Molecular epidemiology; New but impressive.

Molecular epidemiology is a subdivision of medical science and epidemiology that emphases on the involvement of potential environmental and genetic risk factors, recognized at the molecular level, to the etiology and avoidance of sickness through populations. This arena has developed from the combination of molecular biology and traditional epidemiological research. Molecular epidemiology can improve our knowledge about the precise pathogenesis of disease through recognizing particular pathways that affect the risk of developing the disease. Furthermore, it tries to find how the collaborations between genetic characteristics and environmental exposures works in disease occurrence.

HDL inhibits the effects of oxidized phospholipids on endothelial cell gene expression via multiple mechanisms.

Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phospholcholine (OxPAPC) and its component phospholipids accumulate in atherosclerotic lesions and regulate the expression of >1,000 genes, many proatherogenic, in human aortic endothelial cells (HAECs). In contrast, there is evidence in the literature that HDL protects the vasculature from inflammatory insult. We have previously shown that in HAECs, HDL attenuates the expression of several proatherogenic genes regulated by OxPAPC and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. We now demonstrate that HDL reverses >50% of the OxPAPC transcriptional response. Genes reversed by HDL are enriched for inflammatory and vascular development pathways, while genes not affected by HDL are enriched for oxidative stress response pathways. The protective effect of HDL is partially mimicked by cholesterol repletion and treatment with apoA1 but does not require signaling through scavenger receptor class B type I. Furthermore, our data demonstrate that HDL protection requires direct interaction with OxPAPC. HDL-associated platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes short-chain bioactive phospholipids in OxPAPC; however, inhibiting PAF-AH activity does not prevent HDL protection. Our results are consistent with HDL sequestering specific bioactive lipids in OxPAPC, thereby preventing their regulation of select target genes. Overall, this work implicates HDL as a major regulator of OxPAPC action in endothelial cells via multiple mechanisms.

Systemic inflammation, intestine, and paraoxonase-1.

Serum paraoxonase 1 (PON1) has been shown to act as an important guardian against cellular damage from oxidized lipids in low-density lipoprotein (LDL), plasma membrane, against toxic agents such as pesticide residues including organophosphates and against bacterial endotoxin. PON1 associated with circulating high-density lipoprotein (HDL) has the ability to prevent the generation of pro inflammatory oxidized phospholipids by reactive oxygen species. The activities of the HDL-associated PON1 and several other anti-inflammatory factors in HDL are in turn negatively regulated by these oxidized lipids. In rabbits, mice, and humans there appears to be an increase in the formation of these oxidized lipids during the acute phase response. This results in the association of acute phase proteins with HDL and inhibition of the HDL-associated PON1 that renders HDL pro inflammatory.In populations, low serum HDL-cholesterol is a risk factor for atherosclerosis and efforts are directed toward therapies to improve the quality and the relative concentrations of LDL and HDL. Apolipoprotein A-I (apoA-I) has been shown to reduce atherosclerotic lesions in laboratory animals. ApoA-I, however, is a large protein that is costly and needs to be administered parenterally. Our group has developed apoA-I mimetic peptides that are much smaller than apoA-I (18 amino acids long vs 243 in ApoA-I itself). These HDL mimetic peptides are much more effective in removing the oxidized phospholipids and other oxidized lipids. They improve LDL and HDL composition and function and reduce lesion formation in animal models of atherogenesis. Following is a brief description of some of the HDL mimetic peptides that can improve HDL and the effect of the peptide on PON1 activity.

Network for activation of human endothelial cells by oxidized phospholipids: a critical role of heme oxygenase 1.

RATIONALE: Oxidized palmitoyl arachidonyl phosphatidylcholine (Ox-PAPC) accumulates in atherosclerotic lesions, is proatherogenic, and influences the expression of more than 1000 genes in endothelial cells. OBJECTIVE: To elucidate the major pathways involved in Ox-PAPC action, we conducted a systems analysis of endothelial cell gene expression after exposure to Ox-PAPC. METHODS AND RESULTS: We used the variable responses of primary endothelial cells from 149 individuals exposed to Ox-PAPC to construct a network that consisted of 11 groups of genes, or modules. Modules were enriched for a broad range of Gene Ontology pathways, some of which have not been identified previously as major Ox-PAPC targets. Further validating our method of network construction, modules were consistent with relationships established by cell biology studies of Ox-PAPC effects on endothelial cells. This network provides novel hypotheses about molecular interactions, as well as candidate molecular regulators of inflammation and atherosclerosis. We validated several hypotheses based on network connections and genomic association. Our network analysis predicted that the hub gene CHAC1 (cation transport regulator homolog 1) was regulated by the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway, and here we showed that ATF4 directly activates an element in the CHAC1 promoter. We showed that variation in basal levels of heme oxygenase 1 (HMOX1) contribute to the response to Ox-PAPC, consistent with its position as a hub in our network. We also identified G-protein-coupled receptor 39 (GPR39) as a regulator of HMOX1 levels and showed that it modulates the promoter activity of HMOX1. We further showed that OKL38/OSGN1 (oxidative stress-induced growth inhibitor), the hub gene in the blue module, is a key regulator of both inflammatory and antiinflammatory molecules. CONCLUSIONS: Our systems genetics approach has provided a broad view of the pathways involved in the response of endothelial cells to Ox-PAPC and also identified novel regulatory mechanisms.

Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma.

The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.

The effect of HDL mimetic peptide 4F on PON1.

