Sex-related differential susceptibility to ponatinib cardiotoxicity and differential modulation of the Notch1 signalling pathway in a murine model.

Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. We assessed mechanisms of sex-related PON-induced cardiotoxicity and identified rescue strategies in a murine model. PON+scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells (%TdT+6.12 ± 0.17), higher percentage of SA-ß-gal-positive senescent cardiac area (%SA-ß-gal 1.41 ± 0.59) and a lower reactivity degree (RD) for the survival marker Bmi1 [Abs (OD) 5000 ± 703] compared to female (%TdT+3.75 ± 0.35; %SA-ß-gal 0.77 ± 0.02; Bmi1 [Abs (OD) 8567 ± 2173]. Proteomics analysis of cardiac tissue showed downstream activation of cell death in PON+siRNA scrambled compared to vehicle or PON+siRNA-Notch1-treated male mice. Upstream analysis showed beta-oestradiol activation, and downstream analysis showed activation of cell survival and inhibition of cell death in PON+scrambled siRNA compared to vehicle or PON+siRNA-Notch1-treated female mice. PON+scrambled siRNA-treated mice also had a downregulation of cardiac actin-more marked in males-and vessel density-more marked in females. Female hearts showed greater cardiac fibrosis than their male counterparts at baseline, with no significant change after PON treatment. PON+siRNA-scrambled mice had less fibrosis than vehicle or PON+siRNA-Notch1-treated mice. The left ventricular systolic dysfunction showed by PON+scrambled siRNA-treated mice (male %EF 28 ± 9; female %EF 36 ± 7) was reversed in both PON+siRNA-Notch1-treated male (%EF 53 ± 9) and female mice (%EF 52 ± 8). We report sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can help to identify biomarkers and potential mechanisms underlying sex-related differences in PON-induced cardiotoxicity.

Empagliflozin reduces the senescence of cardiac stromal cells and improves cardiac function in a murine model of diabetes.

The sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin reduces heart failure in diabetes, but underlying mechanisms remain elusive. We hypothesized that empagliflozin could counteract the senescence of cardiac stromal cells (CSC), the action of which limits cardiac damage and cardiac fibrosis in diabetic-like conditions in vitro and in vivo. CSC were isolated from murine heart biopsies (n = 5) through cardiosphere (CSp) formation and incubated for 3 or 48 hours with 5.5 mmol/L normal glucose (NG), high glucose (12-5 and 30.5 mmol/L, HG) or a hyperosmolar control of mannitol (HM) in the presence or absence of empagliflozin 100 nmol/L. The senescent CSC status was verified by ß-gal staining and expression of the pro-survival marker Akt (pAkt) and the pro-inflammatory marker p38 (p-P38). The cardiac effects of empagliflozin were also studied in vivo by echocardiography and by histology in a murine model of streptozotocin (STZ)-induced diabetes. Compared to NG, incubations with HG and HM significantly reduced the number of CSps, increased the ß-gal-positive CSC and P-p38, while decreasing pAkt, all reversed by empagliflozin (P < .01). Empagliflozin also reversed cardiac dysfunction, cardiac fibrosis and cell senescence in mice with (STZ)-induced diabetes (P < .01). Empagliflozin counteracts the pro-senescence effect of HG and of hyperosmolar stress on CSC, and improves cardiac function via decreasing cardiac fibrosis and senescence in diabetic mice, possibly through SGLT2 off-target effects. These effects may explain empagliflozin unexpected benefits on cardiac function in diabetic patients.

Co-expression of glycosylated aquaporin-1 and transcription factor NFAT5 contributes to aortic stiffness in diabetic and atherosclerosis-prone mice.

Increased stiffness characterizes the early change in the arterial wall with subclinical atherosclerosis. Proteins inducing arterial stiffness in diabetes and hypercholesterolaemia are largely unknown. This study aimed at determining the pattern of protein expression in stiffening aorta of diabetic and hypercholesterolaemic mice. Male Ins2+/Akita mice were crossbred with ApoE-/- (Ins2+/Akita : ApoE-/- ) mice. Relative aortic distension (relD) values were determined by ultrasound analysis and arterial stiffness modulators by immunoblotting. Compared with age- and sex-matched C57/BL6 control mice, the aortas of Ins2+/Akita , ApoE-/- and Ins2+/Akita :ApoE-/- mice showed increased aortic stiffness. The aortas of Ins2+/Akita , ApoE-/- and Ins2+/Akita :ApoE-/- mice showed greater expression of VCAM-1, collagen type III, NADPH oxidase and iNOS, as well as reduced elastin, with increased collagen type III-to-elastin ratio. The aorta of Ins2+/Akita and Ins2+/Akita :ApoE-/- mice showed higher expression of eNOS and cytoskeletal remodelling proteins, such as F-actin and α-smooth muscle actin, in addition to increased glycosylated aquaporin (AQP)-1 and transcription factor NFAT5, which control the expression of genes activated by high glucose-induced hyperosmotic stress. Diabetic and hypercholesterolaemic mice have increased aortic stiffness. The association of AQP1 and NFAT5 co-expression with aortic stiffness in diabetes and hypercholesterolaemia may represent a novel molecular pathway or therapeutic target.

