As mentioned earlier, the key events in the development of atherosclerosis revolve around macrophages, their transition into subendothelial space and transformation into foam cells ^[1][30]. The role of macrophages in the subendothelium is quite complex and as such is interconnected with the processes of deregulated cholesterol transport, the reduced ability of macrophages to perform apoptosis, phagocytosis, efferocytosis, and the presence of inflammation and oxidative stress ^[2][31].

Cholesterol build-up in the macrophages can be aborted by the efficient reverse cholesterol transport mediated by the HDL. However, this process is often dysregulated during the development of atherosclerosis ^[1][30]. Currently, a number of lncRNAs are found to be implicated in this process. A recent study by Yu et al. ^[3][32] showed that overexpression of the lncRNA Kcnq1 overlapping transcript 1 (KCNQ1OT1) in apoE influences the efficiency of RCT. This occurs through the competitive binding of KCNQ1OT1 to miR-452-3p, resulting in increased HDAC3 expression and decreased ABCA1 expression in macrophages. In contrast, the knockdown of KCNQ1OT1 prevented atherosclerosis in −/− mice thanks to reduced lipid accumulation in THP-1 cells ^[4][26]. On the other hand, the lncRNA MeXis shows a different effect on RTC in macrophages ^[5][33]. Namely, MeXis potentiates LXR-dependent transcription of ABCA1 through loosened actions of the transcriptional coactivator DDX17, which has been shown to be enriched at LXR-binding sites in ABCA1 enhancer regions. Reduced expression of ABCA1 was found in the bone marrow of Ldlr−/− mice after transplantation of MeXis−/− hematopoietic cells, compared to expression in the bone marrow of mice transplanted with wild-type (WT) hematopoietic cells. In addition, mice with transplanted MeXis−/− blood cells have a significantly increased plaque burden after 17 weeks of the Western diet. Another lncRNA with LXR-regulating function is CHROME ^[6][34]. CHROME expression is tightly controlled by dietary and cellular cholesterol levels. Namely, an increase in cholesterol levels can activate the sterol-activated transcription factor LXR, leading to the upregulation of CHROME. Additionally, in THP-1 macrophage cells, this lncRNA expresses its cytosolic functions by sequestering miR-27b, miR-33a, miR-33b, and miR-128 from their target mRNAs. Loss of function of these miRNAs could significantly increase the mRNA levels of ABCA1, OSBPL6 and NPC1, which is an important step in maintaining the efflux of excess cellular cholesterol, especially in macrophages in arterial walls. CHROME expression levels were found to be higher in cholesterol-laden macrophages in vitro and in human atherosclerotic plaques, while CHROME-7WT-expressing THP-1 macrophages showed lower cytoplasmic accumulation of lipid droplets ^[6][34]. Another lncRNA associated with the regulation of ABCA1 expression is Prostate Cancer Antigen 3 (PCA3). Its downregulation has been associated with foam cell formation and the development of atherosclerosis ^[7][35]. More specifically, the atheroprotective effect of this lncRNA is achieved through the PCA3/miR-140-5p/RFX7/ABCA1 axis. PCA3 sponges miR-140-5p, which upregulates RFX7, one of the DNA binding proteins. RFX7, in turn, upregulates the expression of ABCA1 in foam cells of ApoE−/− mice. This leads to increased cholesterol efflux and decreased lipid accumulation in macrophages ^[7][35]. On the other hand, the lncRNA GAS5 showed significant upregulation in the AS mouse model and in ox-LDL-treated macrophages ^[8][36]. This lncRNA acts as a suppressor of miR-135a, which has been shown to have potent anti-atherosclerotic effects. In macrophages treated with ox-LDL, restoration of miR-135a expression reversed the effects of GAS5—particularly those related to dysregulation of lipid metabolism and inflammation ^[8][36]. Meng et al. demonstrated increased lipid accumulation due to overexpression of GAS5 in THP-1 foam cells. By interacting with EZH2, GAS5 can prevent the expression of ABCA1, which was reduced by EZH2 overexpression, resulting in decreased cholesterol efflux ^[9][37].

