.
F U L L T E X T S O U R C E : ScienceDirect
Highlights
• 7KC is mainly a nonenzymatically produced oxysterol known at the moment for its cytotoxicity.
• 7KC is implicated in many age-related diseases.
• 7KC appears to be a good target for disease, age related and otherwise.
• Removal of 7KC from the body could stall and/or reverse disease progression.
Abstract
7-Ketocholesterol (7KC) is a toxic oxysterol that is associated with many diseases and disabilities of aging, as well as several orphan diseases. 7KC is the most common product of a reaction between cholesterol and oxygen radicals and is the most concentrated oxysterol found in the blood and arterial plaques of coronary artery disease patients as well as various other disease tissues and cell types. Unlike cholesterol, 7KC consistently shows cytotoxicity to cells and its physiological function in humans or other complex organisms is unknown. Oxysterols, particularly 7KC, have also been shown to diffuse through membranes where they affect receptor and enzymatic function. Here, we will explore the known and proposed mechanisms of pathologies that are associated with 7KC, as well speculate about the future of 7KC as a diagnostic and therapeutic target in medicine.
1. Introduction
Cholesterol, one of the most abundant and essential molecules in the body, creates functional membranes by influencing fluidity and allows cells to biosynthesize a variety of other important molecules. Cholesterols exist both inside and outside of the cell, as they are important components of all cellular membranes, but these and other nonpolar substances are transported in the plasma via lipoprotein particles (classified by hydrated density) which are otherwise insoluble in blood [1]. Low density lipoprotein (LDL) is the principal carrier of cholesterol to peripheral tissue. LDL is composed of a cholesterol, protein, and phospholipid shell with a core of cholesteryl esters and triglycerides. All of the components of LDL are susceptible to oxidation to produce an oxidized form of LDL (OxLDL). OxLDL has been linked to a variety of pathologies [[1], [2], [3], [4], [5], [6], [7], [8], [9]]. Oxidation of the cholesterol in LDL produces several oxidation products including 7KC, which is the most abundant oxysterol present in OxLDL [9,10]. We believe that it is important to distinguish between the effects of OxLDL and that of unsequestered 7KC, as many studies fail to account for this important difference in how 7KC interacts with the cell.
OxLDL is not the only source of 7KC within the body. 7KC can be produced endogenously by a series of oxidation steps or, much less commonly, enzymatic reactions [[11], [12], [13]]. It can also be ingested directly in food, however the liver is well equipped to process and rid the body of exogenous toxins, so 7KC is not acutely poisonous to ingest [14]. Endogenously produced, unsequestered 7KC can, on the other hand, wreak havoc inside of most cells. Unesterified 7KC can be found within membranes of organelles where it disrupts fluidity and signaling pathways, causing cellular damage via multiple stress-response pathways [[15], [16], [17], [18]]. These stress-response pathways induce a vicious cycle by increasing the population of reactive oxidative species (ROS), which in turn increases the oxidation of cholesterol and production of 7KC. Particularly in people with already-compromised cholesterol pathways, 7KC buildup can be overwhelming and cause significant damage to membranes, pathways, and overall cell function. In this review, we will discuss the chemistry and cell biological effects of 7KC and show how these attributes are critical contributors to a number of important diseases and to the aging process itself.
2. Chemistry of 7-Ketocholesterol
Some oxidized cholesterols (oxysterols) are physiological compounds produced enzymatically and serve as signaling molecules, while others are adventitious products of the nonenzymatic reaction of cholesterol with ROS and are generally cytotoxic. Oxysterols are shown to be consistently 10 to 100 times more reactive than native cholesterol, making biological systems quite sensitive to these oxidized sterols [19,20]. Nonenzymatically-produced oxysterols are present in oxLDL, in atherosclerotic plaques, and in all cells to varying degrees; the predominant and most toxic of these is 7KC [21,22] which forms when cholesterol oxidation occurs on the C7 position. Auto-oxidation of cholesterol can occur via the reaction of O2 oxygen, hydroxyl radicals, peroxides, or superoxide catalyzed by metal, radiation, or heat [11,23] (Fig. 1). The 7 position on cholesterol seems to be the most reactive with oxygen and a carbonyl group the most stable form [11]. Hydroxyl and peroxide groups often form first and then further oxidize into 7KC [24]. Consistent with 7KC being the most chemically stable and common auto-oxidized oxysterol in-vitro, 7KC is also the most prevalent nonenzymatically produced oxysterol in-vivo [25].
