BACKGROUND The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. METHODS We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. RESULTS We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10−14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. CONCLUSIONS We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)
Inducible nitric oxide synthase (iNOS) is a hallmark of chronic inflammation which is also overexpressed in melanoma and other cancers. While iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b+Gr1+ MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with VEGF-depleting antibody and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, where L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS “knockout” mice, suggesting L-NIL acts primarily on tumor-rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend towards increased tumor-infiltrating CD8+ T cells was also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.
To explore the mechanisms by which NO elicits endothelial cell (EC) migration we used murine and bovine aortic ECs in an in vitro wound-healing model. We found that exogenous or endogenous NO stimulated EC migration. Moreover, migration was significantly delayed in ECs derived from endothelial NO synthase-deficient mice compared with WT murine aortic EC. To assess the contribution of matrix metalloproteinase (MMP)-13 to NO-mediated EC migration, we used RNA interference to silence MMP-13 expression in ECs. Migration was delayed in cells in which MMP-13 was silenced. In untreated cells MMP-13 was localized to caveolae, forming a complex with caveolin-1. Stimulation with NO disrupted this complex and significantly increased extracellular MMP-13 abundance, leading to collagen breakdown. Our findings show that MMP-13 is an important effector of NO-activated endothelial migration.angiogenesis ͉ endothelium ͉ vasular remodeling W ound healing is an orchestrated cascade of enzymatic activities that converge toward damage repair. Wound healing involves inflammation and angiogenesis and is tightly regulated by cytokines (1). Vascular endothelial growth factor (VEGF) is a critical cytokine involved in angiogenesis, and nitric oxide (NO) is a downstream effector (2, 3). VEGF increases NO levels in endothelial cells (ECs) by activating endothelial NO synthase (eNOS͞NOS3) (4-7). Recently, the role of eNOS in EC migration has been demonstrated in vivo and in vitro (8, 9) but the precise mechanism by which NO regulates migration is unknown.ECs migrate as a result of an injury and during angiogenesis from preexisting vessels. The process is tightly regulated by matrix turnover, in which matrix metalloproteinases (MMPs) play a pivotal role (10-12). We have previously reported that NO induces MMP-13 expression and activity in bovine aortic ECs (BAECs) (13,14).MMPs are extracellular matrix-degrading endopeptidases. MMP expression and activity can be found in physiological and pathological situations, such as tissue development, atherosclerosis, ovarian function, arthritis, osteoarthritis, cancer, angiogenesis, and wound healing (15). MMP-13 was initially discovered in mammalian cell carcinomas and is also expressed by several cell types, including endothelium (13,(16)(17)(18).Here, we present evidence that MMP-13 is a downstream effector of NO-activated EC movement. We have developed a wound-healing model in BAECs and aortic cells from eNOS WT and eNOS-deficient mice. We show that aortic ECs lacking MMP-13 experience delayed migration, and aortic ECs from eNOS null mice present delayed cell migration and a significant decrease in MMP-13 expression. In addition, we present data showing that MMP-13 exists in association with caveolin-1 in resting cells, and that this complex is disrupted in the presence of NO, leading to the secretion of MMP-13 to the extracellular media. We postulate that NO induces EC movement in part via the disruption of the MMP-13͞caveolin-1 complex, which in turn releases the secretion of active MMP-13 to the ...
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