Free radicals and antioxidants

The oxidation hypothesis of atherosclerosis

Nanoplastics (NPs) exposure is usually linked with abnormal inflammation and oxidative stress, which are high-risk triggers of atherosclerosis; however, whether this exposure causes the development of atherosclerosis is vague. Here, we found that PS NPs co-exposure with ox-LDL induces significant accumulation of lipid, as well as oxidative stress and inflammation in RAW264.7 macrophages. Using an ultrasound biomicroscope (UBM), we observed the emergence of atherosclerotic plaques at the aortic arch of apolipoprotein knockout (ApoE^-/-) mice after being exposed to PS NPs for three months. Oil-red O and hematoxylin-eosin (H&E) staining at the mice’s aortic root also observed the deposition of lipids with plaque formation. Moreover, the development of atherosclerotic disease is associated with disturbances in lipid metabolism and oxidative stress damage in the mice liver. In conclusion, this study provides additional evidence to further understand the possible cardiovascular damage caused by NPs exposure.

Atherosclerosis, formed by fibrofatty lesions in the artery wall, underpins myocardial infarctions, strokes and disabling peripheral artery diseases. Inflammation with multiple maladaptive roles runs through atherosclerotic inception, propagation, and complications. Given degree of inflammation differing among atherosclerotics, inflammation-targeted nanomedicine may produce individualized monitoring and manipulation of inflammatory regulators beyond traditionally slowing down atherosclerosis progression. Equipped with biomimetic membrane, microenvironment-responsive and targeting structures, nanomedicines could escape from the innate immune system and autonomously navigate to targeted lesions, thereby muting inflammation in atherosclerotic sites. Moreover, nanoparticles’ advantages go beyond advancing pharmacology. The optimal physicochemical properties of nanoparticles favor noninvasive imaging function to provide diagnostic and prognostic information of atherosclerotic plaques. Despite efficacious ability of the preclinical anti-inflammation nanotherapeutics to reduce atherosclerotic risks, future translation of inflammation-targeted nanomedicines requires to comprehensively refine the strategies that the nanomedicines forestall inflammatory pathways via systemic exposure, drug-cell interactions, and endothelial barrier reconstruction. In this review, we outline the development of diverse nanomedicines to specifically identify pathophysiological changes and combat advanced atherosclerosis along with dynamically tracing inflammatory markers. Finally, challenges and opportunities of inflammation-targeted nanomedicines are highlighted to pave avenues toward personalized medicine for atherosclerosis to stem the growing global burden of cardiovascular diseases.

Atherosclerotic plaques associated with acute coronary syndromes (ACS), i.e. culprit lesions, frequently feature a ruptured fibrous cap with thrombotic complications. On imaging, these plaques exhibit a low attenuation, lipid-rich, necrotic core containing cholesterol crystals and are inherently unstable. Indeed, cholesterol crystals are causally associated with plaque vulnerability in vivo; their formation results from spontaneous self-assembly of cholesterol molecules. Cholesterol homeostasis is a central determinant of the physicochemical conditions leading to crystal formation, which are favored by elevated membrane free cholesterol content in plaque endothelial cells, smooth muscle cells, monocyte-derived macrophages, and foam cells, and equally by lipid oxidation.

Emerging evidence from imaging trials in patients with coronary heart disease has highlighted the impact of intervention involving the omega-3 fatty acid, eicosapentaenoic acid (EPA), on vulnerable, low attenuation atherosclerotic plaques. Thus, EPA decreased features associated with unstable plaque by increasing fibrous cap thickness in statin-treated patients, by reducing lipid volume and equally attenuating intraplaque inflammation. Importantly, atherosclerotic plaques rapidly incorporate EPA; indeed, a high content of EPA in plaque tissue is associated with decreased plaque inflammation and increased stability. These findings are entirely consistent with the major reduction seen in cardiovascular events in the REDUCE-IT trial, in which high dose EPA was administered as its esterified precursor, icosapent ethyl (IPE); moreover, clinical benefit was proportional to circulating EPA levels.

Eicosapentaenoic acid is efficiently incorporated into phospholipids, where it modulates cholesterol-enriched domains in cell membranes through physicochemical lipid interactions and changes in rates of lipid oxidation. Indeed, biophysical analyses indicate that EPA exists in an extended conformation in membranes, thereby enhancing normal cholesterol distribution while reducing propagation of free radicals. Such effects mitigate cholesterol aggregation and crystal formation. In addition to its favorable effect on cholesterol domain structure, EPA/IPE exerts pleiotropic actions, including antithrombotic, antiplatelet, anti-inflammatory, and proresolving effects, whose plaque-stabilizing potential cannot be excluded.

Docosahexaenoic acid is distinguished from EPA by a higher degree of unsaturation and longer carbon chain length; DHA is thus predisposed to changes in its conformation with ensuing increase in membrane lipid fluidity and promotion of cholesterol aggregation into discrete domains. Such distinct molecular effects between EPA and DHA are pronounced under conditions of high cellular cholesterol content and oxidative stress.

