Arterial Stiffness: From Basic Primers to Integrative Physiology.

The elastic properties of conductance arteries are one of the most important hemodynamic functions in the body, and data continue to emerge regarding the importance of their dysfunction in vascular aging and a range of cardiovascular diseases. Here, we provide new insight into the integrative physiology of arterial stiffening and its clinical consequence. We also comprehensively review progress made on pathways/molecules that appear today as important basic determinants of arterial stiffness, particularly those mediating the vascular smooth muscle cell (VSMC) contractility, plasticity and stiffness. We focus on membrane and nuclear mechanotransduction, clearance function of the vascular wall, phenotypic switching of VSMCs, immunoinflammatory stimuli and epigenetic mechanisms. Finally, we discuss the most important advances of the latest clinical studies that revisit the classical therapeutic concepts of arterial stiffness and lead to a patient-by-patient strategy according to cardiovascular risk exposure and underlying disease.

Vascular Smooth Muscle Cells and Arterial Stiffening: Relevance in Development, Aging, and Disease.

The cushioning function of large arteries encompasses distension during systole and recoil during diastole which transforms pulsatile flow into a steady flow in the microcirculation. Arterial stiffness, the inverse of distensibility, has been implicated in various etiologies of chronic common and monogenic cardiovascular diseases and is a major cause of morbidity and mortality globally. The first components that contribute to arterial stiffening are extracellular matrix (ECM) proteins that support the mechanical load, while the second important components are vascular smooth muscle cells (VSMCs), which not only regulate actomyosin interactions for contraction but mediate also mechanotransduction in cell-ECM homeostasis. Eventually, VSMC plasticity and signaling in both conductance and resistance arteries are highly relevant to the physiology of normal and early vascular aging. This review summarizes current concepts of central pressure and tensile pulsatile circumferential stress as key mechanical determinants of arterial wall remodeling, cell-ECM interactions depending mainly on the architecture of cytoskeletal proteins and focal adhesion, the large/small arteries cross-talk that gives rise to target organ damage, and inflammatory pathways leading to calcification or atherosclerosis. We further speculate on the contribution of cellular stiffness along the arterial tree to vascular wall stiffness. In addition, this review provides the latest advances in the identification of gene variants affecting arterial stiffening. Now that important hemodynamic and molecular mechanisms of arterial stiffness have been elucidated, and the complex interplay between ECM, cells, and sensors identified, further research should study their potential to halt or to reverse the development of arterial stiffness.

Conductance Artery Wall Layers and Their Respective Roles in the Clearance Functions.

Evolutionary organization of the arterial wall into layers occurred concomitantly with the emergence of a highly muscularized, pressurized arterial system that facilitates outward hydraulic conductance and mass transport of soluble substances across the arterial wall. Although colliding circulating cells disperse potential energy within the arterial wall, the different layers counteract this effect: (1) the endothelium ensures a partial barrier function; (2) the media comprises smooth muscle cells capable of endocytosis/phagocytosis; (3) the outer adventitia and perivascular adipocytic tissue are the final receptacles of convected substances. While the endothelium forms a physical and a biochemical barrier, the medial layer is avascular, relying on the specific permeability properties of the endothelium for metabolic support. Different components of the media interact with convected molecules: medial smooth muscle cells take up numerous molecules via scavenger receptors and are capable of phagocytosis of macro/micro particles. The outer layers-the highly microvascularized innervated adventitia and perivascular adipose tissue-are also involved in the clearance functions of the media: the adventitia is the seat of immune response development, inward angiogenesis, macromolecular lymphatic drainage, and neuronal stimulation. Consequently, the clearance functions of the arterial wall are physiologically essential, but also may favor the development of arterial wall pathologies. This review describes how the walls of large conductance arteries have acquired physiological clearance functions, how this is determined by the attributes of the endothelial barrier, governed by endocytic and phagocytic capacities of smooth muscle cells, impacting adventitial functions, and the role of these clearance functions in arterial wall diseases.

The ß 1 -Adrenergic Receptor Contributes to Sepsis-Induced Immunosuppression Through Modulation of Regulatory T-Cell Inhibitory Function.

