Data from experimental aged animal models have portrayed the associations between DNA damage and atherosclerosis [34]. End-replication and oxidative DNA damage mechanisms alter DNA stability; therefore, failures in DNA damage response (DDR) sequences cause genome instability by activating DDR [35, 36]. The result of constant DDR is the activation of cyclin-dependent inhibitor pathways, including p53/p21Cip and p16^INK4A/Rb [37]. With the successful repair of damaged DNA regions, standard cellular mechanisms are resumed [38]. However, unresolved DNA damage induces nuclear events that activate ataxia-telangiectasia mutated (ATM) and post-translational modifications of the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) essential modulator (NEMO), thus activating NF-κB, senescence (characterized by the expression of pro-inflammatory and immune-modulatory chemokines and cytokines, growth factors and matrix metalloproteinases) and, consequently, inflammatory circuits in atherosclerosis [39, 40]. This is evident in aged animal models that have been studied [41, 42]. Another evidence of an association between atherosclerosis, inflammation, and genetic instability involves the immune checkpoints CD80/CD86-CD28 and CTLA4. Breaks in DNA cause the expression of class I MHC and costimulatory molecules (CD80/CD86-CD28) in fibroblasts, together with adhesive molecules such as VCAM1 and ICAM1 [43]. Evidently, genetic deletion of CD80/86 vitiated the development of atherosclerosis in LDLR^-/- mice by lowering the inflammatory feedback driven by T cells [44]. However, in the aortic regions, which comprise more advanced plaques, αCTLA4 antibody (negative regulator CD80/CD86-CD28) increased plaque T cell composition, indicating the importance of this pathway in interventional therapy for accelerated atherosclerosis [44, 45]. Patients with Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) also show DNA detrition, cellular senescence, deregulated mTOR pathways (nutritional sensing pathway), and upregulated inflammatory responses and consequently succumb to complications from atherosclerosis [19, 46]. For instance, administering mTOR inhibitors in HGPS patients delays senescence [47]. Microsatellite instability (MSI) and loss of heterozygosity (LOH) are also commonly observed in atherosclerosis. For example, LOH at the hMSH2, hPMS1, and hMLH1 loci occurs in atherosclerosis. MSI and LOH have also been found on chromosomes 1, 2, 8, 9, and 17 in cerebral atherosclerotic plaques [48]. DNA repair has been shown to be less accurate in atherosclerotic tissues due to changes found in mismatch repair (MMR) genes [48]. The aortic samples from people with atherosclerosis had more damage to mitochondrial DNA (mtDNA) than the samples of healthy donors of identical age. These samples also had metabolic enzymes involved in fatty acid oxidation [49]. Manganese superoxide dismutase 2 (MnSOD2) deficiency and p66sch overexpression increase reactive oxygen species (ROS)-induced damage in mtDNA, accelerating atherosclerosis in ApoE^-/- mice [50, 51]. In the same way, ApoE^-/- mice models that were haploinsufficient for ATM/angiotensin (Ang) II receptor (ATR) repair mechanisms show worsened atherosclerotic lesions together with inflammatory markers in the mitochondria [52, 53]. The telomerase enzyme also ensures stable telomere lengths (TLs) and stability through the telomerase RNA component (TERC), telomerase reverse transcriptase (TERT), and dyskerin [54]. Their activities are reduced with age progression [54]. Despite contradictory results, associations have been observed between shorter TLs and CVDs [55]. For example, data from the West of Scotland Coronary Prevention Study Group (WOSCOPS) trial and other studies indicate a shorter TL as a predictor of cardiovascular risk factors such as atherosclerosis. Fibroblasts derived from patients with HGPS show premature senescence and telomere shortening [56]. VSMC, EC, and fibrous cap leukocytes also show shorter telomeres than usual during aging and plaque progression [57, 58]. The decreased expression of SRY-box transcription factor 4 (SOX4) genes in peripheral blood cells during aging also causes a shortening of TL, which contributes to senescence and atherosclerosis [59]. This is achieved by activating the tumor suppressor p53, reduction in levels of the coactivator 1- and the transcriptional regulator peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1-α (PGC1-α) and PGC1-β leading to mitochondrial dysfunction, deficient apoptosis, or EC senescence [60]. In short, DNA damage involves genomic alterations, adducts, and shortening of the telomere. In the same line, blood cells undergo somatic genetic mutations as we age, leading to expanded leukocyte clones and clonal hematopoiesis of indeterminate potential (CHIP) (another aging mechanism) [61]. Individuals with CHIP have increased cardiovascular related mortality. Data shows that mutations in DNA methyltransferase 3A (DNMT3A), tet methylcytosine dioxygenase 2 (TET2), and ASXL1 facilitate the clonal expansion of leukocytes. Furthermore, Macrophages from TET2-knockout hyperlipidemic mice result in abnormal activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and are absent in melanoma 2 (AIM2) inflammasome and contribute to enhanced atherosclerosis [60, 62, 63].

