Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm.

Peroxisome proliferator-activated receptor x03B3; agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor x03B3; agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation.

Investigating the Effect of a Single Infusion of Reconstituted High-Density Lipoprotein in Patients with Symptomatic Carotid Plaques.

BACKGROUND: Elevation of plasma high-density lipoprotein (HDL) cholesterol concentration reduces cardiovascular mortality and morbidity. HDLs have been shown to possess acute anti-inflammatory, antioxidant, and antithrombotic properties. We hypothesize that HDL therapy can acutely alter local and systemic manifestations of plaque instability. METHODS: Forty patients with early symptomatic carotid disease were randomized to either receive reconstituted HDL (rHDL) 40 mg/kg (n = 20) or placebo (n = 20). Carotid endarterectomies were performed 24 hr later. Plaques were obtained intraoperatively and used for measurement of thrombomodulatory genes expression. Plasma samples were collected before the infusion, 24 and 48 hr later to measure changes in systemic markers of plaque instability. RESULTS: No significant differences were noted in thrombomodulatory genes expression between the 2 groups. Systemic levels of tissue factor, matrix metalloproteinase 9 (MMP-9), and monocyte chemotactic factor-1 (MCP-1) were significantly reduced in the rHDL group. However, the effects on MMP-9 and MCP-1 were abolished in the immediate postoperative period. Although rHDL did not affect plasma interleukin-6 levels 24 hr following the infusion, it prevented the significant postoperative elevation seen in the placebo group. CONCLUSIONS: A single infusion of rHDL can acutely alter plasma biomarkers associated with plaque instability and cardiovascular morbidity.

XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation.

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1α (IRE1α)-dependent manner. Knockdown of XBP1 or IRE1α by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.

Histone deacetylase 3 unconventional splicing mediates endothelial-to-mesenchymal transition through transforming growth factor ß2.

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -ß, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor ß2 (TGFß2) secretion and cleavage. TGFß2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFß2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.

Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.

BACKGROUND: Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. METHODS AND RESULTS: We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3ß phosphorylation, ß-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. CONCLUSIONS: These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis.

Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1.

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 × 10(-5)) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 × 10(-5)). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10(-10), odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression.

Elevation of plasma high-density lipoproteins inhibits development of experimental abdominal aortic aneurysms.

OBJECTIVE: Patients with abdominal aortic aneurysms have lower concentrations of high-density lipoproteins (HDLs), leading us to investigate whether increasing plasma HDLs could influence aneurysm formation. METHODS AND RESULTS: Using the angiotensin II-induced hypercholesterolemic and the CaCl(2)-induced normocholesterolemic mouse model of AAA, we investigated the hypothesis that elevation of HDLs inhibits AAA. HDLs elevated before or at the time of AAA induction reduced AAA formation in both models but had no effect on early ruptures. Analysis of protein lysates from specific aortic segments demonstrated site-specific effects of HDLs on early signal transduction and cellular attrition. We found that HDLs reduced extracellular signal related kinases 1/2 activation in the suprarenal segment, while having no effect on p38 mitogen-associated protein kinase activation in any aortic segment and inhibiting c-Jun N-terminal kinase activation in all aortic segments. In addition, HDL elevation inhibited angiotensin II-induced apoptosis while inducing autophagy in the suprarenal segment of the aorta. Using Illumina gene array profiling we investigated the ability of HDL to modulate basal suprarenal aortic gene expression. CONCLUSIONS: Increasing plasma HDLs inhibit experimental AAA formation, independent of hypercholesterolemia via reduced extracellular signal related kinases 1/2 activation and alteration of the balance of cellular attrition. HDLs modulate genes involved in matrix remodelling, cell migration, and proliferation.

Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3.

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-γ (IFN-γ) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-γ-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-γ also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.

Downregulation of Kv7.4 channel activity in primary and secondary hypertension.

BACKGROUND: Voltage-gated potassium (K(+)) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of 3 structurally different activators of Kv7.2 through Kv7.5 channels (BMS-204352, S-1, and retigabine) on blood vessels from normotensive and hypertensive animals. METHODS AND RESULTS: Precontracted thoracic aorta and mesenteric artery segments from normotensive rats were relaxed by all 3 Kv7 activators, with potencies of BMS-204352=S-1>retigabine. We also tested these agents in the coronary circulation using the Langendorff heart preparation. BMS-204352 and S-1 dose dependently increased coronary perfusion at concentrations between 0.1 and 10 µmol/L, whereas retigabine was effective at 1 to 10 µmol/L. In addition, S-1 increased K(+) currents in isolated mesenteric artery myocytes. The ability of these agents to relax precontracted vessels, increase coronary flow, or augment K(+) currents was impaired considerably in tissues isolated from spontaneously hypertensive rats (SHRs). Of the 5 KCNQ genes, only the expression of KCNQ4 was reduced (≈3.7 fold) in SHRs aorta. Kv7.4 protein levels were ≈50% lower in aortas and mesenteric arteries from spontaneously hypertensive rats compared with normotensive vessels. A similar attenuated response to S-1 and decreased Kv7.4 were observed in mesenteric arteries from mice made hypertensive by angiotensin II infusion compared with normotensive controls. CONCLUSIONS: In 2 different rat and mouse models of hypertension, the functional impact of Kv7 channels was dramatically downregulated.

Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin.

RATIONALE: Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function. OBJECTIVE: This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism. METHODS AND RESULTS: Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of beta-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G(1) phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other beta-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased beta-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced beta-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to beta-catenin and forms a complex with 14-3-3 epsilon, zeta, and eta proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLCgamma-IP3K (phospholipase Cgamma-inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with beta-catenin, disrupting the complex and releasing beta-catenin to translocate into the nucleus. CONCLUSIONS: These findings demonstrate that HDAC7 interacts with beta-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth.

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow.

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE(-/-) mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE(-/-) mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.

Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow.

BACKGROUND: Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development. METHODS AND RESULTS: En face staining of aortas from apolipoprotein E-knockout mice revealed increased expression of HDAC3, specifically in these branching areas in vivo, whereas rapid upregulation of HDAC3 protein was observed in endothelial cells exposed to disturbed flow in vitro. Interestingly, phosphorylation of HDAC3 at serine/threonine was observed in these cells, suggesting that disturbed flow leads to posttranscriptional modification and stabilization of the HDAC3 protein. Coimmunoprecipitation experiments showed that HDAC3 and Akt form a complex. Using a series of constructs harboring deletions, we found residues 136 to 206 of HDAC3 to be crucial in this interaction. Enforced expression of HDAC3 resulted in increased phosphorylation of Akt and upregulation of its kinase activity. In line with these findings, knockdown of HDAC3 with lentiviral vectors (shHDAC3) led to a dramatic decrease in cell survival accompanied by apoptosis in endothelial cells. In aortic isografts of apolipoprotein E-knockout mice treated with shHDAC3, a robust atherosclerotic lesion was formed. Surprisingly, 3 of the 8 mice that received shHDAC3-infected grafts died within 2 days after the operation. Miller staining of the isografts revealed disruption of the basement membrane and rupture of the vessel. CONCLUSIONS: Our findings demonstrated that HDAC3 serves as an essential prosurvival molecule with a critical role in maintaining the endothelial integrity via Akt activation and that severe atherosclerosis and vessel rupture in isografted vessels of apolipoprotein E-knockout mice occur when HDAC3 is knocked down.

Comparative proteomics reveals a systemic vulnerability in the vasculature of patients with abdominal aortic aneurysms.

INTRODUCTION: Abdominal aortic aneurysms (AAA) are associated with inflammation, apoptosis, and matrix degradation. AAA tissue represents the end stage of disease, limiting its utility in identification of factors culpable for initiation of aneurysm development. Recent evidence suggests that AAAs are a local representation of a systemic disease of the vasculature. Morphologic and molecular changes, comparable to those found in the aneurysm wall, have been demonstrated in veins from patients with AAAs. Changes in the vascular tissue proteome of patients with AAAs were investigated, using inferior mesenteric vein (IMV), to gain insight into early molecular changes contributing to AAA development. METHODS: IMV was harvested from 16 patients with AAA and 16 matched controls. Whole IMV lysates were subjected to 2-D difference in gel electrophoresis (2D-DIGE) with quantitative densitometry. Protein spots differentially expressed in AAA were identified using mass spectrometry. Differential protein expression was validated by Western blotting and localized to cell type by immunohistochemistry (IHC). RESULTS: Decreased levels of prohibitin (AAA, 2.00 ± 1.37; controls, 3.81 ± 1.39; 1.9-fold change; P = .02) AAA (7.33 ± 3.9; controls, 14.5 ± 5.6; 2-fold change; P = .001), along with relative increases in a cleaved fragment of vimentin (AAA, 12.9 ± 9; controls, 6.9 ± 4.7; 2-fold change; P = .11) were identified in AAA patients. All proteins were localized to the vascular smooth muscle cells. CONCLUSIONS: Proteins important in combating the injurious effects of oxidative stress and modulating the response to inflammation appear reduced in the vasculature of patients with AAA. These changes may represent early events in AAA formation. Enhancing expression of these proteins might offer a novel therapeutic avenue to inhibit AAA development.

Rosiglitazone reduces the development and rupture of experimental aortic aneurysms.

