Pericyte, but not astrocyte, hypoxia inducible factor-1 (HIF-1) drives hypoxia-induced vascular permeability in vivo

Background Ways to prevent disease-induced vascular modifications that accelerate brain damage remain largely elusive. Improved understanding of perivascular cell signalling could provide unparalleled insight as these cells impact vascular stability and functionality of the neurovascular unit as a whole. Identifying key drivers of astrocyte and pericyte responses that modify cell–cell interactions and crosstalk during injury is key. At the cellular level, injury-induced outcomes are closely entwined with activation of the hypoxia-inducible factor-1 (HIF-1) pathway. Studies clearly suggest that endothelial HIF-1 signalling increases blood–brain barrier permeability but the influence of perivascular HIF-1 induction on outcome is unknown. Using novel mouse lines with astrocyte and pericyte targeted HIF-1 loss of function, we herein show that vascular stability in vivo is differentially impacted by perivascular hypoxia-induced HIF-1 stabilization. Methods To facilitate HIF-1 deletion in adult mice without developmental complications, novel Cre-inducible astrocyte-targeted (GFAP-CreERT2; HIF-1αfl/fl and GLAST-CreERT2; HIF-1αfl/fl) and pericyte-targeted (SMMHC-CreERT2; HIF-1αfl/fl) transgenic animals were generated. Mice in their home cages were exposed to either normoxia (21% O2) or hypoxia (8% O2) for 96 h in an oxygen-controlled humidified glove box. All lines were similarly responsive to hypoxic challenge and post-Cre activation showed significantly reduced HIF-1 target gene levels in the individual cells as predicted. Results Unexpectedly, hypoxia-induced vascular remodelling was unaffected by HIF-1 loss of function in the two astrocyte lines but effectively blocked in the pericyte line. In correlation, hypoxia-induced barrier permeability and water accumulation were abrogated only in pericyte targeted HIF-1 loss of function mice. In contrast to expectation, brain and serum levels of hypoxia-induced VEGF, TGF-β and MMPs (genes known to mediate vascular remodelling) were unaffected by HIF-1 deletion in all lines. However, in agreement with the permeability data, immunofluorescence and electron microscopy showed clear prevention of hypoxia-induced tight junction disruption in the pericyte loss of function line. Conclusion This study shows that pericyte but not astrocyte HIF-1 stabilization modulates endothelial tight junction functionality and thereby plays a pivotal role in hypoxia-induced vascular dysfunction. Whether the cells respond similarly or differentially to other injury stimuli will be of significant relevance.

Daily consumption of wild olive (acebuche) oil reduces blood pressure and ameliorates endothelial dysfunction and vascular remodelling in rats with L-NAME-induced hypertension

Purpose: Despite numerous reports on the beneficial effects of olive oil in the cardiovascular context, very little is known about the olive tree’s wild counterpart (Olea europaea, L. var. sylvestris), commonly known as acebuche (ACE) in Spain. The aim of this study was to analyse the possible beneficial effects of an extra virgin ACE oil on vascular function in a rodent model of arterial hypertension induced by L-NAME (NG-nitro-L-arginine methyl ester). Methods: Four experimental groups of male Wistar rats were studied: 1) normotensive rats (Control group); 2) normotensive rats fed a commercial diet supplemented with 15% (w/w) ACE oil (Acebuche group); 3) rats made hypertensive following administration of L-NAME (L-NAME group); and 4) rats treated with L-NAME and simultaneously supplemented with 15% ACE oil (LN+ACE group). All treatments were maintained for 12 weeks. Results: Besides a significant blood pressure-lowering effect, the ACE oil-enriched diet counteracted the alterations found in aortas from hypertensive rats in terms of morphology and responsiveness to vasoactive mediators. In addition, a decrease in hypertension-related fibrotic and oxidative stress processes was observed in L-NAME-treated rats subjected to ACE oil supplement. Conclusion: Using a model of arterial hypertension via nitric oxide depletion, here we demonstrate the beneficial effects of a wild olive oil based upon its vasodilator, antihypertensive, antioxidant, antihypertrophic and antifibrotic properties. We postulate that regular inclusion of ACE oil in the diet can alleviate the vascular remodelling and endothelial dysfunction processes typically found in arterial hypertension, thus resulting in a significant reduction of blood pressure.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation

Background The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. Objectives The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells’ (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Methods Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. Results This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs’ proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3′UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Conclusion Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

511 Cardio-vascular remodelling during sacubitril/valsartan therapy in patients with heart failure and reduced ejection fraction

Aims Sacubitril/valsartan (S/V) benefits in patients with heart failure and reduced ejection fraction (HFrEF) are partially related to cardiac reverse remodelling, in terms of volumes reduction and function improvement. Effects on vascular remodelling are less investigated. To evaluate cardiac and vascular remodelling in a cohort of patients with HFrEF after 6 months of therapy with S/V. Methods and results 50 patients with HFrEF eligible to start a therapy with sacubitril/valsartan were enrolled. Clinical evaluation and standard and advanced echocardiography were performed at baseline and after 6 months of follow-up (FU). Standard left ventricular dimension and function parameters and global longitudinal strain (GLS) were calculated. Non-invasive pressure-volume curves (P-V loop) estimation was assessed with an off-line dedicated software using ST-E derived time-resolved LV volumes and brachial pressure as input. The following haemodynamic parameters were calculated based on P–V loop curves: left ventricular elastance (Ees), arterial elastance (Ea), and ventricular-arterial coupling (VAC). At 6 months F/U, a reduction of NYHA class in the vast majority of patients was detected (NYHA Class ≥ II, baseline vs. F/U = 100% vs. 50%; P &lt; 0.001). Systolic and diastolic blood pressure were lower, in comparison with baseline values (119 ± 16 vs. 126 ± 11 mmHg; P = 0.002 and 71 ± 8 vs. 78 ± 8 mmHg; P = 0.001, respectively). At echocardiographic evaluation, left ventricular end-diastolic and end-systolic volumes decreased and ejection fraction and GLS significantly improved (Table). Moreover, a significant reduction of Ea and a significant improvement of Ees and VAC was observed (Table). 511 Table 1 Conclusions Therapy with S/V in HFrEF patients determines both cardiac and vascular remodelling reflecting the complex mechanisms behind clinical improvement.

S-glutathionylation, a redox switch disrupting angiogenic balance to promote the preeclampsia phenotype

Background Elevation of circulating anti-angiogenic factors is pivotal in the development of the preeclampsia (PE) phenotype of incomplete vascular remodelling, hypertension and kidney dysfunction during pregnancy. Oxidative stress is explicitly linked to PE with high levels measurable in the placenta. Yet antioxidant therapy has failed, in some cases worsening pregnancy outcomes. The modulation of protein activity by reversible oxidative post-translational modifications (oxPTM) under low levels of reactive oxygen species is emerging as an important “redox-switch” mechanism in cardiovascular diseases, although oxPTM have not been investigated in the context of PE. Of significance, S-glutathionylation is a common oxPTM which reversal by glutaredoxin (Grx) is predominant in preeclamptic placenta and was associated with attenuated revascularisation and sFlt-1 elevation in mice. Purpose We aimed to identify the molecular basis for how S-glutathionylation reversal by Grx may contribute to pregnancy-induced vascular complications by modulating angiogenic signalling at the maternofoetal interface. Methods We combined physiological in vivo assessment with bioinformatics proteomic analysis and exon-level microarray to investigate the role of S-glutathionylation in the development of PE phenotype. In vitro studies using primary endothelial cells (EC) and iPS-derived trophoblasts investigated the effects of oxPTM reversal on angiogenic signalling in individual placental cell types and the functional consequences were assessed in 3D models replicating early-pregnancy events. Results Overexpressing Grx transgenic mice (TG) developed gestational hypertension, kidney dysfunction and elevated plasma levels of the anti-angiogenic factor sFlt-1 compared to their littermate controls (WT) during timed pregnancy. Grx-mediated oxPTM reversal in EC disrupted angiogenic sprouting and promoted anti-angiogenic signals by increasing sFlt-1:PlGF ratio and decreasing endoglin levels. The rise in sFlt-1 was associated with an isoform switch promoting sFlt-e15a over sFlt-i13. In trophoblasts, Grx overexpression inhibited migration and syncytialisation and modulated angiogenic balance in a cell type-specific manner. The sFlt1-e15a:PlGF ratio was increased in syncytiotrophoblasts and decreased in extra-villous trophoblasts, while endoglin expression was decreased in both cell types. A genome-wide exon-level profiling of TG vs WT mice placenta revealed a global alteration of alternative splicing events. Conclusion Grx-mediated removal of oxPTM directly disrupts placental angiogenic balance via dysregulation of sFlt-1 isoforms, which may promote the PE phenotype of impaired vascular remodelling, hypertension and kidney dysfunction during pregnancy. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020 - Marie Skłodowska-Curie grant agreement (iPLACENTA)

