RACK1/TRAF2 regulation of modulator of apoptosis-1 (MOAP-1).

MOAP-1 is a pro-apoptotic tumor suppressor molecule with a growing set of known interacting partners. We have demonstrated that during death receptor-dependent apoptosis, MOAP-1 is recruited to TNF-R1 or TRAIL-R1, followed by RASSF1A and Bax association. MOAP-1/Bax association promotes Bax conformational change resulting in the translocation of Bax into the mitochondrial membrane, mitochondrial membrane insertion and dysregulation resulting in several hallmark events that execute apoptosis. Although a role in apoptosis is established, it is currently unknown how MOAP-1 is regulated and how it links to Bax to promote apoptosis. In this study, we demonstrate robust association with RACK1, a versatile scaffolding protein that responds to activation of protein kinase C. Furthermore, we can demonstrate that RACK1 functions to bring the E3 ligase, TRAF2, to MOAP-1 in order to undergo a K63-dependent ubiquitination. Furthermore, RACK1 associates with MOAP-1 via electrostatic associations similar to those observed between MOAP-1/RASSF1A and MOAP-1/TNF-R1. These events illustrate the complex nature of MOAP-1 regulation and characterizes the important role of the scaffolding protein, RACK1, in influencing MOAP-1 biology.

miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation.

BACKGROUND: MicroRNA-22 (miR-22) has recently been reported to play a regulatory role during vascular smooth muscle cell (VSMC) differentiation from stem cells, but little is known about its target genes and related pathways in mature VSMC phenotypic modulation or its clinical implication in neointima formation following vascular injury. METHODS: We applied a wire-injury mouse model, and local delivery of AgomiR-22 or miR-22 inhibitor, as well, to explore the therapeutic potential of miR-22 in vascular diseases. Furthermore, normal and diseased human femoral arteries were harvested, and various in vivo, ex vivo, and in vitro models of VSMC phenotype switching were conducted to examine miR-22 expression during VSMC phenotype switching. RESULTS: Expression of miR-22 was closely regulated during VSMC phenotypic modulation. miR-22 overexpression significantly increased expression of VSMC marker genes and inhibited VSMC proliferation and migration, whereas the opposite effect was observed when endogenous miR-22 was knocked down. As expected, 2 previously reported miR-22 target genes, MECP2 (methyl-CpG binding protein 2) and histone deacetylase 4, exhibited a regulatory role in VSMC phenotypic modulation. A transcriptional regulator and oncoprotein, EVI1 (ecotropic virus integration site 1 protein homolog), has been identified as a novel miR-22 target gene in VSMC phenotypic modulation. It is noteworthy that overexpression of miR-22 in the injured vessels significantly reduced the expression of its target genes, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries, whereas the opposite effect was observed with local application of a miR-22 inhibitor to injured arteries. We next examined the clinical relevance of miR-22 expression and its target genes in human femoral arteries. We found that miR-22 expression was significantly reduced, whereas MECP2 and EVI1 expression levels were dramatically increased, in diseased in comparison with healthy femoral human arteries. This inverse relationship between miR-22 and MECP2 and EVI1 was evident in both healthy and diseased human femoral arteries. CONCLUSIONS: Our data demonstrate that miR-22 and EVI1 are novel regulators of VSMC function, specifically during neointima hyperplasia, offering a novel therapeutic opportunity for treating vascular diseases.

Novel Pathological Role of hnRNPA1 (Heterogeneous Nuclear Ribonucleoprotein A1) in Vascular Smooth Muscle Cell Function and Neointima Hyperplasia.

OBJECTIVE: hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) plays a variety of roles in gene expression. However, little is known about the functional involvement of hnRNPA1 in vascular smooth muscle cell (VSMC) function and neointima hyperplasia. In this study, we have attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC function, injury-induced vessel remodeling, and human atherosclerotic lesions, as well as discern the molecular mechanisms involved. APPROACH AND RESULTS: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation, and migration were regulated by hnRNPA1. Our data show that hnRNPA1 exerts its effects on VSMC functions through modulation of IQGAP1 (IQ motif containing GTPase activating protein 1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through 2 mechanisms: upregulating microRNA-124 (miR-124) and binding to AU-rich element of IQGAP1 gene. Further evidence suggests that hnRNPA1 upregulates miR-124 by modulating miR-124 biogenesis and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. CONCLUSIONS: Our data show that hnRNPA1 is a critical regulator of VSMC function and behavior in the context of neointima hyperplasia, and the hnRNPA1/miR-124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.

