Objective: We sought to identify and investigate the functional role of the major endothelial cell (EC)-derived factors that control pericyte recruitment to EC tubes and pericyte-induced tube maturation during capillary network formation. Approach and Results: We identify PDGF (platelet-derived growth factor)-BB, PDGF-DD, ET (endothelin)-1, TGF (transforming growth factor)-β, and HB-EGF (heparin-binding epidermal growth factor), as the key individual and combined regulators of pericyte assembly around EC tubes. Using novel pericyte only assays, we demonstrate that PDGF-BB, PDGF-DD, and ET-1 are the primary direct drivers of pericyte invasion. Their addition to pericytes induces invasion as if ECs were present. In contrast, TGF-β and HB-EGF have minimal ability to directly stimulate pericyte invasion. In contrast, TGF-β1 can act as an upstream pericyte primer to stimulate invasion in response to PDGFs and ET-1. HB-EGF stimulates pericyte proliferation along with PDGFs and ET-1. Using EC-pericyte cocultures, individual, or combined blockade of these EC-derived factors, or their pericyte receptors, using neutralizing antibodies or chemical inhibitors, respectively, interferes with pericyte recruitment and proliferation. As individual factors, PDGF-BB and ET-1 have the strongest impact on these events. However, when the blocking reagents are combined to interfere with each of the above factors or their receptors, more dramatic and profound blockade of pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition occurs. Under these conditions, ECs form tubes that become much wider and less elongated as if pericytes were absent. Conclusions: Overall, these new studies define and characterize a functional role for key EC-derived factors controlling pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition during capillary network assembly.

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)-1β, 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-1β 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-1β, TNFα, and thrombin, individually and in combination. Conclusions: Overall, these new studies demonstrate that the major proinflammatory mediators, IL-1β, TNFα, and thrombin, are key regulators of capillary tube regression—a critical pathological process regulating human disease.

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.

Objective: We sought to determine how endothelial cell (EC) expression of the activating k-Ras mutation, k-RasV12, affects their ability to form lumens and tubes and interact with pericytes during capillary assembly Approach and Results: Using defined bioassays where human ECs undergo observable tubulogenesis, sprouting behavior, pericyte recruitment to EC-lined tubes, and pericyte-induced EC basement membrane deposition, we assessed the impact of EC k-RasV12 expression on these critical processes that are necessary for proper capillary network formation. This mutation, which is frequently seen in human ECs within brain arteriovenous malformations, was found to markedly accentuate EC lumen formation mechanisms, with strongly accelerated intracellular vacuole formation, vacuole fusion, and lumen expansion and with reduced sprouting behavior, leading to excessively widened tube networks compared with control ECs. These abnormal tubes demonstrate strong reductions in pericyte recruitment and pericyte-induced EC basement membranes compared with controls, with deficiencies in fibronectin, collagen type IV, and perlecan deposition. Analyses of signaling during tube formation from these k-RasV12 ECs reveals strong enhancement of Src, Pak2 (P21 [RAC1 (Rac family small GTPase 1)] activated kinase 2), b-Raf (v-raf murine sarcoma viral oncogene homolog B1), Erk (extracellular signal–related kinase), and Akt activation and increased expression of PKCε (protein kinase C epsilon), MT1-MMP (membrane-type 1 matrix metalloproteinase), acetylated tubulin and CDCP1 (CUB domain-containing protein 1; most are known EC lumen regulators). Pharmacological blockade of MT1-MMP, Src, Pak, Raf, Mek (mitogen-activated protein kinase) kinases, Cdc42 (cell division cycle 42)/Rac1, and Notch markedly interferes with lumen and tube formation from these ECs. Conclusions: Overall, this novel work demonstrates that EC expression of k-RasV12 disrupts capillary assembly due to markedly excessive lumen formation coupled with strongly reduced pericyte recruitment and basement membrane deposition, which are critical pathogenic features predisposing the vasculature to develop arteriovenous malformations.

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