Browsing Vascular Biology Center: Student Research and Presentations by Submit Date
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Neurovascular Injury After Retinal Ischemia Reperfusion Insult: Contrasting Roles Of Arginase Enzyme IsoformsPurpose: We have previously shown the involvement of arginase enzyme in retinal neurovascular injury. The present study was undertaken to determine the distinct roles of arginase 1 (A1) and arginase 2 (A2) in neurovascular damage following ischemia/reperfusion (I/R) injury. Methods: We used wild type (WT) mice, A2 knock out mice (A2-/-) and mice lacking one copy of A1 (A1+/-). Western blotting, RT-PCR, vascular digests, immunofluorescence, Propidium Iodide (PI) labeling and electroretinography (ERG) were used to evaluate retinal injury and function. Results: I/R injury caused significant increases in A2 expression along with thinning of the neural retina, decreases in NeuN+ GCL neurons and formation of acellular capillaries. Increases in PI labeling and RIP-3 expression showed that cell death occurred by necroptosis. Neurovascular injury was accompanied by microglial activation along with increased expression of GFAP and impairment of the ERG. Neuronal cell loss, capillary degeneration, necroptosis, gliosis and ERG impairment were all significantly reduced by deletion of A2. On the other hand, A1 deletion exacerbated I/R-induced neuronal and vascular injury and further increased necroptosis and gliosis as compared with WT retinas. Conclusions: This study shows for the first time the different roles of arginase isoforms after I/R insult. I/R-induced necrotic cell death and gliosis are mediated by A2, whereas upregulation of A1 may play a role in limiting the pathology.
Molecular Mechanisms Underlying ATP- And Adenosine Induced Microvascular Endothelial Barrier PreservationEndothelial barrier integrity has critical importance in vascular homeostasis. Disruption of the endothelial cell (EC) barrier results in increased vascular permeability. Extracellular purines, ATP and adenosine (Ado) can restore the barrier function, involving the activation of myosin light chain phosphatase (MLCP). Both ATP and Ado increase protein kinase-A (PKA) activity, however a direct link between purine-induced EC barrier enhancement, MLCP and PKA was not described. Here we show that Ado and a stable analog of ATP, ATPγS, induced human lung microvascular EC (HLMVEC) barrier enhancement and PKA activation leads to decrease in MLC and MYPT1T696 phosphorylation. Surprisingly, PKA catalytic subunit (PKAc) depletion attenuates ATPγS, but not Ado-induced increase in transendothelial electrical resistance (TER), indicating that PKA activation is involved in ATP-induced EC barrier enhancement. Depletion of PKAc leads to increase in MLC and MYPTT696 phosphorylation in ATPγS challenged EC supporting the role of PKA in MLCP activation. To elucidate the role of PKA signaling in ATP-induced EC barrier enhancement we depleted several PKA-anchoring proteins (AKAPs). AKAP2 depletion attenuates ATPγS, but not Ado-induced TER increase. Furthermore, AKAP2 co-immunoprecipitates with MYPT1. This interaction was also confirmed by PLA. In conclusion ATP- and Ado-induced barrier enhancement requires different signaling with PKA promoting ATP-, but not Ado-induced EC barrier strengthening.