A growing body of evidence suggests that apoptosis of macrophages present in lesions is closely related to atherosclerosis stages ^[10][38]. A recent study by Simion et al. demonstrated the specific presence of proatherogenic lncRNA MAARS in the macrophages present in plaques. MAARS knockdown resulted in significantly reduced areas of atherosclerotic plaques in the thoraco-abdominal aorta and aortic sinus in Ldlr−/− mice. It is proposed that MAARS may act upon the RNA-binding protein HuR one of the critical mediators of transcript stability and an apoptosis regulator. HuR nucleus-cytosolic shuttling was found to be reduced in macrophages overexpressing MAARS. Additionally, in Ldlr−/− mice, attenuation of interactions between MAARS and HuR significantly decreased pro-apoptotic markers, such as p53, p27, and caspase-3, -8, and -9, whereas anti-apoptotic markers, such as BCL2, Mcl1, and ProtA, increased in the intima of atherosclerotic lesions. MAARS knockdown has also shown the potential to increase macrophage clearance, or efferocytosis through decreased expression of c-Mer tyrosine kinase, which acts as a cell surface receptor and signalling molecule mediating efferocytosis ^[10][38]. Another lncRNA, MIAT, expresses its proatherogenic roles through several mechanisms. One of the recent studies showed that the upregulation of MIAT is stimulated by ox-LDL, particularly in macrophages of advanced atherosclerotic lesions in ApoE−/− mice ^[11][39]. In vivo MIAT knockdown resulted in a decreased necrotic core and increased plaque stability, primarily through increased macrophage-mediated clearance of apoptotic cells. This effect was also observed in vitro. Additionally, inhibition of efferocytosis by MIAT was observed, most likely targeting the miR-149-5p/CD47 axis ^[11][39]. Another important lncRNA with pro-atherogenic function is LIPCAR. In the study by Hu N et al. ^[12][40], overexpression of LIPCAR in oxLDL-treated human acute monocytic leukaemia (THP-1) cells resulted in increased lipid accumulation leading to the formation of foam cells. Additionally, the knockdown of LIPCAR in THP-1 cells could potentially reverse ox-LDL-mediated inhibition of cell proliferation and apoptosis. Another lncRNA that may have a role in macrophage function is PELATON. Namely, this lncRNA is predominantly located in the nucleus of monocytes and monocyte-derived macrophages. In one of the recent studies ^[13][41], knockdown of PELATON in macrophages resulted in the impairment of phagocytosis. Alterations in different phagocytosis processes were observed, such as the uptake of oxLDL particles or production of reactive oxygen species, which are closely associated with the progression of stable plaques into vulnerable or unstable phenotypes. The knockdown of PELATON resulted in the downregulation of CD36, one of the scavenger receptors that recognizes dying cells and oxLDL particles, demonstrating its role as a critical regulator of phagocytosis ^[13][41].

