Unraveling liver complexity from molecular to organ level: challenges and perspectives.

Biological responses are determined by information processing at multiple and highly interconnected scales. Within a tissue the individual cells respond to extracellular stimuli by regulating intracellular signaling pathways that in turn determine cell fate decisions and influence the behavior of neighboring cells. As a consequence the cellular responses critically impact tissue composition and architecture. Understanding the regulation of these mechanisms at different scales is key to unravel the emergent properties of biological systems. In this perspective, a multidisciplinary approach combining experimental data with mathematical modeling is introduced. We report the approach applied within the Virtual Liver Network to analyze processes that regulate liver functions from single cell responses to the organ level using a number of examples. By facilitating interdisciplinary collaborations, the Virtual Liver Network studies liver regeneration and inflammatory processes as well as liver metabolic functions at multiple scales, and thus provides a suitable example to identify challenges and point out potential future application of multi-scale systems biology.

Clinical translation in the virtual liver network.

The liver is the central detoxifying organ, continuously removing xenobiotics from the vascular system. Given its role in drug metabolism, a functional understanding of liver physiology is crucial to optimizing drug efficacy and patient safety. The Virtual Liver Network (VLN), a German national flagship research program, focuses on producing validated computer models of human liver physiology. These models are used to analyze patient-derived data and thereby gain mechanistic insights in the processes underlying drug pharmacokinetics (PK).

Polymorphisms of the human matrix gla protein (MGP) gene, vascular calcification, and myocardial infarction.

The matrix Gla protein (MGP) is an important inhibitor of vessel and cartilage calcification that is strongly expressed in human calcified, atherosclerotic plaques and could modulate plaque calcification and coronary heart disease risk. Using a genetic approach, we explored this possibility by identifying polymorphisms of the MGP gene and testing their possible association with myocardial infarction (MI) and plaque calcification. Eight polymorphisms were identified in the coding and 5'-flanking sequences of the MGP gene. All polymorphisms were investigated in 607 patients with MI and 667 control subjects recruited into the ECTIM Study (Etude Cas-Témoins de l'Infarctus du Myocarde) and in 717 healthy individuals with echographically assessed arterial calcification and atherosclerosis who were participating in the AXA Study. In the ECTIM Study, alleles and genotypes were distributed similarly in patients and controls in the whole study group; in only 1 subgroup of subjects defined as being at low risk for MI were the concordant A-7 and Ala 83 alleles more frequent in patients with MI than in controls (P<0.003). In the AXA Study among subjects with femoral atherosclerosis, the same alleles were more common in the presence than the absence of plaque calcification (P<0.025). The other MGP polymorphisms were not associated with any investigated clinical phenotype. Transient transfection experiments with allelic promoter-reporter gene constructs and DNA-protein interaction assays were carried out to assess possible in vitro functionality of the promoter variants detected at positions -814, -138, and -7 relative to the start of transcription. When compared with the -138 T allele, the minor -138 C: allele consistently conferred a reduced promoter activity of -20% (P<0.0001) in rat vascular smooth muscle cells and of -50% (P<0.004) in a human fibroblast cell line, whereas the other polymorphisms, including -7, displayed no evidence of in vitro functionality. We conclude that the A-7 or Ala 83 alleles of the MGP gene may confer an increased risk of plaque calcification and MI; however, the observed relationships are weak or limited to subgroups of patients and therefore need confirmation.

Genetic diversity in the matrix metalloproteinase family. Effects on function and disease progression.

