The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial ß1-adrenergic receptor expression and signaling.

Reversible ubiquitination of G protein-coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial ß1-adrenergic receptors (ß1ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the ß1AR. ß1AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of ß1AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the ß1AR and failed to deubiquitinate the ß1AR. USP20-KO mice showed normal baseline systolic function but impaired ß1AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20-KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on ß1AR ubiquitination, but USP33 was up-regulated in USP20-KO hearts suggesting compensatory regulation. Myocardial ß1AR expression in USP20-KO was drastically reduced, whereas ß2AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in ß1AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates ß1AR signaling in vitro and in vivo Additionally, ß1AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize ß1AR expression and signaling during pathological insults to the myocardium.

G-Protein-Coupled Receptors in Heart Disease.

GPCRs (G-protein [guanine nucleotide-binding protein]-coupled receptors) play a central physiological role in the regulation of cardiac function in both health and disease and thus represent one of the largest class of surface receptors targeted by drugs. Several antagonists of GPCRs, such as ßARs (ß-adrenergic receptors) and Ang II (angiotensin II) receptors, are now considered standard of therapy for a wide range of cardiovascular disease, such as hypertension, coronary artery disease, and heart failure. Although the mechanism of action for GPCRs was thought to be largely worked out in the 80s and 90s, recent discoveries have brought to the fore new and previously unappreciated mechanisms for GPCR activation and subsequent downstream signaling. In this review, we focus on GPCRs most relevant to the cardiovascular system and discuss traditional components of GPCR signaling and highlight evolving concepts in the field, such as ligand bias, ß-arrestin-mediated signaling, and conformational heterogeneity.

Mechanoactivation of the angiotensin II type 1 receptor induces ß-arrestin-biased signaling through Gαi coupling.

Ligand activation of the angiotensin II type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, stimulates intracellular signaling to mediate a variety of physiological responses. The AT1R is also known to be a mechanical sensor. When activated by mechanical stretch, the AT1R can signal via the multifunctional adaptor protein ß-arrestin, rather than through classical heterotrimeric G protein pathways. To date, the AT1R conformation induced by membrane stretch in the absence of ligand was thought to be the same as that induced by ß-arrestin-biased agonists, which selectively engage ß-arrestin thereby preventing G protein coupling. Here, we show that in contrast to the ß-arrestin-biased agonists TRV120023 and TRV120026, membrane stretch uniquely promotes the coupling of the inhibitory G protein (Gαi ) to the AT1R to transduce signaling. Stretch-triggered AT1R-Gαi coupling is required for the recruitment of ß-arrestin2 and activation of downstream signaling pathways, such as EGFR transactivation and ERK phosphorylation. Our findings demonstrate additional complexity in the mechanism of receptor bias in which the recruitment of Gαi is required for allosteric mechanoactivation of the AT1R-induced ß-arrestin-biased signaling.

MicroRNAs for Restenosis and Thrombosis After Vascular Injury.

Percutaneous revascularization revolutionized the therapy of patients with coronary artery disease. Despite continuous technical advances that substantially improved patients' outcome after percutaneous revascularization, some issues are still open. In particular, restenosis still represents a challenge, even though it was dramatically reduced with the advent of drug-eluting stents. At the same time, drug-eluting stent thrombosis emerged as a major concern because of incomplete or delayed re-endothelialization after vascular injury. The discovery of microRNAs revealed a previously unknown layer of regulation for several biological processes, increasing our knowledge on the biological mechanisms underlying restenosis and stent thrombosis, revealing novel promising targets for more efficient and selective therapies. The present review summarizes recent experimental and clinical evidence on the role of microRNAs after arterial injury, focusing on practical aspects of their potential therapeutic application for selective inhibition of smooth muscle cell proliferation, enhancement of endothelial regeneration, and inhibition of platelet activation after coronary interventions. Application of circulating microRNAs as potential biomarkers is also discussed.

G Protein-coupled Receptor Biased Agonism.

G protein-coupled receptors are the largest family of targets for current therapeutics. The classic model of their activation was binary, where agonist binding induced an active conformation and subsequent downstream signaling. Subsequently, the revised concept of biased agonism emerged, where different ligands at the same G protein-coupled receptor selectively activate one downstream pathway versus another. Advances in understanding the mechanism of biased agonism have led to the development of novel ligands, which have the potential for improved therapeutic and safety profiles. In this review, we summarize the theory and most recent breakthroughs in understanding biased signaling, examine recent laboratory investigations concerning biased ligands across different organ systems, and discuss the promising clinical applications of biased agonism.

Non-coding RNAs: the "dark matter" of cardiovascular pathophysiology.

Large-scale analyses of mammalian transcriptomes have identified a significant number of different RNA molecules that are not translated into protein. In fact, the use of new sequencing technologies has identified that most of the genome is transcribed, producing a heterogeneous population of RNAs which do not encode for proteins (ncRNAs). Emerging data suggest that these transcripts influence the development of cardiovascular disease. The best characterized non-coding RNA family is represented by short highly conserved RNA molecules, termed microRNAs (miRNAs), which mediate a process of mRNA silencing through transcript degradation or translational repression. These microRNAs (miRNAs) are expressed in cardiovascular tissues and play key roles in many cardiovascular pathologies, such as coronary artery disease (CAD) and heart failure (HF). Potential links between other ncRNAs, like long non-coding RNA, and cardiovascular disease are intriguing but the functions of these transcripts are largely unknown. Thus, the functional characterization of ncRNAs is essential to improve the overall understanding of cellular processes involved in cardiovascular diseases in order to define new therapeutic strategies. This review outlines the current knowledge of the different ncRNA classes and summarizes their role in cardiovascular development and disease.

Antisense Oligonucleotides and Small Interfering RNA for the Treatment of Dyslipidemias.

The burden of atherosclerotic disease worldwide necessitates implementing the treatment of its risk factors. Among them, hypercholesterolemia has a central role. In addition to conventional small organic compounds and the recently introduced monoclonal antibodies, new technologies are arising such as the antisense oligonucleotides and small interfering RNAs (siRNAs) that operate upstream, blocking the mRNA translation of the proteins specifically involved in lipid metabolism. In this review, we briefly explain the mechanisms of action of these molecules and discuss the difficulties related to their in vivo use as therapeutical agents. We go over the oligonucleotides tested in clinical trials that could potentially revolutionize the care of patients by acting on proteins involved in the lipoprotein metabolism and regulation, namely: angiopoietin-like protein 3 (ANGPTL3); lipoprotein a (Lp(a)); apolipoprotein B (Apo B); apolipoprotein C III (Apo C-III); and proprotein convertase subtilisin-kexin type 9 (PCSK9). Finally, the differences between ASOs and siRNAs, their future possible clinical applications, and the role of Inclisiran, a siRNA direct against PCSK9 to reduce LDL-C, were reviewed in detail.

Use of a new cold plasma tool for psoriasis treatment: A case report.

Cold atmospheric plasma sources are emerging as new potent tools in different fields of medicine, from oncology to dermatology. Psoriasis is a chronic inflammatory disease characterized by the presence of itchy red plaque on skin, known as psoriatic plaques. In this case report, we evaluate the effectiveness of a cold atmospheric plasma treatment on a psoriatic plaque on the hand of a 20-year-old woman. Two quick applications of the procedure led to a complete disappearance of the cutaneous lesion in 14 days. The results of this case show a potential application of this new technique in the dermatological field, as palmo-plantar psoriasis is usually resistant to traditional treatments. A clinical trial would be necessary in order to evaluate the real effectiveness of this plasma.

Non-coding RNAs in vascular remodeling and restenosis.

Vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are crucial in vascular remodeling. They exert pivotal roles in the development and progression of atherosclerosis, vascular response to injury, and restenosis after transcatheter angioplasty. As a witness of their importance in the cardiovascular system, a large body of evidence has accumulated about the role played by micro RNAs (miRNA) in modulating both VSMCs and ECs. More recently, a growing number of long noncoding RNA (lncRNAs) came beneath the spotlights in this research field. Several mechanisms have been revealed by which lncRNAs are able to exert a relevant biological impact on vascular remodeling. The aim of this review is to provide an integrated summary of ncRNAs that exert a relevant biological function in VSMCs and ECs of the vascular wall, with emphasis on the available clinical evidence of the potential usefulness of these molecules as circulating biomarkers of in-stent restenosis.

Transcoronary concentration gradients of circulating microRNAs in heart failure.

AIMS: Circulating levels of microRNAs (miRNAs) are emergent promising biomarkers for cardiovascular disease. Altered expression of miRNAs has been related to heart failure (HF) and cardiac remodelling. We measured the concentration gradients across the coronary circulation to assess their usefulness to diagnose HF of different aetiologies. METHODS AND RESULTS: Circulating miRNAs were measured in plasma samples simultaneously obtained from the aorta and the coronary venous sinus in patients with non-ischaemic HF (NICM-HF, n = 23) ischaemic HF (ICM-HF, n = 41), and in control patients (n = 11). A differential modulation of circulating levels of miR-423, -34a, -21-3p, -126, -199 and -30a was found across the aetiology groups. Interestingly, a positive transcoronary gradient was found for miR-423 (P < 0.001) and miR-34a (P < 0.001) only in the ICM-HF group. On the contrary, a positive gradient was found for miR-21-3p (P < 0.001) and miR-30a (P = 0.030) only in the NICM-HF group. Finally, no significant variations were observed in the transcoronary gradient of miR-126 or miR-199. CONCLUSIONS: The present findings suggest that circulating levels of miRNAs are differentially expressed in patients with HF of different aetiologies. The presence of a transcoronary concentration gradient suggests a selective release of miRNAs by the failing heart into the coronary circulation. The presence of aetiology-specific transcoronary concentration gradients in HF patients might provide important information to better understand their role in HF, and suggests they could be useful biomarkers to distinguish HF of different aetiologies.