OBJECTIVES: Although cardiovascular benefits of ß 1 -adrenergic receptor blockade have been described in sepsis, little is known about its impact on the adaptive immune response, specifically CD4 T cells. Herein, we study the effects of ß 1 -adrenergic receptor modulation on CD4 T-cell function in a murine model of sepsis. DESIGN: Experimental study. SETTING: University laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: High-grade sepsis was induced by cecal ligation and puncture in wild-type mice (ß 1+/+ ) with or without esmolol (a selective ß 1 -adrenergic receptor blocker) or in ß 1 -adrenergic receptor knockout mice (ß 1-/- ). At 18 hours after surgery, echocardiography was performed with blood and spleen collected to analyze lymphocyte function. MEASUREMENTS AND MAIN RESULTS: At 18 hours, ß 1+/+ cecal ligation and puncture mice exhibited characteristics of high-grade sepsis and three surrogate markers of immunosuppression, namely decreased splenic CD4 T cells, reduced CD4 T-cell proliferation, and increased regulatory T lymphocyte cell proportions. Pharmacologic and genetic ß 1 -adrenergic receptor blockade reversed the impact of sepsis on CD4 T and regulatory T lymphocyte proportions and maintained CD4 T-cell proliferative capacity. ß 1 -adrenergic receptor blocked cecal ligation and puncture mice also exhibited a global decrease in both pro- and anti-inflammatory mediators and improved in vivo cardiovascular efficiency with maintained cardiac power index despite the expected decrease in heart rate. CONCLUSIONS: ß 1 -adrenergic receptor activation enhances regulatory T lymphocyte inhibitory function and thus contributes to sepsis-induced immunosuppression. This can be attenuated by ß 1 -adrenergic receptor blockade, suggesting a potential immunoregulatory role for this therapy in the management of sepsis.

A new pro-thrombotic mechanism of neutrophil extracellular traps in antiphospholipid syndrome: impact on activated protein C resistance.

OBJECTIVES: In APS, precise evaluation of thrombotic risk is a major challenge. Different players, such as activated protein C (APC) resistance or neutrophil extracellular traps (NETs) contribute to the risk of thrombosis. Nevertheless, no study has investigated the interaction between these actors. The main objective of this study was to investigate the relation between NETs and APC resistance. METHODS: We designed a cross-sectional study including APS/antiphospholipid antibodies (aPL) patients and patients with autoimmune diseases (AID). We performed thrombin generation tests without and with APC to determine APC resistance. To evaluate circulating NETs, we measured plasma levels of MPO-DNA complexes and cell-free DNA with ELISA. RESULTS: We recruited 117 patients with definite APS/aPL or AID. We found a positive correlation between NETs and APC resistance, in APS patients and specifically in patients with high thrombotic risk, displaying LA or positivity of all three aPL tests (triple+), or anti-domain I IgG (aDI+). All these patient subgroups had increased NETs concentrations and APC resistance. As the risk profile for thrombosis increased, the relationship between NETs and APC resistance was stronger. CONCLUSION: We have shown that NETs participate in the hypercoagulable state of APS patients by contributing to APC resistance, in particular in high-risk patients. In these most at-risk patients, a targeted action on NETs could reduce APC resistance and constitute a new therapeutic approach in the treatment of APS patients in addition to antithrombotic therapy.

Genome-wide association analysis in dilated cardiomyopathy reveals two new players in systolic heart failure on chromosomes 3p25.1 and 22q11.23.

AIMS: Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. METHODS AND RESULTS: We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10-11 and 7.7 × 10-4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10-8 and 1.4 × 10-3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. CONCLUSION: This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure.

A case-control study indicates that coagulation imbalance is associated with arteriosclerosis and markers of endothelial dysfunction in kidney failure.

Endothelial dysfunction, one of many causes of arterial changes in end-stage kidney disease (kidney failure), is a likely link between early vascular aging and the risk of thrombosis or bleeding in this condition. To evaluate this, we compared links between arterial stiffness and endothelial/coagulation factors in 55 patients receiving hemodialysis therapy and 57 age-/sex-matched control individuals. Arterial stiffness was assessed from carotid-femoral pulse wave velocity, and coagulation status from the endogenous thrombin generating potential. Markers of endothelial dysfunction (von Willebrand factor, tissue factor pathway inhibitor), neutrophil extracellular traps and tissue factor-positive extracellular vesicles were higher in patients with kidney failure. Prothrombin fragments 1 and 2, and D-dimer markers of in vivo coagulation activation were also higher. However, in vitro in the presence of platelets, endogenous thrombin generating potential was lower and its downregulation by activated protein C impaired. Antiplatelet drugs did not affect these parameters. In multiple regression analysis, prothrombin fragments 1 and 2, D-dimer, factor VIII and monocyte-derived tissue factor-positive extracellular vesicles correlated with higher carotid-femoral pulse wave velocity. In patients with kidney failure, in vivo hypercoagulability occurred with reduced thrombin generation in platelet-rich plasma, likely explaining the opposing thrombotic and bleeding tendencies in patients with kidney failure. Importantly, arteriosclerosis is more closely related to a prothrombotic state. Thus, coagulation changes plus arterial stiffness highlight a major therapeutic challenge for anticoagulant and antiplatelet drug use.

Shedding Light on Hemostasis in Patients With Inflammatory Bowel Diseases.

