The interface between cardiovascular disease and inflammation: hydrogen sulfide and annexin A1 pathways as novel pharmacological targets

Cardiovascular diseases are a heterogeneous group of disorders of the heart and the vasculature, in which different factors are responsible for their onset. In particular, among the various factors contributing the development of vascular disorders there are hyperlipidaemia, hyperglycaemia, cigarette smoke, obesity and hyperhomocysteinemia. Many of the pathologies that are part of vascular disorders, as hypertension, diabetes, atherosclerosis and heart failure, are characterized by two main conditions: oxidative stress and excessive prolonged inflammatory response. The interception of these dual aspects delineates the concept of inflammation-based vascular diseases (IBVD). In this context, it is of note to underline the decline of the nitric oxide (NO) and hydrogen sulfide (H2S) levels, molecules that share vasorelaxant and anti-inflammatory properties. Deficiency of these two gaseous mediators, normally produced within the body, targets vascular endothelium resulting into one of the classical IBVD example. In this project, we aimed to dissect the molecular mechanisms underlying the vascular impairment observed in hyperglycaemia and the cross talk between NO and H2S in IBVD, with respect to vascular inflammatory response. Indeed, we used in vitro, ex vivo and in vivo models to perform multiple approaches tackling different sides of disease conditions. We used bovine aortic endothelial cells (BAEC) and aorta rings harvested from mice to study the impact of hyperglycaemia and the effect of a novel mitochondrial-specific H2S donor (AP123). In addition, we also performed specific experiments on human macrophages aiming to evaluate the effect of H2S pathway in the physiological response by specialized pro-resolving molecules (SPMs) in inflammatory conditions. First, we found that the decline NO levels associated to hyperglycaemia is paralleled by similar outcomes in H2S levels. This impairment can be corrected in endothelial cells when AP123 was used as H2S donor and the underlying mechanism is based on a transcriptional effect on eNOS, involving the activation of cAMP response binding protein (CREB) by PI3K. Furthermore, we found that this mechanism is also operative in the whole vascular tissue as demonstrated by ex vivo experiments. In the context of experimental inflammation, we found that CSE pathway is downregulated in endothelial cells following administration of TNF-α. Interestingly, the beneficial role of H2S has been confirmed by administration of AP123. Therefore, the modulation of inflammatory response by H2S has been also shown to be part of the mechanism of action of “unrelated” anti-inflammatory drug such as clodronate. To evaluate whether H2S activity could influence changes in the lipid mediators’ activity, we evaluated the effect of slow-releasing H2S donor, AP123, and a specific CSE inhibitor, PAG, on the concentrations of SPMs in M1 macrophages, stimulated with LPS, at two different time points. We found that the majority of LM are differentially regulated between two treatments, involving pro- and anti-inflammatory mediators. However, the administration of 1nM AP123, at 45 min, mainly triggered the involvement of anti-inflammatory lipid mediators, while H2S donor (100nM and 1μM, 45min) seems to positively modulate the levels of a particular pro-resolving mediator, referred to as RvD2n3-DPA. Overall, our studies suggest that H2S is a crucial vasculoprotective mediator in IBVD at vascular level and H2S-releasing molecules may open new perspectives in the therapy of H2S deficiency conditions with a pleiotropic mechanism not only related to a simple “replacement” effect

Adaptive and Innate Cytotoxic Effectors in Chronic Lymphocytic Leukaemia (CLL) Subjects with Stable Disease

Chronic lymphocytic leukaemia (CLL) is characterised by the expansion of a neoplastic mature B cell clone. CLL clinical outcome is very heterogeneous, with some subjects never requiring therapy and some showing an aggressive disease. Genetic and epigenetic alterations and pro-inflammatory microenvironment influence CLL progression and prognosis. The involvement of immune-mediated mechanisms in CLL control needs to be investigated. We analyse the activation profile of innate and adaptive cytotoxic immune effectors in a cohort of 26 CLL patients with stable disease, as key elements for immune-mediated control of cancer progression. We observed an increase in CD54 expression and interferon (IFN)-γ production by cytotoxic T cells (CTL). CTL ability to recognise tumour-targets depends on human leukocyte antigens (HLA)-class I expression. We observed a decreased expression of HLA-A and HLA-BC on B cells of CLL subjects, associated with a significant reduction in intracellular calnexin that is relevant for HLA surface expression. Natural killer (NK) cells and CTL from CLL subjects show an increased expression of the activating receptor KIR2DS2 and a reduction of 3DL1 and NKG2A inhibiting molecules. Therefore, an activation profile characterises CTL and NK cells of CLL subjects with stable disease. This profile is conceivable with the functional involvement of cytotoxic effectors in CLL control