Fig. 1. 7KC can be nonenzymatically produced via oxidation of cholesterol along multiple different pathways.
7KC is also able to convert into 7β-hydroxycholesterol (7β-OHCh) in vivo via 11β-hydroxysteroid dehydrogenase (11β-HSD), and this reduced product is significantly less toxic than 7KC itself [26]. While this enzyme has been shown to interconvert 7KC and 7α/β-OHCh in some animals [26,27], it seems that human 11β-HSD1 in live cells only functions to stereospecifically reduce 7KC to 7β-OHCh in the liver, thus detoxifying and metabolizing it [26]. The production of 7α-OHCh is generally via the 7α-hydroxylase enzyme, which is essential for the formation of bile acids in the liver [28]. 7α and 7β-OHCh are very similar molecules, 7β-OHCh being the more stable of the two, and they both show some cytotoxic effects. However, it appears that the body is able to efficiently process these reduced derivatives [26], possibly because they are more soluble and/or less damaging than 7KC, as 7KC is generally formed by harmful ROS.
A separate mechanism proposed in the formation of 7KC has been suggested to involve the enzymatic conversion of lanosterol to lathosterol and 7-dehydrocholesterol by acetyl-CoA [13,29]. 7-dehydrocholesterol (the immediate precursor of cholesterol) can then be oxidized by cytochrome P450 7A1 to 7KC13. This mechanism is more plausible for the formation of 7KC in brain tissues than in other tissues, because of the prominence of lanosterol, lathosterol, and 7-dehydrocholesterol in neurons [30].
As with cholesterol, oxysterols can be present in free-form or in esterified form. Generally, unesterified sterols are found in the plasma membrane while esterified sterols are found within lipoproteins. 7KC does not seem to be efficiently esterified and exported from cells to the lipoprotein cholesterol transport system [31] and is normally present at very low levels in the serum. Exceptions to this in certain diseases are discussed in sections 4 and 6.
7KC can also be found in LDL, but generally only when the LDL itself has been oxidized (oxLDL). 7KC accounts for up to 30% of the total sterol in these oxLDLs [9,32], but it is important to note that free 7KC exerts toxicity differently from oxLDL. Free 7KC seems to predominantly permeabilize the plasma membrane by disrupting calcium channels, while cholesterols in oxLDL are taken up by endocytosis and are ultimately sequestered and stored in the lysosome [33,34].
7KC induces apoptosis in its free form by increasing both extracellular Ca2+ influx and intracellular Ca2+ mobilization; free 7KC concentrations of 10–30 μM will trigger apoptosis in a variety of cell types [8,17,[35], [36], [37], [38]]. 7KC-induced Ca2+ increase also activates cytosolic phospholipase A2 (cPLA2) and releases arachidonate from membrane phospholipids. Using this released arachidonate, 7KC can then be esterified by the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) to form 7KC-arachidonate [8]. It has been shown that this esterification of 7KC can decrease the esterification of cholesterol [39], which is required for its loading inside LDL. Thus, 7KC can impair normal cholesterol metabolism and exacerbate conditions which already compromise cholesterol metabolism.
