Bmper, which is orthologous to Drosophila melanogaster crossveinless 2, is a secreted factor that regulates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been postulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. In this paper, we demonstrate that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mechanism leads to the efficient degradation of Bmper and Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4-dependent Smad signaling. We also determined that Noggin and Gremlin, but not Chordin, trigger endocytosis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implications for Bmp signal regulation by secreted mediators.

Background-The efficacy of nitroglycerin (NTG) as a vasodilator is limited by tolerance, which develops shortly after treatment begins. In vascular smooth muscle cells (VSMCs), NTG is denitrated to form nitric oxide (NO), which activates guanylyl cyclase and generates cGMP. cGMP plays a key role in nitrate-induced vasodilation by reducing intracellular Ca 2ϩ concentration. Therefore, one possible mechanism for development of nitrate tolerance would be increased activity of the cGMP phosphodiesterase (PDE), which decreases cGMP levels. Methods and Results-To test this hypothesis, rats were made tolerant by continuous infusion of NTG for 3 days (10 g · kg Ϫ1 · min Ϫ1 SC) with an osmotic pump. Analysis of PDE activities showed an increased function of Ca 2ϩ /calmodulin (CaM)-stimulated PDE (PDE1A1), which preferentially hydrolyzes cGMP after NTG treatment. Western blot analysis for the Ca 2ϩ /CaM-stimulated PDE revealed that PDE1A1 was increased 2.3-fold in NTG-tolerant rat aortas. Increased PDE1A1 was due to mRNA upregulation as measured by relative quantitative reverse transcription-polymerase chain reaction. The PDE1-specific inhibitor vinpocetine partially restored the sensitivity of the tolerant vasculature to subsequent NTG exposure. In cultured rat aortic VSMCs, angiotensin II (Ang II) increased PDE1A1 activity, and vinpocetine blocked the effect of Ang II on decrease in cGMP accumulation. Conclusions-Induction of PDE1A1 in nitrate-tolerant vessels may be one mechanism by which NO/cGMP-mediated vasodilation is desensitized and Ca 2ϩ -mediated vasoconstriction is supersensitized. Inhibiting PDE1A1 expression and/or activity could be a novel therapeutic approach to limit nitrate tolerance.

Many findings suggest that steady laminar flow in blood vessels activates signal transduction events that lead to expression of atheroprotective genes (1, 2). The nature and magnitude of shear stress plays an important role in long-term maintenance of the structure and function of the blood vessel. In ''linear'' areas of the vasculature blood flows in ordered laminar patterns in a pulsatile fashion and endothelial cells (ECs) experience pulsatile shear stress with fluctuations in magnitude that yield a mean positive shear stress. At areas of abrupt curvatures in the vasculature, as in the carotid bifurcation, the laminar flow of blood is disrupted and separate flow patterns result (3-8). The significance of these flow patterns is demonstrated by studies that correlate development of atherosclerotic lesions (fatty streaks and small plaques) with areas of the carotid that experience these flow reversals with low time-averaged shear stress (3, 4). Regions of the carotid bifurcation that experience steady nonoscillatory shear stress as the result of laminar blood flow patterns are relatively protected from atherosclerosis. Examples of the atheroprotective nature of steady laminar flow are inhibition of E-selectin expression and suppression of vascular cell adhesion molecule 1 (VCAM-1) induction by cytokines such as IL-1 and tumor necrosis factor ␣