Anton Razuvaev

Profiling of Atherosclerotic Lesions by Gene and Tissue Microarrays Reveals PCSK6 as a Novel Protease in Unstable Carotid Atherosclerosis

Objective—Carotid plaque instability is a major cause of ischemic stroke, but detailed knowledge about underlying molecular pathways is still lacking. Here, we evaluated large-scale transcriptomic and protein expression profiling in a biobank of carotid endarterectomies followed by characterization of identified candidates, as a platform for discovery of novel proteins differentially regulated in unstable carotid lesions. Approach and Results—Genes highly upregulated in symptomatic versus asymptomatic plaques were selected from Affymetrix microarray analyses (n=127 plaques), and tissue microarrays constructed from 34 lesions were assayed for 21 corresponding proteins by immunohistochemistry. Quantification of stainings demonstrated differential expression of CD36, CD137, and DOCK7 (P<0.05) in unstable versus stable lesions and the most significant upregulation of a proprotein convertase, PCSK6 (P<0.0001). Increased expression of PCSK6 in symptomatic lesions was verified by quantitative real-time polymerase chain reaction (n=233), and the protein was localized to smooth muscle &agr;-actin positive cells and extracellular matrix of the fibrous cap by immunohistochemistry. PCSK6 expression positively correlated to genes associated with inflammation, matrix degradation, and mitogens in microarrays. Stimulation of human carotid smooth muscle cells in vitro with cytokines caused rapid induction of PCSK6 mRNA. Conclusions—Using a combination of transcriptomic and tissue microarray profiling, we demonstrate a novel approach to identify proteins differentially expressed in unstable carotid atherosclerosis. The proprotein convertase PCSK6 was detected at increased levels in the fibrous cap of symptomatic carotid plaques, possibly associated with key processes in plaque rupture such as inflammation and extracellular matrix remodeling. Further studies are needed to clarify the role of PCSK6 in atherosclerosis.

Gene expression signatures, pathways and networks in carotid atherosclerosis

Embolism from unstable atheromas in the carotid bifurcation is a major cause of stroke. Here, we analysed gene expression in endarterectomies from patients with symptomatic (S) and asymptomatic (AS) carotid stenosis to identify pathways linked to plaque instability.

The expression of IGFs and IGF binding proteins in human carotid atherosclerosis, and the possible role of IGF binding protein-1 in the regulation of smooth muscle cell proliferation

OBJECTIVE Proliferation of smooth muscle cells (SMCs) in the fibrous cap of atherosclerotic lesions has been proposed to be important for plaque stability. Since the insulin-like growth factor (IGF) system has been implicated to play a role in atherosclerosis and plaque stability, we investigated the expression of members of the IGF system in carotid plaques, in particular IGFBP-1 and its role in the regulation of SMC proliferation. METHODS AND RESULTS Gene expression profiles of the IGF system in 164 human carotid plaques obtained from our Biobank of Karolinska Endarterectomies (BiKE) were analyzed. Expression of IGFBP-1 mRNA was significantly increased in carotid plaques compared with normal iliac arteries in contrast to IGF-1, IGF-2, and IGFBP-3 to IGFBP-6. The expression of IGFBP-1 mRNA correlated positively to that of CD163, CD68, IL-1β, IL-6, TNFα, IGFBP-4 and IGFBP-5. Immunohistochemistry demonstrated co-localization of IGFBP-1 with SMCs and macrophages. In vitro studies showed that IL-1β, IL-6 and TNFα stimulated IGFBP-1 mRNA expression in SMCs. IGFBP-1 stimulated SMC proliferation through ERK1/2 activation but independently of the IGF-1 receptor. In addition, IGFBP-1 modulated the effect of IGF-1 on SMC proliferation and ERK1/2 activation. CONCLUSIONS Our results demonstrate that IGFBP-1 mRNA and protein is detected at increased levels in human carotid plaques, possibly as a consequence of plaque inflammation. IGFBP-1 affects SMC proliferation and may be involved in the regulation of plaque stability.

Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival

OBJECTIVE Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. METHODS Laminar FSS of arterial level (14 dynes/cm(2)) was applied to SMC cultures for 24 hours in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology, and results further verified by real time polymerase chain reaction (RT-PCR) and immunoblotting. Tissue factor pathway inhibitor-2 (TFPI-2) and caspase-3 protein expression was studied in the rat carotid artery after balloon-injury, and the effect of TFPI-2 on SMC DNA synthesis and apoptosis was examined in vitro. RESULTS Microarrays identified TFPI-2 as one of the most differentially expressed gene by FSS in cultured SMCs (P < .001). Gene set enrichment analysis revealed significant regulation of genes linked to proliferation, apoptosis, and cell cycle regulation. TFPI-2 induction was confirmed by RT-PCR and immunoblotting demonstrating a more than 400-fold (P < .001) increase in TFPI-2 mRNA in SMCs exposed to FSS compared with static controls, and a consistent protein upregulation. Functionally, SMC proliferation was decreased by FSS (P < .001), and recombinant TFPI-2 was found to inhibit SMC proliferation (P < .001) and induce SMC apoptosis as indicated by activation of caspase-3 (P < .01). In vivo, TFPI-2 expression was found to be upregulated 5, 10, and 20 hours (P < .01) after rat carotid balloon injury, and immunohistochemistry demonstrated TFPI-2 protein in FSS-exposed luminal SMCs, co-localized with caspase-3 in the rat carotid neointima. CONCLUSION FSS influenced gene expression associated with cell growth and apoptosis in cultured SMCs and strongly induced expression of TFPI-2 mRNA and protein. TFPI-2 was expressed in luminal, FSS-exposed SMCs together with caspase-3 in the rat carotid neointima after balloon injury. Functionally, TFPI-2 may play a role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 controls SMC function.

