Transcatheter Aortic Valve Implantation Represents an Anti-Inflammatory Therapy Via Reduction of Shear Stress-Induced, Piezo-1-Mediated Monocyte Activation.

BACKGROUND: Aortic valve stenosis is an increasingly prevalent degenerative and inflammatory disease. Transcatheter aortic valve implantation (TAVI) has revolutionized its treatment, thereby avoiding its life-threatening/disabling consequences. Whether aortic valve stenosis is accelerated by inflammation and whether it is itself a cause of inflammation are unclear. We hypothesized that the large shear forces exerted on circulating cells, particularly on the largest circulating cells, monocytes, while passing through stenotic aortic valves result in proinflammatory effects that are resolved with TAVI. METHODS: TAVI provides a unique opportunity to compare the activation status of monocytes under high shear stress (before TAVI) and under low shear stress (after TAVI). The activation status of monocytes was determined with a single-chain antibody, MAN-1, which is specific for the activated ß2-integrin Mac-1. Monocyte function was further characterized by the adhesion of myocytes to stimulated endothelial cells, phagocytic activity, uptake of oxidized low-density lipoprotein, and cytokine expression. In addition, we designed a microfluidic system to recapitulate the shear rate conditions before and after TAVI. We used this tool in combination with functional assays, Ca2+ imaging, siRNA gene silencing, and pharmacological agonists and antagonists to identify the key mechanoreceptor mediating the shear stress sensitivity of monocytes. Last, we stained for monocytes in explanted stenotic aortic human valves. RESULTS: The resolution of high shear stress through TAVI reduces Mac-1 activation, cellular adhesion, phagocytosis, oxidized low-density lipoprotein uptake, and expression of inflammatory markers in monocytes and plasma. Using microfluidics and pharmacological and genetic studies, we could recapitulate high shear stress effects on isolated human monocytes under highly controlled conditions, showing that shear stress-dependent calcium influx and monocyte adhesion are mediated by the mechanosensitive ion channel Piezo-1. We also demonstrate that the expression of this receptor is shear stress dependent and downregulated in patients receiving TAVI. Last, we show monocyte accumulation at the aortic side of leaflets of explanted aortic valves. CONCLUSIONS: We demonstrate that high shear stress, as present in patients with aortic valve stenosis, activates multiple monocyte functions, and we identify Piezo-1 as the mainly responsible mechanoreceptor, representing a potentially druggable target. We demonstrate an anti-inflammatory effect and therefore a novel therapeutic benefit of TAVI.

Pharmacological Inhibition of Factor XIIa Attenuates Abdominal Aortic Aneurysm, Reduces Atherosclerosis, and Stabilizes Atherosclerotic Plaques.

BACKGROUND: 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. METHODS: The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease-angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE-/- model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. RESULTS: Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. CONCLUSION: Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.

Plasma levels of trimethylamine-N-oxide can be increased with 'healthy' and 'unhealthy' diets and do not correlate with the extent of atherosclerosis but with plaque instability.

AIMS: The microbiome-derived metabolite trimethylamine-N-oxide (TMAO) has attracted major interest and controversy both as a diagnostic biomarker and therapeutic target in atherothrombosis. METHODS AND RESULTS: Plasma TMAO increased in mice on 'unhealthy' high-choline diets and notably also on 'healthy' high-fibre diets. Interestingly, TMAO was found to be generated by direct oxidation in the gut in addition to oxidation by hepatic flavin-monooxygenases. Unexpectedly, two well-accepted mouse models of atherosclerosis, ApoE-/- and Ldlr-/- mice, which reflect the development of stable atherosclerosis, showed no association of TMAO with the extent of atherosclerosis. This finding was validated in the Framingham Heart Study showing no correlation between plasma TMAO and coronary artery calcium score or carotid intima-media thickness (IMT), as measures of atherosclerosis in human subjects. However, in the tandem-stenosis mouse model, which reflects plaque instability as typically seen in patients, TMAO levels correlated with several characteristics of plaque instability, such as markers of inflammation, platelet activation, and intraplaque haemorrhage. CONCLUSIONS: Dietary-induced changes in the microbiome, of both 'healthy' and 'unhealthy' diets, can cause an increase in the plasma level of TMAO. The gut itself is a site of significant oxidative production of TMAO. Most importantly, our findings reconcile contradictory data on TMAO. There was no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. However, using a mouse model of plaque instability we demonstrated an association of TMAO plasma levels with atherosclerotic plaque instability. The latter confirms TMAO as being a marker of cardiovascular risk.

Successful renal denervation decreases the platelet activation status in hypertensive patients.

