Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis.

OBJECTIVES: We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. BACKGROUND: Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of HIF. METHODS: To examine the presence of hypoxia in atherosclerotic plaques, the hypoxia marker pimonidazole was infused before carotid endarterectomy in 7 symptomatic patients. Also, the messenger ribonucleic acid (mRNA) and protein expression of HIF1 alpha, HIF2 alpha, HIF-responsive genes (vascular endothelial growth factor [VEGF], glucose transporter [GLUT]1, GLUT3, hexokinase [HK]1, and HK2), and microvessel density were determined in a larger series of nondiseased and atherosclerotic carotid arteries with microarray, quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. RESULTS: Pimonidazole immunohistochemistry demonstrated the presence of hypoxia, especially within the macrophage-rich center of the lesions. Hypoxia correlated with the presence of a thrombus, angiogenesis, and expression of CD68, HIF, and VEGF. The mRNA and protein expression of HIF, its target genes, and microvessel density increased from early to stable lesions, but no changes were observed between stable and ruptured lesions. CONCLUSION: This is the first study directly demonstrating hypoxia in advanced human atherosclerosis and its correlation with the presence of macrophages and the expression of HIF and VEGF. Also, the HIF pathway was associated with lesion progression and angiogenesis, suggesting its involvement in the response to hypoxia and the regulation of human intraplaque angiogenesis.

Dead or alive: gene expression profiles of advanced atherosclerotic plaques from autopsy and surgery.

Since inclusion of atherosclerotic tissues from different sources is often indispensable to study the full atherogenic spectrum, we investigated to what extent the expression profiles of advanced, stable atherosclerotic lesions obtained during autopsy and surgery are comparable. The gene expression profiles of human carotids with advanced atherosclerosis obtained at autopsy and at vascular surgery were studied by microarray analysis. Expression analysis was performed both at the single gene (Rosetta, Gene Ontology) and at the pathway level using Ingenuity and Gene Set Enrichment Analysis. In addition, mRNA and protein expression levels were validated using quantitative (q) RT-PCR and immunohistochemistry on unrelated advanced carotid lesions from autopsy and surgery. Microarray analysis indicated that the 97.2% of genes showed similar expression levels in advanced atherosclerotic lesions from autopsy and surgery. While the expression data revealed no differences in common atherosclerotic related pathways such as lipid metabolism and inflammation, the differentially expressed genes were mainly involved in basal cell metabolism and hypoxia driven pathways. qRT-PCR confirmed the differential expression of hypoxia-driven genes VEGF-A (2.3-fold upward arrow), glucose transporter (GLUT)-1 (2.5-fold upward arrow), GLUT3 (8.3-fold upward arrow), and hexokinase 1 (2.4-fold upward arrow) in autopsy vs. surgical specimens. Immunohistochemistry revealed that the transcriptional differences in these hypoxia-related genes were not reflected at the protein level. The gene expression profiles of advanced atherosclerotic lesions from autopsy and surgery are largely similar. However, >500 genes, mostly involved in basal cell metabolism and hypoxia were differentially expressed at mRNA, but not at the protein level.

Synergistic effect of Toll-like receptor 4 and CD14 polymorphisms on the total atherosclerosis burden in patients with peripheral arterial disease.