BACKGROUND: Development and rupture of aortic aneurysms involve a combination of complex biological processes. Rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, has been shown to have a broad spectrum of effects in vivo. The hypothesis that rosiglitazone would reduce aneurysm expansion or rupture was tested in the angiotensin II (Ang II)-induced hypercholesterolemic mouse model. METHODS AND RESULTS: Apolipoprotein E-deficient mice, 12 months of age, were allocated to 4 groups. Three groups were infused with Ang II (1 microg . min(-1) . kg(-1)), and the fourth was infused with saline. Rosiglitazone was given 1 week before infusion and 1 week after infusion. At day 28, aortic size was measured, and tissues were collected for analyses. Both pretreatment and posttreatment with rosiglitazone inhibited the occurrence of fatal rupture (11 of 30 versus 0 of 30 versus 0 of 15; P=0.0013) and reduced maximal dilatation of the aorta (4.6+/-0.13 versus 2.4+/-0.48 versus 2.15+/-0.46 mm2; P<0.0001). Blood glucose, total cholesterol, body weight, and atherosclerosis did not differ between groups. Pretreatment with rosiglitazone inhibited the Ang II-induced expression of angiotensin type 1a Ang II receptor while having no effect on the angiotensin type 2 Ang II receptor, in addition to reducing Ang II-induced expression of E-selectin, tumor necrosis factor-alpha, and interleukin-6. CONCLUSIONS: Pretreatment or posttreatment with RGZ reduced aortic expansion and rupture in this mouse model. Reduction of lesions in animals pretreated with rosiglitazone is concomitant with decreased expression of inflammatory mediators. Further studies are needed to elucidate the precise mechanism.

Vascular endothelial growth factor enhances angiotensin II-induced aneurysm formation in apolipoprotein E-deficient mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) development is associated with increased angiogenesis and overexpression of vascular endothelial growth factor (VEGF). Inhibition of angiogenesis results in attenuation of experimental aneurysms. This study investigated the effects of recombinant human (rh)VEGF on experimental aneurysms. METHODS: Apolipoprotein E-deficient (apoE(-/-)) mice were assigned to one of four groups: (1) normal saline infusion (sham), (2) angiotensin-II (AngII) infusion, (3) AngII infusion plus 100 microg daily rhVEGF for 14 days (AngII+14dVEGF), or (4) AngII infusion plus 100 microg daily rhVEGF for 21 days (AngII+21dVEGF). Aortic maximum diameter and cross-sectional area were determined by magnetic resonance imaging and microscopy. All mice were sacrificed at day 28. RESULTS: Aneurysms developed in all mice in the AngII+14dVEGF and AngII+21dVEGF groups by day 21 compared with 40% in the AngII group. Treatment with rhVEGF increased maximum aortic diameter (P < .002) and cross-sectional area of aneurysms (P < .005) at day 21. This effect was maintained at day 28 (P < .0005). Decreasing rhVEGF treatment from 21 to 14 days did not attenuate aneurysm formation. Treatment with rhVEGF upregulated matrix metalloproteinase 2 gene expression within the aortic wall (P < .0009). CONCLUSIONS: Treatment with rhVEGF intensified the formation of AngII-induced aneurysms. Further studies are needed to investigate if antiangiogenic therapy may be a valid medical therapy against aneurysm expansion or rupture.

The role of proteomic research in vascular disease.

INTRODUCTION: Arterial diseases including atherosclerosis, intimal hyperplasia and aneurysms have been shown to be a product of genotype and environment. Gene expression pathways rely on protein translation to generate target effects. As a result of alternative splicing and post-translational modifications, one gene does not code for a single protein but for many. Proteomic studies allow quantification of these proteins in a biological system and determination of altered protein expression in disease. Proteomics is a powerful and expanding field of investigation which in combination with other 'omics may enhance understanding of disease pathophysiology and/or identify biomarkers of vascular disease. This review describes the methodology of protein mining and provides an insight into the valuable contributions already made by proteomics to vascular surgery. METHODS: MEDLINE and EMBASE databases were searched for relevant articles. RESULTS: 118 relevant articles were identified. These were subdivided into categories based on the aspect of protein research they reported. The subheadings include methodology, atherosclerosis, intimal hyperplasia, aortic disease and biomarkers. CONCLUSIONS: Disease processes classified as genetic are functionally proteomic. Equally disease pathophysiology is the result of, or leads to alternate protein expression. Understanding the proteome will clarify the pathophysiology of disease. The translation of these findings to clinical practice impacts diagnosis, staging and treatment of disease processes. Biomarker discovery will enable earlier diagnosis of unstable atherosclerotic plaques, it will allow identification of aneurysms more likely to rupture and stratify risk. Proteomic research has enormous potential to modulate many aspects of patient care.