Atherosclerosis is an example of a complex trait, where the course of the disease is influenced by a combination of common variation in a constellation of genes and the effect of a wide range of environmental variables. Thus, the underlying disease mechanisms will be modulated by genetic diversity and the effect this diversity has on an individual's response to environmental challenges such as smoking, diet, and exercise. Unlike the consequences of mutations in severe single-gene disorders on protein function, the impact of individual common, functionally important sequence changes in genes contributing to multifactorial diseases is likely to be very small. The challenge is to dissect the contribution that each of these genes makes to the disease process. We have tackled this by identifying common genetic variants, studying their effects on function, and applying them to the analysis of association in appropriately structured and suitably powered studies. Even with our incomplete understanding of the disease, the list of potential candidate genes we could study is vast; but, we do know from pathological studies that a wide spectrum of structural architecture exists in atherosclerotic plaques, suggesting that remodeling of vascular connective tissue is fundamentally important. Matrix remodeling is controlled by a complex network of cell and matrix interactions, the net outcome of which is the product of a balance between synthetic and degradative processes. Our work has focused on the family of enzymes and inhibitors most directly associated with matrix turnover--the matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs, tissue inhibitors of MPs). We specifically searched for functionally relevant genetic variants that might modulate the delicate control of matrix turnover. Using these molecular genetic strategies to investigate the impact of natural genetic variation on vascular matrix remodeling has begun to shed new light on the importance of these genes in atherogenesis.

Allele-specific regulation of matrix metalloproteinase-12 gene activity is associated with coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease.

Both the processes of atherosclerosis and plaque rupture are indicated to be influenced by matrix metalloproteinase (MMP) activity. We therefore searched for common functional variation in the matrix metalloelastase (MMP-12) gene locus that may be implicated in coronary artery disease. Single-strand conformation polymorphism analysis of DNA from healthy individuals detected a common polymorphism within the MMP-12 gene promoter (an A-to-G substitution at position -82). The frequency of the G allele was 0. 19. The polymorphism influences the binding of the transcription factor activator protein-1 (AP-1) in electromobility shift assay. A higher binding affinity of AP-1 to the A allele was associated with higher MMP-12 promoter activity in vitro in transient transfection studies in U937 and murine lung macrophage (MALU) cells. Phorbol 12-myristate 13-acetate (PMA) and insulin, 2 known activators of AP-1, increased the binding of AP-1 to the MMP-12 promoter, with higher affinity for the A allele. In transfection experiments, both the A and the G alleles responded to insulin and PMA, the A allele showing higher promoter activity than the G allele. Furthermore, Western blot analysis demonstrated that insulin increased MMP-12 protein production. To analyze whether the -82 A/G polymorphism is associated with coronary artery disease, 367 consecutive patients who underwent percutaneous transluminal coronary angiography with stent implantation were genotyped. In patients (n=71) with diabetes, the A allele was associated with a smaller luminal diameter. In conclusion, a common functional polymorphism within the MMP-12 promoter influences coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease.

Genetic variation at the matrix metalloproteinase-9 locus on chromosome 20q12.2-13.1.

Allelic association methods are better suited than linkage analysis for mapping of susceptibility genes that confer modest increases in risk in complex diseases. In both family- and population-based association studies, it is very useful to have prior knowledge of all sequence variants and the degree of linkage disequilibrium in a candidate gene region. In this study, we scanned sequence variants in a 2.2-kb promoter sequence and all 13 exons (totalling 3.3 kb) of the matrix metalloproteinase-9 gene, which is associated with coronary heart disease and a candidate for other diseases involving connective tissue remodelling, such as cancer metastasis. The sequences had a total of ten variable sites, four in the promoter, five in the coding region (three of which alter the amino acid encoded) and one in the 3' untranslated sequence. Sequence inspection suggests that some of the variants will have a functional impact on either level of expression or enzymatic activity. Tight linkage disequilibrium was detected between variants across the entire length of the gene (approximately 9 kb), and frequencies of different haplotypes were determined. The data provide an essential tool for studies of the possible contribution of genetic variation at the matrix metalloproteinase-9 locus to genetically determined susceptibility to a number of important diseases. The results also provide experimental data on the extent of linkage disequilibrium in the general population, which is yet to be resolved.

Human stromelysin gene promoter activity is modulated by transcription factor ZBP-89.