Zebrafish Model for Functional Screening of Flow-Responsive Genes.

OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.

A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization.

Matrix metalloproteinase-8 (MMP8) has been shown to influence various cellular functions. As monocytes and macrophages (Mφ) express MMP8, we investigated if MMP8 played a role in macrophage differentiation and polarization. MMP8 expression was significantly increased during monocyte differentiation into Mφ. Monocyte-derived Mφ from MMP8-deficient mice expressed higher levels of M1-Mφ markers but lower levels of M2-Mφ markers than monocyte-derived Mφ from wild-type mice. Although Mφ from either MMP8-deficient or wild-type mice were inducible by interferon-γ into M1-Mφ, only wild-type Mφ but not MMP8-deficient Mφ could be induced into M2-Mφ by interleukin-4. However, MMP8-deficient Mφ exposed to conditioned culture media of wild-type Mφ developed a M2-Mφ phenotype. Compared with conditioned culture media of wild-type Mφ, conditioned culture media of MMP8-deficient Mφ contained a lower concentration of active transforming growth factor-ß (TGF-ß), an M2-Mφ inducer. Moreover, evidence also showed that the degradation of the TGF-ß sequester, fibromodulin, was modulated by MMP8. The data indicate a previously unknown role of MMP8 in M2-Mφ polarization by cleaving fibromodulin and therefore increasing the bioavailability of the M2-Mφ inducer TGF-ß.

Modulator of apoptosis 1 (MOAP-1) is a tumor suppressor protein linked to the RASSF1A protein.

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-ß, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.

A20 suppresses vascular inflammation by recruiting proinflammatory signaling molecules to intracellular aggresomes.

A20 protects against pathologic vascular remodeling by inhibiting the inflammatory transcription factor NF-κB. A20's function has been attributed to ubiquitin editing of receptor-interacting protein 1 (RIP1) to influence activity/stability. The validity of this mechanism was tested using a murine model of transplant vasculopathy and human cells. Mouse C57BL/6 aortae transduced with adenoviruses containing A20 (or ß-galactosidase as a control) were allografted into major histocompatibility complex-mismatched BALB/c mice. Primary endothelial cells, smooth muscle cells, or transformed epithelial cells (all human) were transfected with wild-type A20 or with catalytically inactive mutants as a control. NF-κB activity and intracellular localization of RIP1 was monitored by reporter gene assay, immunofluorescent staining, and Western blotting. Native and catalytically inactive versions of A20 had similar inhibitory effects on NF-κB activity (-70% vs. -76%; P > 0.05). A20 promoted localization of RIP1 to insoluble aggresomes in murine vascular allografts and in human cells (53% vs. 0%) without altering RIP1 expression, and this process was increased by the assembly of polyubiquitin chains (87% vs. 28%; P < 0.05). A20 captures polyubiquitinated signaling intermediaries in insoluble aggresomes, thus reducing their bioavailability for downstream NF-κB signaling. This novel mechanism contributes to protection from vasculopathy in transplanted organs treated with exogenous A20.

MicroRNA-22 regulates smooth muscle cell differentiation from stem cells by targeting methyl CpG-binding protein 2.

OBJECTIVE: In this study, we attempted to uncover the functional impact of microRNA-22 (miR-22) and its target gene in smooth muscle cell (SMC) differentiation and delineate the molecular mechanism involved. APPROACH AND RESULTS: miR-22 was found to be significantly upregulated during SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells. Enforced expression of miR-22 by its mimic, while knockdown of miR-22 by its antagomiR, promotes or inhibits SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells, respectively. Expectedly, miR-22 overexpression in stem cells promoted SMC differentiation in vivo. Methyl CpG-binding protein 2 (MECP2) was predicted as one of the top targets of miR-22. Interestingly, the gene expression levels of MECP2 were significantly decreased during SMC differentiation, and MECP2 was dramatically decreased in miR-22 overexpressing cells but significantly increased when miR-22 was knockdown in the differentiating stem cells. Importantly, luciferase assay showed that miR-22 substantially inhibited wild-type, but not mutant MECP2-3' untranslated region-luciferase activity. In addition, modulation of MECP2 expression levels affects multiple SMC-specific gene expression in differentiated embryonic stem cells. Mechanistically, our data showed that MECP2 could transcriptionally repress SMC gene expression through modulating various SMC transcription factors, as well as several proven SMC differentiation regulators. Evidence also revealed that enrichment of H3K9 trimethylation around the promoter regions of the SMC differentiation regulators genes were significantly increased by MECP2 overexpression. Finally, miR-22 was upregulated by platelet-derived growth factor-BB and transforming growth factor-ß through a transcriptional mechanism during SMC differentiation. CONCLUSIONS: miR-22 plays an important role in SMC differentiation, and epigenetic regulation through MECP2 is required for miR-22 mediated SMC differentiation.

miRNA-34a reduces neointima formation through inhibiting smooth muscle cell proliferation and migration.

