Sign Inversion in Photopharmacology: Incorporation of Cyclic Azobenzenes in Photoswitchable Potassium Channel Blockers and Openers.

Photopharmacology relies on ligands that change their pharmacodynamics upon photoisomerization. Many of these ligands are azobenzenes that are thermodynamically more stable in their elongated trans-configuration. Often, they are biologically active in this form and lose activity upon irradiation and photoisomerization to their cis-isomer. Recently, cyclic azobenzenes, so-called diazocines, have emerged, which are thermodynamically more stable in their bent cis-form. Incorporation of these switches into a variety of photopharmaceuticals could convert dark-active ligands into dark-inactive ligands, which is preferred in most biological applications. This "pharmacological sign-inversion" is demonstrated for a photochromic blocker of voltage-gated potassium channels, termed CAL, and a photochromic opener of G protein-coupled inwardly rectifying potassium (GIRK) channels, termed CLOGO.

Perivascular CD73+ cells attenuate inflammation and interstitial fibrosis in the kidney microenvironment.

Progressive tubulointerstitial fibrosis may occur after acute kidney injury due to persistent inflammation. Purinergic signaling by 5'-ectonucleotidase, CD73, an enzyme that converts AMP to adenosine on the extracellular surface, can suppress inflammation. The role of CD73 in progressive kidney fibrosis has not been elucidated. We evaluated the effect of deletion of CD73 from kidney perivascular cells (including pericytes and/or fibroblasts of the Foxd1+ lineage) on fibrosis. Perivascular cell expression of CD73 was necessary to suppress inflammation and prevent kidney fibrosis in Foxd1CreCD73fl/fl mice evaluated 14 days after unilateral ischemia-reperfusion injury or folic acid treatment (250 mg/kg). Kidneys of Foxd1CreCD73fl/fl mice had greater collagen deposition, expression of proinflammatory markers (including various macrophage markers), and platelet-derived growth factor recepetor-ß immunoreactivity than CD73fl/fl mice. Kidney dysfunction and fibrosis were rescued by administration of soluble CD73 or by macrophage deletion. Isolated CD73-/- kidney pericytes displayed an activated phenotype (increased proliferation and α-smooth muscle actin mRNA expression) compared with wild-type controls. In conclusion, CD73 in perivascular cells may act to suppress myofibroblast transformation and influence macrophages to promote a wound healing response. These results suggest that the purinergic signaling pathway in the kidney interstitial microenvironment orchestrates perivascular cells and macrophages to suppress inflammation and prevent progressive fibrosis.

CD73 on T Cells Orchestrates Cardiac Wound Healing After Myocardial Infarction by Purinergic Metabolic Reprogramming.

BACKGROUND: T cells are required for proper healing after myocardial infarction. The mechanism of their beneficial action, however, is unknown. The proinflammatory danger signal ATP, released from damaged cells, is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine. Here, we investigate the contribution of CD73-derived adenosine produced by T cells to cardiac remodeling after ischemia/reperfusion and define its mechanism of action. METHODS: Myocardial ischemia (50 minutes followed by reperfusion) was induced in global CD73-/- and CD4-CD73-/- mice. Tissue injury, T-cell purinergic signaling, cytokines, and cardiac function (magnetic resonance tomography at 9.4 T over 4 weeks) were analyzed. RESULTS: Changes in functional parameters of CD4-CD73-/- mice were identical to those in global CD73 knockouts (KOs). T cells infiltrating the injured heart significantly upregulated at the gene (quantitative polymerase chain reaction) and protein (enzymatic activity) levels critical transporters and enzymes (connexin43, connexin37, pannexin-1, equilibrative nucleoside transporter 1, CD39, CD73, ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3, CD157, CD38) for the accelerated release and hydrolysis of ATP, cAMP, AMP, and NAD to adenosine. It is surprising that a lack of CD39 on T cells (from CD39-/- mice) did not alter ATP hydrolysis and very likely involves pyrophosphatases (ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3). Circulating T cells predominantly expressed A2a receptor (A2aR) transcripts. After myocardial infarction, A2b receptor (A2bR) transcription was induced in both T cells and myeloid cells in the heart. Thus, A2aR and A2bR signaling may contribute to myocardial responses after myocardial infarction. In the case of T cells, this was associated with an accelerated secretion of proinflammatory and profibrotic cytokines (interleukin-2, interferon-γ, and interleukin-17) when CD73 was lacking. Cytokine production by T cells from peripheral lymph nodes was inhibited by A2aR activation (CGS-21680). The A2bR agonist BAY 60-6583 showed off-target effects. The adenosine receptor agonist NECA inhibited interferon-γ and stimulated interleukin-6 production, each of which was antagonized by a specific A2bR antagonist (PSB-603). CONCLUSIONS: This work demonstrates that CD73 on T cells plays a crucial role in the cardiac wound healing process after myocardial infarction. The underlying mechanism involves a profound increase in the hydrolysis of ATP/NAD and AMP, resulting primarily from the upregulation of pyrophosphatases and CD73. We also define A2bR/A2aR-mediated autacoid feedback inhibition of proinflammatory/profibrotic cytokines by T cell-derived CD73.

