Molecular and Cellular Mechanisms of Electronegative Lipoproteins in Cardiovascular Diseases

Dysregulation of glucose and lipid metabolism increases plasma levels of lipoproteins and triglycerides, resulting in vascular endothelial damage. Remarkably, the oxidation of lipid and lipoprotein particles generates electronegative lipoproteins that mediate cellular deterioration of atherosclerosis. In this review, we examined the core of atherosclerotic plaque, which is enriched by byproducts of lipid metabolism and lipoproteins, such as oxidized low-density lipoproteins (oxLDL) and electronegative subfraction of LDL (LDL(−)). We also summarized the chemical properties, receptors, and molecular mechanisms of LDL(−). In combination with other well-known markers of inflammation, namely metabolic diseases, we concluded that LDL(−) can be used as a novel prognostic tool for these lipid disorders. In addition, through understanding the underlying pathophysiological molecular routes for endothelial dysfunction and inflammation, we may reassess current therapeutics and might gain a new direction to treat atherosclerotic cardiovascular diseases, mainly targeting LDL(−) clearance.


Introduction
Approximately 1.9 billion people are obese or overweight worldwide [1]. Obesity is associated with excessive calorific intake and microvasculature damage, resulting in atherosclerosis, diabetes, and cardiovascular diseases (CVDs) [2]. The prevalence of CVDs has significantly increased in the past few decades [3]. Current strategies against CVDs mainly focus on lowering the level of low-density lipoprotein cholesterol (LDL-C) [4,5]. Intensive-dose statin therapy has been endorsed for clinical atherosclerotic vascular disease (ASCVD); however, it also increases statin-related side effects and intolerance [6,7]. To figure out this dilemma and find a balanced solution, here we address the

Chemical Properties of LDL(−)
LDL(−) differs from LDL(+) in many aspects [23]. Regarding the lipid components, LDL(−) contains higher concentrations of triglycerides, non-esterified fatty acids (NEFA), Accumulating evidence shows that LDL(−) could be a novel marker for ASCVD, and levels of LDL(−) are positively correlated with the increasing severity of CVDs [20][21][22]. LDL(−) serves as a pivotal target for further studies and clinical development strategies beyond statins therapies. By targeting LDL(−), we summarize its pathophysiological links and highlight the molecular mechanisms of atherogenic lipids in the current review.

Structure Modifications and Enzymatic Functions of Electronegative LDL
Electronegativity and apolipoprotein misfolding are two independent features of LDL(−) [46]. The misfolded apoB100 of LDL(−) shows an increased binding affinity to proteoglycans, which may prolong LDL retention in the arterial wall and trigger inflammatory responses [31]. Stabilizing the LDL's structure through the use of 17-β-estradiol (E2) prevents aggregation; however, it cannot prevent the generation of LDL(−) [46,47]. The structural modifications of apoB100 are associated with phospholipolytic activities and exchange of lipid components [28,48,49]. The sphingomyelinase (SMase)-like activity of LDL(−) may hydrolyze sphingomyelin, which produces apoptotic factor, a ceramide [28,48]. The phospholipase D (PLD) activity of LDL(−) degrades phosphorylcholine, LPC, and sphingomyelin, which is associated with self-aggregation and atherogenic properties. Treatment with 400 µM of chlorpromazine may effectively inhibit both the SMase and PLD activities of LDL(−) [48].

Animal Models Showing Elevated Electronegative LDL
The overproduction of LDL(−) was demonstrated in animal models that consumed a high-fat diet. Lai et al. gave either a standard chow diet or high-fat & high-cholesterol (HFC) diet to each group of 8-week-old male golden Syrian hamsters for six weeks. Plasma LDL-C levels in HFC-diet-fed hamsters were significantly higher than for the control group. Additionally, LDL(−) accounted for 12.5% of all lipoproteins in control hamsters, whereas the value was drastically increased to 42% in HFC-diet-fed hamsters [50]. Recently, Chang et al. distributed an atherogenic diet to sixteen-week-old male New Zealand White rabbits. After six weeks, the LDL(−) from HFC-diet-fed rabbits accounted for about 17.2 ± 5.5% of the LDL fraction. On the other hand, it was almost undetectable in rabbits fed with a control chow diet [51]. Moreover, from the recent publication by Chan et al., LDL(+) and LDL(−) isolated from SLE patients' LDL samples were then injected into eight-week-old apoE knockout mice. Their results showed that only the LDL(−)-injected mice experienced a significant increase in the plasma CX3CL1 level. By observing histological staining results, LDL(−) can trigger endothelial dysfunction and the formation of atherosclerotic lesions in apoE knockout mice [27]. Taken together, we summarized that LDL(−) plays a vital role in atherosclerosis and plaque formation.