AIMS: We have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells in vitro and in vivo. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. METHODS AND RESULTS: Various pathological stimuli regulate miR-34a expression in VSMCs through a transcriptional mechanism, and the P53 binding site is required for miR-34a gene regulation by these stimuli. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodeling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1-3'-UTR-luciferase activity in VSMCs, but not mutant Notch1-3'-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, the expression level of miR-34a was significantly down-regulated in injured arteries, and miR-34a perivascular over-expression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. CONCLUSION: Our data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.

Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination.

RATIONALE: Hypoxia followed by reoxygenation promotes inflammation by activating nuclear factor κB transcription factors in endothelial cells (ECs). This process involves modification of the signaling intermediary tumor necrosis factor receptor-associated factor 6 with polyubiquitin chains. Thus, cellular mechanisms that suppress tumor necrosis factor receptor-associated factor 6 ubiquitination are potential therapeutic targets to reduce inflammation in hypoxic tissues. OBJECTIVE: In this study, we tested the hypothesis that endothelial activation in response to hypoxia-reoxygenation can be influenced by Cezanne, a deubiquitinating enzyme that cleaves ubiquitin from specific modified proteins. METHODS AND RESULTS: Studies of cultured ECs demonstrated that hypoxia (1% oxygen) induced Cezanne via p38 mitogen-activated protein kinase-dependent transcriptional and post-transcriptional mechanisms. Hypoxia-reoxygenation had minimal effects on proinflammatory signaling in unmanipulated ECs but significantly enhanced Lys63 polyubiquitination of tumor necrosis factor receptor-associated factor 6, activation of nuclear factor κB, and expression of inflammatory genes after silencing of Cezanne. Thus, although hypoxia primed cells for inflammatory activation, it simultaneously induced Cezanne, which impeded signaling to nuclear factor κB by suppressing tumor necrosis factor receptor-associated factor 6 ubiquitination. Similarly, ischemia induced Cezanne in the murine kidney in vascular ECs, glomerular ECs, podocytes, and epithelial cells, and genetic deletion of Cezanne enhanced renal inflammation and injury in murine kidneys exposed to ischemia followed by reperfusion. CONCLUSIONS: We conclude that inflammatory responses to ischemia are controlled by a balance between ubiquitination and deubiquitination, and that Cezanne is a key regulator of this process. Our observations have important implications for therapeutic targeting of inflammation and injury during ischemia-reperfusion.

Disturbed flow promotes endothelial senescence via a p53-dependent pathway.

OBJECTIVE: Although atherosclerosis is associated with systemic risk factors such as age, high cholesterol, and obesity, plaque formation occurs predominately at branches and bends that are exposed to disturbed patterns of blood flow. The molecular mechanisms that link disturbed flow-generated mechanical forces with arterial injury are uncertain. To illuminate them, we investigated the effects of flow on endothelial cell (EC) senescence. APPROACH AND RESULTS: LDLR(-/-) (low-density lipoprotein receptor(-/-)) mice were exposed to a high-fat diet for 2 to 12 weeks (or to a normal chow diet as a control) before the assessment of cellular senescence in aortic ECs. En face staining revealed that senescence-associated ß-galactosidase activity and p53 expression were elevated in ECs at sites of disturbed flow in response to a high-fat diet. By contrast, ECs exposed to undisturbed flow did not express senescence-associated ß-galactosidase or p53. Studies of aortae from healthy pigs (aged 6 months) also revealed enhanced senescence-associated ß-galactosidase staining at sites of disturbed flow. These data suggest that senescent ECs accumulate at disturbed flow sites during atherogenesis. We used in vitro flow systems to examine whether a causal relationship exists between flow and EC senescence. Exposure of cultured ECs to flow (using either an orbital shaker or a syringe-pump flow bioreactor) revealed that disturbed flow promoted EC senescence compared with static conditions, whereas undisturbed flow reduced senescence. Gene silencing studies demonstrated that disturbed flow induced EC senescence via a p53-p21 signaling pathway. Disturbed flow-induced senescent ECs exhibited reduced migration compared with nonsenescent ECs in a scratch wound closure assay, and thus may be defective for arterial repair. However, pharmacological activation of sirtuin 1 (using resveratrol or SRT1720) protected ECs from disturbed flow-induced senescence. CONCLUSIONS: Disturbed flow promotes endothelial senescence via a p53-p21-dependent pathway which can be inhibited by activation of sirtuin 1. These observations support the principle that pharmacological activation of sirtuin 1 may promote cardiovascular health by suppressing EC senescence at atheroprone sites.

