Krüppel-Like Factor 15/Interleukin 11 Axis-Mediated Adventitial Remodeling Depends on Extracellular Signal-Regulated Kinases 1 and 2 Activation in Angiotensin II-Induced Hypertension.

Background Adventitial remodeling is a pathological hallmark of hypertension that results in target organ damage. Activated adventitial fibroblasts have emerged as critical regulators in this process, but the precise mechanism remains unclear. Methods and Results Interleukin 11 (IL-11) knockout and wild-type mice were subjected to angiotensin II (Ang II) infusion to establish models of hypertension-associated vascular remodeling. IL-11 mRNA and protein were increased especially in the adventitia in response to Ang II. Compared with wild-type mice, Ang II-treated IL-11 knockout mice showed amelioration of vascular hypertrophy, adventitial fibrosis, macrophage infiltration, and inflammatory factor expression. Recombination mouse IL-11 exacerbated adventitial fibrosis in Ang II-infused wild-type mice. Interestingly, IL-11 neutralizing antibody attenuated adventitial fibrosis, macrophage infiltration, and inflammatory factor expression after Ang II infusion for 7 days. Mechanistically, in primary cultured adventitial fibroblasts, Krüppel-like factor 15 negatively regulated Ang II-induced IL-11 expression. Ang II increased extracellular signal-regulated kinases 1 and 2 activation, especially in adventitia, and caused biphasic extracellular signal-regulated kinases 1 and 2 activation in adventitial fibroblasts. A rapid and early activation increased IL-11 production through decreasing Krüppel-like factor 15 expression, which, in turn, induced the second extracellular signal-regulated kinases 1 and 2 activation, resulting in posttranscriptional profibrotic gene expression. Conclusions These results demonstrate that extracellular signal-regulated kinases 1 and 2 activation is important for Krüppel-like factor 15-mediated IL-11 expression in adventitial fibroblasts to promote adventitial remodeling in Ang II-induced hypertension. Therefore, targeting the Krüppel-like factor 15/IL-11 axis might serve as a new therapeutic strategy for vascular diseases.

Transient Receptor Potential Melastatin 7 Promotes Vascular Adventitial Fibroblasts Phenotypic Transformation and Inflammatory Reaction Induced by Mechanical Stretching Stress via p38 MAPK/JNK Pathway.

Remodeling of the arteries is one of the pathological bases of hypertension. We have previously shown that transient receptor potential melastatin 7 (TRPM7) aggravates the vascular adventitial remodeling caused by pressure overload in the transverse aortic constriction (TAC) model. In this study, we sought to explore the functional expression and downstream signaling of TRPM7 in vascular adventitial fibroblasts (AFs) stimulated by mechanical stretching stress (MSS). The expression of TRPM7 was upregulated with a concomitant translocation to the cytoplasm in the AFs stimulated with 20% MSS. Meanwhile, the expression of α-smooth muscle actin (α-SMA), a marker of transformation from AFs to myofibroblasts (MFs) was also increased. Moreover, AF-conditioned medium caused a significant migration of macrophages after treatment with MSS and contained high levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Pharmacological and RNA interference approaches using the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB) and specific anti-TRPM7 small interfering RNA (si-RNA-TRPM7) abrogated these changes significantly. Further exploration uncloaked that inhibition of TRPM7 reduced the phosphorylation of p38 MAP kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in the AFs stimulated with MSS. Furthermore, inhibition of the phosphorylation of p38MAPK or JNK could also alleviate the MSS-induced expression of α-SMA and secretion of inflammatory factors. These observations indicate that activated TRPM7 participates in the phenotypic transformation and inflammatory action of AFs in response to MSS through the p38MAPK/JNK pathway and suggest that TRPM7 may be a potential therapeutic target for vascular remodeling caused by hemodynamic changes in hypertension.

MicroRNA-122 aggravates angiotensin II-mediated apoptosis and autophagy imbalance in rat aortic adventitial fibroblasts via the modulation of SIRT6-elabela-ACE2 signaling.

