Aims: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H 2 O 2 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 b-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 H 2 O 2 and O 2 .-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 H 2 O 2 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.

Abstract:Aim: to analyze the signaling pathways involved in H₂O₂ vascular responses in hypertension. Methods: Vascular function, thromboxane A₂ (TXA₂) production, oxidative stress and protein expression were determined in mesenteric resistance arteries (MRA) from hypertensive (SHR) and normotensive WKY rats. Results: H₂O₂ and the TP agonist U46619 induced greater contractile responses in MRA from SHR than WKY. Moreover H₂O₂increased TXA₂production more in SHR than in WKY. The cSrc inhibitor PP1 reduced H₂O₂-and U46619-induced contraction and TXA₂ release in both strains. The ERK1/2 inhibitor PD98059 reduced H₂O₂but not U46619-induced contraction only in SHR arteries. The Rho Kinase inhibitor Y26372 reduced H₂O₂and U46619-induced contractions only in SHR arteries. Basal c-Src, ERK1/2 and Rho Kinase expression were greater in MRA from SHR than WKY. In SHR, the combination of PD98059 with the TP antagonist SQ29548 but not with Y27632, inhibited the H₂O₂contraction more than each inhibitor alone. H₂O₂and U46619 increased NAD(P)H Oxidase activity and O₂.-production and decreased mitochondrial membrane potential in vessels from SHR. The effects induced by H₂O₂were abolished by inhibitors of TXA₂synthase, ERK1/2 and c-Src. The mitochondrial antioxidant mitoTEMPO reduced H₂O₂-induced contraction and NAD(P)H Oxidase activation. Conclusions: In arteries from WKY, c-Src mediates H₂O₂ contractile responses by modulating TXA₂release and TXA₂ effect. In SHR, H₂O₂contraction is mediated by c-Src-dependent TXA₂ release which further activates Rho Kinase, c-Src and the relationship between mitochondria and NAD(P)H Oxidase. Moreover, ERK1/2 activation contributes to H₂O₂ contraction in SHR through effects on mitochondria/NAD(P)H Oxidase. We affirm that the present study is not under consideration by another journal, and that no part of the material has been published elsewhere. All persons acknowledged have seen and approved the mention of their name in the paper. We also state that there is no conflict of interest. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationWe hope that the new version of the manuscript fulfills all the requirements for its publication Answers to Reviewer 1We would like to thank the Reviewer for his/her constructive comments. The manuscript has undergone changes to include data from new experiments. All concerns raised have been addressed and a detailed list of changes made is attached. We believe that the paper is significantly improved and hope that it is now acceptable for publication. I have no major concerns regarding this study apart from a couple of considerations:1)The potential "translational" meaning of the observed findings could be more clearly addressed and detailed.The Reviewer raised an interesting aspect. It is now evident from an overwhelming amount of experimental and animal studies, that oxidative stress has a role in the etiology of hypertension. Unfortunately, data in patients are more varying and still no clear picture of this aspect has been drawn. ...

Thrombospondin 4 (TSP-4) expression is induced in the heart and vasculature under pathological conditions, including myocardial infarction, myocardial pressure overload, and hypertension. TSP-4 is linked to remodelling processes, where it may affect extracellular matrix protein organization. In previous work, we studied the role of TSP-4 in small arteries during hypertension using Ang II-treated Thrombospondin 4 knockout (Thbs4) mice. We reported increased heart weight, as well as the occurrence of aortic aneurysms in the Ang II-treated Thbs4 animals. In the present study, we further characterized the hearts and aortas from these animals. Hypertrophy of cardiomyocytes, together with perivascular fibrosis and inflammation was observed in the Ang II-treated Thbs4 hearts. In the aortas, an increase in the aortic wall cross-sectional area (CSA) and wall thickness of the Ang II-treated Thbs4 mice was found. More detailed investigation of the Ang II-treated Thbs4 aortas also revealed the appearance of aortic dissections in the outer medial layer of the arteries, as well as pronounced inflammation. No differences were found in several other extracellular matrix-related parameters, such as number of elastin breaks or stress-strain relationships. However, at the ultrastructural level, collagen fibers showed alterations in diameter in the media and adventitia of the Ang II-treated Thbs4 mice, in the area prone to dissection. In conclusion, we identified TSP-4 as an important protein in the development of cardiac hypertrophy and aortic dissections in Ang II-induced hypertension.

Small arteries are known to develop functional and structural alterations in hypertension. However, the mechanisms of this remodeling are not fully understood. We hypothesized that altered gene expression is associated with the development of hypertension in mesenteric arteries of spontaneously hypertensive rats (SHR). Three sublines of SHR and normotensive Wistar Kyoto rats (WKY) were studied at 6 weeks and 5 months of age. MiRNA and mRNA microarray experiments were performed and analyzed with bioinformatical tools, including Ingenuity Pathway Analysis (IPA). Principal component analysis showed a clear separation in both miRNA and mRNA expression levels between both ages studied, demonstrating strong age-related changes in expression. At the miRNA level, IPA identified differences between SHR and WKY related to metabolic diseases, cellular growth, and proliferation. The mRNAs differentially expressed between SHR and WKY were related to metabolism, cellular movement and proliferation. The most strongly upregulated gene (9.2-fold) was thrombospondin 4 (Thbs4), a protein involved in the endoplasmic reticulum (ER) stress response that activates transcription factor 6α (ATF6α). ATF6α downstream targets were also differentially expressed in SHR vs. WKY. Differential expression of THBS4, the cleaved form of ATF6α, and two of its targets were further confirmed at the protein level by western blot. In summary, these data revealed a number of genes (n = 202) and miRNAs (n = 3) in mesenteric arteries of SHR that had not been related to hypertension previously. The most prominent of these, Thbs4, is related to vascular ER stress that is associated with hypertension.

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout (Thbs4(-/-)) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4(-/-) mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4(-/-) mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4(-/-) mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4(-/-) mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

AimConduction of vasomotor responses may contribute to long‐term regulation of resistance artery function and structure. Most previous studies have addressed conduction of vasoactivity only during very brief stimulations. We developed a novel set‐up that allows the local pharmacological stimulation of arteries in vitro for extended periods of time and studied the conduction of vasomotor responses in rat mesenteric arteries under those conditions.MethodsThe new in vitro set‐up was based on the pressure myograph. The superfusion chamber was divided halfway along the vessel into two compartments, allowing an independent superfusion of the arterial segment in each compartment. Local and remote cumulative concentration‐response curves were obtained for a range of vasoactive agents. Additional experiments were performed with the gap junction inhibitor 18β‐glycyrrhetinic acid and in absence of the endothelium.ResultsPhenylephrine‐induced constriction and acetylcholine‐induced dilation were conducted over a measured distance up to 2.84 mm, and this conduction was maintained for 5 minutes. Conduction of acetylcholine‐induced dilation was inhibited by 18β‐glycyrrhetinic acid, and conduction of phenylephrine‐induced constriction was abolished in absence of the endothelium. Constriction in response to high K+ was not conducted. Absence of remote stimulation dampened the local response to phenylephrine.ConclusionThis study demonstrates maintained conduction of vasoactive responses to physiological agonists in rat mesenteric small arteries likely via gap junctions and endothelial cells, providing a possible mechanism for the sustained functional and structural control of arterial networks.