Primary Ciliary Dyskinesia (PCD) most often arises from loss of the dynein motors that power ciliary beating. Here we show that PF22/DNAAF3, a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes prior to their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in patients from families with situs inversus and defects in assembly of inner and outer dynein arms. Zebrafish dnaaf3 knockdown likewise disrupts dynein arm assembly and ciliary motility, causing PCD phenotypes including hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a null mutant fails to assemble outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests PF22/DNAAF3 acts at a similar stage to other preassembly proteins, PF13/KTU and ODA7/LRRC50, in the dynein preassembly pathway. These results support the existence of a conserved multi-step pathway for cytoplasmic formation of assembly-competent ciliary dynein complexes.

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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.

Vascular dysfunction contributes to the pro-oncogenic tumor microenvironment and impedes the delivery of therapeutics. Normalizing of the tumor vasculature has therefore become a potential therapeutic objective. We previously reported that the secreted glycoprotein, leucine-rich α-2-glycoprotein 1 (LRG1), contributes to the formation of pathogenic neovascularization. Here we show that in mouse models of cancer, Lrg1 is induced in tumor endothelial cells. We demonstrate that the expression of LRG1 impacts on tumor progression as Lrg1 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 significantly 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 (cold) to immune active (hot). LRG1 therefore drives vascular abnormalization and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics.

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.MethodsWe 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.ResultsIncreased 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.

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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. Genomewide 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.

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