Programmed translational readthrough generates antiangiogenic VEGF-Ax.

Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

p38 MAPK activation and STIM1-Orai3 association mediate TRPC6 externalization.

Endothelial cell (EC) migration is critical for the repair of monolayer disruption following angioplasties, but migration is inhibited by lipid oxidation products, including lysophosphatidylcholine (lysoPC), which open canonical transient receptor potential 6 (TRPC6) channels. TRPC6 activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), the source of which is unknown. LysoPC can activate phospholipase A2 to release arachidonic acid (ArA). ArA can activate arachidonic acid-regulated calcium (ARC) channels that are formed by stromal interaction molecule 1 (STIM1) and Orai1 and Orai3 proteins. Both lysoPC and ArA can activate p38 mitogen-activated protein kinase (MAPK) that induces the phosphorylation required for STIM1-Orai3 association. This is accompanied by an increase in [Ca2+]i and TRPC6 externalization. The effect of lysoPC and ArA is not additive, suggesting activation of the same pathway. The increase in [Ca2+]i activates an Src kinase that leads to TRPC6 activation. Downregulation of Orai3 using siRNA blocks the lysoPC- or ArA-induced increase in [Ca2+]i and TRPC6 externalization and preserves EC migration. These data show that lysoPC induces activation of p38 MAPK, which leads to STIM1-Orai3 association and increased [Ca2+]i. This increase in [Ca2+]i activates an Src kinase leading to TRPC6 externalization, which initiates a cascade of events ending in cytoskeletal changes that disrupt EC migration. Blocking this pathway preserves EC migration in the presence of lipid oxidation products.NEW & NOTEWORTHY The major lysophospholipid component in oxidized LDL, lysophosphatidylcholine (lysoPC), can activate p38 MAP kinase, which in turn promotes externalization of Orai3 and STIM1-Orai3 association, suggesting involvement of arachidonic acid-regulated calcium (ARC) channels. The subsequent increase in intracellular calcium activates an Src kinase required for TRPC6 externalization. TRPC6 activation, which has been shown to inhibit endothelial cell migration, is blocked by p38 MAP kinase or Orai3 downregulation, and this partially preserves endothelial migration in lysoPC.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta.

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor" apoA-I forms via the nonenzymatic CNO- pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Hypogastric artery luminal diameter predicts common-external iliac stent patency and major adverse limb events in patients with aortoiliac occlusive disease.

OBJECTIVE: Hypogastric coverage may be required for occlusive disease at the iliac arterial bifurcation. In this study, we sought to determine patency rates of common-external iliac artery (C-EIA) bare metal stents (BMS) spanning the hypogastric origin in patients with aortoiliac occlusive disease (AIOD). In addition, we sought to identify predictors of C-EIA BMS patency loss and major adverse limb events (MALE) in patients requiring hypogastric coverage. We hypothesized that worsening stenosis of the hypogastric origin would negatively influence C-EIA stent patency and freedom from MALE. METHODS: This is a single center, retrospective review of consecutive patients undergoing elective, endovascular treatment of aortoiliac disease (AIOD) between 2010 and 2018. Only patients with C-EIA BMS coverage of a patent IIA origin were included in the study. Hypogastric luminal diameter was determined from preoperative CT angiography. Analysis was performed using Kaplan-Meier survival analysis, univariable and multivariable logistic regression, and receiver operator characteristics (ROC). RESULTS: There were 236 patients (318 limbs) who were included in the study. AIOD was TASC C/D in 236/318 (74.2%) of cases. C-EIA stent primary patency was 86.5% (95% confidence interval: 81.1, 91.9) at 2 years and 79.7% (72.8, 86.7) at 4 years. Freedom from ipsilateral MALE was 77.0% (71.1, 82.9) at 2 years and 68.7% (61.3, 76.2) at 4 years. Luminal diameter of the hypogastric origin was most strongly associated with loss of C-EIA BMS primary patency in multivariable analysis (hazard ratio: 0.81, p = .02). Insulin-dependent diabetes, Rutherford's class IV or above, and stenosis of the hypogastric origin were significantly predictive of MALE in both univariable and multivariable analyses. In ROC analysis, luminal diameter of the hypogastric origin was superior to chance in prediction of C-EIA primary patency loss and MALE. Hypogastric diameter >4.5 mm had a negative predictive value of 0.94 for C-EIA primary patency loss and 0.83 for MALE. CONCLUSIONS: Patency rates of C-EIA BMS are high. Hypogastric luminal diameter is an important and potentially modifiable predictor of C-EIA BMS patency and MALE in patients with AIOD.

