Atherosclerotic Plaque Instability Research Articles

NecroX-7 reduces necrotic core formation in atherosclerotic plaques of Apoe knockout mice

Background and aimsA large necrotic core is a key feature of atherosclerotic plaque instability. Necrotic cellular debris accumulates in the lipid-rich core and promotes inflammation, destabilization and ultimately rupture of the plaque. Although the role of necrosis in atherosclerosis is rather clear-cut, not many strategies have been performed up till now to specifically target plaque necrosis. In the present study, we tested the plaque stabilizing potential of NecroX-7, a novel compound with antioxidative and anti-necrotic properties. MethodsMale apolipoprotein E (Apoe) knockout mice were treated with NecroX-7 (30 mg/kg) or vehicle, 3 times per week, via intraperitoneal injections for 16 weeks. Meanwhile, mice were fed a western-type diet to induce plaque formation. ResultsNecroX-7 reduced total plaque burden in the thoracic aorta as compared to vehicle-treated mice, without affecting total plasma cholesterol. Plaques in the aortic root of NecroX-7-treated mice showed a significant decrease in necrotic core area, 8-oxodG, iNOS and MMP13 expression, while collagen content and minimum fibrous cap thickness were increased. Moreover, NecroX-7 treatment reduced the expression of multiple inflammation markers such as TNFα, IL1β, iNOS, HMGB1 and RAGE in a NF-κB-dependent manner. In vitro, NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced mitochondrial ROS formation, necrosis, iNOS expression and HMGB1 release in primary macrophages. ConclusionsNecroX-7 improves features of plaque stability in Apoe knockout mice by reducing necrotic core formation, oxidative stress and inflammation, and by increasing collagen deposition and fibrous cap thickness. Therefore, NecroX-7 could be a promising pleiotropic drug for the treatment of atherosclerosis.

Increase of Serum CXCL16 Level Correlates Well to Microembolic Signals in Acute Stroke Patients with Carotid Artery Stenosis

BackgroundThe majority of strokes are combined with the instability of atherosclerotic plaques. Microembolic signals (MES) have been considered as evidence of plaque destabilization. We found that increased CXCL16 correlated to atherosclerotic ischemic stroke. Thus, we explored whether CXCL16 correlates to MES. MethodsThe study recruited 104 controls and 118 patients with acute ischemic stroke that has an ipsilateral carotid artery stenosis of >50%. The ipsilateral middle cerebral artery of patients was insonated for 60min using Doppler device within 72h of their clinical presentation. ResultsWe found that CXCL16 was significantly increased in the stroke patients. Furthermore, there was a significant difference in CXCL16 between the MES-positive and MES-negative patients. Using CXCL16 to distinguish the controls and stroke patients, the area under the ROC curve (AUC) was 0.722; the cut-off value was 2.015ng/ml. The sensitivity and specificity were 70.5% and 67.9%, respectively. Furthermore, if we used CXCL16 to distinguish the MES-positive and MES-negative patients, the AUC was 0.736; the cut-off value was 2.115ng/ml. The sensitivity and specificity were 88.5% and 56.5%, respectively. ConclusionsHigher levels CXCL16 may be a biomarker for predicting stroke incidence and might contribute to plaque destabilization.

Circulation Research “In This Issue” Anthology

Circulation Research “In This Issue” Anthology

MafB and the role of macrophage apoptosis in atherosclerosis: A time to kill, a time to heal

MafB and the role of macrophage apoptosis in atherosclerosis: A time to kill, a time to heal

Mast cells are increased in the media of coronary lesions in patients with myocardial infarction and may favor atherosclerotic plaque instability

