Hypercholesterolemia Induces Vascular Cell Dysfunction: Molecular Basis for Atherosclerosis

    

    

    Figure 1:  LDL-induced signaling in EC and mononuclear cells
(A) Both nLDL and oxLDL induce intracellular signaling in EC, which leads to enhanced expression of downstream targets such as cytokines, MMP and adhesion
molecules (indicated by red arrows). This leads to EC dysfunction and apoptosis, two mechanisms contributing to atherosclerosis. (B) Both nLDL and oxLDL affect
mononuclear cell function by influencing downstream signaling cascades and target expression, however, the exact molecular mechanisms remain to be fully
elucidated.
    
    

Studies with ECs revealed that oxLDL induces Rho-Associated Protein Kinase 2 (ROCK 2) via LOX-1. This activates downstream molecules involved in signaling pathways such as Akt, also known as protein kinase B (PKB), PKC, NF-κB and MAPK [45]. There is further evidence that LDL influences other transcription factors. A study revealed a correlation between the oxLDL/LDL ratio and the expression of the zinc finger protein 580 (ZNF 580) and IL-8 [46]. ZNF 580 has been shown to be a transcription factor involved in MMP2 and Vascular Endothelial Growth Factor (VEGF) regulation [47]. High oxLDL/LDL ratios are found in patients with high numbers of atherosclerotic lesions and are associated with an increased IL-8 expression, which is due to down regulation of ZNF 580 and leads to an increased monocyte adhesion on EC [46]. OxLDL not only induces NF-κB, but also the transcription factor Activator Protein-1 (AP-1), which in turn up regulates the expression of inflammatory genes leading to EC dysfunction [48]. It is known, that ROCK2 influences cell motility, the cytoskeleton, as well as the vascular tone and adhesion [49]. Furthermore, it is also linked to atherosclerosis [50]. Activation of signaling pathways by oxLDL induced ROCK2 further leads to increased chemokine/cytokine expression such as interleukin IL-8 by NF-κB. This in turn recruits monocytes to the inflamed endothelium during atherosclerosis [45]. In addition, EC exposed to LDL show reduced migratory responses towards VEGF probably due to internalization and degradation of VEGF receptor VEGFR2 and reduced VEGF downstream signaling pathways such as phosphatidylinositide 3-kinase (PI3K)/PKB and ERK1/2 [51]. These results suggest, that oxLDL, as well as nLDL have an inhibiting effect on angiogenesis. Similar results have been previously described by us in conjunction with DM. Diabetic monocytes show diminished chemotaxis towards VEGF compared to monocyte migration from healthy individuals, which may explain the reduced angiogenesis (healing) after MI in diabetic patients. This phenomenon is called VEGF resistance [52].Taken together, these finding suggest that oxLDL mediates its pathological effects on EC by activating different signaling cascades.

There are many studies on the role of HC/LDL in EC dysfunction, while the effect of HC on leukocyte/monocyte dysfunction is not so clear yet. Nevertheless, some investigations have been made. While oxLDL induces cytokine secretion in EC to recruit monocytes, it can also act as a chemoattractant for monocytes by itself [53]. oxLDL downregulated the expression of the MCP-1 receptor CCR2 on human monocytic cells THP-1, while it increased the expression of peroxisome proliferator-activated receptor-γ (PPARγ), which is known to regulate the inflammatory response in monocytes/ macrophages [54]. In contrast, chemotaxis of monocytes is increased by up regulation of CCR2 expression by nLDL [55]. It is well known, that macrophages internalize oxLDL via their SR and develop into foam cells, a hallmark of atherosclerosis. Nevertheless, both nLDL and oxLDL exert their pathological effects in different ways on leukocytes [13]. HC affects not only the phenotype and therefore the function, but also proliferation of leukocytes. Studies revealed that HC correlated with increasing numbers of circulating neutrophils and monocytes. It has been shown that HC induces proliferation of both hematopoietic stem and progenitor cells [56]. Moreover, oxLDL increases chemotactic activity of monocytes and their adhesion to endothelial cells by enhancing expression of endothelial adhesion molecules. It has been demonstrated that oxLDL induced the expression of growth factors in EC and macrophages and therefore also the proliferation and migration of SMC [57]. In monocytes/macrophages, oxLDL induces apoptosis by inducing DNA fragmentation [58], whereas another study showed that oxLDL reduced apoptosis by activating the ERK1/2 pathways in the monocytic cell line THP-1 [59]. It is known that the oxLDL receptor CD36 activates MAPK via activation of Src kinases [60]. Also nLDL increases adhesion of leukocytes on the endothelium by inducing the expression of adhesion molecules such as P-selectin in human monocytes. The effects of nLDL are mediated by LDLR [26]. OxLDL induces the toll like receptor (TLR 4/6) hetero dimer assembly via CD36 and therefore the NF-κB pathway andIL-1β secretion [61]. LDL induced the p38 signaling cascade after short term incubation in human platelets, which inhibited the Na+/H+antiport and leads to sensitization [62]. All in all, the findings of the different in vitro studies show that the modified forms of LDL have more severe, pathological effects on vascular cell function compared to the effects of nLDL.