Activation of the cytosolic calcium-independent phospholipase A2 ß isoform contributes to TRPC6 externalization via release of arachidonic acid.

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A2 (PLA2), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA2 in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA2 enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA2 involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA2α, cPLA2γ, iPLA2ß, or iPLA2γ. Downregulation of the ß isoform of iPLA2 blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

Inhibition of P110α and P110δ catalytic subunits of PI3 kinase reverses impaired arterial healing after injury in hypercholesterolemic male mice.

Endothelial cell (EC) migration is critical for healing arterial injuries, such as those that occur with angioplasty. Impaired re-endothelialization following arterial injury contributes to vessel thrombogenicity, intimal hyperplasia, and restenosis. Oxidized lipid products, including lysophosphatidylcholine (lysoPC), induce canonical transient receptor potential 6 (TRPC6) externalization leading to increased [Ca2+]i, activation of calpains, and alterations of the EC cytoskeletal structure that inhibit migration. The p110α and p110δ catalytic subunit isoforms of phosphatidylinositol 3-kinase (PI3K) regulate lysoPC-induced TRPC6 externalization in vitro. The goal of this study was to assess the in vivo relevance of those in vitro findings to arterial healing following a denuding injury in hypercholesterolemic mice treated with pharmacologic inhibitors of the p110α and p110δ isoforms of PI3K and a general PI3K inhibitor. Pharmacologic inhibition of the p110α or the p110δ isoform of PI3K partially preserves healing in hypercholesterolemic male mice, similar to a general PI3K inhibitor. Interestingly, the p110α, p110δ, and the general PI3K inhibitor do not improve arterial healing after injury in hypercholesterolemic female mice. These results indicate a potential new role for isoform-specific PI3K inhibitors in male patients following arterial injury/intervention. The results also identify significant sex differences in the response to PI3K inhibition in the cardiovascular system, where female sex generally has a cardioprotective effect. This study provides a foundation to investigate the mechanism for the sex differences in response to PI3K inhibition to develop a more generally applicable treatment option.

P110α and P110δ catalytic subunits of PI3 kinase regulate lysophosphatidylcholine-induced TRPC6 externalization.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC) inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo, in part by activating phosphatidylinositol 3-kinase (PI3K), which increases the externalization of canonical transient receptor potential 6 (TRPC6) channels and the subsequent increase in intracellular calcium. Inhibition of PI3K is a potential method to decrease TRPC6 activation and restore migration, but PI3K is involved in multiple intracellular signaling pathways and has multiple downstream effectors. The goal of this study is to identify the specific p110 catalytic subunit isoforms responsible for lysoPC-induced TRPC6 externalization to identify a target for intervention while minimizing impact on alternative signaling pathways. Down-regulation of the p110α and p110δ isoforms, but not the p110ß or p110γ isoforms, with small interfering RNA significantly decreased phosphatidylinositol (3,4,5)-trisphosphate production and TRPC6 externalization, and significantly improved EC migration in the presence of lysoPC. These results identify an additional role of p110α in EC and reveal for the first time a specific role of p110δ in EC, providing a foundation for subsequent in vivo studies to investigate the impact of p110 isoform inhibition on arterial healing after injury.

Collagen prolyl 4-hydroxylases modify tumor progression.

Collagen is the main component of the extracellular matrix. Hydroxylation of proline residues on collagen, catalyzed by collagen prolyl 4-hydroxylase (C-P4H), is essential for the stability of the collagen triple helix. Vertebrate C-P4H is an α2ß2 tetramer with three isoenzymes differing in the catalytic α-subunits, which are encoded by P4HA1, P4HA2, and P4HA3 genes. In contrast, ß-subunit is encoded by a single gene P4HB. The expressions of P4HAs and P4HB are regulated by multiple cellular factors, including cytokines, transcription factors, and microRNAs. P4HAs and P4HB are highly expressed in many tumors and participate in cancer progression. Several inhibitors of P4HAs and P4HB have been confirmed to have anti-tumor effects, suggesting that targeting C-P4H is a feasible strategy for cancer treatment. Here, we summarize recent progresses on the function and expression of regulatory mechanisms of C-P4H in cancer progression and point out the potential development of therapeutic strategies in targeting C-P4H in the future.

Superoxide-induced Type I collagen secretion depends on prolyl 4-hydroxylases.

Reactive oxygen species (ROS) including superoxide (O2•-) play an important role in a variety of diseases, including Alzheimer's Disease, cancer, and atherosclerosis. Early reports showed that O2•- is a stimulant for collagen synthesis. However, the mechanism remains incompletely understood. Here we showed that LY83583 (6-anilinoquinoline-5,8-quinone), a substance known to induce O2•- production by smooth muscle cell (SMC), increases Type I collagen secretion. This effect could be blocked by treating the cells with Tiron, a scavenger for O2•-. LY83583-induced Type I collagen secretion required P4HA1 and P4HA2. Knockout of either P4ha1 or P4ha2 greatly reduced LY83583-stimulated Type I collagen maturation whereas silencing of both P4ha1 and P4ha2 completely blocked LY83583-induced Type I collagen maturation. Although significantly more hydroxyproline on purified Type I collagen was detected from LY83583 treated mouse embryonic fibroblast (MEF) cells by mass spectrometry, the level of prolyl 4-hydroxylases was not altered. Thus, LY83583 might increase the enzymatic activity of prolyl 4-hydroxylases to increase Type I collagen maturation. In addition, we found that LY83583 activated prolyl 4-hydrolases differed from ascorbate-activated prolyl 4-hydroxylase in two aspects: (1) LY83583 activated both P4HA1 and P4HA2 involved in collagen maturation whereas ascorbate mainly stimulated P4HA1 in collagen maturation; (2) LY83583 did not induce N259 glycosylation on P4HA1 as ascorbate did. The mechanisms remain to be investigated.

Ascorbate inducible N259 glycans on prolyl 4-hydroxylase subunit α1 promote hydroxylation and secretion of type I collagen.

Ascorbic acid (vitamin C, VC) increases the secretion of mature collagen by promoting the activity of prolyl 4-hydroxylase subunit α 1 (P4HA1). To explore the mechanism involved, we investigated the role of N-linked glycosylation, which can regulate enzyme activity. P4HA1 has two glycosylation sites, Asn (N) 113 and N259. Our studies show that glycosylation of N259, but not N113, by STT3B and magnesium transporter 1 (MAGT1) is augmented by VC. N259 glycosylation on P4HA1 correlates with enhanced pepsin-resistant collagen 1α2 secretion. Downregulation of Stt3b and Magt1 reduces N259 glycans on P4HA1. In collagen 1α2 purified from Stt3b-silenced fibroblasts, decreased hydroxylation is found at five specific proline residues, while significantly increased hydroxylation is noted at two proline residues. Similarly, in collagen 1α1, reduced proline hydroxylation is detected at eight sites and increased proline hydroxylation is found at four sites. These results suggest that N-linked glycosylation of P4HA1 can direct hydroxylation at specific proline residues and affect collagen maturation.

Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr(99) or Tyr(138) of CaM was replaced with Phe, generating mutant CaM, Phe(99)-CaM, or Phe(138)-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe(99)-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe(138)-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe(99)-CaM. Blocking phosphorylation of CaM at Tyr(99) also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr(99) by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane.

Integration of TRPC6 and NADPH oxidase activation in lysophosphatidylcholine-induced TRPC5 externalization.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels, and the subsequent increase in intracellular Ca2+ leads to TRPC5 activation. The goal of this study is to elucidate the steps in the pathway between TRPC6 activation and TRPC5 externalization. Following TRPC6 activation by lysoPC, extracellular regulated kinase (ERK) is phosphorylated. This leads to phosphorylation of p47phox and subsequent NADPH oxidase activation with increased production of reactive oxygen species. ERK activation requires TRPC6 opening and influx of Ca2+ as evidenced by the failure of lysoPC to induce ERK phosphorylation in TRPC6-/- endothelial cells. ERK siRNA blocks the lysoPC-induced activation of NADPH oxidase, demonstrating that ERK activation is upstream of NADPH oxidase. The reactive oxygen species produced by NADPH oxidase promote myosin light chain kinase (MLCK) activation with phosphorylation of MLC and TRPC5 externalization. Downregulation of ERK, NADPH oxidase, or MLCK with the relevant siRNA prevents TRPC5 externalization. Blocking MLCK activation prevents the prolonged rise in intracellular calcium levels and preserves endothelial migration in the presence of lysoPC.

Hypercholesterolemia inhibits re-endothelialization of arterial injuries by TRPC channel activation.

OBJECTIVE: After arterial injury, endothelial cell (EC) migration is essential for healing, but lipid oxidation products activate TRPC6 and TRPC5 ion channels, leading to increased intracellular calcium and inhibition of EC migration in vitro. The objective of this study was to further evaluate the role of TRPC channels in EC migration in vitro and to validate in vitro findings in an in vivo model. METHODS: Mouse aortic ECs were cultured, and the effect of lysophosphatidylcholine, the major lysophospholipid in oxidized low-density lipoprotein, on migration was assessed in a razor-scrape assay. EC healing after a carotid injury with electrocautery was evaluated in wild-type (WT), TRPC6(-/-), and TRPC5(-/-) mice receiving either a chow or high-cholesterol (HC) diet. RESULTS: Lysophosphatidylcholine inhibited EC migration of WT ECs to 22% of baseline and of TRPC5(-/-) ECs to 53% of baseline but had minimal effect on TRPC6(-/-) EC migration. Hypercholesterolemia severely impaired EC healing in vivo, with 51.4% ± 1.8% and 24.9% ± 2.0% of the injury resurfaced with ECs at 5 days in chow-fed and HC-fed WT mice, respectively (P < .001). Hypercholesterolemia did not impair healing in TRPC6(-/-) mice, with coverage of 48.4% ± 3.4% and 46.8% ± 1.6% in chow-fed and HC-fed TRPC6(-/-) mice, respectively. Hypercholesterolemia had a reduced inhibitory effect in TRPC5(-/-) mice, with EC coverage of 51.7% ± 3.0% and 37.% ± 1.4% in chow-fed and HC-fed TRPC5(-/-) mice, respectively. CONCLUSIONS: Results suggest that activation of TRPC6 and TRPC5 channels is the key contributor to impaired endothelial healing of arterial injuries in hypercholesterolemic mice.

p85α regulatory subunit isoform controls PI3-kinase and TRPC6 membrane translocation.