Epigenetics and aging mechanisms are linked to age-related diseases [98]. Individuals with advanced epigenetic caducity have an increased cardiovascular age and a higher risk of subclinical atherosclerosis progression [99]. Hypomethylation and DNA hypermethylation have been observed in the atheroma of ApoE^-/- mice and monocyte/macrophage cell lines before and after the onset of atherosclerosis [100–103]. Patients with atherogenesis and familial hypercholesterolemia (FH) often exhibit hypermethylation of promoters and protective genes such as ABCA1 and Krüppel-like factor 4 (KLF4) [104]. Atherogenic enzymes and receptors, including SOD, endothelial nitric oxide synthase (eNOS), and estrogen receptors, are hypomethylated [105, 106]. This overshadows the protective mechanisms. DNMT1 disrupts PPARγ signaling, leading to atherosclerosis. TET2 counteracts this, restoring autophagic activities in EC and VSMC [107]. The somatic mutation TET2, which leads to accelerated atherosclerosis, is associated with NLRP3-mediated IL-1β production in clonal hematopoiesis [108].

Histone deacetylases (HDACs) affect the progression of atherosclerosis in various ways, from lipid metabolism, EC integrity, inflammatory pathways, and plaque disruption to modulation of oxidative pathways. The role of HDACs in atherosclerosis often leads to conflicting results. HDAC3 plays a role in EC integrity, but mice lacking it exhibit more stable plaques, decreased NADPH oxidase 4 (Nox4), reduced lipid content, and an anti-inflammatory phenotype during atherosclerosis. HDAC3 also inhibits PPAR and enhances foam cell formation [109]. HDAC9 interacts with the IκK/NF-κB p65 complex, leading to inflammatory phenotypes in macrophages and ECs linked to atherosclerosis. HDAC9^-/- mice exhibit increased histone H3K9 acetylation at the ABCA1 and ABCG1 promoters, which affects macrophage cholesterol efflux and highlights its significant role in atherosclerosis [110]. HDAC6 causes EC injury and autophagy [111]. HDAC1/HDAC2 disrupts prolyl-4-hydroxylase alpha1 (P4Hα1) transcription, an enzyme in collagen formation, enhancing plaque destabilization. Furthermore, HDAC2 exhibits sexual dimorphic activity when inhibiting the expression of the antioxidant glutaredoxin-1 [111, 112]. HDAC7 suppresses VSMC proliferation and neointima formation by preventing β-catenin nuclear translocation [113, 114]. Recently, data suggest a more protective role for HDAC11 primarily through the expression of IL-10, IL-17, IL-10, IL-12, and TNF [115]. Members of the sirtuin (Sirt) family (Sirt1, Sirt3, and Sirt6) are studied for their antiaging and vasoprotective activities in atherosclerosis. Sirt1 modulates FOXO signaling, upregulates 5’-AMP-activated protein kinase (AMPK) (in low energy conditions), and restores normal autophagic activities (mTOR inhibition) [47], while Sirt6 targets inflammatory factors and loss of telomeres. SIRT3 protects against oxidative stress by upregulating PPAR‐α and eNOS and downregulating iNOS [116–118]. Sirt activities decline with age.

