H3K4 Methyltransferase Smyd3 Mediates Vascular Smooth Muscle Cell Proliferation, Migration, and Neointima Formation.

[Figure: see text].

Postnatal development alters functional compartmentalization of myosin light chain kinase in ovine carotid arteries.

The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC20) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC20. Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of Vmax and Km for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC20. The in situ rate of MLC20 phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the Vmax values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC20, and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

Hypoxic modulation of fetal vascular MLCK abundance, localization, and function.

Changes in vascular contractility are among the most important physiological effects of acute and chronic fetal hypoxia. Given the essential role of myosin light-chain kinase (MLCK) in smooth muscle contractility and its heterogeneous distribution, this study explores the hypothesis that subcellular changes in MLCK distribution contribute to hypoxic modulation of fetal carotid artery contractility. Relative to common carotid arteries from normoxic term fetal lambs (FN), carotids from fetal lambs gestated at high altitude (3,802 m) (FH) exhibited depressed contractility without changes in MLCK mRNA or protein abundance. Patterns of confocal colocalization of MLCK with α-actin and 20-kDa regulatory myosin light chain (MLC20) enabled calculation of subcellular MLCK fractions: 1) colocalized with the contractile apparatus, 2) colocalized with α-actin distant from the contractile apparatus, and 3) not colocalized with α-actin. Chronic hypoxia did not affect MLCK abundance in the contractile fraction, despite a concurrent decrease in contractility. Organ culture for 72 h under 1% O2 decreased total MLCK abundance in FN and FH carotid arteries, but decreased the contractile MLCK abundance only in FH carotid arteries. Correspondingly, culture under 1% O2 depressed contractility more in FH than FN carotid arteries. In addition, hypoxia appeared to attenuate ubiquitin-independent proteasomal degradation of MLCK, as reported for other proteins. In aggregate, these results demonstrate that the combination of chronic hypoxia followed by hypoxic culture can induce MLCK translocation among at least three subcellular fractions with possible influences on contractility, indicating that changes in MLCK distribution are a significant component of fetal vascular responses to hypoxia.

Sickle Cell Anemia Mediates Carotid Artery Expansive Remodeling That Can Be Prevented by Inhibition of JNK (c-Jun N-Terminal Kinase).

OBJECTIVE: Sickle cell anemia (SCA) causes chronic inflammation and multiorgan damage. Less understood are the arterial complications, most evident by increased strokes among children. Proteolytic mechanisms, biomechanical consequences, and pharmaceutical inhibitory strategies were studied in a mouse model to provide a platform for mechanistic and intervention studies of large artery damage due to sickle cell disease. Approach and Results: Townes humanized transgenic mouse model of SCA was used to test the hypothesis that elastic lamina and structural damage in carotid arteries increased with age and was accelerated in mice homozygous for SCA (sickle cell anemia homozygous genotype [SS]) due to inflammatory signaling pathways activating proteolytic enzymes. Elastic lamina fragmentation observed by 1 month in SS mice compared with heterozygous littermate controls (sickle cell trait heterozygous genotype [AS]). Positive immunostaining for cathepsin K, a powerful collagenase and elastase, confirmed accelerated proteolytic activity in SS carotids. Larger cross-sectional areas were quantified by magnetic resonance angiography and increased arterial compliance in SS carotids were also measured. Inhibiting JNK (c-jun N-terminal kinase) signaling with SP600125 significantly reduced cathepsin K expression, elastin fragmentation, and carotid artery perimeters in SS mice. By 5 months of age, continued medial thinning and collagen degradation was mitigated by treatment of SS mice with JNK inhibitor. CONCLUSIONS: Arterial remodeling due to SCA is mediated by JNK signaling, cathepsin proteolytic upregulation, and degradation of elastin and collagen. Demonstration in Townes mice establishes their utility for mechanistic studies of arterial vasculopathy, related complications, and therapeutic interventions for large artery damage due to SCA.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

Chronological Change of Vascular Reactivity to cGMP Generators in the Balloon-Injured Rat Carotid Artery.