Mechanisms of Electronegative LDL on Endothelial Cells
The endothelium regulates fluid and molecule trafficking between the bloodstream and tissues for metabolism [52]. In addition, it inhibits platelet aggregation and adhesions by secreting prostacyclin, NO, and exosomes [53,54]. With LDL(−), the atherogenic components lead to endothelial activation and vascular inflammation. Chemokines such as monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) are released from the damaged endothelium. The vascular adhesion molecules are highly expressed to promote plaque formation [55]. The mechanisms behind this are listed below.

Mitochondria Damage
The basal physiological mechanism of mitochondrial ROS formation is dependent on several factors such as NO, cytosolic Ca2+, and fatty acids [82]. NADPH oxidase 4 (NOX4) in vascular cells inhibits mitochondrial complex I and promotes ROS generation [83]. During the pro-apoptotic conditions, ROS formation is also boosted by growth factor adaptor protein p66Shc, which facilitates the cytochrome c oxidation. Moreover, ROS formation can be further increased by the expression and activation of p66Shc during hyperglycemic conditions [84,85]. LDL(−) inhibits endothelial nitric oxide synthase (eNOS) expression via the Akt signaling pathway, resulting in decreased NO production and leading to endothelial cell apoptosis [86]. Recently, Chen et al. demonstrated that apoE in LDL(−) is responsible for LDL-induced mitochondrial dysfunction. After LDL(−) internalization, apoE translocates from the lysosome to the mitochondria, leading to mitochondrial permeability transition pore (mPTP) opening, dynamin-related protein 1 (DRP1) phosphorylation, and mitochondrial fission [41].

Endoplasmic Reticulum Stress
The intraluminal oxidation in the endoplasmic reticulum (ER) plays a critical role in maintaining calcium concentration and proper folding of transmembrane proteins. The increased amount of lipoprotein promotes a condition known as ER stress, defined by the accumulation of unfolded protein in the ER lumen [87,88]. The molecular mechanism between LDL oxidation and UPR (unfolded protein response)-mediated expression of IL-8, IL-6, and MCP-1 in endothelial cells, which contributes to endothelial dysfunction, is poorly explained [89,90]. Apart from oxidation, glycation of LDL is also found to be a potent marker for dyslipidemia. Studies showed that glycated LDL could initiate nicotinamide adenine dinucleotide phosphate (NADPH) oxidation via ROS production and could induce apoptosis in endothelial cells [91,92]. Therefore, the LDL oxidation and glycation are involved in amplifying endothelial dysfunction and contributing to atherosclerosis.

Mechanisms of Electronegative LDL on Immune Cells
Alongside endothelial cells, immune cells play a significant role in the pathogenesis of atherosclerosis. Monocytes and T lymphocytes create an inflammatory milieu by releasing several cytokines and growth factors. As LDL(−) concentration is elevated in the blood plasma, it tend to interacts with these monocytes and lymphocytes via cytokines and growth factors [93,94]. LDL(−) impregnates the process of oxidation via the feedback loop mechanism shown in Figure 2 and enhances inflammation. The NEFA and ceramide in LDL(−) also show atherogenic properties [93,[95][96][97]. The detailed mechanisms behind this are listed below.

Monocytes
Numerous studies have described the effects of LDL(−) on inducing cytokine release from monocytes, which may be important in atherosclerosis [25,98]. Remodeling of the vascular extracellular matrix (ECM) seemed to be an important landmark of atherosclerosis. LDL(−) induces the release of matrix metalloproteinase (MMP)-9 and tissue inhibitors of metalloproteinase (TIMP)-1 from monocytes through the TLR4/CD14 inflammatory pathway [45]. Additionally, the downstream signal cascade of TLR4/CD14 will then trigger PI3K/Akt signaling and promote p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, leading to LDL(−)-induced cytokine release from monocytes [99]. The elevated levels of those cytokines may regulate and contribute to vascular plaque formation.

