Are we ready for cell-specific therapies in atherosclerosis?

Atherosclerotic cardiovascular diseases (CVD) represent the major cause of death worldwide. Atherosclerosis is a chronic non-resolving low-grade inflammatory disease elicited by accumulation of low-density lipoprotein (LDL) in the arterial sites that are exposed to turbulent blood flow. Current systemically delivered therapies have been associated with complications resulting in limited efficacy in ameliorating atherosclerotic CVD. Cell-specific drug delivery offers advantages to overcome these limitations.

Current available therapies

Current clinical intervention focuses on reducing disease-promoting factors including hypercholesterolaemia, hyperglycaemia, and hypertension. Pharmacological intervention targeting dyslipidaemia includes cholesterol-lowering drugs such as statins, ezetimibe, and proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors. Additional therapeutic agents that target the neurohormonal system and thrombosis include angiotensin-converting enzyme (ACE) inhibitors, beta blockers, anticoagulants, and antiplatelet therapies. Moreover, approved therapeutic agents for diabetes, which is associated with accelerated atherosclerosis, including sodium–glucose cotransporter-2 (SGLT-2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonist not only improve glucose control but also reduce cardiovascular (CV) events in patients with Type 2 diabetes. Interestingly, these clinical benefits have also been observed in heart failure patients in the absence of diabetes.¹

New therapies in clinical development

Recent large-scale clinical trials involving colchicine (COLCOT and LoDoCo2) have demonstrated the benefits of anti-inflammatory therapy for atherosclerotic CVD re-purposing a relatively safe and economical treatment. It is anticipated that the use of the colchicine may be considered in forthcoming international guidelines.

New nucleic acid–based therapeutics including small interfering RNAs (siRNA) and anti-sense oligonucleotides target gene suppression of proteins such as PCSK9 and lipoprotein(a) [Lp(a)] that play an important role in lipoprotein production or removal. Acetylgalactosamine (GalNAc) conjugation delivers these RNA molecules specifically to hepatocytes resulting in reduced drug toxicity. Acetylgalactosamine-conjugated siRNA against PCSK9, inclisiran, has recently been approved by the Food and Drug Administration (FDA).²

Need for cell-specific therapy

Systemically administered drugs have multiple limitations including suboptimal bioavailability, poor stability, and accelerated clearance as well as off-target distribution of the drug. Cell-specific therapeutic delivery is an alternative drug delivery approach that overcomes these limitations. However, cell-specific therapy in atherosclerosis faces several challenges including targeting specific cell type at different stages of the disease and specifying a unique cell antigen that is only expressed on diseased cells. Recently, hepatocyte-specific PCSK9 inhibition via inclisiran provides long-term therapeutic effects with only one injection in 6 months indicating the superiority of cell-specific therapy. In addition, beneficial CV outcomes of PCSK9 inhibition directed towards hepatocytes indicate that cells other than at the plaque site can be targeted. However, anti-inflammatory therapies should probably be directed to the vascular endothelium due the side effects of systemic inhibition of inflammation.

Cell-specific drug delivery systems

Currently, nanoparticles (NPs) are the most widely used drug delivery system for the cell-specific therapy. Cell-specific drug delivery can be achieved by displaying ligands for cell-specific disease-associated antigens at the surface of nanocarriers. The NPs offer several advantages including drug stability, enhanced drug bioavailability, delivery of therapeutic agents to the injured site in a targeted manner, and prevention of off-target drug toxicity. Nanoparticles are a diverse group of nanocarriers ranging from one to several hundred nanometres in size and include polymeric, HDL-like, LDL-like, inorganic NPs, and liposomes. A range of active therapeutic agents including epigenetic drugs (epi-drugs) can be encapsulated in the NPs. Epigenetics, through a cell-specific gene regulatory network, mediate environmental effects in the aetiology of several diseases including atherosclerosis.³ Application of systemic epigenetic therapy is often limited due to the toxicity of epigenetic drugs that can be overcome by cell-directed epi-drug delivery using NPs.

Another cell-specific drug delivery system is GalNAc conjugation. Acetylgalactosamine is an amino sugar derivate of galactose. The asialoglycoprotein is highly expressed on hepatocytes. It is a perfect candidate for hepatocyte-directed targeted drug delivery due to its high capacity for substrate clearance from serum by receptor-mediated endocytosis. Acetylgalactosamine binds to the asialoglycoprotein receptor resulting in efficient receptor-mediated endocytosis in hepatocytes. Many GalNAc-conjugated siRNA are currently being tested in clinical trials.

Directed therapies to the cells of interest in atherosclerosis

Each vascular cell type plays significantly different role at different stages of atherosclerosis (Figure 1). Due to their critical role in atherosclerosis, endothelial cells and macrophages have been tested intensively in cell-specific therapies. Chronically activated inflamed endothelial cells show increased expression of cell surface molecules including Intercellular Adhesion Molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), Platelet endothelial cell adhesion molecule-1 (PECAM-1), E-selectin, and P-selectin. These proteins have been targeted using NPs in multiple studies. For example, Receptor for Advanced Glycation Endproducts (RAGE) shRNA was delivered using P-selectin–targeted lipoplexes that alleviated RAGE-associated inflammation resulting in atherosclerosis attenuation in Apoe^−/− mice.⁴ More recently, the anti-inflammatory drug rapamycin, which was encapsulated in nanocarriers with self-delivery properties consisting of low-molecular-weight heparin conjugated with unsaturated fatty acids, was shown to blunt the vascular inflammation directed at P-selectin.⁵ Since turbulent blood flow mediates transcriptomic changes in vascular endothelial cells via mechanosensitive transcriptional regulators, targeting these factors may provide additional therapeutic options for atherosclerosis.

