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Aim: Atherosclerosis is a major complication of diabetes mellitus and a leading cause of mortality in diabetic patients. Vascular interventions in diabetic patients can lead to complications attributed to defective vascular remodeling and impaired healing response. In this study, we aim to elucidate the physiological and molecular differences in the vascular healing response over time using a rat model of arterial injury applied in healthy and diabetic conditions. Methods: Wistar (healthy) and Goto-Kakizaki (GK, diabetic) rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 24 hours, 5 days, 2, 4 and 6 weeks. Non-invasive morphological and physiological assessment, and microarray profiling of the CCA was performed for each timepoint. Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database and Cytoscape software. Results: Significant increase in the neointimal thickness (p Conclusions: In this study, we investigated the effect of diabetes on vessel wall healing. We demonstrate that diabetic rats exhibit impaired arterial remodelling characterised by a delayed healing response. These results further corroborate the higher prevalence of restenosis in diabetic patients and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes.

Rationale: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Objective: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Methods and Results: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6 −/− versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6 −/− mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. Conclusions: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

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.

Diabetic patients suffer an increased risk of restenosis and late stent thrombosis after angioplasty, complications which are related to a defective reendothelialization. Dipeptidyl peptidase-4 inhibitors have been suggested to exert a direct effect on endothelial and smooth muscle cells (SMCs). Therefore, the objective was to study if the dipeptidyl peptidase-4 inhibitor linagliptin could influence vascular repair and accelerate reendothelialization after arterial injury in healthy and diabetic animals. Diabetic Goto-Kakizaki and healthy Wistar rats were subjected to arterial injury and treated with linagliptin or vehicle. Vessel wall healing was monitored noninvasively using ultrasound, and on sacrifice, with Evans blue staining and immunohistochemistry. The effect of linagliptin on SMCs was also studied in vitro. We found that linagliptin reduced the proliferation and dedifferentiation of SMCs in vitro, and modulated the inflammatory response in the SMCs after arterial injury in vivo. However, these effects of linagliptin did not affect the neointima formation or the reendothelialization under normal and diabetic conditions. Although linagliptin did not influence vessel wall healing, it seems to possess a desirable antiproliferative influence on SMCs in vitro and an antiinflammatory effect in vivo. These pharmacological properties might carry a potential significance for favorable outcome after vascular interventions in diabetic patients.

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 r >0.8, P 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.

Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines, but their function in the vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques from symptomatic patients, localization to smooth muscle cells (SMCs) in the fibrous cap and positive correlations with inflammation, extracellular matrix remodeling and cytokines. Here, we hypothesize that PCSK6 could be involved in flow-mediated vascular remodeling and aim to evaluate its role in the physiology of this process using knockout mice. Pcsk6&minus, /&minus, and wild type mice were randomized into control and increased blood flow groups and induced in the right common carotid artery (CCA) by ligation of the left CCA. The animals underwent repeated ultrasound biomicroscopy (UBM) examinations followed by euthanization with subsequent evaluation using wire myography, transmission electron microscopy or histology. The Pcsk6&minus, mice displayed a flow-mediated increase in lumen circumference over time, assessed with UBM. Wire myography revealed differences in the flow-mediated remodeling response detected as an increase in lumen circumference at optimal stretch with concomitant reduction in active tension. Furthermore, a flow-mediated reduction in expression of SMC contractile markers SMA, MYH11 and LMOD1 was seen in the Pcsk6&minus, media. Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow.

OBJECTIVES Ultrasound biomicroscopy (UBM), or ultra high-frequency ultrasound, is a technique used to assess the anatomy of small research animals. In this study, UBM was used to assess differences in intimal hyperplasia thickness as a surrogate measurement of the re-endothelialization process after carotid artery balloon injury in rats. METHODS Ultrasound biomicroscopic data from 3 different experiments and rat strains (Sprague Dawley, Wistar, and diabetic Goto-Kakizaki) were analyzed. All animals were subjected to carotid artery balloon injury and examined with UBM (30-70 MHz) 2 and 4 weeks after injury. Re-endothelialization on UBM was defined as the length from the carotid bifurcation to the most distal visible edge of the intimal hyperplasia. En face staining with Evans blue dye was performed at euthanasia 4 weeks after injury, followed by tissue harvesting for histochemical and immunohistochemical evaluations. RESULTS A significant correlation (Spearman r = 0.63; P

