Defining the Functional Influence of Endothelial Cell-Expressed Oncogenic Activating Mutations on Vascular Morphogenesis and Capillary Assembly.

This study sought to define key molecules and signals controlling major steps in vascular morphogenesis, and how these signals regulate pericyte recruitment and pericyte-induced basement membrane deposition. The morphogenic impact of endothelial cell (EC) expression of activating mutants of Kirsten rat sarcoma virus (kRas), mitogen-activated protein kinase 1 (Mek1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Akt serine/threonine kinase 1 (Akt1), Ras homolog enriched in brain (Rheb) Janus kinase 2 (Jak2), or signal transducer and activator of transcription 3 (Stat3) expression versus controls was evaluated, along with EC signaling events, pharmacologic inhibitor assays, and siRNA suppression experiments. Primary stimulators of EC lumen formation included kRas, Akt1, and Mek1, whereas PIK3CA and Akt1 stimulated a specialized type of cystic lumen formation. In contrast, the key drivers of EC sprouting behavior were Jak2, Stat3, Mek1, PIK3CA, and mammalian target of rapamycin (mTor). These conclusions are further supported by pharmacologic inhibitor and siRNA suppression experiments. EC expression of active Akt1, kRas, and PIK3CA led to markedly dysregulated lumen formation coupled to strongly inhibited pericyte recruitment and basement membrane deposition. For example, activated Akt1 expression in ECs excessively stimulated lumen formation, decreased EC sprouting behavior, and showed minimal pericyte recruitment with reduced mRNA expression of platelet-derived growth factor-BB, platelet-derived growth factor-DD, and endothelin-1, critical EC-derived factors known to stimulate pericyte invasion. The study identified key signals controlling fundamental steps in capillary morphogenesis and maturation and provided mechanistic details on why EC activating mutations induced a capillary deficiency state with abnormal lumens, impaired pericyte recruitment, and basement deposition: predisposing stimuli for the development of vascular malformations.

Elucidating the Morphogenic and Signaling Roles of Defined Growth Factors Controlling Human Endothelial Cell Lumen Formation Versus Sprouting Behavior.

Five growth factors [ie, insulin, fibroblast growth factor-2 (FGF-2), stem cell factor, IL-3, and stromal-derived factor 1α] in combination are necessary for human endothelial cells (ECs) to undergo tube morphogenesis, a process requiring both lumen formation and sprouting behavior. This study investigated why these factors are required by subdividing the factors into 4 separate groups: insulin-only, insulin and FGF-2, no FGF-2 (all factors but without FGF-2), and all factors. The study found that the insulin-only condition failed to support EC morphogenesis or survival, the insulin and FGF-2 condition supported primarily EC lumen formation, and the no FGF-2 condition supported EC sprouting behavior. By comparison, the all-factors condition more strongly stimulated both EC lumen formation and sprouting behavior, and signaling analysis revealed prolonged stimulation of multiple promorphogenic signals coupled with inhibition of proregressive signals. Pharmacologic inhibition of Jak kinases more selectively blocked EC sprouting behavior, whereas inhibition of Raf, phosphatidylinositol 3-kinase, and Akt kinases showed selective blockade of lumen formation. Inhibition of Src family kinases and Notch led to increased sprouting coupled to decreased lumen formation, whereas inhibition of Pak, Mek, and mammalian target of rapamycin kinases blocked both sprouting and lumen formation. These findings reveal novel downstream biological and signaling activities of defined factors that are required for the assembly of human EC-lined capillary tube networks.

Extracellular Matrix Remodeling in Vascular Disease: Defining Its Regulators and Pathological Influence.

Because of structural and cellular differences (ie, degrees of matrix abundance and cross-linking, mural cell density, and adventitia), large and medium-sized vessels, in comparison to capillaries, react in a unique manner to stimuli that induce vascular disease. A stereotypical vascular injury response is ECM (extracellular matrix) remodeling that occurs particularly in larger vessels in response to injurious stimuli, such as elevated angiotensin II, hyperlipidemia, hyperglycemia, genetic deficiencies, inflammatory cell infiltration, or exposure to proinflammatory mediators. Even with substantial and prolonged vascular damage, large- and medium-sized arteries, persist, but become modified by (1) changes in vascular wall cellularity; (2) modifications in the differentiation status of endothelial cells, vascular smooth muscle cells, or adventitial stem cells (each can become activated); (3) infiltration of the vascular wall by various leukocyte types; (4) increased exposure to critical growth factors and proinflammatory mediators; and (5) marked changes in the vascular ECM, that remodels from a homeostatic, prodifferentiation ECM environment to matrices that instead promote tissue reparative responses. This latter ECM presents previously hidden matricryptic sites that bind integrins to signal vascular cells and infiltrating leukocytes (in coordination with other mediators) to proliferate, invade, secrete ECM-degrading proteinases, and deposit injury-induced matrices (predisposing to vessel wall fibrosis). In contrast, in response to similar stimuli, capillaries can undergo regression responses (rarefaction). In summary, we have described the molecular events controlling ECM remodeling in major vascular diseases as well as the differential responses of arteries versus capillaries to key mediators inducing vascular injury.

Increased Matrix Metalloproteinase-1 Activation Enhances Disruption and Regression of k-RasV12-Expressing Arteriovenous Malformation-Like Vessels.