Additionally, 7KC increases the production of ROS by activation of NADPH oxidase and triggers an apoptotic stress response [40]. Studies have shown that 7KC has a primary role in intracellular ROS overproduction via stimulation of NADPH-oxidase (NOX). Nearly full protection by multiple selective NADPH-oxidase inhibitors suggests that 7KC can interact directly and quickly with this enzyme to trigger ROS overproduction [18]. 7KC has also been shown to trigger ROS, and ROS alters lysosomal activity which results in diminished mitochondrial turnover [5,16].
7KC can be metabolized in the liver by enzymes like 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which is why it is not acutely toxic to ingest foods high in 7KC. Reductive enzymes like 11β-HSD1 convert 7KC to 7β- and 7α-hydroxycholesterol [26,41,42], while sterol sulfotransferases (specifically SULT2B1b) have been shown to sulfonate oxysterols like 7KC (and its reduced derivatives), making them significantly less cytotoxic [43]. However, these enzymes are largely restricted to the liver, and do not meaningfully metabolize 7KC in other tissues.
In summary, 7KC is produced by the oxidation of cholesterol at the C7 position primarily nonenzymatically by ROS or alternatively by CYT-mediated oxidation. Intracellularly 7KC partitions into membranes in its free form or is esterified for transport in lipoproteins. If 7KC is consumed and absorbed from the digestive tract, 7KC can be reduced by enzymes in the liver or sulfonated elsewhere in the body by sulfotransferases [43,44].
3. Biology of 7KC
3.1. General cellular effects of 7KC
Cholesterol is one of the most common molecules in the human body – it is found in all cellular membranes and is essential for maintaining elasticity, regulating membrane trafficking, and organizing signaling molecules at the cell surface [42,45]. Likewise, 7KC can be found anywhere that cholesterol can be found, and this has many biological implications. Cholesterol-rich lipid rafts are particularly affected because 7KC creates surface packing defects in the cell membrane, leading to increased permeability due to increased hydrophilicity of the C7 carbonyl group. This increase in permeability causes the opening of calcium channels, allowing calcium to enter the cell and induce apoptosis, and can also inhibit cholesterol efflux [32,34,42,46]. Conversely, the closely related 7β-OHCh does not easily enter and disrupt cell membranes, which likely accounts for its relatively reduced cytotoxicity compared to 7KC [47].
Perhaps the most notable effect of 7KC on cells is its ability to activate NADPH oxidase (NOX), NADPH oxidase (NOX), leading to rapid ROS generation and eventually cell death by apoptosis [18]. 7KC has been shown to promote ROS production in neutrophils by enhancing the translocation of cytosolic NOX components to the cell membrane, where NOX is active to increase ROS production [48]. The oxidative stress induced by 7KC also affects membrane permeability of the lysosomes and other organelles [49]. 7KC-induced apoptosis via NOX-mediated ROS production and oxidative stress, caspase activation, lysosomal degradation, and phospholipidosis has been demonstrated in human promonocytic U937 cells [20,37]. 7KC has also been shown to induce expression of interleukin-1B (IL-1B), IL-6, IL-8, MCP-1, tumor necrosis factor α (TNF-α), and macrophage inflammatory protein-1B (MIP-1B) in human macrophages/monocytes, showing that 7KC can also affect how cells grow and communicate with one another [50]. 7KC can also cause myelin figure formation and polar lipid accumulation; there is strong evidence that this is dependent on the PI3–K signaling pathway [37].
7KC has also been shown to activate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). This pathway is a key homeostatic regulator in cells. The UPR is initiated when the ER is under stress, the canonical stressor being the presence of excess misfolded proteins. This instigates a number of signals which slow down protein synthesis, increase protein folding machinery, and increase degradation of incorrectly folded proteins. If these alterations do not alleviate the ER stress, these same pathways can initiate cell death. Importantly, 7KC is able to perturb the ER membrane, activating UPR and eventually destroying the cell [20,[51], [52], [53]].