Non coding RNAs (ncRNAs) play a crucial role in atherogenesis, with their expression linked to EC dysfunction, inflammation, lipogenesis, and macrophage deregulation in atherosclerosis, with knockdown mice models demonstrating alleviation. Long ncRNAs (LncRNAs) and circular RNAs (circRNAs) modulate atherosclerotic progression via microRNAs (miRNAs) at the transcriptional and post-transcriptional levels. In cardiovascular development, three novel LncRNAs (TERMINATOR, ALIEN, and PUNISHER) have attracted great attention [119]. LncRNA H19 promotes the area of atherosclerotic plaque area in ApoE^-/- by inhibiting cholesterol efflux mechanisms and upregulating NF-κB-mediated inflammatory responses in vascular ECs by miR-130b [120]. The lncRNA RP5–833A20.1 THP-1 was found to increase oxidized LDL uptake, lower cholesterol efflux, and increase the production of inflammatory mediators like TNF-α, IL-1β, and IL-6 in macrophages [121]. However, the expression of LncRNA-DYNLRB2-2 lowered the amount of cholesterol in macrophages by increasing the expression of G-protein-coupled receptor 119 (GPR119) and the cholesterol transporter ABCA1 [122]. LncRNA SMILR (smooth muscle-induced LncRNA) enhances replication and aggravates atherosclerosis by targeting the miR-10b-3p/KLF5 axis [123, 124]. LncRNA-p21 improves atherosclerosis by regulating the miR-221/SIRT1/Pcsk9 axis [125]. LncRNA-MALAT1 sponges miR-330-5p to activate the NF-κB signal pathway in THP-1 macrophage-derived foam cells in atherosclerosis pathogenesis [126]. Similarly, circRNAs are differentially expressed in atherosclerotic regions. Li et al. [127] found that 52 circRNAs were up-regulated more than twice and 47 circRNAs were down-regulated more than 1.5 times in atherosclerotic aortic vessels and of the circRNA examined, Mmu_circRNA_36781 (circRNA ABCA1) and 37699 (circRNA KHDRBS1) were significantly up-regulated in atherosclerotic aortic vessels and H₂O₂-treated mouse aortic ECs. They potentially regulated atherosclerosis and vascular endothelial injury by targeting the miR-30d-3p-TP53RK and miR-140-3p-MKK6 axes [127, 128].

Protein homeostasis declines with age, leading to abnormalities in the neurological and cardiovascular systems [129]. Numerous studies show that one of the main factors influencing atherogenesis is cell death [130, 131]. The signaling channels involved include the ubiquitin- proteasome, chaperons, and lysosome-dependent autophagy, with evidence found in human and animal cardiovascular models leading to atherosclerosis [132]. Madrigal-Matute et al. [129] found a decrease in activities and elevated inflammatory responses after chaperone-mediated autophagy (CMA) blockage in a fluorescent reporter mouse model, consistent with the results observed by Qiao et al. [133] (2021). Subclinical models demonstrate the essential involvement of the ubiquitin proteasome system (UPS) in NF-κB activation, where impaired UPS is associated with uncontrolled NF-κB pathways that drive inflammation in inflammatory-related disorders [131, 134]. Exploring the connection between atherosclerosis and UPS has revealed both pro-atherogenic and potentially preventive impacts. Increased inhibition of the proteasome accelerates the progression of atherosclerosis, while reduced inhibition enhances cholesterol efflux through ABCA1 and ABCG1 [135]. UPS is essential for the intracellular breakdown of proteins in all cells. Under stress, these proteasome activities are replaced by immunoproteasomes (IP), which mediate immune responses in various inflammatory diseases. Nevertheless, in LDLR^-/-LMP7^-/- and LDLR^-/- mice fed on a Western-type diet for 6 weeks or 24 weeks to induce early and advanced stage atherosclerosis, respectively, depleted IP subunit β5i/LMP7 did not alter protein homeostasis and macrophage polarization, thus not aggravating atherogenesis in LDLR^-/- mice [96]. The degradation of perilipin (protects lipid droplets from lipase activities) through the UPS pathway leads to the progression of atherosclerosis, while inhibition of adiponectin receptor 1 (ADR1) protects atherosclerotic models by altering the generation of foam cells [136]. Ubiquitin-specific peptidase 9 X-linked (USP9X), a deubiquitinating enzyme, inhibits macrophage lipid uptake by downregulating SR-A1, preventing foam cell formation [137]. Furthermore, ubiquitin C-terminal hydrolase L1 (UCHL1) and USP14 promote CD36 autophagic degradation, but their mechanical effects have opposite results: Inhibition of USP14 leads to the suppression of foam cells while deubiquitinating UCHL1 promotes the formation and stabilization of foam cells [138, 139]. Heat shock protein 60 (HSP 60) is the most recognized HSP 60 with known direct atherogenic potential. However, HSP 70, HSP 90, and HSP 47 have shown the potential to aggravate atherosclerosis in ApoE^-/- mice and THP-1 derived macrophage models as reviewed by various authors [139–144]. In the same line, HSP 27 demonstrates atheroprotective activities in vitro [142].