Patients with inflammatory bowel diseases (IBD) have an increased risk of thrombosis, possibly due to changes in blood cells and molecules involved in hemostasis. They have increased platelet counts and reactivity as well as increased platelet-derived large extracellular vesicles. Coagulation is continuously activated in patients with IBD, based on measured markers of thrombin generation, and the anticoagulant functions of endothelial cells are damaged. Furthermore, fibrinogen is increased and fibrin clots are denser. However, pathogenesis of thrombosis in patients with IBD appears to differ from that of patients without IBD. Patients with IBD also take drugs that might contribute to risk of thrombosis, complicating the picture. We review the features of homeostasis that are altered in patients with IBD and possible mechanisms of this relationship.

Elastase and exacerbation of neutrophil innate immunity are involved in multi-visceral manifestations of COVID-19.

BACKGROUND: Many arguments suggest that neutrophils could play a prominent role in COVID-19. However, the role of key components of neutrophil innate immunity in severe forms of COVID-19 has deserved insufficient attention. We aimed to evaluate the involvement of neutrophil elastase, histone-DNA, and DNases in systemic and multi-organ manifestations of COVID-19. METHODS: We performed a multicenter study of markers of neutrophil innate immunity in 155 cases consecutively recruited in a screening center, local hospitals, and two regional university hospitals. The cases were evaluated according to clinical and biological markers of severity and multi-organ manifestations and compared to 35 healthy controls. RESULTS: Blood neutrophil elastase, histone-DNA, myeloperoxidase-DNA, and free dsDNA were dramatically increased, and DNase activity was decreased by 10-fold, compared with controls. Neutrophil elastase and histone-DNA were associated with intensive care admission, body temperature, lung damage, and markers of cardiovascular outcomes, renal failure, and increased interleukin-6 (IL-6), IL-8, and CXCR2. Neutrophil elastase was an independent predictor of the computed tomography score of COVID-19 lung damage and the number of affected organs, in multivariate analyses. The increased blood concentrations of NE and neutrophil extracellular traps were related to exacerbation of neutrophil stimulation through IL-8 and CXCR2 increased concentrations and increased serum DAMPs, and to impaired degradation of NETs as a consequence of the dramatic decrease in blood DNase activity. CONCLUSION: Our results point out the key role of neutrophil innate immunity exacerbation in COVID-19. Neutrophil elastase and DNase could be potential biomarkers and therapeutic targets of severe systemic manifestations of COVID-19.

Mechanisms of Arterial Stiffening: From Mechanotransduction to Epigenetics.

Arterial stiffness is a major independent risk factor for cardiovascular complications causing isolated systolic hypertension and increased pulse pressure in the microvasculature of target organs. Stiffening of the arterial wall is determined by common mechanisms including reduced elastin/collagen ratio, production of elastin cross-linking, reactive oxygen species-induced inflammation, calcification, vascular smooth muscle cell stiffness, and endothelial dysfunction. This brief review will discuss current biological mechanisms by which other cardiovascular risk factors (eg, aging, hypertension, diabetes mellitus, and chronic kidney disease) cause arterial stiffness, with a particular focus on recent advances regarding nuclear mechanotransduction, mitochondrial oxidative stress, metabolism and dyslipidemia, genome mutations, and epigenetics. Targeting these different molecular pathways at different time of cardiovascular risk factor exposure may be a novel approach for discovering drugs to reduce arterial stiffening without affecting artery strength and normal remodeling.

Systematic Review of Antiphospholipid Antibodies in COVID-19 Patients: Culprits or Bystanders?

PURPOSE OF REVIEW: COVID-19 patients have a procoagulant state with a high prevalence of thrombotic events. The hypothesis of an involvement of antiphospholipid antibodies (aPL) has been suggested by several reports. Here, we reviewed 48 studies investigating aPL in COVID-19 patients. RECENT FINDINGS: Prevalence of Lupus Anticoagulant (LA) ranged from 35% to 92% in ICU patients. Anti-cardiolipin (aCL) IgG and IgM were found in up to 52% and up to 40% of patients respectively. Anti-ß2-glycoprotein I (aß2-GPI) IgG and IgM were found in up to 39% and up to 34% of patients respectively. Between 1% and 12% of patients had a triple positive aPL profile. There was a high prevalence of aß2-GPI and aCL IgA isotype. Two cohort studies found few persistent LA but more persistent solid phase assay aPL over time. aPL determination and their potential role is a real challenge for the treatment of this disease.

C-glyco"RGD" as αIIbß3 and αvß integrin ligands for imaging applications: Synthesis, in vitro evaluation and molecular modeling.

The design of conjugates displaying simultaneously high selectivity and high affinity for different subtypes of integrins is a current challenge. The arginine-glycine-aspartic acid amino acid sequence (RGD) is one of the most efficient short peptides targeting these receptors. We report herein the development of linear and cyclic fluoro-C-glycoside"RGD" conjugates, taking advantage of the robustness and hydrophilicity of C-glycosides. As attested by in vitro evaluation, the design of these C-glyco"RGD" with a flexible three-carbon triazolyl linker allows distinct profiles towards αIIbß3 and αvß3 integrins. Molecular-dynamics simulations confirm the suitability of cyclic C-glyco-c(RGDfC) to target αvß3 integrin. These C-glyco"RGD" could become promising biological tools in particular for Positron Emission Tomography imaging.