Matrix metalloproteinase expression is under strict regulation in physiological conditions. Disruption of the regulatory mechanisms can lead to tissue destruction and is associated with tumour invasion and metastasis. Using the one-hybrid assay technique with a cis-element in the promoter region of the stromelysin (matrix metalloproteinase-3) gene, a cDNA encoding a transcription factor termed ZBP-89 was obtained. The interaction between ZBP-89 and the stromelysin promoter element was confirmed by electrophoretic mobility shift assays with a recombinant ZBP-89. Reporter gene expression under the control of the stromelysin promoter in transiently transfected cells was significantly increased when the cells were cotransfected with a ZBP-89 expression construct. These results indicate that ZBP-89 interacts with the stromelysin promoter and upregulates its activity. As ZBP-89 expression is known to be increased in gastric carcinoma cells, induction of stromelysin expression may be a significant factor in tumour metastasis.

Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis.

BACKGROUND: Gelatinase B, a matrix metalloproteinase that has proteolytic activity against connective tissue proteins, has been suggested to be important in the connective tissue remodeling processes associated with atherogenesis and plaque rupture. This study tested the hypothesis that sequence variation in the promoter region of the gelatinase B gene influences its expression, predisposing individuals carrying certain genetic variants to more severe atherosclerosis. METHODS AND RESULTS: Single-strand conformation polymorphism analysis was carried out to search the promoter region of the gene encoding gelatinase B for naturally occurring genetic variation. As a result, an unreported common polymorphism was detected, which arose from a cytosine (C) to thymidine (T) transition at position -1562 relative to the start of transcription. Transient transfection experiments and DNA-protein interaction assays indicated that the T allele had a higher promoter activity than the C allele, which appeared to be due to preferential binding of a putative transcription repressor protein to the C allelic promoter. A sample of 584 male patients with myocardial infarction and 645 age-matched male healthy control subjects were genotyped. The allele frequencies were not significantly different between the cases and control subjects. However, in 374 patients with available angiographic data, 26% of those carrying 1 or 2 copies of the T allele had >50% stenosis in 3 coronary arteries, whereas only 15% of C/C homozygotes had triple-vessel disease. CONCLUSIONS: These data suggest that this functional genetic variation influences gelatinase B gene promoter activity in an allele-specific manner and has an effect on atherosclerotic phenotype.

Effect of the stromelysin-1 promoter on efficacy of pravastatin in coronary atherosclerosis and restenosis.

It has proved difficult to identify high-risk patients for atherosclerosis and to determine how they might respond to medication. Recently, a common promoter variant of the human stromelysin-1 gene has been reported, which has been shown to affect the transcription. We investigated whether this polymorphism had any impact on the risk of events, especially restenosis and progression of coronary artery disease and whether the effect was modulated by treatment with pravastatin. The stromelysin-1 genotype was determined for 496 men with coronary artery disease and cholesterol levels between 4.0 and 8.0 mmol/L, participating in the Regression Growth Evaluation Statin Study (REGRESS) study, a clinical trial assessing the effect of the lipid-lowering drug pravastatin on the progression of atherosclerosis. Patients in the placebo group with 5A6A or 6A6A genotypes had more clinical events than patients with the 5A5A genotype (26% and 12%, respectively, p = 0.03). In the pravastatin group, the risk of clinical events in patients with 5A6A or 6A6A genotypes was lower, compared with placebo, whereas it was unchanged in those with a 5A5A genotype (p value for interaction: 0.038). Also, the incidence of repeat angioplasty in the placebo group was greater in patients with the 6A6A or 5A6A genotypes, compared with 5A homozygotes (38% and 40%, respectively, vs 11%, p = 0.09). Again, treatment substantially reduced the incidence in heterozygotes and 6A homozygotes (0% and 15%, respectively), whereas it was unchanged in 5A homozygotes (28%, p for interaction: 0.002). These effects were independent of the effects of pravastatin on the lipid levels. Thus, this study suggests that the stomelysin-1 promoter polymorphism confers a genotype-specific response to medication in determining clinical event-free survival and the risk for symptom-driven repeat angioplasty. This variant may therefore act as a predictor, not only of disease progression, but also of response to therapy and risk of restenosis.