ObjectivesMast cells (MCs) may play an important role in plaque destabilization and atherosclerotic coronary complications. Here, we have studied the presence of MCs in the intima and media of unstable and stable coronary lesions at different time points after myocardial infarction (MI). MethodsCoronary arteries were obtained at autopsy from patients with acute MI (up to 5 days old; n=27) and with chronic MI (5–14 days old; n=18), as well as sections from controls without cardiac disease (n=10). Herein, tryptase-positive MCs were quantified in the intima and media of both unstable and stable atherosclerotic plaques in infarct-related and non-infarct-related coronary arteries. ResultsIn the media of both acute and chronic MI patients, the number of MCs was significantly higher than in controls. This was also found when evaluating unstable and stable plaques separately. In patients with chronic MI, the number of MCs in unstable lesions was significantly higher than in stable lesions. This coincided with a significant increase in the relative number of unstable plaques in patients with chronic MI compared with control and acute MI. No differences in MC density were found between infarct-related and non-infarct-related coronary arteries in patients with MI. ConclusionThe presence of MCs in the media of both stable and unstable atherosclerotic coronary lesions after MI suggests that MCs may be involved in the onset of MI and, on the other hand, that MI triggers intra-plaque infiltration of MCs especially in unstable plaques, possibly increasing the risk of re-infarction.

The role of macrophage transcription factor MafB in atherosclerotic plaque stability

Background and aimsMacrophage differentiation is associated with the development of atherosclerosis and plaque vulnerability and is regulated by transcription factor MafB. We previously reported that MafB attenuates macrophage apoptosis, which is associated with atherosclerotic plaque instability. The aim of this study was to elucidate the role of MafB in the progression of atherosclerotic plaque. MethodsWe generated macrophage-specific dominant-negative (DN) MafB transgenic mice and intercrossed DN-MafB mice with apolipoprotein E (ApoE) knockout (KO) mice. ResultsThere was no significant difference in advanced atherosclerotic lesion area between DN-MafB/ApoE KO mice and littermate control ApoE KO mice 9 weeks after high-cholesterol diet. However, DN-MafB/ApoE KO mice showed significantly larger necrotic cores and lower collagen content in atherosclerotic plaques than ApoE KO mice. Although there was no difference in intraplaque macrophage infiltration and efferocytosis, DN-MafB/ApoE KO mice showed significantly more apoptotic macrophages at the plaque edges than did ApoE KO mice. Real-time PCR analysis revealed that peritoneal macrophages of DN-MafB/ApoE KO mice had a greater increase in matrix metalloproteinase-9 and mRNA expression of inflammatory/M1 macrophage markers (tissue necrosis factor-α, interleukin-6, CD11c, and p47phox) after lipopolysaccharide stimulation than those of ApoE KO mice. ConclusionMacrophage-specific inhibition of MafB may destabilize atherosclerotic plaques in advanced lesions.

Abstract 110: Carotid Plaque Instability is Associated with an Increase in the Serum Ratio of circularRNA-284 to microRNA-221

Introduction: Atherosclerotic cap thinning and plaque instability occur as a result of a decrease in vascular smooth muscle cell proliferation, which is partly regulated by alterations in the expression of non-coding RNAs in the arterial wall. We recently reported that miR-221 expression in the carotid plaque shoulder is reduced immediately following a carotid-related ischemic cerebrovascular event and returns to normal levels after seven days. We hypothesized that changes in the expression of non-coding RNAs within carotid plaques are reflected in the serum of asymptomatic and acutely symptomatic patients with carotid disease. Methods: Serum levels of microRNA (miR) -221 and a circular RNA with potential binding sites for miR-221 (circR-284), were measured using real-time polymerase chain reaction in 41 patients undergoing carotid endarterectomy. Patients were grouped into those who were asymptomatic and those with an acute ischemic cerebrovascular event within the previous 5 days (urgent). Results: miR-221 was significantly lower (0.25 ± 0.11 vs. 1.00 ± 0.31, p = 0.01) while circR-284 was significantly elevated (2.96 ± 1.16 vs. 1.00 ± 0.37, p = 0.06) in the serum of the urgent compared to the asymptomatic group. Serum levels of these RNAs alone did not exhibit favorable sensitivity and specificity for use as a biomarker indicative of carotid-related ischemic stroke. The ratio of serum circR-284:miR-221, however, was significantly elevated in the urgent group [11.7 ± 0.48 vs. 1.0 ± 0.6, p = 0.0002 (Figure, A)]. Furthermore, receiver operator curve analysis of circR-284:miR-221 ratio demonstrated favorable sensitivity and specificity (Figure, B) for detecting carotid plaque rupture and ischemic stroke. Conclusions: Increases in the ratio of serum circR-284:miR-221 has potential as a diagnostic biomarker of carotid-related ischemic stroke. This data also supports the use and development of functionally related pairs of circulating non-coding RNAs as biomarkers.