Background: Autophagy is a cell survival mechanism, which has been implicated in atherogenesis in mouse models by studying core autophagy machinery proteins using knock-out models. MAP1LC3A and MAP1LC3B play a key role in autophagy activity and have been implicated as prognostic factors in several human cancers. However, data on the involvement of autophagy in human atherosclerotic disease and plaque vulnerability are still sparse and completely lacking with regards to the involvement of MAP1LC3. Approach and Results: Using two independent biobanks of human carotid atherosclerotic plaques, we observe that MAP1LC3A mRNA and protein levels are decreased in plaques from patients with symptomatic disease compared to asymptomatic. Notably, MAP1LC3A mRNA levels strongly correlate with vascular smooth muscle cell markers, while MAP1LC3B does not. In in vivo models, we show that MAP1LC3A mRNA is downregulated during the progression of atherosclerosis in hypercholesterolemic mice as well as upon hyperplasia induced by balloon-injury in rats. In vitro, we show that ablation of MAP1LC3A in human carotid VSMC induces a transient compensatory increase in myocardin, a master regulator of vascular smooth muscle cell phenotypic switch. Conclusions: Taken together, these results demonstrate that reduced MAP1LC3A expression is a relevant marker of vulnerable plaque phenotype, suggesting an impact on vascular smooth muscle cell biology in the context of atherogenesis.

Objective: Understanding molecular processes behind carotid plaque instability is necessary to develop methods that can identify patients and lesions at risk of stroke. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound (US). Results: Plaque echogenicity measured by US was correlated to microarray profiles from lesions retrieved at surgery (n=96). Pathway analyses highlighted enrichment of cell apoptosis and proliferation, and BCLAF1 (BCL2 associated factor 1) as the most significantly dysregulated gene (adjusted pIn vitro , stimulation of SMCs with pro-survival factors EGF, bFGF, PDGFB resulted in induction of BCLAF1, while it was suppressed by macrophage-conditioned medium. Moreover, BCLAF1 silencing in SMCs led to downregulation of BCL2 and SMC markers, and a decrease in proliferation and adhesion (p Conclusions: Carotid plaque echogenicity correlated with enrichment of molecular pathways associated with cell survival and apoptosis and identified BCLAF1, previously not described in atherosclerosis, as the most dysregulated gene. Functionally, BCLAF1 appeared to promote SMC survival by transdifferentiation into macrophage-like phenotype, by interacting with BCL2 and THRAP3.

Background 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. Methods Microarrays were prepared from plaques (n = 127) and peripheral blood samples (n = 96) of S and AS patients. Gene set enrichment, pathway mapping and network analyses of differentially expressed genes were performed. Results These studies revealed upregulation of haemoglobin metabolism (P = 2.20E-05) and bone resorption (P = 9.63E-04) in S patients. Analysis of subgroups of patients indicated enrichment of calcification and osteoblast differentiation in S patients on statins, as well as inflammation and apoptosis in plaques removed >1 month compared to

Neoadjuvant chemotherapeutic regimens for metastatic colorectal cancer are now so effective that they can cause "vanishing" lesions. With new advances such as local ablation, intra-arterial treatments in bolus with pumps or with beads, and isolation of hepatic perfusion, the need for a working channel to the liver may be warranted, ideally reducing the risk of spreading neoplastic cells.The endovascular trans-vessel wall Extroducer device makes it possible to gain direct access to the liver parenchyma. The distal tip is then detached, to act as both a marker and a securing plug in the vessel defect. We used ex vivo and in vivo tests to evaluate the device as a working channel for local administration of substances to the parenchyma and as a marker for detection with both transabdominal and intraoperative ultrasonography.We could deploy the Extroducer device without any hemorrhagic or thromboembolic complications in vivo, and we were able to detect all markers ex vivo and in vivo using both transabdominal and intraoperative ultrasonography. Furthermore, we found that it is possible to administer substances to the liver parenchyma using the catheter.The trans-vessel wall technique can be used to establish a working channel to the liver parenchyma for administration of any substance, such as chemotherapeutic agents or cells. The detached device can also be used as a marker for ultrasound-guided partial liver resection in "vanishing lesions." The technique should have a low risk of seeding of neoplastic cells. This study in large animals forms a strong basis for translation to clinical studies.