Detection of mutations and DNA polymorphisms in genes involved in cardiovascular diseases by polymerase chain reaction-single-strand conformation polymorphism analysis.

Over the last 15 years, there has been remarkably rapid progress in defining the molecular basis of inherited disorders. Many disease genes (the majority of which are genes responsible for monogenic Mendelian diseases) have now been identified, predominately through linkage analysis and positional cloning approaches. With the continuing expansion in this research area, the number of genes to be screened for disease-causing mutations will continue to increase, especially as there are now worldwide efforts aiming to identify the gene lesions that contribute to complex diseases, such as hypertension, diabetes mellitus, and coronary artery diseases, each of which involves many susceptibility genes.

Matrix metalloproteinases: implication in vascular matrix remodelling during atherogenesis.

1. The matrix metalloproteinases are a family of at least 16 zinc-dependent endopeptidases possessing catalytic activity against extracellular matrix components. Some members of this family have been implicated in vascular matrix remodelling in the pathogenesis of atherosclerosis. 2. A common, naturally occurring variant has been identified in the promoter of the stromelysin gene with one allele having a run of five adenosines (5A) and the other having six adenosines (6A). Functional analyses have shown that the 6A allele has a lower promoter activity than the 5A allele, which is probably attributable to preferential binding of a putative transcriptional repressor protein. 3. In patients with coronary artery disease, the 6A allele has been found to be associated with progression of atherosclerosis assessed by sequential quantitative angiography. 4. In conclusion, the matrix metalloproteinases may be over-expressed in certain locations in atherosclerotic plaques, which might contribute to local destruction of connective tissue and thus plaque rupture. In the majority of lesional areas, however, matrix synthesis is likely to outstrip matrix degradation, because matrix accumulation is a major feature of most atheromas. This imbalance favouring matrix deposition is likely to be exacerbated in individuals with the 6A6A genotype in whom stromelysin expression is lower due to the weaker stromelysin promoter.

An association between arterial pulse pressure and variation in the fibrillin-1 gene.

OBJECTIVE: To investigate whether variation in the fibrillin-1 gene was associated with blood pressure in healthy middle aged men, as had been observed in patients with abdominal aortic aneurysm. DESIGN, SETTING, AND PATIENTS: Middle aged men (n = 245), aged 50 to 61 years, were recruited from one of the nine general practices participating in the second Northwick Park heart study. Blood samples were obtained for the preparation of genomic DNA and analysis of plasma variables. MAIN OUTCOME MEASURES: Systolic, diastolic, and pulse pressures; fibrillin-1 genotype characterised with a four allele variable tandem nucleotide repeat polymorphism in intron 28. RESULTS: In healthy middle aged men only three common genotypes were observed: 2-2 (frequency 54.1%), 2-3 (16%) and 2-4 (15%). The mean arterial systolic (and pulse) pressure varied according to fibrillin-1 genotype: 2-4 genotype, 126-3 (47.6) mm Hg; 2-2 genotype, 131.0 (51.3) mm Hg; and 2-3 genotype, 135.5 (54.2) mm Hg. The median pulse pressure was 50 mm Hg. Distribution of men around the median pulse pressure, according to genotype, showed a significant trend for patients of 2-4 genotype to have the lowest pulse pressures, those of 2-2 genotype to have intermediate pressures, and those of 2-3 genotype to have the highest pulse pressures (p = 0.003 for healthy men). CONCLUSIONS: There appears to be a significant association between fibrillin-1 genotype and arterial pulse pressure in men aged 50 to 61 years.

Expression of matrix metalloproteinases and their inhibitor TIMP-1 in the rat carotid artery after balloon injury.