AIMS: To determine whether renal denervation (RDN) in hypertensive patients affects the platelet activation status. METHODS AND RESULTS: We investigated the effect of RDN on the platelet activation status in 41 hypertensive patients undergoing RDN. Ambulatory blood pressure (BP), plasma sympathetic neurotransmitter Neuropeptide Y, and platelet activation markers were measured at baseline, at 3 months, and 6 months after RDN. RDN significantly decreased BP at 3 months (150.6 ± 11.3/80.9 ± 11.4 mmHg to 144.7 ± 12.0/77.1 ± 11.1 mmHg; P < 0.01) and at 6 months (144.3 ± 13.8/78.3 ± 11.1 mmHg; P < 0.01). Plasma levels of the sympathetic neurotransmitter Neuropeptide Y, an indicator of sympathetic nerve activity, were significantly decreased at 3 months (0.29 ± 0.11 ng/mL to 0.23 ± 0.11 ng/mL; P < 0.0001) and at 6 months (0.22 ± 0.12 ng/mL; P < 0.001) after RDN. This was associated with a reduction in platelet membrane P-selectin expression (3 months, P < 0.05; 6 months, P < 0.05), soluble P-selectin (6 months, P < 0.05), circulating numbers of platelet-derived extracellular vesicles (EVs) (3 months, P < 0.001; 6 months, P < 0.01), and phosphatidylserine expressing EVs (3 months, P < 0.001; 6 months, P < 0.0001), indicative of a reduction in platelet activation status and procoagulant activity. Only patients who responded to RDN with a BP reduction showed inhibition of P-selectin expression at 3 months (P < 0.05) and 6 months (P < 0.05) as well as reduction of glycoprotein IIb/IIIa activation at 3 months (P < 0.05). Notably, 13 patients who took aspirin did not show significant reduction in platelet P-selectin expression following RDN. CONCLUSION: Our results imply a connection between the sympathetic nervous system and the platelet activation status and provide a potential mechanistic explanation by which RDN can have favourable effects towards reducing cardiovascular complications.

The bidirectional interaction between the sympathetic nervous system and immune mechanisms in the pathogenesis of hypertension.

Over the last few years, evidence has accumulated to suggest that hypertension is, at least in part, an immune-mediated inflammatory disorder. Many links between immunity and hypertension have been established and provide a complex framework of mechanistic interactions contributing to the rise in BP. These include immune-mediated inflammatory processes affecting regulatory brain nuclei and interactions with other mediators of cardiovascular regulation such as the sympathetic nervous system. Sympathoexcitation differentially regulates T-cells based upon activation status of the immune cell as well as the resident organ. Exogenous and endogenous triggers activate signalling pathways in innate and adaptive immune cells resulting in pro-inflammatory cytokine production and activation of T-lymphocytes in the cardiovascular and renal regions, now considered major factors in the development of essential hypertension. The inflammatory cascade is sustained and exacerbated by the immune flow via the brain-bone marrow-spleen-gastrointestinal axis and thereby further aggravating immune-mediated pathways resulting in a vicious cycle of established hypertension and target organ damage. This review summarizes the evidence and recent advances in linking immune-mediated inflammation, sympathetic activation and their bidirectional interactions with the development of hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Lysophosphatidylcholine is a Major Component of Platelet Microvesicles Promoting Platelet Activation and Reporting Atherosclerotic Plaque Instability.

BACKGROUND: Microvesicles (MVs) are small cell-derived vesicles, which are mainly released by activated cells. They are part of a communication network delivering biomolecules, for example, inflammatory molecules, via the blood circulation to remote cells in the body. Platelet-derived MVs are known to induce vascular inflammation. Research on the mediators and mechanisms of their inflammatory effects has attracted major interest. We hypothesize that specific lipids are the mediators of vascular inflammation caused by platelet-derived MVs. METHODS AND RESULTS: Liquid chromatography electrospray ionization-tandem mass spectrometry was used for lipid profiling of platelet-derived MVs. Lysophosphatidylcholine (LPC) was found to be a major component of platelet-derived MVs. Investigating the direct effects of LPC, we found that it induces platelet activation, spreading, migration and aggregation as well as formation of inflammatory platelet-monocyte aggregates. We show for the first time that platelets express the LPC receptor G2AR, which mediates LPC-induced platelet activation. In a mouse model of atherosclerotic plaque instability/rupture, circulating LPC was detected as a surrogate marker of plaque instability. These findings were confirmed by matrix-assisted laser desorption ionization imaging, which showed that the LPC concentration of human plaques was highest in vulnerable plaque regions. CONCLUSION: LPC is a major component of platelet-derived MVs and via its interaction with G2AR on platelets contributes to platelet activation, spreading, migration and aggregation and ultimately to vascular inflammation. Circulating LPC reports on atherosclerotic plaque instability in mice and is significantly increased in unstable areas of atherosclerotic plaques in both mice and humans, linking LPC to plaque instability.

