Annexin A3 is necessary for parallel artery-vein alignment in the mouse retina.

BACKGROUND: Annexin A3 (Anxa3) is a member of the calcium-regulated, cell membrane-binding family of annexin proteins. We previously confirmed that Anxa3 is expressed in the endothelial lineage in vertebrates and that loss of anxa3 in Xenopus laevis leads to embryonic blood vessel defects. However, the biological function of Anxa3 in mammals is completely unknown. In order to investigate Anxa3 vascular function in mammals, we generated an endothelial cell-specific Anxa3 conditional knockout mouse model (Anxa3f/f ;Tie2-Cre). RESULTS: Anxa3f/f ;Tie2-Cre mice are born at Mendelian ratios and display morphologically normal blood vessels during development. However, loss of Anxa3 leads to artery-vein (AV) misalignment characterized by atypical AV crossovers in the postnatal and adult retina. CONCLUSIONS: Anxa3 is not essential for embryonic blood vessel formation but is required for proper parallel AV alignment in the murine retina. AV crossovers associated with Anxa3f/f ;Tie2-Cre mice are similar to AV intersections observed in patients with branch retinal vein occlusion (BRVO), although we did not observe occluded vessels. This new Anxa3 mouse model may provide a basis for understanding AV crossover formation associated with BRVO.

Endothelial cell polarity and extracellular matrix composition require functional ATP6AP2 during developmental and pathological angiogenesis.

The (Pro)renin receptor ([P]RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell-specific (EC-specific) Atp6ap2-KO mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity, and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2-deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration, and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the ERK pathway. Furthermore, Atp6ap2-deficient retinas exhibited reduced revascularization in an oxygen-induced retinopathy model. Collectively, our results demonstrate a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.

Discovery of Bifunctional Oncogenic Target Inhibitors against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3-Kinase (PI3K).

The synthesis of a series of single entity, bifunctional MEK1/PI3K inhibitors achieved by covalent linking of structural analogs of the ATP-competitive PI3K inhibitor ZSTK474 and the ATP-noncompetitive MEK inhibitor PD0325901 is described. Inhibitors displayed potent in vitro inhibition of MEK1 (0.015 < IC50 (nM) < 56.7) and PI3K (54 < IC50 (nM) < 341) in enzymatic inhibition assays. Concurrent MEK1 and PI3K inhibition was demonstrated with inhibitors 9 and 14 in two tumor cell lines (A549, D54). Inhibitors produced dose-dependent decreased cell viability similar to the combined administration of equivalent doses of ZSTK474 and PD0325901. In vivo efficacy of 14 following oral administration was demonstrated in D54 glioma and A549 lung tumor bearing mice. Compound 14 showed a 95% and 67% inhibition of tumor ERK1/2 and Akt phosphorylation, respectively, at 2 h postadministration by Western blot analysis, confirming the bioavailability and efficacy of this bifunctional inhibitor strategy toward combined MEK1/PI3K inhibition.