Several lines of evidence indicate that serum paraoxonase 1 (PON1) acts as an important guardian against cellular damage from oxidized lipids in plasma membrane, in low-density lipoprotein (LDL), against bacterial endotoxin and against toxic agents such as pesticide residues including organophosphates. In circulation, the high-density lipoprotein (HDL)-associated PON1 has the ability to prevent the formation of proinflammatory oxidized phospholipids. These oxidized phospholipids negatively regulate the activities of the HDL-associated PON1 and several other anti-inflammatory factors in HDL. During the acute phase response in rabbits, mice, and humans, there appears to be an increase in the formation of these oxidized lipids that results in the inhibition of the HDL-associated PON1 and an association of acute phase proteins with HDL that renders HDL proinflammatory. Low serum HDL is a risk factor for atherosclerosis and attempts are directed toward therapies to improve the quality and the relative concentrations of LDL and HDL. Apolipoprotein A-I (apoA-I) has been shown to reduce atherosclerotic lesions in laboratory animals. ApoA-I, however, is a large protein and needs to be administered parenterally, and it is costly. We have developed apoA-I mimetic peptides that are much smaller than apoA-I, and much more effective in removing the oxidized phospholipids and other oxidized lipids. These mimetic peptides improve LDL and HDL composition and function and reduce lesion formation in animal models of atherogenesis. Following is a brief description of some of the HDL mimetic peptides that can improve HDL and the effect of the peptide on PON1 activity.

Proatherogenic high-density lipoprotein, vascular inflammation, and mimetic peptides.

Atherosclerosis is an example of an inflammatory disorder. During the acute phase and under inflammatory conditions, high-density lipoprotein (HDL), which is normally anti-inflammatory, can become proinflammatory. Reactive oxygen species generated by several enzyme systems can modify phospholipids and sterols, producing oxidized phospholipids and oxidized sterols that reduce the capacity of HDL to protect against undesirable oxidative modifications of molecules. In animal models of dyslipidemia, diabetes, vascular inflammation, and chronic rejection, it is observed that reducing oxidative and inflammatory pressure will help HDL regain its protective role. One way to accomplish this is through the use of apolipoprotein A-I mimetic peptides, which remove oxidation products from lipoproteins and cell membranes, returning normal structure and function to low-density lipoprotein and HDL. These mimetic peptides markedly reduce atherosclerosis in animal models. Published studies of apolipoprotein mimetic peptides in models of inflammatory disorders other than atherosclerosis suggest that they have efficacy in a wide range of inflammatory conditions.

Endothelial NOTCH1 is suppressed by circulating lipids and antagonizes inflammation during atherosclerosis.

Although much progress has been made in identifying the mechanisms that trigger endothelial activation and inflammatory cell recruitment during atherosclerosis, less is known about the intrinsic pathways that counteract these events. Here we identified NOTCH1 as an antagonist of endothelial cell (EC) activation. NOTCH1 was constitutively expressed by adult arterial endothelium, but levels were significantly reduced by high-fat diet. Furthermore, treatment of human aortic ECs (HAECs) with inflammatory lipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [Ox-PAPC]) and proinflammatory cytokines (TNF and IL1ß) decreased Notch1 expression and signaling in vitro through a mechanism that requires STAT3 activation. Reduction of NOTCH1 in HAECs by siRNA, in the absence of inflammatory lipids or cytokines, increased inflammatory molecules and binding of monocytes. Conversely, some of the effects mediated by Ox-PAPC were reversed by increased NOTCH1 signaling, suggesting a link between lipid-mediated inflammation and Notch1. Interestingly, reduction of NOTCH1 by Ox-PAPC in HAECs was associated with a genetic variant previously correlated to high-density lipoprotein in a human genome-wide association study. Finally, endothelial Notch1 heterozygous mice showed higher diet-induced atherosclerosis. Based on these findings, we propose that reduction of endothelial NOTCH1 is a predisposing factor in the onset of vascular inflammation and initiation of atherosclerosis.

Chronic inflammatory disorders and accelerated atherosclerosis: chronic kidney disease.

Increasing evidence points to the fact that plasma HDL cholesterol levels do not always accurately predict HDL function including reverse cholesterol transport and modulation of inflammation. These functions appear to have evolved as part of our innate immune system. HDL is anti inflammatory in healthy individuals in the absence of systemic oxidative stress and inflammation. In those with chronic illnesses such as renal failure however, HDL may become dysfunctional and actually promote inflammation. HDL may be thought of as a shuttle whose size can be estimated by HDL cholesterol levels. The content of the shuttle however, is what determines the anti inflammatory potential of HDL and can change from one, supporting reverse cholesterol transport to one that is less efficient in carrying out this function. Chronic kidney disease (CKD), and inflammatory disorder, is associated with development of accelerated atherosclerosis and premature death from coronary artery disease (CAD). Patients with CKD present with dyslipidemia, oxidative stress and systemic inflammation. Among the abnormalities in lipid metabolism in these patients is reduced levels and protective capacity of HDL. Recent studies have shown that HDL from patients with end stage renal disease is not capable of preventing LDL oxidation and that it induces monocyte migration in artery wall model systems. Treatment of plasma from these patients, with an HDL mimetic peptide improved the anti inflammatory properties of patient's HDL and made LDL more resistant to oxidative modification. Animal models of kidney disease also had proinflammatory HDL and treatment with the peptide mimetic improved markers of inflammation and anti inflammatory capacity of HDL. Whether HDL mimetic peptides will have therapeutic benefit in patients with renal failure will have to be determined in clinical studies.