Platelet-Derived Microvesicles in Cardiovascular Diseases.

Microvesicles (MVs) circulating in the blood are small vesicles (100-1,000 nm in diameter) derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs), the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their "cargo" of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.

A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology.

Progress in pharmaceutical development is highly-dependent on preclinical in vivo animal studies. Small animal imaging is invaluable for the identification of new disease markers and the evaluation of drug efficacy. Here, we report for the first time the use of a three-dimensional fluorescence bioimager called FLuorescence Emission Computed Tomography (FLECT) for the detection of a novel recombinant fluoroprobe that is safe, easily prepared on a large scale and stably stored prior to scan. This novel fluoroprobe (Targ-Cy7) comprises a single-chain antibody-fragment (scFvTarg), which binds exclusively to activated-platelets, conjugated to a near-infrared (NIR) dye, Cy7, for detection. Upon mouse carotid artery injury, the injected fluoroprobe circulates and binds within the platelet-rich thrombus. This specific in vivo binding of the fluoroprobe to the thrombus, compared to its non-targeting control-fluoroprobe, is detected by the FLECT imager. The analyzed FLECT image quantifies the NIR signal and localizes it to the site of vascular injury. The detected fluorescence is further verified using a two-dimensional IVIS® Lumina scanner, where significant NIR fluorescence is detected in vivo at the thrombotic site, and ex vivo, at the injured carotid artery. Furthermore, fluorescence levels in various organs have also been quantified for biodistribution, with the highest fluoroprobe uptake shown to be in the injured artery. Subsequently, this live animal imaging technique is successfully employed to monitor the response of the induced thrombus to treatment over time. This demonstrates the potential of using longitudinal FLECT scanning to examine the efficacy of candidate drugs in preclinical settings. Besides intravascular thrombosis, we have shown that this non-invasive FLECT-imaging can also detect in vivo pulmonary embolism. Overall, this report describes a novel fluorescence-based preclinical imaging modality that uses an easy-to-prepare and non-radioactive recombinant fluoroprobe. This represents a unique tool to study mechanisms of thromboembolic diseases and it will strongly facilitate the in vivo testing of antithrombotic drugs. Furthermore, the non-radiation nature, low-cost, high sensitivity, and the rapid advancement of optical scanning technologies make this fluorescence imaging an attractive development for future clinical applications.

Renal Denervation Reduces Monocyte Activation and Monocyte-Platelet Aggregate Formation: An Anti-Inflammatory Effect Relevant for Cardiovascular Risk.

Overactivation of renal sympathetic nervous system and low-grade systemic inflammation are common features of hypertension. Renal denervation (RDN) reduces sympathetic activity in patients with resistant hypertension. However, its effect on systemic inflammation has not been examined. We prospectively investigated the effect of RDN on monocyte activation and inflammation in patients with uncontrolled hypertension scheduled for RDN. Ambulatory blood pressure, monocyte, and monocyte subset activation and inflammatory markers were assessed at baseline, 3 months, and 6 months after procedure in 42 patients. RDN significantly lowered blood pressure at 3 months (150.5±11.2/81.0±11.2 mm Hg to 144.7±11.8/77.9±11.0 mm Hg), which was sustained at 6 months (144.7±13.8/78.6±11.0 mm Hg). Activation status of monocytes significantly decreased at 3 months (P<0.01) and 6 months (P<0.01) after the procedure. In particular, classical monocyte activation was reduced at 6 months (P<0.05). Similarly, we observed a reduction of several inflammatory markers, including monocyte-platelet aggregates (3 months, P<0.01), plasma monocyte chemoattractant protein-1 levels (3 months, P<0.0001; 6 months, P<0.05), interleukin-1ß (3 months, P<0.05; 6 months, P<0.05), tumor necrosis factor-α (3 months, P<0.01; 6 months, P<0.05), and interleukin-12 (3 months, P<0.01; 6 months, P<0.05). A positive correlation was observed between muscle sympathetic nerve activity and monocyte activation before and after the procedure. These results indicate that inhibition of sympathetic activity via RDN is associated with a reduction of monocyte activation and other inflammatory markers in hypertensive patients. These findings point to a direct interaction between the inflammatory and sympathetic nervous system, which is of central relevance for the understanding of beneficial cardiovascular effects of RDN.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators.

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.