Nuclear factor-κB (NFκB) mediates inflammation-driven angiogenesis, which promotes growth of atherosclerotic plaques and tumors. The deubiquitinase ubiquitin-specific peptidase 20 (USP20) suppresses NFκB activation in vascular smooth muscle cells (SMCs) and attenuates atherosclerosis, but the role of USP20 in endothelial cells (ECs) was undefined. We tested whether USP20 activity diminishes NFκB signaling in ECs and thereby diminishes angiogenesis. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 -/- mice as compared with ECs from wild-type (WT) mice. Similarly, cytokine-induced NFκB activity was elevated in mouse coronary endothelial cells (MCECs) expressing catalytically inactive USP20 (USP20-DN) or phospho-mimetic USP20(S334D). In contrast, cytokine stimulation of MCECs expressing WT USP20 (USP20-WT) or phospho-resistant USP20(S334A) produced blunted NFκB activity. Assessed by scratch-wound healing and spheroid assays, migration and angiogenesis of MCECs, respectively, were (a) increased by USP20-DN or USP20(S334D), and (b) decreased by USP20-WT or USP20(S334A). Angiogenesis assessed by the aortic ring assay was significantly increased in Usp20 -/- mice and was suppressed by TPCA-1, an inhibitor of NFκB signaling. Angiogenesis was augmented in USP20(S334D) mouse aortic rings but reduced in USP20(S334A) mice. By screening known angiogenesis factors, we identified matrix metalloproteinase 3 (MMP3), a transcriptional target of NFκB, as a gene that is also regulated by USP20 expression and Ser334 phosphorylation. Inhibiting MMP3 reduced angiogenic sprouting in the Usp20 -/- mouse aortic rings. We conclude that USP20 expression inversely correlates with the extent of angiogenesis, and that inhibiting USP20 Ser334 phosphorylation could be a useful strategy to constrain inflammation-driven angiogenesis under pathological circumstances, like cancer and atherosclerosis.

BACKGROUND:Reactive oxygen species (ROS) augment the activation of vascular smooth muscle cells (SMCs) and promote neointimal hyperplasia evoked by arterial injury or atherogenesis. We have previously shown that small nucleolar RNAs (snoRNAs) from the Rpl13a locus are key regulators of cellular ROS levels. METHODS:Using mice deficient in the Rpl13a snoRNAs, we tested whether these snoRNAs regulate SMC activation in vitro and in vivo. Carotid endothelial denudation was used to provoke neointimal hyperplasia in wild-type (WT) and snoRNA knockout (snoKO) mice, which lack all four intronically-encoded Rpl13a snoRNAs. Primary SMCs from WT and snoKO mice were used for in vitro functional and proteomic analyses. HEK293T cells with specific snoRNA deletions were used to test for snoRNA-guided 2'-O-methylation of mRNA. RESULTS:Arterial ROS levels, inflammation, and carotid artery neointimal hyperplasia were reduced in snoKO compared with WT mice. In vitro, snoKO SMCs demonstrated lower ROS levels and less migration, proliferation, and inflammatory signaling than WT SMCs. Reduced ROS levels in snoKO SMCs and aortas correlated with upregulation of the mitochondrial protein COX4I2, which is associated with reduced mitochondrial ROS under normoxic conditions. Deleting the snoRNA U32A in human HEK293T cells decreased 2'-O-methylation of COX4I2 mRNA and upregulated COX4I2 protein without changing COX4I2 mRNA levels. Silencing Cox4i2 in snoKO SMCs upregulated SMC ROS to WT levels. CONCLUSIONS:Rpl13a snoRNAs are important drivers of SMC activation and neointimal hyperplasia. Rpl13a snoRNAs augment SMC ROS levels, at least in part, by post-transcriptional downregulation of COX4I2 expression.

