A vascular biology network model focused on inflammatory processes to investigate atherogenesis and plaque instability.

BACKGROUND: Numerous inflammation-related pathways have been shown to play important roles in atherogenesis. Rapid and efficient assessment of the relative influence of each of those pathways is a challenge in the era of "omics" data generation. The aim of the present work was to develop a network model of inflammation-related molecular pathways underlying vascular disease to assess the degree of translatability of preclinical molecular data to the human clinical setting. METHODS: We constructed and evaluated the Vascular Inflammatory Processes Network (V-IPN), a model representing a collection of vascular processes modulated by inflammatory stimuli that lead to the development of atherosclerosis. RESULTS: Utilizing the V-IPN as a platform for biological discovery, we have identified key vascular processes and mechanisms captured by gene expression profiling data from four independent datasets from human endothelial cells (ECs) and human and murine intact vessels. Primary ECs in culture from multiple donors revealed a richer mapping of mechanisms identified by the V-IPN compared to an immortalized EC line. Furthermore, an evaluation of gene expression datasets from aortas of old ApoE-/- mice (78 weeks) and human coronary arteries with advanced atherosclerotic lesions identified significant commonalities in the two species, as well as several mechanisms specific to human arteries that are consistent with the development of unstable atherosclerotic plaques. CONCLUSIONS: We have generated a new biological network model of atherogenic processes that demonstrates the power of network analysis to advance integrative, systems biology-based knowledge of cross-species translatability, plaque development and potential mechanisms leading to plaque instability.

Mechanism of an indirect effect of aqueous cigarette smoke extract on the adhesion of monocytic cells to endothelial cells in an in vitro assay revealed by transcriptomics analysis.

The adhesion of monocytic cells to the "dysfunctional" endothelium constitutes a critical step in the initiation of atherosclerosis. Cigarette smoke (CS) has been shown to contribute to this process, the complex mechanism of which still needs to be unraveled. We developed an in vitro adhesion assay to investigate the CS-induced adhesion of monocytic MM6 cells to human umbilical vein endothelial cells (HUVECs) following exposure to an aqueous CS extract (smoke-bubbled phosphate buffered saline: sbPBS), reasoning that in vivo monocytes and endothelial cells are exposed primarily to soluble constituents from inhaled CS absorbed through the lung alveolar wall. MM6 cell adhesion was increased exclusively by the conditioned medium from sbPBS-exposed MM6 cells, not by direct sbPBS exposure of the HUVECs within a range of sbPBS doses. Using a transcriptomics approach followed by confirmation experiments, we identified different exposure effects on both cell types and a key mechanism by which sbPBS promoted the adhesion of MM6 cells to HUVECs. While sbPBS provoked a strong oxidative stress response in both cell types, the expression of E-selectin, VCAM-1 and ICAM-1, responsible for the adhesion of MM6 cells to HUVECs, was induced in the latter through a proinflammatory paracrine effect. We confirmed that this effect was driven mainly by TNFα produced by MM6 cells exposed to sbPBS. In conclusion, we have elucidated an indirect mechanism by which sbPBS increases the adhesion of monocytic cells to endothelial cells in this in vitro assay that was designed for tobacco product risk assessment while mimicking the in vivo exposure conditions as closely as possible.

Cigarette-smoke-induced atherogenic lipid profiles in plasma and vascular tissue of apolipoprotein E-deficient mice are attenuated by smoking cessation.

Tobacco smoke exerts perturbations on lipid metabolism and arterial cell function that accelerate atherosclerosis. Lipidomics has emerged as a key technology in helping to elucidate the lipid-related mechanisms of atherosclerosis. In this study, we investigated the effects of smoking cessation on plaque development and aortic arch content of various lipid molecular classes and species. Apolipoprotein E-deficient mice were exposed to fresh air (sham) or to mainstream cigarette smoke (CS) for 6 months, or to CS for 3 months followed by sham for 3 months (cessation group). Lipids from plasma and aortic arches, plasma lipoprotein profiles and plaque morphometry measurements were analyzed. We already showed that CS exposure accelerated plaque size and total cholesterol content of the aortic arch at 3 and 6 months. Marked increases were seen in the relative enrichment of cholesteryl esters, phospholipids, sphingomyelins, and glycosphingolipids. Smoking cessation slowed plaque progression and resulted in lower levels of many lipid species in plasma and aortic arch. While CS exposure promoted rapid lipid accumulation in mouse aorta, smoking cessation translated into a slow removal of lipids from the vessel wall. Despite the smoking cessation-dependent metabolic changes leading to increased animal body weight, accumulation of proatherogenic lipids in the vessel was halted after exposure cessation, indicating that the clinical benefits of smoking cessation translate directly to the vessel wall and its lipid makeup.