Phenolic Compounds of Red Wine \u3ci\u3eAglianico del Vulture\u3c/i\u3e Modulate the Functional Activity of Macrophages via Inhibition of \u3ci\u3eNF-κB\u3c/i\u3e and the Citrate Pathway

Phenolic compounds of red wine powder (RWP) extracted from the Italian red wine Aglianico del Vulture have been investigated for the potential immunomodulatory and anti-inflammatory capacity on human macrophages. These compounds reduce the secretion of IL-1β, IL-6, and TNF-α proinflammatory cytokines and increase the release of IL-10 anti-inflammatory cytokine induced by lipopolysaccharide (LPS). In addition, RWP restores Annexin A1 levels, thus involving activation of proresolutive pathways. Noteworthy, RWP lowers NF-κB protein levels, promoter activity, and nuclear translocation. As a consequence of NF-κB inhibition, reduced promoter activities of SLC25A1—encoding the mitochondrial citrate carrier (CIC)—and ATP citrate lyase (ACLY) metabolic genes have been observed. CIC, ACLY, and citrate are components of the citrate pathway: in LPS-activated macrophages, the mitochondrial citrate is exported by CIC into the cytosol where it is cleaved by ACLY in oxaloacetate and acetyl-CoA, precursors for ROS, NO⋅, and PGE2 inflammatory mediators. We identify the citrate pathway as a RWP target in carrying out its anti-inflammatory activity since RWP reduces CIC and ACLY protein levels, ACLY enzymatic activity, the cytosolic citrate concentration, and in turn ROS, NO⋅, PGE2, and histone acetylation levels. Overall findings suggest that RWP potentially restores macrophage homeostasis by suppressing inflammatory pathways and activating proresolutive processes

Anti-Inflammatory Effect of the Natural H2S-Donor Erucin in Vascular Endothelium

Vascular inflammation (VI) represents a pathological condition that progressively affects the integrity and functionality of the vascular wall, thus leading to endothelial dysfunction and the onset of several cardiovascular diseases. Therefore, the research of novel compounds able to prevent VI represents a compelling need. In this study, we tested erucin, the natural isothiocyanate H2S-donor derived from Eruca sativa Mill. (Brassicaceae), in an in vivo mouse model of lipopolysaccharide (LPS)-induced peritonitis, where it significantly reduced the amount of emigrated CD11b positive neutrophils. We then evaluated the anti-inflammatory effects of erucin in LPS-challenged human umbilical vein endothelial cells (HUVECs). The pre-incubation of erucin, before LPS treatment (1, 6, 24 h), significantly preserved cell viability and prevented the increase of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-alpha) levels. Moreover, erucin downregulated endothelial hyperpermeability and reduced the loss of vascular endothelial (VE)-Cadherin levels. In addition, erucin decreased vascular cell adhesion molecule 1 (VCAM-1), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E-synthase 1 (mPGES-1) expression. Of note, erucin induced eNOS phosphorylation and counteracted LPS-mediated NF-kappa B nuclear translocation, an effect that was partially abolished in the presence of the eNOS inhibitor L-NAME. Therefore, erucin can control endothelial function through biochemical and genomic positive effects against VI

Phenolic Compounds of Red Wine Aglianico del Vulture Modulate the Functional Activity of Macrophages via Inhibition of NF-κB and the Citrate Pathway

