Mihaela Nitulescu

Inhibition of Tumor Necrosis Factor-{alpha} Reduces Atherosclerosis in Apolipoprotein E Knockout Mice.

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-&agr; (TNF-&agr;), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-&agr; in atherogenesis. Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-&agr; were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-&agr; exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoEo mice with apoEotnf-&agr;o bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). Conclusions—These findings demonstrate that TNF-&agr; is actively involved in the progression of atherosclerosis. Accordingly, TNF-&agr; represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

Inhibition of Tumor Necrosis Factor-α Reduces Atherosclerosis in Apolipoprotein E Knockout Mice

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-&agr; (TNF-&agr;), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-&agr; in atherogenesis. Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-&agr; were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-&agr; exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoEo mice with apoEotnf-&agr;o bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). Conclusions—These findings demonstrate that TNF-&agr; is actively involved in the progression of atherosclerosis. Accordingly, TNF-&agr; represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

Evidence Supporting a Key Role of Lp-PLA2-Generated Lysophosphatidylcholine in Human Atherosclerotic Plaque Inflammation

Objectives—To determine whether the level of lysophosphatidylcholine (lysoPC) generated by lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with severity of inflammation in human atherosclerotic plaques. Elevated plasma Lp-PLA2 is associated with increased cardiovascular risk. Lp-PLA2 inhibition reduces atherosclerosis. Lp-PLA2 hydrolyzes low-density lipoprotein–oxidized phospholipids generating lysoPCs. According to in vitro studies, lysoPCs are proinflammatory but the association between their generation and plaque inflammation remains unknown. Methods and Results—Inflammatory activity in carotid plaques (162 patients) was determined immunohistochemically and by analyzing cytokines in homogenates (multiplex immunoassay). LysoPCs were quantified using mass spectrometry and Lp-PLA2 and the lysoPC metabolite lysophosphatidic acid (LPA) by ELISA. There was a strong correlation among lysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 in plaques. LysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 correlated with interleukin-1&bgr;, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1&bgr;, regulated on activation normal T-cell expressed and secreted, and tumor necrosis factor-&agr; in plaques. High lysoPC and Lp-PLA2 correlated with increased plaque macrophages and lipids and with low content of smooth muscle cells, whereas LPA only correlated with plaque macrophages. Lp-PLA2, lysoPC 16:0, 18:0, and 18:1, but not LPA were higher in symptomatic than in asymptomatic plaques. Conclusions—The associations among Lp-PLA2, lysoPCs, LPA, and proinflammatory cytokines in human plaques suggest that lysoPCs play a key role in plaque inflammation and vulnerability. Our findings support Lp-PLA2 inhibition as a possible strategy for the prevention of cardiovascular disease.

Inhibition of Tumor Necrosis Factor- Reduces Atherosclerosis in Apolipoprotein E Knockout Mice

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-&agr; (TNF-&agr;), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-&agr; in atherogenesis. Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-&agr; were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-&agr; exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoEo mice with apoEotnf-&agr;o bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). Conclusions—These findings demonstrate that TNF-&agr; is actively involved in the progression of atherosclerosis. Accordingly, TNF-&agr; represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

Soluble Urokinase Plasminogen Activator Receptor is Associated With Inflammation in the Vulnerable Human Atherosclerotic Plaque.

