Dual-Specificity Phosphatase 6 Deficiency Attenuates Arterial-Injury-Induced Intimal Hyperplasia in Mice.

In response to injury, vascular smooth muscle cells (VSMCs) of the arterial wall dedifferentiate into a proliferative and migratory phenotype, leading to intimal hyperplasia. The ERK1/2 pathway participates in cellular proliferation and migration, while dual-specificity phosphatase 6 (DUSP6, also named MKP3) can dephosphorylate activated ERK1/2. We showed that DUSP6 was expressed in low baseline levels in normal arteries; however, arterial injury significantly increased DUSP6 levels in the vessel wall. Compared with wild-type mice, Dusp6-deficient mice had smaller neointima. In vitro, IL-1ß induced DUSP6 expression and increased VSMC proliferation and migration. Lack of DUSP6 reduced IL-1ß-induced VSMC proliferation and migration. DUSP6 deficiency did not affect IL-1ß-stimulated ERK1/2 activation. Instead, ERK1/2 inhibitor U0126 prevented DUSP6 induction by IL-1ß, indicating that ERK1/2 functions upstream of DUSP6 to regulate DUSP6 expression in VSMCs rather than downstream as a DUSP6 substrate. IL-1ß decreased the levels of cell cycle inhibitor p27 and cell-cell adhesion molecule N-cadherin in VSMCs, whereas lack of DUSP6 maintained their high levels, revealing novel functions of DUSP6 in regulating these two molecules. Taken together, our results indicate that lack of DUSP6 attenuated neointima formation following arterial injury by reducing VSMC proliferation and migration, which were likely mediated via maintaining p27 and N-cadherin levels.

Cysteine-rich protein 2 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in mice.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a relatively common and often fatal condition. A major histopathological hallmark of AAA is the severe degeneration of aortic media with loss of vascular smooth muscle cells (VSMCs), which are the main source of extracellular matrix (ECM) proteins. VSMCs and ECM homeostasis are essential in maintaining structural integrity of the aorta. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed protein; however, the role of CRP2 in AAA formation is unclear. METHODS: To investigate the function of CRP2 in AAA formation, mice deficient in Apoe (Apoe-/-) or both CRP2 (gene name Csrp2) and Apoe (Csrp2-/-Apoe-/-) were subjected to an angiotensin II (Ang II) infusion model of AAA formation. Aortas were harvested at different time points and histological analysis was performed. Primary VSMCs were generated from Apoe-/- and Csrp2-/-Apoe-/- mouse aortas for in vitro mechanistic studies. RESULTS: Loss of CRP2 attenuated Ang II-induced AAA incidence and severity, accompanied by preserved smooth muscle α-actin expression and reduced elastin degradation, matrix metalloproteinase 2 (MMP2) activity, deposition of collagen, particularly collagen III (Col III), aortic tensile strength, and blood pressure. CRP2 deficiency decreased the baseline MMP2 and Col III expression in VSMCs and mitigated Ang II-induced increases of MMP2 and Col III via blunting Erk1/2 signaling. Rescue experiments were performed by reintroducing CRP2 into Csrp2-/-Apoe-/- VSMCs restored Ang II-induced Erk1/2 activation, MMP2 expression and activity, and Col III levels. CONCLUSIONS: Our results indicate that in response to Ang II stimulation, CRP2 deficiency maintains aortic VSMC density, ECM homeostasis, and structural integrity through Erk1/2-Col III and MMP2 axis and reduces AAA formation. Thus, targeting CRP2 provides a potential therapeutic strategy for AAA.

Modulation of cysteine-rich protein 2 expression in vascular injury and atherosclerosis.

Vascular smooth muscle cells (VSMCs) of the arterial wall normally display a differentiated and contractile phenotype. In response to arterial injury, VSMCs switch to a synthetic phenotype, contributing to vascular remodeling. Cysteine-rich protein 2 (CRP2) is a cytoskeletal protein expressed in VSMCs and blunts VSMC migration in part by sequestering the scaffolding protein p130Cas at focal adhesions. CRP2 deficiency in mice increases neointima formation following arterial injury. The goal of this study was to use Csrp2 promoter-lacZ transgenic mice to analyze CRP2 expression during VSMC phenotypic modulation. In a neointima formation model after carotid artery cessation of blood flow, lacZ reporter activity and smooth muscle (SM) α-actin expression in the media were rapidly downregulated 4 days after carotid ligation. Fourteen days after ligation, there was a high level expression of both Csrp2 promoter activity and SM α-actin protein expression in neointimal cells. In atherosclerosis prone mice fed an atherogenic diet, Csrp2 promoter activity was detected within complex atherosclerotic lesions. Interestingly, Csrp2 promoter activity was also present in the fibrous caps of complicated atherosclerotic lesions, indicating that CRP2 might contribute to plaque stability. These findings support the concept that CRP2 contributes to the phenotypic modulation of VSMCs during vascular disease. Modulating transcription to increase CRP2 expression during vascular injury might attenuate vascular remodeling. In addition, increased CRP2 expression at the fibrous caps of advanced lesions might also serve to protect atherosclerotic plaques from rupture.

Essential role of nephrocystin in photoreceptor intraflagellar transport in mouse.

Nephrocystin mutations account for the vast majority of juvenile nephronophthisis, the most common inherited cause of renal failure in children. Nephrocystin has been localized to the ciliary transition zone of epithelial cells or its analogous structure, connecting cilium of retinal photoreceptors. Thus, the retinal degeneration associated with nephronophthisis may be explained by a functional ciliary defect. However, the function of nephrocystin in cilium assembly and maintenance of common epithelial cells and photoreceptors is still obscure. Here, we used Nphp1-targeted mutant mice and transgenic mice expressing EmGFP-tagged nephrocystin to demonstrate that nephrocystin located at connecting cilium axoneme can affect the sorting mechanism and transportation efficiency of the traffic machinery between inner and outer segments of photoreceptors. This traffic machinery is now recognized as intraflagellar transport (IFT); a microtubule-based transport system consisting of motors, IFT particles and associated cargo molecules. Nephrocystin seems to control some of the IFT particle components moving along the connecting cilia so as to regulate this inter-segmental traffic. Our novel findings provide a clue to unraveling the regulatory mechanism of nephrocystin in IFT machinery.

Progressive renal distortion by multiple cysts in transgenic mice expressing artificial microRNAs against Pkd1.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening inherited diseases, and the PKD1 gene is responsible for most cases of this disease. Previous efforts to establish a mouse model that recapitulates the phenotypic characteristics of ADPKD, which have used conventional or conditional knockout of the mouse orthologue Pkd1, have been unsuccessful or unreliable. In a previous study, we described the generation of a novel Pkd1 hypomorphic allele, in which Pkd1 expression was significantly reduced but not totally blocked. These Pkd1 homozygous mutant mice rapidly developed renal cystic disease, supporting the hypothesis that 'haploinsufficiency' explains development of the ADPKD phenotype. In the present study, we further investigated the Pkd1 haploinsufficiency effect by generating Pkd1 knockdown transgenic mice with co-cistronic expression of two miRNA hairpins specific to Pkd1 transcript and an Emerald GFP reporter driven by a human ubiquitin B promoter. Two transgenic lines which had ∼60-70% reduction of Pkd1 expression developed severe renal cystic disease at a rate similar to that of human ADPKD. These results further support the haploinsufficiency hypothesis, and suggest that the onset and progression of the renal cystic diseases are correlated with the level of Pkd1 expression. The two novel mutant lines of mice appear to be ideal models for the study of ADPKD.

Effects of isolated soy protein and strength exercise training on exercise performance and biochemical profile in postpartum mice.

OBJECTIVE: Postpartum women are at an increased risk for obesity and metabolic diseases because of excessive weight gain during pregnancy and weight retention after delivery. Maintenance of good nutrition and regular physical activity is used as a therapeutic approach for promotion of health and well-being in postpartum women. The aim of this study is to assess the independent and additive effects of isolated soy protein (ISP) and strength exercise training (ET) on weight management, exercise performance and health maintenance in postpartum mice. DESIGN AND METHODS: Thirty-two postpartum mice (ICR, 14-weeks old) were divided into four groups (n = 8 per group): Group 1 mice were the sedentary control with vehicle (SC), Group 2 mice were the sedentary control with ISP supplementation (8.95 g·kg-1, SC + ISP), Group 3 mice received vehicle with exercise training (ET) and Group 4 mice received isolated soy protein with exercise training (ISP + ET). Animals in the ET and ISP + ET groups underwent strength exercise training for 6 weeks, 5 days a week. Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time, as well as by changes in body composition and biochemical parameters at the end of the experiment. RESULTS: Combined intervention of ISP and ET increased lean muscle mass and prevented body weight and fat elevation. The grip strength and exhaustive swimming time of the ISP + ET group were significantly higher than the other groups. The ISP + ET group showed significantly decreased serum levels of lactate, ammonia and creatinine phosphate kinase (CPK), and increased glucose level after the 15-min swimming test. The serum levels of aspartate transaminase (AST), triglyceride (TG) and creatinine after sacrifice were significantly decreased in the ET + ISP group. ISP combined with ET promoted fat oxidation in brown adipose tissue (BAT) as evidenced from the increased utilization of plasma and BAT tissue triglyceride. CONCLUSIONS: We suggest that long-term supplementation with ISP can have a wide spectrum of bioactivities on health promotion, performance improvement and fitness. ISP with ET conferred better energy utilization, improved biochemical profiles and may be an effective ergogenic aid in strength training.

