Background: Patients with nonalcoholic fatty liver disease are at increased risk to develop atherosclerotic cardiovascular diseases. FXR and GPBAR1 are 2 bile acid–activated receptors exploited in the treatment of nonalcoholic fatty liver disease: whether dual GPBAR1/FXR agonists synergize with statins in the treatment of the liver and cardiovascular components of nonalcoholic fatty liver disease is unknown. Methods and Results: Investigations of human aortic samples obtained from patients who underwent surgery for aortic aneurysms and Gpbar1 ^−/− , Fxr ^−/− , and dual Gpbar1 ^−/‐ Fxr ^−/− mice demonstrated that GPBAR1 and FXR are expressed in the aortic wall and regulate endothelial cell/macrophage interactions. The expression of GPBAR1 in the human endothelium correlated with the expression of inflammatory biomarkers. Mice lacking Fxr and Gpbar1 ^−/− / Fxr ^−/− display hypotension and aortic inflammation, along with altered intestinal permeability that deteriorates with age, and severe dysbiosis, along with dysregulated bile acid synthesis. Vasomotor activities of aortic rings were altered by Gpbar1 and Fxr gene ablation. In apolipoprotein E ^−/− and wild‐type mice, BAR502, a dual GPBAR1/FXR agonist, alone or in combination with atorvastatin, reduced cholesterol and low‐density lipoprotein plasma levels, mitigated the development of liver steatosis and aortic plaque formation, and shifted the polarization of circulating leukocytes toward an anti‐inflammatory phenotype. BAR502/atorvastatin reversed intestinal dysbiosis and dysregulated bile acid synthesis, promoting a shift of bile acid pool composition toward FXR antagonists and GPBAR1 agonists. Conclusions: FXR and GPBAR1 maintain intestinal, liver, and cardiovascular homeostasis, and their therapeutic targeting with a dual GPBAR1/FXR ligand and atorvastatin holds potential in the treatment of liver and cardiovascular components of nonalcoholic fatty liver disease.

Background and Purpose: l‐cysteine or hydrogen sulfide (H₂S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 μM followed by vasodilatation at 30–100 μM. Here, we have investigated the signalling involved in the H₂S‐induced contraction.Experimental Approach: Vascular response to NaHS or l‐cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ‐lyase (CSE^−/−), soluble guanylyl cyclase (sGC_α1^−/−) and endothelial nitric oxide synthase (eNOS^−/−) knock‐out mice. The cAMP, cGMP and inosine 3′,5′‐cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated.Key Results: CSE‐derived H₂S‐induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H₂S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell‐permeable analogue of cIMP elicits concentration‐dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE^−/− mice, confirms that H₂S‐induced contraction involves cIMP.Conclusion and Implications: The endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE‐derived H₂S that is mediated by cIMP.

Objectives: Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor proteolytically activated by trypsin, tryptase or factor Xa. Alternatively, PAR2 can be activated by synthetic peptides whose sequence mimics the tethered ligand exposed after receptor cleavage. It is known that PAR2 modulates vascular reactivity, both in vitro and in vivo. The present study was designed to investigate the role of basal nitric oxide and cyclic nucleotides, adenosine 3′5′cyclic monophosphate (cAMP) and guanosine 3′5′ cyclic monophosphate (cGMP), in the vasorelaxation induced by a PAR2-activating peptide (PAR2-AP; SLIGRL-NH₂) on rat aorta in vitro. Methods: A concentration–response curve to PAR2-AP was performed on rat thoracic aorta with or without a functional endothelium, and the effect of inhibitors was evaluated. The effect of PAR2-AP (10⁻⁷–3 × 10⁻⁵ M) on endothelium-denuded aorta was also evaluated after tissue incubation with sodium nitroprusside. Results: PAR2-AP (10⁻⁷–3 × 10⁻⁵ M) caused an endothelium-dependent relaxation abolished by N^ω-nitro-l-arginine methyl ester, L-NAME, and by the guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), but unaffected by geldanamycin. Vasorelaxant effect of PAR2-AP was only partially inhibited by the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), and it was increased by tissue incubation with the phosphodiesterase inhibitor, rolipram. On tissue without endothelium, PAR2-AP did not cause vasorelaxation, up to a concentration of 10⁻⁴ M. However, after tissue incubation with sodium nitroprusside (SNP, 3 × 10⁻⁹ M), the vasorelaxant effect of PAR2-AP was restored. Following tissue incubation with PAR2-AP, cAMP levels were significantly increased compared to control values. Conclusions: Our results suggest that vasorelaxation induced by PAR2-AP is modulated by basal nitric oxide with an involvement of both cyclic nucleotides, cGMP and cAMP.

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2′-deoxyguanosine (G^Br) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G^LNA) in the analogue TBABL, 2’-O-methylguanosine (G^OMe) in TBABM, and 2’-F-riboguanosine (G^F) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (T_m approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer.

In this paper, we study the biological properties of two TBA analogs containing one and two extra G-tetrads, namely TBAG3 and TBAG4, respectively, and two further derivatives in which one of the small loops at the bottom (TBAG41S) or the large loop at the top (TBAG4GS) of the TBAG4 structure has been completely modified by replacing all loop residues with abasic site mimics. The therapeutical development of the TBA was hindered by its low thermodynamic and nuclease stability, while its potential as an anticancer/antiproliferative molecule is also affected by the anticoagulant activity, being a side effect in this case. In order to obtain suitable TBA analogs and to explore the involvement of specific aptamer regions in biological activity, the antiproliferative capability against DU 145 and MDAMB 231 cancer cell lines (MTT), the anticoagulant properties (PT), the biological degradability (nuclease stability assay) and nucleolin (NCL) binding ability (SPR) of the above described TBA derivatives have been tested. Interestingly, none of the TBA analogs exhibits an anticoagulant activity, while all of them show antiproliferative properties to the same extent. Furthermore, TBAG4 displays extraordinary nuclease stability and promising antiproliferative properties against breast cancer cells binding NCL efficiently. These results expand the range of G4-structures targeting NCL and the possibility of developing novel anticancer and antiviral drugs.