Correlations between clinical variables and gene-expression profiles in carotid plaque instability.

OBJECTIVE Strokes, a major cause of disability, are often caused by embolism from unstable carotid plaques. The aim of this study was to validate a biobank of human carotid endarterectomies as a platform for further exploration of pathways for plaque instability. For this purpose, we investigated the relationship between clinical parameters of plaque instability and expression of genes previously shown to be associated with either plaque instability or healing processes in the vessel wall. METHODS A database of clinical information and gene-expression microarray data from 106 carotid endarterectomies were used. RESULTS Expression of matrix metalloproteinase (MMP)-9 and MMP-7 was 100-fold higher in plaques than in normal artery. In general, genes associated with inflammation (such as RANKL and CD68) were overexpressed in symptomatic compared with asymptomatic plaques. Plaques obtained from patients undergoing surgery within 2 weeks after an embolic event showed up-regulation of genes involved in healing reactions in the vessel wall (including elastin and collagen). Statin treatment, as well as echodense lesions, were associated with a more stable phenotype. CONCLUSION Here, we demonstrate that gene-expression profiles reflect clinical parameters. Our results suggest that microarray technology and clinical variables can be used for the future identification of central molecular pathways in plaque instability.

Noninvasive real-time imaging of intima thickness after rat carotid artery balloon injury using ultrasound biomicroscopy

OBJECTIVES High frequency ultrasound imaging for small animal research (ultrasound biomicroscopy, UBM) has recently become available. Here, we evaluated the possibility to determine intima thickness in the rat carotid artery after balloon injury and to monitor intimal hyperplasia formation by UBM during pharmacological treatment. METHODS Balloon injury of the left carotid artery was performed on Sprague-Dawley rats. Carotid arteries of all animals were examined by Vevo 770 UBM (VisualSonics Inc.) using 55 MHz probe at day 1, 7, 14 and 21 after the injury. Whole vessel wall, intima and media thicknesses as well as lumen diameter were measured at different levels. Histomorphometric analyses were performed on day 14 and 21. A group of animals were treated with picropodophyllin, an insulin-like growth factor-1 receptor inhibitor. RESULTS Ex-vivo comparison of UBM and histology demonstrated an excellent correspondence of intimal tears, and the internal and external elastic membranes could be identified. We found also an agreement (Bland-Altman test) between histological measurements and UBM delineations of the rat carotid artery wall layers, with a significant correlation for intima-media thickness (r=0.97; p<0.0001) and intima measurements. We were able to follow changes in the vessel wall structure and vessel diameter as a response to balloon injury in real time. Furthermore, the therapeutic effect of picropodophyllin could be assessed using UBM. CONCLUSIONS UBM provides a reliable noninvasive, in-vivo visualization of rat vasculature. It allows us to perform longitudinal studies of intimal thickness progression and regression as well as lumen changes in individual animals.

MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions

Rationale: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. Objective: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Methods and Results: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction–based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. Conclusions: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.

Serine protease inhibitor A3 in atherosclerosis and aneurysm disease

Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta.

Endogenous control genes in complex vascular tissue samples

BackgroundGene expression microarrays and real-time PCR are common methods used to measure mRNA levels. Each method has a fundamentally different approach of normalization between samples. Relative quantification of gene expression using real-time PCR is often done using the 2^(-ΔΔCt) method, in which the normalization is performed using one or more endogenous control genes. The choice of endogenous control gene is often arbitrary or bound by tradition. We here present an analysis of the differences in expression results obtained with microarray and real-time PCR, dependent on different choices of endogenous control genes.ResultsIn complex tissue, microarray data and real-time PCR data show the best correlation when endogenous control genes are omitted and the normalization is done relative to total RNA mass, as measured before reverse transcription.ConclusionWe have found that for real-time PCR in heterogeneous tissue samples, it may be a better choice to normalize real-time PCR Ct values to the carefully measured mass of total RNA than to use endogenous control genes. We base this conclusion on the fact that total RNA mass normalization of real-time PCR data shows better correlation to microarray data. Because microarray data use a different normalization approach based on a larger part of the transcriptome, we conclude that omitting endogenous control genes will give measurements more in accordance with actual concentrations.

Glucagon-Like Peptide-1 Receptor Activation Does Not Affect Re-Endothelialization but Reduces Intimal Hyperplasia via Direct Effects on Smooth Muscle Cells in a Nondiabetic Model of Arterial Injury

Diabetic patients have an increased risk of restenosis and late stent thrombosis after angioplasty, i.e. complications that are related to a defective re-endothelialization. Exendin-4, a stable glucagon-like peptide (GLP)-1 receptor agonist, has been suggested to influence the formation of intimal hyperplasia and to increase endothelial cell proliferation in vitro. Thus, the aim of this study was to investigate the mechanisms by which treatment with exendin-4 could influence re-endothelialization and intimal hyperplasia after vascular injury. Methods: Sprague-Dawley rats were subjected to balloon injury of the left common carotid artery and treated for 4 weeks with exendin-4 or vehicle. Intimal hyperplasia and vessel wall elasticity were monitored noninvasively by high-frequency ultrasound, and re-endothelialization was evaluated upon sacrifice using Evans blue dye. Results and Conclusion: Exendin-4 selectively reduced the proliferation of smooth muscle cells (SMCs) and intimal hyperplasia in vivo without affecting the re-endothelialization process, but treatment with exendin-4 improved arterial wall elasticity. Our data also show that exendin-4 significantly decreased the proliferation and increased the apoptosis of SMCs in vitro, effects that appear to be mediated through cAMP signaling and endothelial nitric oxide synthase following GLP-1 receptor activation. Together, these effects of exendin-4 are highly desirable and may lead to an improved outcome for patients undergoing vascular interventions.