Medical Hypotheses 84 (2015) 141–144
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Atherosclerosis regulation via media lipid-driven VSMC cholesterol efflux switch q G.V. Chepelenko N.A. Semashko Central Clinical Hospital, Moscow, Russia
a r t i c l e
i n f o
Article history: Received 16 June 2014 Accepted 4 December 2014
a b s t r a c t It is known, that initial events in atherosclerosis arise in the intima with a parallel influx of inflammatory cells. I propose the opposite – that the disease onset begins from the media vascular smooth muscle cell (VSMC) involvement and through its utilization of modified low-density lipoproteins (LDL), and free or esterified cholesterol. Other oxidized lipoprotein molecules remain in the media which are non-removed by high-density lipoproteins (HDL), owing to their structural damages after local vasa vasorum and adventitia lymphatic disorders. Mechanism by which VSMC ingulf and degrade them includes lipiddriven activation of VSMC reverse cholesterol transport pathways from the media to macrophages, and from the last – to plasma HDL (non-damaged) and apoA-1 or – directly to them. When some of the pathways are impaired, its demands a reprogramming of the existing cholesterol removal route to another, or selective gene involvements and transcriptional regulation of inflammatory signaling. Intima cell call-effects may be linked after their down-regulation with the expression of cytokines, chemokines by migrating VSMC to stem cells for dose-dependant proliferation, VSMC and macrophage maturation in non- and inflammatory phases of early or late atherosclerosis. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction It is known, that initial events in atherosclerosis arise in the intima, where plasma low-density lipoproteins (LDL) are retained and modified [1] with vascular smooth muscle cell (VSMC) involvement. Molecular after-effects may be explained by responseto-injury [2], LDL oxidation [3] and response-to-retention [4] hypotheses. However, none of these hypotheses consider the different efflux pathways for low- and high density lipoproteins (HDL) from the lumen through the entire artery wall and the quantity that enters via vasa vasorum [5]. Both LDL and HDL particles enter the intima from the plasma. The intimal clearance of HDL is larger than that of LDL [5]. Both these separate lipoprotein flows leave the arterial wall via the lymphatica vasa vasorum. Adventitia lymphatic pathways are overloaded [6] in response to risk factors (elevated and modified LDL particles, free radicals, caused by cigarette smoking, hypertension and others), acting via arterial vasa vasorum response and ischemia-induced mechanisms, that may create their efflux delay, and disturbing natural routes for cholesterol LDL and HDL particles to the adventitia. Accumulation of non- and modified lipoproteins in artery compartments demands their removal. However, the molecular mechanism lipid transformation and q
No grant support. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.mehy.2014.12.002 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
cholesterol transfer from the media layer to the intima remain unknown. Factors that account for separate cell response in the intima, media remain largely unclear. The pathogenesis of selective lipoprotein delay and media/intima cell interactions also remains obscure. Nothing is known regarding mechanisms that control lipid accumulation and regulate selective immune response, promoting atherosclerosis attenuation in the early phase of the disease. Hypothesis I propose that irrespective of cause, leading to atherosclerosis progress, early molecular events appear after media lipoprotein accumulation, when the major part that enters the intima could not to leave the wall because the lymphatica vasa vasorum was overloaded. The primary impulses are induced by the direct influence of various forms of lipoproteins on VSMCs receptor activation (by free cholesterol, LDL and HDL) and after their enzymatic modulation, especially of HDL that deteriorates free cholesterol and LDL cholesterol uptake and their transfer through the media compartment. I suggest that increased uptake of lipoproteins by VSMCs leads to initial non-macrophage reverse cholesterol transport (RCT) that more precisely describes whole body cholesterol efflux from the peripheral tissue and its removal from the cholesterol-loaded macrophages in the intima. Beside macrophage RCT in intima arise new pathways for the removal of cholesteryl esters,
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G.V. Chepelenko / Medical Hypotheses 84 (2015) 141–144
modified and oxidized HDL, intact LDL, and their different particles – by smooth muscle cell RCT. This mechanism, activated by them, leading to VSMCs migration, directs on uptake non-removal lipoproteins, their transport from media to macrophages, and from them to plasma HDL (non-damaged) or directly to HDL and apoA-1. VSMC migration are thought to be an important response to atherogenic lipoproteins through molecular events, when migrating cells became transported with signals toward the intima for macrophage proliferation, their invitation for lipoprotein utilization and for macrophage RCT pathways and cholesterol removal out of the artery wall. The mechanism by which VSMCs utilize lipoproteins includes an activation of different receptor types and intracellular switches of their trafficking and signaling pathways for selective macrophage invitation and further cholesterol presentation to HDL and apoA-1. Overloaded VSM cells modulate lipoprotein particle removal. When an efflux function is over, that leads to the expression of VSMC chemokines to the intima and promotes endothelial cell activation with adhesion molecules for monocytes. Next independent pathway for cholesterol excess removal are their cytokine production, some of the most important are: plateletderived growth factor (PDGF), transforming growth factor b (TGF-b), macrophage migration inhibitory factor (MMIF), stem cell activation factor and others. These mitotic and trophic factors define the variability and diversity of the VSMC reverse cholesterol transport pathways to macrophage receptors and their reprogramming for cholesterol removal from one intra- and intercellular pathway to another, when some of them are impaired, including increased uptake function of newly-born media/intima cells. Thus, the pathogenesis of early and late forms of atherosclerosis involves a pathway that was first selective, then became completely impaired with a delay of cholesterol efflux and when there appeared a two-directed cytokine and chemokine signaling between VSMC and intima cells. Such a media-related mechanism with VSMCs as an active carrier of lipoproteins from the media to the intima was previously proposed in local atherosclerotic plaque origin hypothesis in patients without a plasma hyperlipidemia [7] with the unknown way of their transfer for macrophages and plasma HDL. Later on, it was revealed that a component for possible lipid transfer by transporting VSM cells – their reverse cholesterol transport pathway, included the ATP binding cassette (ABC) transporters ABCA1 and ABCG1 [8]. These findings support my media-related mechanism of cholesterol removal from the middle layer of the artery and may also explain the onset and regulation of its accumulation in the intima. Stressful influence of non-removed native and oxidized lipoproteins on media VSMCs results in selective activation of some pathways, and may lead to a marked upregulation of one of the cholesterol removal routes (ABCA1) and to a moderate reduction in ABCG1 protein levels. As the first step of the cholesterol export regulation is the reprogramming of VSMC RCT pathways from one transporter to another, or from one VSMC pool to non-overloaded cells. Irregular activation patterns link with dose-dependent activation of various pathways after selective expression of various types of lipoprotein receptors for the uptake of lipoproteins and their intracellular degradation. When activation of transport pathways of mature VSMCs was downregulated due to their overload, the next events link with the activation of VSMC signaling to increase their proliferation in the media and the intima, and macrophages, other types of cells – in the intima. Their selective inclusion in the inflammatory process enhances cholesterol uptake and its removal from media. Recent studies show that such rate-limiting events of foam cell growth may be regulated via new partners: vascular multipotent stem cells (VMSCs) [9] and LDL upregulated CCL20 expression in media VSMCs, that induce lymphocyte migration into the intima [10]. Such CCL20 and other signaling molecules are upregulated
by LDL, when smooth muscle cell RCT pathways are violated in vasa vasorum traps, non-removed extracellular lipoproteins can act directly on stem cells to induce their VSMC differentiation, to cell lineages that respond differently to the stimuli that generate atherosclerotic lesions. Weak and strong signals for cell pool regulation Transporting VSMC controls a delayed inflammatory reaction in the subintimal space, more willingly stimulating VSMC-macrophage interactions during lipid transfer from the media to the intima, to HDL and apoA-1 in the early phase, but poorly modulating other cell activity in the vessel. Leukocyte–endothelial cell interactions arise much later, than the same between VSMC and macrophages. Macrophages may have a role in the apparent regression of cholesterol and oxidized LDL high levels, that arrived from the media, and as an initial additional source of their reutilization and reversal of their trafficking out of the artery wall. Much later, via VSMCs and macrophages may be formed an amplification mechanism for leukocyte recruitment into growing atheromatous plaque. Details of invitation, wall margination and leukocyte distribution in the subintimal space according to the hypothesis may be only proposed. Leukocyte-endothelial cell interactions have a secondary origin and are being formed by weak or strong signals from VSMCs and macrophages, overloaded by different lipid classes, where they are not completely utilized and removed during their trafficking by these cells. In such a situation, when endothelial cells are stimulated by lipids and inflammatory signals inside of the intima space, they rapidly move the preformed adhesion molecules, stored in cytoplasmatic granules, to the surface. Such adhesion molecule expression on endothelial cells is preferentially dense at the margins or edges of individual cells and this denseness helps to conventionally localize them at the inter-endothelial cell junctions, where they can eventually force their way out of the bloodstream into the subendothelial space. Adhesiveness of endothelial cells according to the hypothesis may depend on the strength of inflammatory stimuli and be modulated. Weak and strong signals promote delayed or accelerated inflammatory responses. Therefore, the initial stages of atherosclerosis with a small leukocyte pool and their insignificant toxic action can occur in the vascular wall without overt endothelial denudation, and which could not be an obligatory condition of atherosclerosis onset, and even of its progression. Moreover, a prolonged period of an early lipid media/intima trafficking out of the wall may arise without the activation of plasma monocytes and lymphocytes with a compensatory increase of their removal by newly-born and proliferated macrophages from the activation of multipotent stem