Novel pharmacological approaches in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a severe vascular disease and a major public health issue with an unmet medical need for therapy. This disease is featured by a progressive dilation of the abdominal aorta, boosted by atherosclerosis, ageing, and smoking as major risk factors. Aneurysm growth increases the risk of aortic rupture, a life-threatening emergency with high mortality rates. Despite the increasing progress in our knowledge about the etiopathology of AAA, an effective pharmacological treatment against this disorder remains elusive and surgical repair is still the unique available therapeutic approach for high-risk patients. Meanwhile, there is no medical alternative for patients with small aneurysms but close surveillance. Clinical trials assessing the efficacy of antihypertensive agents, statins, doxycycline, or anti-platelet drugs, among others, failed to demonstrate a clear benefit limiting AAA growth, while data from ongoing clinical trials addressing the benefit of metformin on aneurysm progression are eagerly awaited. Recent preclinical studies have postulated new therapeutic targets and pharmacological strategies paving the way for the implementation of future clinical studies exploring these novel therapeutic strategies. This review summarises some of the most relevant clinical and preclinical studies in search of new therapeutic approaches for AAA.

PDE4 Phosphodiesterases in Cardiovascular Diseases: Key Pathophysiological Players and Potential Therapeutic Targets.

3',5'-cyclic adenosine monophosphate (cAMP) is a second messenger critically involved in the control of a myriad of processes with significant implications for vascular and cardiac cell function. The temporal and spatial compartmentalization of cAMP is governed by the activity of phosphodiesterases (PDEs), a superfamily of enzymes responsible for the hydrolysis of cyclic nucleotides. Through the fine-tuning of cAMP signaling, PDE4 enzymes could play an important role in cardiac hypertrophy and arrhythmogenesis, while it decisively influences vascular homeostasis through the control of vascular smooth muscle cell proliferation, migration, differentiation and contraction, as well as regulating endothelial permeability, angiogenesis, monocyte/macrophage activation and cardiomyocyte function. This review summarizes the current knowledge and recent advances in understanding the contribution of the PDE4 subfamily to cardiovascular function and underscores the intricate challenges associated with targeting PDE4 enzymes as a therapeutic strategy for the management of cardiovascular diseases.

Upregulation of NOR-1 in calcified human vascular tissues: impact on osteogenic differentiation and calcification.

Cardiovascular calcification is a significant public health issue whose pathophysiology is not fully understood. NOR-1 regulates critical processes in cardiovascular remodeling, but its contribution to ectopic calcification is unknown. NOR-1 was overexpressed in human calcific aortic valves and calcified atherosclerotic lesions colocalizing with RUNX2, a factor essential for osteochondrogenic differentiation and calcification. NOR-1 and osteogenic markers were upregulated in calcifying human valvular interstitial cells (VICs) and human vascular smooth muscle cells (VSMCs). Gain- and loss-of-function approaches demonstrated that NOR-1 negatively modulates the expression of osteogenic genes relevant for the osteogenic transdifferentiation (RUNX2, IL-6, BMP2, and ALPL) and calcification of VICs. VSMCs from transgenic mice overexpressing NOR-1 in these cells (TgNOR-1VSMC) expressed lower basal levels of osteogenic genes (IL-6, BMP2, ALPL, OPN) than cells from WT littermates, and their upregulation by a high-phosphate osteogenic medium (OM) was completely prevented by NOR-1 transgenesis. Consistently, this was associated with a dramatic reduction in the calcification of both transgenic VSMCs and aortic rings from TgNOR-1VSMC mice exposed to OM. Atherosclerosis and calcification were induce in mice by the administration of AAV-PCSK9D374Y and a high-fat/high-cholesterol diet. Challenged-TgNOR-1VSMC mice exhibited decreased vascular expression of osteogenic markers, and both less atherosclerotic burden (assessed in whole aorta and lesion size in aortic arch and brachiocephalic artery) and less vascular calcification (assessed either by near-infrared fluorescence imaging or histological analysis) than WT mice. Our data indicate that NOR-1 negatively modulates the expression of genes critically involved in the osteogenic differentiation of VICs and VSMCs, thereby restraining ectopic cardiovascular calcification.

