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Aims Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. Methods and results Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55 +), Cxcl chemokine 14 (Cxcl14 +), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. Conclusion We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo , associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD. [ABSTRACT FROM AUTHOR]

Aims Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo , cellular and molecular alterations. Methods and results We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. Conclusion Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death. [ABSTRACT FROM AUTHOR]

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation. [ABSTRACT FROM AUTHOR]

Aims After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI. Methods and results Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1–4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-β as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function. Conclusion LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery. [ABSTRACT FROM AUTHOR]

Aims Diastolic dysfunction is central to the development of heart failure. To date, there is no effective treatment and only limited understanding of its molecular basis. Recently, we showed that the transmembrane proteoglycan syndecan-4 increases in the left ventricle after pressure overload in mice and man, and that syndecan-4 via calcineurin/nuclear factor of activated T-cells (NFAT) promotes myofibroblast differentiation and collagen production upon mechanical stress. The aim of this study was to investigate whether syndecan-4 affects collagen cross-linking and myocardial stiffening in the pressure-overloaded heart. Methods and results Aortic banding (AB) caused concentric hypertrophy and increased passive tension of left ventricular muscle strips, responses that were blunted in syndecan-4-/- mice. Disruption of titin anchoring by salt extraction of actin and myosin filaments revealed that the effect of syndecan-4 on passive tension was due to extracellular matrix remodelling. Expression and activity of the cross-linking enzyme lysyl oxidase (LOX)increased with mechanical stress and was lower in left ventricles and cardiac fibroblasts from syndecan-4-/- mice, which exhibited less collagen cross-linking after AB. Expression of osteopontin (OPN), a matricellular protein able to induce LOX in cardiac fibroblasts, was up-regulated in hearts after AB, in mechanically stressed fibroblasts and in fibroblasts overexpressing syndecan-4, calcineurin, or NFAT, but down-regulated in fibroblasts lacking syndecan-4 or after NFAT inhibition. Interestingly, the extracellular domain of syndecan-4 facilitated LOX-mediated collagen cross-linking. Conclusions Syndecan-4 exerts a dual role in collagen cross-linking, one involving its cytosolic domain and NFAT signalling leading to collagen, OPN, and LOX induction in cardiac fibroblasts; the other involving the extracellular domain promoting LOX-dependent cross-linking. [ABSTRACT FROM AUTHOR]

Aims We investigated whether the pro-fibrotic matricellular protein osteopontin (OPN) is associated with the enzymes involved in the extracellular synthesis of fibril-forming collagen type I (i.e. procollagen C-proteinase, PCP) and its cross-linking to form insoluble fibrils (i.e. lysyl oxidase, LOX) in heart failure (HF) of hypertensive origin. Methods and results OPN, PCP, and LOX were assessed by histochemical and molecular methods in the myocardium of 21 patients with hypertensive heart disease (HHD) and HF. Whereas the myocardial expression of OPN was very scarce in control hearts (n = 10), it was highly expressed in HF patients (P < 0.0001). OPN was directly correlated with LOX (r = 0.460, P = 0.041), insoluble collagen (r = 0.534, P = 0.015), pulmonary capillary wedge pressure (r = 0.558; P = 0.009), and left-ventricular (LV) chamber stiffness (r = 0.458, P = 0.037), and inversely correlated with LV ejection fraction (r = −0.513, P = 0.017) in all patients. OPN did not correlate with PCP and other parameters assessing collagen synthesis by fibroblasts or degradation by matrix metalloproteinases. In vitro studies showed that OPN significantly (P < 0.05) increases the expression and activity of LOX in human cardiac and dermal fibroblasts. Conclusion An excess of OPN is associated with increased LOX and insoluble collagen, as well as with LV stiffness and systolic dysfunction in patients with HHD and HF. In addition, OPN up-regulates LOX in human fibroblasts. It is suggested that the OPN–LOX axis might facilitate the formation of insoluble collagen (i.e. stiff and resistant to degradation) and the subsequent alteration in LV mechanical properties and function in patients with HHD and HF. [ABSTRACT FROM PUBLISHER]

Lysyl oxidase (LOX) plays a crucial role in the maintenance of extracellular matrix stability and could participate in vascular remodelling associated with cardiovascular diseases. Evidence from in vitro and in vivo studies shows that LOX downregulation is associated with the endothelial dysfunction characteristic of earlier stages of the atherosclerotic process. Conversely, upregulation of this enzyme in vascular cells could induce neointimal thickening in atherosclerosis and restenosis. In fact, LOX is chemotactic for vascular smooth muscle cells and monocytes, is modulated by proliferative stimulus in these cells, and could control other cellular processes such as gene expression and cell transformation. Furthermore, it is conceivable that LOX downregulation could underlie plaque instability and contribute to the destructive remodelling that takes place during aneurysm development. Overall, LOX could play a key role in vascular homeostasis and, hence, it emerges as a new player in cardiovascular diseases. This review addresses the experimental evidence related to the role of LOX in vascular disorders and the potential benefits of controlling its expression and function. [ABSTRACT FROM PUBLISHER]