Platelet aggregation impacts thrombin generation assessed by calibrated automated thrombography.

A calibrated automated thrombogram (CAT) is performed usually with human platelet-free plasma (PFP) but may be more relevant with platelet-rich plasma (PRP). In this case, platelets are not stimulated by subendothelial molecules like collagen. Our aim was to assess the consequence of strong (collagen) or weak (ADP) induction of platelet release and aggregation on thrombin generation. Platelet aggregation in PRP was triggered with 10 µg/mL collagen or 10 µM ADP using a lumi-aggregometer. Thrombin generation curves were monitored by CAT in different conditions: PRP, PRP with activated platelets (actPRP), aggregated PRP (agPRP), aggregated platelets resuspended in autologous PFP (resPRP), PFP and PFP obtained after aggregation (agPFP). We found a 3-fold shortening of the lag time and time to peak and a marked increase in velocity and thrombin peak without changes in endogenous thrombin potential (ETP) in agPRP with both agonists compared with PRP. The same holds true in agPFP but with a marked increase in ETP compared with PFP. Similar changes in the kinetics of thrombin generation were observed with actPRP-collagen and to a lesser extent in resPRP-collagen compared with PRP. By contrast, there were no modifications of the thrombin generation curves in actPRP-ADP. Alpha-2-macroglobin-thrombin complexes were unchanged in the different PRP conditions but were increased in PFP prepared from agPFP compared to control PFP. Platelet aggregation during activation by agonists other than thrombin did not increase thrombin generation but accelerated its kinetics mainly via platelet content release and platelet-derived extracellular vesicules formation. In diseases characterized by altered platelet granule content or release as well as altered platelet activation, a platelet aggregation step prior to CAT analysis may be clinically relevant to improve laboratory estimation of the bleeding/thrombotic balance.

Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries.

OBJECTIVE: The hypothesis that hypertension induces a hypercoagulable state arises from the complications associated with hypertension: stroke and myocardial infarction. Here, we determine whether hypertension causes changes in the thrombin-generating capacity of the vascular wall. APPROACH AND RESULTS: We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aortic rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared with equivalent rings from Wistar. This increase occurred in 12- but not 5-week-old rats and was prevented by an angiotensin II-converting enzyme inhibitor, indicating that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher on aortic smooth muscle cells (SMCs) from SHR than on those from Wistar. Exposure of negatively charged phospholipids was higher on SHR than on Wistar rings, as well as on cultured SMCs. Tissue factor activity was higher in SHR SMCs. Twelve-week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries, and the resulting occlusive thrombi were disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombin-induced proliferation than Wistar SMCs. This effect was totally abolished by a protease-activated receptor 1 inhibitor. CONCLUSIONS: The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which was sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Aldosterone up-regulates MMP-9 and MMP-9/NGAL expression in human neutrophils through p38, ERK1/2 and PI3K pathways.

Aldosterone and mineralocorticoid receptors are important regulators of inflammation. During this process, chemokines and extracellular matrix degradation by matrix metalloproteases, such as MMP-9, help leukocytes reaching swiftly and infiltrating the injured tissue, two processes essential for tissue repair. Leukocytes, such as neutrophils, are a rich source of MMP-9 and possess mineralocorticoid receptors (MR). The aim of our study was to investigate whether aldosterone was able to regulate proMMP-9, active MMP-9 and MMP-9/NGAL production in human neutrophils. Here we show that aldosterone increased MMP-9 mRNA in a dose- and time-dependent manner. This hormone up-regulated also dose-dependently proMMP-9 and active MMP-9 protein release as well as the MMP-9/NGAL protein complex. PI3K, p38 and ERK1/2 inhibition diminished these aldosterone-induced neutrophil productions. Furthermore, spironolactone, a MR antagonist, counteracted aldosterone-induced increases of proMMP-9, active MMP-9 and MMP-9/NGAL complex. These findings indicate that aldosterone could participate in tissue repair by modulating neutrophil activity and favoring extracellular matrix degradation.

Role of smooth muscle cell mineralocorticoid receptor in vascular tone.

Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MR(SMKO)), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MR(SMKO) mice, contractions induced by potassium chloride and calcium (Ca(2+)) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca(2+) suggests that the VSMC-specific deficiency of the MR modifies VSM Ca(2+) signaling but without altering the intracellular Ca(2+) store handling. The relaxation induced by acetylcholine is not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MR(SMKO) mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca(2+) signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca(2+) signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling.