Biliverdin Reductase B Is a Plasma Biomarker for Intraplaque Hemorrhage and a Predictor of Ischemic Stroke in Patients with Symptomatic Carotid Atherosclerosis

Background: Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with IPH. Objective: We explored the biomarker potential of plasma BLVRB through (1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI), and with recurrent ischemic stroke, and (2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting. Methods: Plasma BLVRB levels were measured in patients with symptomatic carotid atherosclerosis from the PARISK study (n = 177, 5 year follow-up) with and without IPH as indicated by MRI. Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2). Results: Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity (MFI), p = 0.033), but had no association with baseline clinical and biological parameters. Plasma BLVRB levels were also significantly higher in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs. 582.07 ± 545.34 MFI, HR = 1.600, CI [1.092–2.344]; p = 0.016). Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts (1189 ± 258.73 vs. 1752 ± 366.84 MFI; p = 0.004). Conclusions: Plasma BLVRB was associated with IPH and increased risk of recurrent ischemic stroke in patients with symptomatic low- to moderate-grade carotid stenosis, indicating the capacity to monitor the efficacy of IPH-preventive pharmacotherapy in an animal model. Together, these results suggest the utility of plasma BLVRB as a biomarker for atherosclerotic plaque instability

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

Biliverdin Reductase B Is a Plasma Biomarker for Intraplaque Hemorrhage and a Predictor of Ischemic Stroke in Patients with Symptomatic Carotid Atherosclerosis

BACKGROUND: Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with IPH. OBJECTIVE: We explored the biomarker potential of plasma BLVRB through (1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI), and with recurrent ischemic stroke, and (2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting. METHODS: Plasma BLVRB levels were measured in patients with symptomatic carotid atherosclerosis from the PARISK study ( n = 177, 5 year follow-up) with and without IPH as indicated by MRI. Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2). RESULTS: Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity (MFI), p = 0.033), but had no association with baseline clinical and biological parameters. Plasma BLVRB levels were also significantly higher in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs. 582.07 ± 545.34 MFI, HR = 1.600, CI [1.092-2.344]; p = 0.016). Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts (1189 ± 258.73 vs. 1752 ± 366.84 MFI; p = 0.004). CONCLUSIONS: Plasma BLVRB was associated with IPH and increased risk of recurrent ischemic stroke in patients with symptomatic low- to moderate-grade carotid stenosis, indicating the capacity to monitor the efficacy of IPH-preventive pharmacotherapy in an animal model. Together, these results suggest the utility of plasma BLVRB as a biomarker for atherosclerotic plaque instability

Dipeptidyl peptidase-4 is increased in the abdominal aortic aneurysm vessel wall and is associated with aneurysm disease processes.

BACKGROUND:Abdominal aortic aneurysm (AAA) is a potentially life-threatening disease, and until today there is no other treatment available than surgical intervention. Dipeptidyl peptidase-4 (DPP4)-inhibitors, used clinically to treat type 2 diabetes, have in murine models been shown to attenuate aneurysm formation and decrease aortic wall matrix degradation, inflammation and apoptosis. Our aim was to investigate if DPP4 is present, active and differentially expressed in human AAA. METHODS AND RESULTS:DPP4 gene expression was elevated in both media and adventitia of AAA tissue compared with control tissue, as measured by microarrays and qPCR, with consistent findings in external data. The plasma activity of DPP4 was however lower in male patients with AAA compared with age- and gender-matched controls, independently of comorbidity or medication. Immunohistochemical double staining revealed co-localization of DPP4 with cells positive for CD68, CD4 and -8, CD20, and SMA. Gene set enrichment analysis demonstrated that expression of DPP4 in AAA tissue correlated with expression of biological processes related to B- and T-cells, extracellular matrix turnover, peptidase activity, oxidative stress and angiogenesis whereas it correlated negatively with muscle-/actin-related processes. CONCLUSION:DPP4 is upregulated in both media and adventitia of human AAA and correlates with aneurysm pathophysiological processes. These results support previous murine mechanistic studies and implicate DPP4 as a target in AAA disease

Transcriptomic and physiological analyses reveal temporal changes contributing to the delayed healing response to arterial injury in diabetic rats

Objective: Atherosclerosis is a leading cause of mortality in the rapidly growing population with diabetes mellitus. Vascular interventions in patients with diabetes can lead to complications attributed to defective vascular remodeling and impaired healing response in the vessel wall. In this study, we aim to elucidate the molecular differences in the vascular healing response over time using a rat model of arterial injury applied to 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 20 hours, 5 days, and 2, 4, and 6 weeks. Noninvasive morphological and physiological assessment of the CCA was performed with ultrasound biomicroscopy (Vevo 2100) and corroborated with histology. Total RNA was isolated from the injured CCA at each timepoint, and microarray profiling was performed (n = 3 rats per timepoint; RaGene-1_0-st-v1 platform). Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database, and Cytoscape software. Results: Significant increase in the neointimal thickness (P < .01; two-way analysis of variance) as well as exaggerated negative remodeling was observed after 2 weeks of injury in GK rats compared with heathy rats, which was confirmed by histological analyses. Bioinformatic analyses showed defective expression patterns for smooth muscle cells and immune cell markers, along with reduced expression of key extracellular matrix-related genes and increased expression of pro-thrombotic genes, indicating potential faults on cell regulation level. Transcription factor–protein-protein interaction analysis provided mechanistic evidence with an array of transcription factors dysregulated in diabetic rats. Conclusions: In this study, we have demonstrated that diabetic rats exhibit impaired arterial remodeling characterized by a delayed healing response. We show that increased contractile smooth muscle cell marker expression coincided with decreased matrix metalloproteinase expression, indicating a potential mechanism for a lack of extracellular matrix reorganization in the impaired vascular healing in GK rats. These results further corroborate the higher prevalence of restenosis in patients with diabetes and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes. Moreover, the presented study provides the research community with the valuable longitudinal gene expression data bank for further exploration of diabetic vasculopathy. : Clinical Relevance: Vascular interventions causes injury to the arterial wall, which in turn induces a healing response to restore vessel wall homeostasis. However, in patients with diabetes, such interventions lead to exaggerated healing response and defective remodeling. There is a need to understand the molecular mechanisms underlying the defective healing response in diabetes. In this study, ultrasound biomicroscopy, histology, and microarray profiling were used to demonstrate the transcriptional and physiological changes at various timepoints following arterial injury in healthy Wistar and diabetic GK rats. This study also provides a database of longitudinal transcriptional changes for the research community to study vascular healing in diabetes

Lack of PCSK6 Increases Flow-Mediated Outward Arterial Remodeling in Mice

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-/- 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-/- 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-/- media. Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow

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