Proteomic analysis of the secretome of adipose tissue-derived murine mesenchymal cells overexpressing telomerase and myocardin.

RATIONALE: Understanding mechanisms of the therapeutic effects of stem/progenitor cells, among which adipose tissue-derived mesenchymal stromal cells (AT-MSCs), has important implications for clinical use. Since the majority of such cells die within days or weeks after transplantation and do not persist in the transplanted organ or tissue, their effects appear to be largely mediated by paracrine signaling pathways, and are enhanced by overexpression of the antisenescent protein telomerase reverse transcriptase (TERT), and the anti-apoptotic transcription factor myocardin (MYOCD). AIM: By a proteomic approach combining two-dimensional gel electrophoresis (2DE) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry, we aimed at analyzing how soluble and vesicular secretomes of aged murine AT-MSCs and their angiogenic function are modulated by the overexpression of TERT and MYOCD. METHODS: We cultured murine mock-transduced AT-MSCs and "rejuvenated" AT-MSCs overexpressing TERT and MYOCD (rTMAT-MSCs) harvested from 1-year-old male C57BL/6 mice. We established proteomes from 3 mock-transduced AT-MSCs and rTMAT-MSCs cultures in serum-free conditions, as well as their corresponding conditioned medium (CM) and exosome-enriched fractions (Exo+). RESULTS AND CONCLUSIONS: Proteomic analysis revealed a 2-fold increase of matrix metalloproteinase-2 (MMP-2) and its inhibitor metalloproteinase inhibitor 2 (TIMP2) in the CM - but not in the Exo + - of rTMAT-MSCs as compared to mock-transduced AT-MSCs. At the functional level, rTMAT-MSCs-CM, and - to a lesser extent - its Exo + fraction, increased tube formation of human vein endothelial cells (HUVECs), which could be blocked by anti-MMP2 and enhanced by anti-TIMP2 antibodies, respectively. Altogether, our results identify MMP2 and its inhibitor TIMP2 as novel candidates by which rTMAT-MSCs can support angiogenesis. Our strategy also illustrates the usefulness of comparative targeted proteomic approach to decipher molecular pathways underlying rTMAT-MSCs properties.

Ponatinib Induces Vascular Toxicity through the Notch-1 Signaling Pathway.

Ponatinib, a third-generation tyrosine kinase inhibitor (TKI), is the only approved TKI that is effective against T315I mutations in patients with chronic myeloid leukemia (CML). Specific activation of Notch signaling in CML cells by ponatinib can be considered as the "on-target effect" on the tumor and represents a therapeutic approach for CML. Nevertheless, ponatinib-induced vascular toxicity remains a serious concern, with underlying mechanisms being poorly understood. We aimed to determine the mechanisms of ponatinib-induced vascular toxicity, defining associated signaling pathways and identifying potential rescue strategies. We exposed human umbilical endothelial cells (HUVECs) to ponatinib or vehicle in the presence or absence of the neutralizing factor anti-Notch-1 antibody for exposure times of 0-72 h. Label-free proteomics and network analysis showed that protein cargo of HUVECs treated with ponatinib triggered apoptosis and inhibited vasculature development. We validated the proteomic data showing the inhibition of matrigel tube formation, an up-regulation of cleaved caspase-3 and a downregulation of phosphorylated AKT and phosphorylated eNOS. We delineated the signaling of ponatinib-induced vascular toxicity, demonstrating that ponatinib inhibits endothelial survival, reduces angiogenesis and induces endothelial senescence and apoptosis via the Notch-1 pathway. Ponatinib induced endothelial toxicity in vitro. Hyperactivation of Notch-1 in the vessels can lead to abnormal vascular development and vascular dysfunction. By hyperactivating Notch-1 in the vessels, ponatinib exerts an "on-target off tumor effect", which leads to deleterious effects and may explain the drug's vasculotoxicity. Selective blockade of Notch-1 prevented ponatinib-induced vascular toxicity.