Hindlimb Ischemia Impairs Endothelial Recovery and Increases Neointimal Proliferation in the Carotid Artery.

Peripheral ischemia is associated with higher degree of endothelial dysfunction and a worse prognosis after percutaneous coronary interventions (PCI). However, the role of peripheral ischemia on vascular remodeling in remote districts remains poorly understood. Here we show that the presence of hindlimb ischemia significantly enhances neointima formation and impairs endothelial recovery in balloon-injured carotid arteries. Endothelial-derived microRNAs are involved in the modulation of these processes. Indeed, endothelial miR-16 is remarkably upregulated after vascular injury in the presences of hindlimb ischemia and exerts a negative effect on endothelial repair through the inhibition of RhoGDIα and nitric oxide (NO) production. We showed that the repression of RhoGDIα by means of miR-16 induces RhoA, with consequent reduction of NO bioavailability. Thus, hindlimb ischemia affects negative carotid remodeling increasing neointima formation after injury, while systemic antagonizzation of miR-16 is able to prevent these negative effects.

Down-regulation of miR-23b induces phenotypic switching of vascular smooth muscle cells in vitro and in vivo.

AIMS: Phenotypic switch of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of different vascular diseases, such as atherosclerosis and restenosis after coronary intervention. MicroRNAs have been identified as key regulators of VSMC biology. The miR-23b is highly expressed in VSMCs and it is involved in differentiation, proliferation, and migration of several non-vascular cell types. However, the role of miR-23b in vascular disease is currently unknown. Thus, the aim of the present study was to evaluate the role of miR-23b on VSMC phenotypic switch in vitro and after vascular injury in vivo. METHODS AND RESULTS: To determine the changes of miR-23b expression in the injured arterial wall, we used the standard rat carotid artery balloon injury model. In vivo studies demonstrated that miR-23b is down-regulated after vascular injury. Gain-of-function studies showed that overexpression of miR-23b inhibited VSMC proliferation and migration, whereas the opposite effect was obtained with the in vitro inhibition of miR-23b. We further demonstrated that miR-23b can significantly promote the expression of VSMC marker genes such as smooth muscle α-actin (ACTA2) and smooth muscle myosin heavy chain (MYH11). Overexpression of miR-23b in balloon-injured arteries by Ad-miR-23b markedly decreased neointimal hyperplasia. Finally, miR-23b specifically suppresses urokinase-type plasminogen activator, SMAD family member 3, and transcription factor forkhead box O4 (FoxO4) expression in phenotypically modulated VSMCs. By luciferase reporter assay, we validated the transcription factor FoxO4 as a direct target of miR-23b in VSMCs. CONCLUSIONS: We identify miR-23b as a novel regulator of VSMC phenotypic switch in vitro and following vascular injury in vivo.

Carbonic anhydrase activation is associated with worsened pathological remodeling in human ischemic diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus (DM) has multifactorial detrimental effects on myocardial tissue. Recently, carbonic anhydrases (CAs) have been shown to play a major role in diabetic microangiopathy but their role in the diabetic cardiomyopathy is still unknown. METHODS AND RESULTS: We obtained left ventricular samples from patients with DM type 2 (DM-T2) and nondiabetic (NDM) patients with postinfarct heart failure who were undergoing surgical coronary revascularization. Myocardial levels of CA-I and CA-II were 6- and 11-fold higher, respectively, in DM-T2 versus NDM patients. Elevated CA-I expression was mainly localized in the cardiac interstitium and endothelial cells. CA-I induced by high glucose levels hampers endothelial cell permeability and determines endothelial cell apoptosis in vitro. Accordingly, capillary density was significantly lower in the DM-T2 myocardial samples (mean±SE=2152±146 versus 4545±211/mm(2)). On the other hand, CA-II was mainly upregulated in cardiomyocytes. The latter was associated with sodium-hydrogen exchanger-1 hyperphosphorylation, exaggerated myocyte hypertrophy (cross-sectional area 565±34 versus 412±27 µm(2)), and apoptotic death (830±54 versus 470±34 per 10(6) myocytes) in DM-T2 versus NDM patients. CA-II is activated by high glucose levels and directly induces cardiomyocyte hypertrophy and death in vitro, which are prevented by sodium-hydrogen exchanger-1 inhibition. CA-II was shown to be a direct target for repression by microRNA-23b, which was downregulated in myocardial samples from DM-T2 patients. MicroRNA-23b is regulated by p38 mitogen-activated protein kinase, and it modulates high-glucose CA-II-dependent effects on cardiomyocyte survival in vitro. CONCLUSIONS: Myocardial CA activation is significantly elevated in human diabetic ischemic cardiomyopathy. These data may open new avenues for targeted treatment of diabetic heart failure.