Rolipram impacts on redox homeostasis and cellular signaling in an experimental model of abdominal aortic aneurysm. / Impacto del tratamiento con rolipram sobre la homeostasis rédox y la señalización celular en un modelo experimental de aneurisma de aorta abdominal.

INTRODUCTION: Cyclic nucleotide phosphodiesterases (PDEs) of the PDE4 subfamily are responsible for the hydrolysis and subcellular compartmentalization of cAMP, a second messenger that modulates vascular functionality. We had shown that PDE4B is induced in abdominal aortic aneurysms (AAA) and that PDE4 inhibition by rolipram limits experimental aneurysms. In this study we have delved into the mechanisms underlying the beneficial effect of rolipram on AAA. METHODS: AAA were induced in ApoE-/- mice by angiotensin II (Ang II) infusion. Aneurysm formation was evaluated by ultrasonography. The expression of enzymes involved in rédox homeostasis was analyzed by real-time RT-PCR and the activation of signaling pathways by Western blot. RESULTS: Induction of PDE4B in human AAA has been confirmed in a second cohort of patients. In Ang II-infused ApoE-/- mice, rolipram increased the percentage of animals free of aneurysms without affecting the percentage of aortic ruptures. Quantitative analyses determined that this drug significantly attenuated aortic collagen deposition. Additionally, rolipram reduced the increased Nox2 expression triggered by Ang II, exacerbated Sod1 induction, and normalized Sod3 expression. Likewise, PDE4 inhibition decreased the activation of both ERK1/2 and the canonical Wnt pathway, while AKT activity was not altered. CONCLUSIONS: The inhibition of PDE4 activity modulates the expression of enzymes involved in rédox homeostasis and affects cell signaling pathways involved in the development of AAA.

Lysyl oxidase-dependent extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease.

Extracellular matrix (ECM) is an active player in cardiovascular calcification (CVC), a major public health issue with an unmet need for effective therapies. Lysyl oxidase (LOX) conditions ECM biomechanical properties; thus, we hypothesized that LOX might impact on mineral deposition in calcific aortic valve disease (CAVD) and atherosclerosis. LOX was upregulated in calcified valves from two cohorts of CAVD patients. Strong LOX immunostaining was detected surrounding calcified foci in calcified human valves and atherosclerotic lesions colocalizing with RUNX2 on valvular interstitial cells (VICs) or vascular smooth muscle cells (VSMCs). Both LOX secretion and organized collagen deposition were enhanced in calcifying VICs exposed to osteogenic media. ß-aminopropionitrile (BAPN), an inhibitor of LOX, attenuated collagen deposition and calcification. VICs seeded onto decellularized matrices from BAPN-treated VICs calcified less than cells cultured onto control scaffolds; instead, VICs exposed to conditioned media from cells over-expressing LOX or cultured onto LOX-crosslinked matrices calcified more. Atherosclerosis was induced in WT and transgenic mice that overexpress LOX in VSMC (TgLOXVSMC) by AAV-PCSK9D374Y injection and high-fat feeding. In atherosclerosis-challenged TgLOXVSMC mice both atherosclerosis burden and calcification assessed by near-infrared fluorescence (NIRF) imaging were higher than in WT mice. These animals also exhibited larger calcified areas in atherosclerotic lesions from aortic arches and brachiocephalic arteries. Moreover, LOX transgenesis exacerbated plaque inflammation, and increased VSMC cellularity, the rate of RUNX2-positive cells and both connective tissue content and collagen cross-linking. Our findings highlight the relevance of LOX in CVC and postulate this enzyme as a potential therapeutic target for CVC.