Abstract 3: Targeting Macrophage Necroptosis for Therapeutic and Diagnostic Interventions to Treat Atherosclerosis

Background: Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. As such, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that in contrast to apoptosis, induces a pro-inflammatory state. We hypothesize that atherogenic ligands within the plaque promote macrophage necroptosis and this process underlies necrotic core formation and drives atherosclerotic plaque instability. Results: In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying plaque necroptosis showed that macrophages treated with oxidized LDL have increased expression of necroptotic genes RIP3 and MLKL through ROS-dependent activation of the promoter region and increased RIP3 and MLKL phosphorylation. Combined treatment with oxLDL and DAMPs (damage associated molecular patterns) amplified macrophage necroptotic cell death, indicating that additional inflammatory stimuli present in the lesion could act synergistically to promote necroptosis. Using a radiotracer developed with the necroptosis inhibitor Nec-1, we show that 123 I-Nec1 localizes specifically to atherosclerotic plaques in Apoe-/- mice, and its uptake is tightly correlated to lesion areas by ex vivo nuclear imaging. Furthermore, treatment of Apoe-/- mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation. Conclusions: Our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.

Treatment with a GnRH receptor agonist, but not the GnRH receptor antagonist degarelix, induces atherosclerotic plaque instability in ApoE(-/-) mice.

Androgen-deprivation therapy (ADT) for prostate cancer has been associated with increased risk for development of cardiovascular events and recent pooled analyses of randomized intervention trials suggest that this primarily is the case for patients with pre-existing cardiovascular disease treated with gonadotropin-releasing hormone receptor (GnRH-R) agonists. In the present study we investigated the effects of the GnRH-R agonist leuprolide and the GnRH-R antagonist degarelix on established atherosclerotic plaques in ApoE−/− mice. A shear stress modifier was used to produce both advanced and more stable plaques in the carotid artery. After 4 weeks of ADT, increased areas of necrosis was observed in stable plaques from leuprolide-treated mice (median and IQR plaque necrotic area in control, degarelix and leuprolide-treated mice were 0.6% (IQR 0–3.1), 0.2% (IQR 0–4.4) and 11.0% (IQR 1.0-19.8), respectively). There was also evidence of increased inflammation as assessed by macrophage immunohistochemistry in the plaques from leuprolide-treated mice, but we found no evidence of such changes in plaques from control mice or mice treated with degarelix. Necrosis destabilizes plaques and increases the risk for rupture and development of acute cardiovascular events. Destabilization of pre-existing atherosclerotic plaques could explain the increased cardiovascular risk in prostate cancer patients treated with GnRH-R agonists.

Atorvastatin attenuates atherosclerotic plaque destabilization by inhibiting endoplasmic reticulum stress in hyperhomocysteinemic mice.

Endoplasmic reticulum (ER) stress has been suggested to play a role in the progression of plaque vulnerability and the occurrence of acute complications of coronary atherosclerosis. Atorvastatin is known to exert pleiotropic effects on the cardiovascular system. The present study aimed to examine the stabilizing effects of atorvastatin on vulnerable plaques within hyperhomocysteinemic apolipoprotein E‑deficient (ApoE‑/‑) mice, and to investigate the potential mechanisms underlying ER stress in ApoE‑/‑ mice and macrophages. In the present study, ApoE‑/‑ mice were administrated methionine or atorvastatin, and were sacrificed after 2 months. Necrotic core size, collagen content and inflammatory cytokine infiltration were subsequently measured in the aortic lesions, in order to investigate plaque stability. Treatment with atorvastatin decreased the number and size of necrotic cores, increased collagen content, and downregulated tumor necrosis factor (TNF)‑α and matrix metalloproteinase (MMP)‑9 mRNA expression, as compared with the methionine group. Immunohistochemical analysis indicated that atorvastatin administration prevented ER stress activation in aortic lesions of hyperhomocysteinemic mice. Furthermore, macrophages were challenged with homocysteine (Hcy) in the presence or absence of atorvastatin and thapsigargin (an ER stress inducer). Atorvastatin suppressed Hcy‑induced ER stress, and downregulated TNF‑α and MMP‑9 mRNA expression in the macrophages. Conversely, thapsigargin attenuated the inhibitory effects of atorvastatin against Hcy‑induced TNF‑α and MMP‑9 expression. These results indicated that hyperhomocysteinemia may promote atherosclerotic plaque development and instability. In addition, atorvastatin was able to improve atherosclerotic plaque stability in hyperhomocysteinemic mice by inhibiting ER stress.