Plasma tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9: novel indicators of left ventricular remodelling and prognosis after acute myocardial infarction.

AIMS: Matrix metalloproteinase (MMP) activity is central to the development of left ventricular (LV) remodelling and dysfunction after acute myocardial infarction (AMI). We assessed the relationships with LV structure and function and outcome, of tissue inhibitors of metalloproteinase-1 (TIMP-1) and MMP-9, and compared with N-terminal pro-B-type natriuretic peptide (NTproBNP). METHODS AND RESULTS: We studied 404 patients with AMI. Primary outcome measures were the associations of TIMP-1, MMP-9, and NTproBNP with death or heart failure, and with LV dimensions, function and remodelling (ΔLVEDV, change in LV end-diastolic volume between discharge and follow-up). Cut-off concentrations for prediction of death or heart failure were identified from receiver operator characteristic (ROC) curves. In multivariable analysis, TIMP-1 and NTproBNP had predictive value for LV ejection fraction pre-discharge (TIMP-1 P = 0.023; N-BNP P = 0.007) and at follow-up (TIMP-1 P = 0.001; N-BNP P = 0.003). MMP-9, TIMP-1, and NTproBNP correlated directly with LV volumes. MMP-9 (P = 0.005) and TIMP-1 (P = 0.036), but not NTproBNP, correlated with ΔLVEDV. For the combined endpoint of death or heart failure the area under the ROC curve was 0.640 for MMP-9, 0.799 for NTproBNP and 0.811 for TIMP-1. Patients with TIMP-1 > 135 ng/mL (P < 0.001) or NTproBNP >1472 fmol/mL (P < 0.001) had increased risk of endpoint. Consideration of both NTproBNP and TIMP-1 further improved risk stratification. CONCLUSION: TIMP-1 and MMP-9 correlate with echocardiographic parameters of LV dysfunction and remodelling after AMI and may identify patients at risk of subsequent LV remodelling and adverse prognosis.

Abdominal aortic aneurysm rupture is associated with increased medial neovascularization and overexpression of proangiogenic cytokines.

OBJECTIVE: Matrix metalloproteinase (MMP) activity has been linked to abdominal aortic aneurysm (AAA) rupture. Medial neovascularization (MNV), a histopathologic characteristic of AAAs, involves proteolytic degradation of extracellular matrix by MMPs to facilitate endothelial cell migration. The role of MNV in aneurysm rupture is unknown. This study investigated whether MNV is increased in aneurysm rupture. METHODS AND RESULTS: Biopsy samples from aneurysm rupture edge were compared with control biopsy samples from aneurysm wall at the level of rupture and from anterior sac in 12 ruptured AAAs. Further controls were obtained from anterior sac of 10 nonruptured AAAs. MNV, microvessel diameter, maturity index, and inflammatory infiltrate were quantified using morphometric analyses following immunohistochemistry. Expression of proangiogenic mediators was quantified using quantitative real-time-polymerase chain reaction. Compared with anterior sac and aneurysm wall at level of rupture, MNV was increased (P<0.001) in rupture edge biopsy samples and consisted of smaller diameter (P<0.001) and more immature microvessels (P<0.001). mRNA expression of alpha(v)-integrin, vascular endothelial growth factor, vascular endothelial-cadherin, monocyte chemoattractant protein-1, and vimentin was increased (P<0.05) in rupture edge biopsy samples. CONCLUSIONS: This study demonstrated increased medial neovascularization and overexpression of proangiogenic cytokines at aneurysm rupture edge. Further investigations into whether this angiogenic response was a causative factor of aneurysm rupture are needed.

Pharmacotherapy of abdominal aortic aneurysms.

Aortic aneurysms account for 10,000 deaths annually in the UK, due to rupture. At present the only effective therapeutic strategy to treat abdominal aortic aneurysms is to surgically repair them; this carries an elective mortality of up to 10%. Recent advances in vascular biology have led to a greater understanding of the pathophysiological process that causes aortic aneurysms to expand and rupture. Key pathological processes include widespread aortic inflammation, proteolytic degradation of the extracellular matrix, neovascularisation and generation of reactive oxygen species. Identification of these processes has lead to pharmacological strategies to prevent aneurysm expansion and rupture. Many of these strategies have undergone proof of concept in animal models and some have now entered clinical trials. This review outlines current thinking regarding the molecular events leading to aneurysm expansion and explains how these processes may be inhibited. Experimental data on agents retarding aneurysm expansion in animal models are discussed. A significant proportion of the review details pharmacological agents that have undergone or are undergoing clinical trials. Pharmacological treatment for abdominal aneurysms is urgently required given the number of small aneurysms being diagnosed by screening programmes. This is a rapidly evolving field and one in which translation from experimental research to clinical practice is anticipated within 5 years.