Jens Kroll

A Dual Inhibitor of Platelet-Derived Growth Factor β-Receptor and Src Kinase Activity Potently Interferes With Motogenic and Mitogenic Responses to PDGF in Vascular Smooth Muscle Cells A Novel Candidate for Prevention of Vascular Remodeling

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin

Histone deacetylases (HDACs) deacetylate histones and non‐histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6‐deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild‐type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α‐tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin‐remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.

Dissociation of angiogenesis and tumorigenesis in follistatin- and activin-expressing tumors

The transforming growth factor-beta superfamily member activin and its antagonist, follistatin, act as a pleiotropic growth factor system that controls cell proliferation, differentiation, and apoptosis. Activin inhibits fibroblast growth factor 2-induced sprouting angiogenesis in vitro (spheroidal angiogenesis assay) and in vivo (Matrigel assay). To further study the role of the activin/follistatin system during angiogenesis and tumor progression, activin- and follistatin-expressing R30C mammary carcinoma cells were studied in mouse tumor experiments. Surprisingly, activin-expressing tumors grew much faster than follistatin-expressing tumors although they failed to induce increased angiogenesis (as evidenced by low microvessel density counts). Conversely, follistatin-expressing tumors were much smaller but had a dense network of small-diameter capillaries. Qualitative angioarchitectural analyses (mural cell recruitment, perfusion) revealed no major functional differences of the tumor neovasculature. Analysis of activin- and follistatin-expressing R30C cells identified a cell autonomous role of this system in controlling tumor cell growth. Whereas proliferation of R30C cells was not altered, follistatin-expressing R30C cells had an enhanced susceptibility to undergo apoptosis. These findings in experimental tumors are complemented by an intriguing case report of a human renal cell carcinoma that similarly shows a dissociation of angiogenesis and tumorigenesis during tumor progression. Collectively, the data shed further light into the dichotomous stimulating and inhibiting roles that the activin/follistatin system can exert during angiogenesis and tumor progression. Furthermore, the experiments provide a critical proof-of-principle example for the dissociation of angiogenesis and tumorigenesis, supporting the concept that tumor growth may not be dependent on increased angiogenesis as long as a minimal intratumoral microvessel density is maintained.

Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole.

Vascular endothelial growth factor‐α (VEGF‐A) plays a major role in tumor angiogenesis and raises the concentration of intracellular free calcium ([Ca2+]i). Carboxyamidotriazole (CAI), an inhibitor of calcium influx and of angiogenesis, is under investigation as a tumoristatic agent. We studied the effect of CAI and the role of [Ca2+]i in VEGF‐α signaling in human endothelial cells. VEGF‐α induced a biphasic [Ca2+]i signal. VEGF‐α increased the level of intracellular inositol 1,4,5‐trisphosphate (IP3), which suggests that VEGF‐A releases Ca2+ from IP3‐sensitive stores and induces store‐operated calcium influx. Reduction of either extracellular or intracellular free Ca2+ inhibited VEGF‐A‐induced proliferation. CAI inhibited IP3 formation, both phases of the calcium signal, nitric oxide (NO) release, and proliferation induced by VEGF‐A. CAI prevented neither activation of VEGF receptor‐2 (VEGFR‐2) (KDR/Flk‐1), phospholipase C‐γ, or mitogen‐activated protein kinase (MAP kinase) nor translocation of nuclear factor of activated T cells (NFAT). We conclude that calcium signaling is necessary for VEGF‐A‐induced proliferation. MAP kinase activation occurs independently of [Ca2+]i but is not sufficient to induce proliferation in the absence of calcium signaling. Inhibition of the VEGF‐Ainduced [Ca2+]i signal and proliferation by CAI can be explained by inhibition of IP3 formation and may contribute to the antiangiogenic action of CAI. Calcium‐dependent NO formation may represent a link between calcium signaling and proliferation.

Wnt2 acts as a cell type–specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross‐stimulating the VEGF pathway