Involvement of Angiotensin II Type 1 and 2 Receptors in Gelatinase Regulation in Human Carotid Atheroma in vitro.

Matrix metalloproteinases (MMPs), angiotensin II (AII) and its receptors are implicated in atherosclerotic plaque instability, however the roles of the two receptor subtypes, ATR1 and ATR2, in MMP regulation remain uncertain. In this study, we investigated the effect of ATR1 and ATR2 blockade on the expression and activity of MMP-2, MMP-3 and MMP-9, in human carotid atheroma. Atheroma samples (n=36) were obtained from patients undergoing carotid endarterectomy. The effects of ATR1 (irbesartan), ATR2 (PD123319) and combined ATR1 and ATR2 blockade on the expression and activity of the MMPs and the expression of tissue inhibitors of metalloproteinases (TIMPs) were investigated in explant culture experiments. Paired atheroma samples were incubated with the intervention or media control for 4 days. Protein levels (MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, TIMP-4, ATR1 and ATR2) were determined by ELISA. Overall gelatinase activity and specific activation were measured by chromogenic activity assays and zymography, respectively. ATR1 blockade, but not ATR2 blockade significantly reduced TIMP-1, TIMP-2 and TIMP-4 expression in atheroma supernatant. Combined ATR1 and ATR2 blockade significantly reduced MMP-2, MMP-3 and MMP-9 expression. MMP-2 and MMP-9 relative activation, and overall MMP-9 catalytic capacity were significantly increased by ATR1 blockade. Our findings suggest that ATR1 blockade reduces TIMP expression and increases gelatinase activity in human carotid atheroma.

Regulation and involvement of matrix metalloproteinases in vascular diseases.

Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases whose main function is to degrade and deposit structural proteins within the extracellular matrix (ECM). A dysregulation of MMPs is linked to vascular diseases. MMPs are classified into collagenases, gelatinases, membrane-type, metalloelastase, stromelysins, matrilysins, enamelysins, and unclassified subgroups. The production of MMPs is stimulated by factors such as oxidative stress, growth factors and inflammation which lead to its up- or down-regulation with subsequent ECM remodeling. Normally, excess activation of MMPs is controlled by their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). An imbalance of MMPs and TIMPs has been implicated in hypertension, atherosclerotic plaque formation and instability, aortic aneurysms and varicose vein wall remodeling. Also, recent evidence suggests epigenetic regulation of some MMPs in angiogenesis and atherosclerosis. Over the years, pharmacological inhibitors of MMPs have been used to modify or prevent the development of the disease with some success. In this review, we discuss recent advances in MMP biology, and their involvement in the manifestation of vascular disease.

Paramagnetic Manganese in the Atherosclerotic Plaque of Carotid Arteries.

The search for adequate markers of atherosclerotic plaque (AP) instability in the context of assessment of the ischemic stroke risk in patients with atherosclerosis of the carotid arteries as well as for solid physical and chemical factors that are connected with the AP stability is extremely important. We investigate the inner lining of the carotid artery specimens from the male patients with atherosclerosis (27 patients, 42–64 years old) obtained during carotid endarterectomy by using different analytical tools including ultrasound angiography, X-ray analysis, immunological, histochemical analyses, and high-field (3.4 T) pulse electron paramagnetic resonance (EPR) at 94 GHz. No correlation between the stable and unstable APs in the sense of the calcification is revealed. In all of the investigated samples, the EPR spectra of manganese, namely, Mn2+ ions, are registered. Spectral and relaxation characteristics of Mn2+ ions are close to those obtained for the synthetic (nano) hydroxyapatite species but differ from each other for stable and unstable APs. This demonstrates that AP stability could be specified by the molecular organization of their hydroxyapatite components. The origin of the obtained differences and the possibility of using EPR of Mn2+ as an AP stability marker are discussed.