vascular cells discovered by Song Li. We note, that endothelial dysfunction may not be an early atherosclerotic marker as in the classical atherosclerosis onset model and can occur as secondary during the initial media VSMC activation as the response to removal delay of different lipid classes and after these cell overloads. Only after their incomplete removal lipid-driven switches redirected the signals from the activation of ABCA1 and ABCG1 to an inflammatory response and invitation of cell different pools in the subendothelial space to their response to extracellular lipids for their possible degradation and reverse cholesterol transport increase out of the artery wall. Such signals are being included into the cross-talk signaling net for the regulation of atherosclerosis. Types of VSMC switches: inflammatory reaction trigger Multi-vector, cross-talk signaling net with VSMC lipid-driven switches of different lipid class trafficking are represented by: (1) different membrane class receptors for the selective uptake of each
G.V. Chepelenko / Medical Hypotheses 84 (2015) 141–144
lipid poll, which classifies them directly into compartments, corresponding to their types, (2) inter-compartmental lipid interactions with selective receptor-dependent and non receptor up- and downregulation signals via intracellular switches to nucleus transcriptional factors, which regulate gene transcription, encoding enzymes, inflammatory molecules under overload circumstances, (3) transcriptional nuclear factor kappa B (NF-kB)-dependent and without the signaling pathway of nuclear receptor activation (liver X receptor, LXR; peroxisome proliferator-activated receptor c, PPARc) by direct lipoprotein action with ABCA1 and ABCG1 expression for cholesterol and oxLDL reversal trafficking. The role of LXR- and PPARc-mediated dysregulation of the sterol-responsive element-binding protein (SREBP) on the blockage of different VSMC receptors in cases of a cell lipid overload and inflammatory signal increase, and how incomplete efflux of modified lipoproteins could influence constant production of different cytokines, chemokines, and growth factors for prolonged inflammatory response in subintimal space after atherosclerosis attenuation periods and lipid removal control, and via what switch mechanisms remains unclear. Previous oscillatory LXR and PPARc levels with transcriptional intermittent cascades of ABCA1 and ABCG1 expressions, and temporary inflammatory signals via NF-kB fluctuation may be blocked with harmful consequences for VSMC reversal cholesterol transport, VSMC and macrophage interactions, with endothelial and inflammatory cell interaction increase. Before these events, possible, lipid-driven switches may be involved in media VSMC lineage proliferation. Such new VSMCs may take part in the safety mechanism, when media is lipid-overloaded. Such mechanisms may arise at early and late atherosclerosis stages, when ABCA1 and ABCG1 poorly coordinate cholesterol and lipid removal. Invitation of VSMCs, and later – inflammatory and immune cells to the endothelial space, decrease a lipid burden around foam cells. NF-kB may indeed be regulating levels of inflammatory signals from media. In proper time this pathway activates processing for ABCA1 and ABCG1 expression and, when they are overloaded, partly or completely blocked, directs signals for recruitment of additional pools of different cells by quickening its up-regulation for the expression of proinflammatory signals to the inside surface of the endothelial cells. I propose, that a multi-wave of NF-kB activation or with inflammatory silencing may be principal in atherosclerosis and lipid level regulation. The exact role of inflammatory and antiinflammatory effects via LXR and PPARc receptors to different lipid classes in media VSMCs, and their multi-vector signals via lipid-driven switches remains to be elucidated. Evidence for hypothesis VSMCs have been demonstrated to express a variety of cholesterol uptake receptors, including the LDL and VLDL receptors, CD36, type I and type II scavenger receptors [11], promoting foam cell formation [12]. In the presence of various forms of cholesterol VSMCs can became lipid-laden [13]. In vitro, the LDL receptors on VSMCs can mediate the uptake of unmodified LDL [14], acetylated LDL [15], and enzymatically modified LDL [16]. The scavenger receptors are also associated with the uptake of oxidized LDL into human VSMCs [17]. Minimally modified LDL can activate toll-like receptors-4 (TLR-4) and participate in lipid uptake and subsequent immune response [18]. In addition of uptake receptors, they also express necessary components of a reverse cholesterol transport pathway, including the ATP binding cassette (ABC) transporters, ABCA1 and ABCG1 [8], suggesting a differential reprogramming of VSMC RCT pathways for many lipoprotein forms. Apart from this, subpopulation of VSM cells in primary culture differently respond to the LDL overload [19]. After initial cholesterol loading VSMCs upregulate cholesterol transporters [13]. This may be evidence that both activated pathways are involved in the regulation
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of VSMC lipid accumulation and may contribute to atherosclerosis attenuation. Thus, the media-induced reprogramming of VSMC reverse cholesterol transport pathways seems to be more important in the early stage of atherosclerosis than macrophage RCT because the last pathways are dependent on the media VSM cell activation by non- removed lipoproteins. Collectively, these data support the hypothesis, that their signals and functional effects evoked by lipoproteins may serve to limit magnitude and duration of their accumulation levels by selective invitation and proliferation of some types of inflammatory cells.