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.

In the search for markers and modulators of vascular disease, miRNAs have emerged as potent therapeutic targets. We investigated miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we profiled miRNA expression in symptomatic versus asymptomatic patients with high-grade carotid artery stenosis. A PCR-based miRNA of plasma, sampled at the carotid lesion site, identified eight deregulated miRNAs (miR-15b, -29c, -30c/d, -150, -191, -210 and -500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser-capture microdissection as well as in situ hybridization revealed a distinct localization of miR-210 in the fibrous caps of atherosclerotic lesions and showed reduced miR-210 expression in the unstable fibrous cap. We confirmed that miR-210 directly targets the tumor suppressor gene adenomatous polyposis coli (APC), thereby affecting Wnt signaling and regulating vascular smooth muscle cell survival, as well as differentiation, in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in two 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. We discovered that 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 vascular smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic disease.

Objective: Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines but their function in vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques, localization to smooth muscle cells in the fibrous cap and positive correlation to inflammation, extracellular matrix remodeling and cytokines. Here, our aim was to evaluate the effects of PCSK6 on flow-mediated vascular remodeling in mice using high-frequency ultrasound and myography. Materials and Methods: PCSK6 -/- and C57Bl/6J mice were compared in this study divided in baseline control and increased flow groups. Increased flow was created in the right common carotid artery (CCA) by ligation of the left CCA. All animals were subjected to high-frequency ultrasound examination prior to surgery, at 3 and 5 weeks after surgery. Upon euthanization 6 weeks post-surgery the right CCA was harvested for myography evaluation, subsequently fixed at optimal stretch and prepared for histological evaluation. Results: The vascular circumference at optimal stretch in myography was strongly correlated (Pearson r=0.74, p Conclusion: Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow.

Objective: Ultrasound BioMicroscopy (UBM), or high-frequency ultrasound, is a novel technique used for assessment of anatomy and physiology small research animals. In this study, we evaluate the UBM assessment of the re-endothelialization process following denudation of the carotid artery in rats. Methods: Ultrasound BioMicroscopy data from three different experiments were analyzed. A total of 66 rats of three different strains (Sprague-Dawley, Wistar and Goto-Kakizaki) were included in this study. All animals were subjected to common carotid artery balloon injury and examined with UBM 2 and 4 weeks after injury. Re-endothelialization in UBM was measured as the length from the carotid bifurcation to the distal edge of the intimal hyperplasia. En face staining with Evans-blue dye was performed upon euthanization at 4 weeks after injury followed by tissue harvest for morphological and immunohistochemical evaluation. Results: A significant correlation (Spearman r=0.63,pen face staining. Analysis by animal strain revealed a similar pattern and a significant growth in re-endothelialization length measured in UBM from 2 to 4 weeks could be identified. Immunohistochemical staining for von Willebrand factor confirmed the presence of endothelium in the areas detected as re-endothelialized by the ultrasound assessment. Conclusion: Ultrasound BioMicroscopy can be used for longitudinal in vivo assessment of the re-endothelialization following arterial injury in rats.

Proprotein convertases (PCSKs) process matrix metalloproteases (MMPs) and cytokines. Apart from PCSK9, the role of these enzymes in vascular disease is largely unknown. Previously, we demonstrated upregulation of PCSK6 in carotid atherosclerosis, primarily localized to smooth muscle cells (SMCs) and positively correlated to inflammation, extracellular matrix remodeling and cytokines. Here, we extended these findings to determine the role of PCSK6 in vascular development and disease. Increased expression of PCSK6 in vascular disease was validated by microarrays from two non-overlapping cohorts of carotid plaques vs. non-atherosclerotic arteries (n=50 patients and n=32 patients, p0.7, p0.5, p-/- mice did not present an obvious vascular phenotype but showed reduced intimal hyperplasia compared to wild-type mice after carotid artery ligation (p=0.015). In vitro, PCSK6 overexpression markedly increased SMC migration upon PDGFBB stimulation (p

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.