Oxysterol sensors, specifically the Liver X Receptor (LXR) and oxysterol-binding-proteins (OSBPs), regulate many of the functions of oxysterols and their roles in regulation of lipid metabolism, cholesterol homeostasis, development, and cell growth. For instance, LXRs control the expression of the ATP-binding cassette (ABC) transporters, and activation of these transporters has implications in tumor growth, immunosuppression, cholesterol metabolism, and apoptosis/phagocytosis [[54], [55], [56], [57], [58], [59], [60], [61]]. Some research suggests that one role for LXR activation is to provide an adaptive response to oxidative stress, therefore protecting cells from further damage. LXR can be activated by multiple different oxysterols, including potentially 7KC. However, the role of 7KC in this pathway is still tenuous. It has been shown that 7KC is a weak agonist for the LXR when LXR-mediated transcription is activated [62]. This would suggest that a cell already responding to oxidative stress could be more affected by 7KC buildup via the LXR pathway. More recently, there is evidence that LXR activation can protect cells from 7KC toxicity, particularly in the brain [57]. It is also debated whether 7KC can effectively bind OSBPs, which are used by the body to transport unesterified oxysterols. However, expression levels of ABCA1 (a key regulator of intracellular sterol levels and sterol efflux) is controlled via both LXR and OSBP, suggesting that oxysterol receptors could make a good target for the control of cholesterol efflux from macrophages [63,64]. Establishing the importance of 7KC in these pathways requires further work, but is suggested as a fruitful avenue of exploration.
Peroxisome proliferator-activated receptor gamma (PPARγ) also regulates expression of ABCA1 genes, inducing cholesterol removal in a transcriptional cascade mediated by the LXR [65]. OxLDLs and especially 7KC have been shown to be activating ligands for PPARγ, resulting in upregulation of proline oxidase (POX), initiating apoptosis [55,66].
PPARγ and 7KC have been linked to poly-ADP-ribose (PARP) formation, which has implications in many age-related diseases including cancer, atherosclerosis, and Alzheimer's disease [[67], [68], [69], [70]]. Furthermore, PARP has been shown to repress LXR expression, possibly counteracting activation of LXR by oxysterols and acting as a negative feedback mechanism to control ABCA1 gene expression in response to cholesterol metabolism and efflux [71]. More research is clearly necessary to fully elucidate the cross-talk between these many different pathways, and the exact role(s) which 7KC and oxysterols play is yet to be determined. However, there are a number of known effects and these are a key subject of this review.
Because the enzymes necessary to detoxify 7KC are mainly expressed in the liver, 7KC in other tissues can remain toxic, leading to ROS generation and increased 7KC formation. Some studies suggest that at least some of these responses are adaptive, where oxidative stress signals trigger an endogenous stress response and kill malfunctioning cells [55,66,72]. Paradoxically though, a significant accumulation of dead cells can cause more harm than good.
From the above it is clear that increased ROS, cell recruitment, and membrane disruption creates a multi-faceted pathway for 7KC-induced cell death. Interference with biological processes including enzymatic reactions, endothelial functions, oxidation of proteins and other molecules, and induction of apoptosis are just a few of the direct consequences from 7KC accumulation in cells. The result is inflammation in many forms, with the potential for causing significant damage to tissues all over the body. This complex combination of oxidation, apoptosis, and autophagy has been dubbed “oxiapoptophagy” [73] and has been observed in multiple types of cells including human monocytes, embryonic kidney cells, macrophages, endothelial cells, smooth muscle cells, as well as mouse embryonic fibroblasts, neutrophils, and oligodendrocytes [43,[74], [75], [76], [77]].
The downstream biochemical and metabolic impacts of any primary toxin are always numerous and diverse, particularly with one as ubiquitous as 7KC and we do not intend to attempt to summarize them all here. The downstream homeostatic impacts of 7KC and other oxysterols have been reviewed elsewhere [20,30,37,41,42,46,58,[78], [79], [80], [81], [82], [83], [84], [85], [86]], however it is worth highlighting the effect of 7KC-induced lysosomal, peroxisomal, and mitochondrial dysfunction.