Early atherosclerosis is characterized by oxidative stress and inflammation. Macrophages play an important role in the establishment of a chronic inflammatory state seen in the development and progression of atherosclerosis. Inflammation is always accompanied by the increased production of reactive oxygen species, and subsequent oxidative damage. Several lncRNAs have been shown to regulate the inflammatory response in macrophages. A recent study by An JH et al. ^[14][42] found that the lncRNA SNHG16 is upregulated in patients with AS and promotes inflammatory responses in THP-1 macrophages. They also discovered that overexpression of this lncRNA leads to a marked downregulation of miR-17-5p, which in turn activates the NF-κB pathway and triggers an inflammatory response in AS patients and THP-1 macrophages ^[14][42]. Another lncRNA that has the ability to activate proinflammatory macrophages via the NF-κB pathway is MIAT ^[15][43]. Elevated levels of MIAT have also been found in the sera and plasma of symptomatic patients ^[11][16][17][39,44,45]. Elevated MIAT levels correlated positively with serum levels of TNFα, IL-6 ^[17][45] and IL-8 ^[16][44] and were also associated with lower levels of IL-10 ^[16][44]. Consistent with this, different polymorphisms in the promoter region of MIAT could potentially confer a higher risk of developing myocardial infarction ^[18][46]. NEAT1 can also contribute to atherosclerosis progression through oxidative stress and inflammatory pathways ^[19][47]. Chen et al. demonstrated that stimulation of macrophages with ox-LDL leads to upregulation of NEAT1. Furthermore, NEAT1 silencing in macrophages led to the downregulation of CD36, IL-6, IL-1β, TNF-α, a decrease in foam cell formation, and suppression of reactive oxygen species (ROS) and malondialdehyde (MDA) levels through an increase in superoxide dismutase (SOD) activity. The authors speculated that these effects could be dependent on sponging of miR-128. NEAT1 inhibition also helps to suppress inflammation through significant reduction in levels of IL6, IL1, cyclooxygenase 2 (COX2), and TNF-α levels in THP1 cells ^[19][47].

Endothelial-to-mesenchymal transition (EndMT) is the process in which endothelial cells are given mesenchymal cell properties, such as VSMCs or fibroblasts ^[20][48]. This transition is a critical step in the development and progression of atherosclerotic plaques and the subsequent transition into unstable plaques. MALAT1 may exhibit proatherogenic effects by influencing EndMT. In human umbilical vascular endothelial cells (HUVECs) treated with ox-LDL particles, a significant decrease in endothelial markers such as CD31 and vWF and a significant increase in mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin was observed, which is a strong indicator of ox-LDL induced EndMT. MALAT1 silencing in HUVECs resulted in a morphologic transition and increase in endothelial markers. EndMT induced by oxidized LDL is most likely mediated by the ability of MALAT1 to activate the Wnt/β-catenin pathway. Overexpression of MALAT1 was found in arterial tissues of atherosclerotic mice, indicating its possible involvement and effect in the development of unstable plaques ^[20][48].

Endothelial cell migration and proliferation contribute to the progression of atherosclerosis by promoting the formation of atherosclerotic plaques. As the migrating endothelial cells penetrate the vessel wall, they initiate an inflammatory response and recruit immune cells. Several lncRNAs have been shown to influence this process. LncRNA at the INK4 locus (ANRIL, CDKN2BAS) plays an important role in atherogenesis since it is involved in almost every mechanism of atherosclerotic initiation and progression, including migration, proliferation, apoptosis of endothelial cells ^[21][49]. Activation of NF-κB by TNF-α increases the expression of ANRIL that forms a functional complex with YY1 and consequently upregulates IL-6 and IL-8 causing endothelial damage ^[22][50]. In addition, the expression of ANRIL is positively correlated with the expression of CARD8 (a member of the caspase recruitment domain (CARD)–containing family) ^[23][51]. CARD8 plays a significant role in endothelial activation by regulating the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions. Furthermore, ANRIL also increases the expression of VEGF, an angiogenic factor, which induces migration and proliferation of endothelial cells and increases vascular permeability through interaction with p300, PRC2 and miR-200b ^[24][52]. The proliferation of endothelial cells is also controlled by NEAT1 ^[25][53]. In oxLDL-treated HUVECs, NEAT1 knockdown promotes proliferation while suppressing apoptosis and inflammation by upregulating miR-30c-5p and reducing expression of T-cell-specific transcription factor-1 (transcription factor implicated in advances of several chronic diseases). In contrast to ANRIL and NEAT1, lincRNA-p21 reduce atherosclerosis progression by influencing miR-221/SIRT1/Pcsk9 axis. Namely, lincRNA-p21 acts as a sponge for mir-221 sequestering it from its target SIRT1 gene that is involved in cell proliferation and fibrosis ^[26][54].

