Adipose tissue-derived WNT5A regulates vascular redox signaling in obesity via USP17//RAC1-mediated activation of NADPH oxidases

Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2–mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans

Molecular and functional characterization of ANKRD1, a candidate gene for total anomalous pulmonary venous return.

Total Anomalous Pulmonary Venous Return (TAPVR, OMIM %106700) is a rare congenital heart defect in which the pulmonary veins fail to enter the left atrium and drain instead into the right atrium or one of its venous tributaries. Previously, a TAPVR patient bearing a t(10;21) de novo balanced translocation was found in our laboratory and the ANKRD1/CARP gene, mapping on chromosome 10 about 130 Kb proximally to the breakpoint, was defined as a good TAPVR candidate gene. Significantly, we found highly increased ANKRD1 expression levels in lymphoblastoid cell lines derived from both the translocation-bearing proband and a second independent sporadic TAPVR patient, suggesting that disruption of the normal ANKRD1 expression pattern is associated with TAPVR. Moreover, an ANKRD1 non-conservative missense mutation was found in a third sporadic TAPVR patient. Such mutation was subsequently found to confer an increased stability to the ANKRD1 protein in vitro and to affect its function as a repressor of cardiac-specific gene expression. The aim of this doctoral project was to characterize at the molecular and functional level the ANKRD1 gene, in order to provide new evidences for its involvement in TAPVR pathogenesis. First, ANKRD1 function as a negative regulator of cardiac genes was further investigated, and its role as a repressor of Nkx2.5 and Pitx2cmediated activation of the cardiac-specific ANF promoter was demonstrated. Moreover, in situ hybridization assays revealed that the expression pattern of the murine Ankrd1 gene strongly overlaps with that of both Nkx2.5 and Pitx2 genes, supporting both the occurrence of a functional interaction between these genes and a role for ANKRD1 in TAPVR pathogenesis. Co-immunoprecipitation assays added a further support for this hypothesis, by showing a physical interaction between the ANKRD1 and Nkx2.5 proteins. The intracellular pathways involved in ANKRD1 turnover were also investigated. ANKRD1 was shown to behave as a short-lived protein in vivo, with the PEST motif playing a crucial role in its turnover rate. Moreover, intracellular degradation of ANKRD1 was found to be carried out mainly by the ubiquitin proteasome system, through several independent degron elements. These data defined for the first time both the intracellular pathway responsible for ANKRD1 degradation and the functional relevance of the PEST motif in this process. Finally, ANKRD1-overexpressing transgenic mice were generated in the attempt to develop an animal model for TAPVR. Several independent transgenic lines are currently under investigation in order to better define the role of ANKRD1 in heart development

microRNA-34a: a new player in arterial inflammaging: DOI: 10.14800/rd.757

Arterial inflammaging highly contributes to cardiovascular morbidity and mortality. As vascular cells age they become senescent and sustain a chronic low grade sterile inflammation by acquiring a senescence-associated secretory phenotype (SASP). The molecular mechanisms leading to the phenotypic changes affecting endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are also relevant for the pathogenesis of vascular diseases, such as atherosclerosis and hypertension. Therefore, unravelling the etiology of vascular inflammaging becomes of crucial importance. MicroRNAs (miRNAs) are small non-coding negative post-transcriptional regulator that are emerging as promising drug targets. MicroRNA-34a (miR-34a) had been implicated in tissues aging and endothelial and endothelial progenitor cells senescence. Our recent work showed that this miRNA is upregulated in aged mouse aortas as well as in senescent VSMCs. Conversely, its target SIRT1 is downregulated in the same specimens. We also found that miR-34a can inhibit VSMCs proliferation and induce VSMCs senescence, the latter by the direct regulation of SIRT1. Notably, for the first time, we demonstrated that miR-34a is also able to modulate the SASP by inducing the transcriptional expression of a subset of pro-inflammatory factors in a SIRT1-independent manner. These data support a model in which the age-dependent upregulation of miR-34a, by affecting senescence and inflammation of vascular cells, could play a causal role to arterial dysfunctions. Hence, further studies are necessary to unravel miR-34a-dependent mechanisms leading to arterial inflammaging in order to develop an effective strategy to age-related cardiovascular complications

MicroRNA-34a: the bad guy in age-related vascular diseases

The age-related vasculature alteration is the prominent risk factor for vascular diseases (VD), namely, atherosclerosis, abdominal aortic aneurysm, vascular calcification (VC) and pulmonary arterial hypertension (PAH). The chronic sterile low-grade inflammation state, alias inflammaging, characterizes elderly people and participates in VD development. MicroRNA34-a (miR-34a) is emerging as an important mediator of inflammaging and VD. miR-34a increases with aging in vessels and induces senescence and the acquisition of the senescence-associated secretory phenotype (SASP) in vascular smooth muscle (VSMCs) and endothelial (ECs) cells. Similarly, other VD risk factors, including dyslipidemia, hyperglycemia and hypertension, modify miR-34a expression to promote vascular senescence and inflammation. miR-34a upregulation causes endothelial dysfunction by affecting ECs nitric oxide bioavailability, adhesion molecules expression and inflammatory cells recruitment. miR-34a-induced senescence facilitates VSMCs osteoblastic switch and VC development in hyperphosphatemia conditions. Conversely, atherogenic and hypoxic stimuli downregulate miR-34a levels and promote VSMCs proliferation and migration during atherosclerosis and PAH. MiR34a genetic ablation or miR-34a inhibition by anti-miR-34a molecules in different experimental models of VD reduce vascular inflammation, senescence and apoptosis through sirtuin 1 Notch1, and B-cell lymphoma 2 modulation. Notably, pleiotropic drugs, like statins, liraglutide and metformin, affect miR-34a expression. Finally, human studies report that miR-34a levels associate to atherosclerosis and diabetes and correlate with inflammatory factors during aging. Herein, we comprehensively review the current knowledge about miR-34a-dependent molecular and cellular mechanisms activated by VD risk factors and highlight the diagnostic and therapeutic potential of modulating its expression in order to reduce inflammaging and VD burn and extend healthy lifespan

Intracellular ANKRD1 protein levels are regulated by 26S proteasome-mediated degradation

The ANKRD1/CARP gene encodes a muscle-specific protein which has been implicated in transcriptional regulation and myofibrillar assembly. Several features at both the mRNA and protein levels define ANKRD1 as a gene whose expression is tightly regulated, and deregulated expression of this protein has been recently associated to human congenital heart disease. It is therefore crucial to define the intracellular pathways that regulate the ANKRD1 protein's steady-state levels. Here, we show that ANKRD1 is a short-lived protein whose levels are tightly regulated by the 26S proteasome. In addition, a critical role for a putative PEST motif was established, although other degrons within the ANKRD1 protein are likely implicated in the control of its intracellular level