Mechanisms of vascular smooth muscle cell investment and phenotypic diversification in vascular diseases

In contrast with the heart, the adult mammalian vasculature retains significant remodelling capacity, dysregulation of which is implicated in disease development. In particular, vascular smooth muscle cells (VSMCs) play major roles in the pathological vascular remodelling characteristic of atherosclerosis, restenosis, aneurysm and pulmonary arterial hypertension. Clonal lineage tracing revealed that the VSMC-contribution to disease results from the hyperproliferation of few pre-existing medial cells and suggested that VSMC-derived cells from the same clone can adopt diverse phenotypes. Studies harnessing the powerful combination of lineage tracing and single-cell transcriptomics have delineated the substantial diversity of VSMC-derived cells in vascular lesions, which are proposed to have both beneficial and detrimental effects on disease severity. Computational analyses further suggest that the pathway from contractile VSMCs in healthy arteries to phenotypically distinct lesional cells consists of multiple, potentially regulatable, steps. A better understanding of how individual steps are controlled could reveal effective therapeutic strategies to minimise VSMC functions that drive pathology whilst maintaining or enhancing their beneficial roles. Here we review current knowledge of VSMC plasticity and highlight important questions that should be addressed to understand how specific stages of VSMC investment and phenotypic diversification are controlled. Implications for developing therapeutic strategies in pathological vascular remodelling are discussed and we explore how cutting-edge approaches could be used to elucidate the molecular mechanisms underlying VSMC regulation.

Myeloid cell-specific deletion of inducible nitric oxide synthase protects against smoke-induced pulmonary hypertension in mice

Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD), associated with increased mortality and morbidity. Intriguingly, pulmonary vascular alterations have been suggested to drive emphysema development. We previously identified inducible nitric oxide synthase (iNOS) as an essential enzyme for development and reversal of smoke-induced PH and emphysema, and showed that iNOS expression in bone-marrow-derived cells drives pulmonary vascular remodelling, but not parenchymal destruction. In this study, we aimed to identify the iNOS-expressing cell type driving smoke-induced PH and to decipher pro-proliferative pathways involved.To address this question we used 1) myeloid cell-specific iNOS knockout mice in chronic smoke exposure, 2) co-cultures of macrophages and pulmonary artery smooth muscle cells (PASMC) to decipher underlying signalling pathways.Myeloid cell-specific iNOS knockout prevented smoke-induced PH but not emphysema in mice. Moreover, iNOS deletion in myeloid cells ameliorated the increase in expression of CD206, a marker of M2 polarisation, on interstitial macrophages. Importantly, the observed effects on lung macrophages were hypoxia-independent, as these mice developed hypoxia-induced PH. In vitro, smoke-induced PASMC proliferation in co-cultures with M2-polarised macrophages could be abolished by iNOS deletion in phagocytic cells, as well as by ERK inhibition in PASMC. Crucially, CD206-positive and iNOS-positive macrophages accumulated in proximity of remodelled vessels in the lungs of COPD patients, as shown by immunohistochemistry.In summary, our results demonstrate that iNOS deletion in myeloid cells confers protection against PH in smoke-exposed mice and provide evidence for an iNOS-dependent communication between M2-like macrophages and PASMC in underlying pulmonary vascular remodelling.