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.

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.

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.

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.

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.

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.

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.

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.

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.

Canonical transient receptor potential 6 channel deficiency promotes smooth muscle cells dedifferentiation and increased proliferation after arterial injury.

OBJECTIVE: Previous studies showed the benefit of canonical transient receptor potential 6 (TRPC6) channel deficiency in promoting endothelial healing of arterial injuries in hypercholesterolemic animals. Long-term studies utilizing a carotid wire-injury model were undertaken in wild-type (WT) and TRPC6-/- mice to determine the effects of TRPC6 on phenotypic modulation of vascular smooth muscle cells (SMC) and neointimal hyperplasia. We hypothesized that TRPC6 was essential in the maintenance or reexpression of a differentiated SMC phenotype and minimized luminal stenosis following arterial injury. METHODS: The common carotid arteries (CCA) of WT and TRPC6-/- mice were evaluated at baseline and 4 weeks after wire injury. At baseline, CCA of TRPC6-/- mice had reduced staining of MYH11 and SM22, fewer elastin lamina, luminal dilation, and wall thinning. After carotid wire injury, TRPC6-/- mice developed significantly more pronounced luminal stenosis compared with WT mice. Injured TRPC6-/- CCA demonstrated increased medial/intimal cell number and active cell proliferation when compared with WT CCA. Immunohistochemistry suggested that expression of contractile biomarkers in medial SMC were essentially at baseline levels in WT CCA at 28 days after wire injury. By contrast, at 28 days after injury medial SMC from TRPC6-/- CCA showed a significant decrease in the expression of contractile biomarkers relative to baseline levels. To assess the role of TRPC6 in systemic arterial SMC phenotype modulation, SMC were harvested from thoracic aortae of WT and TRPC6-/- mice and were characterized. TRPC6-/- SMC showed enhanced proliferation and migration in response to serum stimulation. Expression of contractile phenotype biomarkers, MYH11 and SM22, was attenuated in TRPC6-/- SMC. siRNA-mediated TRPC6 deficiency inhibited contractile biomarker expression in a mouse SMC line. CONCLUSIONS: These results suggest that TRPC6 contributes to the restoration or maintenance of arterial SMC contractile phenotype following injury. Understanding the role of TRPC6 in phenotypic modulation may lead to mechanism-based therapies for attenuation of IH.

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.

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.

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.

Apolipoprotein A-I mimetic peptide reverses impaired arterial healing after injury by reducing oxidative stress.

OBJECTIVE: Endothelial cell (EC) migration is essential for healing of arterial injuries caused by angioplasty, but a high cholesterol diet inhibits endothelial repair. In vivo studies suggest that apolipoprotein A-I (apoA-I), the major protein constituent of HDL, is essential for normal healing of arterial injuries. ApoA-I mimetics, including 4F, have been designed to mimic the amphipathic portion of the apoA-I molecule. This study was undertaken to determine if 4F improves endothelial migration and healing. METHODS: A razor scrape assay was used to analyze the effect of 4F on EC migration in vitro. Endothelial healing in vivo was assessed following electrical injury of carotid arteries in mice. Markers of oxidative stress were also examined. RESULTS: Lipid oxidation products inhibited EC migration in vitro, but preincubation with L-4F preserved EC migration. Endothelial healing of carotid arterial injuries in mice on a high cholesterol diet was delayed compared with mice on a chow diet with 27.8% vs. 48.2% healing, respectively, at 5 days. Administration of D-4F improved endothelial healing in mice on a high cholesterol diet to 43.4%. D-4F administration had no effect on lipid levels but decreased markers of oxidation. In vivo, there was a significant inverse correlation between endothelial healing and plasma markers of oxidative stress. CONCLUSION: These studies suggested that an apoA-I mimetic can improve endothelial healing of arterial injuries by decreasing oxidative stress.

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.

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.

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.

Stimulus-dependent phosphorylation of profilin-1 in angiogenesis.

Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr 129 is critical for endothelial cell migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induces Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Conditional endothelial knock-in of phosphorylation-deficient Pfn1(Y129F) in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischaemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signalling events to initiate endothelial cell migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.

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.