Mar Orriols

Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1α)-dependent Mechanism

Hypoxia modulates gene expression and affects multiple aspects of endothelial cell biology. Fibulin-5 (FBLN5) is an extracellular matrix protein essential for elastic fiber assembly and vasculogenesis that participates in vascular remodeling and controls endothelial cell adhesion, motility, and proliferation. In this context, we aimed to analyze FBLN5 regulation by hypoxia in endothelial cells. Hypoxia (1% O2) increased FBLN5 mRNA levels in endothelial cells in a time-dependent manner. Maximal induction (∼2.5-fold) was achieved after 24 h of hypoxia. This effect paralleled an increase in both intracellular and extracellular FBLN5 protein levels. The increase in FBLN5 mRNA levels observed in hypoxic cells was blocked by inhibitors of the PI3K/Akt/mTOR pathway (LY294002 and rapamycin) and mimicked by dimethyl oxal glycine, which prevents proline hydroxylase-mediated degradation of HIF-1α. Silencing of HIF-1α completely prevented hypoxia-induced FBLN5 up-regulation. Accordingly, both hypoxia and HIF-1α overexpression increased FBLN5 transcriptional activity. Serial promoter deletion and mutagenesis studies revealed the involvement of a putative hypoxia response element (HRE) located at −78 bp. In fact, EMSA and ChIP assays demonstrated increased HIF-1 binding to this site in hypoxic cells. Interestingly, the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia increased in FBLN5 knockdown cells, suggesting that hypoxia-induced FBLN5 expression contributes to preserve cell survival. These results provide evidence that HIF-1 signaling underlies the increase of FBLN5 expression elicited by hypoxia in endothelial cells and suggest that FBLN5 induction could be involved in the adaptive survival response of endothelial cells to hypoxia.

Hypoxia upregulates PGI-synthase and increases PGI2 release in human vascular cells exposed to inflammatory stimuli

Hypoxia affects vascular function and cell metabolism, survival, growth, and motility; these processes are partially regulated by prostanoids. We analyzed the effect of hypoxia and inflammation on key enzymes involved in prostanoid biosynthesis in human vascular cells. In human vascular smooth muscle cells (VSMC), hypoxia and interleukin (IL)-1β synergistically increased prostaglandin (PG)I2 but not PGE2 release, thereby increasing the PGI2/PGE2 ratio. Concomitantly, these stimuli upregulated cyclooxygenase-2 (COX-2) expression (mRNA and protein) and COX activity. Interestingly, hypoxia enhanced PGI-synthase (PGIS) expression and activity in VSMC and human endothelial cells. Hypoxia did not significantly modify the inducible microsomal-PGE-synthase (mPGES)-1. Hypoxia-inducible factor (HIF)-1α-silencing abrogated hypoxia-induced PGIS upregulation. PGIS transcriptional activity was enhanced by hypoxia; however, the minimal PGIS promoter responsive to hypoxia (-131 bp) did not contain any putative hypoxia response element (HRE), suggesting that HIF-1 does not directly drive PGIS transcription. Serial deletion and site-directed mutagenesis studies suggested several transcription factors participate cooperatively. Plasma levels of the stable metabolite of PGI2 and PGIS expression in several tissues were also upregulated in mice exposed to hypoxia. These data suggest that PGIS upregulation is part of the adaptive response of vascular cells to hypoxic stress and could play a role in counteracting the deleterious effect of inflammatory stimuli.

Lysyl oxidase (LOX) in vascular remodelling. Insight from a new animal model.

Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.

HuR mediates the synergistic effects of angiotensin II and IL‐1β on vascular COX‐2 expression and cell migration

Angiotensin II (AngII) and IL‐1β are involved in cardiovascular diseases through the induction of inflammatory pathways. HuR is an adenylate‐ and uridylate‐rich element (ARE)‐binding protein involved in the mRNA stabilization of many genes. This study investigated the contribution of HuR to the increased expression of COX‐2 induced by AngII and IL‐1β and its consequences on VSMC migration and remodelling.

Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK

AIMS Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H2O2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. RESULTS Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with β-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H2O2 and O2.- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H2O2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. INNOVATION We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. CONCLUSION LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27, 379-397.

Down-regulation of Fibulin-5 is associated with aortic dilation: role of inflammation and epigenetics

AIMS Destructive remodelling of extracellular matrix (ECM) and inflammation lead to dilation and ultimately abdominal aortic aneurysm (AAA). Fibulin-5 (FBLN5) mediates cell-ECM interactions and elastic fibre assembly and is critical for ECM remodelling. We aimed to characterize FBLN5 regulation in human AAA and analyse the underlying mechanisms. METHODS AND RESULTS FBLN5 expression was significantly decreased in human aneurysmatic aortas compared with healthy vessels. Local FBLN5 knockdown promoted aortic dilation and enhanced vascular expression of inflammatory markers in Ang II-infused C57BL/6J mice. Inflammatory stimuli down-regulated FBLN5 expression and transcriptional activity in human aortic vascular smooth muscle cells (VSMC). Further, aortic FBLN5 expression was reduced in LPS-challenged mice. A SOX response element was critical for FBLN5 promoter activity. The SOX9 expression pattern in human AAA parallels that of FBLN5, and like FBLN5, it was reduced in TNFα-stimulated VSMC. Interestingly, SOX9 over-expression prevented the cytokine-mediated reduction of FBLN5 expression and transcription. The inhibition of Class I histone deacetylases (HDACs) by MS-275 or gene silencing attenuated the inflammation-mediated decrease of FBLN5 expression in VSMC and in the vascular wall. Consistently, HDAC inhibition counteracted the reduction of SOX9 expression induced by inflammatory stimuli and prevented the TNFα-mediated decrease in the binding of SOX9 to FBLN5 promoter normalizing FBLN5 expression. CONCLUSION We evidence the deregulation of FBLN5 in human AAA and identify a SOX9/HDAC-dependent mechanism involved in the down-regulation of FBLN5 by inflammation. HDAC inhibitors or pharmacological approaches that aimed to preserve FBLN5 could be useful to prevent the disorganization of ECM induced by inflammation in AAA.

Induction of histone deacetylases (HDACs) in human abdominal aortic aneurysm: therapeutic potential of HDAC inhibitors

ABSTRACT Clinical management of abdominal aortic aneurysm (AAA) is currently limited to elective surgical repair because an effective pharmacotherapy is still awaited. Inhibition of histone deacetylase (HDAC) activity could be a promising therapeutic option in cardiovascular diseases. We aimed to characterise HDAC expression in human AAA and to evaluate the therapeutic potential of class I and IIa HDAC inhibitors in the AAA model of angiotensin II (Ang II)-infused apolipoprotein-E-deficient (ApoE−/−) mice. Real-time PCR, western blot and immunohistochemistry evidenced an increased expression of HDACs 1, 2 (both class I), 4 and 7 (both class IIa) in abdominal aorta samples from patients undergoing AAA open repair (n=22) compared with those from donors (n=14). Aortic aneurysms from Ang-II-infused ApoE−/− mice exhibited a similar HDAC expression profile. In these animals, treatment with a class I HDAC inhibitor (MS-275) or a class IIa inhibitor (MC-1568) improved survival, reduced the incidence and severity of AAA and limited aneurysmal expansion evaluated by Doppler ultrasonography. These beneficial effects were more potent in MC-1568-treated mice. The disorganisation of elastin and collagen fibres and lymphocyte and macrophage infiltration were effectively reduced by both inhibitors. Additionally, HDAC inhibition attenuated the exacerbated expression of pro-inflammatory markers and the increase in metalloproteinase-2 and -9 activity induced by Ang II in this model. Therefore, our data evidence that HDAC expression is deregulated in human AAA and that class-selective HDAC inhibitors limit aneurysm expansion in an AAA mouse model. New-generation HDAC inhibitors represent a promising therapeutic approach to overcome human aneurysm progression. Summary: This study reports the upregulation of HDACs in human AAA, evidences that HDAC inhibitors limit aneurysm progression in a preclinical model and suggests the therapeutic interest of HDAC inhibition in AAA.

Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II–induced hypertrophy

Lysyl oxidase (LOX) controls matrix remodeling, a key process that underlies cardiovascular diseases and heart failure; however, a lack of suitable animal models has limited our knowledge with regard to the contribution of LOX to cardiac dysfunction. Here, we assessed the impact of LOX overexpression on ventricular function and cardiac hypertrophy in a transgenic LOX (TgLOX) mouse model with a strong cardiac expression of human LOX. TgLOX mice exhibited high expression of the transgene in cardiomyocytes and cardiofibroblasts, which are associated with enhanced LOX activity and H2O2 production and with cardiofibroblast reprogramming. LOX overexpression promoted an age‐associated concentric remodeling of the left ventricle and impaired diastolic function. Furthermore, LOX transgenesis aggravated angiotensin II (Ang II)–induced cardiac hypertrophy and dysfunction, which triggered a greater fibrotic response that was characterized by stronger collagen deposition and cross‐linking and high expression of fibrotic markers. In addition, LOX transgenesis increased the Ang II–induced myocardial inflammatory infiltrate, exacerbated expression of proinflammatory markers, and decreased that of cardioprotective factors. Mechanistically, LOX overexpression enhanced oxidative stress and potentiated the Ang II–mediated cardiac activation of p38 MAPK while reducing AMPK activation. Our findings suggest that LOX induces an age‐dependent disturbance of diastolic function and aggravates Ang II–induced hypertrophy, which provides novel insights into the role of LOX in cardiac performance.—Galán, M., Varona, S., Guadall, A., Orriols, M., Navas, M., Aguiló, S., deDiego, A., Navarro, M. A., García‐Dorado, D., Rodríguez‐Sinovas, A., Martínez‐González, J., Rodriguez, C. Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II–induced hypertrophy. FASEB J. 31, 3787–3799 (2017). www.fasebj.org—Galán, María, Varona, Saray, Guadall, Anna, Orriols, Mar, Navas, Miquel, Aguiló, Silvia, de Diego, Alicia, Navarro, María A., García‐Dorado, David, Rodríguez‐Sinovas, Antonio, Martínez‐González, José, Rodriguez, Cristina Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II–induced hypertrophy. FASEB J. 31, 3787–3799 (2017)

La inflamación inhibe la expresión vascular de la fibulina-5: implicación del factor de transcripción SOX9 ☆

INTRODUCTION Fibulin-5 (FBLN5) is an elastogenic protein critically involved in extracellular matrix (ECM) remodelling, a key process in abdominal aortic aneurysm (AAA). However, the possible contribution of FBLN5 to AAA development has not been addressed. METHODS Expression levels were determined by real-time PCR and Western blot in human abdominal aorta from patients with AAA or healthy donors, as well as in human aortic vascular smooth muscle cells (VSMC). Lentiviral transduction, transient transfections, and chromatin immunoprecipitation (ChIP) assays were also performed. RESULTS The expression of FBLN5 in human AAA was significantly lower than in healthy donors. FBLN5 mRNA and protein levels and their secretion to the extracellular environment were down-regulated in VSMC exposed to inflammatory stimuli. Interestingly, FBLN5 transcriptional activity was inhibited by TNFα and lipopolysaccharide (LPS), and depends on a SOX response element. In fact, SOX9 expression was reduced in VMSC induced by inflammatory mediators and in human AAA, and correlated with that of FBLN5. Furthermore, SOX9 over-expression limited the reduction of FBLN5 expression induced by cytokines in VSMC. Finally, it was observed that SOX9 interacts with FBLN5 promoter, and that this binding was reduced upon TNFα exposure. CONCLUSIONS FBLN5 downregulation in human AAA could contribute to extracellular matrix remodelling induced by the inflammatory component of the disease.