Defining an Upstream VEGF (Vascular Endothelial Growth Factor) Priming Signature for Downstream Factor-Induced Endothelial Cell-Pericyte Tube Network Coassembly.

OBJECTIVE: In this work, we have sought to define growth factor requirements and the signaling basis for different stages of human vascular morphogenesis and maturation. Approach and Results: Using a serum-free model of endothelial cell (EC) tube morphogenesis in 3-dimensional collagen matrices that depends on a 5 growth factor combination, SCF (stem cell factor), IL (interleukin)-3, SDF (stromal-derived factor)-1α, FGF (fibroblast growth factor)-2, and insulin (factors), we demonstrate that VEGF (vascular endothelial growth factor) pretreatment of ECs for 8 hours (ie, VEGF priming) leads to marked increases in the EC response to the factors which includes; EC tip cells, EC tubulogenesis, pericyte recruitment and proliferation, and basement membrane deposition. VEGF priming requires VEGFR2, and the effect of VEGFR2 is selective to the priming response and does not affect factor-dependent tubulogenesis in the absence of priming. Key molecule and signaling requirements for VEGF priming include RhoA, Rock1 (Rho-kinase), PKCα (protein kinase C α), and PKD2 (protein kinase D2). siRNA suppression or pharmacological blockade of these molecules and signaling pathways interfere with the ability of VEGF to act as an upstream primer of downstream factor-dependent EC tube formation as well as pericyte recruitment. VEGF priming was also associated with the formation of actin stress fibers, activation of focal adhesion components, upregulation of the EC factor receptors, c-Kit, IL-3Rα, and CXCR4 (C-X-C chemokine receptor type 4), and upregulation of EC-derived PDGF (platelet-derived growth factor)-BB, PDGF-DD, and HB-EGF (heparin-binding epidermal growth factor) which collectively affect pericyte recruitment and proliferation. CONCLUSIONS: Overall, this study defines a signaling signature for a separable upstream VEGF priming step, which can activate ECs to respond to downstream factors that are necessary to form branching tube networks with associated mural cells.

Proinflammatory Mediators, IL (Interleukin)-1ß, TNF (Tumor Necrosis Factor) α, and Thrombin Directly Induce Capillary Tube Regression.

OBJECTIVE: In this work, we examine the molecular basis for capillary tube regression and identify key proregressive factors, signaling pathways, and pharmacological antagonists of this process. Approach and Results: We demonstrate that the proinflammatory mediators, IL (interleukin)-1ß, TNF (tumor necrosis factor) α, and thrombin, singly and in combination, are potent regulators of capillary tube regression in vitro. These proregressive factors, when added to endothelial cell-pericyte cocultures, led to selective loss of endothelial cell-lined tube networks, with retention and proliferation of pericytes despite the marked destruction of adjacent capillary tubes. Moreover, treatment of macrophages with the TLR (toll-like receptor) agonists Pam3CSK4 and lipopolysaccharide generates conditioned media with marked proregressive activity, that is completely blocked by a combination of neutralizing antibodies directed to IL-1ß and TNFα but not to other factors. The same combination of blocking antibodies, as well as the anti-inflammatory cytokine IL-10, interfere with macrophage-dependent hyaloid vasculature regression in mice suggesting that proinflammatory cytokine signaling regulates capillary regression in vivo. In addition, we identified a capillary regression signaling signature in endothelial cells downstream of these proregressive agents that is characterized by increased levels of ICAM-1 (intercellular adhesion molecule-1), phospho-p38, and phospho-MLC2 (myosin light chain-2) and decreased levels of phospho-Pak2, acetylated tubulin, phospho-cofilin, and pro-caspase3. Finally, we identified combinations of pharmacological agents (ie, FIST and FISTSB) that markedly rescue the proregressive activities of IL-1ß, TNFα, and thrombin, individually and in combination. CONCLUSIONS: Overall, these new studies demonstrate that the major proinflammatory mediators, IL-1ß, TNFα, and thrombin, are key regulators of capillary tube regression-a critical pathological process regulating human disease.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTPγS or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.