ObjectiveVascular 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.MethodsLaminar FSS of arterial level (14 dynes/cm2) 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.ResultsMicroarrays 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.ConclusionFSS 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.Clinical RelevanceIn the arterial wall, endothelial cells are exposed to fluid shear stress imposed by the flowing blood. However, after vascular interventions, where the endothelial layer is denuded and in intimal hyperplasia that develops, luminal smooth muscle cells are exposed to shear stress. We show that TFPI-2 expression is strongly augmented in smooth muscle cells exposed to shear stress and that TFPI-2 co-localizes with caspase-3 in vivo. In addition, TFPI-2 inhibits smooth muscle cell proliferation and induces apoptosis in vitro. The adaption of smooth muscle cells to shear stress is of interest in understanding the pathophysiology behind intimal hyperplasia and restenosis.

Smooth muscle cell (SMC) activation is a hallmark of vascular remodeling associated with downregulation of SMC specific genes and altered cell function. Our aim was to identify a novel set of genes silenced following SMC activation in vascular disease. We compared microarrays from carotid plaques (n=177) vs. undiseased arteries and symptomatic vs. asymptomatic patients and found that ‘muscle contraction’, ‘muscle differentiation’ and ‘cytoskeleton based migration’ were the most significantly downregulated pathways (p=6.06e-23; 5.22e-11; 4.62e-11 respectively). Genes clustered in these categories were known SMC markers together with a novel set of genes functionally coupled to actin cytoskeleton: SYNPO2, SYNM, LMOD1, PDLIM7 and PLN. Transcription factors regulating the expression of these genes showed enrichment of SRF, MYOD1 and MYOGENIN, known to control muscle differentiation. Downregulation of these genes was validated in independent microarrays from carotid (n=21) and coronary plaques (n=38). Their expression was positively correlated to typical SMC markers both in human plaques (Pearson r>0.8, p0.9, p We identified a panel of novel SMC proteins, which are lost in vascular disease and reflect the altered phenotype of SMCs in vascular remodeling. Our results indicate that these could be early and sensitive markers of the SMCs dedifferentiation in atherosclerosis.

Background/Aims: Diabetic patients have an increased risk of restenosis and late stent thrombosis after angioplasty, complications that are related to a defective re-endothelialization. Exendin-4, a stable 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 the study was to investigate the mechanisms by which treatment with exendin-4 could influence re-endothelialization and intimal hyperplasia after vascular injury. Methods: 40 male Sprague-Dawley rats were subjected to balloon injury of the left common carotid artery (LCCA) and treated for four weeks with exendin-4 (1 nmol/day) or vehicle. Intimal hyperplasia and vessel wall elasticity were monitored non-invasively by high frequency ultrasound and re-endothelialization was evaluated upon sacrifice using Evans blue dye. The LCCA was then sectioned for subsequent morphometric and immunohistochemical analyses. To further investigate if and how smooth muscle cells (SMCs) are directly affected by exendin-4 treatment, we studied proliferation and apoptosis of SMCs in vitro. Results and Conclusion: Exendin-4 selectively reduced proliferation of SMCs and intimal hyperplasia in vivo without affecting the re-endothelialization process, however 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 improved outcome for patients undergoing vascular interventions.

Proprotein convertases (PCSKs) are conserved among species and involved in processing of MMPs and growth factors, but poorly characterised in atherosclerosis. Previously we demonstrated upregulation of PCSK6 in a large cohort of plaques from symptomatic vs. asymptomatic patients. This protease localized to smooth muscle cells (SMCs) and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines in plaques. Here we aimed at elucidating its role in vascular development and disease. In zebrafish embryos PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. Increased expression of PCSK6 in vascular pathologies was validated by microarrays from carotid plaques vs. undiseased arteries (n=32 patients, p0.7, p We identified a functional link between elevated expression of PCSK6 and vascular remodeling characterized by SMC activation. The present study establishes PCSK6 as one of the key modulators of pathological processes in relation to plaque vulnerability.