Evidence for an alternative genomic structure, mRNA and protein sequence of human ABCA13.

ABC transporters form one of the major families of transport proteins. In humans, the ABC family comprises seven subfamilies named A to G, of which the A subfamily contains twelve members. Among these are several well-characterized transporters, including ABCA1, which is involved in cellular cholesterol transport and HDL formation, and ABCA4, which is a transporter for vitamin A derivatives in photoreceptor cells. The function of another subfamily member termed ABCA13 is unknown. The human ABCA13 gene has been reported to span 450kb of genomic DNA at chromosomal locus 7p12.3 and to encode a 5058 amino acid protein that includes two unusually large exons close to the N-terminus. We now show that the gene as well as the corresponding mRNA and protein may be considerably shorter than previously thought. We used PCR and RACE to identify a genomic sequence spanning about 350kb and encoding a protein of 2323 amino acids. This corresponds to the C-terminal half of the previously reported ABCA13 protein but lacks the residues reportedly encoded by the two very big N-terminal exons. Using immunoprecipitation and Western blot analyses we identified a protein of about 260kDa in size likely representing the shorter protein proposed here. Computer analyses showed that our proposed sequence contains all the structural elements of an ABCA protein and agrees well with the mouse ABCA13 protein sequence. Additionally, we identified a putative promoter region containing well-conserved TATA and CAAT boxes just upstream of our transcription start site. Overall, our data provide good evidence for an alternative human ABCA13 transcript and protein.

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase.

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions ((·)O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.

Industrial methodology for process verification in research (IMPROVER): toward systems biology verification.

MOTIVATION: Analyses and algorithmic predictions based on high-throughput data are essential for the success of systems biology in academic and industrial settings. Organizations, such as companies and academic consortia, conduct large multi-year scientific studies that entail the collection and analysis of thousands of individual experiments, often over many physical sites and with internal and outsourced components. To extract maximum value, the interested parties need to verify the accuracy and reproducibility of data and methods before the initiation of such large multi-year studies. However, systematic and well-established verification procedures do not exist for automated collection and analysis workflows in systems biology which could lead to inaccurate conclusions. RESULTS: We present here, a review of the current state of systems biology verification and a detailed methodology to address its shortcomings. This methodology named 'Industrial Methodology for Process Verification in Research' or IMPROVER, consists on evaluating a research program by dividing a workflow into smaller building blocks that are individually verified. The verification of each building block can be done internally by members of the research program or externally by 'crowd-sourcing' to an interested community. www.sbvimprover.com IMPLEMENTATION: This methodology could become the preferred choice to verify systems biology research workflows that are becoming increasingly complex and sophisticated in industrial and academic settings.

Cigarette smoke enhances abdominal aortic aneurysm formation in angiotensin II-treated apolipoprotein E-deficient mice.

Cigarette smoke, hyperlipidemia, and hypertension with the risk of development and progression of atherosclerosis and associated pathologies such as abdominal aortic aneurysm (AAA) are correlated. We examined the interaction of cigarette mainstream smoke (MS) and angiotensin-II (Ang II)-induced hypertension in the atherosclerotic process using hyperlipidemic apolipoprotein E-knockout (ApoE(-/-)) mice. ApoE(-/-) mice were treated with Ang II for 4 weeks and then further exposed to MS or to fresh air for 4 weeks. AAA formation was observed in all mice treated with Ang II, regardless of smoke exposure; however, smoke exposure increased the incidence of AAA in these mice. Ang II treatment resulted in higher gene expression of matrix metalloproteinases (MMP)-2, -3, -8, -9, and -12 in the abdominal aortas, which was further increased by MS exposure. The proteolytic activity of MMP-2 and MMP-9 was also enhanced in Ang II-treated mice exposed to MS, but only minor changes were seen with either smoke exposure or Ang II treatment alone. This study shows for the first time that both formation and severity of AAA in hypertensive ApoE(-/-) mice are accelerated by exposure to MS and that the proteolytic activity of MMPs is enhanced by the combination of Ang II and MS.

Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity.

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

Cloning, genomic organization, and tissue-specific expression of the RASL11B gene.

