Dapagliflozin protects the kidney in a non-diabetic model of cardiorenal syndrome.

Cardiorenal syndrome encompasses a spectrum of disorders involving heart and kidney dysfunction, and sharing common risk factors, such as hypertension and diabetes. Clinical studies have shown that patients with and without diabetes may benefit from using sodium-glucose cotransporter 2 inhibitors to reduce the risk of heart failure and ameliorate renal endpoints. Because the underlying mechanisms remain elusive, we investigated the effects of dapagliflozin on the progression of renal damage, using a model of non-diabetic cardiorenal disease. Dahl salt-sensitive rats were fed a high-salt diet for five weeks and then randomized to dapagliflozin or vehicle for the following six weeks. After treatment with dapagliflozin, renal function resulted ameliorated as shown by decrease of albuminuria and urine albumin-to-creatinine ratio. Functional benefit was accompanied by a decreased accumulation of extracellular matrix and a reduced number of sclerotic glomeruli. Dapagliflozin significantly reduced expression of inflammatory and endothelial activation markers such as NF-κB and e-selectin. Upregulation of pro-oxidant-releasing NADPH oxidases 2 and 4 as well as downregulation of antioxidant enzymes were also counteracted by drug treatment. Our findings also evidenced the modulation of both classic and non-classic renin-angiotensin-aldosterone system (RAAS), and effects of dapagliflozin on gene expression of ion channels/transporters involved in renal homeostasis. Thus, in a non-diabetic model of cardiorenal syndrome, dapagliflozin provides renal protection by modulating inflammatory response, endothelial activation, fibrosis, oxidative stress, local RAAS and ion channels.

Development of 1,2,3-Triazole-Based Sphingosine Kinase Inhibitors and Their Evaluation as Antiproliferative Agents.

Two series of N-(aryl)-1-(hydroxyalkyl)pyrrolidine-2-carboxamides (2a-2g and 3a-3g) and 1,4-disubstituted 1,2,3-triazoles (5a-5h and 8a-8h) were synthesized. All the compounds, containing a lipophilic tail and a polar headgroup, were evaluated as sphingosine kinase (SphK) inhibitors by assessing their ability to interfere with the acetylcholine (Ach) induced relaxation of aortic rings pre-contracted with phenylephrine. Moreover, their antiproliferative activity was tested on several cell lines expressing both SphK1 and SphK2. Compounds 5h and 8f, identified as the most efficient antiproliferative agents, showed a different selectivity profile, with 8f being selective for SphK1.

Disodium cromoglycate inhibits asthma-like features induced by sphingosine-1-phosphate.

Compelling evidence suggests the involvement of sphingosine-1-phosphate (S1P) in the pathogenesis of asthma. The systemic administration of S1P causes asthma like features in the mouse involving mast cells. In this study we investigated whether disodium cromoglycate (DSCG), administered as a preventative treatment as in human therapy, could affect S1P effects on airways. BALB/c mice, treated with DSCG, received subcutaneous administration of S1P. Bronchi and pulmonary tissues were collected and functional, molecular and cellular studies were performed. DSCG inhibited S1P-induced airway hyper-reactivity as well as pulmonary inflammation. DSCG decreased the recruitment of solely mast cells and B cells in the lung. IgE serum levels, prostaglandin D2, mucus production and IL-13 were also reduced when mice were pretreated with DSCG. S1P induced pulmonary expression of CD23 on T and B cells, that was reversed by DSCG. Conversely, S1P failed to upregulate CD23 in mast cell-deficient Kit W-sh/W-sh mice. In conclusion we have shown that DSCG inhibits S1P-induced asthma like features in the mouse. This beneficial effect is due to a regulatory action on mast cell activity, and in turn to an inhibition of IgE-dependent T and B cells responses.

Glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter 2 inhibitors, anti-diabetic drugs in heart failure and cognitive impairment: potential mechanisms of the protective effects.

Heart failure and cognitive impairment emerge as public health problems that need to be addressed due to the aging global population. The conditions that often coexist are strongly related to advancing age and multimorbidity. Epidemiological evidence indicates that cardiovascular disease and neurodegenerative processes shares similar aspects, in term of prevalence, age distribution, and mortality. Type 2 diabetes increasingly represents a risk factor associated not only to cardiometabolic pathologies but also to neurological conditions. The pathophysiological features of type 2 diabetes and its metabolic complications (hyperglycemia, hyperinsulinemia, and insulin resistance) play a crucial role in the development and progression of both heart failure and cognitive dysfunction. This connection has opened to a potential new strategy, in which new classes of anti-diabetic medications, such as glucagon-like peptide-1 receptor (GLP-1R) agonists and sodium-glucose cotransporter 2 (SGLT2) inhibitors, are able to reduce the overall risk of cardiovascular events and neuronal damage, showing additional protective effects beyond glycemic control. The pleiotropic effects of GLP-1R agonists and SGLT2 inhibitors have been extensively investigated. They exert direct and indirect cardioprotective and neuroprotective actions, by reducing inflammation, oxidative stress, ions overload, and restoring insulin signaling. Nonetheless, the specificity of pathways and their contribution has not been fully elucidated, and this underlines the urgency for more comprehensive research.