Targeting mitochondrial stress with Szeto-Schiller 31 prevents experimental abdominal aortic aneurysm: Crosstalk with endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Mitochondrial dysfunction and inflammation contribute to a myriad of cardiovascular diseases. Deleterious crosstalk of mitochondria and persistent endoplasmic reticulum (ER) stress triggers oxidative stress, which is involved in the development of vascular diseases. This study determined if inhibition of mitochondrial stress reduces aneurysm development in angiotensin II (Ang II)-infused apolipoprotein-E-deficient (ApoE-/- ) mice and its effect on ER stress. EXPERIMENTAL APPROACH: The mitochondria-targeted tetrapeptide, Szeto-Schiller 31 (SS31), ameliorated mitochondrial dysfunction and the enhanced expression of ER stress markers triggered by Ang II in ApoE-/- mice, and limited plasmatic and vascular reactive oxygen species (ROS) levels. Interestingly, SS31 improved survival, reduced the incidence and severity of abdominal aortic aneurysm (AAA), and the Ang II-induced increase in aortic diameter as evaluated by ultrasonography, resembling the response triggered by the classic ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyrate (PBA). KEY RESULTS: Disorganization of the extracellular matrix, increased expression of metalloproteinases and pro-inflammatory markers and infiltration of immune cells induced by Ang II in the abdominal aorta were effectively reduced by SS31 and ER inhibitors. Further, C/EBP homologous protein (CHOP) deficiency in ApoE-/- mice attenuated Ang II-mediated increase in vascular diameter and incidence of AAA, suggesting its contribution to the favourable response induced by ER stress inhibition. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that inhibition of mitochondrial stress by SS31 limits AAA formation and increases survival through a reduction of vascular remodelling, inflammation and ROS, and support that attenuation of ER stress contributes to the favourable response elicited by SS31.

Serum from Stroke Patients with High-Grade Carotid Stenosis Promotes Cyclooxygenase-Dependent Endothelial Dysfunction in Non-ischemic Mice Carotid Arteries.

Atherosclerosis is responsible for 20% of ischemic strokes, and severe carotid stenosis is associated with a higher incidence of first-ever and recurrent strokes. The release of pro-inflammatory mediators into the blood in severe atherosclerosis may aggravate endothelial dysfunction after stroke contributing to impair disease outcomes. We hypothesize that environments of severe carotid atherosclerotic disease worsen endothelial dysfunction in stroke linked to enhanced risk of further cerebrovascular events. We mounted nonischemic common carotid arteries from 2- to 4-month-old male Oncins France 1 mice in tissue baths for isometric contraction force measurements and exposed them to serum from men with a recent ischemic stroke and different degrees of carotid stenosis: low- or moderate-grade stenosis (LMGS; < 70%) and high-grade stenosis (HGS; ≥ 70%). The results show that serum from stroke patients induced an impairment of acetylcholine relaxations in mice carotid arteries indicative of endothelium dysfunction. This effect was more pronounced after incubation with serum from patients with a recurrent stroke or vascular death within 1 year of follow-up. When patients were stratified according to the degree of stenosis, serum from HGS patients induced more pronounced carotid artery endothelial dysfunction, an effect that was associated with enhanced circulating levels of IL-1ß. Mechanistically, endothelial dysfunction was prevented by both nonselective and selective COX blockade. Altogether, the present findings add knowledge on the understanding of the mechanisms involved in the increased risk of stroke in atherosclerosis and suggest that targeting COX in the carotid artery wall may represent a potential novel therapeutic strategy for secondary stroke prevention.

Human Lysyl Oxidase Over-Expression Enhances Baseline Cardiac Oxidative Stress but Does Not Aggravate ROS Generation or Infarct Size Following Myocardial Ischemia-Reperfusion.