The gut microbiome and the immune system are interdependent, and disruptions can increase vulnerability to disease and influence drug candidates’ pharmacokinetic or pharmacodynamic results. Studies show that as we age, the diversity of the gut microbiome decreases due to a loss in organisms’ physiological and symbiotic capacities, leading to reduced longevity and age-related anomalies [145]. Reprogramming of the gut microbiome induced by trimethylamine N-oxide (TMAO) is a typical example that serves as a prognostic index for CVDs and senescence [146, 147]. In aged animal models, TMAO is elevated and accelerates cell senescence by causing mitochondrial damage, superoxide formation (ROS), and promoting the generation of pro-inflammatory factors such as caspase-1 and IL-1β through the ROS-TXNIP-NLRP3 signaling pathway, thus aggravating atherosclerosis [148, 149]. It can also stop the SIRT3-SOD2-mitochondrial ROS signaling pathway, which damages ECs and leads to the development of atherosclerosis [148]. Zhu et al. [150] found that an enriched titanium dioxide diet (E71)/TMAO in ApoE^-/- mice caused the appearance of lesions, mediated by E71, which transformed choline into pro-atherogenic TMAO. Consistently, Traughber et al. [151] reversed cadmium and high-fat-induced atherosclerosis by inhibiting TMAO production, NLRP3 machinery, and macrophage polarization in the gut microbiota of mice. TMAO also reduces bile acid formation, increases foam cell formation, and promotes oxidative stress-induced mitochondrial damage, leading to pro-inflammatory factors, senescence, and atherosclerosis during aging [148]. The alteration of the intestinal microbiome reduces butyrate-producing bacteria, increasing intestinal permeability and lipopolysaccharide levels, which activate inflammatory pathways, primarily in gram-negative species [152]. The intestinal microbiomes also produce hydrogen sulfide (H₂S), which supports vascular tone, VSMC, cholesteryl ester (CE) activities, and energy production under normal conditions, but under strenuous conditions, H₂S becomes pro-atherogenic [153–156]. Specifically, H₂S is produced by three key enzymes; cystathionine β-lyase (CBE), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST)/cysteine aminotransferase (CAT) [157]. CSE is predominantly in the cardiovascular system [147]. These enzymes can be suppressed under conditions of high glucose, oxidative stress, and high fat diet (HFD) thus leading to a reduction in H₂S production [156, 158]. For example, inhibition of MST by 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) resulted in adipocyte differentiation and lipid accumulation with increased inflammation of adipose tissue [159]. CSE exhibits its antiatherosclerotic effect through 3 mechanisms, namely reduction of the chemoattractant factor, ICAM1, and CX3C CCR 1 (CX3CR1), inhibition of macrophage lipid uptake, and induction of VSMCs apoptosis through the MAPK pathway [157]. Although it has not been clinically tested, H₂S has excellent potential as a treatment for this pathophysiological condition. Figure 3 below summarizes vascular mechanisms associated with aging and atherosclerosis materialization.