Oxidative stress and cellular dysfunction: Oxidative stress is the imbalance between ROS and mechanisms of a biological system to act against or clear these substances [63]. ROS are not only free radicals (superoxide), but also other oxidants like hydrogen peroxide. Oxidative stress is associated with the progression and presence of CVD [7]. In addition, it has been shown, that oxidative stress leads to cellular dysfunction. ROS could increase the permeability of human EC in vitro [64]. Furthermore, ROS induces cell dysfunction by damaging different cellular components such as proteins, DNA and mitochondria [65]. Three enzymes produce the major amount of ROS, called nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), xanthine oxidase and MPO. HC leads to their up regulation. NOX isoforms catalyze the reaction from superoxide to hydrogen peroxide, whereas NOX2 is the most important isoform in the vascular wall. Xanthine oxidase catalyzes reactions to superoxide and hydrogen peroxide. MPO, which is known to oxidize LDL as described above, catalyzed the production of hypochlorous acid [13]. Enhanced ROS production can lead to oxidative damage of proteins, lipids or DNA. Further oxidative stress induces signaling cascades such as NF-κB or MAPK pathways leading to an up regulation of inflammatory conditions. Also intracellular calcium (Ca²⁺), which is an important molecule for many signaling processes within the cell, increases due to oxidative stress [7].

Incubation of polymorph nuclear leukocytes with oxLDL but not nLDL increased the intracellular calcium levels [66]. It has also been shown that oxLDL increased NOX activity in both THP-1 and in U937-derived macrophages supporting the fact that HC enhances oxidative stress [67]. Furthermore, it is known that ROS production is induced by oxLDL and LOX-1 interaction leading to upregulation of adhesion molecules in EC and enhanced inflammation [68]. OxLDL induces NOX2 mRNA expression and superoxide production in human EC supporting the role of HC in oxidative stress [69]. Not only oxLDL but also glycated LDL can induce oxidative stress by up regulating the NOX subunits p²²phox and NOX4 in EC by activating the NF-κB and the p³⁸ MAPK pathway [70]. Moreover, oxidative stress induced by HC leads to a decrease in NO bioavailability in EC. This is due to increased circulating levels of the endothelial NO synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA). Since NO as an endothelium-derived relaxing factor is important for maintenance of the vascular tone a reduced NO bioavailability leads to EC dysfunction [71]. Besides its functional impact, ADMA has been shown to be a valid marker for oxidative damage [72]. Taken together, these results suggest that the pathology of HC and LDL respectively is due to a combination of its inflammatory and oxidative properties.

Role of HC in development of atherosclerosis in preclinical models: Animals, such as mice, which have naturally low LDLcholesterol and high HDL levels, do not develop atherosclerosis by themselves [56]. The first successful deletion of a protein in mice resulted in the apoE knockout (KO) mice, which develop spontaneous atherosclerosis, when they are fed with a high fat diet [73]. In addition, this is associated with elevated levels of circulating LDL. It has been shown that dietary plant sterols can prevent HC and atherosclerosis in apoE KO mice [74]. A study with apoE-/- mice fed with Western diet revealed that HC induced monocytosis of monocytes, which were found in lesions at later time points during atherosclerosis and shown to adhere on the endothelium and differentiate to macrophages [75]. In addition, it has been shown that there was increased adhesion of leukocytes on EC in apoE-/- mice after feeding high cholesterol compared to wild type mice. These effects were reversed by simvastatin, a commonly used statin [76]. The effects of HC on the endothelium are due to activation of the NF-κB pathway, since inhibition of this pathway in EC resulted in reduced atherosclerosis in apoE-/- mice fed with a cholesterol rich diet [77]. Another prominent mouse model for studying atherosclerosis is the LDLR KO mouse [78]. LDLR-/- mice develop atherosclerosis as LDL-cholesterol cannot be cleared from the circulation. It has been shown, that overexpression of 15-lipooxigenase in the vascular wall contributes to atherosclerotic lesion formation in these mice [79]. A study with wild-type (WT) and cJun N-terminal kinase (JNK) KO mice fed with high cholesterol diet revealed that JNK deletion protected against endothelial dysfunction. The mechanism is dependent on HC/JNK-induced ROS production [80]. Cholesterol rich diet induced expression of adhesion molecule VCAM-1 in rabbit EC in vivo. This supports the hypothesis that HC induces inflammation [81]. Watanabe Heritable Hyperlipidemic (WHHL) rabbits are a model organism for familial HC since they are characterized by natural LDLR deficiency and high LDL levels in the circulation [82]. It was shown, that hind limb ischemia induced capillary formation was significantly lower in WHHL rabbits compared to control animals [83]. This can be due to defects in VEGF signaling since EC migration and proliferation induced by VEGF is impaired by nLDL in vitro [51]. Studies with hyperlipidemic hamsters demonstrated an increase of nLDL transport in aortic fatty steaks, a preliminary stage of the atherosclerotic plaque, whereas in control animals nLDL was not detectable in these areas [60]. In mice deficient for SR-AI and SR-AII, prominent receptors for oxLDL, a reduced formation of atherosclerotic changes could be seen as well as a reduced number of adherent monocytes, further supporting the role of oxLDL in the progression of atherosclerosis [84].