Background and Purpose— Recently, plasma soluble urokinase plasminogen activator receptor (suPAR) has gained interest as a marker of cardiovascular risk. suPAR is released through the cleavage of urokinase plasminogen activator receptor (uPAR), which is found in monocytes, activated T-lymphocytes and endothelial cells, all involved in atherosclerosis. suPAR levels have been well studied in plasma, but no studies have focused on suPAR in human atherosclerotic plaques. The aim of this study was to determine whether suPAR measured in the plaque is associated with symptomatic plaques and plaque inflammation. Methods— Plasma and carotid plaques from 162 patients were analyzed. Lipids, collagen, uPAR, and macrophages were measured histologically. Cytokines and suPAR were measured in homogenized plaque extracts using multiplex immunoassay and ELISA, respectively. Plasma levels of suPAR were analysed with ELISA. CD3, CD4, as well as uPAR mRNA expression were assessed with quantitative real-time polymerase chain reaction in plaque homogenates from 123 patients. Results— Plaque and plasma suPAR levels were higher in symptomatic patients compared with asymptomatic patients. Plaque suPAR levels correlated with plaque content of lipids and macrophages and with proinflammatory chemokines and cytokines monocyte chemoattractant protein 1, tumor necrosis factor &agr;, interleukin 1&bgr;, interleukin 6, platelet-derived growth factor AB/BB, monocyte inflammatory protein 1&bgr;, regulated on activation normal T-cell expressed and secreted, and s-CD40L. uPAR mRNA and histological staining for uPAR correlated with plaque content of suPAR. Conclusion— This study shows that suPAR in human carotid plaques and plasma is associated with the presence of symptoms and that plaque suPAR is associated with the vulnerable inflammatory plaque. These findings strengthen the hypothesis of suPAR as a future marker of vulnerable atherosclerotic plaques.

Dating Components of Human Atherosclerotic Plaques

Rationale: Atherosclerotic plaques that give rise to acute clinical symptoms are typically characterized by degradation of the connective tissue and plaque rupture. Experimental studies have shown that mechanisms to repair vulnerable lesions exist, but the rate of remodeling of human plaque tissue has not been studied. Objective: In the present study, we determined the biological age of different components of advanced human atherosclerotic plaques by analyzing tissue levels of 14C released into the atmosphere during the nuclear weapons tests in the late 1950s and early 1960s. Methods and Results: Atherosclerotic plaques were obtained from 10 patients (age 46 to 80 years) undergoing carotid surgery. Different regions of the plaques were dissected and analyzed for 14C content using accelerator mass spectrometry. At the time of surgery, the mean biological age of the cap region was 6.4±3.2 years, which was significantly lower than that of the shoulder region (12.9±3.0 years, P<0.01), the interface toward the media (12.4±3.3 years, P<0.01), and the core (9.8±4.5 years, P<0.05). Analysis of proliferative activity and rate of apoptosis showed no signs of increased cellular turnover in the cap, suggesting that the lower 14C content reflected a more recent time of formation. Conclusions: These results show that the turnover time of human plaque tissue is very long and may explain why regression of atherosclerotic plaque size rarely is observed in cardiovascular intervention trials.

