Regulation of NKX2-5 in blood vessels

NKX2-5 is a transcription factor required for the formation of the heart and vessels during development. Postnatal expression is significantly downregulated, and then re-activated in diseased conditions characterised by vascular remodelling. However, the mechanisms regulating NKX2-5 activation in diseased vessels remain unknown. The aim of this thesis is to identify these mechanisms and provide information on how the gene contributes to cardiovascular pathologies, such as sclerodermaassociated pulmonary hypertension. A case-control genetic association study was performed in two independent cohorts of scleroderma patients. Associated SNPs located in the NKX2-5 genomic region were cloned into reporter vectors, and transcriptional activity was assessed by reporter-gene assays. Associated SNPs were further evaluated through proteinDNA binding assays, chromatin immunoprecipitation and RNA silencing. Signalling mechanisms activating NKX2-5 expression were investigated in vascular endothelial and smooth muscle cells using a panel of selective inhibitors. Meta-analysis across the two independent cohorts revealed that rs3131917 was associated with scleroderma. Rs3132139, downstream of NKX2-5, was significantly associated with pulmonary hypertension in both cohorts. The region containing rs3132139 and rs3131917 was shown to be a novel functional enhancer, which increased NKX2-5 transcriptional activity through the binding of GATA6, c-JUN, and MEF-2c. An activator TEAD/YAP1 complex was shown to bind at rs3095870, another functional SNP upstream of NKX2-5 transcription start site, which showed marginal association with scleroderma. Signalling mechanisms, involving TGF-β, ERK5, AKT and hypoxia, stimulated NKX2-5 expression during phenotypic modulation of vascular endothelial and smooth muscle cells. Overall, the data showed that NKX2-5 is genetically associated with scleroderma and pulmonary hypertension. Functional evidence revealed a regulatory mechanism, activated by TGF-β, which results in NKX2-5 transcription in human vascular smooth muscle cells through the interaction of an upstream promoter and a novel downstream enhancer. These regulatory mechanisms can act as a model for NKX2-5 activation in cardiovascular disease characterised by vascular remodellin

Epigenome-wide methylation profile of chronic kidney disease-derived arterial DNA uncovers novel pathways in disease-associated cardiovascular pathology

Chronic kidney disease (CKD) related cardiovascular disease (CVD) is characterized by vascular remodelling with well-established structural and functional changes in the vascular wall such as arterial stiffness, matrix deposition, and calcification. These phenotypic changes resemble pathology seen in ageing, and are likely to be mediated by sustained alterations in gene expression, which may be caused by epigenetic changes such as tissue-specific DNA methylation. We aimed to investigate tissue specific changes in DNA methylation that occur in CKD-related CVD. Genome-wide DNA methylation changes were examined in bisulphite converted genomic DNA isolated from the vascular media of CKD and healthy arteries. Methylation-specific PCR was used to validate the array data, and the association between DNA methylation and gene and protein expression was examined. The DNA methylation age was compared to the chronological age in both cases and controls. Three hundred and nineteen differentially methylated regions (DMR) were identified spread across the genome. Pathway analysis revealed that DMRs associated with genes were involved in embryonic and vascular development, and signalling pathways such as TGFβ and FGF. Expression of top differentially methylated gene HOXA5 showed a significant negative correlation with DNA methylation. Interestingly, DNA methylation age and chronological age were highly correlated, but there was no evidence of accelerated age-related DNA methylation in the arteries of CKD patients. In conclusion, we demonstrated that differential DNA methylation in the arterial tissue of CKD patients represents a potential mediator of arterial pathology and may be used to uncover novel pathways in the genesis of CKD-associated complications

NKX2-5 regulates vessel remodelling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodelling in vivo. NKX2-5 is up-regulated in scleroderma (SSc) patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells, halted vascular smooth muscle proliferation and migration, enhanced contractility and blocked the expression of the extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodelling and halted the progression to hypertension in the mouse chronic hypoxia mouse model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGFβ and further enhanced by hypoxia. The effect of TGFβ was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodelling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics

Understanding bifurcation of slow versus fast cyber-attackers

Anecdotally, the distinction between fast “Smash-and-Grab” cyber-attacks on the one hand and slow attacks or “Advanced Persistent Threats” on the other hand is well known. In this article, we provide an explanation for this phenomenon as the outcome of an optimization from the perspective of the attacker. To this end, we model attacks as an interaction between an attacker and a defender and infer the two types of behavior observed based on justifiable assumptions on key variables such as detection thresholds. On the basis of our analysis, it follows that bi-modal detection capabilities are optimal