Cardiomyocyte calcineurin is required for the onset and progression of cardiac hypertrophy and fibrosis in adult mice

Previous studies have demonstrated that activation of calcineurin induces pathological cardiac hypertrophy. In these studies, loss-of-function was mostly achieved by systemic administration of the calcineurin inhibitor cyclosporin A. The lack of conditional knockout models for calcineurin function has impeded progress toward defining the role of this protein during the onset and the development of cardiac hypertrophy in adults. Here, we exploited a mouse model of cardiac hypertrophy based on the infusion of a hypertensive dose of angiotensin II (AngII) to model the role of calcineurin in cardiac hypertrophy in adulthood. AngII-induced cardiac hypertrophy in adult mice was reduced by treatment with cyclosporin A, without affecting the associated increase in blood pressure, and also by induction of calcineurin deletion in adult mouse cardiomyocytes, indicating that cardiomyocyte calcineurin is required for AngII-induced cardiac hypertrophy. Surprisingly, cardiac-specific delection of calcineurin, but not treatment of mice with cyclosporin A, significantly reduced AngII-induced cardiac fibrosis and apoptosis. Analysis of pro-fibrotic genes revealed that AngII-induced expression of Tgfβ-family members and Lox was not inhibited by cyclosporin A but was markedly reduced by cardiac-specific calcineurin deletion. These results show that AngII induces a direct, calcineurin-dependent pro-hypertrophic effect in cardiomyocytes, as well as a systemic hypertensive effect that is independent of calcineurin activity

Selective inhibition of plasma membrane calcium ATPase 4 improves angiogenesis and vascular reperfusion

Aims Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis. Methods and results We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs. Conclusions Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors

Aortic disease in Marfan syndrome is caused by overactivation of sGC-PRKG signaling by NO

AbstractThoracic aortic aneurysm, as occurs in Marfan syndrome, is generally asymptomatic until dissection or rupture, requiring surgical intervention as the only available treatment. Here, we show that nitric oxide (NO) signaling dysregulates actin cytoskeleton dynamics in Marfan Syndrome smooth muscle cells and that NO-donors induce Marfan-like aortopathy in wild-type mice, indicating that a marked increase in NO suffices to induce aortopathy. Levels of nitrated proteins are higher in plasma from Marfan patients and mice and in aortic tissue from Marfan mice than in control samples, indicating elevated circulating and tissue NO. Soluble guanylate cyclase and cGMP-dependent protein kinase are both activated in Marfan patients and mice and in wild-type mice treated with NO-donors, as shown by increased plasma cGMP and pVASP-S239 staining in aortic tissue. Marfan aortopathy in mice is reverted by pharmacological inhibition of soluble guanylate cyclase and cGMP-dependent protein kinase and lentiviral-mediated Prkg1 silencing. These findings identify potential biomarkers for monitoring Marfan Syndrome in patients and urge evaluation of cGMP-dependent protein kinase and soluble guanylate cyclase as therapeutic targets.</jats:p

Papel de las actividades MAPK (ERK y JNK) en procesos de diferenciación y apoptosis en células de neuroblastoma N2A

Tesis doctoral inédita de la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 20-07-200

Opposite effects of the Hsp90 inhibitor Geldanamycin: induction of apoptosis in PC12, and differentiation in N2A cells

The inhibitor of the Hsp90 chaperone Geldanamycin has been reported to have several cellular effects, such as inhibition of v-src activity or destabilization of Raf-1 among others. We show now that Geldanamycin treatment induces different phenotypes in different cell lines. In PC12 cells, it triggers apoptosis, whereas in the murine neuroblastoma N2A, it induces differentiation with neurite outgrowth. Geldanamycin effects cannot be mimicked by inhibition of the c-src protein tyrosine kinases, and nerve growth factor does not protect PC12 cells from apoptosis. Mitogen-activated protein kinase activities ERK and JNK are activated differently according to cell type: in PC12 cells JNK is activated, and its inhibition abolishes apoptosis, but not ERK; in N2A cells, both ERK and JNK are activated, but with peak activities at different times.This work was supported by Grant SAF97-0163 from CICYT and 08.9/0005/1998 from Comunidad de Madrid, Spain.Peer Reviewe

Versican accumulation drives Nos2 induction and aortic disease in Marfan syndrome via Akt activation.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening condition associated with Marfan syndrome (MFS), a disease caused by fibrillin-1 gene mutations. While various conditions causing TAAD exhibit aortic accumulation of the proteoglycans versican (Vcan) and aggrecan (Acan), it is unclear whether these ECM proteins are involved in aortic disease. Here, we find that Vcan, but not Acan, accumulated in Fbn1C1041G/+ aortas, a mouse model of MFS. Vcan haploinsufficiency protected MFS mice against aortic dilation, and its silencing reverted aortic disease by reducing Nos2 protein expression. Our results suggest that Acan is not an essential contributor to MFS aortopathy. We further demonstrate that Vcan triggers Akt activation and that pharmacological Akt pathway inhibition rapidly regresses aortic dilation and Nos2 expression in MFS mice. Analysis of aortic tissue from MFS human patients revealed accumulation of VCAN and elevated pAKT-S473 staining. Together, these findings reveal that Vcan plays a causative role in MFS aortic disease in vivo by inducing Nos2 via Akt activation and identify Akt signaling pathway components as candidate therapeutic targets.We thank S. Bartlett for English language editing; V Labrador for advice on confocal imaging and immunofluorescent experiments; the CNIC Facilities of histology, microscopy, and advanced imaging; and AI Torralbo for excellent technical support and advice. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), the CBMSO is supported by Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid. CBMSO and CNIC are Severo Ochoa Centers of Excellence (grants CEX2021-001154-S and CEX2020-001041-S, respectively) funded by MICIN/AEI/10.13039/ 501100011033. The project leading to these results has received funding from “La Caixa” Banking Foundation under project codes HR18-00068 (to MRC and JMR); Spanish Ministerio de Ciencia e Innovación grant RTI2018-099246-BI00 (MICIU/AEI/FEDER, UE) to JMR, and grants PID2020-115217RB-100 and PID2021-122388OB-100 to MRC and JMR, respectively, funded by MCIN/AEI/ 10.13039/501100011033; Instituto de Salud Carlos III (CIBER-CV CB16/11/ 00264 and CB16/11/00479; and grants PI17/00381 to GT-T and PI21/00084 (co-funded by Fondo Europeo de Desarrollo Regional (FEDER)) to JFN); Fundacio La Marato TV3 (20151330 to JMR); Instituto de Investigación Sanitaria Marqués de Valdecilla (IDIVAL) (INNVAL 21/24) to JFN; The Marfan Foundation USA Faculty grant 2017 MRF/1701 (to JMR); Fundación MERCKFundación Española de Enfermedades Raras 2022 and V-Ayudas “Muevete por ́ los que no pueden 2021” (to JO); and Spanish Ministerio de Ciencia e Innovación contracts FPI (BES-2016-077649) to MJR-R; Sara Borrell (CD18/ 00028) and Juan de la Cierva (IJC2020-044581-I) to MT; Ramón y Cajal (RYC2021-033343-I) to JO; and FPU (20/04814) to IA-R.S

Plasma membrane calcium ATPase isoform 4 inhibits vascular endothelial growth factor-mediated angiogenesis through interaction with calcineurin.

Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far. Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF. Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis