Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB 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 ∼50% 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 NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. 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-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB 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-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

Drebrin attenuates atherosclerosis by limiting smooth muscle cell transdifferentiation.

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+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/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 Dbnflox/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 Dbnflox/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 Dbnflox/flox SMCs. CONCLUSION: We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells.

USP20 (Ubiquitin-Specific Protease 20) Inhibits TNF (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates Atherosclerosis.

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-1ß (interleukin-1ß). 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-1ß-evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.

Drebrin regulates angiotensin II-induced aortic remodelling.

Aims: The 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 results: Angiotensin 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. Conclusions: We conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.

Regulation of inflammation by ß-arrestins: Not just receptor tales.

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

Interleukin-9 mediates chronic kidney disease-dependent vein graft disease: a role for mast cells.

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.

C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways.

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 G αi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G αi or G αs activity. ß-arrestin (ßarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in ßarr2 conformation. ßarr2 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 ßarr1/2 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 ßarr 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 Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia.

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of ß-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation.

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ß-arrestin2. Although ß-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ß-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of ß-arrestin2 activity could be dictated by ß-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ß-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20in vivoandin vitro Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that ß-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ß-arrestin2 was augmented when ß-arrestin2 ubiquitination was prevented and reduced when ß-arrestin2 ubiquitination was rendered constitutive. Constitutive ß-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of ß-arrestin2 are determined by ß-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ß-arrestin2.

Kruppel-like factor 15 is critical for vascular inflammation.

Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle-specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.

Immunoglobulin-like domain containing receptor 1 mediates fat-stimulated cholecystokinin secretion.

Cholecystokinin (CCK) is a satiety hormone produced by discrete enteroendocrine cells scattered among absorptive cells of the small intestine. CCK is released into blood following a meal; however, the mechanisms inducing hormone secretion are largely unknown. Ingested fat is the major stimulant of CCK secretion. We recently identified a novel member of the lipoprotein remnant receptor family known as immunoglobulin-like domain containing receptor 1 (ILDR1) in intestinal CCK cells and postulated that this receptor conveyed the signal for fat-stimulated CCK secretion. In the intestine, ILDR1 is expressed exclusively in CCK cells. Orogastric administration of fatty acids elevated blood levels of CCK in wild-type mice but not Ildr1-deficient mice, although the CCK secretory response to trypsin inhibitor was retained. The uptake of fluorescently labeled lipoproteins in ILDR1-transfected CHO cells and release of CCK from isolated intestinal cells required a unique combination of fatty acid plus HDL. CCK secretion secondary to ILDR1 activation was associated with increased [Ca2+]i, consistent with regulated hormone release. These findings demonstrate that ILDR1 regulates CCK release through a mechanism dependent on fatty acids and lipoproteins and that absorbed fatty acids regulate gastrointestinal hormone secretion.

Kalirin promotes neointimal hyperplasia by activating Rac in smooth muscle cells.

OBJECTIVE: Kalirin is a multifunctional protein that contains 2 guanine nucleotide exchange factor domains for the GTPases Rac1 and RhoA. Variants of KALRN have been associated with atherosclerosis in humans, but Kalirin's activity has been characterized almost exclusively in the central nervous system. We therefore tested the hypothesis that Kalirin functions as a Rho-guanine nucleotide exchange factor in arterial smooth muscle cells (SMCs). APPROACH AND RESULTS: Kalirin-9 protein is expressed abundantly in aorta and bone marrow, as well as in cultured SMCs, endothelial cells, and macrophages. Moreover, arterial Kalirin was upregulated during early atherogenesis in apolipoprotein E-deficient mice. In cultured SMCs, signaling was affected similarly in 3 models of Kalirin loss-of-function: heterozygous Kalrn deletion, Kalirin RNAi, and treatment with the Kalirin Rho-guanine nucleotide exchange factor -1 inhibitor 1-(3-nitrophenyl)-1H-pyrrole-2,5-dione. With reduced Kalirin function, SMCs showed normal RhoA activation but diminished Rac1 activation, assessed as reduced Rac-GTP levels, p21-activated kinase autophosphorylation, and SMC migration. Kalrn(-/+) SMCs proliferated 30% less rapidly than wild-type SMCs. Neointimal hyperplasia engendered by carotid endothelial denudation was ≈60% less in Kalrn(-/+) and SMC-specific Kalrn(-/+) mice than in control mice. CONCLUSIONS: Kalirin functions as a guanine nucleotide exchange factor for Rac1 in SMCs, and promotes SMC migration and proliferation both in vitro and in vivo.

MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound ß(2)-adrenergic receptors.

Lysosomal degradation of ubiquitinated ß(2)-adrenergic receptors (ß(2)ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the ßAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed ß(2)ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-ß(2)ARs in HEK-293 cells. Carvedilol prevented ß(2)AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography-tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound ß(2)AR. The association of MARCH2 with internalized ß(2)ARs was stabilized by carvedilol and did not involve ß-arrestin. Small interfering RNA-mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous ß(2)ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to ß blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface ß(2)AR expression and, consequently, its signaling.

Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft-extrinsic cells.

OBJECTIVE: Because vein graft neointimal hyperplasia engenders vein graft failure, and because most vein graft neointimal cells derive from outside the vein graft, we sought to determine whether vein graft neointimal hyperplasia is affected by activity of the CXC chemokine receptor-4 (CXCR4), which is important for bone marrow-derived cell migration. METHODS: In congenic Cxcr4(-/+) and wild-type (WT) recipient mice, we performed interposition grafting of the common carotid artery with the inferior vena cava (IVC) of either Cxcr4(-/+) or WT mice to create four surgically chimeric groups of mice (n ≥ 5 each), characterized by vein graft donor/recipient: WT/WT; Cxcr4(-/+)/WT; WT/Cxcr4(-/+); and Cxcr4(-/+)/Cxcr4(-/+); vein grafts were harvested 6 weeks postoperatively. RESULTS: The agonist for CXCR4 is expressed by cells in the arterializing vein graft. Vein graft neointimal hyperplasia was reduced by reducing CXCR4 activity in vein graft-extrinsic cells, but not in vein graft-intrinsic cells: the rank order of neointimal hyperplasia was WT/WT ≈ Cxcr4(-/+)/WT > WT/Cxcr4(-/+) ≈ Cxcr4(-/+)/Cxcr4(-/+); CXCR4 deficiency in graft-extrinsic cells reduced neointimal hyperplasia by 39% to 47% (P < .05). Vein graft medial area was equivalent in all grafts except Cxcr4(-/+)/Cxcr4(-/+), in which the medial area was 60% ± 20% greater (P < .05). Vein graft re-endothelialization was indistinguishable among all three vein graft groups. However, the prevalence of medial leukocytes was 40% ± 10% lower in Cxcr4(-/+)/Cxcr4(-/+) than in WT/WT vein grafts (P < .05), and the prevalence of smooth muscle actin-positive cells was 45% ± 20% higher (P < .05). CONCLUSIONS: We conclude that CXCR4 contributes to vein graft neointimal hyperplasia through mechanisms that alter homing to the vein graft of graft-extrinsic cells, particularly leukocytes. CLINICAL RELEVANCE: The utility of autologous vein grafts is severely reduced by neointimal hyperplasia, which accelerates subsequent graft atherosclerosis. Our study demonstrates that vein graft neointimal hyperplasia is aggravated by activity of the cell-surface "CXC" chemokine receptor-4 (CXCR4), which is critical for recruitment of bone marrow-derived cells to sites of inflammation. Our model for CXCR4 deficiency used mice with heterozygous deficiency of Cxcr4. Consequently, our results suggest the possibility that a CXCR4 antagonist--like plerixafor, currently in clinical use--could be applied to vein grafts periadventitially, and perhaps achieve beneficial effects on vein graft neointimal hyperplasia.

G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors.