Abnormal aortic adventitial fibroblasts (AFs) play essential roles in the development of vascular remodeling and disorders. Previous studies revealed that microRNA-122 (miR-122) levels were elevated in the aortic adventitia of hypertensive rats with vascular injury. Here, we aim to evaluate the biological effects and underlying mechanisms of miR-122 in rat AFs. Exposure to angiotensin II (ATII) in rat AFs resulted in decreased levels of sirtuin 6 (SIRT6), elabela (ELA), and angiotensin-converting enzyme 2 (ACE2). Additionally, stimulation with ATII contributed to a decline in autophagic flux and obvious increases in cellular migration, oxidative stress, and apoptosis, which were exacerbated by the transfection of miR-122-5p mimic but were rescued by miR-122-5p inhibitor, exogenous replenishment of ELA, and recombinant adeno-associated virus expressing SIRT6 (rAAV-SIRT6), respectively. Moreover, stimulation with miR-122-5p mimic led to a marked reduction in the levels of SIRT6 and ELA in rat AFs, which were elevated by stimulation with rAAV-SIRT6. Furthermore, miR-122-5p inhibitor-mediated pro-autophagic, anti-oxidant and anti-apoptotic effects in rat AFs were partially suppressed by 3-methyladenine, SIRT6 small interfering RNA (siRNA) and ELA siRNA, which were linked with the downregulation in the protein levels of LC3-II, beclin-1, and ACE2 and the upregulation of p62 expression and bax/bcl-2 ratio. Our findings indicated that miR-122-5p inhibition prevented ATII-mediated loss of autophagy, and the promotion of apoptosis and oxidative stress via activating the SIRT6-ELA-ACE2 signaling. MiR-122-5p may be a novel predictive biomarker of adventitial injury, and targeting the SIRT6-ELA-ACE2 signaling may have the potential therapeutic importance of controlling vascular remodeling and disorders.

Macrophage-derived MMP-8 determines smooth muscle cell differentiation from adventitia stem/progenitor cells and promotes neointima hyperplasia.

AIMS: Emerging evidence has suggested that adventitia stem/progenitor cells (AdSPCs) migrate into the intima of arteries in response to injury, where they differentiate towards smooth muscle cells (SMCs) and participate in neointimal hyperplasia. We have previously identified matrix metalloproteinase-8 (MMP8) as a key player in atherogenesis. In this study, we aimed to investigate the functional roles of macrophage-derived MMP8 in AdSPC differentiation and injury-induced arterial remodelling. METHODS AND RESULTS: We first observed an important role for MMP8 in SMC differentiation from embryonic stem cells, but this effect was not seen in AdSPCs. Instead, through macrophages/AdSPCs co-culture and macrophage conditional culture medium studies, we have demonstrated that the MMP8 protein secreted from macrophages promotes SMC differentiation from AdSPCs. Mechanistically, we showed that macrophage-derived MMP8 promotes SMC differentiation from AdSPCs through modulating transforming growth factor-ß activity and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)/Notch1 signalling. We further demonstrated that the binding site for CBF1, Suppressor of Hairless, and Lag-1 (CSL) within SMC gene promoters is responsible for Notch1 mediated SMC differentiation. Finally, we demonstrated that macrophage-derived MMP8 increased injury-induced neointimal SMC hyperplasia by activating ADAM10/Notch1 signalling. CONCLUSIONS: We have identified macrophage-derived MMP8 as a regulator in SMC differentiation from AdSPCs and neointimal SMC hyperplasia in response to injury. Our data provide new insights into the roles of MMP8 in AdSPC differentiation and the pathogenesis of neointima formation in the context of angiographic restenosis, and therefore may aid in the development of novel therapeutic agents for the prevention of this disease.

CYLD Deubiquitinates Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Contributing to Adventitial Remodeling.

OBJECTIVE: Transdifferentiation of adventitial fibroblasts (AFs) into myofibroblasts plays a critical role during the vascular remodeling that occurs during atherosclerosis, restenosis, and aortic aneurysm. The ubiquitination/deubiquitination regulatory system is essential for the quality control of proteins. The involvement of ubiquitination/deubiquitination during AF transdifferentiation remains largely unknown. In this study, we determined the role of cylindromatosis (CYLD), a deubiquitinase, in the process of AF differentiation and activation in vitro and in vivo. APPROACH AND RESULTS: Transforming growth factor-ß1 and homocysteine, 2 known inducers of AF transdifferentiation, greatly upregulated CYLD expression in a time- and dose-dependent manner. The silencing of CYLD significantly inhibited AF transdifferentiation and activation as evidenced by the expression of contractile proteins, the production of the proinflammatory cytokines MCP-1 (monocyte chemotactic protein 1) and IL-6 (interleukin-6), the deposition of extracellular matrix, and cell migration. We further asked whether CYLD mediates AF activation via the regulation of nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) as it is an essential factor during AF transdifferentiation. Indeed, the silencing of CYLD repressed transforming growth factor-ß1-induced and homocysteine-induced Nox4 upregulation and reactive oxygen species production, whereas Nox4 overexpression greatly rescued the inhibitory effect on AF activation by CYLD silencing. Most interestingly, transforming growth factor-ß1 and homocysteine repressed Nox4 ubiquitination and prolonged the half-life of Nox4. Moreover, Nox4 was deubiquitinated via a direct interaction with the ubiquitin-specific protease domain of CYLD. In accordance, hyperhomocysteinemia significantly increased adventitial CYLD and Nox4 expression, promoted AF transdifferentiation, and aggravated CaPO4-induced abdominal aortic aneurysm in mice. These effects were abolished in CYLD-/- mice. CONCLUSIONS: CYLD contributes to the transdifferentiation of AFs via deubiquitinating Nox4 and may play a role in vascular remodeling.

Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).

RATIONALE: In human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene was associated with hypertension. Lower SIK1 activity in vascular smooth muscle cells (VSMCs) leads to decreased sodium-potassium ATPase activity, which associates with increased vascular tone. Also, SIK1 participates in a negative feedback mechanism on the transforming growth factor-ß1 signaling and downregulation of SIK1 induces the expression of extracellular matrix remodeling genes. OBJECTIVE: To evaluate whether reduced expression/activity of SIK1 alone or in combination with elevated salt intake could modify the structure and function of the vasculature, leading to higher blood pressure. METHODS AND RESULTS: SIK1 knockout (sik1(-/-)) and wild-type (sik1(+/+)) mice were challenged to a normal- or chronic high-salt intake (1% NaCl). Under normal-salt conditions, the sik1(-/-) mice showed increased collagen deposition in the aorta but similar blood pressure compared with the sik1(+/+) mice. During high-salt intake, the sik1(+/+) mice exhibited an increase in SIK1 expression in the VSMCs layer of the aorta, whereas the sik1(-/-) mice exhibited upregulated transforming growth factor-ß1 signaling and increased expression of endothelin-1 and genes involved in VSMC contraction, higher systolic blood pressure, and signs of cardiac hypertrophy. In vitro knockdown of SIK1 induced upregulation of collagen in aortic adventitial fibroblasts and enhanced the expression of contractile markers and of endothelin-1 in VSMCs. CONCLUSIONS: Vascular SIK1 activation might represent a novel mechanism involved in the prevention of high blood pressure development triggered by high-salt intake through the modulation of the contractile phenotype of VSMCs via transforming growth factor-ß1-signaling inhibition.

NADPH oxidase 4 is expressed in pulmonary artery adventitia and contributes to hypertensive vascular remodeling.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. APPROACH AND RESULTS: Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. CONCLUSION: These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

Correlation of bacterial coinfection versus matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 expression in aortic aneurysm and atherosclerosis.

BACKGROUND: We searched for any relationship between Chlamydophila pneumoniae, Mycoplasma pneumoniae, matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) in aneurysmatic atherosclerotic lesions, and whether this relationship differed from that in atherosclerotic nonaneurysmatic lesions. METHODS: Twenty-eight tissue samples paired by age and sex were grouped as follows: group 1 included 14 nonaneurysmal atherosclerotic fragments obtained from abdominal aortas collected from necropsies; group 2 included 14 aneurysmatic atherosclerotic aortic fragments obtained from patients during corrective surgery. Immunohistochemistry reactions were evaluated for C pneumoniae, M pneumoniae, MMP-9, and TIMP-1 antigens. Both groups were compared using the Mann-Whitney test, and the correlations among variables were obtained using the Spearman correlation test. P ≤ 0.05 was considered statistically significant. RESULTS: C pneumoniae and M pneumoniae antigens were detected in 100% of cases. A higher amount of C pneumoniae (P = 0.005), M pneumoniae (P = 0.002), and MMP-9 (P = 0.021) was found in adventitia of group 2 with aneurysm. A positive correlation was found in the aneurysm group, as follows: intima C pneumoniae versus adventitia thickness (r = 0.70; P = 0.01), media C pneumoniae versus adventitia C pneumoniae (r = 0.75; P = 0.002), intima C pneumoniae versus media C pneumoniae (r = 0.8; P = 0.00), and adventitia C pneumoniae versus intima M pneumoniae (r = 0.54; P = 0.05); negative correlations were as follows: adventitia thickness and adventitia M pneumoniae (r = -0.65; P = 0.01), media MMP-9 and media thickness (r = -0.55; P = 0.04), TIMP-1 media versus adventitia C pneumoniae (r = -0.86; P = 0.00), and TIMP-1 media versus M pneumoniae intima (r = -0.67; P = 0.03). Nonaneurysmal atherosclerotic group 1 results are as follows: adventitia C pneumoniae versus TIMP-1 media (r = 0.75; P = 0.01) and media C pneumoniae and adventitia C pneumoniae (r = 0.59; P = 0.03). CONCLUSIONS: The present work favors a role for coinfection of both M pneumoniae and C pneumoniae in the development of aortic atherosclerotic aneurysm, with increased adventitial inflammation, inhibition of TIMP-1 activity, and increased collagen degradation.