The deubiquitinase ubiquitin-specific peptidase 20 (USP20) reverses lysine-63 ubiquitination of the signaling adaptor TRAF6 and suppresses nuclear-factor-κB (NFκB) activation induced by proatherogenic stimuli in vascular smooth muscle cells (SMCs). Dephosphorylation of USP20 at Ser 334 augments USP20/TRAF6 binding and USP20-mediated TRAF6 deubiquitination, consequently decreasing NFκB signaling. SMC USP20 activity also attenuates atherosclerosis in Ldlr -/- mice. NFκB is a key mediator of vascular endothelial cell (EC) activation and inflammation-driven angiogenesis, which promotes growth and rupture of atherosclerotic plaques. We tested the hypothesis that USP20 attenuates EC NFκB signaling and decreases angiogenesis, with both in vitro and ex vivo models to study the role of USP20 in EC activation and ensuing angiogenesis. We analyzed experimental data by one-way ANOVA followed by Holm-Šídák's multiple comparisons test. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 -/- mice as compared with ECs from congenic wild type (WT) mice (N=3, p<0.001). Assessed by scratch-wound healing and spheroid assays, respectively, migration and angiogenesis of mouse coronary endothelial cells (MCECs) transduced with recombinant adenoviruses was (a) increased by inactive USP20 or phospho-mimetic USP20(S334D) (migration, N=4, P<0.05; angiogenesis, N=6, P<0.01), and (b) decreased by transduction with WT USP20 or phospho-resistant USP20(S334A) (migration, N=4, P< 0.05; angiogenesis, N=6, P<0.01). Additionally, chemical inhibition of NFκB activation with TPCA-1 (which inhibits the upstream kinase IKK-2) attenuated MCEC migration (N=4, P<0.0001) and angiogenesis (N=6, P<0.005), as compared with untreated cells. Angiogenesis assessed by aortic ring sprouting was increased in Usp20 -/- compared with WT specimens (males, N=3, P<0.0001; females, N=2, P<0.0001), and it was also suppressed by TPCA-1 (males, N=2, WT; N=3 Usp20 -/- , P<0.0001; females, N=2 WT; N=3, Usp20 -/- P<0.0005). Angiogenesis was enhanced in aortic rings from USP20(S334D) CRISPR/Cas9 gene-edited mice (males, N=3, P<0.01; females, N=3 WT & N=4, USP20(S334D) P<0.0001), but reduced in aortic rings from USP20(S334A) mice (males, N=3, P<0.05; females, N=3 WT & N=5 USP20(S334A) P<0.0001). We conclude that preserving EC USP20 activity by preventing phosphorylation of USP20 on Ser 334 diminishes endothelial activation and angiogenic sprouting, which can decrease atherosclerosis.

Specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene elevate cellular levels of reactive oxygen species (ROS), and ROS are known to aggravate atherosclerosis (athero). We found that genetic deficiency of Rpl13a -snoRNAs reduces brachiocephalic artery athero in Apoe -/- mice by 50%. We therefore tested the hypothesis that athero could be reduced by acute pharmacologic intervention targeting Rpl13a -snoRNAs in Apoe -/- mice. To that end, we injected male and female Apoe -/- mice subcutaneously with 48 mg/kg antisense oligonucleotides (ASOs) targeting (a) the four Rpl13a -snoRNAs (ASO-snoRNA), or (b) green fluorescent protein (ASO-control). We tested two distinct strategies: ( i ) preventive, in which ASO injections and Western diet were started in 10-wk-old mice, with 4 weekly and 5 bi-weekly ASO treatments over 14 wk, then sacrifice; and ( ii ) “therapeutic,” in which ASO injections were started in 24-wk-old mice that had been fed Western diet for 14 wk and then were continued on Western diet with 6 weekly ASO treatments and then sacrificed. ASO injections were administered as indicated until 1 wk prior to sacrifice. Compared with saline-injected mice, ASO-control and ASO-snoRNA showed no hepatotoxicity (as assessed by serum ALT and AST). Compared with ASO-control, ASO-snoRNA administration for 6 wk reduced Rpl13a -snoRNA levels in the aorta and spleen by 54-80% for snoRNAs U32a , U33 , and U34 (RT-qPCR). In the “therapeutic” cohort, brachiocephalic artery cross-sectional athero was reduced by 38% in ASO-snoRNA-treated as compared with ASO-control-treated mice (n=7/group, p <0.03). Additionally, male ASO-snoRNA-treated mice exhibited a 98.1% decrease in circulating IL1-β transcripts (n=6-8/group, p<0.003). In the “preventive” cohort, aortic athero was reduced by 40% (13±5 vs 7±3 % aortic lesion area, n= 11-13/group, p <0.02) in ASO-snoRNA-treated as compared with ASO-control-treated mice. We conclude that therapeutic targeting of Rpl13a -snoRNAs with ASOs mitigates athero, both during the early and later stages of atherogenesis.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitinspecific peptidase 20 (USP20) reduces NFKB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed X50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFKB activation, VCAM-1 expression, and SMC proliferation than USP20S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1 beta. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1 beta induced less K63-linked polyubiquitination of TRAF6 and less downstream NFKB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1 beta-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1 beta-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFKB activation, SMC inflammation, and neointimal hyperplasia.