RASL11B is a member of the small GTPase protein family with a high degree of similarity to RAS proteins. Cloning of RASL11B mRNA and in silico analyses revealed that the human RASL11B gene spans about 4.5 kb and comprises four exons on chromosomal locus 4q12. The proximal 5'-flanking region of the gene lacks a TATA box but is GC-rich and contains a CCAAT box and several Sp1 sites. Consistent with this, the RASL11B gene was found to be expressed in all tissues investigated, with highest levels in placenta and in primary macrophages. The predicted RASL11B protein has no typical prenylation signal, indicating that it is probably not anchored to cellular membranes. RASL11B was induced during maturation of THP-1 monocytic cells into macrophage-like cells and in coronary artery smooth muscle cells after treatment with TGF-beta1. These results indicate that RASL11B may play a role in TGF-beta1-mediated developmental processes and in pathophysiologies such as inflammation, cancer, and arteriosclerosis.

Cloning, cellular localization, genomic organization, and tissue-specific expression of the TGFbeta1-inducible SMAP-5 gene.

SMAP-5 is a member of the five-pass transmembrane protein family localizing in the Golgi apparatus and the endoplasmic reticulum. These proteins have been implicated in intracellular trafficking, in secretion and in vesicular transport. Phylogenetic analyses revealed that SMAP-5 is a member of a small Rab GTPase interacting factor protein family. The human SMAP-5 gene spans about 12.5 kb and comprises 6 exons on chromosomal locus 5q32. The proximal 5'-flanking region of the gene lacks a TATA box and is highly GC rich. Consistent with this, the SMAP-5 gene is expressed in all tissues. The highest level of expression was found in coronary smooth muscle cells, in which expression of the SMAP-5 gene was induced by transforming growth factor beta1, thus indicating that this protein may play an important role in inflammation.

Laser microdissection-based analysis of mRNA expression in human coronary arteries with intimal thickening.

Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed by ISH and protein expression was detected by IHC. LMD plus RT-PCR found similar levels of SRF mRNA in intimal and medial SMCs, while medial mRNA levels for calponin-1 and smoothelin were higher. ISH confirmed that smoothelin mRNA levels in media exceeded those in intima, whereas SRF mRNA levels were similar at both sites. For calponin-1 and smoothelin, protein levels mirrored respective mRNA levels. By contrast, more medial than intimal SRF protein was present. Our results indicate that intimal SMCs exhibit a largely synthetic phenotype, perhaps reflecting lower intimal levels of SRF protein; ISH and LMD plus real-time RT-PCR provide comparable results; as a valuable alternative to ISH, LMD plus RT-PCR allows parallel measurement of several transcripts; and tissue gene expression studies must measure both protein and mRNA levels.

Proinflammatory cytokines regulate LOX-1 expression in vascular smooth muscle cells.

OBJECTIVE: Atherogenesis represents a type of chronic inflammation and involves elements of the immune response, eg, the expression of proinflammatory cytokines. In advanced atherosclerotic lesions, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs). In vitro, the expression of LOX-1 is induced by inflammatory cytokines like TNF-alpha and transforming growth factor (TGF)-beta. Therefore, LOX-1 is thought to be upregulated locally in response to cytokines in vivo. METHODS AND RESULTS: We determined by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis whether the mediators of the acute phase response in inflammation, IL-1alpha, IL-1beta, and TNF-alpha, regulate LOX-1 expression in cultured SMC, and whether this regulation is influenced by peroxisome proliferator-activated receptor gamma (PPARgamma). We studied by immunohistochemistry whether these cytokines are spatially correlated with LOX-1 expression in advanced atherosclerotic lesions. We found upregulation of LOX-1 expression in SMC in a dose- and time-dependent manner after incubation with IL-1alpha, IL-1beta, and TNF-alpha. Simultaneous incubation with these cytokines at saturated concentrations had an additive effect on LOX-1 expression. The PPARgamma activator, 15d-PGJ(2), however, inhibited IL-1beta-induced upregulation of LOX-1. In the intima of atherosclerotic lesions regions of IL-1alpha, IL-1beta, and TNF-alpha expression corresponded to regions of LOX-1 expression. CONCLUSIONS: We suppose that upregulated LOX-1 expression in SMC of advanced atherosclerotic lesions is a response to these proinflammatory cytokines. Moreover, the proinflammatory effects of these cytokines can be decreased by the antiinflammatory effect of PPARgamma.