Purpose: We have previously reported that leptin can participate in the cardiovascular fibrosis associated with obesity. One of the key steps in the synthesis and maturation of the ECM involves the enzyme lysyl oxidase (LOX), which allows the crosslinking of collagen and elastin fibers and the formation of a stable and insoluble ECM. Therefore, we explore the effects of an inhibitor of LOX activity, beta-aminopropionitrile (BAPN), on cardiovascular alterations in a model of diet-induced obesity in rats, and whether or not these effects could involve the modulation of the profibrotic effects of leptin.Methods and Results: Male Wistar rats were fed either a high-fat diet (HFD; 33.5% fat), or a standard diet (3.5% fat) for 6 weeks. Half of the animals of each group were treated with BAPN (100 mg·kg-1 ·day-1) in drinking water. The inhibition of LOX prevented the increase in body weight observed in the HFD group (p<0.05). No differences were found in echocardiographic parameters of cardiac structure and systolic function among any group. As compared with CT animals, HFD rats showed an increase in relative heart weight and thickening of aorta. HFD rats showed cardiac and vascular fibrosis (picrosirius red staining), and an increase in superoxide anion (O2.-) production as evaluated by dihydroethidium staining. Inhibition of LOX reduced the production of both O2.- and collagen, especially the non-soluble one. Cardiac levels of collagen I and TGF-β were higher in HFD than in controls, while in the aorta, HFD showed high levels of collagen I, fibronectin, TGF-β and CTGF. All of these effects were prevented by the inhibition of LOX activity except the cardiac increase in cardiac galectin-3 levels. The inhibition of LOX reduced the increase in circulating leptin levels in HFD. Leptin (100 ng/ml) increased protein levels of collagen I, TGF-β and CTGF and enhanced O2.- production in both cardiac myofibroblasts and VSMCs. Leptin was also able to increase fibronectin in VSMCs. These effects were prevented by the presence of BAPN (250 μmol/L) in the culture media.Conclusions: LOX inhibition modulates the profibrotic effect of leptin in cardiac and vascular cells through the inhibition of TGF-β. BAPN improves the altered ECM by reducing collagen synthesis and limits the induction of oxidative stress observed in both myocardium and aorta of obese animals. [ABSTRACT FROM AUTHOR]

Purpose: Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that could play a critical role in vascular remodelling. 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.Methods: We designed a construct in which the full-length cDNA of human LOX was under the control of the SM22α proximal promoter and generated a transgenic mouse model (TgLOX) by conventional methods. LOX expression was assessed by real-time PCR, western-blot and immunohistochemistry and LOX activity by a fluorimetric assay. Collagen content and assembly were also analyzed. [3H]-thymidine incorporation into DNA, was used as an index of mitogenic activity and the carotid artery ligation model allowed to evaluate neointimal formation in TgLOX mice.Results: The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated. 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. [3H]-thymidine incorporation into DNA was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of both SMemb (marker of SMC phenotypic switching) and PCNA (marker of cell proliferation), 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-immunopositive VSMC.Conclusions: Our results 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. [ABSTRACT FROM AUTHOR]

: Aims Statins are lipid-lowering drugs widely used in the management of vascular diseases. Clinical and experimental evidence suggest that statins improve endothelial function by both cholesterol-lowering-dependent and -independent mechanisms. We have previously shown that endothelial dysfunction induced by risk factors and proinflammatory cytokines is associated with down-regulation of lysyl oxidase (LOX), a key enzyme modulating extracellular matrix maturation and vascular integrity. Our aim was to analyse whether statins could normalize LOX expression impaired by proatherogenic risk factors. : Methods and results We observed that pharmacological concentrations of statins (atorvastatin and simvastatin) modulated LOX transcriptional activity, counteracting the down-regulation of LOX (at the mRNA, protein, and activity level) caused by tumour necrosis factor-α (TNFα) in porcine, bovine, and human aortic endothelial cells. Geranylgeraniol but not farnesol reversed this effect, suggesting the involvement of geranylgeranylated proteins. In accordance, inhibitors of RhoA/Rho kinase also counteracted LOX down-regulation caused by TNFα, and over-expression of a RhoA dominant-negative mutant mimicked statin effects. Statins were also able to counteract the decrease in LOX expression produced by atherogenic concentrations of LDL by a similar mechanism and to partially prevent the increase in endothelial permeability elicited by these lipoproteins. Finally, in the in vivo porcine model of hypercholesterolaemia, we observed that statins abrogated the reduction of vascular LOX expression triggered by high plasma levels of LDL. : Conclusion These data indicate that statins normalize vascular LOX expression altered by atherogenic risk factors through a RhoA/Rho kinase-dependent mechanism. Thus, modulation of LOX by statins could contribute to vascular protection and to the cardiovascular risk reduction achieved by this therapy. [ABSTRACT FROM AUTHOR]