Reactive oxygen species (ROS) exacerbate atherosclerosis (athero). ROS levels are elevated by specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene. We therefore tested the hypothesis that these snoRNAs promote athero, using “snoKO” mice deficient in Rpl13a snoRNAs, but not in Rpl13a itself. ROS levels assessed by CellROX Orange were 35% lower in snoKO than snoRNA +/+ aorta frozen sections ( p <0.01). After 14 wk on Western diet, female snoKO/ Apoe -/- unexpectedly showed total cholesterol levels 20% higher than Apoe -/- mice in (1,046 vs 869 mg/dl, p <0.05). Despite this, neointimal lesions in brachiocephalic artery (BCA) cross-sections were 50% smaller in snoKO/ Apoe -/- than in Apoe -/- mice, and lumen size was 45% larger (both p <0.01, n=8/group). Similar data were obtained in males: snoKO/ Apoe -/- mice had 40% smaller BCA lesion areas ( p <0.02, n=8/group). After being stained for cholesteryl ester with BODIPY, for ACTA2 by immunofluorescence and for DNA (Hoechst), BCAs from female snoKO/ Apoe -/- mice (n=9) demonstrated 50% less foam cell-positive and 95% more ACTA2 + area, 40% less necrotic core area, and a 60% lower prevalence of ACTA2 + foam cells ( p <0.05 for each). Thus, Rpl13a snoRNAs promote vascular ROS and athero. Assessed by MitoSOX Red, ROS levels were 25% lower in snoKO than WT M1-polarized bone marrow-derived Mϕs in vitro (n=3, p<0.05). To identify mechanisms linking the Rpl13a snoRNAs to ROS and athero, we performed LC-MS/MS on WT and snoKO aortic SMCs. COX4I2 was expressed 5.7-fold higher in snoKO than WT SMCs by MS/MS, and 2.5-fold higher in snoKO SMCs by immunoblot (n=3/group, p <0.05). As part of mitochondrial complex IV, COX4I2 lowers cellular ROS levels. We used CRISPR/Cas9 to create 293T cells lacking either the RPL13a -snoRNA U34A or the irrelevant snoRNA U25 . With mRNA from these cells we performed reverse transcription at low [dNTP] followed by qPCR (RTL-P) for COX4I2 . Inversely proportional to the degree of mRNA 2’- O -methylation (mediated by snoRNAs), the RTL-P efficiency was 4-fold higher in U34A -knockout than control cells (3 clones/genotype, p <0.01). Thus, COX4I2 mRNA appears to be regulated by RPL13A -snoRNA-guided 2’- O -methylation in a manner that could link Rpl13a snoRNAs, vascular ROS, and athero.

Aims The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation, and pro-inflammatory signalling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis. Methods and results SM22-Cre(+)/Dbn(flox/flox)/Ldlr(-)(/)(-) (SMC-Dbn(-)(/)(-)/Ldlr(-)(/)(-)) and control mice (SM22-Cre(+)/Ldlr(-)(/)(-), Dbn(flox/flox)/Ldlr(-)(/)(-), and Ldlr(-)(/)(-)) were fed a western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn (-)(/)(-)/Ldlr(-)(/)(-) mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 weeks, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn(-)(/)(-)/Ldlr(-)(/)(-) mice. SMC-Dbn(-)(/)(-)/Ldlr(-)(/)(-) lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn(-)(/)(-) arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn(-)(/)(-) SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbn(flox/flox) SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn(-)(/)(-) and Dbn(flox/flox) SMCs. During early atherogenesis, SMC-Dbn(-)(/)(-)/Ldlr(-)(/)(-) aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn(-)(/)(-) SMCs were reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn(-)(/)(-) and Dbn(flox/flox) SMCs. Conclusion We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells.