Testing the hypothesis To prove this hypothesis, several sets of experiments are required. First, to explore the relevance of media VSMCs to the uptake of lipoproteins, they must be used as models of hypercholesterolemia in apolipoprotein E-deficient (apo E / ) mice without and with a lymphatic vessel separation following a high-cholesterol feeding during 12 months. LDL and HDL accumulation across the arterial wall must be determined by in situ immunolabeling and microscopy in each experimental mice group. Next, findings must show and illustrate that the media lipoprotein environment varies in VSMCs ability to support their reverse cholesterol transport pathways from initial (non-inflammatory) signals for the effective removal of media lipoproteins into the plasma (via HDL) or their inclusions into macrophages to switching them to inflammatory signal increase to promote stem cell activation in the intima for selective immune response. It is important to define the signaling increase to the intima, when they are lipid-overloaded compared with media VSMCs isolated within 1–4 months in a high cholesterol diet, when the accumulation of LDL and HDL in arterial walls is absent against a background of plasma cholesterol increase during these terms. Such findings might also show activation or decrease of RCT pathways in VSMCs and macrophages. To test the hypothesis in vitro, a possible transfer mechanism of various forms of lipoproteins via receptors or by endocytic manner, it is needed to obtain a cholesterol ester-rich inclusion from cultured media VSMCs and incubate these lipids with intima macrophages. Level measurements with gas–liquid-chromatography must confirm that their uptake is both time- and concentrationdependant, that lipids stored by macrophages in response to exposure to VSMC inclusions are also metabolically available for macrophage RCT to plasma HDL. Next experiments might also show an improved and impaired mechanism of VSMC RCT in initial (compensated) and late (decompensated) phases of plaque formation without and with selective immune signals from media VSMCs to intima stem cells and elucidate whether intimal proliferation of macrophages and media/intima new-born VSMCs are capable to participate in new-inflammatory regulation of lipid accumulation and removal of their excess from the artery wall when media maternal VSMC RCT pathways are broken. Next, effects of native and oxidized LDL on LDL and scavenger receptor promoter activity and mRNA expression using flow cytometry could be studied and confirmed by comparative expression or downregulation of inflammatory-related genes. The same must be done in the elucidation of various receptor involvement of different media and intima cultured stem cell lineage for synthetic and mature types of VSMCs, proving their participation in lipoprotein degradation and removal, promoting signal increase for intima lymphocyte invitation. Confirmation of the mediainduced molecular events by primary signals from media VSMCs must support the non-inflammatory phase of atherosclerosis (early) with foam cell formation and secondary inflammatory (selective) response, providing a novel mechanism for macrophage
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and newly-born intima VSMCs from stem cells by signals from media mature smooth muscle cells. The next set of experiments would aim to identify the native LDL, free cholesterol, minimally oxidized and oxidized lipoproteins that lead to VSMC receptor activation and induce RCT pathway expression for the regulation of their media/intima removal without signals and with inflammatory intima response via expression of chemokines, cytokines and adhesion molecules by media VSM cells. The hypothesis predicts that high lipoprotein uptake indices would be correlated with down-regulation of VSMC RCT pathway receptors and demonstrates dose-dependent expression of inflammatory genes and will show successively their expression in cholesterol, cholesterol esters, minimally oxidized LDL, very low density lipoproteins (VLDL), and oxidized LDL treated cells. We need to show that these non-inflammatory (requiring the expression of ABCA1 and ABCG1) and inflammatory pathways of VSMC signaling could be used for quantitative real-time RT-PCR for mRNA level definition for specific gene involvement in their transcriptional regulation. Confirmation of ATP-binding cassette transporter activation would note the inflammatory signal silencing, showing lipid-driven expression. A promotor sequence analysis might also show several conserved putative binding sites for transcriptional factors potential regulated by native and oxidized LDL. Conflicts of interest statement I have no conflict of interest: I am the only author and have no sources of funding. References [1] Oloffson SO, Boren J. Apolipoprotein B: a clinically important apolipoprotein which assembles atherogenic lipoproteins and promote the development of atherosclerosis. J Intern Med 2005;258:395–410. [2] Ross R, Glomset J. The pathogenesis of atherosclerosis: part I. N Engl J Med 1976;24:2211–8. [3] Chisolm GM, Steinberg D. The oxidative modifications hypothesis of atherosclerosis: an overviews. Free Radic Biol Med 2000;28:1815–26. [4] Williams KJ, Tabas I. The response-to-retention hypothesis of atherogenesis reinforced. Curr Opin Lipidol 1998;9:471–4.
[5] Nordestgaard BG, Hjelms E, Stender S, Kjeldsen K. Different efflux pathways for high and low density lipoproteins from porcine aortic intima. Artheriosclerosis 1990;10(3):477–85. [6] Lim HY, Ritkowski JM, Helft J, Reddy ST, Swartz MA, Randolf GJ, et al. Hypercholesterolemic mice exhibit lymphatic vessel dysfunction and degeneration. Am J Pathol 2009;175(3):1328–37. [7] Chepelenko GV. Pathogenesis of atherosclerosis in patients without lipid metabolism disturbances: hypothesis on cholesterol utilization and atheromatous plaque formation. Angiol Vasc Surg (Russia) 2003;3:20–5. [8] Nagao S, Murao K, Imachi H, Cao WM, Yu X, Li J, et al. Platelet derived growth factor regulates ABCA1 expression is vascular smooth muscle cells. FEBS Lett 2006;580:4371–6. [9] Tang Z, Wang A, Yuan F, Van Z, Liu B, Chu JS, et al. Differentiation of multipotent vascular stem cells contribute to vascular diseases. Nat Commun 2012 [V3: article number 875, 06 June]. [10] Calvayrac O, Rodrigues-Calvo R, Alons J, Orbe J, Marton-Ventura JL, Guadal A, et al. CCL20 