BACKGROUND/AIMS: Soluble guanylate cyclase (sGC) exists as reduced, oxidized, and heme-free forms. Currently, it is unclear whether endovascular mechanical stenosis has an impact on vascular tone control by drugs targeting sGC, namely cGMP generators. METHODS: Pharmacological responses to acidified sodium nitrite (reduced sGC stimulant) and BAY 60-2770 (oxidized/heme-free sGC stimulant) were studied in balloon-injured rat carotid arteries at several time points. In addition, sGC expression was detected by immunohistochemistry. RESULTS: At 1 day after injury, acidified sodium nitrite-induced relaxation was attenuated in the injured artery, whereas BAY 60-2770-induced relaxation was augmented. Similar attenuation of response to acidified sodium nitrite was seen at 7 and 14 days after injury. On the other hand, the augmentation of response to BAY 60-2770 disappeared at 7 and 14 days after injury. At 1 day after injury, the immunohistochemical expression pattern of sGC in the smooth muscle layer of the injured artery was not different from that of the uninjured artery. However, in the injured artery, the intensity of sGC staining was weak at 7 and 14 days after injury. CONCLUSION: Balloon injury alters vascular responsiveness to cGMP generators, which seems to be associated with the form and/or expression of sGC.

RhoA inhibitor-eluting stent attenuates restenosis by inhibiting YAP signaling.

OBJECTIVE: Current drug-eluting stent (DES) treatment is promising, but it still has the drawback of in-stent restenosis, which remains a clinically relevant problem. Efforts should be made to discover new signaling molecules and novel potential targets for the prevention of arterial restenosis. In this study, we fabricated a novel DES targeting the RhoA pathway and further examined this promising strategy in vitro and in a rabbit carotid model. METHODS: Active RhoA expression is correlated with the synthetic smooth muscle phenotype, and the RhoA inhibitor rhosin suppresses this phenotypic modulation at both transcriptional and translational levels. We further demonstrated that the RhoA inhibitor rhosin might act through the YAP pathway in smooth muscle cell phenotype modulation by a gain-of-function assay. Moreover, we fabricated a RhoA inhibitor-eluting stent and tested it in a rabbit carotid model. RESULTS: Compared with a bare-metal stent, the RhoA inhibitor-eluting stent significantly attenuated neointimal formation at 6 months. However, overexpression of YAP by lentivirus blocked the antirestenosis effect of the RhoA inhibitor-eluting stent and repressed smooth muscle-specific genes. CONCLUSIONS: RhoA inhibitor-eluting stents attenuate neointimal formation through inhibition of the YAP signaling pathway. This novel DES may represent a potential strategy for the treatment of in-stent restenosis.

MicroRNA-24 attenuates vascular remodeling in diabetic rats through PI3K/Akt signaling pathway.

BACKGROUND AND AIMS: The vascular remodeling plays a crucial role in pathogenesis of diabetic cardiovascular complications. In this study, we intended to explore the effects and potential mechanisms of microRNA-24 (miR-24) on vascular remodeling under diabetic conditions. METHODS AND RESULTS: MiR-24 recombinant adenovirus (Ad-miR-24-GFP) was used to induce miR-24 overexpression either in carotid arteries or high glucose (HG)-induced vascular smooth muscle cells (VSMCs). Cell proliferation was analyzed using CCK-8 method. Cell migration was examined using wound-healing and transwell assay. mRNA and protein expressions of critical factors were, respectively, measured by real-time PCR and western blot as follows: qRT-PCR for the levels of miR-24, PIK3R1; western blot for the protein levels of PI3K (p85α), Akt, p-Akt, mTOR, p-mTOR, 4E-BP1, p-4E-BP1, p70s6k, p-p70s6k, MMP 2, MMP 9, collagen Ⅰ, as well as collagen Ⅲ. Carotid arteries in diabetic rats suffered balloon injury were harvested and examined by HE, immunohistochemical and Masson trichrome staining. The expression of miR-24 was decreased in HG-stimulated VSMCs and balloon-injured carotid arteries of diabetic rats, accompanied by increased mRNA expression of PIK3R1. The up-regulation of miR-24 suppressed VSMCs proliferation, migration, collagen deposition not only induced by HG in vitro, but also in balloon-injured diabetic rats, which were related to inactivation of PI3K/Akt signaling pathway. CONCLUSION: The up-regulation of miR-24 significantly attenuated vascular remodeling both in balloon-injured diabetic rats and HG-stimulated VSMCs via suppression of proliferation, migration and collagen deposition by acting on PIK3R1 gene that modulated the PI3K/Akt/mTOR axes.