Reactive oxygen species (ROS) contribute to atherogenesis. An unusual mechanism that increases cellular ROS levels and oxidative stress involves 4 ubiquitously expressed noncoding small nucleolar RNAs (snoRNAs) from introns of the ribosomal protein L13a ( Rpl13a ) locus: U32a , U33 , U34 , and U35a . We tested the hypothesis that these snoRNAs promote aortic smooth muscle cell (SMC) activation and vascular inflammation, by using “snoKO” mice with targeted deletion of the 4 snoRNAs (but not Rpl13a ). Compared with congenic WT SMCs, snoKO SMCs showed 40±20% lower ROS levels, assessed by DCF fluorescence ( p <0.02). Congruently, ROS levels were 35±5% lower in snoKO than WT aorta and carotid frozen sections ( p <0.01), assessed by CellROX Orange fluorescence. Proliferation and migration evoked by FBS and PDGF-BB, respectively, were each 30±10% less in snoKO than WT SMCs ( p <0.01 for each). To assess SMC migration and proliferation in vivo, we performed carotid artery endothelial denudation. Before injury, snoKO and WT carotid arteries were morphologically equivalent. Four wk after injury, carotid neointimal hyperplasia was 57±9% less and luminal area was 40±20 % more in snoKO than in WT mice ( p <0.01). WT and snoKO mice had equivalent heart rates and systolic blood pressures by tail-cuff plethysmography: 480±20 vs 420±80 beats/min; 133±5, 132±7 mm Hg, respectively (n=5/group). To test whether snoRNAs affect atherosclerosis, we orthotopically transplanted carotid arteries from WT and snoKO mice into congenic Apoe -/- mice. Six wk post-op, atherosclerotic neointima was 70±10% smaller in snoKO than in WT carotids ( p <0.01). To assess SMC-to-foam-cell transdifferentiation, which is ROS-dependent, carotid cross-sections were stained for apoE to identify graft-derived cells and for cholesteryl ester with BODIPY. BODIPY + foam cells comprised 21±3% and 11±7% of neointimal area in WT and snoKO carotids, respectively ( p <0.05). Confocal co-localization of apoE and BODIPY (optical slice thickness 1 μm) showed that graft-derived foam cells were 2.0±0.6-fold more prevalent in WT than in snoKO carotids ( p <0.01). We conclude that Rpl13a snoRNAs promote SMC ROS levels, proliferation and migration in vitro and in vivo, and that these snoRNAs augment atherosclerosis.

AimsThe actin-binding protein Drebrin is up-regulated in response to arterial injury and reduces smooth muscle cell (SMC) migration and proliferation through its interaction with the actin cytoskeleton. We, therefore, tested the hypothesis that SMC Drebrin inhibits angiotensin II-induced remodelling of the proximal aorta.Methods and resultsAngiotensin II was administered via osmotic minipumps at 1000 ng/kg/min continuously for 28 days in SM22-Cre+/Dbnflox/flox (SMC-Dbn-/-) and control mice. Blood pressure responses to angiotensin II were assessed by telemetry. After angiotensin II infusion, we assessed remodelling in the proximal ascending aorta by echocardiography and planimetry of histological cross sections. Although the degree of hypertension was equivalent in SMC-Dbn-/- and control mice, SMC-Dbn-/- mice nonetheless exhibited 60% more proximal aortic medial thickening and two-fold more outward aortic remodelling than control mice in response to angiotensin II. Proximal aortas demonstrated greater cellular proliferation and matrix deposition in SMC-Dbn-/- mice than in control mice, as evidenced by a higher prevalence of proliferating cell nuclear antigen-positive nuclei and higher levels of collagen I. Compared with control mouse aortas, SMC-Dbn-/- aortas demonstrated greater angiotensin II-induced NADPH oxidase activation and inflammation, evidenced by higher levels of Ser-536-phosphorylated NFκB p65 subunits and higher levels of vascular cell adhesion molecule-1, matrix metalloproteinase-9, and adventitial macrophages.ConclusionsWe conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.