The mechanisms regulating the growth and differentiation of hepatic sinusoidal endothelial cells (HSECs) are not well defined. Because Wnt signaling has become increasingly important in developmental processes such as vascular and hepatic differentiation, we analyzed HSEC‐specific Wnt signaling in detail. Using highly pure HSECs isolated by a newly developed protocol selecting against nonsinusoidal hepatic endothelial cells, we comparatively screened the multiple components of the Wnt pathway for differential expression in HSECs and lung microvascular endothelial cells (LMECs) via reverse‐transcription polymerase chain reaction (RT‐PCR). As confirmed via quantitative RT‐PCR and northern and western blotting experiments, Wnt2 (and less so Wnt transporter wls/evi) and Wnt coreceptor Ryk were overexpressed by HSECs, whereas Wnt inhibitory factor (WIF) was strongly overexpressed by LMECs. Exogenous Wnt2 superinduced proliferation of HSECs (P < 0.05). The Wnt inhibitor secreted frizzled‐related protein 1 (sFRP1) (P < 0.005) and transfection of HSECs with Wnt2 small interfering RNA (siRNA) reduced proliferation of HSECs. These effects were rescued by exogenous Wnt2. Tube formation of HSECs on matrigel was strongly inhibited by Wnt inhibitors sFRP1 and WIF (P < 0.0005). Wnt signaling in HSECs activated the canonical pathway inducing nuclear translocation of β‐catenin. GST (glutathione transferase) pull‐down and co‐immunoprecipitation assays showed Fzd4 to be a novel Wnt2 receptor in HSECs. Gene profiling identified vascular endothelial growth factor receptor‐2 (VEGFR‐2) as a target of Wnt2 signaling in HSECs. Inhibition of Wnt signaling down‐regulated VEGFR‐2 messenger RNA and protein. Wnt2 siRNA knock‐down confirmed Wnt2 specificity of VEGFR‐2 regulation in HSECs. Conclusion: Wnt2 is an autocrine growth and differentiation factor specific for HSECs that synergizes with the VEGF signaling pathway to exert its effects. (HEPATOLOGY 2008;47:1018–1031.)

Neuropilin-1 and neuropilin-2 enhance VEGF121 stimulated signal transduction by the VEGFR-2 receptor

The neuropilin‐1 (np1) receptor binds the 165 amino‐acid form of vascular endothelial growth factor165 (VEGF165) and functions as an enhancer that potentiates VEGF165 signaling via the VEGFR‐2 ty‐rosine‐kinase receptor. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF165 mutant (VEGF165KF) that binds to neuropilins but displays a much lower affinity toward VEGFR‐1 and VEGFR‐2. VEGF165KF failed to induce VEGFR‐2 phosphorylation in cells lacking neuropilins. However, in the presence of np1, VEGF165KF bound weakly to VEGFR‐2, induced VEGFR‐2 phosphorylation, and activated ERK1/2. Interestingly, VEGF165KF did not promote formation of VEGFR‐2/np1 complexes nor did high concentrations of VEGF165KF inhibit VEGF165 induced formation of such complexes, suggesting that VEGF165 does not stabilize VEGFR‐2/ np1 complexes by forming bridges spanning VEGFR‐2 and np1. VEGF121 is a VEGF form that does not bind to neuropilins. Surprisingly, both np1 and neuropilin‐2 (np2) enhanced VEGF121‐induced phosphorylation of VEGFR‐2 and VEGF121‐induced proliferation of endo‐thelial cells. The enhancement of VEGF121 activity by np1 was accompanied by a 10‐fold increase in binding affinity to VEGFR‐2 and was not associated with the formation of new VEGFR‐2/np1 complexes. These observations suggest that neuropilins enhance the activity of VEGF forms that do not bind to neuropilins, indicate that np2 is a functional VEGF receptor, and imply that spontaneously formed VEGFR‐2/np1 complexes suffice for efficient neuropilin mediated enhancement of VEGF activity.—Shraga‐Heled, N., Kessler, O., Prahst, C., Kroll, J., Augustin, H., Neufeld, G. Neuropilin‐1 and neuropilin‐2 enhance VEGF121 stimulated signal transduction by the VEGFR‐2 receptor. FASEB J. 21, 915–926 (2007)

Histone Deacetylase 9 Promotes Angiogenesis by Targeting the Antiangiogenic MicroRNA 17–92 Cluster in Endothelial Cells

Objective—Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear. Methods and Results—Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17–92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17–92. Inhibition of miR-17–20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos. Conclusion—We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17–92 cluster.

Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis

Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho-dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H-1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK787/ZK222584. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H-1152 and ROCK I/II-specific small-interfering RNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor kinase insert domain receptor phosphorylation and extracellular signal-regulated kinase 1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho-dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo.

MicroRNA-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting Vascular Endothelial Growth Factor Signaling

Rationale: Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood. Objective: To identify microRNAs that regulate angiogenesis. Methods and Results: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling. Conclusions: Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.

The Rac1 Regulator ELMO1 Controls Vascular Morphogenesis in Zebrafish

Rationale: Angiogenesis is regulated by the small GTPase Rac1. The ELMO1/DOCK180 complex forms a guanine nucleotide exchange factor for Rac1, regulating its activation during cell migration in different biological systems. Objective: To investigate the function of ELMO1/DOCK180 in vascular development. Methods and Results: In situ hybridization studies for elmo1 identified a vascular and neuronal expression in zebrafish. Morpholino-based expression silencing of elmo1 severely impaired the formation of the vasculature, including intersomitic vessels, the dorsal longitudinal anastomotic vessel, the parachordal vessel, and the development of the thoracic duct in tg(fli1:EGFP) embryos. Mechanistically, we identified Netrin-1 and its receptor Unc5B as upstream activators of the ELMO1/DOCK180 complex, regulating its functional interaction and leading to Rac1 activation in endothelial cells and vessel formation in zebrafish. Conclusions: Our data have identified a novel signaling cascade regulating vasculature formation in zebrafish.