CLINICAL SIGNIFICANCE OF PROTEIN GROWTH AND INJURY FACTORS IN ACUTE CORONARY SYNDROME

Aim . As fundamentals for current study we layed upon the comparison tasks for concentration of pregnancy associated plasma protein (PAPP-A) and insulin-like growth factor 1 (IGF-1) in blood plasma of patients with acute coronary syndrome (ACS); confirmation of biological role of PAPP-A and IGF-1 as interacting components of intravascular injury and reparation of atherosclerotic plaque; clinical significance of PAPP-A and IGF-1 in ACS patients. Material and methods . We measured concentrations of PAPP-A and IGF-1 in 71 patient with ACS, mean age 57 year old. The study group had a diagnosed myocardial infarction (MI) in 44 patients, and unstable angina in 27; 9 death cases. Synchronically, we measured PAPP-A and IGF-1 in the control and comparison group. Comparison group consisted of 40 arterial hypertension patients and with stable forms of ischemic heart disease. Controls were 20 almost healthy persons. Results . The concentrations of PAPP-A and IGF-1 in ACS patients that were different from controls and comparison. In unstable angina patients PAPP-A is higher than in controls 3,56 times, and in MI 11,6 times. PAPP-A in MI is 3,2 times higher than in unstable angina. In unstable angina the highest found levels of IGF-1 are 1,2 times higher than in controls. The lowest IGF-1 were in lethal cases of MI, and were 1,27 lower than in controls. Conclusion. PAPP-A and IGF-I are new biochemical markers of intravascular injury and reparation of atherosclerotic plaques, and can be applied in urgent cardiology as analyzers of destruction and morphological instability of atherosclerotic plaques in ACS.

Calcification stimulates inflammatory signalling pathways in human vascular smooth muscle cells

Introduction: Calcification is associated with ageing blood vessels and with diseases including atherosclerosis and osteoarthritis. In particular, small calcified particles correlate with atherosclerotic plaque instability and the reason for this is unknown. Some studies suggest that calcium phosphate (CaP) particles cause mechanical stress in the fibrous cap, while others imply that CaP can be engulfed by macrophages leading to caspase-1 activation and secretion of mature IL-1β. Our own studies suggest that small CaP particles are taken up into vascular smooth muscle cell (VSMC) and can cause cell death. Methods: In this study, the effects of CaP particles on VSMC inflammatory pathway activity was investigated by Western analysis for caspase-1 and Syk, and for IL-1β release by ELISA. Results: We found that CaP particles stimulated the secretion of IL-1β from growing and senescent human VSMCs, with higher levels of IL-1β release seen in senescent cells (up to 1.5ng/ml). IL-1β secretion required caspase-1 activity, but unlike macrophages, priming of cells was not required. IL-1β secretion was blocked by R406, a highly specific inhibitor of the Syk tyrosine kinase. Syk is known to be required for NLRP3 inflammasome activation, and Western blot analysis demonstrated that CaP particles induced Syk phosphorylation after 10 minutes of exposure to VSMCs. Additionally, inhibitors of particle uptake, wortmannin and chlorpromazine reduced CaP particle-induced IL-1β release. Conclusions: Together, these studies indicate that CaP particles enter cells via endocytic mechanisms and activate Syk kinase and caspase-1 upstream of IL-1β release, and highlight that VSMCs have the potential to generate pro-inflammatory signals.

Differential effects of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 on atherosclerosis and monocyte/macrophage invasion.