Activation of phosphatidylinositol 3-kinase (PI3K) by lipid oxidation products, including lysophosphatidylcholine (lysoPC), increases the externalization of canonical transient receptor potential 6 (TRPC6) channels leading to a subsequent increase in intracellular calcium that contributes to cytoskeletal changes which inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo. The PI3K p110α and p110δ catalytic subunit isoforms regulate lysoPC-induced TRPC6 externalization in vitro, but have many other functions. The goal of the current study is to identify the PI3K regulatory subunit isoform involved in TRPC6 externalization to potentially identify a more specific treatment regimen to improve EC migration and arterial healing, while minimizing off-target effects. Decreasing the p85α regulatory subunit isoform protein levels, but not the p85ß and p55γ regulatory subunit isoforms, with small interfering RNA inhibits lysoPC-induced translocation of the PI3K catalytic subunit to the plasma membrane, dramatically decreased phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production and TRPC6 externalization, and significantly improves EC migration in the presence of lysoPC. These results identify the important and specific role of p85α in controlling translocation of PI3K from the cytosol to the plasma membrane and PI3K-mediated TRPC externalization by oxidized lipids. Current PI3K inhibitors block the catalytic subunit, but our data suggest that the regulatory subunit is a novel therapeutic target to promote EC migration and healing after arterial injuries that occur with angioplasty.

iPLA2 inhibition blocks LysoPC-induced TRPC6 externalization and promotes Re-endothelialization of carotid injuries in hypercholesterolemic mice.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), accumulate at the site of arterial injury after vascular interventions and hinder re-endothelization. LysoPC activates calcium-permeable channels, specifically canonical transient receptor potential 6 (TRPC6) channels that induce a sustained increase in intracellular calcium ion concentration [Ca2+]i and contribute to dysregulation of the endothelial cell (EC) cytoskeleton. Activation of TRPC6 leads to inhibition of EC migration in vitro and delayed re-endothelization of arterial injuries in vivo. Previously, we demonstrated the role of phospholipase A2 (PLA2), specifically calcium-independent PLA2 (iPLA2), in lysoPC-induced TRPC6 externalization and inhibition of EC migration in vitro. The ability of FKGK11, an iPLA2-specific pharmacological inhibitor, to block TRPC6 externalization and preserve EC migration was assessed in vitro and in a mouse model of carotid injury. Our data suggest that FKGK11 prevents lysoPC-induced PLA2 activity, blocks TRPC6 externalization, attenuates calcium influx, and partially preserves EC migration in vitro. Furthermore, FKGK11 promotes re-endothelization of an electrocautery carotid injury in hypercholesterolemic mice. FKGK11 has similar arterial healing effects in male and female mice on a high-fat diet. This study suggests that iPLA2 is a potential therapeutic target to attenuate calcium influx through TRPC6 channels and promote EC healing in cardiovascular patients undergoing angioplasty.

Hypercholesterolemia and oxidative stress inhibit endothelial cell healing after arterial injury.

OBJECTIVE: Endothelial cell (EC) migration is essential for arterial healing after angioplasty. Oxidized low-density lipoproteins and oxidative stress decrease EC migration in vitro. The objective of this study was to determine the effect of hypercholesterolemia and oxidative stress on EC healing after an arterial injury. METHODS: C57BL/6 wild-type mice were placed in one of eight groups: chow diet (n = 11), high-cholesterol (HC) diet (n = 11), chow diet plus paraquat (n = 11), HC diet plus paraquat (n = 11), chow diet plus N-acetylcysteine (NAC) (n = 11), HC diet plus NAC (n = 11), chow diet plus paraquat and NAC (n = 11), and HC diet plus paraquat and NAC (n = 11). After 2 weeks on the assigned diet with or without NAC, the carotid artery was injured using electrocautery. Animals in the paraquat groups were given 1 mg/kg intraperitoneally to increase oxidative stress. After 120 hours, Evans Blue dye was infused intravenously to stain the area of the artery that remained deendothelialized. This was used to calculate the percentage of re-endothelialization. Plasma and tissue samples were analyzed for measures of oxidative stress. RESULTS: The HC diet increased oxidative stress and reduced EC healing compared with a chow diet, with EC covering 26.8% ± 2.8% and 48.1% ± 5.2% (P < .001) of the injured area, respectively. Administration of paraquat decreased healing in both chow and HC animals to 18.1% ± 3.5% (P < .001) and 9.8% ± 4.6% (P < .001), respectively. Pretreatment with NAC (120 mmol/L in drinking water) for 2 weeks prior to injury, to decrease oxidative stress, improved EC healing to 39.9% ± 5.7% (P < .001) in hypercholesterolemic mice and to 30.7% ± 3.6% (P < .001) in the paraquat group. NAC treatment improved healing to 24.6% ± 3.4% (P < .001) in hypercholesterolemic mice treated with paraquat. CONCLUSION: Re-endothelialization of arterial injuries is reduced in hypercholesterolemic mice and is inversely correlated with oxidative stress. An oral antioxidant decreases oxidative stress and improves EC healing. CLINICAL RELEVANCE: Vascular injury following cardiovascular intervention, including cardiac and peripheral arterial angioplasty and stenting, is associated with inflammation and oxidative stress. Hypercholesterolemia is also associated with increased oxidative stress. Oxidative stress, regardless of the source, induces cellular dysfunction in endothelial and smooth muscle cells that reduce healing after arterial injury. Decreasing oxidative stress with an exogenously administered antioxidant can improve endothelial cell healing, and this is important to control intimal hyperplasia and reduce the thrombogenicity of the vessel.