This study sought to identify potential mechanisms by which k-RasV12-expressing endothelial cell (EC) tubes demonstrate an increased propensity to regress compared with controls. Activated k-Ras mutations play a role in a variety of pathological conditions, including arteriovenous malformations, which are prone to bleed, causing serious hemorrhagic complications. ECs expressing active k-RasV12 demonstrate markedly excessive lumen formation with widened and shortened tubes accompanied by reduced pericyte recruitment and basement membrane deposition, leading to deficient capillary network assembly. The current study showed that active k-Ras-expressing ECs secreted greater amounts of MMP-1 proenzyme compared with control ECs, and readily converted it to increased active MMP-1 levels through the action of plasmin or plasma kallikrein (generated from their added zymogens). Active MMP-1 degraded three-dimensional collagen matrices, leading to more rapid and extensive regression of the active k-Ras-expressing EC tubes, in conjunction with matrix contraction, compared with control ECs. Under conditions where pericytes protect control EC tubes from plasminogen- and MMP-1-dependent tube regression, this failed to occur with k-RasV12 ECs, due to reduced pericyte interactions. In summary, k-RasV12-expressing EC vessels showed an increased propensity to regress in response to serine proteinases through accentuated levels of active MMP-1, a novel pathogenic mechanism that may underlie hemorrhagic events associated with arteriovenous malformation lesions.

Molecular basis for pericyte-induced capillary tube network assembly and maturation.

Here we address the functional importance and role of pericytes in capillary tube network assembly, an essential process that is required for vascularized tissue development, maintenance, and health. Healthy capillaries may be directly capable of suppressing human disease. Considerable advances have occurred in our understanding of the molecular and signaling requirements controlling EC lumen and tube formation in 3D extracellular matrices. A combination of SCF, IL-3, SDF-1α, FGF-2 and insulin ("Factors") in conjunction with integrin- and MT1-MMP-induced signaling are required for EC sprouting behavior and tube formation under serum-free defined conditions. Pericyte recruitment to the abluminal EC tube surface results in elongated and narrow tube diameters and deposition of the vascular basement membrane. In contrast, EC tubes in the absence of pericytes continue to widen and shorten over time and fail to deposit basement membranes. Pericyte invasion, recruitment and proliferation in 3D matrices requires the presence of ECs. A detailed analysis identified that EC-derived PDGF-BB, PDGF-DD, ET-1, HB-EGF, and TGFß1 are necessary for pericyte recruitment, proliferation, and basement membrane deposition. Blockade of these individual factors causes significant pericyte inhibition, but combined blockade profoundly interferes with these events, resulting in markedly widened EC tubes without basement membranes, like when pericytes are absent.

Endothelial k-RasV12 Expression Induces Capillary Deficiency Attributable to Marked Tube Network Expansion Coupled to Reduced Pericytes and Basement Membranes.

OBJECTIVE: We sought to determine how endothelial cell (EC) expression of the activating k-Ras (kirsten rat sarcoma 2 viral oncogene homolog) 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 (Src proto-oncogene, non-receptor tyrosine kinase), 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 (AK strain transforming) 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.

Selective and Marked Blockade of Endothelial Sprouting Behavior Using Paclitaxel and Related Pharmacologic Agents.

Whether alterations in the microtubule cytoskeleton affect the ability of endothelial cells (ECs) to sprout and form branching networks of tubes was investigated in this study. Bioassays of human EC tubulogenesis, where both sprouting behavior and lumen formation can be rigorously evaluated, were used to demonstrate that addition of the microtubule-stabilizing drugs, paclitaxel, docetaxel, ixabepilone, and epothilone B, completely interferes with EC tip cells and sprouting behavior, while allowing for EC lumen formation. In bioassays mimicking vasculogenesis using single or aggregated ECs, these drugs induce ring-like lumens from single cells or cyst-like spherical lumens from multicellular aggregates with no evidence of EC sprouting behavior. Remarkably, treatment of these cultures with a low dose of the microtubule-destabilizing drug, vinblastine, led to an identical result, with complete blockade of EC sprouting, but allowing for EC lumen formation. Administration of paclitaxel in vivo markedly interfered with angiogenic sprouting behavior in developing mouse retina, providing corroboration. These findings reveal novel biological activities for pharmacologic agents that are widely utilized in multidrug chemotherapeutic regimens for the treatment of human malignant cancers. Overall, this work demonstrates that manipulation of microtubule stability selectively interferes with the ability of ECs to sprout, a necessary step to initiate and form branched capillary tube networks.

An inhibitor of endothelial ETS transcription factors promotes physiologic and therapeutic vessel regression.

During the progression of ocular diseases such as retinopathy of prematurity and diabetic retinopathy, overgrowth of retinal blood vessels results in the formation of pathological neovascular tufts that impair vision. Current therapeutic options for treating these diseases include antiangiogenic strategies that can lead to the undesirable inhibition of normal vascular development. Therefore, strategies that eliminate pathological neovascular tufts while sparing normal blood vessels are needed. In this study we exploited the hyaloid vascular network in murine eyes, which naturally undergoes regression after birth, to gain mechanistic insights that could be therapeutically adapted for driving neovessel regression in ocular diseases. We found that endothelial cells of regressing hyaloid vessels underwent down-regulation of two structurally related E-26 transformation-specific (ETS) transcription factors, ETS-related gene (ERG) and Friend leukemia integration 1 (FLI1), prior to apoptosis. Moreover, the small molecule YK-4-279, which inhibits the transcriptional and biological activity of ETS factors, enhanced hyaloid regression in vivo and drove Human Umbilical Vein Endothelial Cells (HUVEC) tube regression and apoptosis in vitro. Importantly, exposure of HUVECs to sheer stress inhibited YK-4-279-induced apoptosis, indicating that low-flow vessels may be uniquely susceptible to YK-4-279-mediated regression. We tested this hypothesis by administering YK-4-279 to mice in an oxygen-induced retinopathy model that generates disorganized and poorly perfused neovascular tufts that mimic human ocular diseases. YK-4-279 treatment significantly reduced neovascular tufts while sparing healthy retinal vessels, thereby demonstrating the therapeutic potential of this inhibitor.

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.