The disruptive role of LRG1 on the vasculature and perivascular microenvironment.

The establishment of new blood vessels, and their subsequent stabilization, is a critical process that facilitates tissue growth and organ development. Once established, vessels need to diversify to meet the specific needs of the local tissue and to maintain homeostasis. These processes are tightly regulated and fundamental to normal vessel and tissue function. The mechanisms that orchestrate angiogenesis and vessel maturation have been widely studied, with signaling crosstalk between endothelium and perivascular cells being identified as an essential component. In disease, however, new vessels develop abnormally, and existing vessels lose their specialization and function, which invariably contributes to disease progression. Despite considerable research into the vasculopathic mechanisms in disease, our knowledge remains incomplete. Accordingly, the identification of angiocrine and angiopathic molecules secreted by cells within the vascular microenvironment, and their effect on vessel behaviour, remains a major research objective. Over the last decade the secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1), has emerged as a significant vasculopathic molecule, stimulating defective angiogenesis, and destabilizing the existing vasculature mainly, but not uniquely, by altering both canonical and non-canonical TGF-ß signaling in a highly cell and context dependent manner. Whilst LRG1 does not possess any overt homeostatic role in vessel development and maintenance, growing evidence provides a compelling case for LRG1 playing a pleiotropic role in disrupting the vasculature in many disease settings. Thus, LRG1 has now been reported to damage vessels in various disorders including cancer, diabetes, chronic kidney disease, ocular disease, and lung disease and the signaling processes that drive this dysfunction are being defined. Moreover, therapeutic targeting of LRG1 has been widely proposed to re-establish a quiescent endothelium and normalized vasculature. In this review, we consider the current status of our understanding of the role of LRG1 in vascular pathology, and its potential as a therapeutic target.

NKX2-5 regulates vessel remodeling 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 remodeling in vivo. NKX2-5 is upregulated in scleroderma 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 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 remodeling and halted the progression to hypertension in a mouse chronic hypoxia 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 TGF-ß and further enhanced by hypoxia. The effect of TGF-ß 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 remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway.

Leucine-rich α-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that under physiological conditions is produced predominantly by the liver. In disease, its local induction promotes pathogenic neovascularisation while its inhibition leads to reduced dysfunctional angiogenesis. Here we examine the role of interleukin-6 (IL-6) in defective angiogenesis mediated by LRG1. IL-6 treatment induced LRG1 expression in endothelial cells and ex vivo angiogenesis cultures and promoted vascular growth with reduced mural cell coverage. In Lrg1-/- explants, however, IL-6 failed to stimulate angiogenesis and vessels exhibited improved mural cell coverage. IL-6 activated LRG1 transcription through the phosphorylation and binding of STAT3 to a conserved consensus site in the LRG1 promoter, the deletion of which abolished activation. Blocking IL-6 signalling in human lung endothelial cells, using the anti-IL6 receptor antibody Tocilizumab, significantly reduced LRG1 expression. Our data demonstrate that IL-6, through STAT3 phosphorylation, activates LRG1 transcription resulting in vascular destabilisation. This observation is especially timely in light of the potential role of IL-6 in COVID-19 patients with severe pulmonary microvascular complications, where targeting IL-6 has been beneficial. However, our data suggest that a therapy directed towards blocking the downstream angiopathic effector molecule LRG1 may be of greater utility.

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. FUNDING: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

"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 TGFß 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.

Molecular Basis for Dysregulated Activation of NKX2-5 in the Vascular Remodeling of Systemic Sclerosis.

OBJECTIVE: NKX2-5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down-regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2-5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)-associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications. METHODS: We explored NKX2-5 expression in biopsy samples from patients with SSc-associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease-associated putative functional single-nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein-DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses. RESULTS: Increased NKX2-5 expression in biopsy samples from patients with SSc-associated PH was localized to remodeled vessels and PASMCs. Meta-analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease-associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA-6, c-Jun, and myocyte-specific enhancer factor 2C. We also characterized an activator/coactivator transcription-enhancer factor domain 1 (TEAD1)/Yes-associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5. CONCLUSION: NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2-5 activation in cardiovascular disease characterized by vascular remodeling.

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia.

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

Genetic variation in the visfatin (PBEF1/NAMPT) gene and type 2 diabetes in the Greek population.

Visfatin (NAMPT formerly known as PBEF1) is an adipokine that is strongly expressed in visceral fat and has caused much debate among researchers, regarding its involvement in glucose homeostasis and insulin resistance. It was initially isolated from bone marrow cells, and its involvement in inflammatory procedures such as sepsis and acute lung inflammation is now evident. Several studies have also reported an association of plasma visfatin levels with obesity. We undertook an evaluation of the involvement of the NAMPT gene in the development of type 2 diabetes (T2DM) in the Greek population. We studied 178 patients with T2DM and 177 controls that were matched for sex, age and body mass index. We genotyped three tagging SNPs selected from the HapMap II CEPH European population as reference for the Greek population. These three SNPs tag another 12 SNPs over the entire NAMPT gene with a mean r(2) of 0.92. No indications of association with disease status were found with any of the tested variants or the inferred haplotypes. Results were also negative when the quantitative traits of weight and BMI were tested. Although our study covers common variants across the NAMPT gene, the possible involvement of rare variants in T2DM etiology cannot be ruled out and will require the investigation of very large numbers of cases and controls.