Relationship between ADAMTS4 and carotid atherosclerotic plaque vulnerability in humans.

BACKGROUND: Rupture of atherosclerotic plaques and the resulting thrombosis are vital causes of clinical ischemic events. Recent studies have shown that ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) is a pathogenic factor of plaque vulnerability in mice. However, the relationship between ADAMTS4 and carotid atherosclerotic vulnerable plaques in humans remains unclear. METHODS: Forty-eight carotid atherosclerotic plaque specimens were obtained from 48 carotid artery stenosis inpatients undergoing carotid endarterectomy. We performed hematoxylin and eosin and Movat pentachrome staining for histologic characteristics; immunohistochemical staining for ADAMTS4, versican, and macrophages; and serologic tests for ADAMTS4. Patients were divided into stable and vulnerable groups on the basis of histologic characterization according to the classification criteria of the American Heart Association. Comparison between the groups was carried out using SPSS 17.0 (SPSS Inc, Chicago, Ill). RESULTS: Expression of ADAMTS4 in the plaque and its serum concentration were significantly higher in the vulnerable group compared with the stable one (P = .004 and P = .021, respectively), whereas the expression of versican was lower in the vulnerable group than in the stable group (P = .015). Univariate analysis revealed that the incidence of symptomatic cerebral ischemic events and ADAMTS4 serum levels were statistically higher in the vulnerable group compared with the stable group (P = .021 and P = .029, respectively). Multivariate analysis showed that ADAMTS4 was an independent risk factor (odds ratio, 1.14; P = .038). CONCLUSIONS: Our study revealed that ADAMTS4 expression was upregulated during carotid atherosclerotic plaque development. Serum levels of ADAMTS4 were associated with increased plaque vulnerability in both symptomatic and asymptomatic patients with carotid artery stenosis. ADAMTS4 may be a potential biomarker for plaque vulnerability.

CDKN2B Regulates TGFß Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels.

RATIONALE: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. OBJECTIVE: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. METHODS AND RESULTS: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor ß (TGFß) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFß1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFß activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFß1-induced-1, which is a TGFß-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. CONCLUSIONS: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFß signaling and hypoxic neovessel maturation.

Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling.

OBJECTIVE: Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. APPROACH AND RESULTS: Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. CONCLUSIONS: Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Synergistic Expression of Histone Deacetylase 9 and Matrix Metalloproteinase 12 in M4 Macrophages in Advanced Carotid Plaques.

OBJECTIVE/BACKGROUND: Expression patterns and association with cell specific gene expression signatures of the epigenetic regulator histone deacetylase 9 (HDAC9) and matrix metalloproteinase 12 (MMP12) in human plaque are not known. METHODS: This was a prospective cohort study. Genome wide expression analysis was performed in carotid, femoral, aortic plaques (n = 68) and left internal thoracic (LITA) controls (n = 28) and plaque histological severity assessed. Correlation and hierarchical cluster analysis was utilised. RESULTS: HDAC9 was associated with MMP12 expression in carotid plaques (r = .46, p = .012) and controls (r = -.44, p = .034). HDAC9 and MMP12 clustered with inflammatory macrophage markers but not with smooth muscle cell (SMC) rich markers. In plaques from all arterial sites, MMP12 but not HDAC9 showed positive correlation (p < .05) with M2 and M4 polarized macrophage markers, and negative correlation with SMC rich signatures. In the carotid plaques, all M4 macrophage markers associated with MMP12 and HDAC9. The negative association of MMP12 with SMC rich signatures was pronounced in the carotid plaques. Neither HDAC9 nor MMP12 associated consistently with plaque stabilisation or thrombosis related genes. Immunohistochemistry further supported the association between HDAC9 and MMP12 in atherosclerotic plaques. CONCLUSION: M4 macrophages are a possible source for HDAC9 and MMP12 expression in advanced human plaques.

Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1.