Objective— Signaling that activates NFκB (nuclear factor κB) in smooth muscle cells (SMCs) is integral to atherosclerosis and involves reversible ubiquitination that activates proteins downstream of proatherogenic receptors. Deubiquitination of these proteins is mediated by USP20 (ubiquitin-specific protease 20), among other deubiquitinases. We sought to determine whether USP20 activity in SMCs decreases atherosclerosis. Approach and Results— To address this question, we used male Ldlr −/− mice without (control) or with SMC-specific expression of murine USP20 (SMC-USP20-transgenic) or its dominant-negative (DN; C154S/H643Q) mutant (SMC-DN-USP20-transgenic). Before the appearance of intimal macrophages, NFκB activation in aortic medial SMCs was greater in SMC-DN-USP20-transgenic than in control mice. After 16 weeks on a Western diet, SMC-DN-USP20-transgenic mice had 46% greater brachiocephalic artery atheroma area than control mice. Congruently, aortic atherosclerosis assessed en face was 21% greater than control in SMC-DN-USP20-transgenic mice and 13% less than control in SMC-USP20-transgenic mice. In response to TNF (tumor necrosis factor), SMCs from SMC-DN-USP20-transgenic mice showed ≈3-fold greater NFκB activation than control SMCs. Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFκB activation by ≈50% in response to either TNF or IL-1β (interleukin-1β). Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor-interacting protein kinase 1), a critical checkpoint in TNF-induced NFκB activation and inflammation. TNF evoked ≈2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro. Conclusions— USP20 attenuates TNF- and IL-1β–evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.

The actin-binding protein, Drebrin, is upregulated in response to arterial injury and reduces smooth muscle cell migration/proliferation through its interaction with the actin cytoskeleton. Because hypertensive aortic remodeling involves smooth muscle cell (SMC) activation and synthesis of extracellular matrix, we tested the hypothesis that Drebrin inhibits this process. To determine the effect of Drebrin deficiency in SMCs on hypertensive aortic remodeling, we induced hypertension by implanting osmotic mini-pumps to infuse angiotensin II (Ang II, 1000 ng/kg/min) or vehicle (0.9% NaCl) continuously for 28 days in SM22-α Cre +/- ; Dbn flox/flox mice (SMC- Dbn -/- mice) and controls. Blood pressure (BP) responses to Ang II treatment were assessed by telemetry. After completion of Ang II infusion, the degree of aortic remodeling was assessed by computerized tomography of histologic cross-sections of the proximal ascending aorta. Despite observing no difference in the extent of Ang II-induced hypertension in SMC- Dbn -/- mice compared with controls, SMC- Dbn -/- mice exhibited a significant increase in medial hypertrophy and outward remodeling. Wall thickness/body weight was increased by 61 ± 2% (p<0.01) in SMC- Dbn -/- mice compared with controls, and lumen area/body weight was increased by 102 ± 9% (p<0.01). Cellular proliferation and matrix deposition were increased in the proximal aortas of Ang II-treated SMC- Dbn -/- mice compared with controls as evidenced by increased immunoreactivity for PCNA, p-ERK, and Collagen I. SMC loss of Drebrin also resulted in increased Ang II-induced pro-inflammatory signaling as evidenced by increased expression of VCAM-1, p-P65 and MMP-9 and increased CD68 positive cellular proliferation in response to chronic Ang II infusion. We conclude that SMC loss of Drebrin augments adverse aortic remodeling in angiotensin II-induced hypertension.

