing transcriptomics, proteomics, metabolomics, and lipidomics—are increasingly being integrated into animal studies to deliver systems-level insights into lipid regulation and pharmacological responses. Non-invasive imaging modalities, advanced biomarker profiling, and computational modeling further elevate the precision of disease monitoring and therapeutic evaluation 14. The present review aims to provide a comprehensive and critical overview of the animal models employed in antihyperlipidemic drug screening. Emphasis is placed on their biological relevance, mechanistic utility, and translational potential, whilst also identifying persistent gaps and opportunities for refining the predictive validity of preclinical lipid research.

Methods: Literature Search and Study Selection

Study Selection and Data Extraction

The initial database search yielded 2,453 records. Following the removal of 658 duplicate records utilizing reference management software (Zotero), 1,795 unique records advanced to title and abstract screening. During this phase, 1,303 records were excluded, categorized as in vitro or non-animal studies (n = 543), conference abstracts or editorials (n = 236), and non-relevant disease models (n = 524). Subsequently, 492 full-text articles underwent rigorous eligibility assessment.

At the full-text evaluation stage, an additional 205 articles were excluded, predominantly due to inadequate methodological detail, an absence of pertinent lipid-related outcomes, or publication in a language other than English (n = 72). Ultimately, 287 studies fulfilled all inclusion criteria and were incorporated into the qualitative synthesis.

Data extraction systematically captured the animal species and strain, model induction methodology (e.g., dietary, chemical, genetic, microbiota-modulated, or large-animal), pharmacological intervention, evaluated lipid parameters, and translational relevance. The selected studies were qualitatively synthesized by categorizing the models into diet-induced, chemically induced, genetic, microbiota-modulated, and large-animal paradigms. Any discrepancies arising during the screening or data extraction processes were resolved via consensus among the authors. The comprehensive study selection workflow is visually detailed in Figure 1 (PRISMA Flow Diagram).

Overview of Lipid Transport

Lipids—encompassing triglycerides (TG), cholesterol, phospholipids, and cholesteryl esters—are hydrophobic molecules essential for cellular architecture, membrane integrity, and energy metabolism. Due to their restricted solubility in aqueous environments, lipids necessitate specialized transport mechanisms to circulate within the bloodstream. This critical physiological function is fulfilled by lipoproteins 13,14. Lipoproteins are spherical macromolecular complexes comprising a hydrophobic lipid core—predominantly containing triglycerides and cholesteryl esters—enveloped by an amphipathic shell of phospholipids, unesterified cholesterol, and apolipoproteins 14,15,16. Beyond lipid transport, lipoproteins govern various physiological processes, including cellular lipid uptake, reverse cholesterol transport, steroidogenesis, and the maintenance of cellular membrane fluidity 16,17,18.

Structure and Components of Lipoproteins

Although lipoproteins exhibit heterogeneity in size and composition, they share a conserved structural architecture. The hydrophobic core houses neutral lipids, specifically triglycerides and cholesteryl esters, whereas the surface monolayer is enriched with phospholipids, unesterified cholesterol, and apolipoproteins. Apolipoproteins are integral to maintaining structural stability, facilitating receptor-mediated interactions, and modulating the activity of key metabolic enzymes. For instance, apolipoprotein B-100 (ApoB-100) is obligatory for the assembly and secretion of very-low-density lipoprotein (VLDL) and its subsequent binding to the LDL receptor. Apolipoprotein A-I (ApoA-I) serves as the primary structural protein of high-density lipoprotein (HDL) and acts as a vital activator for lecithin-cholesterol acyltransferase (LCAT). Furthermore, apolipoprotein C-II activates lipoprotein lipase (LPL), whereas apolipoprotein E (ApoE) mediates the hepatic clearance of remnant lipoproteins 16,18.

Classification of Lipoproteins

Lipoproteins are systematically classified based on their hydrated density, particle diameter, lipid-to-protein ratio, electrophoretic mobility, and specific apolipoprotein composition. According to these physicochemical properties, lipoproteins are categorized into chylomicrons, VLDL, intermediate-density lipoproteins (IDL), LDL, and HDL (Table 1) 2,5,15,16.

Each lipoprotein class fulfills a specialized physiological role. Chylomicrons are responsible for transporting dietary triglycerides from the intestinal lumen to peripheral tissues. VLDL particles distribute endogenously synthesized triglycerides from the liver to peripheral sites. LDL functions as the principal carrier of cholesterol to peripheral cells, while HDL mediates reverse cholesterol transport by mobilizing excess peripheral cholesterol back to the liver for biliary excretion.

Functional Pathways of Lipoprotein Transport

Lipoprotein metabolism is meticulously regulated via three primary transport pathways.

Key Enzymes Regulating Lipoprotein Metabolism

Several key enzymes exert critical control over lipoprotein remodeling and systemic lipid homeostasis (Table 2). LPL catalyzes the hydrolysis of triglycerides sequestered in chylomicrons and VLDL, thereby facilitating lipid clearance. Hepatic lipase modifies HDL and IDL particles, significantly influencing hepatic cholesterol turnover. Furthermore, LCAT is indispensable for HDL maturation, whereas CETP governs the dynamic distribution of cholesterol across distinct lipoprotein fractions. Intracellularly, acyl-CoA:cholesterol acyltransferase (ACAT) dictates cholesterol esterification, a process central to macrophage foam cell formation 16,18. Pathological alterations in the activities of these enzymes can predispose individuals to lipid accumulation, atherogenesis, and systemic metabolic dysfunction (Table 2).

Clinical and Experimental Implications

Dysregulated lipoprotein metabolism forms the fundamental pathological basis for numerous cardiometabolic disorders. Elevated concentrations of circulating LDL and oxidatively modified LDL actively promote endothelial dysfunction and macrophage foam cell generation, acting as the primary instigators of atherosclerosis. Moreover, increased levels of VLDL and small, dense LDL particles are hallmark features of insulin-resistant states and diabetic dyslipidemia. Conversely, diminished HDL concentrations compromise reverse cholesterol transport and attenuate its inherent atheroprotective properties 7,9.

These distinct, species-specific variations in lipoprotein metabolism must meticulously guide animal model selection during experimental design. Models inherently lacking CETP activity, such as wild-type mice, inadequately recapitulate human HDL dynamics. In contrast, CETP-expressing species—including rabbits, hamsters, and non-human primates—offer substantially improved translational relevance for the preclinical evaluation of HDL-targeted therapeutic interventions 15,19,20.

Pathophysiological Stages of Atherogenesis

The development of atherosclerosis proceeds through a continuum of overlapping pathophysiological stages intricately linked to underlying lipid abnormalities (Table 3) 15,16,17,18.

Immunological Mechanisms in Atherosclerosis

Atherosclerosis is increasingly characterized as a complex immunometabolic disorder governed by both innate and adaptive immune responses. In the innate arm, macrophages, dendritic cells, and neutrophils heavily contribute to localized cytokine production and the propagation of the necrotic core. Pro-inflammatory cytokines, notably IL-1β, TNF-α, and MCP-1, serve to amplify lesion progression. Adaptive immune responses are equally critical; for instance, Th1-polarized T cells secrete interferon-γ (IFN-γ), which potently activates macrophages while simultaneously inhibiting collagen synthesis by VSMCs, thereby fostering plaque instability. Consequently, targeting inflammatory pathways has emerged as a vital complementary strategy in cardiovascular therapeutics, a paradigm exemplified by the CANTOS trial, which demonstrated a significant reduction in cardiovascular events following IL-1β inhibition, independent of lipid-lowering efficacy 17,26,27.

Role of Lipoproteins in Atherogenesis

Distinct lipoprotein classes contribute differentially to plaque pathogenesis. LDL serves as the primary atherogenic particle, with its oxidative modification acting as a requisite initiating event in lesion formation. Conversely, HDL exerts profound atheroprotective effects, primarily mediated through reverse cholesterol transport and intrinsic anti-inflammatory properties. Lipoprotein(a) [Lp(a)], which contains the highly homologous apolipoprotein(a), possesses dual pro-atherogenic and pro-thrombotic properties and is frequently elevated in patients with familial hyperlipidemia. Importantly, species-specific variations in baseline lipoprotein profiles significantly dictate susceptibility to atherosclerosis, thereby determining the translational relevance of specific animal models employed in drug screening 28.

Relevance in Animal Models

The accurate biological reproduction of atherosclerotic pathology is a prerequisite for the rigorous evaluation of antihyperlipidemic agents. When subjected to high-fat diets, ApoE⁻/⁻ and LDLR⁻/⁻ murine models reliably develop atherosclerotic lesions localized to the aorta and aortic root; however, the architectural complexity of these plaques remains somewhat limited relative to human disease 28. In contrast, WHHL rabbits and cholesterol-fed New Zealand White rabbits consistently develop foam cell-rich vascular lesions that closely parallel early-stage human plaques 10. Larger models, including pigs and non-human primates, are capable of exhibiting advanced plaques characterized by distinct necrotic cores, calcification, and mature fibrous caps, thereby offering superior physiological parallels to human atherosclerosis 28,29.

Limitations in Modelling Atherosclerosis

Despite their undeniable utility, animal models harbor inherent translational limitations. Spontaneous plaque rupture and subsequent occlusive thrombosis remain exceedingly rare events in most non-human species. Fundamental discrepancies in lipoprotein metabolism, immunologic repertoire, and relatively abbreviated lifespans further constrain the fidelity of long-term disease modeling. Specifically, the complete absence of CETP expression in rodents fundamentally restricts their utility in reliably replicating human HDL biology. Consequently, rational model selection must be meticulously guided by the specific pathophysiological features and therapeutic mechanisms under active investigation to ensure maximal translational relevance 29.

Diet-Induced Hyperlipidemia Models

Diet-induced models are among the most widely utilized systems for studying hyperlipidemia and its associated metabolic disorders. These models typically employ high-fat diets (HFD), high-cholesterol diets (HCD), or combinatorial diets enriched with saturated fats, sucrose, or fructose to induce dyslipidemia. Rodents fed an HFD or HCD exhibit elevated plasma triglycerides, total cholesterol, and LDL-C, alongside reduced HDL-C. These metabolic alterations are frequently accompanied by insulin resistance, hepatic steatosis, and low-grade systemic inflammation, closely mimicking the features of human metabolic syndrome 31. However, diet composition varies considerably across studies with respect to fat source, cholesterol content, and duration of feeding. Such heterogeneity introduces significant variability and complicates cross-study comparisons. Furthermore, rodents require supraphysiological dietary cholesterol to induce hypercholesterolemia, inherently limiting their ability to fully replicate human lipid metabolism 32.

Chemically Induced Hyperlipidemia Models

Chemical induction models utilize pharmacological agents to disrupt systemic lipid homeostasis. Triton WR-1339 (tyloxapol) is commonly employed to inhibit lipoprotein lipase activity, resulting in an acute elevation of plasma triglycerides and cholesterol. Similarly, poloxamer 407 induces hyperlipidemia through the dual inhibition of lipoprotein clearance and the upregulation of hepatic lipid synthesis 33. These models are particularly advantageous for the rapid, short-term screening of lipid-lowering compounds and for mechanistic studies involving triglyceride metabolism. However, chemically induced hyperlipidemia fails to replicate the chronic inflammatory and complex vascular features characteristic of human dyslipidemia. Consequently, their translational relevance for evaluating long-term cardiovascular outcomes remains highly limited 34,35.

Genetically Modified Animal Models

Genetically modified models provide invaluable insights into inherited dyslipidemias and the specific molecular pathways regulating lipid metabolism. Apolipoprotein E-deficient (ApoE⁻/⁻) and LDL receptor-deficient (LDLR⁻/⁻) mice are extensively utilized in atherosclerosis research due to their profound propensity to develop hypercholesterolemia and vascular lesions 36. ApoE⁻/⁻ mice develop spontaneous atherosclerosis even when maintained on standard chow diets, whereas LDLR⁻/⁻ mice require dietary cholesterol supplementation to accelerate lesion formation. Despite their robust utility, these models differ substantially from human lipid metabolism, particularly regarding lipoprotein distribution and the absence of CETP activity 37. Recent advances in CRISPR/Cas9 technology have enabled the generation of humanized models expressing human ApoB, PCSK9, or CETP, thereby significantly improving their translational relevance. Nonetheless, the high cost and technical complexity associated with generating and maintaining such models continue to limit their widespread adoption 38.