Restenosis is a major complication after coronary angioplasty and stenting. The major cause of restenosis is neointimal hyperplasia, which results from an excessive proliferative response of vascular smooth muscle cells (VSMC) to mechanical injury. Platelet derived growth factor (PDGF) family members (A, B, C, D) are known to be related to vascular remodeling. However whether this role is specific for each one or overlapping remains to be elucidated. Aim: To assess the specific role of PDGF family members (A, B, C, D) in vascular remodeling after injury. Methods: We used an established model of balloon injury in rat carotid artery. The endothelium of the intima is mechanically removed. The animals (n=10/group) were sacrificed at different time points after injury (0-2-20 hours, 2-5-15 days, 6-12 weeks). mRNA from carotid arteries were isolated for gene expression studies using microarray gene expression. Results: PDGFs are differentially expressed in vascular remodeling (mRNA, A adj P val=3.28E-06, B adj P val=4.52E-8, C adj P val=5,91E-15, D adj P val=2,64E-18). Also the expression profile differs among them. We selected the genes highly correlated with each of the PDGFs (Spearman correlation, │rs >0.7│) and identified the most preeminent biological pathways associated to each one. PDGF-A positively correlates with program cell death. On the other hand, PDGF-B and C have some overlapping biological processes. There is positive correlation with blood vessel morphogenesis and angiogenesis (B), cell differentiation (B and C), DNA replication (B and C), antigen presentation and T-cell activation/differentiation (B and C). However, there is negative correlation with platelet activation (B) and cell adhesion (B and C). PDGF-D positively correlates with blood vessel morphogenesis and angiogenesis (like B) and cell differentiation (B, C), but is negatively correlated with T-cell activation/proliferation (opposite effect to B and C), apoptosis (opposite effect to A) and platelet activation (B). Conclusion: We identified specific biological processes for PDGF- A, B, C and D. Despite some overlapping, each one plays a specific role within vascular remodeling.

Background and aims: Diabetic patients have an increased risk of late stent thrombosis formation and restenosis, after angioplasty. It is hypothesized that delayed re-endothelization is a major underlying problem for late-stent thrombosis and also that rapid re-endothelization is essential for preventing restenosis. Exendin-4 is a stable GLP-1 receptor agonist, which is applied in clinical treatment of diabetes. Beside its insulinotropic action, it may also exert direct beneficial effects on endothelial function. We previously reported that exendin-4 is able to activate the GLP-1 receptor on human coronary artery endothelial cells, leading to increased cell proliferation and decreased apoptosis. Therefore, our aim was to study if exendin-4 can influence endothelialization and decrease neointima formation after vascular injury. Materials and Methods: Balloon injury of the left common carotid artery (LCCA) was performed on Sprague-Dawley rats. Rats were randomized into two groups and treated for four weeks with exendin-4 (1 nmol/day) or saline. Intimal hyperplasia and endothelialization was monitored non-invasively by high frequency ultrasound and upon sacrifice with Evans blue, respectively. The LCCA was then sectioned for subsequent morphometric and immunohistochemical analyses. To further investigate if and how smooth muscle cells (SMCs) are directly affected by exendin-4 treatment, we studied proliferation and apoptosis of SMCs in vitro . Results and conclusions: Our findings show that exendin-4 selectively reduces SMC proliferation and intimal hyperplasia through activation of cAMP signaling and endothelial nitric oxide synthase without influencing re-endothelialization. Treatment with exendin-4 did however improve arterial wall elasticity. Together, these effects of exendin-4 are highly desirable and may lead to improved outcome for patients undergoing vascular interventions

Recently we demonstrated upregulation of the proprotein convertase PCSK6 in a large cohort of human carotid atherosclerotic plaques (n=127) compared to normal arteries and in symptomatic vs. asymptomatic lesions. PCSK6 was localized to smooth muscle cells (SMCs) in the fibrous cap and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines. Here, we aimed at elucidating the role of this protease in vascular disease by examining its expression in different human pathologies and in animal models. Increased expression of PCSK6 in vascular diseases was validated in public microarray datasets and other available human cohorts. PCSK6 was upregulated in carotid atherosclerotic plaques vs. controls (n=32 patients, pvs. normal tissue (n=14, pvs. tricuspid patients (n=244, p=0.012). By eQTL analyses, several SNPs in the PCSK6 genomic region were shown to influence its expression in carotid plaques and TAA tissue. Among these, rs6598465 showed a mild association to progression of maximum intima-media thickness in the left and right arteries in a separate cohort of high-risk coronary artery disease subjects (n=3400, p=0.037). By immunohistochemistry, PCSK6 localized mainly to SMCs in carotid plaques, AAA and TAA tissue, but was also found to be expressed by CD68 and CD163+ macrophages. Investigation of mouse, rat and human artery tissues with pronounced intimal hyperplasia revealed strong expression of PCSK6 in proliferating SMCs. In rat carotid artery balloon injury, PCSK6 was downregulated in the early phases after injury mostly defined by inflammatory response, while upregulated in later phases with prominent SMC activation, and consistently localized in SMCs. Expression of PCSK6 in this model was strongly positively correlated solely to PDGFB and IGF1 (p We established a functional link between elevated expression of PCSK6 and vascular diseases characterized by inflammation and SMC proliferation. Further investigations in vitro are necessary to provide mechanistic insight into the role of this protease in vascular disease.