Laminar flow regulates a plethora of protective vascular genes, which have a significant impact on the normal morphology and functions of ECs ^[26][54]. Leisegang et al. showed that the downregulation of MANTIS leads to a significant alteration of the expression levels of flow-induced genes ICAM1, LINC00920, Collagen Type III Alpha 1 Chain (COL3A1), and Semaphorin 3A (SEMA3A) in HUVECs ^[26][54]. Furthermore, the authors demonstrated that overexpression of MANTIS in the laminar flow-induced inertial state of HUVECs inhibited transcription of ICAM1, whereas downregulation of MANTIS mediated nearly five-fold increased adhesion of THP-1 cells. Laminar flow conditions increased MAP-kinase-5 activity, which phosphorylates the transcription factor MEF2. Phosphorylated MEF2 activates KLF2 and KLF4, which leads to the expression of MANTIS. MANTIS was found to be significantly downregulated in carotid plaques. Interestingly, MANTIS expression levels in carotid plaques of patients on statin therapy were higher than in those not on statin therapy. These beneficial statin effects were diminished in unstable plaques ^[26][54].

The inflammatory response of endothelial cells plays a critical role in the pathogenesis of atherosclerosis. Dysfunctional endothelium upregulates the expression of adhesion molecules leading to the recruitment of immune cells and progression of atherosclerosis. LncRNA AK136714 atherogenic effects are related to its ability to induce inflammatory response in endothelial cells. AK136714 binds to HuR, also known as ELAVL1. HuR is a widely expressed RNA-binding protein that increases the stability of TNF-α, IL-1β and IL-6 mRNA, which promotes inflammation ^[27][55]. In addition, Ming-Peng and colleagues indicated the potential significance of the COLCA1 (colorectal cancer associated 1)/miR-371a-5p/SPP1 axis in atherosclerosis-related inflammation ^[28][56]. Stimulation of coronary endothelial vascular cells with ox-LDL particles was followed by upregulation of COLCA1, which led to downregulation of miR-371a-5p, and consequently to upregulation of SPP1 (also known as osteopontin). SPP1 is a proinflammatory cytokine that stimulates the production of IFN-γ and IL-12. All these changes prevent endothelial cells from wound healing, making the vascular endothelium susceptible to plaque formation and progression ^[28][56].