Microbiota-Modulated Models

Emerging evidence highlights the critical role of the gut microbiota in systemic lipid metabolism and cardiovascular risk. Interventions utilizing probiotics, prebiotics, and synbiotics have been shown to modulate lipid profiles via bile acid metabolism, short-chain fatty acid production, and the regulation of host gene expression 39,40. Animal models incorporating microbiota modulation, including antibiotic-treated or germ-free rodents, are increasingly used to elucidate complex host–microbe interactions in hyperlipidemia. While highly promising, such studies often exhibit significant methodological variability in microbial strain selection, dosing regimens, and intervention duration, which complicates reproducibility and translational interpretation 41. Therefore, critical appraisal of these studies is essential, particularly when sample sizes are restricted or appropriate control groups are lacking.

Alternative and Emerging Models

Zebrafish (Danio rerio) have gained considerable attention as alternative models for lipid research due to their optical transparency, genetic manipulability, and rapid lipid metabolism. Zebrafish larvae fed high-cholesterol diets exhibit pronounced lipid accumulation and vascular lipid deposition, providing an efficient platform for the high-throughput screening of prospective antihyperlipidemic compounds. However, fundamental evolutionary differences in cardiovascular anatomy, lipoprotein composition, and immunological responses severely limit their applicability for modeling advanced, complex atherosclerosis. Consequently, zebrafish models are optimally positioned for early-stage discovery and screening rather than for definitive translational studies 42.

Small Animal Models

Small animals—predominantly mice, rats, and hamsters—are extensively utilized due to their cost-effectiveness, ease of handling, rapid reproductive cycles, and the vast availability of genetic manipulation tools. These models are particularly advantageous for mechanistic investigations, target validation, and early-stage pharmacological screening 43. However, they possess substantial limitations. Notably, most rodents inherently lack CETP activity, exhibit disparate baseline lipoprotein distributions compared to humans, and rarely manifest spontaneous plaque rupture or occlusive thrombosis. Furthermore, a pervasive sex bias remains in preclinical research, as numerous studies are conducted exclusively on male cohorts, thereby significantly restricting the clinical generalizability of the findings (Figure 3).

Large Animal Models

Large animal models, including rabbits, pigs, and non-human primates, provide a superior physiological resemblance to human lipid metabolism, vascular architecture, and atherosclerotic disease progression. Rabbits naturally express CETP and readily develop cholesterol-enriched lipoproteins alongside foam cell-dense vascular lesions. Pigs and non-human primates are capable of developing complex, advanced atherosclerotic plaques characterized by mature fibrous caps and necrotic cores 44. Despite these distinct translational advantages, the utility of large animal models is frequently constrained by prohibitive costs, extended study durations, heightened ethical scrutiny, and restricted biological availability. Additionally, stringent regulatory requirements and substantial infrastructural demands further limit their routine implementation in lipid research (Figure 3) 38,44.

Strategic Integration of Models

Employing a tiered, strategic approach that integrates both small and large animal models substantially enhances the probability of translational success. Small animals are optimally suited for initial hypothesis generation, fundamental mechanistic exploration, and preliminary efficacy screening. Conversely, large animals are requisite for late-stage translational validation, comprehensive safety assessments, and precise dose optimization. A judicious, sequentially aligned combination of both model archetypes—tailored specifically to the given research phase (e.g., early discovery versus advanced preclinical validation)—ensures scientific robustness, ethical compliance, and maximal translational relevance (Figure 3) 45.

Quality Grading of Evidence

The strength of evidence derived from animal studies was qualitatively graded based on experimental reproducibility, consistency across species, mechanistic plausibility, and alignment with human pathophysiology. Models demonstrating conserved lipid pathways, endogenous CETP activity, and plaque characteristics comparable to human atheromas were considered to provide strong evidence (++ evidence). Conversely, models primarily suited for mechanistic or exploratory studies were graded as providing moderate evidence (+ evidence). Rodent models, for instance, offer robust mechanistic insights but demonstrate limited translational accuracy for evaluating HDL-targeted therapies. In contrast, rabbit and non-human primate models provide a stronger predictive value for LDL-centric interventions, although their utility is frequently constrained by ethical and logistical considerations 47.

Methodological Limitations in Primary Studies

Many of the primary studies included in this review report positive lipid-lowering outcomes without adequately addressing underlying experimental limitations. Common methodological issues include small sample sizes, an absence of sex-based analyses, abbreviated intervention durations, and insufficient reporting of randomization or blinding procedures. These factors collectively inflate the risk of bias and can lead to the overestimation of therapeutic efficacy 48. Furthermore, in microbiota-modulation studies, profound heterogeneity in microbial strain selection, dosing regimens, and treatment duration further complicates data interpretation. The pervasive absence of standardized outcome measures across independent laboratories remains a major contributor to the irreproducibility often observed in lipid research.