Embolism from atherosclerotic plaques in the carotid artery is a major cause of stroke, disability and death. The aim of this project was to explore gene expression signatures and clinical parameters of carotid plaque instability in the Biobank of Karolinska Endarterectomies (BiKE). Whole transcriptome profiling by microarrays was performed on n=127 plaques from n=40 asymptomatic (AS) and n=87 symptomatic (S) patients, and on n=10 normal arteries. After normalization and gender adjustments, differential gene expression was assessed by multiple t-tests with Bonferroni correction. Gene set enrichment and pathway analyses were done using DAVID software. Genes significantly upregulated between S and AS plaques were associated with oxidative stress, immune system activation, exopeptidases, lipoprotein particle clearance and bone resorption, Transcription factors and markers of smooth muscle cells were downregulated in this comparison. When S patients with transitory ischemic attacks and minor stroke were compared with amaurosis fugax , most enriched GO terms were angiogenesis, oxidoreductases and inflammation. Patients on statins showed enrichment of calcification, osteoblast differentiation and negative regulation of angiogenesis. Prolonged immune response, lymphocyte activation and apoptosis were processes ongoing in plaques obtained >1 month compared to 0.90, p In conclusion, analysis of the largest existing global transcriptome database of carotid atherosclerosis reveals novel genes and pathways related to patient phenotype, confirms the important role of inflammation and lipid metabolism, and highlights the role of repair processes and lymphocyte activation in human 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 P 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.

Restenosis caused by intimal hyperplasia (IH) remains a significant drawback for vascular interventions. It is crucial to understand the molecular mechanisms that control activation of smooth muscle cells (SMCs) after the injury in order to develop strategies to prevent IH. The purpose of the present study was to investigate the early alterations in arterial-wall gene expression after balloon injury in the rat carotid artery with focus on the induction of an inflammatory response.Twenty-four male Sprague-Dawley rats were subjected to injury of the left common carotid artery by using a 2-F Fogarty catheter. The arteries were harvested 5, 10, and 20 hours after injury. Uninjured arteries from an additional eight rats were used as controls. RNA was isolated and used for genome-wide microarray expression analysis, followed by validation of selected genes with quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed on the cross-sectioned vessels.Analysis of gene expression by microarrays showed that the most differentially expressed genes were primarily associated with inflammation, cell proliferation, migration, and adhesion. As confirmed by qRT-PCR, microarray data showed a significant (P.005) upregulation of cytokines and chemokines (IL-6, CCL2, CXCL1, AIMP1, and CD44) just 5 hours after injury. Immunohistochemistry demonstrated that CCL2 and the adhesion receptor CD44 were expressed by SMCs in the early response to injury and in the absence of leukocyte infiltration.Arterial injury is followed by an early induction of inflammatory genes in the vessel wall that appears to be confined to SMCs.

Objective High-throughput immunohistochemistry (IHC) techniques are not commonly applied in studies of vascular diseases. Tissue microarrays (TMAs) are collections of multiple tissue cores placed in parallel in a single acceptor paraffin block, thereby enabling large-scale expression analyses of many samples concomitantly. Our aim was to evaluate this approach in qualitative and quantitative studies of candidate proteins implicated in atherosclerotic carotid stenosis. Methods and Results We constructed TMAs from a collection of 34 carotid plaques (6 asymptomatic/28 symptomatic patients) and assayed them for expression of 21 proteins by IHC. Majority of proteins chosen for the study were previously identified through microarray profiling of symptomatic vs. asymptomatic plaques (n=150) from the Biobank of Karolinska Endarterectomies. Quantification of stainings highlighted differential expression of several proteins in unstable compared to stable lesions (ADIPOR1, CD36, CD137, DOCK7), the most significantly upregulated being a secreted member of the proprotein convertase family, PCSK6 (p Conclusions Here we demonstrate the feasibility of a scaled-up method for simultaneous and standardized expression studies in a large collection of human atherosclerotic lesions. Our results show that PCSK6, a novel protease previously not associated with atherosclerosis, is detected at increased levels in symptomatic carotid plaques, possibly in association with inflammation and extracellular matrix remodeling leading to plaque rupture. Further mechanistic studies are needed to dissect the role of PCSK6 in progression of atherosclerosis.