AIMS:Chronic kidney disease (CKD) is a powerful independent risk factor for cardiovascular events, including vein graft failure. Because CKD impairs the clearance of small proteins, we tested the hypothesis that CKD exacerbates vein graft disease by elevating serum levels of critical cytokines that promote vein graft neointimal hyperplasia.METHODS AND RESULTS:We modelled CKD in C57BL/6 mice with 5/6ths nephrectomy, which reduced glomerular filtration rate by 60%, and we modelled vein grafting with inferior-vena-cava-to-carotid interposition grafting. CKD increased vein graft neointimal hyperplasia four-fold, decreased vein graft re-endothelialization two-fold, and increased serum levels of interleukin-9 (IL-9) five-fold. By quantitative immunofluorescence and histochemical staining, vein grafts from CKD mice demonstrated a ∼two-fold higher prevalence of mast cells, and a six-fold higher prevalence of activated mast cells. Concordantly, vein grafts from CKD mice showed higher levels of TNF and NFκB activation, as judged by phosphorylation of NFκB p65 on Ser536 and by expression of VCAM-1. Arteriovenous fistula veins from humans with CKD also showed up-regulation of mast cells and IL-9. Treating CKD mice with IL-9-neutralizing IgG reduced vein graft neointimal area four-fold, increased vein graft re-endothelialization ∼two-fold, and reduced vein graft total and activated mast cell levels two- and four-fold, respectively. Treating CKD mice with the mast cell stabilizer cromolyn reduced neointimal hyperplasia and increased re-endothelialization in vein grafts. In vitro, IL-9 promoted endothelial cell apoptosis but had no effect on smooth muscle cell proliferation.CONCLUSION:CKD aggravates vein graft disease through mechanisms involving IL-9 and mast cell activation.

The actin-binding protein Drebrin constrains smooth muscle cell (SMC) migration and proliferation in vitro and in vivo, and thereby reduces neointimal hyperplasia in response to arterial injury. We...

Kalirin is a 270-kDa scaffolding protein comprising 2 GTP exchange factors (GEFs). KALRN has been associated with human atherosclerosis (athero) in multiple genetic studies, and carotid injury prod...

Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated Gαi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable Gαi or Gαs activity. β-arrestin (βarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in βarr2 conformation. βarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by βarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of βarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.

The ubiquitously expressed, multifunctional scaffolding proteins β-arrestin1 and β-arrestin2 each affect inflammatory signaling in a variety of cell lines. In addition to binding the carboxyl-terminal tails of innumerable 7-transmembrane receptors, β-arrestins scaffold untold numbers of other plasma membrane and cytoplasmic proteins. Consequently, the effects of β-arrestins on inflammatory signaling are diverse, and context-specific. This review highlights the roles of β-arrestins in regulating canonical activation of the pro-inflammatory transcription factor NFκB.

Integral to atherosclerosis (athero) is smooth muscle cell (SMC) NFκB-activating signaling, which involves reversible ubiquitination that enhances protein activity downstream of pro-atherogenic tum...

beta-Arrestin1 and beta-arrestin2 are homologous adaptor proteins that are ubiquitously expressed in mammalian cells. They belong to a four-member family of arrestins that regulate the vast family of seven-transmembrane receptors that couple to heterotrimeric G proteins (7TMRs or GPCRs), and that modulate 7TMR signal transduction. beta-Arrestins were originally identified in the context of signal inhibition via the 7TMRs because they competed with and thereby blocked G protein coupling to 7TMRs. Currently, in addition to their role as desensitizers of signaling, beta-arrestins are appreciated as multifunctional adaptors that mediate trafficking and signal transduction of not only 7TMRs, but a growing list of additional receptors, ion channels, and nonreceptor proteins. beta-Arrestins' interactions with their multifarious partners are based on their dynamic conformational states rather than particular domain-domain interactions. beta-Arrestins adopt activated conformations upon 7TMR association. In addition, beta-arrestins undergo various posttranslational modifications that are choreographed by activated 7TMRs, including phosphorylation, ubiquitination, acetylation, nitrosylation, and SUMOylation. Ubiquitination of beta-arrestins is critical for their high-affinity interaction with 7TMRs as well as with endocytic adaptor proteins and signaling kinases. beta-Arrestins also function as critical adaptors for ubiquitination and deubiquitination of various cellular proteins, and thereby affect the longevity of signal transducers and the intensity of signal transmission.