OBJECTIVE: G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-κB (NF-κB). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. METHODS AND RESULTS: Grk5(-/-)/Apoe(-/-) mice developed 50% greater aortic atherosclerosis than Apoe(-/-) mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe(-/-) mice, carotid interposition grafts from Grk5(-/-) mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5(-/-) with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-ß in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-κB-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-κB activity in macrophages. CONCLUSIONS: GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-κB activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-ß.

Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis.

OBJECTIVE: To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. METHODS AND RESULTS: Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface α(5)ß(1) integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdc(scid)/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression. CONCLUSIONS: The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.

Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice.

AIMS: Vein graft endothelial damage is a key step in the development of neointimal hyperplasia, leading to vein graft failure. We sought to determine whether exogenous endothelial progenitor cells could promote vein graft re-endothelialization, and thereby ameliorate neointimal hyperplasia. METHODS AND RESULTS: Carotid artery interposition grafting was performed with syngeneic inferior vena cavae in mice with severe combined immunodeficiency (SCID). Lineage-negative human umbilical cord blood (hUCB) cells (or medium alone) were injected into vein-grafted mice intra-operatively and 2 weeks post-operatively. In vein grafts from hUCB cell-injected mice, we found human HLA-expressing endothelial cells, as well as increased levels of VEGF and FGF-2. Furthermore, hUCB cells secreted VEGF and FGF-2 in vitro. The markedly enhanced endothelial regeneration, likely resulting from both direct engraftment and paracrine actions of hUCB cells, inhibited inflammatory response, diminished intimal cell proliferation, and reduced neointimal hyperplasia in the vein grafts. CONCLUSIONS: hUCB cells may accelerate vein graft re-endothelialization via both direct differentiation into endothelial cells and release of paracrine factors to enhance endothelial regeneration and reduce inflammation. These data highlight a potential therapeutic role for cellular therapy in vessel injury.

Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies.

Aging is believed to be among the most important contributors to atherosclerosis, through mechanisms that remain largely obscure. Serum levels of tumor necrosis factor (TNF) rise with aging and have been correlated with the incidence of myocardial infarction. We therefore sought to determine whether genetic variation in the TNF receptor-1 gene (TNFR1) contributes to aging-related atherosclerosis in humans and whether Tnfr1 expression aggravates aging-related atherosclerosis in mice. With 1330 subjects from a coronary angiography database, we performed a case-control association study of coronary artery disease (CAD) with 16 TNFR1 single-nucleotide polymorphisms (SNPs). Two TNFR1 SNPs significantly associated with CAD in subjects >55 years old, and this association was supported by analysis of a set of 759 independent CAD cases. In multiple linear regression analysis, accounting for TNFR1 SNP rs4149573 significantly altered the relationship between aging and CAD index among 1811 subjects from the coronary angiography database. To confirm that TNFR1 contributes to aging-dependent atherosclerosis, we grafted carotid arteries from 18- and 2-month-old wild-type (WT) and Tnfr1(-/-) mice into congenic apolipoprotein E-deficient (Apoe(-/-)) mice and harvested grafts from 1 to 7 weeks post-operatively. Aged WT arteries developed accelerated atherosclerosis associated with enhanced TNFR1 expression, enhanced macrophage recruitment, reduced smooth muscle cell proliferation and collagen content, augmented apoptosis and plaque hemorrhage. In contrast, aged Tnfr1(-/-) arteries developed atherosclerosis that was indistinguishable from that in young Tnfr1(-/-) arteries and significantly less than that observed in aged WT arteries. We conclude that TNFR1 polymorphisms associate with aging-related CAD in humans, and TNFR1 contributes to aging-dependent atherosclerosis in mice.

Neuropeptide Y gene polymorphisms confer risk of early-onset atherosclerosis.

Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58-2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46-1.65, p = 0.01-0.05), showing stronger association in youngest cases (OR 1.84-2.20, p = 0.0004-0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79-2.06, p = 0.003-0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor-antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.