Role of HC in human CVD: High oxLDL levels are associated with CVD [2]. OxLDL expresses immunogenic epitopes which induce the generation of antibodies against them. These antibodies can be detected in the serum of patients with advanced atherosclerosis and they are used as in vivo markers for LDL oxidation [57]. A study from our group using monocytes from HC patients revealed that the chemotactic response of these cells towards VEGF was attenuated, an observation described as VEGF resistance [85]. Similarly, MCP-1 induced responses were functionally impaired in monocytes from patients with HC [85]. In diabetic patients this is due to increased baseline activation of p38 and PI3K/PKB signaling [86]. This leads to the assumption, that the same mechanism may be active under hypercholesterolemic conditions, which deserves verification. Furthermore, it has been shown, that monocytes from hypercholesterolemic patients treated with simvastatin show reduced release of cytokines such as IL-1β, IL-6 or MCP-1 ex vivo, which leads to reduced activation of these cells [87]. In line with results from preclinical models [56], it has been shown, that there are increased numbers of monocytes and macrophages in patients with familial or non-familial HC [88]. Moreover, monocytes isolated from hypercholesterolemic patients have been shown to produce increasing amounts of ROS, which is due to elevated oxLDL levels [89]. Another study on monocytes from patients with HC revealed that incubation of these cells with nLDL ex vivo led to an increase in expression of the adhesion molecule CD11b compared to monocytes from healthy individuals. The increase was reversed by simvastatin treatment [90]. There are also different studies on the effect of HC on endothelium dependent vasodilation, i.e. means the widening of blood vessels. This is impaired in hypercholesterolemic patients but can be reverted by the use of statins [91-93]. It has also been shown that inhibition of p38 MAPK improves HC-induced impairment of endothelial vasodilation [94]. Platelets from patients with HC are activated and aggregate to a higher degree. NLDL as well as oxLDL have an influence on platelets. Platelets are able to internalize oxLDL via their SR CD36 and thereby induce foam cell formation in macrophages. In that way activated platelets contribute to atherosclerotic plaque formation [95].

Therapeutic Strategies

Lipid lowering strategies: Statins and other lipid lowering agents

The most commonly used medication to treat HC is hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors better known as statins. Their potential to reduce the risk of CVD has been verified in multiple studies [96]. The HMG-CoA reductase catalyzes the last and rate limiting step during cholesterol synthesis. Statins inhibit protein isoprenylation, which plays important roles in signaling processes in the cardiovascular system by interfering with Guanosine Triphosphate (GTP) binding proteins such as Ras, Rho, and Rac. Statins are thought to act beneficial against CVD not only because of their lipid lowering function, but also because of their antioxidant and anti-inflammatory properties, previously referred to as “pleiotropic effects” by reducing oxidative stress [97]. It remains to be demonstrated whether these pleiotropic effects are indeed independent of cholesterol modification.

