As far as major factors influencing atherosclerotic traits are concerned, our study was based on a homogeneous cohort of patients. Patients and controls were matched for age, as ageing influences the vascular substrate and the development and progression of atherosclerosis. LAD patients were 59 years old ± 9.7, while controls were 54.7 years ± 7.5. Datasets of gene expression in carotid plaques were also obtained from patients with similar age. Moreover, all atherosclerotic patients had comparable stenosis. Due to this homogeneity of experimental sources, the differentially expressed genes that we have found should be considered as generally involved in the molecular events establishing and maintaining atherosclerosis.
The atherogenes network described in Figure , shows the importance of both the inflammatory response and caveolae system, the first controlled by central node STAT1 (inducing also the over-expression of the heat shock proteins HSP47[17
] and HO-1), the second related to steroid hormone receptors.
STATs are cytosolic proteins that, upon activation, translocate in to the nucleus to activate target genes. Originally, this pathway was found to be activated by INFs, but a number of cytokines, growth factors, and hormonal factors were successively found as activators of JAK and/or STAT proteins (see Additional file 2
, Figure S3). In particular, INF-g activates STAT1 one of the central nodes of our atherogene network (Figure ) and this is in accordance with the high level of the interferon protein found in patients plasma (Figure ). It is noteworthy that the anti-inflammatory cytokine IL-10, which is not altered in the patient blood, also activates JAK/STAT protein[18
]. Its receptor results among the up-regulated atherogenes (Figure ), confirming the importance of this molecule in the response to the pathological status to translate signals by IL-10 molecule. These results confirm previous data obtained in the atherosclerotic tissues but allow in addition the link between gene expression and plasma proteins. The importance of the STAT, INFs and IL-10 signals is strengthened by our results, since we were able to detected it in atherosclerotic plaques of different vessels in a series of 25 patients and 29 samples (Table ).
Estrogen receptors (ESR) 1 and 2, expressed in normal and atherosclerotic arteries, mediate the protective action of estrogen to artery wall. ESR1 genotype is a predictor of complex lesions, of coronary thrombosis, and has a role in rising HDL cholesterol level[19
]. Glucocorticoids and their receptor (NR3C1) are also involved in atheroprotective effect. Our data suggest that the protective effect is impaired by GR down-regulation in atherosclerotic plaques from coronaries and carotid (Figure and ).
The activation of HSL is compromised by the down-regulation of both the hormone receptors and HSL activator PRKAB2 (Figure ). Decrease of adipocyte HSL activity has been demonstrated in familial combined hyperlipidemia, characterized by excessive concentrations of serum-free fatty acids[20
]. All patients showed an elevated cholesterol concentration in the blood with index of cardiovascular risk (total cholesterol concentration/HDL) ≥ 5 for males and 4.5 for females.
Glucocorticoid receptor interacts with BMPR2[14
], a component of the TGFBR superfamily. BMPR2 is located within caveolae of endothelial cell membranes suggesting potential dynamic regulatory structural relationships[21
] lost in plaque (Figure and ). BMPR2 gene defect is involved in the development of pulmonary arterial hypertension (PAH). PAH is characterized by the reduction of the lumen of pulmonary arteries and serum IL-6 increase. Additionally, IL-6 level was dramatically increased in transgenic mice expressing a mutant BMPR2 which spontaneously developed PAH[22
]. IL-6 is a cytokine with a wide variety of biological functions. We focalized our attention on its involvement in JAK/STAT pathway because it appeared significantly compromised in both coronary and carotid plaques (see Additional file 2
, Figure S3). It is considered a stress responsive signaling cascade, involved in down-regulation of matrix genes[23
] that could promote plaque rupture. In fact, a reduced production of collagen and its uncontrolled degradation may affect the stability of the vessel wall especially in atherosclerotic plaques by making them prone to aneurysm and rupture. Type IV, XV, XVIII and XIX collagens are structural elements associated with the vascular basement membranes. Each individual SMC is surrounded by a basement membrane. Thus, all membrane-associated collagens are probably involved in the regulation of intercommunication between vessel lumen and wall or between individual SMCs in the intimal and medial layers. This intercommunication appears to loosen in the coronaries and carotids, and this probably leads to de-differentiation of SMC highlighted by down-regulation of CSRP2. This gene is highly expressed in aorta and is drastically down-regulated in response to PDGF-BB (Figure ) or cell injury promoting SMC proliferation and de-differentiation[24
]. A dominant-negative CSRP2 mutant blocked proepicardial cells from differentiating into SMC[25
]. The possible de-differentiation of SMC is also supported by the down-regulation of ACTA2 and PDLIM5 genes in both carotids and coronaries. Proliferative effect of PDGF on SMC could be inhibited by APOER which presents both a protective role in the vasculature and an atherogenic function[26
] (Figure ). APOER up-regulation could balance de-differentiative effects induced by PDGF on SMC, but could allow SMC transformation to foam cells and impairment of vessel structure. This correlation was already evidenced by the work of King Y.J. et al.[6
] but, in addition to this, we have correlated the extracellular matrix with the expression of two important LIM proteins involved in the development of muscle cells: CSRP2 and PDLIM5. These genes could become important targets to modulate the effects of the atherosclerotic lesion at its initial stage.
