Differences in activation of intracellular signaling in primary human retinal endothelial cells between isoforms of VEGFA 165.

Purpose: There are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). Methods: To determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR. Results: Activation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA 165 a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA 165 b. Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA 165 b was only half of the maximum activation from VEGFA 165 a. At a lower intermediate dose, where VEGFA 165 a activated intracellular signaling stronger than VEGFA 165 b, the changes in VEGFA target gene expression were generally greater with VEGFA 165 a. Conclusions: In primary HRMECs, VEGFA 165 a could maximally activate MAPK and AKT at lower concentrations where VEGFA 165 b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA 165 a and VEGFA 165 b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.

Cloning and characterization of glycogen branching and debranching enzymes from the parasitic protist Trichomonas vaginalis.

The protist Trichomonas vaginalis is an obligate parasite of humans and the causative agent of trichomoniasis, a common sexually transmitted infection. The organism has long been known to accumulate glycogen, a branched polymer of glucose, and to mobilize this reserve in response to carbohydrate limitation. However, the enzymes required for the synthesis and degradation of glycogen by T. vaginalis have been little studied. Previously, we characterized T. vaginalis glycogen synthase and glycogen phosphorylase, the key enzymes of glycogen synthesis and degradation, respectively. We determined that their regulatory properties differed from those of well-characterized animal and fungal enzymes. Here, we turn our attention to how glycogen attains its branched structure. We first determined that the glycogen from T. vaginalis resembled that from a related organism, T. gallinae. To determine how the branched structure of T. vaginalis glycogen arose, we identified open reading frames encoding putative T. vaginalis branching and debranching enzymes. When the open reading frames TVAG_276310 and TVAG_330630 were expressed recombinantly in bacteria, the resulting proteins exhibited branching and debranching activity, respectively. Specifically, recombinant TVAG_276310 had affinity for polysaccharides with long outer branches and could add branches to both amylose and amylopectin. TVAG_330630 displayed both 4-α-glucanotransferase and α1,6-glucosidase activity and could efficiently debranch phosphorylase limit dextrin. Furthermore, expression of TVAG_276310 and TVAG_330630 in yeast cells lacking endogenous glycogen branching or debranching enzyme activity, restored normal glycogen accumulation and branched structure. We now have access to the suite of enzymes required for glycogen synthesis and degradation in T. vaginalis.