Sphingolipids Contribute to Human Atherosclerotic Plaque Inflammation

Objective— Lipids are central to the development of atherosclerotic plaques. Specifically, which lipids are culprits remains controversial, and promising targets have failed in clinical studies. Sphingolipids are bioactive lipids present in atherosclerotic plaques, and they have been suggested to have both proatherogenic and antiatherogenic. However, the biological effects of these lipids remain unknown in the human atherosclerotic plaque. The aim of this study was to assess plaque levels of sphingolipids and investigate their potential association with and contribution to plaque vulnerability. Approach and Results— Glucosylceramide, lactosylceramide, ceramide, dihydroceramide, sphingomyelin, and sphingosine-1-phosphate were analyzed in homogenates from 200 human carotid plaques using mass spectrometry. Inflammatory activity was determined by analyzing plaque levels of cytokines and plaque histology. Caspase-3 was analyzed by ELISA technique. Expression of regulatory enzymes was analyzed with RNA sequencing. Human coronary artery smooth muscle cells were used to analyze the potential role of the 6 sphingolipids as inducers of plaque inflammation and cellular apoptosis in vitro. All sphingolipids were increased in plaques associated with symptoms and correlated with inflammatory cytokines. All sphingolipids, except sphingosine-1-phosphate, also correlated with histological markers of plaque instability. Lactosylceramide, ceramide, sphingomyelin, and sphingosine-1-phosphate correlated with caspase-3 activity. In vitro experiments revealed that glucosylceramide, lactosylceramide, and ceramide induced cellular apoptosis. All analyzed sphingolipids induced an inflammatory response in human coronary artery smooth muscle cells. Conclusions— This study shows for the first time that sphingolipids and particularly glucosylceramide are associated with and are possible inducers of plaque inflammation and instability, pointing to sphingolipid metabolic pathways as possible novel therapeutic targets.Objective— Lipids are central to the development of atherosclerotic plaques. Specifically, which lipids are culprits remains controversial, and promising targets have failed in clinical studies. Sphingolipids are bioactive lipids present in atherosclerotic plaques, and they have been suggested to have both proatherogenic and antiatherogenic. However, the biological effects of these lipids remain unknown in the human atherosclerotic plaque. The aim of this study was to assess plaque levels of sphingolipids and investigate their potential association with and contribution to plaque vulnerability. Approach and Results— Glucosylceramide, lactosylceramide, ceramide, dihydroceramide, sphingomyelin, and sphingosine-1-phosphate were analyzed in homogenates from 200 human carotid plaques using mass spectrometry. Inflammatory activity was determined by analyzing plaque levels of cytokines and plaque histology. Caspase-3 was analyzed by ELISA technique. Expression of regulatory enzymes was analyzed with RNA sequencing. Human coronary artery smooth muscle cells were used to analyze the potential role of the 6 sphingolipids as inducers of plaque inflammation and cellular apoptosis in vitro. All sphingolipids were increased in plaques associated with symptoms and correlated with inflammatory cytokines. All sphingolipids, except sphingosine-1-phosphate, also correlated with histological markers of plaque instability. Lactosylceramide, ceramide, sphingomyelin, and sphingosine-1-phosphate correlated with caspase-3 activity. In vitro experiments revealed that glucosylceramide, lactosylceramide, and ceramide induced cellular apoptosis. All analyzed sphingolipids induced an inflammatory response in human coronary artery smooth muscle cells. Conclusions— This study shows for the first time that sphingolipids and particularly glucosylceramide are associated with and are possible inducers of plaque inflammation and instability, pointing to sphingolipid metabolic pathways as possible novel therapeutic targets.

Impaired Fibrous Repair A Possible Contributor to Atherosclerotic Plaque Vulnerability in Patients With Type II Diabetes

Objective— Diabetes mellitus (DM) type II is increasing rapidly worldwide. Patients with DM II have a greater atherosclerotic burden and higher risk of developing cardiovascular complications. Inflammation has been proposed as the main cause for the high risk of atherosclerotic disease in DM II. In this study, we compared markers of inflammation and fibrous repair in plaques from subjects with and without DM II. Approach and Results— Carotid endarterectomy specimens were obtained from 63 patients with and 131 without DM. Plaque structure, connective tissue proteins, inflammatory cells, and markers were analyzed by immunohistochemistry, ELISA, Mesoscale, and Luminex technology. Carotid plaques from diabetics had lower levels of extracellular matrix proteins, elastin, and collagen, which are critical for plaque stability. Plaques from diabetics had reduced levels of platelet-derived growth factor and matrix metalloproteinase-2, both important for tissue repair. No differences were observed in inflammatory markers in plaques from diabetic and nondiabetic patients. Conclusion— This study suggests that atherosclerotic plaques in subjects with DM II are more prone to rupture because of impaired repair responses rather than to increased vascular inflammation. Although this study did not have a mechanistic design, our findings suggest that targeting impaired repair responses in carotid plaques may help to increase our understanding of atherosclerotic plaque development and vulnerability in patients with DM II. # Significance {#article-title-31}Objective— Diabetes mellitus (DM) type II is increasing rapidly worldwide. Patients with DM II have a greater atherosclerotic burden and higher risk of developing cardiovascular complications. Inflammation has been proposed as the main cause for the high risk of atherosclerotic disease in DM II. In this study, we compared markers of inflammation and fibrous repair in plaques from subjects with and without DM II. Approach and Results— Carotid endarterectomy specimens were obtained from 63 patients with and 131 without DM. Plaque structure, connective tissue proteins, inflammatory cells, and markers were analyzed by immunohistochemistry, ELISA, Mesoscale, and Luminex technology. Carotid plaques from diabetics had lower levels of extracellular matrix proteins, elastin, and collagen, which are critical for plaque stability. Plaques from diabetics had reduced levels of platelet-derived growth factor and matrix metalloproteinase-2, both important for tissue repair. No differences were observed in inflammatory markers in plaques from diabetic and nondiabetic patients. Conclusion— This study suggests that atherosclerotic plaques in subjects with DM II are more prone to rupture because of impaired repair responses rather than to increased vascular inflammation. Although this study did not have a mechanistic design, our findings suggest that targeting impaired repair responses in carotid plaques may help to increase our understanding of atherosclerotic plaque development and vulnerability in patients with DM II.