is increased in hypercholesterolemic subjects and is upregulated by LDL in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2011;31:2733–41. [11] Lacolley P, Regnault V, Nicoletti A, Li Z, Michel J-B. The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles. Cardiovasc Res 2012;95(2):194–204. [12] Doran AC, Meller N, McNamara GA. The role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol 2008;28(5):815–9. [13] Rong JX, Shapiro M, Trogan E, Fisher EA. Transdifferentiation of mouse smooth muscle cells to a macrophage-like state after cholesterol loading. Proc Natl Acad Sci USA 2003;100:13531–6. [14] Ruan XZ, Moorhead JF, Tac JL, Mak L, Wheeler DC, Powis SH, et al. Mechanisms of dysregulation of low-density lipoprotein receptor expression in vascular smooth muscle cells by inflammatory cytokines. Arterioscler Thromb Vasc Biol 2006;26:1150–5. [15] Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis: beyond lipid uptake. Arterioscler Thromb Vasc Biol 2006;26:1702–11. [16] Klouche M, Rose-John S, Scmiedt W, Bhakdi S. Enzymatically degraded, nonoxidized LDL induces human vascular smooth muscle cell activation foam cell transformation, and proliferation. Circulation 2000;101:1799–805. [17] Wagsater D, Olofsson PS, Norgren L, Stenberg B, Sirsjo A. The chemokines and scavenger receptor CXCL/SR-PSOX is expressed in human vascular smooth muscle cells and is induced by interferon gamma. Biochem Biophys Res Commun 2004;325:1187–93. [18] Choi SH, Harkewicz R, Lee JH, Boullier A, Almazan F, Li AC, et al. Lipoprotein accumulation in macrophages via toll-like receptors-4-dependant fluid phase uptake. Circ Res 2009;104:1355–63. [19] Argamann CA, Sawyez CG, Li S, Nong Z, Hegele RA, Pickering JG, et al. Human smooth muscle cells subpopulation differently accumulate cholesteryl ester when exposed to naive and oxidized lipoproteins. Arterioscler Thromb Vasc Biol 2004;24:1290–6.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Atherosclerosis regulation via media lipid-driven VSMC cholesterol efflux switch q G.V. Chepelenko N.A. Semashko Central Clinical Hospital, Moscow, Russia
a r t i c l e
i n f o
Article history: Received 16 June 2014 Accepted 4 December 2014
a b s t r a c t It is known, that initial events in atherosclerosis arise in the intima with a parallel influx of inflammatory cells. I propose the opposite – that the disease onset begins from the media vascular smooth muscle cell (VSMC) involvement and through its utilization of modified low-density lipoproteins (LDL), and free or esterified cholesterol. Other oxidized lipoprotein molecules remain in the media which are non-removed by high-density lipoproteins (HDL), owing to their structural damages after local vasa vasorum and adventitia lymphatic disorders. Mechanism by which VSMC ingulf and degrade them includes lipiddriven activation of VSMC reverse cholesterol transport pathways from the media to macrophages, and from the last – to plasma HDL (non-damaged) and apoA-1 or – directly to them. When some of the pathways are impaired, its demands a reprogramming of the existing cholesterol removal route to another, or selective gene involvements and transcriptional regulation of inflammatory signaling. Intima cell call-effects may be linked after their down-regulation with the expression of cytokines, chemokines by migrating VSMC to stem cells for dose-dependant proliferation, VSMC and macrophage maturation in non- and inflammatory phases of early or late atherosclerosis. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction It is known, that initial events in atherosclerosis arise in the intima, where plasma low-density lipoproteins (LDL) are retained and modified [1] with vascular smooth muscle cell (VSMC) involvement. Molecular after-effects may be explained by responseto-injury [2], LDL oxidation [3] and response-to-retention [4] hypotheses. However, none of these hypotheses consider the different efflux pathways for low- and high density lipoproteins (HDL) from the lumen through the entire artery wall and the quantity that enters via vasa vasorum [5]. Both LDL and HDL particles enter the intima from the plasma. The intimal clearance of HDL is larger than that of LDL [5]. Both these separate lipoprotein flows leave the arterial wall via the lymphatica vasa vasorum. Adventitia lymphatic pathways are overloaded [6] in response to risk factors (elevated and modified LDL particles, free radicals, caused by cigarette smoking, hypertension and others), acting via arterial vasa vasorum response and ischemia-induced mechanisms, that may create their efflux delay, and disturbing natural routes for cholesterol LDL and HDL particles to the adventitia. Accumulation of non- and modified lipoproteins in artery compartments demands their removal. However, the molecular mechanism lipid transformation and q
No grant support. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.mehy.2014.12.002 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
cholesterol transfer from the media layer to the intima remain unknown. Factors that account for separate cell response in the intima, media remain largely unclear. The pathogenesis of selective lipoprotein delay and media/intima cell interactions also remains obscure. Nothing is known regarding mechanisms that control lipid accumulation and regulate selective immune response, promoting atherosclerosis attenuation in the early phase of the disease. Hypothesis I propose that irrespective of cause, leading to atherosclerosis progress, early molecular events appear after media lipoprotein accumulation, when the major part that enters the intima could not to leave the wall because the lymphatica vasa vasorum was overloaded. The primary impulses are induced by the direct influence of various forms of lipoproteins on VSMCs receptor activation (by free cholesterol, LDL and HDL) and after their enzymatic modulation, especially of HDL that deteriorates free cholesterol and LDL cholesterol uptake and their transfer through the media compartment. I suggest that increased uptake of lipoproteins by VSMCs leads to initial non-macrophage reverse cholesterol transport (RCT) that more precisely describes whole body cholesterol efflux from the peripheral tissue and its removal from the cholesterol-loaded macrophages in the intima. Beside macrophage RCT in intima arise new pathways for the removal of cholesteryl esters,
142