The temporal relationship of matrix metalloproteinases (MMPs) and a specific tissue inhibitor (TIMP-1) has been examined by reverse transcription-polymerase chain reaction and substrate zymography, after balloon catheter angioplasty of the rat carotid artery. The contralateral uninjured carotid artery was used as a comparative control. Of the MMPs examined, only MMP-2 (72-kDa gelatinase) was produced constitutively by normal uninjured arteries. After injury, MMP-2 mRNA levels fell compared with the uninjured arteries; by 24 hours, levels had increased 2-fold. Zymography showed that the inactive form of MMP-2 predominated in uninjured vessels, but after injury, the level of the active form was increased. MMP-9 (92-kDa gelatinase) mRNA levels and activity peaked at 6 hours after injury and were still detectable at 7 days. MMP-3 (stromelysin) expression was detectable at low levels as early as 2 hours after injury and showed an approximate 2-fold increase of expression at 7 days. The presence of the active protein paralleled the mRNA expression. The inhibitor TIMP-1 mRNA was first detected 6 hours after injury and showed a marked peak of expression at 24 hours; however, no expression was detected by 7 days. The presence of a constitutively expressed, low molecular weight caseinolytic enzyme (27 kDa) was observed, and the induction of a caseinolytic enzyme (30 kDa) was noted that was induced as early as 2 hours after injury, peaked at 6 hours, and was barely detectable by 7 days. These results demonstrate that the process of extracellular matrix breakdown by MMPs after balloon catheter-induced injury is controlled by a tightly regulated temporal response by the genes responsible for the production of these enzymes and their inhibitor and by post-translational activation of the proenzymes.

Interaction between fibrillin genotype and blood pressure and the development of aneurysmal disease.

Debate as to whether abdominal aortic aneurysms (AAA) are caused by atherosclerosis or whether they have a strong genetic etiology continues. We have investigated the hypothesis that risk factors are likely to be strongest in patients with generalized aneurysmal disease. We screened 232 consecutive AAA patients for popliteal aneurysm and investigated cardiovascular and genetic risk factors in these patients. Ultrasonography demonstrated the presence of a popliteal aneurysm in 24 of 232 (10%) patients. Multivariate analysis identified four independent factors associated with popliteal aneurysm: age (p = 0.013), height (p = 0.017), triglyceride concentration (p = 0.009), and systolic blood pressure (p = 0.037). In the AAA patients a significant association of fibrillin-1 genotype was present, determined by a tandem repeat polymorphism, with both systolic and pulse pressure. The genotypes associated with the highest pressures were significantly more common among the patients with popliteal aneurysm, p = 0.03. Following these findings we investigated whether there was an association between fibrillin-1 genotype and blood pressure in a healthy population, 245 men aged 50-61 years. Again we found a significant association between fibrillin genotype and pulse pressure, p = 0.003. We suggest that a strong interaction occurs between fibrillin genotype and blood pressure which contributes to the development of aneurysmal disease.

Unravelling the familial tendency to aneurysmal disease: popliteal aneurysm, hypertension and fibrillin genotype.

PURPOSE: To screen patients with abdominal aortic aneurysm for popliteal aneurysm and investigate cardiovascular and genetic risk factors associated with aneurysmal disease at more than one site (generalised aneurysmal disease). SUBJECTS, DESIGN AND SETTING: All patients referred to the Regional Vascular Surgical Service at Charing Cross Hospital with unruptured abdominal aortic aneurysm between 1989 and 1993 were screened for popliteal aneurysms, using ultrasonography. MAIN OUTCOME MEASURES: Palpation of a popliteal aneurysm or ultrasonographic detection of popliteal dilatation, where the ratio maximum popliteal fossa diameter/suprageniculate popliteal diameter was > or = 1.5, in relation to cardiovascular and genetic risk factors. RESULTS: Clinical examination detected popliteal aneurysms in only 11/232 patients (5%), but ultrasonography demonstrated the presence of popliteal aneurysm in a further 13 patients, 24/232 in total (10%). Multivariate regression identified four independent factors associated with popliteal dilatation disease: age (p = 0.046), height (p = 0.006), systolic hypertension (p = 0.037) and triglyceride concentration (p = 0.009). Generalised aneurysmal disease and systolic blood pressure were associated with polymorphic variation in the fibrillin-1 gene, but not with variations in the apolipoprotein B and type III collagen genes. CONCLUSIONS: Few patients with abdominal aortic aneurysm (10%) also have popliteal aneurysms: the risk of popliteal dilatation increases with age, height, systolic blood pressure, triglyceride concentration and fibrillin genotype. The strong interaction between fibrillin genotype and blood pressure may contribute to the familial tendency to aortic aneurysm.

Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression.

There is a common polymorphism in the promoter sequence of the human stromelysin-1 gene, with one allele having a run of six adenosines (6A) and the other five adenosines (5A). We have previously reported, in a 3-year follow-up study of patients with coronary atherosclerosis, that those patients who are homozygous for the 6A allele show a more rapid progression of the disease. In this study, we have investigated whether the 5A/6A promoter polymorphism plays a role in the regulation of stromelysin-1 gene expression. In transient transfection experiments, a stromelysin-1 promoter construct with 6A at the polymorphic site was found to express less of the chloramphenicol acetyltransferase reporter gene than a construct containing 5A. Electrophoretic mobility shift assay and DNase I footprinting revealed the interaction of one or more nuclear protein(s) with the DNA sequence at the 5A/6A polymorphic site. The binding of one of the nucleoprotein factors was more readily detectable with an oligonucleotide probe corresponding to the 6A allele as compared with a probe corresponding to the 5A allele. Replacing the core binding sequence with a random DNA sequence abolished the interaction between the nuclear protein(s) and the probe and also increased reporter gene expression in transiently transfected cells. Thus, the common 5A/6A polymorphism of the human stromelysin-1 promoter appears to play an important role in regulating stromelysin-1 gene expression and may be involved in the progression of coronary heart disease.

Preliminary report: genetic variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis.

Stromelysin is a member of the family of metalloproteinases that degrade extracellular matrix. In situ hybridisation and histopathological studies suggest that stromelysin activity may be important in the connective tissue remodelling processes associated with atherogenesis and plaque rupture. Single strand conformation polymorphism analysis identified a common polymorphism in the stromelysin gene promoter located 1171 bp upstream from the start of transcription in which one allele has a run of six adenosines (6A) and another has five adenosines (5A). 72 men with coronary heart disease, were genotyped. They were participants in the St Thomas' Atherosclerosis Regression Study who were randomised to receive usual care (UC), dietary intervention (D), or diet plus cholestyramine (DC), with angiography at baseline and at 39 months. In these patients the frequency of the 5A allele was 0.49 (95% CI from 0.41 to 0.57) and was not significantly different from that in a sample of 354 healthy UK men. In the UC group, patients who were homozygous for the 6A allele showed greater progression of angiographic disease than those with other genotypes: the minimum absolute width of coronary segments decreased by 0.04 (SEM 0.10) mm for 5A5A, 0.20 (0.07) mm for 5A6A, and 0.67 (0.19) mm for 6A6A (P < 0.01). The findings were similar but slightly less significant for the change in mean absolute width of coronary segments (P < 0.05). No significant associations were seen in patients in the D or DC groups. In data pooled from the three treatment groups, the 6A6A genotype was significantly associated with greater progression of coronary atherosclerosis than other genotypes in patients with baseline percentage diameter stenosis less than 20% (P < 0.05), but not in those with baseline percentage diameter stenosis greater than or equal to 20%. These results provide the first evidence of a link between genetic variation in stromelysin and progression of coronary atherosclerosis and support the hypothesis that connective tissue remodeling mediated by metalloproteinases contribute to the pathogenesis of atherosclerosis.