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Supplemental Digital Content is available in the text., Objective: The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)–induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II–stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II–treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. Conclusions: These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II–induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.

Supplemental Digital Content is available in the text., Objective: Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-β1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up. Conclusions: Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS.

Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic extracellular matrix (ECM). Evidence is emerging that ECM processing by members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family could play an important role in the progression of aortic dilatation. In particular, ADAMTS-1 and -4 have been implicated in TAA. This study aimed to investigate the contribution of ADAMTS-5 to TAA development. A model of aortic dilatation by angiotensin II (AngII) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5 Δcat ). Adamts5 Δcat mice showed an attenuated rise in blood pressure whilst displaying increased dilatation of the ascending aorta. Interestingly, a proteomics comparison of the aortic ECM from AngII-treated wildtype and Adamts5 Δcat mice revealed versican as the most up-regulated ECM protein in Adamts5 Δcat mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine), an increase in TGFβ and a decrease of low-density lipoprotein-related protein 1 (LRP1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of ADAMTS5 , attenuated the generation of versikine but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5 Δcat mice, but was not sufficient to maintain versican processing and prevent aortic dilatation. Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. A compensatory rise in ADAMTS-1 could not prevent aortic dilatation in Adamts5 Δcat mice. LRP1, which has been involved in aneurysm pathology in both human and mice, appears to be linked to ADAMTS-5 and ADAMTS-5-mediated versican cleavage. Further studies are needed to explore the ADAMTS protease family as target for therapeutic approaches aimed to reduce or halt dilatation of the aorta resulting in aneurysm.

Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic ECM (extracellular matrix). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development.A model of aortic dilatation by AngII (angiotensin II) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. Further studies are needed to define the ECM substrates of the different ADAMTS proteases and their contribution to TAA formation.

Objective— Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non–vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II. Approach and Results— Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E -deficient ( ApoE −/− ) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE −/− mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient ( ApoE −/− /Bgn −/0 ) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE −/− /Bgn −/0 mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE −/− /Bgn −/0 mice with the thrombin inhibitor argatroban. Ultimately, ApoE −/− /Bgn −/0 mice developed aggravated atherosclerosis. Conclusions— The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation.

Rationale: Extracellular matrix (ECM) remodeling is a key function of vascular smooth muscle cells (SMCs). MicroRNAs (miRNAs), in particular the miR-29 family and miR-195, have been implicated in the control of ECM secretion. Objective: To perform a proteomics comparison of miRNA effects on ECM production by vascular SMCs. Methods and Results: Murine SMCs were transfected with miRNA mimics and antimiRs of miR-29b and miR-195, and their conditioned medium was analyzed by mass spectrometry. Both miRNAs targeted a cadre of ECM proteins, including proteoglycans, collagens, proteases, elastin and proteins associated with elastic microfibrils, albeit miR-29 showed a stronger effect. The proteomics findings were subsequently validated at the transcription level using quantitative polymerase chain reaction. Similar to miR-29, in vivo inhibition of miR-195 by intraperitoneal injection of cholesterol bound antagomiRs led to significant alterations of elastin expression in murine aortas. Since elastin degradation is a key event in aortic aneurysm formation, we investigated miR-195 expression in patients. In human aortic aneurysmal tissue, miR-195 expression was reduced compared to non-aneurysmal tissue. In plasma, a comparison between male patients with abdominal aortic aneurysms and controls matched for diabetes and hypertension returned a panel of five highly correlated miRNAs: miR-195, miR-125b, miR-148a, miR-20a and miR-340 showed significant inverse associations with the presence of abdominal aortic aneurysms and aortic diameter, with miR-195 dominating in terms of association strength. Conclusions: Using proteomic analysis, we compared the effect of miR-29 and miR-195 on ECM secretion by vascular SMCs and identified novel miRNA targets. Findings in patients support an important role for miR-195 in vascular remodeling as evidenced by reduced miR-195 expression in human aneurysmal tissue and an inverse correlation between plasma miR-195 levels and aortic diameter.

Objective— We investigated the role of matrix metalloproteinase-8 (MMP8) in neointima formation and in vascular smooth muscle cell (VSMC) migration and proliferation. Approach and Results— After carotid artery wire injuring, MMP8 –/– /apoE –/– mice had fewer proliferating cells in neointimal lesions and smaller lesion sizes. Ex vivo assays comparing VSMCs isolated from MMP8 knockout and wild-type mice showed that MMP8 knockout decreased proliferation and migration. Proteomics analysis revealed that a disintegrin and metalloproteinase domain–containing protein 10 (ADAM10) had lower concentrations in MMP8 knockout VSMC culture media than in MMP8 wild-type VSMC culture media. Western blot, flow cytometric, and immunocytochemical analyses showed that MMP8 knockout VSMCs contained more pro-ADAM10 but less mature ADAM10, more N-cadherin, and β-catenin in the plasma membrane but less β-catenin in the nucleus and less cyclin D1. Treatment of MMP8 wild-type VSMCs with an ADAM10 inhibitor, GI254023X, or siRNA knockdown of ADAM10 in MMP8 wild-type VSMCs inhibited proliferation and migration, increased N-cadherin and β-catenin in the plasma membrane, reduced β-catenin in the nucleus, and decreased cyclin D1 expression. Incubation of MMP8 knockout VSMCs with a recombinant ADAM10 rescued the proliferative and migratory ability of MMP8 knockout VSMCs and increased cyclin D1 expression. Furthermore, immunohistochemical analyses showed colocalization of ADAM10 with VSMCs and N-cadherin, and nuclear accumulation of β-catenin in the neointima in apoE –/– /MMP8 +/+ mice. Conclusions— MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and β-catenin–mediated pathway and plays an important role in neointima formation.

Objective— Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear. Methods and Results— Here we demonstrated that chromobox protein homolog 3 (Cbx3) is crucial for SMC differentiation from stem cells and that the chromodomain and chromoshadow domain of Cbx3 are responsible for Cbx3-induced SMC differentiation. Moreover, we identified that 4 amino acids (165 to 168) within the chromoshadow domain of Cbx3 are key elements for Cbx3 interaction with Dia-1- and Cbx3-induced SMC differentiation. Mechanistically, we found that Cbx3 mediates SMC differentiation through modulating serum response factor (SRF) recruitment to the promoters of SMC genes, in which the interaction between Cbx3 and Dia-1/SRF plays a crucial role in this process. Moreover, our in vivo study demonstrated that the misexpression of Cbx3 within neural crest cells of chick embryos resulted in the death of chick embryos at early stages because of the maldevelopment of branchial arch arteries. Conclusion— Our findings suggest that the interaction between Cbx3 and Dia-1/SRF is essential for SMC differentiation from stem cells and for the development of functional cardiovascular system.