HDL mediate cholesterol clearance from foam cells but, recently, raising HDL as a treatment for cardiovascular disease has been disputed because of the failure of the torcetrapib therapy[27
]. Despite this negative data, HDL-based therapies such as reconstituted HDL, phospholipids and APOA1Milano
have shown some promising results focusing the importance of lipids homeostasis in atherosclerosis. LXR has a central role in the lipid homeostasis regulating ABC transporter, PLTP and ApoE/C-I/C-IV-CII gene cluster expression (Figure ). Coronary and carotid gene expression profiles evidenced the up-regulation of the ABCC3 gene, a new member of the ATP-binding-cassette family associated with atherosclerosis in mouse[28
]. ABC transporter regulates efflux of cholesterol and phospholipids from peripheral tissues and macrophages mediating reverse cholesterol transport (RCT). RCT is considered to be the primary mechanism through which HDL protects against atherosclerosis, but its production is limited in LAD patients by LPL down-regulation and PLTP up-regulation. Moreover, the index of cardiovascular risk was ≥ 5 and 4.5 for males and females respectively indicating low HDL in patient blood. PLTP over-expression appears to be connected to a decrease of HDL and to an increase of ApoE that prevents its inactivation. PLTP has been already identified like one of the "candidate genes" for atherosclerosis[29
] and this is confirmed by our meta-analysis. On the other hand LPL is an enzyme present in capillary surface, and lack of it causes familial hyperlipoproteinemia type I, characterized by massive hypertriglyceridemia. The impairment of the lipolysis process could support low HDL production by this pathway.
Fatty acids and lipoproteins are removed from circulation by scavenger receptors. CD36 and MSR1 genes are down-regulated in atherosclerotic vessels and this could contribute to the impairment of clearance process and cause the elevated values in the analyzed patients (Table ). CD36 is a scavenger receptor for modified forms of LDL and its loss leads to a pro-atherogenic lipid profile in mice[31
] while MSR1 scavenger increases clearance of modified lipoproteins, protecting the arterial wall against the pro-atherogenic action of LP[32
]. The down-regulation of CD36 and MSR1 could result in a increasing of atherosclerotic lesion in vessel as it was demonstrated in studies on SR-BI-/-
mouse model with microdeletions in scavenger genes[33
]. Our data are coherent with the dyslipidemia of atherosclerotic patients (Figure ) that could be associated with the subsequent development of hypertension causing damage to the inner walls of arteries. These data could apparently appear in contrast with the supposed pro-atherogenic function of CD36. However this protein could play very different roles due to the variety of functions that have been associated to it. In fact, it is involved also in the regulation of the angiogenesis in association with thrombospondin (TSP)[34
]. Angiogenesis is the body response to ischemia and vascular injury and is inhibited by the interaction of CD36 with TSP-1. CD36-TSP-1 interaction is also important in the maintenance of an anti-inflammatory milieu in the uptake of apoptotic cells as a homeostatic process, and during wound resolution. The down regulation of CD36 may be also important to maintain the ability of producing novel vessels important for the plaque maintaining supplying nutrition to grow[35
CD36 and SR are related to Caveolin-1 (Cav-1)[36
] which is a cholesterol-binding protein transporting cholesterol from the endoplasmic reticulum to the plasma membrane. Before this work, there was limited evidence of altered Cav-1 levels in human atherosclerotic lesions[37
]. Loss of caveolin-1 in ApoE-/-
mouse resulted in a pro-atherogenic lipid profile, similar to that seen in CD36-/-
mice bread to an ApoE background[31
]. Absence of Cav-1 and caveolae in endothelial cell (EC) lead to increased eNOS activity and NO release, resulting in reduced vascular tone[38
]. Furthermore, Caveolin-1 is a negative regulator of EC proliferation but promotes cellular differentiation[38
]. However, angiogenic factors such as VEGF have been shown to induce down-regulation of caveolin-1, which was suggested to be important for the mitogenic effects of growth factors in EC[38
]. Caveolin genes down-regulation therefore could lead to decreased inhibition of EC proliferation and to a negative effect on SMC differentiation. Dyslipidemia evidenced in Cav-1-/-
mice is congruent once more with our hypothesis on involvement of Caveolin genes in the control of cholesterol homeostasis.
In summary, the analysis of the atherogenes network that we have outlined shows that the atherosclerotic phenotype of arterial vessels is maintained by altered pathways of hormone receptors, lipid homeostasis and caveolae system that are highly interconnected through specific nodal genes. This is a novel interconnection evidenced in the atherosclerotic plaque that has been confirmed in different patients (different genetic background) and different atherosclerotic sites. Before our work, there were no evidences for the relationship between caveolae, BMPR2 and hormone receptor genes that are in turn related, in our network, to genes involved in the apoptotic process (Figure ). The involvement of the caveolae system in human lipid homeostasis and atherosclerosis has been confirmed. This is a central node linking the network of down regulated genes (left in Figure ) to the up regulated one (right in Figure ). This connection is sustained by APP (amyloid beta precursor protein) and APOE, previously discussed. Perhaps it should be considered the possibility that amyloid-β (Aβ) might be involved in vascular pathology. In fact, APP is involved in blood clotting[40
] and Aβ drains from the brain along the walls of the microvasculature[41
]. Down regulation of APP gene could be involved in thrombosis effect[40
] related to the advanced status and unstable condition of the plaques studied in this work (Figure ).
The portion of the network with over expressed genes is characterized by the inflammatory response regulated by JAK/STAT pathway and is integrated by the data on plasma proteins involved in this process. These data confirm the known involvement of JAK/STAT pathway in atherosclerosis, but our study has validated for the first time the common deregulation of these genes in different atherosclerotic sites and correlated to the hormone receptor, caveolae system and APP connected to the instability of the plaque.