Variants in ALOX5, ALOX5AP and LTA4H are not associated with atherosclerotic plaque phenotypes: the Athero-Express Genomics Study.

BACKGROUND: The eicosanoid genes ALOX5, ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes. METHODS: We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N = 109), and the Athero-Express Biobank Study (AE, N = 1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5, ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage). RESULTS: We replicate a known cis-eQTL (rs6538697, p = 1.96 × 10(-6)) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p > 5.79 × 10(-4)). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p > 1.67 × 10(-4)). CONCLUSIONS: We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5, ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease.

The CD200-CD200 receptor inhibitory axis controls arteriogenesis and local T lymphocyte influx.

The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in arteriogenesis, we hypothesized that disruption of the CD200-CD200R axis promotes arteriogenesis in a murine hindlimb ischemia model. Female Cd200-/- and wildtype (C57Bl/6J) mice underwent unilateral femoral artery ligation. Perfusion recovery was monitored over 7 days using Laser-Doppler analysis and was increased in Cd200-/- mice at day 3 and 7 after femoral artery ligation, compared to wildtype. Histology was performed on hindlimb muscles at baseline, day 3 and 7 to assess vessel geometry and number and inflammatory cell influx. Vessel geometry in non-ischemic muscles was larger, and vessel numbers in ischemic muscles were increased in Cd200-/- mice compared to wildtype. Furthermore, T lymphocyte influx was increased in Cd200-/- compared to wildtype. CD200R agonist treatment was performed in male C57Bl/6J mice to validate the role of the CD200-CD200R axis in arteriogenesis. CD200R agonist treatment after unilateral femoral artery ligation resulted in a significant decrease in vessel geometry, perfusion recovery and T lymphocyte influx at day 7 compared to isotype treatment. In this study, we show a causal role for the CD200-CD200R inhibitory axis in arteriogenesis in a murine hindlimb ischemia model. Lack of CD200R signaling is accompanied by increased T lymphocyte recruitment to the collateral vasculature and results in enlargement of preexisting collateral arteries.

The multifaceted functions of CXCL10 in cardiovascular disease.

C-X-C motif ligand 10 (CXCL10), or interferon-inducible protein-10, is a small chemokine belonging to the CXC chemokine family. Its members are responsible for leukocyte trafficking and act on tissue cells, like endothelial and vascular smooth muscle cells. CXCL10 is secreted by leukocytes and tissue cells and functions as a chemoattractant, mainly for lymphocytes. After binding to its receptor CXCR3, CXCL10 evokes a range of inflammatory responses: key features in cardiovascular disease (CVD). The role of CXCL10 in CVD has been extensively described, for example for atherosclerosis, aneurysm formation, and myocardial infarction. However, there seems to be a discrepancy between experimental and clinical settings. This discrepancy occurs from differences in biological actions between species (e.g. mice and human), which is dependent on CXCL10 signaling via different CXCR3 isoforms or CXCR3-independent signaling. This makes translation from experimental to clinical settings challenging. Furthermore, the overall consensus on the actions of CXCL10 in specific CVD models is not yet reached. The purpose of this review is to describe the functions of CXCL10 in different CVDs in both experimental and clinical settings and to highlight and discuss the possible discrepancies and translational difficulties. Furthermore, CXCL10 as a possible biomarker in CVD will be discussed.

Leukotriene B4 levels in human atherosclerotic plaques and abdominal aortic aneurysms.

BACKGROUND: Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome. METHODS: Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls. RESULTS: LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017). CONCLUSIONS: LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

Absence of chemokine (C-x-C motif) ligand 10 diminishes perfusion recovery after local arterial occlusion in mice.

OBJECTIVE: In arteriogenesis, pre-existing anastomoses undergo enlargement to restore blood flow in ischemic tissues. Chemokine (C-X-C motif) ligand 10 (CXCL10) is secreted after Toll-like receptor activation. Toll-like receptors are involved in arteriogenesis; however, the role of CXCL10 is still unclear. In this study, we investigated the role for CXCL10 in a murine hindlimb ischemia model. APPROACH AND RESULTS: Unilateral femoral artery ligation was performed in wild-type (WT) and CXCL10(-/-) knockout (KO) mice and perfusion recovery was measured using laser-Doppler perfusion analysis. Perfusion recovery was significantly lower in KO mice compared with WT at days 4 and 7 after surgery (KO versus WT: 28±5% versus 81±13% at day 4; P=0.003 and 57±12% versus 107±8% at day 7; P=0.003). Vessel measurements of α-smooth muscle actin-positive vessels revealed increasing numbers in time after surgery, which was significantly higher in WT when compared with that in KO. Furthermore, α-smooth muscle actin-positive vessels were significantly larger in WT when compared with those in KO at day 7 (wall thickness, P<0.001; lumen area, P=0.003). Local inflammation was assessed in hindlimb muscles, but this did not differ between WT and KO. Chimerization experiments analyzing perfusion recovery and histology revealed an equal contribution for bone marrow-derived and circulating CXCL10. Migration assays showed a stimulating role for both intrinsic and extrinsic CXCL10 in vascular smooth muscle cell migration. CONCLUSIONS: CXCL10 plays a causal role in arteriogenesis. Bone marrow-derived CXCL10 and tissue-derived CXCL10 play a critical role in accelerating perfusion recovery after arterial occlusion in mice probably by promoting vascular smooth muscle cell recruitment and maturation of pre-existing anastomoses.

Toll-like receptor 4 inhibitor TAK-242 treatment does not influence perfusion recovery in tissue ischemia.