Disturbed blood flow induces RelA expression via c-Jun N-terminal kinase 1: a novel mode of NF-κB regulation that promotes arterial inflammation.

RATIONALE: The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. OBJECTIVE: We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. METHODS AND RESULTS: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. CONCLUSIONS: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK-NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

Dexamethasone arterializes venous endothelial cells by inducing mitogen-activated protein kinase phosphatase-1: a novel antiinflammatory treatment for vein grafts?

BACKGROUND: Vein grafting in coronary artery surgery is complicated by a high restenosis rate resulting from the development of vascular inflammation, intimal hyperplasia, and accelerated atherosclerosis. In contrast, arterial grafts are relatively resistant to these processes. Vascular inflammation is regulated by signaling intermediaries, including p38 mitogen-activated protein (MAP) kinase, that trigger endothelial cell (EC) expression of chemokines (eg, interleukin-8, monocyte chemotactic protein-1) and other proinflammatory molecules. Here, we have tested the hypothesis that p38 MAP kinase activation in response to arterial shear stress (flow) may occur more readily in venous ECs, leading to greater proinflammatory activation. METHODS AND RESULTS: Comparative reverse-transcriptase polymerase chain reaction and Western blotting revealed that arterial shear stress induced p38-dependent expression of monocyte chemotactic protein-1 and interleukin-8 in porcine jugular vein ECs. In contrast, porcine aortic ECs were protected from shear stress-induced expression of p38-dependent chemokines as a result of rapid induction of MAP kinase phosphatase-1. However, we observed with both cultured porcine jugular vein ECs and perfused veins that venous ECs can be protected by brief treatment with dexamethasone, which induced MAP kinase phosphatase-1 to suppress proinflammatory activation. CONCLUSIONS: Arterial but not venous ECs are protected from proinflammatory activation in response to short-term exposure to high shear stress by the induction of MAP kinase phosphatase-1. Dexamethasone pretreatment arterializes venous ECs by inducing MAP kinase phosphatase-1 and may protect veins from inflammation.

Inhibition of NF-κB signaling in human dendritic cells by the enteropathogenic Escherichia coli effector protein NleE.

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by ß-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-ß, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer's patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.

Cezanne is a critical regulator of pathological arterial remodelling by targeting ß-catenin signalling.

AIMS: Pathological arterial remodelling including neointimal hyperplasia and atherosclerosis is the main underlying cause for occluding arterial diseases. Cezanne is a novel deubiquitinating enzyme, functioning as a NF-кB negative regulator, and plays a key role in renal inflammatory response and kidney injury induced by ischaemia. Here we attempted to examine its pathological role in vascular smooth muscle cell (VSMC) pathology and arterial remodelling. METHODS AND RESULTS: Cezanne expression levels were consistently induced by various atherogenic stimuli in VSMCs, and in remodelled arteries upon injury. Functionally, VSMCs over-expressing wild-type Cezanne, but not the mutated catalytically-inactive Cezanne (C209S), had an increased proliferative ability and mobility, while the opposite was observed in VSMCs with Cezanne knockdown. Surprisingly, we observed no significant effects of Cezanne on VSMC apoptosis, NF-κB signalling, or inflammation. RNA-sequencing and biochemical studies showed that Cezanne drives VSMC proliferation by regulating CCN family member 1 (CCN1) by targeting ß-catenin for deubiquitination. Importantly, local correction of Cezanne expression in the injured arteries greatly decreased VSMC proliferation, and prevented arterial inward remodelling. Interestingly, global Cezanne gene deletion in mice led to smaller atherosclerotic plaques, but with a lower level of plaque stability. Translating, we observed a similar role for Cezanne in human VSMCs, and higher expression levels of Cezanne in human atherosclerotic lesions. CONCLUSION: Cezanne is a key regulator of VSMC proliferation and migration in pathological arterial remodelling. Our findings have important implications for therapeutic targeting Cezanne signalling and VSMC pathology in vascular diseases.

Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

c-Jun N-terminal kinase primes endothelial cells at atheroprone sites for apoptosis.