Tryptophan metabolite 5-methoxytryptophan ameliorates arterial denudation-induced intimal hyperplasia via opposing effects on vascular endothelial and smooth muscle cells.

Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, particularly among older adults. Despite the advent of medical technology, restenosis is still an issue after interventional procedures. Tryptophan metabolite 5-methoxytryptophan (5-MTP) has recently been shown to protect against systemic inflammatory responses. This study aimed to investigate the function and mechanisms of 5-MTP in interventional procedure-induced restenosis. We found that after mouse femoral artery denudation with a guide wire, 5-MTP accelerated recovery of endothelium in the denuded area and reduced vascular leakage and intimal thickening. 5-MTP increased endothelial cell proliferation in the denuded arteries and rescued TNF-α-reduced endothelial cell proliferation and migration, likely via maintaining vascular endothelial growth factor receptor 2 activation. In contrast, 5-MTP preserved differentiated phenotype of medial vascular smooth muscle cells (VSMCs) and decreased VSMC proliferation and migration. Furthermore, 5-MTP maintained expression levels of critical transcription factors for VSMC marker gene expressions via attenuated activation of p38 MAPK and NFκB-p65. Our findings uncover a novel protective mechanism of 5-MTP in restenosis. In response to denudation injury, 5-MTP attenuates intimal hyperplasia via concerted but opposing actions on endothelial cells and VSMCs. Taken together, our results suggest that 5-MTP is a valuable therapeutic target for arterial injury-induced restenosis.

A Novel Protective Function of 5-Methoxytryptophan in Vascular Injury.

5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of tryptophan metabolism, was recently shown to suppress inflammatory mediator-induced cancer cell proliferation and migration. However, the role of 5-MTP in vascular disease is unknown. In this study, we investigated whether 5-MTP protects against vascular remodeling following arterial injury. Measurements of serum 5-MTP levels in healthy subjects and patients with coronary artery disease (CAD) showed that serum 5-MTP concentrations were inversely correlated with CAD. To test the role of 5-MTP in occlusive vascular disease, we subjected mice to a carotid artery ligation model of neointima formation and treated mice with vehicle or 5-MTP. Compared with vehicle-treated mice, 5-MTP significantly reduced intimal thickening by 40% 4 weeks after ligation. BrdU incorporation assays revealed that 5-MTP significantly reduced VSMC proliferation both in vivo and in vitro. Furthermore, 5-MTP reduced endothelial loss and detachment, ICAM-1 and VCAM-1 expressions, and inflammatory cell infiltration in the ligated arterial wall, suggesting attenuation of endothelial dysfunction. Signaling pathway analysis indicated that 5-MTP mediated its effects predominantly via suppressing p38 MAPK signaling in endothelial and VSMCs. Our data demonstrate a novel vascular protective function of 5-MTP against arterial injury-induced intimal hyperplasia. 5-MTP might be a therapeutic target for preventing and/or treating vascular remodeling.

Cysteine-rich protein 2 alters p130Cas localization and inhibits vascular smooth muscle cell migration.

AIMS: Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, is expressed in vascular smooth muscle cells (VSMCs) of blood vessels and functions to repress VSMC migration and vascular remodelling. The goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration. METHODS AND RESULTS: Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 associated with the actin cytoskeleton. In response to chemoattractant stimulation, Csrp2 (mouse CRP2 gene symbol)-deficient (Csrp2(-/-)) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2(-/-) VSMCs following chemoattractant stimulation. Mammalian 2-hybrid and co-immunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibres in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs, whereas CRP2 was restricted to FAs and stress fibres. Furthermore, we demonstrated that p130Cas expression and phosphorylation promote neointima formation following arterial injury. CONCLUSION: These studies demonstrate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.