Toll-like receptors (TLRs) are important in innate immune responses, which are crucial in collateral artery formation (arteriogenesis). TLR4⁻/⁻ mice undergoing hind limb ischemia show decreased perfusion recovery accompanied by an impaired infiltration of inflammatory cells. TLR antagonists are currently developed and tested with the objective to inhibit acute exacerbation of organ damaging immune responses. However, systemic inhibition of innate immune responses may negatively influence arteriogenesis. In this study, we evaluated if TLR4 inhibition by a potent TLR4 inhibitor (TAK-242) would negatively influence perfusion recovery in a mouse model for arteriogenesis. Whole blood from human and mouse origin was stimulated with the TLR4 ligand lipopolysaccharide following TAK-242 incubation. After stimulation, cellular TLR4 activation was measured using fluorescence-activated cell sorting and tumor necrosis factor alpha release was measured using enzyme-linked immunosorbent assay. Next, the effect of TAK-242 was tested in a mouse model for arteriogenesis on perfusion recovery. TLR4 responses measured by tumor necrosis factor alpha levels were inhibited by TAK-242 in human and mouse blood after long-term stimulation. TAK-242 attenuated TLR4 responses in vivo but did not inhibit perfusion recovery in mice. In conclusion, TAK-242 does not negatively influence perfusion recovery following hind limb ischemia despite its TLR4 inhibiting properties.

Arterial occlusion induces systemic changes in leucocyte composition.

BACKGROUND: Lack of tissue perfusion because of arterial occlusion can result in mortality and morbidity. In response to local tissue ischaemia, extravasation of leucocytes into the region at risk is initiated to facilitate matrix remodelling and subsequent perfusion recovery. However, it is unknown if local tissue ischaemia also induces a more generalized response of leucocyte trafficking and compartmentalization. This study was designed to gain insight into the temporal changes in circulating and bone marrow-derived leucocyte fractions following peripheral arterial occlusion in mice. MATERIALS AND METHODS: Mouse peripheral blood and bone marrow samples were collected at baseline and subsequently at day 1, 2, 3, 4 and 7 after femoral artery ligation. Leucocyte and bone marrow cell subsets were quantified using flow cytometry. RESULTS: After arterial occlusion, peripheral blood leucocyte numbers did not vary significantly over time. However, significant intrinsic temporal changes in cell numbers were observed for monocytes, lymphocytes, neutrophils and their subsets with fluctuations of > 50%. Granulocytes, for example, showed an initial upregulation, while monocytes and lymphocytes numbers initially decreased. These variations in the circulation were largely preceded by changes in the corresponding bone marrow lineages. Progenitor cells of the myeloid and lymphoid lineage in the bone marrow were upregulated after the decrease in the numbers of their progeny in the peripheral blood. CONCLUSIONS: Local arterial occlusion results in an orchestrated systemic response of leucocyte trafficking. This response substantiates the pivotal role of leucocytes as mediators of processes leading to perfusion recovery and tissue remodelling.

High neutrophil numbers in human carotid atherosclerotic plaques are associated with characteristics of rupture-prone lesions.

OBJECTIVE: To score the number of plaque neutrophils and relate the score to plaque morphology and inflammatory status. METHODS AND RESULTS: Neutrophils are inflammatory cells with tissue destruction capabilities that have been found at the site of an atherosclerotic plaque rupture or erosion. Poor evidence exists for neutrophil infiltration in human carotid atherosclerotic plaques, and its association with plaque morphology has not yet been described. A set of 355 human carotid plaques was stained for the neutrophil marker CD66b. High neutrophil numbers were found in plaques with a large lipid core, high macrophage numbers, and low collagen amount and smooth muscle cell numbers. High neutrophil numbers were associated with high interleukin 8 (P<0.001) and matrix metalloproteases 8 (P=0.005) and 9 (P<0.001) plaque levels. High microvessel density within plaques was correlated with high neutrophil numbers (P=0.01). In addition, low numbers of neutrophils were associated with female sex and use of beta-blockers. CONCLUSIONS: For the first time to our knowledge, these results show that neutrophil numbers are strongly associated with the histopathologic features of rupture-prone atherosclerotic lesions and suggest a role for neutrophils in plaque destabilization.

Neonatal dexamethasone treatment in the rat leads to kidney damage in adulthood.

UNLABELLED: Recently, concern has been raised that corticosteroid treatment of preterm neonates might be associated with adverse effects later in life, including early development of hypertension. Here, we investigate the impact of neonatal dexamethasone (Dex) treatment on early renal cell proliferation and nephron number. We analyzed mitotic activity in renal cortex of rat pups neonatally treated with Dex. Nephron number was measured and possible renal damage was quantified by counting inflammatory foci, ED-1 positive cells (macrophages), and the desmin score (activated podocytes). Mitotic activity was 34 and 29% lower on d 2 and 4 in Dex-treated rats compared with saline-treated controls. The number of glomeruli was lower at 4 wk, but nephron size was unchanged after Dex treatment, as calculated from glomerular density and (lower) body- and kidney weight. At wk 50, the glomerular number was significantly lower in Dex-treated rats, whereas body and kidney weight were the same as in Sal controls. Dex rats also showed more kidney damage, manifested by a approximately 3.5-fold increase in inflammation foci/mm and in ED-1 positive cells/mm and a approximately 4.3-fold increased desmin score. Temporary suppression of mitotic activity during neonatal Dex treatment leads to reduction of nephron number and more kidney damage later in life. ABBREVIATIONS: :