Proximal Tubule CD73 Is Critical in Renal Ischemia-Reperfusion Injury Protection.

CD73-derived adenosine plays an anti-inflammatory role in various organs. However, its role in renal ischemia-reperfusion injury (IRI) is controversial. We targeted CD73 mutant mice to determine the function of CD73 expressed by various renal cell types under mild IRI conditions. Mice with CD73 deletion in proximal tubules exhibited exacerbated IRI, comparable with that of CD73-/- mice compared with WT mice. Mice with CD73 deletions in other cell types, including cortical type 1 fibroblast-like cells, mesangial cells, macrophages, and dendritic cells, showed small or no increases in injury above control mice when subjected to threshold levels of ischemia. Results from adoptive transfer experiments between WT and CD73-/- mice and pharmacologic studies modulating enzymatic activity of CD73 and extracellular adenosine levels supported a critical role of adenosine generated by proximal tubule CD73 expression in abrogating IRI. Renal adenosine levels were lower before and after ischemia in CD73-deficient mice. However, reduction in total acid-extractable renal adenosine levels was inadequate to explain the marked difference in kidney injury in these CD73-deficient mice. Furthermore, CD73 inhibition and enzyme replacement studies showed no change in total kidney adenosine levels in treated mice compared with vehicle-treated controls. Protection from IRI in neutrophil-depleted WT recipients was sustained by repopulation with bone marrow neutrophils from WT mice but not by those lacking adenosine 2a receptors (from Adora2a-/- mice). These data support the thesis that local adenosine generated by cells at the injury site is critical for protection from IRI through bone marrow-derived adenosine 2a receptors.

Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation.

The WT1 gene encodes a zinc finger transcription factor important for normal kidney development. WT1 is a suppressor for Wilms tumour development and an oncogene for diverse malignant tumours. We recently established cell lines from primary Wilms tumours with different WT1 mutations. To investigate the function of mutant WT1 proteins, we performed WT1 knockdown experiments in cell lines with a frameshift/extension (p.V432fsX87 = Wilms3) and a stop mutation (p.P362X = Wilms2) of WT1, followed by genome-wide gene expression analysis. We also expressed wild-type and mutant WT1 proteins in human mesenchymal stem cells and established gene expression profiles. A detailed analysis of gene expression data enabled us to classify the WT1 mutations as gain-of-function mutations. The mutant WT1(Wilms2) and WT1(Wilms3) proteins acquired an ability to modulate the expression of a highly significant number of genes from the G2/M phase of the cell cycle, and WT1 knockdown experiments showed that they are required for Wilms tumour cell proliferation. p53 negatively regulates the activity of a large number of these genes that are also part of a core proliferation cluster in diverse human cancers. Our data strongly suggest that mutant WT1 proteins facilitate expression of these cell cycle genes by antagonizing transcriptional repression mediated by p53. We show that mutant WT1 can physically interact with p53. Together the findings show for the first time that mutant WT1 proteins have a gain-of-function and act as oncogenes for Wilms tumour development by regulating Wilms tumour cell proliferation.