AimsMMPs contribute to atherosclerotic plaque progression and instability, but the relative potency of their endogenous tissue inhibitors of metalloproteinases (TIMPs) as protective factors has not been defined. We therefore investigated the impact of TIMP-1 and TIMP-2 knockout on atherosclerotic plaque burden and composition in apolipoprotein E-knockout (Apoe−/−) mice and studied the underlying effects on monocyte/macrophage behaviour.Methods and resultsAnalysis of brachiocephalic artery plaques revealed comparable atherosclerotic lesion areas between TIMP-1−/− Apoe−/− or TIMP-2−/− Apoe−/− double deficient mice and relevant age-matched, strain-matched Apoe−/− controls after 8 weeks of high-fat feeding. However, lesions from TIMP-2−/− Apoe−/− mice had higher levels of markers associated with plaque vulnerability, including increased macrophage: vascular smooth muscle cell ratios, larger necrotic core areas, reduced collagen contents, increased macrophage proliferation, and apoptosis frequencies, compared with TIMP-1−/−Apoe−/− and controls. In contrast, TIMP-1−/− Apoe−/− animals only had a significant reduction in vascular smooth muscle cell content compared with Apoe−/− controls. In vitro and in vivo findings implicated heightened monocyte/macrophage invasion in the detrimental effects observed on atherosclerotic plaque composition in TIMP-2−/− Apoe−/− mice. Moreover, TIMP-2 specifically decreased MMP-14-dependent monocyte/macrophage infiltration into sites of experimentally induced inflammation and established atherosclerotic lesions.ConclusionOur data demonstrate that TIMP-2 plays a greater protective role than TIMP-1 during the pathogenesis of atherosclerosis, in part by suppressing MMP-14-dependent monocyte/macrophage accumulation into plaques.

Abstract 13246: The Role of Kca3.1 in Monocyte-macrophage Polarization and Its Relevance to Atherosclerotic Plaque Instability

Background Emerging evidence indicates that pro-inflammatory monocyte-macrophage polarization imbalance play key role in the atherosclerotic plaque progression and instability. The calcium-activated potassium channel KCa3.1 is critically involved in macrophage activation and function. However, the role of KCa3.1 in transcriptional regulation of monocyte-macrophage polarization and its relation to plaque instability is still unknown. Methods THP-1 monocytes were differentiated into macrophages using phorbol myristate acetate (PMA) and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4. Monocytes/macrophages were treated with KCa3.1 blocker TRAM-34 during differentiation or polarization. Animal model of plaque instability was induced by combined partial ligation of the left renal artery and left common carotid artery in apolipoprotein E knockout mice. From 6 weeks after surgery, the animals were treated with subcutaneous injection of TRAM-34 (120 mg/kg/d), or vehicle (miglyol 812) for 4 weeks. Phenotypic markers of macrophages and histological plaque characteristics were analysed. Results Significant up-regulation of KCa3.1 expression was found during the differentiation of THP-1 monocytes to macrophages. TRAM-34 treatment reduced the polarization of THP-1-derived macrophages to proinflammatory M1 macrophage while increasing the polarization of THP-1-derived macrophages to anti-inflammatory M2 macrophages. Further mechanistic studies indicated that KCa3.1 blocker promoted an M2 macrophage phenotype through up-regulation the activation of PPAR-γ. In animal models, KCa3.1 blockade therapy with TRAM-34 strikingly reduced the incidence of plaque rupture and luminal thrombus in carotid arteries. Moreover, macrophages within atherosclerotic lesions of mice receiving TRAM-34 injection showed decreased expression of markers associated with M1 macrophage polarization and enhanced expression of the M2 markers. Conclusion These results suggest that blockade of KCa3.1 suppresses plaque instability at the advanced stage of atherosclerosis by promoting polarization of macrophages toward an M2 phenotype.