BACKGROUND: Genes involved in the regulation of immune responses, such as Toll-like receptor 4 (TLR4) and CD14, show genetic variations with potential functional implications. Because atherosclerosis is an inflammatory process apparently modulated by chronic infections, we studied the effect of single nucleotide polymorphisms (SNPs) in TLR4 and CD14 on the extent of clinically relevant atherosclerosis in patients with peripheral arterial disease (PAD). METHODS: Using an in-house-developed polymerase chain reaction-based restriction length polymorphism assay, we determined the genotype, allele frequency, and carrier traits of the TLR4 +896 A>G and the CD14 -260 C>T SNPs in 607 white Dutch patients with PAD. The extent of clinically relevant atherosclerosis was determined on the basis of the number of vascular territories involved, ie, coronary, cerebral, aortic, and peripheral. RESULTS: A total of 55% of the patients had PAD only. Approximately one third of the patients had two and 11% had three vascular territories affected by clinically relevant atherosclerosis. The TLR4 +866 G allele frequency was 11%, and the CD14 -260 T allele frequency was approximately 74%. Among PAD patients, TLR4 +896 G allele carriership was univariantly associated with extensive (more than two vascular territories affected) atherosclerotic disease (odds ratio, 2.22; P = .020; chi(2) test), whereas CD14 -260 C>T carriership/homozygosity was not. Trend analysis showed that the TLR4 +866 G allele frequency increased with the number of vascular territories affected by clinically relevant atherosclerosis (P trend, .0074). In a multivariate logistic regression analysis including cardiovascular risk factors and TLR4 and CD14 SNPs, only the interaction variable "TLR4 +896 G allele carriership/CD14 -260 TT genotype" survived as an independent predictor of extensive atherosclerotic disease (P = .031; odds ratio, 4.2; 95% confidence interval, 1.1-15.4). CONCLUSIONS: The carrier trait TLR4 G allele/CD14 TT genotype, rather than each SNP individually, is associated with the extent of clinically relevant atherosclerotic disease. Considering the importance of immune responses in atherogenesis and the genetic variation of immune regulatory genes, our data provide an explanation for interindividual differences in susceptibility to atherosclerosis and demonstrate the need to take a wider approach in analyzing relevant carrier traits instead of individual polymorphisms in relation to atherosclerosis.

C-reactive protein in peripheral arterial disease: relation to severity of the disease and to future cardiovascular events.

BACKGROUND: Serum C-reactive protein (CRP) has proven to be an independent marker of the extent of atherosclerosis in patients with coronary, cerebrovascular, and peripheral arterial disease. In this prospective observational study, we wanted to assess the relationship between serum CRP and extent of disease transversely and longitudinally in time, as well as future cardiovascular complications in patients with peripheral arterial disease (PAD). Hypothesizing that CRP not only is a marker of but also actively participates in atherogenesis, we explored the possibility of CRP production by femoral atherosclerotic plaques. METHODS: Serum CRP was measured as highly sensitive (hsCRP) in 387 patients with PAD attending the vascular clinic of a university and 2 affiliated teaching hospitals. Serum hsCRP was related to the ankle-brachial pressure index (ABPI) as an indication of severity of disease at inclusion and at 12 months' follow-up and to future events (death and coronary, cerebral, and peripheral arterial events). In femoral plaques, the production of CRP was analyzed with reverse transcription-polymerase chain reaction, and CRP plaque localization was assessed with immunostaining on serial tissue sections with antibodies toward CRP, smooth muscle cells, T cells, and macrophages. RESULTS: The hsCRP (average +/- SD) was 3.26 +/- 2.41 mg/L. Serum hsCRP showed a correlation with baseline and 12-month follow-up ABPI (Spearman rank correlation; P < .05 for both correlations). When the patients were divided into three equally sized groups according to baseline serum hsCRP, the ABPI at baseline and at 12 months decreased significantly from the low- to the high-hsCRP group (baseline ABPI: 0.70, 0.65, and 0.57, P < .01; 12-month follow-up ABPI: 0.78, 0.70, and 0.65, P < .01). These associations persisted after correction for conventional risk factors. Furthermore, serum hsCRP was related to the combined end point "death and/or any cardiovascular event" (log-rank test; P = .04) during a median 24-month follow-up period. Reverse transcription-polymerase chain reaction analysis showed CRP production in 4 of 14 femoral plaques. CRP was detected in all femoral plaques, but not in healthy brachial arteries. Immunoreactivity for CRP was observed in smooth muscle cells, macrophages, and T cells. CONCLUSIONS: Serum hsCRP was related to the severity of PAD, showing a relation to future hemodynamic function and cardiovascular events in PAD patients. In addition to coronary plaques, aneurysmal aortas, and failed venous coronary bypasses, femoral plaques also produce CRP, thus illustrating that the production of CRP may represent a universal response to vascular injury and suggesting that vascular CRP may contribute to plaque development.

Proteomic analysis of differential protein expression in human atherosclerotic plaque progression.

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole-mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin-beta and alpha1-antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin-beta and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin-beta in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus-containing plaques. In conclusion, vinexin-beta is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus-containing plaques.