Effects of sacubitril-valsartan on aging-related cardiac dysfunction.

Heart failure (HF) remains a huge medical burden worldwide, with aging representing a major risk factor. Here, we report the effects of sacubitril/valsartan, an approved drug for HF with reduced EF, in an experimental model of aging-related HF with preserved ejection fraction (HFpEF). Eighteen-month-old female Fisher 344 rats were treated for 12 weeks with sacubitril/valsartan (60 mg/kg/day) or with valsartan (30 mg/kg/day). Three-month-old rats were used as control. No differential action of sacubitril/valsartan versus valsartan alone, either positive or negative, was observed. The positive effects of both sacubitril/valsartan and valsartan on cardiac hypertrophy was evidenced by a significant reduction of wall thickness and myocyte cross-sectional area. Contrarily, myocardial fibrosis in aging heart was not reduced by any treatment. Doppler echocardiography and left ventricular catheterization evidenced diastolic dysfunction in untreated and treated old rats. In aging rats, both classical and non-classical renin-angiotensin-aldosterone system (RAAS) were modulated. In particular, with respect to untreated animals, both sacubitril/valsartan and valsartan showed a partial restoration of cardioprotective non-classical RAAS. In conclusion, this study evidenced the favorable effects, by both treatments, on age-related cardiac hypertrophy. The attenuation of cardiomyocyte size and hypertrophic response may be linked to a shift towards cardioprotective RAAS signaling. However, diastolic dysfunction and cardiac fibrosis persisted despite of treatment and were accompanied by myocardial inflammation, endothelial activation, and oxidative stress.

Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

AIMS: Growing evidence correlate the accrual of the sphingolipid ceramide in plasma and cardiac tissue with heart failure (HF). Regulation of sphingolipid metabolism in the heart and the pathological impact of its derangement remain poorly understood. Recently, we discovered that Nogo-B, a membrane protein of endoplasmic reticulum, abundant in the vascular wall, down-regulates the sphingolipid de novo biosynthesis via serine palmitoyltransferase (SPT), first and rate liming enzyme, to impact vascular functions and blood pressure. Nogo-A, a splice isoform of Nogo, is transiently expressed in cardiomyocyte (CM) following pressure overload. Cardiac Nogo is up-regulated in dilated and ischaemic cardiomyopathies in animals and humans. However, its biological function in the heart remains unknown. METHODS AND RESULTS: We discovered that Nogo-A is a negative regulator of SPT activity and refrains ceramide de novo biosynthesis in CM exposed to haemodynamic stress, hence limiting ceramide accrual. At 7 days following transverse aortic constriction (TAC), SPT activity was significantly up-regulated in CM lacking Nogo-A and correlated with ceramide accrual, particularly very long-chain ceramides, which are the most abundant in CM, resulting in the suppression of 'beneficial' autophagy. At 3 months post-TAC, mice lacking Nogo-A in CM showed worse pathological cardiac hypertrophy and dysfunction, with ca. 50% mortality rate. CONCLUSION: Mechanistically, Nogo-A refrains ceramides from accrual, therefore preserves the 'beneficial' autophagy, mitochondrial function, and metabolic gene expression, limiting the progression to HF under sustained stress.

Initial Phase of Anthracycline Cardiotoxicity Involves Cardiac Fibroblasts Activation and Metabolic Switch.

The application of doxorubicin (DOX) is hampered by cardiotoxicity, with diastolic dysfunction as the earliest manifestation. Fibrosis leads to impaired relaxation, but the mechanisms that operate shortly after DOX exposure are not clear. We asked whether the activation of cardiac fibroblasts (CFs) anticipates myocardial dysfunction and evaluated the effects of DOX on CF metabolism. CFs were isolated from the hearts of rats after the first injection of DOX. In another experiment, CFs were exposed to DOX in vitro. Cell phenotype and metabolism were determined. Early effects of DOX consisted of diastolic dysfunction and unchanged ejection fraction. Markers of pro-fibrotic remodeling and evidence of CF transformation were present immediately after treatment completion. Oxygen consumption rate and extracellular acidification revealed an increased metabolic activity of CFs and a switch to glycolytic energy production. These effects were consistent in CFs isolated from the hearts of DOX-treated animals and in naïve CFs exposed to DOX in vitro. The metabolic switch was paralleled with the phenotype change of CFs that upregulated markers of myofibroblast differentiation and the activation of pro-fibrotic signaling. In conclusion, the metabolic switch and activation of CFs anticipate DOX-induced damage and represent a novel target in the early phase of anthracycline cardiomyopathy.

Lack of Ecto-5'-Nucleotidase Protects Sensitized Mice against Allergen Challenge.