G.V. Chepelenko / Medical Hypotheses 84 (2015) 141–144
modified and oxidized HDL, intact LDL, and their different particles – by smooth muscle cell RCT. This mechanism, activated by them, leading to VSMCs migration, directs on uptake non-removal lipoproteins, their transport from media to macrophages, and from them to plasma HDL (non-damaged) or directly to HDL and apoA-1. VSMC migration are thought to be an important response to atherogenic lipoproteins through molecular events, when migrating cells became transported with signals toward the intima for macrophage proliferation, their invitation for lipoprotein utilization and for macrophage RCT pathways and cholesterol removal out of the artery wall. The mechanism by which VSMCs utilize lipoproteins includes an activation of different receptor types and intracellular switches of their trafficking and signaling pathways for selective macrophage invitation and further cholesterol presentation to HDL and apoA-1. Overloaded VSM cells modulate lipoprotein particle removal. When an efflux function is over, that leads to the expression of VSMC chemokines to the intima and promotes endothelial cell activation with adhesion molecules for monocytes. Next independent pathway for cholesterol excess removal are their cytokine production, some of the most important are: plateletderived growth factor (PDGF), transforming growth factor b (TGF-b), macrophage migration inhibitory factor (MMIF), stem cell activation factor and others. These mitotic and trophic factors define the variability and diversity of the VSMC reverse cholesterol transport pathways to macrophage receptors and their reprogramming for cholesterol removal from one intra- and intercellular pathway to another, when some of them are impaired, including increased uptake function of newly-born media/intima cells. Thus, the pathogenesis of early and late forms of atherosclerosis involves a pathway that was first selective, then became completely impaired with a delay of cholesterol efflux and when there appeared a two-directed cytokine and chemokine signaling between VSMC and intima cells. Such a media-related mechanism with VSMCs as an active carrier of lipoproteins from the media to the intima was previously proposed in local atherosclerotic plaque origin hypothesis in patients without a plasma hyperlipidemia [7] with the unknown way of their transfer for macrophages and plasma HDL. Later on, it was revealed that a component for possible lipid transfer by transporting VSM cells – their reverse cholesterol transport pathway, included the ATP binding cassette (ABC) transporters ABCA1 and ABCG1 [8]. These findings support my media-related mechanism of cholesterol removal from the middle layer of the artery and may also explain the onset and regulation of its accumulation in the intima. Stressful influence of non-removed native and oxidized lipoproteins on media VSMCs results in selective activation of some pathways, and may lead to a marked upregulation of one of the cholesterol removal routes (ABCA1) and to a moderate reduction in ABCG1 protein levels. As the first step of the cholesterol export regulation is the reprogramming of VSMC RCT pathways from one transporter to another, or from one VSMC pool to non-overloaded cells. Irregular activation patterns link with dose-dependent activation of various pathways after selective expression of various types of lipoprotein receptors for the uptake of lipoproteins and their intracellular degradation. When activation of transport pathways of mature VSMCs was downregulated due to their overload, the next events link with the activation of VSMC signaling to increase their proliferation in the media and the intima, and macrophages, other types of cells – in the intima. Their selective inclusion in the inflammatory process enhances cholesterol uptake and its removal from media. Recent studies show that such rate-limiting events of foam cell growth may be regulated via new partners: vascular multipotent stem cells (VMSCs) [9] and LDL upregulated CCL20 expression in media VSMCs, that induce lymphocyte migration into the intima [10]. Such CCL20 and other signaling molecules are upregulated
by LDL, when smooth muscle cell RCT pathways are violated in vasa vasorum traps, non-removed extracellular lipoproteins can act directly on stem cells to induce their VSMC differentiation, to cell lineages that respond differently to the stimuli that generate atherosclerotic lesions. Weak and strong signals for cell pool regulation Transporting VSMC controls a delayed inflammatory reaction in the subintimal space, more willingly stimulating VSMC-macrophage interactions during lipid transfer from the media to the intima, to HDL and apoA-1 in the early phase, but poorly modulating other cell activity in the vessel. Leukocyte–endothelial cell interactions arise much later, than the same between VSMC and macrophages. Macrophages may have a role in the apparent regression of cholesterol and oxidized LDL high levels, that arrived from the media, and as an initial additional source of their reutilization and reversal of their trafficking out of the artery wall. Much later, via VSMCs and macrophages may be formed an amplification mechanism for leukocyte recruitment into growing atheromatous plaque. Details of invitation, wall margination and leukocyte distribution in the subintimal space according to the hypothesis may be only proposed. Leukocyte-endothelial cell interactions have a secondary origin and are being formed by weak or strong signals from VSMCs and macrophages, overloaded by different lipid classes, where they are not completely utilized and removed during their trafficking by these cells. In such a situation, when endothelial cells are stimulated by lipids and inflammatory signals inside of the intima space, they rapidly move the preformed adhesion molecules, stored in cytoplasmatic granules, to the surface. Such adhesion molecule expression on endothelial cells is preferentially dense at the margins or edges of individual cells and this denseness helps to conventionally localize them at the inter-endothelial cell junctions, where they can eventually force their way out of the bloodstream into the subendothelial space. Adhesiveness of endothelial cells according to the hypothesis may depend on the strength of inflammatory stimuli and be modulated. Weak and strong signals promote delayed or