The ability of vascular smooth muscle cells (VSMCs) to switch phenotypes seems to be critical in atherosclerosis progression, as they migrate, proliferate, and contribute to intimal hyperplasia, arterial wall degeneration, and restenosis. Dysregulated VSMC functions, including proliferation and migration, play key roles in vascular remodelling and the development of atherosclerotic plaques, which are significant in arteriosclerosis. The proliferation, migration, senescence, and apoptosis of VSMCs are all influenced by the ab-errant expression of ANRIL ^[29][30][31][57,58,59]. ANRIL and SUZ12, a vital part of the PRC2 complex, work together to silence p15INK4, responsible for necrotic debris accumulation in the plaque ^[29][57]. On the other hand, as a result of the interaction between ANRIL and the chromodomain of chromobox homolog 7 (CBX7, a subunit of PRC1), p16INK4, an inhibitor of kinases responsible for cell apoptosis, and PRC1 are combined ^[30][31][58,59]. Both processes lead to VSMC proliferation and the development of plaque ^[29][30][31][57,58,59]. On the contrary, in the apoE−/− mouse model, metformin treatment resulted in the upregulation of ANRIL, which consequentially inhibited phenotype switching of VSMCs and the development of atherosclerotic plaques. These alterations in phenotype switching were mediated through ANRIL’s activation of AMP-activated protein kinase (AMPK) ^[32][60]. LncRNA H19 may promote vulnerable plaque formation also by influencing the proliferation and migration of VSMCs. Knockdown of H19 may induce apoptosis of VSMCs in a p53-dependent manner, thus slowing the progression of atherosclerosis ^[33][61]. The upregulation of H19 in apoE−/− mice could also promote the formation of vulnerable plaques by downregulating PKD1 via the recruitment of CTCF. Moreover, it has been shown that knockdown of H19 leads to downregulation of MMP-2, VEGF, and p53 and upregulation of TIMP-1 expression ^[34][62]. There is growing evidence that LIPCAR also plays a critical role in phenotype switching and overall differentiation, proliferation, and migration of human VSMC ^[35][63]. A significant increase in the expression of the lncRNA LIPCAR was observed in human VSMCs treated with ox-LDL particles and PDGF-BB ^[35][63]. In addition to LIPCAR, these cells showed increased expression of PCNA and cyclin D2, which exhibit potent proliferative effects, whereas the expression of p21, one of the major anti-proliferative genes, was significantly decreased. Upregulation of LIPCAR resulted in increased expression of MMP2 and MMP9, which regulate VSMC migration. Finally, this study demonstrated that upregulation of LIPCAR could potentially lead to facilitated migration of VSMCs via CDK2/PCNA upregulation ^[12][40]. LncRNA MIAT is involved in the activation of the ERK-ELK1-EGR1 pathway, which is a major contributor to vascular smooth muscle cell (VSMC) proliferation ^[16][44]. LncRNA AL355711 acts as an enhancer of atherogenesis by promoting the migration of VSMCs ^[36][64]. The overexpression of AL355711 leads to the overexpression of ABCG1, which in turn promotes the expression of MMP3. Moreover, silencing of AL355711 inhibits the migration of VMSCs ^[36][64]. LncRNA ZNF800 shows its atheroprotective role by inhibiting migration and proliferation of VSMCs by increasing PTEN protein expression, which leads to the inhibition of the AKT/mTOR signalling pathway and inhibition of the VSCMs proliferation ^[37][65]. LncRNA NEXN-AS1 that is significantly downregulated in atherosclerotic plaques mitigates atherosclerosis by regulating the actin-binding protein NEXN, and through inhibition of the TLR4/NF-κB signalling pathway results in decreased migration of VSCMs ^[38][66].

Several lncRNAs influence VSCMs functions by acting as miRNA sponges. H19 act as a competing endogenous RNA via interaction with let-7a miRNA, which promotes the expression of cyclin D1 and favours the proliferation of VSMCs ^[39][67]. LINC0123, showed a potent pro-atherosclerotic effect acting through the miR-1277-5p/KLF5 axis and causing migration and proliferation of VMSCs ^[40][68]. LncRNA SNHG8 promotes the proliferation and migration of VSMCs by via sponging of miR-224-3p ^[41][69], while lncRNA SNHG7-003 decrease proliferation, migration and invasion of VSMCs by binding miR-1306-5p/SIRT7 ^[42][70].

Apoptosis of VSMCs is one of the critical mechanisms that leads to the destabilization of atherosclerotic plaques. In the complex of pathological molecular mechanisms of atherosclerosis, the loss of function in lncRNA CERNA1 might play a pivotal role in plaque destabilization ^[43][71]. Despite the fact that CERNA1 overexpression had no effect on lipid droplet accumulation, there was a significant increase in the number of VSMCs and anti-inflammatory macrophages in the plaques of the CERNA1-overexpressed group of apoE−/− mice ^[43][71]. Furthermore, CERNA1 overexpression led to a significant decrease in MMP2/9 activity and IL6 expression levels. Increase in cell numbers and anti-inflammatory effects are most likely mediated by CERNA1′s ability to induce the expression of API5, which in turn inhibits VSMCs and anti-inflammatory macrophage apoptosis, which ultimately led to the stabilization of atherosclerotic plaques in the apoE−/− mouse model ^[43][71]. In contrast to handful of studies showing pro-atherogenic effects of NEAT1, overexpression of NEAT1 inhibits VSCMs apoptosis by increasing expression of Bmal1/Clock and decreasing levels of Bax, cytochrome c, and cleaved caspa-se-3 expression ^[44][72]. LncRNA-SNHG14 could potentially inhibit VSMCs proliferation, but induce apoptosis through its sponging of miR-19a-3p, which results in overexpression of RORα ^[45][73].