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Transforming growth factor-␤1 (TGF-␤1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations. Platelets contain 40 times as much TGF-␤1 as other cells and secrete it as an inactive (latent) form in complex with latency-associated peptide (LAP), which is disulfide bonded via Cys33 to latent TGF-␤ binding protein 1 (LTBP-1). Little is known about how latent TGF-␤1 becomes activated in vivo.Here we show that TGF-␤1 released from platelets or fibroblasts undergoes dramatic activation when subjected to stirring or shear forces, providing a potential mechanism for physiologic control. Thioldisulfide exchange appears to contribute to the process based on the effects of thiol-reactive reagents and differences in thiol labeling of TGF-␤1 before and after stirring or shear. Activation required the presence of LTBP, as TGF-␤1 contained in complex with only LAP could not be activated by stirring when studied as either a recombinant purified protein complex or in the platelet releasates or sera of mice engineered to contain an LAP C33S mutation. Release IntroductionTransforming growth factor-␤1 (TGF-␤1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations, including cells of the immune and hematopoietic systems, as well as malignant cells and fibroblasts, 1 with the latter responding with increased collagen production leading to tissue fibrosis. 2 Nearly all cellular TGF-␤1 exists, however, in a biologically inactive (latent) form in a noncovalent complex with the remaining portion of its precursor molecule, latency-associated peptide (LAP), which is disulfide bonded to a latent TGF-␤ binding protein 3,or 4), forming the large latent complex (LLC). 3 Although much is known about the signaling mechanisms and downstream effects of TGF-␤1, 4 and several latent TGF-␤1 activating mechanisms have been identified in vitro (reviewed in Annes et al 3 ), little is known about the physiologic mechanisms that control latent TGF-␤1 activation in vivo. Recent data support activation of LLC TGF-␤1 via a traction mechanism, 3,[5][6][7][8][9][10][11] with LAP binding to integrins ␣V␤5, ␣V␤6, and ␣V␤8, and LTBP-1 binding to extracellular matrix.Blood platelets are a rich source of TGF-␤1, containing 40 to 100 times as much as other cells, 12 and releasing it when activated by a variety of agents, including thrombin, which is produced during blood clotting. [13][14][15][16][17][18][19] Virtually all of the TGF-␤1 released from platelets is in the LLC. 3,16 Because platelet latent TGF-␤1 is released into the circulation, and because traction on the molecule has been proposed as a mechanism of latent TGF-␤1 activation, 5,6 we hypothesized that intravascular shear force may serve as an analog of traction mediated by cellular contraction and contribute to the activation of latent TGF-␤1 released from platelets. We therefore tested this hypothesis by both in vitro and in vivo experiments. Methods Antibodies and reagentsAnti-TGF-␤1 (polyclonal chi...

The vasculature of the central nervous system (CNS) forms a selective barrier termed the blood-brain barrier (BBB). Disruption of the BBB may contribute to various CNS diseases. Conversely, the intact BBB restricts efficient penetration of CNS-targeted drugs. Here, we report the BBB-regulatory role of endothelial sphingosine 1-phosphate (S1P) receptor-1, a G protein-coupled receptor known to promote the barrier function in peripheral vessels. Endothelial-specific S1pr1 knockout mice (S1pr1 iECKO ) showed BBB breach for small-molecular-mass fluorescence tracers (<3 kDa), but not larger tracers (>10 kDa). Chronic BBB leakiness was associated with cognitive impairment, as assessed by the novel object recognition test, but not signs of brain inflammation. Brain microvessels of S1pr1 iECKO mice showed altered subcellular distribution of tight junctional proteins. Pharmacological inhibition of S1P 1 function led to transient BBB breach. These data suggest that brain endothelial S1P 1 maintain the BBB by regulating the proper localization of tight junction proteins and raise the possibility that endothelial S1P 1 inhibition may be a strategy for transient BBB opening and delivery of small molecules into the CNS.blood-brain barrier | sphingosine 1-phosphate | endothelium | tight junction | drug delivery

Endothelial dysfunction, a hallmark of vascular disease, is restored by plasma high-density lipoprotein (HDL). However, a generalized increase in HDL abundance is not beneficial, suggesting that specific HDL species mediate protective effects. Apolipoprotein M–containing HDL (ApoM+HDL), which carries the bioactive lipid sphingosine 1-phosphate (S1P), promotes endothelial function by activating G protein–coupled S1P receptors. Moreover, HDL-bound S1P is limiting in several inflammatory, metabolic, and vascular diseases. We report the development of a soluble carrier for S1P, ApoM-Fc, which activated S1P receptors in a sustained manner and promoted endothelial function. In contrast, ApoM-Fc did not modulate circulating lymphocyte numbers, suggesting that it specifically activated endothelial S1P receptors. ApoM-Fc administration reduced blood pressure in hypertensive mice, attenuated myocardial damage after ischemia/reperfusion injury, and reduced brain infarct volume in the middle cerebral artery occlusion model of stroke. Our proof-of-concept study suggests that selective and sustained targeting of endothelial S1P receptors by ApoM-Fc could be a viable therapeutic strategy in vascular diseases.

Our findings indicate that S1P signaling in ECs modulates vascular responses to IC deposition. S1P agonists and ApoM-Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end-organ damage.

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Background Transforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-β1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-β1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa). Methods Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels. Results Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-β1 levels (r=0.38, p=0.05 for PLAs and r=−0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=−0.42, p=0.03 for PLAs and r=−0.37, p=0.06 for single leukocytes). Conclusions p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-β1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-β1.