Figure 1.

Atherosclerosis plaque development is initiated with endothelial cell activation and elicited with LDL retention in the subendothelial space at the arterial sites that are exposed to turbulent blood flow. Mechanical forces associated with turbulent blood flow induce endothelial cell activation. Important transcriptional events that reflect endothelial cell response to turbulent blood flow include the activation of mechanosensitive transcriptional regulators including transcription factors and epigenetic modifiers that may represent novel site-specific targets. Hepatocyte-directed acetylgalactosamine-conjugated lipid-lowering therapies have shown promising outcomes in the clinical settings. Activated endothelial cells express inflammatory molecules that have been used to deliver anti-inflammatory drugs using nanoparticles to target vascular inflammation. Activated endothelial cells recruit monocytes to the atherosclerotic lesion that can be targeted with nanoparticles in early atherosclerosis. Macrophage-specific nanotherapy targets multiple pathological processes including inflammation, cholesterol efflux, proliferation, efferocytosis, and apoptosis using drug delivery systems including HDL-like particles, nanoparticles encapsulating small interfering RNAs, antibodies, and small-molecule drugs. Vascular smooth muscle cells-targeting cell-specific therapies need careful evaluation due to their contradictory roles in the plaque development including phenotypic switching to cells such as macrophage-like cells and their role in fibrous cap formation and plaque stability. Thrombosis causes most acute clinical events. Thrombolytic drugs can be delivered using nanoparticles. TBF, turbulent blood flow; GalNAc, acetylgalactosamine; NPs, nanoparticles; siRNA, small interfering RNAs; Abs, antibodies; SMD, small-molecule drugs; VSMCs, vascular smooth muscle cells; RBC, red blood cells.

Monocyte recruitment blockade has been successfully tested in preclinical studies and is an attractive target but only in early stage of the disease. Macrophage-directed therapeutic efforts have been focusing on multiple cellular processes including macrophage proliferation, efferocytosis, inflammation, apoptosis, and cholesterol efflux. In a preclinical study, when poly(lactic-co-glycolic acid) (PLGA) copolymer NPs loaded with rapamycin and coated with macrophage membrane were administered to high-fat diet-fed Apoe^−/− mice, these biomimetic NPs accumulated within the plaque, released rapamycin, and resulted in reduced atherosclerosis.⁶ In another study, single-walled carbon nanotubes were loaded with an inhibitor for the antiphagocytic CD47-Signal Regulatory Protein alpha (SIRPα) signalling axis. When administered in Apoe ^−/− mice, these nanotubes accumulated within the atherosclerotic lesion and reactivated lesional phagocytosis resulting in decreased plaque burden.⁷ More recently, Stablin-2 (S2P) peptide that recognized macrophage receptor stabilin-2 was used for macrophage-specific delivery of lipid NPs encapsulating siRNA for epsins, a family of endocytic adaptors. This nanotherapy reduced CD36-mediated lipid uptake and increased ATP Binding Cassette Subfamily G Member 1 (ABCG-1)–mediated cholesterol efflux resulting in plaque regression in Apoe^−/− mice.⁸ Although these examples indicate successful targeting of plaque macrophages, targeting newly identified macrophage subpopulations such as Trem2^hi macrophages discovered with single-cell sequencing needs to be tested using cell-specific therapeutic approaches.

Multiple macrophage-directed nanotherapies have been tested in the clinical settings. Although several clinical trials using either HDL mimetic NP CER-001 have produced disappointing results in TANGO, CARAT, and CHI-SQUARE and MDCO-216 in the MILANO-PILOT study, clinical outcomes with human plasma-derived apolipoprotein A-I CSL112 are promising which is currently in Phase 3 of clinical development (AEGIS-II).⁹

Concluding remarks

Despite the advances in preclinical studies, some limitations of cell-specific therapies need to be addressed for their clinical translation for atherosclerotic CVD. For example, NP-associated challenges including industrial-scale production, batch-to-batch reproducibility, biosafety, and immunogenicity need to be considered. In addition, pathophysiological complexity of atherosclerotic CVD represents a major challenge of identifying a unique cell-specific antigen that is only expressed on diseased cells. Although recent single-cell multiomics studies have provided improved information of disease-specific cellular populations and novel molecular targets, while targeting specific cell populations, the atherosclerotic plaque developmental stage needs to be considered. It is the right time to ask, ‘are we there yet’. Although many preclinical studies successfully used cell-specific treatment to attenuate atherosclerosis, clinical studies are limited. With technological advances and an improved understanding of the pathophysiology of atherosclerosis, the near future will witness research breakthroughs overcoming limitations of cell-specific therapies for atherosclerotic CVD.

Author contributions

Abdul Waheed Khan, PhD

Data availability

No new data were generated or analysed in support of this research.

Funding

A.W.K. is a National Heart Foundation of Australia research fellow (102492).