Mariette Lengquist

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.

NLRP3 Inflammasome Expression and Activation in Human Atherosclerosis

Background The NLR family, pyrin domain containing 3 (NLRP3) inflammasome is an interleukin (IL)‐1β and IL‐18 cytokine processing complex that is activated in inflammatory conditions. The role of the NLRP3 inflammasome in the pathogenesis of atherosclerosis and myocardial infarction is not fully understood. Methods and Results Atherosclerotic plaques were analyzed for transcripts of the NLRP3 inflammasome, and for IL‐1β release. The Swedish First‐ever myocardial Infarction study in Ac‐county (FIA) cohort consisting of DNA from 555 myocardial infarction patients and 1016 healthy individuals was used to determine the frequency of 4 single nucleotide polymorphisms (SNPs) from the downstream regulatory region of NLRP3. Expression of NLRP3, Apoptosis‐associated speck‐like protein containing a CARD (ASC), caspase‐1 (CASP1), IL1B, and IL18 mRNA was significantly increased in atherosclerotic plaques compared to normal arteries. The expression of NLRP3 mRNA was significantly higher in plaques of symptomatic patients when compared to asymptomatic ones. CD68‐positive macrophages were observed in the same areas of atherosclerotic lesions as NLRP3 and ASC expression. Occasionally, expression of NLRP3 and ASC was also present in smooth muscle cells. Cholesterol crystals and ATP induced IL‐1β release from lipopolysaccharide‐primed human atherosclerotic lesion plaques. The minor alleles of the variants rs4266924, rs6672995, and rs10733113 were associated with NLRP3 mRNA levels in peripheral blood mononuclear cells but not with the risk of myocardial infarction. Conclusions Our results indicate a possible role of the NLRP3 inflammasome and its genetic variants in the pathogenesis of 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.

Increased expression of heparanase in symptomatic carotid atherosclerosis

OBJECTIVE Proliferation of smooth muscle cells (SMCs) can stabilize atherosclerotic lesions but the molecular mechanisms that regulate this process in humans are largely unknown. We have previously shown that heparan sulfate proteoglycans (HSPGs), such as perlecan, regulate SMC growth in animal models by modulating heparin-binding mitogens. Since perlecan is expressed at low levels in human atherosclerosis, we speculated that the effect of heparan sulfate (HS) in human disease was rather influenced by HS degradation and investigated the expression of heparanase (HPSE) in human carotid endarterectomies. METHODS AND RESULTS Gene expression analysis from 127 endarterectomies in the BiKE database revealed increased expression of HPSE in carotid plaques compared with normal arteries, and a further elevation in symptomatic lesions. Increased HPSE protein expression in symptomatic plaque tissue was verified by tissue microarrays. HPSE mRNA levels correlated positively with expression of inflammatory markers IL-18, RANTES and IL-1β, and also T-cell co-stimulatory molecules, such as B7.2, CD28, LFA-1 and 4-1BB. Previously reported single nucleotide polymorphisms within HPSE were associated with differential mRNA expression in plaques. Immunohistochemistry revealed that inflammatory cells were major producers of HPSE in plaque tissue. HPSE immunoreactivity was also observed in SMCs adjacent to the necrotic core and was co-localized to deposits of fibrin. CONCLUSIONS This study demonstrates increased expression of HPSE in human atherosclerosis associated with inflammation, coagulation and plaque instability. Since HS can regulate SMC proliferation and influence plaque stability, the findings suggest that HPSE degradation of HS take part in the regulation of SMC function in human atherosclerosis.

Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques

BACKGROUND. The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the “vulnerable plaque,” necessitating an improved approach for predicting onset of symptoms. METHODS. We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS. Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION. The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING. UK: British Heart Foundation (BHF); King’s BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London in partnership with King’s College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

Antenatal imatinib treatment reduces pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia

The pathophysiology of congenital diaphragmatic hernia (CDH) is constituted by pulmonary hypoplasia and pulmonary hypertension (PH). We previously reported successful treatment with imatinib of a patient with CDH. This study examines the effect of antenatal imatinib administration on the pulmonary vasculature in a rat model of CDH. Pregnant rats were given nitrofen to induce CDH. Controls were given olive oil. Half of the CDH fetuses and half of the controls were treated with imatinib antenatally E17-E21, rendering four groups: Control, Control+Imatinib, CDH, and CDH+Imatinib. Lung sections were obtained for morphometry and immunohistochemistry, and protein was purified for Western blot. Effects of nitrofen and imatinib on Ki-67, caspase-3, PDGF-B, and PDGF receptors were analyzed. Imatinib significantly reduced medial wall thickness in pulmonary arteries of rats with CDH. It also normalized lumen area and reduced the proportion of fully muscularized arteries. Imatinib also caused medial thinning in the control group. Cell proliferation was increased in CDH, and this proliferation was significantly reduced by imatinib. PDGF-B and PDGFR-β were upregulated in CDH, and imatinib treatment resulted in a downregulation. PDGFR-α remained unchanged in CDH but was significantly downregulated by imatinib. Antenatal imatinib treatment reduces development of medial wall thickness and restores lumen area in pulmonary arteries in nitrofen-induced CDH. The mechanism is reduced cell proliferation. Imatinib is an interesting candidate for antenatal therapy for PH in CDH, but potential side effects need to be investigated and more specific targeting of PDGF signaling is needed.