Statins reduce LDL-cholesterol levels in familial hypercholesterolemic patients up to 50%. Nevertheless, statins have only limited effects on LDL levels in some patient groups [98]. For example, in patients with extremely high baseline LDLcholesterol the treatment with statins could not show a sufficient reduction of these levels [99]. Another problem in daily practice is statin intolerance [100]. Therefore, other lipid lowering strategies are a current topic of research and development. Most of them are used in addition to statins since this is the accepted and established standard therapy for reducing LDL-cholesterol [98]. Among them are cholesterol absorption inhibitors like ezetimibe (IMPROVE-IT) [9]. In combination with statins they have been shown to reduce cardiovascular events by 27% [101]. Another approach exploits microsomal transfer protein (MTP) inhibitors, which inhibit the transport of cholesterol and triglycerides, which in turn stabilizes nascent VLDL particles. They show an efficiency of more than 50% reduction of LDL-cholesterol levels by using high concentrations [102]. Nevertheless, since these inhibitors also increase cholesterol pools in the liver, which in turn leads to fatty liver development only the use of one MTP inhibitor (lomitapide) with less side effects is approved and allowed in humans [102]. Other current developments refer to the possibility to reduce LDL via antisense oligonuclotides. MipomersenTM is used as an antisense therapy and reduces apoB und therefore LDL in human plasma from 20% to 65% [103]. This drug is currently marketed [104].

A novel and very promising LDL lowering therapy is via Preprotein convertase subtilisin kexin-9 (PCSK-9) inhibitors. PCSK-9 is involved in intracellular and extracellular expression of LDLR. Binding of PCSK-9 to LDLR leads to its internalization. It has been proven in different studies and clinical trials that statins lead to upregulation of PCSK [105]. Antibodies against PCSK-9 have been shown to increase LDLR expression and therefore reduce LDL-cholesterol level in human plasma from about 30% to 50% [95]. Although the use of monoclonal antibodies is the most common treatment against PCSK also antisense oligonucleotides and small interfering RNAs have been shown to reduce PCSK and therefore LDL-cholesterol levels [105]. Important clinical trial data are expected soon, especially on Alirocumab, which is being tested in the Odyssey trial [106].

Other anti-inflammatory agents

Statins are thought to be beneficial against CVD not only because of their lipid lowering but also because of their anti-inflammatory potential. Phytosterols are plant derived sterols or stanols [107]. Their structure is similar to the one of cholesterol. They have been shown to reduce not only LDL-cholesterol but also total cholesterol in hypercholesterolemic subjects which is probably due to interaction in micelle formation. Nevertheless, phytosterols also exert antiinflammatory properties [108]. Studies with apoE-KO mice could show a reduced expression of proinflammatory cytokines in atherosclerotic lesions after phytosterol treatment [109]. Clinical data supports these findings [108]. Furthermore, immunization plays a role in therapeutic strategies against CVD. It is known that circulating auto antibodies against LDL exist in human [110]. A study by Nielson and colleagues found a modest inverse correlation between autoantibody titers and the intima-media thickness in healthy people [111]. Monoclonal IgG antibodies against LDL have been shown to reduce atherosclerotic lesion in LDLR deficient mice. However, this antibody failed to achieve the expected results in phase II of a clinical study [110]. By eating moderately, meaning especially reducing the uptake of animal fats and salts and by increasing the consumption of fruits and vegetables, patients with or without cardiovascular problems were able to reduce their weight and LDL-cholesterol levels while improving their HDL levels. Furthermore, also reduced the risk of a cardiovascular event [112]. This shows that not only medication/ drugs can lower lipid levels but also a healthy lifestyle (low fat diet) can contribute to that.

Conclusion

Atherosclerosis is the underlying process of the most important CVDs. HC is one of the most important and modifiable cardiovascular risk factors for atherosclerosis. High LDL plasma levels result in increased inflammation and enhanced oxidative stress as well as cellcell adhesion thereby facilitating the infiltration of leukocytes into the vessel wall and enhances plaque progression. Moreover, HC induces dysfunction of vascular cells, such as EC, SMC and leukocytes by activation intracellular signaling cascades and expression of inflammatory molecules, which further contribute to atherosclerosis. Statins are established drugs to reduce the risk of MI in patients with CVD, as well as to reducing progression of atherosclerosis. Other more potent drugs to reduce HC are currently under development. While much needs to be investigated on the molecular mechanisms that underlie the role of HC in cellular dysfunction and development of atherosclerosis, the recent advances provide ample opportunities for the development of new therapies for CVD.

Acknowledgement

Our studies on the role of growth factor signaling in CVD are supported by the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) of the Medical Faculty of the University of Münster, Germany and by the ‘Innovative Medizinische Forschung’ (IMF PA121004) program of the Medical Faculty of the University of Münster, Germany. Furthermore, our studies are supported by Cells-in- Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster, Germany.

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