accelerated inflammatory responses. Therefore, the initial stages of atherosclerosis with a small leukocyte pool and their insignificant toxic action can occur in the vascular wall without overt endothelial denudation, and which could not be an obligatory condition of atherosclerosis onset, and even of its progression. Moreover, a prolonged period of an early lipid media/intima trafficking out of the wall may arise without the activation of plasma monocytes and lymphocytes with a compensatory increase of their removal by newly-born and proliferated macrophages from the activation of multipotent stem vascular cells discovered by Song Li. We note, that endothelial dysfunction may not be an early atherosclerotic marker as in the classical atherosclerosis onset model and can occur as secondary during the initial media VSMC activation as the response to removal delay of different lipid classes and after these cell overloads. Only after their incomplete removal lipid-driven switches redirected the signals from the activation of ABCA1 and ABCG1 to an inflammatory response and invitation of cell different pools in the subendothelial space to their response to extracellular lipids for their possible degradation and reverse cholesterol transport increase out of the artery wall. Such signals are being included into the cross-talk signaling net for the regulation of atherosclerosis. Types of VSMC switches: inflammatory reaction trigger Multi-vector, cross-talk signaling net with VSMC lipid-driven switches of different lipid class trafficking are represented by: (1) different membrane class receptors for the selective uptake of each
G.V. Chepelenko / Medical Hypotheses 84 (2015) 141–144
lipid poll, which classifies them directly into compartments, corresponding to their types, (2) inter-compartmental lipid interactions with selective receptor-dependent and non receptor up- and downregulation signals via intracellular switches to nucleus transcriptional factors, which regulate gene transcription, encoding enzymes, inflammatory molecules under overload circumstances, (3) transcriptional nuclear factor kappa B (NF-kB)-dependent and without the signaling pathway of nuclear receptor activation (liver X receptor, LXR; peroxisome proliferator-activated receptor c, PPARc) by direct lipoprotein action with ABCA1 and ABCG1 expression for cholesterol and oxLDL reversal trafficking. The role of LXR- and PPARc-mediated dysregulation of the sterol-responsive element-binding protein (SREBP) on the blockage of different VSMC receptors in cases of a cell lipid overload and inflammatory signal increase, and how incomplete efflux of modified lipoproteins could influence constant production of different cytokines, chemokines, and growth factors for prolonged inflammatory response in subintimal space after atherosclerosis attenuation periods and lipid removal control, and via what switch mechanisms remains unclear. Previous oscillatory LXR and PPARc levels with transcriptional intermittent cascades of ABCA1 and ABCG1 expressions, and temporary inflammatory signals via NF-kB fluctuation may be blocked with harmful consequences for VSMC reversal cholesterol transport, VSMC and macrophage interactions, with endothelial and inflammatory cell interaction increase. Before these events, possible, lipid-driven switches may be involved in media VSMC lineage proliferation. Such new VSMCs may take part in the safety mechanism, when media is lipid-overloaded. Such mechanisms may arise at early and late atherosclerosis stages, when ABCA1 and ABCG1 poorly coordinate cholesterol and lipid removal. Invitation of VSMCs, and later – inflammatory and immune cells to the endothelial space, decrease a lipid burden around foam cells. NF-kB may indeed be regulating levels of inflammatory signals from media. In proper time this pathway activates processing for ABCA1 and ABCG1 expression and, when they are overloaded, partly or completely blocked, directs signals for recruitment of additional pools of different cells by quickening its up-regulation for the expression of proinflammatory signals to the inside surface of the endothelial cells. I propose, that a multi-wave of NF-kB activation or with inflammatory silencing may be principal in atherosclerosis and lipid level regulation. The exact role of inflammatory and antiinflammatory effects via LXR and PPARc receptors to different lipid classes in media VSMCs, and their multi-vector signals via lipid-driven switches remains to be elucidated. Evidence for hypothesis VSMCs have been demonstrated to express a variety of cholesterol uptake receptors, including the LDL and VLDL receptors, CD36, type I and type II scavenger receptors [11], promoting foam cell formation [12]. In the presence of various forms of cholesterol VSMCs can became lipid-laden [13]. In vitro, the LDL receptors on VSMCs can mediate the uptake of unmodified LDL [14], acetylated LDL [15], and enzymatically modified LDL [16]. The scavenger receptors are also associated with the uptake of oxidized LDL into human VSMCs [17]. Minimally modified LDL can activate toll-like receptors-4 (TLR-4) and participate in lipid uptake and subsequent immune response [18]. In addition of uptake receptors, they also express necessary components of a reverse cholesterol transport pathway, including the ATP binding cassette (ABC) transporters, ABCA1 and ABCG1 [8], suggesting a differential reprogramming of VSMC RCT pathways for many lipoprotein forms. Apart from this, subpopulation of VSM cells in primary culture differently respond to the LDL overload [19]. After initial cholesterol loading VSMCs upregulate cholesterol transporters [13]. This may be evidence that both activated pathways are involved in the regulation
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of VSMC lipid accumulation and may contribute to atherosclerosis attenuation. Thus, the media-induced reprogramming of VSMC reverse cholesterol transport pathways seems to be more important in the early stage of atherosclerosis than macrophage RCT because the last pathways are dependent on the media VSM cell activation by non- removed lipoproteins. Collectively, these data support the hypothesis, that their signals and functional effects evoked by lipoproteins may serve to limit magnitude and duration of their accumulation levels by selective invitation and proliferation of some types of inflammatory cells.