Sirolimus stimulates vascular stem/progenitor cell migration and differentiation into smooth muscle cells via epidermal growth factor receptor/extracellular signal-regulated kinase/ß-catenin signaling pathway.

OBJECTIVE: Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown. APPROACH AND RESULTS: Using an established technique, vascular progenitor cells were isolated from adventitial tissues of mouse vessel grafts and purified with microbeads specific for stem cell antigen-1. We provide evidence that vascular progenitor cells treated with sirolimus resulted in an induction of their migration in both transwell and wound healing models, clearly mediated by CXCR4 activation. We confirmed the sirolimus-mediated increase of migration from the adventitial into the intima side using an ex vivo decellularized vessel scaffold, where they form neointima-like lesions that expressed high levels of smooth muscle cell (SMC) markers (SM-22α and calponin). Subsequent in vitro studies confirmed that sirolimus can induce SMC but not endothelial cell differentiation of progenitor cells. Mechanistically, we showed that sirolimus-induced progenitor-SMC differentiation was mediated via epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 activation that lead to ß-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal-regulated kinase 1/2, or ß-catenin attenuated sirolimus-induced SM-22α promoter activation and SMC differentiation. CONCLUSIONS: These findings provide direct evidence of sirolimus-induced progenitor cell migration and differentiation into SMC via CXCR4 and epidermal growth factor receptor/extracellular signal-regulated kinase/ß-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.

p38 MAPK inhibitor, CBS3830 limits vascular remodelling in arterialised vein grafts.

OBJECTIVE: Following bypass surgery vein grafts undergo a remodelling process that can lead to restenosis and ultimately vein graft failure. Signalling through mitogen activated protein kinases (MAPKs) is a key mechanism involved in vein graft failure. Here, we investigated whether CBS3830 (c-a-i-r biosciences GmbH, Tübingen, Germany), a new highly selectively inhibitor of p38 MAPK, has a significant effect on inhibiting intimal, medial and adventitial hyperplasia. METHODS: Sixty specific pathogen free Sprague Dawley male rats were randomly divided into three groups. The control group with a reversed right jugular vein, which is common to carotid artery interposition graft, was compared with sham-operated, and CBS3830 treated animals. Intimal, medial and adventitia morphometric examinations and expression of proliferating cell nuclear antigen (PCNA) were analysed after one, two and four weeks for vein grafts. RESULTS: Intimal, medial and adventitia thickening in CBS3830 group were significantly lower than in the control group at each time point. Moreover, CBS3830 significantly reduced the phosphorylation of p38 MAPK and PCNA expression compared to the control. CONCLUSION: On the basis of the present work, intima, media and adventitia of saphenous vein grafts undergo vascular remodelling after surgery. The new, highly selective p38 MAPK inhibitor, CBS3830, ameliorates intimal, medial, and adventitial remodelling by varying degrees.

Urotensin II promotes the production of LTC4 in rat aortic adventitial fibroblasts through NF-κB-5-LO pathway by p38 MAPK and ERK activations.