Macrophages
Macrophages play a crucial role in the early stage pathogenesis of atherosclerosis [100]. Circulating monocytes undergo differentiation into macrophages and further polarization into classically activated (M1) or alternatively activated (M2) states in order to withstand environmental stimuli. M1 macrophages are responsible for pro-inflammatory properties, whereas M2 macrophages exert opposing anti-inflammatory properties [101].
In addition to human macrophages, in treating LDL(+) and LDL(−) with RAW264.7 cell, the results showed that only LDL(−) can induce the expression of CD95 death receptor (Fas), its ligand CD95 L (FasL), and tumor necrosis factor ligand member 10 (Tnfsf10), which stimulate the activation of the caspases, resulting in cell apoptosis [105].

Macrophages
Macrophages play a crucial role in the early stage pathogenesis of atherosclerosis [100]. Circulating monocytes undergo differentiation into macrophages and further polarization into classically activated (M1) or alternatively activated (M2) states in order to withstand environmental stimuli. M1 macrophages are responsible for pro-inflammatory properties, whereas M2 macrophages exert opposing anti-inflammatory properties [101].
In addition to human macrophages, in treating LDL(+) and LDL(−) with RAW264.7 cell, the results showed that only LDL(−) can induce the expression of CD95 death receptor (Fas), its ligand CD95 L (FasL), and tumor necrosis factor ligand member 10 (Tnfsf10), which stimulate the activation of the caspases, resulting in cell apoptosis [105].

Platelets
Apart from monocytes and macrophages, accumulating evidence has shown that LDL(−) may trigger platelet activation and aggregation. Platelet hyperreactivity is the most direct evidence contributing to thrombosis in the leading causes of cardiovascular diseases, such as STEMI [106] and stroke [43,107]. As above, Chan et al. separated LDL(+) and LDL(−) from patients with STEMI, with the results illustrating that only LDL(−) was augmented in patients compared to healthy controls. Treating LDL(−) to platelets enhanced their aggregation and adhesion to damaged human aortic endothelial cells (HAECs), which was through LOX-1 and PAFR activation [37]. Furthermore, LDL(−)-induced amyloid β (Aβ) release via IκB kinase 2 (IKK2) in human platelets was reported by Shen et al. in 2016. Besides, LDL(−) works synergistically with Aβ to induce glycoprotein IIb/IIIa receptor activation and phosphorylation of IKK2, IkBa, p65, and c-Jun N-terminal kinase 1 in order to enhance platelet aggregation. These results can be attenuated by inhibiting IKK2, LOX-1, or NF-kB with their inhibitors BMS-345541, TS92, and Bay 117-82, respectively [43]. To conclude, high levels of LDL(−) in patients can trigger platelet activation and aggregation through LOX-1 and PAFR receptors. Figure 2 demonstrates the lipid and lipoprotein metabolism in the liver, blood, and peripheral tissues. Nutritional overload increases fatty acids via the overexpression of cluster of differentiation 36 (CD36) and peroxisome proliferator-activated receptor (PPAR-γ) [108][109][110]. This phenomenon is highly contrasted to the de novo synthesis pathway, although FFAs from either source in the liver are indistinguishable. The elevated level of free fatty acids ultimately increases triglyceride through esterification. Combined with apoB100 and triglyceride, the efflux of VLDL into circulation promotes the pro-atherogenic metabolic state. VLDL particles deliver lipids hydrolyzed by lipoprotein lipase (LPL) and release FFAs in plasma [111][112][113].
The presence of LDL(−) in circulation correlates with atherosclerosis progression and endothelial dysfunction-mediated cardiovascular diseases. LDL(−) levels are significantly higher in frequent smokers, diabetic patients, and hypercholesterolemia patients [33,34,40,59]. In addition, LDL(−) levels were 10-times higher in STEMI and stroke patients, even though the LDL-C levels were similar to healthy controls [37,43].

Current Treatment Strategies Targeting Electronegative LDL
The diagnosis and treatment for endothelial damage are dependent on the ankle-brachial index, vascular imaging, surgery, and revascularization [126][127][128]. Currently, treatment for dyslipidemia and the prevention of microvasculature damage mainly revolve around reducing LDL-C levels [129][130][131].
In combination with other well-known markers of inflammation, namely metabolic diseases, we concluded that LDL(−) can be a novel prognostic tool for these lipid disorders. Regarding treatment for the prevention of ASCVD, even though statins can partially reduce the concentration, finding a way to clear LDL(−) remains of utmost importance [22]. In particular, a method involving hydrolyzing atherogenic lipids in LDL(−) and producing harmless metabolites might be a novel therapeutic approach in the future.

Conflicts of Interest:
The authors declare no conflict of interest.