Circulating Bmp10 acts through endothelial Alk1 to mediate flow-dependent arterial quiescence.

Blood flow plays crucial roles in vascular development, remodeling and homeostasis, but the molecular pathways required for transducing flow signals are not well understood. In zebrafish embryos, arterial expression of activin receptor-like kinase 1 (alk1), which encodes a TGFß family type I receptor, is dependent on blood flow, and loss of alk1 mimics lack of blood flow in terms of dysregulation of a subset of flow-responsive arterial genes and increased arterial endothelial cell number. These data suggest that blood flow activates Alk1 signaling to promote a flow-responsive gene expression program that limits nascent arterial caliber. Here, we demonstrate that restoration of endothelial alk1 expression to flow-deprived arteries fails to rescue Alk1 activity or normalize arterial endothelial cell gene expression or number, implying that blood flow may play an additional role in Alk1 signaling independent of alk1 induction. To this end, we define cardiac-derived Bmp10 as the crucial ligand for endothelial Alk1 in embryonic vascular development, and provide evidence that circulating Bmp10 acts through endothelial Alk1 to limit endothelial cell number in and thereby stabilize the caliber of nascent arteries. Thus, blood flow promotes Alk1 activity by concomitantly inducing alk1 expression and distributing Bmp10, thereby reinforcing this signaling pathway, which functions to limit arterial caliber at the onset of flow. Because mutations in ALK1 cause arteriovenous malformations (AVMs), our findings suggest that an impaired flow response initiates AVM development.

Reduced glyoxalase 1 activity in carotid artery plaques of nondiabetic patients with increased hemoglobin A1c level.

OBJECTIVE: Glyoxalase 1 (GLO1) is ubiquitously expressed in the cytosol of the cell and is the major opponent against the reactive metabolite methylglyoxal, which is involved in the development of atherosclerosis. Nondiabetic individuals with an increased hemoglobin A1c (HbA1c) level are at higher risk for development of cardiovascular diseases. As such, this study investigated whether there was an association between reduced GLO1 activity in atherosclerotic lesions of nondiabetic patients with an increased HbA1c level. METHODS: HbA1c level was determined in venous blood of patients with carotid artery disease. Protein level of GLO1 was measured in endarterectomy-derived carotid artery plaques by Western blotting. Activity was measured by spectrophotometric assay in the plaques as well as in the erythrocytes; GLO1 activity in erythrocytes was compared with that in a cohort of healthy individuals (n = 15; 33% men; average age, 60 years). RESULTS: There were 36 patients with carotid artery disease (69% men; average age, 69 years) included in this study and divided into two equal groups: group I, HbA1c < 5.7% (<39 mmol/mol); and group II, 5.7% ≤ HbA1c < 6.5% (39 mmol/mol ≤ HbA1c < 48 mmol/mol). GLO1 activity in carotid plaques was reduced by 29% in group II compared with group I (P = .048), whereas protein expression was unchanged (P = .25). Analysis of GLO1 activity in erythrocytes revealed no difference between the groups (P = .36) or in comparison to healthy controls (P = .15). Examination of clinical parameters showed an increased amount of patients with concomitant peripheral arterial disease in group II (44% vs 10%; P = .020). CONCLUSIONS: Reduction of GLO1 activity in atherosclerotic lesions of nondiabetic patients with increased HbA1c is associated with a functional involvement of this protective enzyme in atherogenesis. Systemic GLO1 activity seems to be independent of both HbA1c and localized atherosclerosis as it was unchanged between group I and group II as well as compared with healthy controls, respectively.

Human urine kininogenase attenuates balloon-induced intimal hyperplasia in rabbit carotid artery through transforming growth factor ß1/Smad2/3 signaling pathway.