This review will focus on the formation and role of cholesterol monohydrate crystals in destabilizing atherosclerotic plaques, and on the potential of EPA as a therapeutic agent to attenuate the formation of deleterious cholesterol membrane domains and of cholesterol crystals. Such a therapeutic approach may translate to enhanced plaque stability and ultimately to reduction in cardiovascular risk.

The omega-3 fatty acids (n3-FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) rapidly incorporate into cell membranes where they modulate signal transduction pathways, lipid raft formation, and cholesterol distribution. Membrane n3-FAs also form specialized pro-resolving mediators and other intracellular oxylipins that modulate inflammatory pathways, including T-cell differentiation and gene expression. Cardiovascular (CV) trials have shown that EPA, administered as icosapent ethyl (IPE), reduces composite CV events, along with plaque volume, in statin-treated, high-risk patients. Mixed EPA/DHA regimens have not shown these benefits, perhaps as the result of differences in formulation, dosage, or potential counter-regulatory actions of DHA. Indeed, EPA and DHA have distinct, tissue-specific effects on membrane structural organization and cell function. This review summarizes: (1) results of clinical outcome and imaging trials using n3-FA formulations; (2) membrane interactions of n3-FAs; (3) effects of n3-FAs on membrane oxidative stress and cholesterol crystalline domain formation during hyperglycemia; (4) n3-FA effects on endothelial function; (5) role of n3-FA-generated metabolites in inflammation; and (6) ongoing and future clinical investigations exploring treatment targets for n3-FAs, including COVID-19.

Pathological eye involvement represents a quite common finding in a broad spectrum of autoimmune rheumatic diseases (ARDs). Ocular signs, often occur as early manifestations in ARDs, ranging from symptoms related to the mild dry eye disease to sight-threatening pathologies, linked to the immune response against retinal and choroidal vessels. Retinovascular damage driven by markedly inflammatory reactivity need a prompt diagnosis and treatment.

Immune-complexes formation, complement activation and antibody-mediated endothelial damage seem to play a key role, particularly, in microvascular damage and ocular symptoms, occurring in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjögren's syndrome (SS).

Conversely, early alterations of retinal and choroidal vessels in the asymptomatic patient, often detectable coincidentally, might be indicators of widespread vascular injury in other connective tissue diseases. Particularly, endothelin-induced hypoperfusion and pathological peri-choroidal extracellular matrix deposition, might be responsible for the micro-architectural alterations and loss of capillaries detected in systemic sclerosis (SSc). Instead, interferon alpha-mediated microvascular rarefaction, combined with endothelial lesions caused by specific autoantibodies and immune-complexes, appear to play a significant role in retinal vasculopathy associated to inflammatory idiopathic myopathies (IIM).

The immuno-pathophysiological mechanisms of ocular microcirculatory damage associated with the major ARDs will be discussed under the light of the most recent achievements.

Oxidation of small dense low-density lipoprotein (sdLDL) and membranes is causally related to atherosclerosis. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA, 20:5, ω-3) significantly reduced oxidized LDL in patients with hypertriglyceridemia by unknown mechanisms. We compared EPA effects to related FAs of varying chain length and unsaturation on oxidation of sdLDL and model membranes, and on cholesterol crystal domains. We compared EPA to the FAs: stearic (SA, 18:0), oleic (OA, 18:1, ω-9), linoleic (LA, 18:2, ω-6), alpha-linolenic (ALA, 18:3, ω-3), eicosanoic (EA, 20:0), eicosatrienoic (ETE, 20:3, ω-3), arachidonic (AA, 20:4, ω-6), docosapentaenoic (DPA, 22:5, ω-3), and docosahexaenoic (DHA, 22:6, ω-3).

Human sdLDL or model membranes of cholesterol and 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine [18:2(cis)PC or DLPC] were preincubated with FAs followed by copper-induced oxidation. Malondialdehyde (MDA) or lipid hydroperoxides (LOOH) levels measured oxidation; small-angle X-ray diffraction assessed cholesterol domain formation.

After 40 min, EPA reduced MDA levels 70% compared to vehicle (p < 0.001). Lesser inhibition was observed with DHA, DPA, ETE, and ALA (33%, 34%, 32%, and 16%, respectively; all p < 0.001 versus vehicle). Similar relative FA effects were observed in model membranes where EPA more substantially inhibited cholesterol crystal domain formation.

We observed relationships between hydrocarbon length and unsaturation with antioxidant activity and membrane cholesterol domain formation. EPA had the most favorable molecular structure, likely contributing to membrane stability, improved lipoprotein clearance, and reduced inflammation.

Insight is provided into FA hydrocarbon length and unsaturation relationships with antioxidant activity in lipoproteins and membranes, and cholesterol crystal domains formation.