Abstract 15140: VEGFR2 Blockade in Murine Vein Grafts Reduces Intraplaque Hemorrhage due to Enhanced Plaque Neovessel Maturation

Introduction: Vein graft patency rates are poor due to the development of extensive neointimal thickening, atherosclerosis and lesion instability. We have previously shown that murine vein graft lesions exhibit leaky neovessels and that intraplaque hemorrhage induces lesion growth. Immature plaque neovessels contribute to atherosclerotic plaque instability by leakage of erythrocytes and leukocytes (intraplaque haemorrhage) in the plaque. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), together with the angiopoietin (Ang)-Tie2 system, regulates the maturation of neovessels. Hypothesis: VEGFR2 blockade induces plaque neovessel maturation resulting in reduced intraplaque hemorrhage and plaque instability. Methods: Donor caval veins were engrafted in carotid arteries of recipient hypercholesterolemic ApoE3*Leiden mice (n=14/group). Mice were treated at day 14, 17, 21 and 25 with VEGFR2 blocking antibodies (DC101) or control IgG antibodies. At day 28 vein grafts were harvested for histological and RNA analysis. Results: Morphometric analysis revealed a striking 50% decrease in vein graft segments that expressed intraplaque hemorrhage in the DC101 treated group. This was accompanied by a significant 25-fold decrease in extravasated erythrocytes. DC101 treatment resulted in a decrease in VEGF expression whereas VEGFR1 and VEGFR2 expression was comparable between the two groups. Connexin 37 and 43 expression was also comparable between the groups, indicating that gap junctions were not affected upon DC101 treatment. However, lesions that exhibit intraplaque hemorrhage showed a strong increase in destabilizing Ang-2. The vein graft lesion area was significantly reduced in the DC101 group, which was accompanied by an increased plaque stability phenotype as a result of a significant reduction in macrophage content and increases in smooth muscle cells and collagen content. Conclusions: Blockade of VEGFR2 leads to reduced intraplaque hemorrhage, decreased vein graft lesion area and increased plaque stability as a result of increased neovessel maturation. This identifies plaque neovascularization as an attractive target for the treatment of unstable atherosclerotic diseases.

Distribution of Matrix Metalloproteinases in Human Atherosclerotic Carotid Plaques and Their Production by Smooth Muscle Cells and Macrophage Subsets.

PurposeIn this study, the potential of matrix metalloproteinase (MMP) sense for detection of atherosclerotic plaque instability was explored. Secondly, expression of MMPs by macrophage subtypes and smooth muscle cells (SMCs) was investigated.ProceduresTwenty-three consecutive plaques removed during carotid endarterectomy were incubated in MMPSense™ 680 and imaged with IVIS® Spectrum. mRNA levels of MMPs, macrophage markers, and SMCs were determined in plaque specimens, and in in vitro differentiated M1 and M2 macrophages.ResultsThere was a significant difference between autofluorescence signals and MMPSense signals, both on the intraluminal and extraluminal sides of plaques. MMP-9 and CD68 messenger RNA (mRNA) expression was higher in hot spots, whereas MMP-2 and αSMA expression was higher in cold spots. In vitro M2 macrophages had higher mRNA expression of MMP-1, MMP-9, MMP-12, and TIMP-1 compared to M1 macrophages.ConclusionMMP-9 is most dominantly MMP present in atherosclerotic plaques and is produced by M2 rather than M1 macrophages.

Therapeutic Role of Innovative Anti-Inflammatory Medications in the Prevention of Acute Coronary Syndrome.

An improved understanding of the pathogenesis of acute coronary syndromes and its relationship to atherosclerotic plaque rupture and thrombosis has contributed to the investigation of novel therapies for prevention and treatment. New data ascribe an increasingly important role of active inflammation in contributing to thinning of the atherosclerotic fibrous cap and plaque instability. Despite this understanding, there are currently no therapeutic approaches to specifically target the unstable plaque. Multiple randomized trials investigating treatment strategies have recently been completed or are currently being conducted, using anti-inflammatory medications, such as methotrexate, colchicine, darapladib, varespladib, losmapimod, and canakinumab, to reduce the incidence of cardiovascular events including acute coronary syndromes. These anti-inflammatory medications differ in their mechanism of action from having widespread targets (as is the case for methotrexate and colchicine) to having specific targets (as is the case for darapladib, varespladib, losmapimod, and canakinumab). The trials investigating the efficacy of darapladib in reducing cardiovascular events revealed no significant benefit when compared with the current standard of care. The varespladib studies were terminated early because of adverse outcomes. However, the outcomes of the remaining drug studies may still contribute to novel therapeutic approaches in the treatment of patients with unstable coronary artery disease.