OBJECTIVE: Effective treatments against restenosis after percutaneous transluminal angioplasty and stenting are largely lacking. Human tissue kallikrein gene transfer has been shown to be able to attenuate neointima formation induced by balloon catheter. As a tissue kallikrein in vivo, human urinary kininogenase (HUK) is widely used to prevent ischemia-reperfusion injury. However, the effects of HUK on neointima formation have not been explored. We therefore investigated whether HUK could alleviate balloon catheter-induced intimal hyperplasia in rabbits fed with high-fat diets. METHODS: The effects of HUK on neointima and atherosclerosis formation were analyzed by hematoxylin-eosin staining and immunohistochemical staining in balloon-injured carotid arteries of rabbits. Local inflammatory response was evaluated by detecting the gene expression of tumor necrosis factor α and interleukin 1ß with real-time quantitative polymerase chain reaction plus the invasion of macrophages with immunohistochemical staining. Western blotting was employed to investigate the effects of HUK on activities of endothelial nitric oxide synthase (eNOS), transforming growth factor ß1 (TGF-ß1), and Smad signaling pathway. The long-term effect of HUK on intimal hyperplasia of the injured carotid artery was assessed by angiography. RESULTS: Quantitative image analysis showed that intravenous administration of HUK for 14 days significantly decreased the intimal areas and intima area/media area ratios (day 14, 54% decrease in intimal area and 58% decrease in intima area/media area ratios; day 28, 63% and 85%). Significant decreases were also noted in macrophage foam cell-positive area after 7-day or 14-day administration of HUK (day 7, 69% decrease in intimal area and 78% decrease in media area; day 14, 79% and 60%; day 28, 68% and 44%). Actin staining for smooth muscle cells in neointima at 2 months showed similar results (vascular smooth muscle cell-positive area of neointima, 28.21% ± 5.58% vs 43.78% ± 8.36%; P < .05). Real-time quantitative polymerase chain reaction or Western blot analysis showed that HUK reduced expression of tumor necrosis factor α, interleukin 1ß, TGF-ß1, and p-Smad2/3 but increased the expression of p-eNOS. Angiography analysis showed that 14-day administration of HUK significantly decreased the degree of stenosis (26.8% ± 7.1% vs 47.9% ± 5.7%; P < .01) at 2 months after balloon injury. CONCLUSIONS: Our results indicate that HUK is able to attenuate atherosclerosis formation and to inhibit intimal hyperplasia by downregulating TGF-ß1 expression and Smad2/3 phosphorylation, upregulating eNOS activity. HUK may be a potential therapeutic agent to prevent stenosis after vascular injury.

Nitric oxide differentially affects proteasome activator 28 after arterial injury in type 1 and type 2 diabetic rats.

BACKGROUND: Diabetic patients display aggressive restenosis after vascular interventions, likely because of proproliferative influences of hyperglycemia and hyperinsulinemia. We have shown that nitric oxide (NO) inhibits neointimal hyperplasia in type 2, but not in type 1, diabetic rats. Here, we examined proteasome activator 28 (PA28) after arterial injury in different diabetic environments, with or without NO. We hypothesize that NO differentially affects PA28 levels based on metabolic environment. MATERIALS AND METHODS: Vascular smooth muscle cell (VSMC) lysates from male, nondiabetic Lean Zucker (LZ) and Zucker Diabetic Fatty (ZDF) rats were assayed for 26S proteasome activity with or without PA28 and S-nitroso-N-acetylpenicillamine. LZ and ZDF VSMCs were treated with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate for 24 h. Balloon-injured carotid arteries from LZ, streptozotocin-injected LZ (STZ, type 1), and ZDF (type 2) rats treated with disodium 1-[2-(carboxylato)pyrrolidin-1-iyl]diazen-1-ium-1,2-diolate were harvested at 3 or 14 d. PA28α was assessed by Western blotting and immunofluorescent staining. RESULTS: S-nitroso-N-acetylpenicillamine reversed PA28-stimulated increases in 26S proteasome activity in LZ and ZDF VSMCs. Increased (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate lowered PA28α in LZ VSMCs but increased PA28α in ZDF VSMCs. At 3 d after injury, disodium 1-[2-(carboxylato)pyrrolidin-1-iyl]diazen-1-ium-1,2-diolate potentiated injury-induced PA28α decreases in LZ, STZ, and ZDF rats, suggesting VSMCs, depleted at this early time point, are major sources of PA28α. At 14 d after injury, total PA28α staining returned to baseline. However, although intimal and medial PA28α staining increased in injured STZ rats, adventitial PA28α staining increased in injured ZDF rats. CONCLUSIONS: PA28 dysregulation may explain the differential ability of NO to inhibit neointimal hyperplasia in type 1 versus type 2 diabetes.

