Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta signaling

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.     

RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia

Although a number of genetic defects are commonly associated with acute myeloid leukemia (AML), a large percentage of AML cases are cytogenetically normal. This suggests a functional screen for transforming genes is required to identify genetic mutations that are missed by cytogenetic analyses. We utilized a retrovirus-based cDNA expression system to identify transforming genes expressed in cytogenetically normal AML patients. We identified a new member of the Ras guanyl nucleotide-releasing protein (RasGRP) family of Ras guanine nucleotide exchange factors, designating it RasGRP4. Subsequently, cDNA sequences encoding rodent and human RasGRP4 proteins were deposited in GenBank. RasGRP4 contains the same protein domain structure as other members of the RasGRP family, including a Ras exchange motif, a CDC25 homology domain, a C1/diacyglycerol-binding domain, and putative calcium-binding EF hands. We show that expression of RasGRP4 induces anchorage-independent growth of Rat1 fibroblasts. RasGRP4 is a Ras-specific activator and, interestingly, is highly expressed in peripheral blood leukocytes and myeloid cell lines. Unlike other RasGRP proteins, RasGRP4 is not expressed in the brain or in lymphoid cells. We demonstrated that 32D myeloid cells expressing RasGRP4 have elevated levels of activated Ras compared with control cells, and phorbol 12-myristate 13-acetate (PMA) treatment greatly enhanced Ras activation. PMA induced membrane localization of RasGRP4 and 32D cells expressing RasGRP4 were capable of cytokine-independent proliferation in the presence of PMA. We conclude that RasGRP4 is a member of the RasGRP family of Ras guanine nucleotide exchange factors that may play a role in myeloid cell signaling growth regulation pathways that are responsive to diacylglycerol levels.     

Lipopolysaccharide-induced expression of the competence gene KC in vascular endothelial cells is mediated through protein kinase C

The KC gene is a cell cycle-dependent competence gene originally identified in platelet-derived growth factor-stimulated BALB/c-3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady-state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA-stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS-treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation-independent process. Second, unlike LPS-induced KC expression in macrophages and platelet-derived growth factor-induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to require activation of protein kinase C.     

Evidence of a role for TRPC channels in VEGF-mediated increased vascular permeability in vivo

Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.     

Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites

Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.     

Differential roles of PKCalpha and PKCepsilon in controlling the gene expression of Nox4 in human endothelial cells

NADPH oxidases are major sources of superoxide in the vascular wall. This study investigates the role of protein kinase C (PKC) in regulating gene expression of NADPH oxidases. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate led to a PKC-dependent biphasic expression of the gp91phox homolog Nox4. A downregulation of Nox4 was observed at 6 h and an upregulation at 48 h after phorbol ester treatment. The early Nox4 downregulation was associated with a reduced superoxide production, whereas the late Nox4 upregulation was accompanied by a clear enhancement of superoxide. PMA activated the PKC isoforms alpha and epsilon in HUVEC and EA.hy 926 cells. Knockdown of PKCepsilon by siRNA prevented the early downregulation of Nox4, whereas knockdown of PKCalpha selectively abolished the late Nox4 upregulation. Vascular endothelial growth factor (VEGF), which activates PKCalpha but not PKCepsilon in HUVEC, increased Nox4 expression without the initial downregulation. VEGF-induced Nox4 upregulation was associated with an enhanced proliferation and angiogenesis of HUVEC. Both effects could be reduced by inhibition of NADPH oxidase. Thus, a selective inhibition/knockdown of PKCalpha may represent a novel therapeutic strategy for vascular disease.     

RasGRPs Are Targets of the Anti-Cancer Agent Ingenol-3-Angelate

Ingenol-3–angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A’s effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP’s C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin’s lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.     

Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.     

Activation of human TRPC6 channels by receptor stimulation

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K(0.5) of approximately 10 microm and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30-300 microm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 microm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.     

Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells.

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).