Ecto-5'-nucleotidase (CD73), the ectoenzyme that together with CD39 is responsible for extracellular ATP hydrolysis and adenosine accumulation, regulates immune/inflammatory processes by controlling innate and acquired immunity cell functions. We previously demonstrated that CD73 is required for the assessment of a controlled allergic sensitization, in mice. Here, we evaluated the response to aerosolized allergen of female-sensitized mice lacking CD73 in comparison with their wild type counterpart. Results obtained show, in mice lacking CD73, the absence of airway hyperreactivity in response to an allergen challenge, paralleled by reduced airway CD23+B cells and IL4+T cells pulmonary accumulation together with reduced mast cells accumulation and degranulation. Our findings indicate CD73 as a potential therapeutic target for allergic asthma.

Exacerbation of Allergic Airway Inflammation in Mice Lacking ECTO-5'-Nucleotidase (CD73).

The airways are a target tissue of type I allergies and atopy is the main etiological factor of bronchial asthma. A predisposition to allergy and individual response to allergens are dependent upon environmental and host factors. Early studies performed to clarify the role of extracellular adenosine in the airways highlighted the importance of adenosine-generating enzymes CD73, together with CD39, as an innate protection system against lung injury. In experimental animals, deletion of CD73 has been associated with immune and autoimmune diseases. Our experiments have been performed to investigate the role of CD73 in the assessment of allergic airway inflammation following sensitization. We found that in CD73-/- mice sensitization, induced by subcutaneous ovalbumin (OVA) administration, increased signs of airway inflammation and atopy developed, characterized by high IgE plasma levels and increased pulmonary cytokines, reduced frequency of lung CD4+CD25+Foxp3+ T cells, but without bronchial hyperreactivity, compared to sensitized wild type mice. Our results provide evidence that the lack of CD73 causes an uncontrolled allergic sensitization, suggesting that CD73 is a key molecule at the interface between innate and adaptive immune response. The knowledge of host immune factors controlling allergic sensitization is of crucial importance and might help to find preventive interventions that could act before an allergy develops.

Functional contribution of sphingosine-1-phosphate to airway pathology in cigarette smoke-exposed mice.

BACKGROUND AND PURPOSE: A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH: C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS: Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS: S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.

Pressure overload leads to coronary plaque formation, progression, and myocardial events in ApoE-/- mice.

Hypercholesterolemia and hypertension are two major risk factors for coronary artery diseases, which remain the major cause of mortality in the industrialized world. Current animal models of atherosclerosis do not recapitulate coronary plaque disruption, thrombosis, and myocardial infarction occurring in humans. Recently, we demonstrated that exposure of the heart to high pressure, by transverse aortic constriction (TAC), induced coronary lesions in ApoE-/- mice on chow diet. The aim of this study was to characterize the magnitude and location of coronary lesions in ApoE-/- mice after TAC and to assess the susceptibility of coronary plaque to disruption, leading to myocardial events. Here, we describe a reliable pathological condition in mice characterized by the development of coronary lesions and its progression, leading to myocardial infarction; this model better recapitulates human disease. Following TAC surgery, about 90% of ApoE-/- mice developed coronary lesions, especially in the left anterior descending artery, with 59% of the mice manifesting a different magnitude of LAD stenosis. Myocardial events, identified in 74% of the mice, were mainly due to coronary plaque thrombosis and occlusion. That TAC-induced development and progression of coronary lesions in ApoE-/- mice, leading to myocardial events, represents a potentially novel and important tool to investigate the development of coronary lesions and its sequelae in a setting that better resemble human conditions.

Toll-Like Receptor 4 Is Essential for the Expression of Sphingosine-1-Phosphate-Dependent Asthma-Like Disease in Mice.

Sphingosine-1-phosphate (S1P) levels significantly increase in bronchoalveolar lavage (BAL) of asthmatic patients following segmental allergen challenge and this increase well correlates with pulmonary inflammation. Epidemiological, genetic, clinical, and experimental data indicate a potential for the toll-like receptor 4 (TLR4) to initiate and exacerbate allergic airway diseases. The aim of this study was to evaluate the contribution of TLR4 in S1P-dependent asthma-like disease in mice. BALB/c or TLR4 defective (C3H/HeJ) mice received S1P (10 ng/mouse), LPS (0.1 µg/mouse) or S1P + LPS. Furthermore, S1P-treated BALB/c mice were injected with the purified rabbit anti-TLR4 antibody (10 µg/mouse). S1P administration induced airway hyperreactivity and pulmonary inflammation associated to an increase in the percentage of dendritic cells (DCs) and macrophages into the lung of BALB/c mice. These effects were coupled to a reduction of DCs in the mediastinic lymph node. All these S1P-mediated effects were absent in TLR4 defective mice or reversed by treatment with a purified rabbit anti-TLR4 antibody. Confocal analysis of pulmonary sections showed a significant increase in TLR4+ cells and a similar presence of S1P1 and TLR4 following S1P challenge. Accordingly, the immunoprecipitation evidenced an increased S1P1/TLR4 interaction. In conclusion, our findings suggest that a functional interaction between S1P1 and TLR4 leads to an enhanced allergic inflammatory response. Thus, S1P pathway contributes to the sentinel role played by innate immunity providing new targets for prevention and treatment of allergic airway diseases.