Testing the hypothesis To prove this hypothesis, several sets of experiments are required. First, to explore the relevance of media VSMCs to the uptake of lipoproteins, they must be used as models of hypercholesterolemia in apolipoprotein E-deficient (apo E / ) mice without and with a lymphatic vessel separation following a high-cholesterol feeding during 12 months. LDL and HDL accumulation across the arterial wall must be determined by in situ immunolabeling and microscopy in each experimental mice group. Next, findings must show and illustrate that the media lipoprotein environment varies in VSMCs ability to support their reverse cholesterol transport pathways from initial (non-inflammatory) signals for the effective removal of media lipoproteins into the plasma (via HDL) or their inclusions into macrophages to switching them to inflammatory signal increase to promote stem cell activation in the intima for selective immune response. It is important to define the signaling increase to the intima, when they are lipid-overloaded compared with media VSMCs isolated within 1–4 months in a high cholesterol diet, when the accumulation of LDL and HDL in arterial walls is absent against a background of plasma cholesterol increase during these terms. Such findings might also show activation or decrease of RCT pathways in VSMCs and macrophages. To test the hypothesis in vitro, a possible transfer mechanism of various forms of lipoproteins via receptors or by endocytic manner, it is needed to obtain a cholesterol ester-rich inclusion from cultured media VSMCs and incubate these lipids with intima macrophages. Level measurements with gas–liquid-chromatography must confirm that their uptake is both time- and concentrationdependant, that lipids stored by macrophages in response to exposure to VSMC inclusions are also metabolically available for macrophage RCT to plasma HDL. Next experiments might also show an improved and impaired mechanism of VSMC RCT in initial (compensated) and late (decompensated) phases of plaque formation without and with selective immune signals from media VSMCs to intima stem cells and elucidate whether intimal proliferation of macrophages and media/intima new-born VSMCs are capable to participate in new-inflammatory regulation of lipid accumulation and removal of their excess from the artery wall when media maternal VSMC RCT pathways are broken. Next, effects of native and oxidized LDL on LDL and scavenger receptor promoter activity and mRNA expression using flow cytometry could be studied and confirmed by comparative expression or downregulation of inflammatory-related genes. The same must be done in the elucidation of various receptor involvement of different media and intima cultured stem cell lineage for synthetic and mature types of VSMCs, proving their participation in lipoprotein degradation and removal, promoting signal increase for intima lymphocyte invitation. Confirmation of the mediainduced molecular events by primary signals from media VSMCs must support the non-inflammatory phase of atherosclerosis (early) with foam cell formation and secondary inflammatory (selective) response, providing a novel mechanism for macrophage
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and newly-born intima VSMCs from stem cells by signals from media mature smooth muscle cells. The next set of experiments would aim to identify the native LDL, free cholesterol, minimally oxidized and oxidized lipoproteins that lead to VSMC receptor activation and induce RCT pathway expression for the regulation of their media/intima removal without signals and with inflammatory intima response via expression of chemokines, cytokines and adhesion molecules by media VSM cells. The hypothesis predicts that high lipoprotein uptake indices would be correlated with down-regulation of VSMC RCT pathway receptors and demonstrates dose-dependent expression of inflammatory genes and will show successively their expression in cholesterol, cholesterol esters, minimally oxidized LDL, very low density lipoproteins (VLDL), and oxidized LDL treated cells. We need to show that these non-inflammatory (requiring the expression of ABCA1 and ABCG1) and inflammatory pathways of VSMC signaling could be used for quantitative real-time RT-PCR for mRNA level definition for specific gene involvement in their transcriptional regulation. Confirmation of ATP-binding cassette transporter activation would note the inflammatory signal silencing, showing lipid-driven expression. A promotor sequence analysis might also show several conserved putative binding sites for transcriptional factors potential regulated by native and oxidized LDL. Conflicts of interest statement I have no conflict of interest: I am the only author and have no sources of funding. References [1] Oloffson SO, Boren J. Apolipoprotein B: a clinically important apolipoprotein which assembles atherogenic lipoproteins and promote the development of atherosclerosis. J Intern Med 2005;258:395–410. [2] Ross R, Glomset J. The pathogenesis of atherosclerosis: part I. N Engl J Med 1976;24:2211–8. [3] Chisolm GM, Steinberg D. The oxidative modifications hypothesis of atherosclerosis: an overviews. Free Radic Biol Med 2000;28:1815–26. [4] Williams KJ, Tabas I. The response-to-retention hypothesis of atherogenesis reinforced. Curr Opin Lipidol 1998;9:471–4.
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