The discovery of an entirely new method of recognition and regulation of gene expression and protein translation by microRNAs (miRs) has provided new hope for innovative disease discovery and therapy. Research in recent years has assigned miRs a key regulatory role during various pathological processes. Lately, their potential as biomarkers in disease received a lot of attention. Aim of the present study was to explore the role of miRs as potential regulators and plasma biomarkers in patients with carotid stenosis and at risk of stroke. Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we were able to profile the regulation of miRs in patients with asymptomatic vs. symptomatic carotid stenosis undergoing carotid endarterectomy. A PCR-based microRNA array of plasma, sampled at the carotid lesion site, identified eight miRs (miRs-15b, -29c, -30c/d, -150, -191, -210 and -500) being substantially altered in symptomatic vs. asymptomatic patients. In peripheral plasma samples, the expression of miRs-150 and -191 remained significantly different. Using an in vitro cell streamer system, we subjected human carotid smooth muscle cells, as well as human carotid endothelial cells, to physiologic and pathologic flow conditions. Interestingly and in accordance with our findings from the initial plasma profiling study, laminar (physiologic) flow induced miR-210 up-regulation, whereas turbulent (pathologic) flow suppressed miR-210 expression in both cell lines. In addition, similar significant changes in miR-210 regulation were detectable in a model of vascular remodelling, balloon injury of the carotid artery in Sprague Dawley rats. Of importance, miR-210 targets several key genes and signalling pathways involved in vascular remodelling and atherosclerotic plaque stability. The present study explores the diagnostic, prognostic and therapeutic potential of miR regulation in patients with symptomatic carotid stenosis and increased risk of stroke. We were able to identify miRs-150 and -191 as potential diagnostic plasma markers, and miR-210 as a key modulator of pathologic processes in atherosclerosis-related vascular remodelling.

Insulin like growth factor-1 (IGF-1) has been shown to have a prominent effect on smooth muscle cell proliferation, migration and apoptosis. We and others have previously shown that inhibition of IGF-1 related signaling can attenuate intimal hyperplasia by attenuating smooth muscle cell proliferation and promoting apoptosis after vascular injury. We hypothesized with support from preliminary work in our laboratory that IGF-1 inhibition will decrease atherosclerosis burden, but may negatively affect plaque stability by increasing SMC apoptosis. Picropodophyllin(PPP) is a specific inhibitor of the IGF-1 receptor phosphorylation and is currently under development as an anti-cancer drug. The aim of this study was to study the short and long term effects of IGF-1 inhibition using PPP in ApoE deficient mice Methods and Results 12 week old Apo E deficient mice (n=64) were treated with an oral preparation of PPP or vehicle (controls) for a period of 10 and 18 weeks. Blood samples were collected before sacrifice and aortas, brachiocephalic trunks and liver samples harvested. Semi-quantitative analysis of the aortic and brachiocephalic trunks using Sudan IV enface staining showed an increase in plaque burden and was significant after 18 weeks. There were no significant differences in weight, serum creatinine, albumin or glucose levels between the two groups. However, we noted 18% lower levels of IGF-1, doubled levels of growth hormone, 30% increase in serum cholesterol levels in the PPP treated group (p Conclusion In contrast to our hypothesis, PPP treatment increased plaque burden but led to plaques with a more stable phenotype. The proposed mechanism is via an increase in serum cholesterol, however further work has to be performed to confirm the pathways involved. In summary, our results suggest that the effects of inhibition of IGF-1R phosphorylation in atherosclerotic animals cannot be extrapolated from results in healthy subjects and has to be considered when designing drugs in the future.

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.

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.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.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; p0.0001) 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.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.