OBJECTIVE: Atherosclerosis is a focal disease that occurs predominantly at branches and bends of the arterial tree. Endothelial cells (EC) at atherosusceptible sites are prone to injury, which can contribute to lesion formation, whereas EC at atheroprotected sites are resistant. The c-Jun N-terminal kinase (JNK) is activated constitutively in EC at atherosusceptible sites but is inactivated at atheroprotected sites by mitogen-activated protein kinase phosphatase-1 (MKP-1). Here, we examined the effects of JNK activation on EC physiology at atherosusceptible sites. METHODS AND RESULTS: We identified transcriptional programs regulated by JNK by applying a specific pharmacological inhibitor to cultured EC and assessing the transcriptome using microarrays. This approach and subsequent validation by gene silencing revealed that JNK positively regulates the expression of numerous proapoptotic molecules. Analysis of aortae of wild-type, JNK1(-/-), and MKP-1(-/-) mice revealed that EC at an atherosusceptible site express proapoptotic proteins and are primed for apoptosis and proliferation in response to lipopolysaccharide through a JNK1-dependent mechanism, whereas EC at a protected site expressed lower levels of proapoptotic molecules and were protected from injury by MKP-1. CONCLUSIONS: Spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries.

Distribution and contamination assessment of trace metals in surface sediments of the South China Sea, Vietnam.

The distribution and enrichment of trace metals in sediments of the South China Sea along the entire coast of Vietnam were described. The concentrations of Cr, Ni, Zn, and Pb in the sediments showed a significant positive correlation with fine-sized fractions and TOC. In contrast, the concentration of As was not positively correlated with particle size and other metals. The relatively positive correlations of Cd with Fe, Al, Ti, Sc, TOC, P, Cr, Ni, Zn, and Pb indicated that it comes from different sources. Ecotoxicological indexes of all elements showed low values, except for Cd in the southwestern part of the South China Sea area, which is likely related to the influx of suspended matter from the Mekong River.

Role of nuclear factor kappaB in cardiovascular health and disease.

Cardiovascular pathologies are still the primary cause of death worldwide. The molecular mechanisms behind these pathologies have not been fully elucidated. Unravelling them will bring us closer to therapeutic strategies to prevent or treat cardiovascular disease. One of the major transcription factors that has been linked to both cardiovascular health and disease is NF-kappaB (nuclear factor kappaB). The NF-kappaB family controls multiple processes, including immunity, inflammation, cell survival, differentiation and proliferation, and regulates cellular responses to stress, hypoxia, stretch and ischaemia. It is therefore not surprising that NF-kappaB has been shown to influence numerous cardiovascular diseases including atherosclerosis, myocardial ischaemia/reperfusion injury, ischaemic preconditioning, vein graft disease, cardiac hypertrophy and heart failure. The function of NF-kappaB is largely dictated by the genes that it targets for transcription and varies according to stimulus and cell type. Thus NF-kappaB has divergent functions and can protect cardiovascular tissues from injury or contribute to pathogenesis depending on the cellular and physiological context. The present review will focus on recent studies on the function of NF-kappaB in the cardiovascular system.

Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinflammatory activation at sites that are resistant to atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of arteries. It is triggered by proinflammatory mediators which induce adhesion molecules (eg, vascular cell adhesion molecule [VCAM]-1) in endothelial cells (ECs) by activating p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases by phosphorylation. Blood flow influences atherosclerosis by exerting shear stress (mechanical drag) on the inner surface of arteries, a force that alters endothelial physiology. Regions of the arterial tree exposed to high shear are protected from endothelial activation, inflammation, and atherosclerosis, whereas regions exposed to low or oscillatory shear are susceptible. We examined whether MAP kinase phosphatase (MKP)-1, a negative regulator of p38 and JNK, mediates the antiinflammatory effects of shear stress. We observed that expression of MKP-1 in cultured ECs was elevated by shear stress, whereas the expression of VCAM-1 was reduced. MKP-1 induction was shown to be necessary for the antiinflammatory effects of shear stress because gene silencing of MKP-1 restored VCAM-1 expression in sheared ECs. Immunostaining revealed that MKP-1 is preferentially expressed by ECs in a high-shear, protected region of the mouse aorta and is necessary for suppression of EC activation at this site, because p38 activation and VCAM-1 expression was enhanced by genetic deletion of MKP-1. We conclude that MKP-1 induction is required for the antiinflammatory effects of shear stress. Thus, our findings reveal a novel molecular mechanism contributing to the spatial distribution of vascular inflammation and atherosclerosis.