Contribution of Endothelial Injury and Inflammation in Early Phase to Vein Graft Failure: The Causal Factors Impact on the Development of Intimal Hyperplasia in Murine Models

Objectives Autologous veins are preferred conduits in by-pass surgery. However, long-term results are hampered by limited patency due to intimal hyperplasia. Although mechanisms involved in development of intimal hyperplasia have been established, the role of inflammatory processes is still unclear. Here, we studied leukocyte recruitment and intimal hyperplasia in inferior vena cava grafts transferred to abdominal aorta in mice. Methods and Results Several microscopic techniques were used to study endothelium denudation and regeneration and leukocyte recruitment on endothelium. Scanning electron microscopy demonstrated denudation of vein graft endothelium 7 days post-transfer and complete endothelial regeneration by 28 days. Examination of vein grafts transferred to mice transgenic for green fluorescent protein under Tie2 promoter in endothelial cells showed regeneration of graft endothelium from the adjacent aorta. Intravital microscopy revealed recruitment of leukocytes in vein grafts at 7 days in wild type mice, which had tapered off by 28 days. At 28 and 63 days there was significant development of intimal hyperplasia. In contrast; no injury, leukocyte recruitment nor intimal hyperplasia occurred in arterial grafts. Leukocyte recruitment was reduced in vein grafts in mice deficient in E- and P-selectin. In parallel, intimal hyperplasia was reduced in vein grafts in mice deficient in E- and P-selectin and in wild type mice receiving P-selectin/E-selectin function-blocking antibodies. Conclusion The results show that early phase endothelial injury and inflammation are crucial processes in intimal hyperplasia in murine vein grafts. The data implicate endothelial selectins as targets for intervention of vein graft disease.

Phenotypic Modulation of Smooth Muscle Cells in Atherosclerosis Is Associated With Downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM

Objective—Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. Approach and Results—Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7, and PLN expression positively correlated to typical SMC markers in plaques (Pearson r>0.6, P<0.0001) and in rat intimal hyperplasia (r>0.8, P<0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN, and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. Conclusions—We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

Differential expression of sex hormone receptors in abdominal aortic aneurysms

OBJECTIVE Male sex is a significant risk factor for abdominal aortic aneurysm (AAA). Female sex hormones have been reported to prevent aneurysm formation in animal models. The study aims to describe the expression profile of sex hormone receptors in the aneurysm wall of men and women with AAA and compare with unaffected controls. METHODS Aneurysm wall biopsies were obtained during elective open repair of AAA in men and women (n=16+16). Aortic vessel wall from controls were obtained at organ donation (n=6). Western blot-, mRNA expression- and immunohistochemical analyses were performed to assess the expression profile of the sex hormone receptors - androgen receptor (AR), progesterone receptor (PR), estrogen receptor α (ERα) and β (ERβ). RESULTS The mRNA- and protein expression levels of AR were higher in AAA compared with control aorta (7.26 vs. 5.14, P=0.001). mRNA- and protein expression levels of ERβ were lower in AAA compared with control aorta (9.15 vs. 12.29, P<0.001). mRNA expression levels of PR were higher in AAA compared with control aorta (8.73 vs. 6.21, P=0.003), but could not be confirmed on protein level. The expression profile of sex hormone receptors in men and women with AAA was similar. CONCLUSION Expression of sex hormone receptors differs in the aneurysmal aorta compared with unaffected aorta in men and women. A higher expression of AR and a lower expression of ERβ suggest that sex hormone activity could be associated with aneurysm development.

Platelet adhesion on endothelium early after vein grafting mediates leukocyte recruitment and intimal hyperplasia in a murine model

Intimal hyperplasia (IH) is the substrate for accelerated atherosclerosis and limited patency of vein grafts. However, there is still no specific treatment targeting IH following graft surgery. In this study, we used a mouse model of vein grafting to investigate the potential for early intervention with platelet function for later development of graft IH. We transferred the inferior vena cava (IVC) from donor C57BL/6 mice to the carotid artery in recipients using a cuff technique. We found extensive endothelial injury and platelet adhesion one hour following grafting. Adhesion of leukocytes was distinct in areas of platelet adhesion. Platelet and leukocyte adhesion was strongly reduced in mice receiving a function-blocking antibody against the integrin αIIbβ3. This was followed by a reduction of IH one month following grafting. Depletion of platelets using antiserum also reduced IH at later time points. These findings indicate platelets as pivotal to leukocyte recruitment to the wall of vein grafts. In conclusion, the data also highlight early intervention of platelets and inflammation as potential treatment for later formation of IH and accelerated atherosclerosis following bypass surgery.