Phenolic compounds of red wine powder (RWP) extracted from the Italian red wine Aglianico del Vulture have been investigated for the potential immunomodulatory and anti-inflammatory capacity on human macrophages. These compounds reduce the secretion of IL-1β, IL-6, and TNF-α proinflammatory cytokines and increase the release of IL-10 anti-inflammatory cytokine induced by lipopolysaccharide (LPS). In addition, RWP restores Annexin A1 levels, thus involving activation of proresolutive pathways. Noteworthy, RWP lowers NF-κB protein levels, promoter activity, and nuclear translocation. As a consequence of NF-κB inhibition, reduced promoter activities of SLC25A1—encoding the mitochondrial citrate carrier (CIC)—and ATP citrate lyase (ACLY) metabolic genes have been observed. CIC, ACLY, and citrate are components of the citrate pathway: in LPS-activated macrophages, the mitochondrial citrate is exported by CIC into the cytosol where it is cleaved by ACLY in oxaloacetate and acetyl-CoA, precursors for ROS, NO⋅, and PGE2 inflammatory mediators. We identify the citrate pathway as a RWP target in carrying out its anti-inflammatory activity since RWP reduces CIC and ACLY protein levels, ACLY enzymatic activity, the cytosolic citrate concentration, and in turn ROS, NO⋅, PGE2, and histone acetylation levels. Overall findings suggest that RWP potentially restores macrophage homeostasis by suppressing inflammatory pathways and activating proresolutive processes

Anti-Inflammatory Effect of the Natural H2S-Donor Erucin in Vascular Endothelium

Vascular inflammation (VI) represents a pathological condition that progressively affects the integrity and functionality of the vascular wall, thus leading to endothelial dysfunction and the onset of several cardiovascular diseases. Therefore, the research of novel compounds able to prevent VI represents a compelling need. In this study, we tested erucin, the natural isothiocyanate H2S-donor derived from Eruca sativa Mill. (Brassicaceae), in an in vivo mouse model of lipopolysaccharide (LPS)-induced peritonitis, where it significantly reduced the amount of emigrated CD11b positive neutrophils. We then evaluated the anti-inflammatory effects of erucin in LPS-challenged human umbilical vein endothelial cells (HUVECs). The pre-incubation of erucin, before LPS treatment (1, 6, 24 h), significantly preserved cell viability and prevented the increase of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) levels. Moreover, erucin downregulated endothelial hyperpermeability and reduced the loss of vascular endothelial (VE)-Cadherin levels. In addition, erucin decreased vascular cell adhesion molecule 1 (VCAM-1), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E-synthase 1 (mPGES-1) expression. Of note, erucin induced eNOS phosphorylation and counteracted LPS-mediated NF-κB nuclear translocation, an effect that was partially abolished in the presence of the eNOS inhibitor L-NAME. Therefore, erucin can control endothelial function through biochemical and genomic positive effects against VI

Anti-Inflammatory Effect of the Natural H2S-Donor Erucin in Vascular Endothelium

Vascular inflammation (VI) represents a pathological condition that progressively affects the integrity and functionality of the vascular wall, thus leading to endothelial dysfunction and the onset of several cardiovascular diseases. Therefore, the research of novel compounds able to prevent VI represents a compelling need. In this study, we tested erucin, the natural isothiocyanate H2S-donor derived from Eruca sativa Mill. (Brassicaceae), in an in vivo mouse model of lipopolysaccharide (LPS)-induced peritonitis, where it significantly reduced the amount of emigrated CD11b positive neutrophils. We then evaluated the anti-inflammatory effects of erucin in LPS-challenged human umbilical vein endothelial cells (HUVECs). The pre-incubation of erucin, before LPS treatment (1, 6, 24 h), significantly preserved cell viability and prevented the increase of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-alpha) levels. Moreover, erucin downregulated endothelial hyperpermeability and reduced the loss of vascular endothelial (VE)-Cadherin levels. In addition, erucin decreased vascular cell adhesion molecule 1 (VCAM-1), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E-synthase 1 (mPGES-1) expression. Of note, erucin induced eNOS phosphorylation and counteracted LPS-mediated NF-kappa B nuclear translocation, an effect that was partially abolished in the presence of the eNOS inhibitor L-NAME. Therefore, erucin can control endothelial function through biochemical and genomic positive effects against VI

Hydrogen sulfide donor AP123 restores endothelial nitric oxide-dependent vascular function in hyperglycemia via a CREB-dependent pathway

Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE−/− mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response