Interleukin-5 is a potential mediator of angiotensin II-induced aneurysm formation in apolipoprotein E knockout mice.

BACKGROUND: The aim of this study was to evaluate alterations in Th1 and Th2 cytokines during experimental abdominal aortic aneurysm (AAA) formation. METHODS: AAAs were induced in apolipoprotein E null mice by infusing angiotensin II (Ang II, 1000 ng/kg/min). Aortic homogenates were assessed at 0, 7, 14, and 28 d (n = 11/time point) for select Th1 and Th2 cytokines by ELISA. Additional mice had co-administration of anti-IgG (n = 20) or anti-IL-5 (n = 20) and were assessed at 28 d for AAA. Aortic homogenates were assessed for MMP-2 and MMP-9 expression. Mouse aortic SMC (MASMC) and peritoneal-derived macrophages were treated with IL-5 (0-40 ng/mL), and cell extracts and media (0-48 h) were assessed for MMP-2 and MMP-9 expression. RESULTS: Ang II infusion was associated with a 3.4-fold (P < 0.01) and 3.6-fold (P < 0.01) increase in IL-5 and IL-10 (respectively), and a 0.6-fold reduction in IL-6, by 7 d. Anti-IL-5, but not anti-IgG, ameliorated Ang II-induced AAA formation. Up-regulation of MMP-2 and MMP-9 was observed in aneurysmal aortas, but not in the aortas obtained from mice treated with anti-IL-5. IL-5 stimulation of MASMC increased MMP-2 and MMP-9 mRNA (2.1-fold and 2.7-fold, respectively, P < 0.01) and protein (1.6-fold and 1.9-fold, respectively, P < 0.01) by 24 h. IL-5 stimulation of macrophages did not alter MMP expression. CONCLUSIONS: Ang II induces increased Th2 cytokines IL-5 and IL-10 early in the course of experimental AAA formation, and inhibition of IL-5 prevents AAA formation suggesting an important role. While IL-5 is capable of up-regulating MMP-2 and MMP-9 expression in MASMC, investigations into alternate roles in AAA formation is warranted.

Local and systemic alterations in signal transducers and activators of transcription (STAT) associated with human abdominal aortic aneurysms.