Lysyl oxidase (LOX) is an enzyme critically involved in collagen maturation, whose activity releases H2O2 as a by-product. Previous studies demonstrated that LOX over-expression enhances reactive oxygen species (ROS) production and exacerbates cardiac remodeling induced by pressure overload. However, whether LOX influences acute myocardial infarction and post-infarct left ventricular remodeling and the contribution of LOX to myocardial oxidative stress following ischemia-reperfusion have not been analyzed. Isolated hearts from transgenic mice over-expressing human LOX in the heart (TgLOX) and wild-type (WT) littermates were subjected to global ischemia and reperfusion. Although under basal conditions LOX transgenesis is associated with higher cardiac superoxide levels than WT mice, no differences in ROS production were detected in ischemic hearts and a comparable acute ischemia-reperfusion injury was observed (infarct size: 56.24 ± 9.44 vs. 48.63 ± 2.99% of cardiac weight in WT and TgLOX, respectively). Further, similar changes in cardiac dimensions and function were observed in TgLOX and WT mice 28 days after myocardial infarction induced by transient left anterior descending (LAD) coronary artery occlusion, and no differences in scar area were detected (20.29 ± 3.10 vs. 21.83 ± 2.83% of left ventricle). Our data evidence that, although LOX transgenesis induces baseline myocardial oxidative stress, neither ROS production, infarct size, nor post-infarction cardiac remodeling were exacerbated following myocardial ischemia-reperfusion.

Sex-Dependent End-of-Life Mental and Vascular Scenarios for Compensatory Mechanisms in Mice with Normal and AD-Neurodegenerative Aging.

Life expectancy decreases with aging, with cardiovascular, mental health, and neurodegenerative disorders strongly contributing to the total disability-adjusted life years. Interestingly, the morbidity/mortality paradox points to females having a worse healthy life expectancy. Since bidirectional interactions between cardiovascular and Alzheimer's diseases (AD) have been reported, the study of this emerging field is promising. In the present work, we further explored the cardiovascular-brain interactions in mice survivors of two cohorts of non-transgenic and 3xTg-AD mice, including both sexes, to investigate the frailty/survival through their life span. Survival, monitored from birth, showed exceptionally worse mortality rates in females than males, independently of the genotype. This mortality selection provided a "survivors" cohort that could unveil brain-cardiovascular interaction mechanisms relevant for normal and neurodegenerative aging processes restricted to long-lived animals. The results show sex-dependent distinct physical (worse in 3xTg-AD males), neuropsychiatric-like and cognitive phenotypes (worse in 3xTg-AD females), and hypothalamic-pituitary-adrenal (HPA) axis activation (higher in females), with higher cerebral blood flow and improved cardiovascular phenotype in 3xTg-AD female mice survivors. The present study provides an experimental scenario to study the suggested potential compensatory hemodynamic mechanisms in end-of-life dementia, which is sex-dependent and can be a target for pharmacological and non-pharmacological interventions.

Stenosis coexists with compromised α1-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) is a rare disorder caused by a heterozygous deletion of 26-28 contiguous genes that affects the brain and cardiovascular system. Here, we investigated whether WBS affects aortic structure and function in the complete deletion (CD) mouse model harbouring the most common deletion found in WBS patients. Thoracic aortas from 3-4 months-old male CD mice and wild-type littermates were mounted in wire myographs or were processed for histomorphometrical analysis. Nitric oxide synthase (NOS) isoforms and oxidative stress levels were assessed. Ascending aortas from young adult CD mice showed moderate (50%) luminal stenosis, whereas endothelial function and oxidative stress were comparable to wild-type. CD mice showed greater contractions to KCl. However, α1-adrenergic contractions to phenylephrine, but not with a thromboxane analogue, were compromised. Decreased phenylephrine responses were not affected by selective inducible NOS blockade with 1400 W, but were prevented by the non-selective NOS inhibitor L-NAME and the selective neuronal NOS inhibitor SMTC. Consistently, CD mice showed increased neuronal NOS expression in aortas. Overall, aortic stenosis in CD mice coexists with excessive nNOS-derived NO signaling that compromises ascending aorta α1-adrenergic contractions. We suggest that increased neuronal NOS signaling may act as a physiological 'brake' against the detrimental effects of stenosis.