3.2. 7KC in the lysosome
7KC is a potent lysosomal poison. It has been shown to alter lysosome function by directly increasing membrane permeability and inducing the buildup of unesterified cholesterol in the lysosome [15,87,88]. Accumulation of cholesterol causes further impairment of the lysosomal membrane which in turn prevents fusion with endosomes or autophagophores [89]. Lysosomal aggregates are highly implicated in many age-related diseases including macular degeneration, neurodegeneration, and atherosclerosis [90].
Additionally, 7KC causes lysosomal dysfunction by inhibiting proteolytic enzymes such as cathepsin D and B [17]. Even if lysosomes are capable of fusing with other vesicles or organelles, 7KC impairs the normal hydrolytic activity of the lysosome, thus inhibiting the turnover/recycling of biomolecules and cellular organelles. The buildup of oxysterols in the lysosome can also lead to the accumulation of ceroid/lipofuscin which is a well-characterized marker of aging in postmitotic cells [91]. Furthermore, disruption of normal lysosomal activity/structure results in decreased mitochondrial turnover and subsequent increase in ROS production from damaged mitochondria that are not efficiently cleared [16]. Lysosomal degradation such as that described here is considered pivotal in accelerating otherwise “normal” cellular decay, resulting in premature deterioration of many related cellular processes due to the inability to process toxins [90,91].
The cholesterol transporter protein ABCG1 has been proposed to exert a protective effect against 7KC in the lysosome. While ABCG1 overexpression fails to rescue cells from 7KC toxicity [92], ABCG1 null macrophages are particularly susceptible to 7KC induced toxicity [93]. It was further shown that ABCG1 can exert a cytoprotective effect by facilitating export of 7KC to HDL [94].
3.3. 7KC in the peroxisome
The peroxisome is an essential component of the cell for, among other things, the oxidation of fatty acids. Because of this, the peroxisome is also a potentially potent source of ROS in cells [95]. Closely associated with the mitochondria and endoplasmic reticulum, functioning peroxisomes are essential for cellular homeostasis.
Treatment of microglial BV-2 and 158 N cells with 7KC significantly alters the integrity, size, and function of peroxisomes in a dose-dependent manner [96,97]. 7KC treated cells express both p62 and ABCD3 (expression of both of these markers is considered pexophagic) in the peroxisomal membrane, suggesting 7KC triggers selective autophagy of the peroxisome [97].
Dysfunction of peroxisomes has been linked to various diseases, specifically due to downstream unregulated autophagy in the mitochondria and ER [96,97]. While it is unclear whether 7KC-induced autophagy is protective or responsive, it is known that 7KC can increase accumulation of VLCFA (very long chain fatty acids). These fatty acids likely accumulate because the peroxisomes are unable to sufficiently oxidize them, and these VLCFAs are known to favor oxidative stress and cell death [97].
Better models are necessary to fully elucidate the direct and/or indirect effects of 7KC on the peroxisome, but it is clear that the peroxisome plays an important role in 7KC-induced cellular damage and aging [98].
3.4. 7KC in the mitochondria
One might expect a direct effect of 7KC on mitochondrial function and therefore free-radical release. In fact, 7KC has been shown to cause mitochondrial dysfunction in cultured cells, namely a loss of mitochondrial membrane potential leading to reduced oxidative phosphorylation and reduced ATP production [97]. We see no evidence in the literature, however, that 7KC accumulates or is produced in the mitochondria. This could be due to the fact that mitochondrial membranes are low in cholesterol [99].
Does that mean that the mitochondria plays no role in generating 7KC? As mitochondria are the primary source of ROS and the reaction of ROS with cholesterol is the primary source of 7KC (Fig. 1) this seems unlikely. One possibility is that ROS leaks out of the mitochondria and reacts with cholesterol elsewhere. Another possibility is that the little cholesterol that is present in the mitochondrial membranes does form 7KC readily, but that mitochondria with significant amounts of 7KC are rapidly subjected to mitophagy. In this case the 7KC would bioaccumulate in the lysosome rather than the mitochondria, even if the mitochondria was the source of much of the cell's 7KC. Certainly mitochondria generate ROS, ROS leads to more 7KC, and 7KC causes greater ROS production. To what extent these are direct or indirect phenomena requires greater experimental attention. Perhaps additional work with more sensitive tools will reveal such a role for an interplay between mitochondria and 7KC.
4. Pathology of 7KC
It is clear that oxiapoptophagy can occur in many different cell types all over the body. However, cell death has varied pathologic effects depending on the type and location of the cells affected. Thus, 7KC is highly implicated in many different diseases with many different pathologies.
4.1. Cardiovascular and arterial diseases
Perhaps the most well-known 7KC-related disease is atherosclerosis, the accumulation of cholesterol-laden macrophages (foam cells), lipids, calcium, cholesterol crystals, and connective tissue in the inner layer of the arterial wall [100]. Atherosclerotic foam cells accumulated in plaques also show many characteristics of acquired lysosomal storage disorder [101], which could be attributed to high levels of 7KC (Fig. 2). Additionally, macrophages exposed to oxLDLs accumulate cholesterol esters within their lysosomes. It is thought that cholesterol accumulation causes lysosomes to lose proper pH, so they can no longer efficiently hydrolyze and process cellular debris due to lysosomal proton pump inhibition [102,103]. Anything that blocks or reduces the clearance of sterol from lysosomes can contribute to proton inhibition, thus 7KC accumulation in lysosomes has the potential to exacerbate the disruption of lysosome pH maintenance. As discussed above, 7KC can also activate a stress response via LXR signaling. The LXR is also active in macrophages [96] and thus aberrant LXR signaling due to an excess of 7KC [62,104] could be responsible for part of the mechanism by which 7KC is toxic to macrophages, promoting inflammation in atherosclerotic plaques.
Fig. 2. Formation of foam cells and arterial plaque due to 7KC-induced differentiation and buildup of ROS.
In hypercholesterolemic patients, 57% of the plasma oxysterols are reported to be 7KC, followed by 7-α/βOHC (21%, a direct product of 7KC metabolism). In arterial plaques, 55% of oxysterols are reported to be 7KC, with the second and third most abundant being cholestane-3β,5α,6β-triol and 7-α/βOHC at 13% and 12%, respectively [25]. While it is controversial whether hypercholesterolemia alone is sufficient to elevate circulating 7KC levels [105,106], cross-sectional studies demonstrate that circulating 7KC levels are elevated in patients with atherosclerosis in proportion to the severity of their disease [4,[106], [107], [108], [109]]. Moreover, prospective studies find that higher levels of circulating 7KC are associated with greater future risk of cardiovascular events and total mortality [48,100,110,111]. Highly elevated 7KC in red blood cells (RBCs) is strongly correlated with heart failure [112]. It is notable that the strong disease correlation in heart failure is associated with intracellular 7KC and not serum 7KC levels, as in atherosclerosis.
Endothelial cells form the inner barrier layer of the blood vessel and are key components of normal arterial function. An initiating event in atherogenesis is disruption of endothelial cell function allowing the atherosclerotic plaque to form and accumulate foam cells directly underneath the endothelial layer [5,113]. 7KC is a pro-inflammatory, pro-oxidant, pro-apoptotic, and fibrogenic molecule that alters endothelial cell function by disrupting cell membranes and critical ion transport pathways for vasodilatory response [5,20,27,35,79,114]. 7KC has also been shown to up-regulate adhesive molecules such as ICAM, VCAM, and E-selectin, suggesting that 7KC has an important role in the initial association of leukocytes and monocytes which penetrate through the endothelial barrier, ultimately resulting in atherosclerotic plaque formation [38,115,116].
.../...
.
Edited by Engadin, 09 January 2020 - 08:52 PM.