BACKGROUND: Signal transducers and activators of transcription (STAT) proteins are transcription factors that, when activated by phosphorylation, regulate gene expression and cellular activity. The aim of this study was to evaluate the local and systemic expression and activation of STAT proteins associated with abdominal aortic aneurysms (AAA). METHODS: Expression and activation of STAT proteins were assessed in aortic wall samples obtained from patients undergoing repair of AAA (n = 9) and from non-aneurysmal (NA) donors (n = 17). Aortic samples were evaluated for mRNA and protein expression for STAT1, 2, 3, 4, 5a, and 5b using RT-PCR and immunoblot (WB) assays and normalized to ß-actin (expressed as arbitrary units). STAT activation was assessed with WB assays using phosphorylated (p)-STAT-specific antibodies. Alterations in STAT activation were calculated by normalizing pSTAT proteins to corresponding total STAT levels. Immunohistochemistry was performed on AAA and NA samples using the total and pSTAT antibodies. Systemic alterations in STAT activation were assessed by evaluating circulating leukocytes for the presence of pSTAT from patients with AAA (AAA, n = 8), repaired aneurysm (RA, n = 8), or age/gender matched controls with no AAA (CT, n = 8). Flow cytometry was performed to assess for circulating levels of STAT1 (pY701), STAT3 (pY705), and STAT5a (pY694) in monocytes, granulocytes, and lymphocytes. Assessments were made at baseline and in response to in vitro stimulation with IFN-γ (50 ng/mL) or IL-6 (100 ng/mL). Results were analyzed using Student's t-test and are expressed as mean ± SEM. RESULTS: In AAA tissue compared with NA, STAT-1 (1.08 ± 0.09 versus 0.62 ± 0.07), -2 (0.98 ± 0.07 versus 0.55 ± 0.08), and -4 (0.89 ± 0.12 versus 0.35 ± 0.11) mRNA levels were elevated (P < 0.01, all). Corresponding increases in STAT protein were only observed for STAT1 (2.77 ± 0.93 versus 0.93 ± 0.08, P < 0.05). Increases in activation were observed in AAA compared with NA in pSTAT2 (0.77 ± 0.1 versus 0.1 ± 0.02, P < 0.01), pSTAT3 (1.6 ± 0.3 versus 0.2 ± 0.06, P < 0.02) and pSTAT5 (0.57 ± 0.03 versus 0.2 ± 0.03, P < 0.05) levels. Phosphorylated STAT1, 2, 3, and 5 were observed in inflammatory cells invading the AAA adventitia. In addition, STAT3 was observed in the media of AAA and NA, but pSTAT3 was only observed in the media of AAA. There were no differences in baseline levels of pSTAT-positive circulating leukocytes. IFN-γ stimulation decreased STAT-5a (pY694)-positive CT lymphocytes to 40% ± 13% of baseline, but had no effect on AAA or RA lymphocytes (116% ± 35%, 102% ± 19%, respectively; P = 0.01). STAT-5a (pY694)-positive CT granulocytes also decreased to 62% ± 18% of baseline compared with AAA or RA granulocytes (122% ± 25%, 126% ± 17%, respectively; P = 0.01). Alterations in STAT1 (pY701) and STAT3 (pY705) were not observed in leukocytes following cytokine stimulation. CONCLUSIONS: STAT proteins are important regulators of transcriptional activity and have been linked to cardiovascular disease. The present data suggest that altered levels of phosphorylated STATs are associated with AAA. Understanding their role may provide further insight into the mechanisms of AAA formation and allow for the development of medical treatment options.

A diagnostic mystery solved by electron microscopy: a case of an "atypical" lymphoproliferative disorder.

An elderly woman with a previous diagnosis of atypical chronic lymphocytic leukemia (CLL) was noted to have a strikingly abnormal blood film, with the lymphocytes displaying numerous large cytoplasmic granules. This appearance had not been described before in the literature to the best of the authors' knowledge. After a series of investigations, electron microscopy was eventually performed, which demonstrated that the abnormal granules were composed of immunoglobulin crystals. The immunofixation study confirmed that they were monoclonal IgM paraprotein. These results led to a change of diagnosis to lymphoplasmacytic lymphoma. This report illustrates how electron microscopy can be used as a valuable additional diagnostic tool in difficult cases.

Membrane microviscosity regulates endothelial cell motility.

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.