Circulating cytokines reflect the expression of pro-inflammatory cytokines in atherosclerotic plaques.

AIMS Inflammation is a key factor in the development of plaque rupture and acute cardiovascular events. Although imaging techniques can be used to identify vulnerable atherosclerotic plaques, we are lacking non-invasive methods, such as plasma markers of plaque inflammation that could help to identify presence of vulnerable plaques. The aim of the present study was to investigate whether increased plasma levels of pro-inflammatory cytokines reflects inflammatory activity within atherosclerotic plaques. METHODS AND RESULTS Cytokines were measured using Luminex immunoassay in 200 homogenized plaque extracts and plasma, obtained from 197 subjects undergoing carotid surgery. Plasma levels of macrophage inflammatory protein-1β (MIP-1β), tumor necrosis factor- α (TNF-α) and fractalkine correlated significantly, not only with plaque levels of the same cytokines but also with the abundance of several pro-inflammatory and atherogenic cytokines assessed in plaque tissue. High plasma levels (upper tertile) of MIP-1β, TNF-α and fractalkine identified the presence of a plaque with high inflammation (above median of a score based on the plaque content of MIP-1β, TNF-α, interferon-γ (IFN-γ) and fractalkine) with a sensitivity between 65 and 67% and a specificity between 78 and 83%. Furthermore, this study shows that high plasma levels of MIP-1β, TNF-α and fractalkine predict future transient ischemic attacks. CONCLUSIONS Our findings show that the plasma levels of MIP-1β, TNF-α and fractalkine reflect the levels of several pro-atherogenic cytokines in plaque tissue and might be possible plasma markers for a vulnerable atherosclerotic disease. We thereby propose that these cytokines can be used as surrogate markers for the identification of patients with high-risk plaques.

Identification of the target for therapeutic recombinant anti-apoB-100 peptide antibodies in human atherosclerotic lesions.

PURPOSE Accumulation of oxidized LDL in the arterial wall is believed to play a key role in the development of atherosclerosis. Experimental studies have identified the presence of immune responses against epitopes in oxidized LDL that protects against atherosclerosis. We have produced human recombinant IgG against one of these epitopes (aldehyde-modified apoB-100 amino acids 661-680) and demonstrated that treatment with this human IgG1 2D03 antibody markedly reduces atherosclerosis in hypercholesterolemic mice. METHODS In the present study, we screened a panel of 25 carotid plaques associated with clinical symptoms and 26 clinically silent plaques obtained at surgery for presence of the aldehyde-modified apoB-100 peptide defined by the 2D03 antibody and compared the expression of this epitope with other plaque constituents, plasma lipoproteins levels, plasma oxidized LDL and autoantibodies against apoB-100 peptides. RESULTS We demonstrated that the epitope is commonly expressed in human atherosclerotic plaques and that plaques associated with clinical symptoms have an almost three-fold higher content of this epitope (8.6+/-4.9% versus 22.1+/-12.2% immunostaining of total plaque area, p<0.0005). There was also a significant association between 2D03 epitope staining and the plaque content of cholesterol esters (r=0.43, p<0.05), whereas there was no association with plasma oxidized LDL and autoantibodies against apoB-100 peptides. CONCLUSIONS By demonstrating the presence of the 2D03 epitope in human atherosclerotic lesions our findings support the possibility that treatment with this antibody may have beneficial effects also in humans. Furthermore, they suggest the possibility to use these or other similar antibodies for diagnostic imaging of atherosclerotic plaques in humans.