Involvement of interleukin-1 receptor-associated kinase-1 in vascular smooth muscle cell proliferation and neointimal formation after rat carotid injury.

OBJECTIVE: Reduced frequency of atherosclerotic plaques is observed in interleukin-1 receptor-associated kinase-1 (IRAK1)-deficient mice; however, the underlying mechanism is not clear. Therefore, this study investigate the role of IRAK1 in vascular smooth muscle cell proliferation and neointimal hyperplasia. APPROACH AND RESULTS: Stimulation of rat primary vascular smooth muscle cells with fetal bovine serum (10%) or platelet-derived growth factor-BB (20 ng/mL) for 15 minutes to 24 hours induced a time-dependent increase in IRAK1 and extracellular signal-regulated kinase (ERK) activation, proliferating cell nuclear antigen upregulation and p27Kip1 downregulation as assessed by Western blotting. Inhibitors of ERK pathway (U0126, 10 µmol/L), IRAK (IRAK1/4, 3 µmol/L), protein kinase C (PKC; Ro-31-8220, 1 µmol/L), siRNA of toll-like receptor-4 (200 nmol/L), and PKC-ε (200 nmol/L) significantly attenuated these changes. Platelet-derived growth factor induced endogenous IRAK-ERK-PKC-ε association in a toll-like receptor-4 and PKC-ε-dependent manner. A time-dependent increase in IRAK1 and ERK activation was observed after 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours of carotid balloon injury in rats. Balloon injury induced endogenous IRAK-ERK-PKC-ε interaction. Perivascular application of IRAK1/4 inhibitor (100 µmol/L), U0126 (100 µmol/L), and IRAK1 siRNA (220 and 360 nmol/L) in pluronic gel abrogated balloon injury-induced ERK phosphorylation, activation, and p27Kip1 downregulation. Hematoxylin and eosin staining and immunohistochemistry of proliferating cell nuclear antigen and smooth muscle actin demonstrated that balloon injury-induced intimal thickening and neointimal vascular smooth muscle cell proliferation were significantly abrogated in the presence of IRAK1/4 inhibitor, IRAK1 siRNA, and U0126. CONCLUSIONS: IRAK1 mediates vascular smooth muscle cell proliferation and neointimal hyperplasia by regulating PKC-ε-IRAK1-ERK axis.

Nucleoside diphosphate kinase B-activated intermediate conductance potassium channels are critical for neointima formation in mouse carotid arteries.

OBJECTIVE: Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca(2+)-activated K(+) (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (ISK4) in proliferating VSMC and neointima formation. APPROACH AND RESULTS: Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased ISK4, whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in ISK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. CONCLUSIONS: NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

Leukocyte cathepsin C deficiency attenuates atherosclerotic lesion progression by selective tuning of innate and adaptive immune responses.

OBJECTIVE: The protein degrading activity of cathepsin C (CatC), combined with its role in leukocyte granule activation, suggests a contribution of this cystein protease in atherosclerosis. However, no experimental data are available to validate this concept. APPROACH AND RESULTS: CatC gene and protein expression were increased in ruptured versus advanced stable human carotid artery lesions. To assess causal involvement of CatC in plaque progression and stability, we generated LDLr(-/-)//CatC(-/-) chimeras by bone marrow transplantation. CatC(-/-) chimeras presented attenuated plaque burden in carotids, descending aorta, aortic arch and root, at both the early and advanced plaque stage. CatC was abundantly expressed by plaque macrophages and foam cells. CatC expression and activity were dramatically downregulated in plaques of CatC(-/-) chimeras, supporting a hematopoietic origin of plaque CatC. Our studies unveiled an unexpected feedback of CatC deficiency on macrophage activation programs and T helper cell differentiation in as much as that CatC expression was upregulated in M1 macrophages, whereas its deficiency led to combined M2 (in vitro) and Th2 polarization (in vivo). CONCLUSIONS: Our data implicate CatC has a role in the selective tuning of innate and adaptive immune responses, relevant to a chronic immune disease, such as atherosclerosis.