Uric Acid Treatment After Stroke Prevents Long-Term Middle Cerebral Artery Remodelling and Attenuates Brain Damage in Spontaneously Hypertensive Rats.

Hypertension is the most important modifiable risk factor for stroke and is associated with poorer post-stroke outcomes. The antioxidant uric acid is protective in experimental normotensive ischaemic stroke. However, it is unknown whether this treatment exerts long-term protection in hypertension. We aimed to evaluate the impact of transient intraluminal middle cerebral artery (MCA) occlusion (90 min)/reperfusion (1-15 days) on brain and vascular damage progression in adult male Wistar-Kyoto (WKY; n = 36) and spontaneously hypertensive (SHR; n = 37) rats treated (i.v./120 min post-occlusion) with uric acid (16 mg kg-1) or vehicle (Locke's buffer). Ischaemic brain damage was assessed longitudinally with magnetic resonance imaging and properties of MCA from both hemispheres were studied 15 days after stroke. Brain lesions in WKY rats were associated with a transitory increase in circulating IL-18 and cerebrovascular oxidative stress that did not culminate in long-term MCA alterations. In SHR rats, more severe brain damage and poorer neurofunctional outcomes were coupled to higher cortical cerebral blood flow at the onset of reperfusion, a transient increase in oxidative stress and long-lasting stroke-induced MCA hypertrophic remodelling. Thus, stroke promotes larger brain and vascular damage in hypertensive rats that persists for long-time. Uric acid administered during early reperfusion attenuated short- and long-term brain injuries in both normotensive and hypertensive rats, an effect that was associated with abolishment of the acute oxidative stress response and prevention of stroke-induced long-lasting MCA remodelling in hypertension. These results suggest that uric acid might be an effective strategy to improve stroke outcomes in hypertensive subjects.

Uric acid treatment after stroke modulates the Krüppel-like factor 2-VEGF-A axis to protect brain endothelial cell functions: Impact of hypertension.

Uric acid (UA) is a promising protective treatment in ischaemic stroke, but the precise molecular targets underlying its in vivo beneficial actions remain unclear. High concentrations of UA inhibit angiogenesis of cultured endothelial cells via Krüppel-like factor 2 (KLF)-induced downregulation of vascular endothelial growth factor (VEGF), a pro-angiogenic mediator that is able to increase blood-brain barrier (BBB) permeability in acute stroke. Here, we investigated whether UA treatment after ischaemic stroke protects brain endothelial cell functions and modulates the KLF2-VEGF-A axis. Transient intraluminal middle cerebral artery (MCA) occlusion/reperfusion was induced in adult male spontaneously hypertensive (SHR) rats and corresponding normotensive Wistar-Kyoto (WKY) rats. Animals received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at reperfusion. BBB permeability was evaluated by Evans blue extravasation to the brain and in human cerebral endothelial hCMEC/D3 cells under oxygen-glucose deprivation/re-oxygenation. Circulating VEGF-A levels were measured in rats and acute ischaemic stroke patients from the URICO-ICTUS trial. Angiogenesis progression was assessed in Matrigel-cultured MCA. Worse post-stroke brain damage in SHR than WKY rats was associated with higher hyperaemia at reperfusion, increased Evans blue extravasation, exacerbated MCA angiogenic sprouting, and higher VEGF-A levels. UA treatment reduced infarct volume and Evans blue leakage in both rat strains, improved endothelial cell barrier integrity and KLF2 expression, and lowered VEGF-A levels in SHR rats. Hypertensive stroke patients treated with UA showed lower levels of VEGF-A than patients receiving vehicle. Consistently, UA prevented the enhanced MCA angiogenesis in SHR rats by a mechanism involving KLF2 activation. We conclude that UA treatment after ischaemic stroke upregulates KLF2, reduces VEGF-A signalling, and attenuates brain endothelial cell dysfunctions leading to neuroprotection.