Adventitia is the outer part of the arterial wall where the inflammatory response often occurs. Urotensin II (UII) is a potent vasoconstrictive peptide that also promotes the inflammatory process in patients with cardiovascular disease. Leukotriene C4 (LTC4), a lipid mediator, was recently found to play a role in the inflammatory process in the artery. We hypothesized that the adventitia is one of the resources of LTC4 and that UII may promote LTC4 production through the 5-LO (5-lipoxygenase) pathway in adventitial fibroblasts. Rat adventitial fibroblasts were isolated and incubated in serum-free medium with either UII alone or in combination with inhibitors of p38 MAPK, ERK, and UII receptors. The expression of 5-LO was detected using real-time polymerase chain reaction and Western blot. The translocation and binding activity of nuclear factor (NF)-κB were measured using immunofluorescence and electrophoretic mobility shift assay, respectively. The production of LTC4 was measured by enzyme-linked immunosorbent assay. The results indicated that: (1) adventitial fibroblasts were a source of LTC4 production; (2) UII increased the expression of the 5-LO mRNA and the protein by NF-κB activation through p38 MAPK and ERK pathways; and (3) UII promoted the LTC4 release in fibroblasts through the 5-LO pathway by p38 MAPK and ERK activations. The 5-LO pathway mediates LTC4 production, which may be a new mechanism in the pathogenesis of the vascular adventitial inflammation caused by UII.

Cellular repressor E1A-stimulated genes controls phenotypic switching of adventitial fibroblasts by blocking p38MAPK activation.

AIMS: Phenotypic modulation of adventitial fibroblasts (AFs) plays an important role in the pathogenesis of proliferative vascular diseases. The current study aimed to identify the role of cellular repressor E1A-stimulated genes (CREG), a critical mediator in the maintenance of vascular homeostasis, in AF phenotypic modulation and adventitial remodeling. METHOD AND RESULTS: Using in situ double-immunofluorescence staining, we ascertained that CREG expression was significantly down-regulated in the adventitia after vascular injury, and its expression pattern was conversely correlated with the expression of smooth muscle α-actin (α-SMA), a marker for differentiation of AFs into myofibroblasts. In vitro data confirmed the association of CREG in angiotensin II (Ang II)-induced AF differentiation. Additionally, overexpression of CREG attenuated Ang II-induced α-SMA expression in AFs. CREGoverexpressing AFs showed decreased levels of proliferation on days 2-5 following stimulation by Ang II compared with controls, with changes in the cell cycle profile as shown by BrdU incorporation assay and fluorescence activated cell sorting analysis. Moreover, wound healing assay and transwell migration model demonstrated that upregulation of CREG expression inhibited Ang II-induced AF migration. We found that CREG-mediated its counterbalancing effects in Ang II-induced phenotypic modulation, proliferation and migration by inhibition of the p38MAPK signaling pathway, validated by pharmacological blockade of p38MAPK with SB 203580 and by overexpression of p38MAPK with transfectants expressing constitutively active p38αMAPK. CONCLUSION: Our findings suggest that CREG is a novel AF phenotypic modulator in a p38MAPK-dependent manner. Modulating CREG on the local vascular wall may become a new therapeutic target against proliferative vascular diseases.

Hypoxia induces unique proliferative response in adventitial fibroblasts by activating PDGFß receptor-JNK1 signalling.

AIMS: Pulmonary hypertension (PH) is a devastating condition for which no disease-modifying therapies exist. PH is recognized as proliferative disease of the pulmonary artery (PA). In the experimental newborn calf model of hypoxia-induced PH, adventitial fibroblasts in the PA wall exhibit a heightened replication index. Because elevated platelet-derived growth factor ß receptor (PDGFß-R) signalling is associated with PH, we tested the hypothesis that the activation of PDGFß-R contributes to fibroblast proliferation and adventitial remodelling in PH. METHODS AND RESULTS: Newborn calves were exposed to either ambient air (P(B) = 640 mmHg) (Neo-C) or high altitude (P(B) = 445 mm Hg) (Neo-PH) for 2 weeks. PDGFß-R phosphorylation was markedly elevated in PA adventitia of Neo-PH calves as well as in cultured PA fibroblasts isolated from Neo-PH animals. PDGFß-R activation with PDGF-BB stimulated higher replication in Neo-PH cells compared with that of control fibroblasts. PDGF-BB-induced proliferation was dependent on reactive oxygen species generation and extracellular signal-regulated kinase1/2 activation in both cell populations; however, only Neo-PH cell division via PDGFß-R activation displayed a unique dependence on c-Jun N-terminal kinase1 (JNK1) stimulation as the blockade of JNK1 with SP600125, a pharmacological antagonist of the JNK pathway, and JNK1-targeted siRNA selectively blunted Neo-PH cell proliferation. CONCLUSIONS: Our data strongly suggest that hypoxia-induced modified cells engage the PDGFß-R-JNK1 axis to confer distinctively heightened proliferation and adventitial remodelling in PH.