Nerea Méndez-Barbero

Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome

Heritable thoracic aortic aneurysms and dissections (TAAD), including Marfan syndrome (MFS), currently lack a cure, and causative mutations have been identified for only a fraction of affected families. Here we identify the metalloproteinase ADAMTS1 and inducible nitric oxide synthase (NOS2) as therapeutic targets in individuals with TAAD. We show that Adamts1 is a major mediator of vascular homeostasis, given that genetic haploinsufficiency of Adamts1 in mice causes TAAD similar to MFS. Aortic nitric oxide and Nos2 levels were higher in Adamts1-deficient mice and in a mouse model of MFS (hereafter referred to as MFS mice), and Nos2 inactivation protected both types of mice from aortic pathology. Pharmacological inhibition of Nos2 rapidly reversed aortic dilation and medial degeneration in young Adamts1-deficient mice and in young or old MFS mice. Patients with MFS showed elevated NOS2 and decreased ADAMTS1 protein levels in the aorta. These findings uncover a possible causative role for the ADAMTS1–NOS2 axis in human TAAD and warrant evaluation of NOS2 inhibitors for therapy.

A major role for RCAN1 in atherosclerosis progression

Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe−/− mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL‐mediated inhibition of macrophage migration and increased lesional IL‐10 and mannose receptor expression. Moreover, Apoe−/−Rcan1−/− macrophages expressed higher‐than‐Apoe−/− levels of anti‐inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe−/−Rcan1−/− bone‐marrow (BM) cells into Apoe−/− recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.

Deficiency of MMP17/MT4-MMP Proteolytic Activity Predisposes to Aortic Aneurysm in Mice

RATIONALE Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. OBJECTIVE To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. METHODS AND RESULTS Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. CONCLUSIONS Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.

A Novel Systems-Biology Algorithm for the Analysis of Coordinated Protein Responses Using Quantitative Proteomics

The coordinated behavior of proteins is central to systems biology. However, the underlying mechanisms are poorly known and methods to analyze coordination by conventional quantitative proteomics are still lacking. We present the Systems Biology Triangle (SBT), a new algorithm that allows the study of protein coordination by pairwise quantitative proteomics. The Systems Biology Triangle detected statistically significant coordination in diverse biological models of very different nature and subjected to different kinds of perturbations. The Systems Biology Triangle also revealed with unprecedented molecular detail an array of coordinated, early protein responses in vascular smooth muscle cells treated at different times with angiotensin-II. These responses included activation of protein synthesis, folding, turnover, and muscle contraction – consistent with a differentiated phenotype—as well as the induction of migration and the repression of cell proliferation and secretion. Remarkably, the majority of the altered functional categories were protein complexes, interaction networks, or metabolic pathways. These changes could not be detected by other algorithms widely used by the proteomics community, and the vast majority of proteins involved have not been described before to be regulated by AngII. The unique capabilities of The Systems Biology Triangle to detect functional protein alterations produced by the coordinated action of proteins in pairwise quantitative proteomics experiments make this algorithm an attractive choice for the biological interpretation of results on a routine basis.

Nonlinear Optical 3-Dimensional Method for Quantifying Atherosclerosis Burden

Changes in atherosclerosis burden provide an experimental measure of the effectiveness of therapeutic strategies. Unfortunately, none of the methods used in mice to assess atherosclerosis burden ex vivo is sufficiently accurate and fast enough to process the number of samples required in preclinical studies. We aimed to design an easy-to-implement and relatively fast method for accurate volumetric quantification of atheroma plaque burden in mice. The apolipoprotein E–deficient mouse ( Apoe −/−) is a widely used preclinical model that reproducibly develops hypercholesterolemia and atherosclerosis.1 Two widely used methods for ex vivo assessment of atherosclerosis burden in mice are histopathologic analysis2 and the en face method.3 The histological method requires serial axial sectioning of arteries and subsequent histopathologic staining. This approach is labor intensive, and its inherent destructiveness undermines the accuracy of lesion-volume determinations. In en face analysis, the aorta is opened longitudinally and stained to reveal lipid-laden plaques; the stained area is quantified from photographs. The speed and ease of en face analysis have made it the most widely used method for measuring atherosclerotic plaques ex vivo , but this method gives only a 2-dimensional (2D) measure of plaque burden. Here, we describe the nonlinear optical 3D (NLO-3D) method. Aortas from euthanized Apoe −/− mice fed a high-fat diet were dissected, stained with oil red O as described,4 and placed in a holder to permit measurement by either NLO-3D imaging or the en face method (Figure IA in the Data Supplement). We used a single excitation wavelength (800 nm) of a commercially available 2-photon instrument to simultaneously generate 3 confocal signals from these aortas (Figure IB in the Data Supplement): second harmonic generation from collagen and 2-photon excitation fluorescence from elastin and from oil red. A Zeiss 780 upright microscope equipped with a ×10 air objective (Zeiss Plan …

Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis

Polo-like kinase 1 (PLK1), an essential regulator of cell division, is currently undergoing clinical evaluation as a target for cancer therapy. We report an unexpected function of Plk1 in sustaining cardiovascular homeostasis. Plk1 haploinsufficiency in mice did not induce obvious cell proliferation defects but did result in arterial structural alterations, which frequently led to aortic rupture and death. Specific ablation of Plk1 in vascular smooth muscle cells (VSMCs) led to reduced arterial elasticity, hypotension, and an impaired arterial response to angiotensin II in vivo. Mechanistically, we found that Plk1 regulated angiotensin II–dependent activation of RhoA and actomyosin dynamics in VSMCs in a mitosis-independent manner. This regulation depended on Plk1 kinase activity, and the administration of small-molecule Plk1 inhibitors to angiotensin II–treated mice led to reduced arterial fitness and an elevated risk of aneurysm and aortic rupture. We thus conclude that a partial reduction of Plk1 activity that does not block cell division can nevertheless impair aortic homeostasis. Our findings have potentially important implications for current approaches aimed at PLK1 inhibition for cancer therapy.

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

ABSTRACT Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1β, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPβ transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPβ proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPβ signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPβ transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease.

Regulator of calcineurin 1 modulates vascular contractility and stiffness through the upregulation of COX-2-derived prostanoids

Graphical abstract Figure. No Caption available. &NA; Cyclooxygenase‐2 (COX‐2) derived‐prostanoids participate in the altered vascular function and mechanical properties in cardiovascular diseases. We investigated whether regulator of calcineurin 1 (Rcan1) participates in vascular contractility and stiffness through the regulation of COX‐2. For this, wild type (Rcan1+/+) and Rcan1‐deficient (Rcan1−/−) mice untreated or treated with the COX‐2 inhibitor rofecoxib were used. Vascular function and structure were analysed by myography. COX‐2 and phospo‐p65 expression were studied by western blotting and immunohistochemistry and TXA2 production by ELISA. We found that Rcan1 deficiency increases COX‐2 and IL‐6 expression and NF‐&kgr;B activation in arteries and vascular smooth muscle cells (VSMC). Adenoviral‐mediated re‐expression of Rcan1.4 in Rcan1−/− VSMC normalized COX‐2 expression. Phenylephrine‐induced vasoconstrictor responses were greater in aorta from Rcan1−/− compared to Rcan1+/+ mice. This increased response were diminished by etoricoxib, furegrelate, SQ 29548, cyclosporine A and parthenolide, inhibitors of COX‐2, TXA2 synthase, TP receptors, calcineurin and NF‐&kgr;B, respectively. Endothelial removal and NOS inhibition increased phenylephrine responses only in Rcan1+/+ mice. TXA2 levels were greater in Rcan1−/− mice. In small mesenteric arteries, vascular function and structure were similar in both groups of mice; however, vessels from Rcan1−/− mice displayed an increase in vascular stiffness that was diminished by rofecoxib. In conclusion, our results suggest that Rcan1 might act as endogenous negative modulator of COX‐2 expression and activity by inhibiting calcineurin and NF‐kB pathways to maintain normal contractility and vascular stiffness in aorta and small mesenteric arteries, respectively. Our results uncover a new role for Rcan1 in vascular contractility and mechanical properties.