Brian Joseph Limberg

Vascular Endothelial Growth Factor Receptor-2 and Neuropilin-1 Form a Receptor Complex That Is Responsible for the Differential Signaling Potency of VEGF165 and VEGF121

The two most abundant secreted isoforms of vascular endothelial growth factor A (VEGF165 and VEGF121) are formed as a result of differential splicing of the VEGF-A gene. VEGF165 and VEGF121 share similar affinities at the isolated VEGF receptor (VEGFR)-2 but have been previously demonstrated to have differential ability to activate VEGFR-2-mediated effects on endothelial cells. Herein we investigate whether the recently described VEGF165 isoform-specific receptor neuropilin-1 (Npn-1) is responsible for the difference in potency observed for these ligands. We demonstrate that although VEGFR-2 and Npn-1 form a complex, this complex does not result in an increase in VEGF165 binding affinity. Therefore, the differential activity of VEGF165 and VEGF121cannot be explained by a differential binding affinity for the complex. Using an antagonist that competes for VEGF165 binding at the VEGFR-2·Npn-1 complex, we observe specific antagonism of VEGF165-meditated phosphorylation of VEGFR-2 without affecting the VEGF121 response. These data indicate that the formation of the complex is responsible for the increased potency of VEGF165 versus VEGF121. Taken together, these data suggest a receptor-clustering role for Npn-1, as opposed to Npn-1 behaving as an affinity-converting subunit.

β−Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder

Abstractβ3-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte β3-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three β-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for β1, β2, and β3 were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The β3-receptor in the human urothelium appears to be functional, as two different selective β3-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in UROtsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the β2-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that β3-receptor expression is maintained with age, but may function in concert with other β-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.

Urothelial beta‐3 adrenergic receptors in the rat bladder

To investigate the distribution of beta‐3 adrenergic receptors (β3ARs) in the rat bladder and to examine the contribution of urothelial β3ARs to agonist‐induced suppression of bladder reflexes and relaxation of smooth muscle.

The Y2 receptor mediates increases in collateral-dependent blood flow in a model of peripheral arterial insufficiency

We have utilized a rat model of peripheral artery disease (PAD) to examine whether the known angiogenic activity of the Y(2) receptor would translate into a meaningful increase in collateral blood flow. The maximal increase in collateral blood flow capacity of approximately 60% (p<0.001) was obtained with a 10microg/kgday (IA infusion, 14 days) of either PYY or PYY(3-36) and did not differ from that obtained with a maximally angiogenic dose of VEGF(165). Pharmacodynamic modeling based upon single dose pharmacokinetic plasma profiles of both agonists suggests that E(max) is reached when the Y(2) receptor is occupied by >or=50%. Furthermore, for PYY(3-36), occupancy of the Y(2) receptor is sufficient to promote a significant benefit in collateral blood flow.