Publication Bias and Reporting Gaps

Publication bias represents a significant yet frequently underappreciated limitation within preclinical lipid research. Studies that demonstrate positive or novel findings are disproportionately more likely to be published, whereas neutral or negative results often remain underreported. This inherent bias can profoundly distort the perceived efficacy of emerging antihyperlipidemic strategies, particularly those evaluated in genetically modified or novel dietary models 49. Selective outcome reporting, coupled with incomplete methodological descriptions, further diminishes study reproducibility. Greater adherence to standardized reporting frameworks, such as the ARRIVE guidelines, alongside the pre-registration of animal studies, could substantially improve the transparency and scientific credibility of the field.

Replacement Strategies

Whenever feasible, alternative methodologies—such as in vitro lipid assays, organ-on-a-chip platforms, computational modeling, and zebrafish larval systems—should be prioritized to replace higher-order animals during early-stage pharmacological screening. Integrating these alternative approaches substantially diminishes animal utilization while simultaneously facilitating rapid and efficient hypothesis testing 51.

Reduction of Animal Use

Rigorous statistical planning, the implementation of longitudinal study designs, and the utilization of shared control cohorts can significantly curtail the number of animals required without compromising statistical power or data integrity. Furthermore, the integration of non-invasive imaging modalities and the serial measurement of circulating biomarkers actively minimize the need for repeated animal sacrifice 52.

Refinement of Experimental Procedures

Refinement strategies are explicitly designed to minimize animal suffering and optimize overall welfare. These strategies encompass the provision of enriched housing conditions, the use of highly refined dietary formulations, the establishment of stringent humane endpoints, and the administration of robust analgesic protocols. Importantly, methodological refinement not only ensures ethical compliance but also bolsters data reliability by mitigating stress-induced metabolic and physiological variability 53. Collectively, strict ethical adherence and scientific rigor operate interdependently, ultimately reinforcing the translational validity of preclinical findings.

Failure of Promising Therapeutics

Numerous pharmacological agents that demonstrated robust efficacy in animal models have ultimately failed to achieve favorable clinical outcomes. CETP inhibitors represent a prominent example of this paradigm; substantial HDL elevation observed in preclinical and early-phase clinical studies did not consistently translate into a reduction in cardiovascular risk and, in certain instances, paradoxically increased adverse events 55. These clinical failures highlight the profound limitations of relying exclusively on biochemical surrogate endpoints—such as isolated alterations in LDL-C or HDL-C—without adequately evaluating plaque stability, systemic inflammation, or the long-term risk of clinical cardiovascular events.

Over-Reliance on Surrogate Endpoints

Preclinical investigations frequently prioritize the biochemical reduction of circulating lipids as their primary efficacy endpoint. However, the lowering of lipid levels does not invariably correlate with atherosclerotic plaque regression or a proportional decrease in clinical cardiovascular events. Integrating non-invasive vascular imaging, robust inflammatory biomarkers, and functional assessments of plaque stability into preclinical protocols may substantially improve translational accuracy 56.

Need for Standardization and Decision-Guided Model Selection

The pervasive lack of standardized experimental protocols governing diet composition, intervention duration, and specific endpoint measurements remains a major obstacle to ensuring cross-study comparability. Consequently, implementing a decision-tree framework that closely aligns specific research questions with the most appropriate animal models is essential. For example, mechanistic studies are optimally conducted using genetically modified rodents; lipoprotein profiling necessitates CETP-expressing species; and the comprehensive evaluation of plaque stability requires large-animal models. Such structured, evidence-based model selection fundamentally enhances both reproducibility and predictive value.

Future Directions

Future research in antihyperlipidemic pharmacology should prioritize the following key areas:

• The development and utilization of humanized and multi-gene animal models.

• The integration of multi-omics-based biomarkers to comprehensively profile metabolic responses.

• The implementation of rigorously sex-inclusive experimental designs.

• Strict ethical compliance with the 3Rs principles.

• The adoption of multimodal endpoints that extend significantly beyond basic lipid quantification.

Collectively, these strategic approaches will support a more reliable, predictive, and ethically responsible translational pipeline (Table 5).