Angiogenin Activates Endothelial Cell Phospholipase C (Diacylglycerol/Second Messenger/Angiogenesis) Roy BICKNELL and BERT L

Home , Angiogenin, Pancreatic ribonuclease, Ribonuclease

Proc. Natl. Acad. Sci. USA Vol. 85, pp. 5961-5965, August 1988 Cell Biology

Angiogenin activates endothelial cell phospholipase C (diacylglycerol/second messenger/angiogenesis) RoY BICKNELL AND BERT L. VALLEE Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115 Contributed by Bert L. Vallee, May 2, 1988

ABSTRACT Low concentrations of angiogenin activate human plasma but comparable to those that elicit angiogenic the inositol-specific phospholipase C of cultured pulmonary responses in the chorioallantoic membrane assay. artery, umbilical vein, and capillary endothelial cells, promot- ing a transient increase in the intracellular levels of 1,2- diacylglycerol and inositol trisphosphate. The response is MATERIALS AND METHODS strongly dose dependent with a maximum in the ng/ml Materials. Angiogenin was isolated from either normal concentration range and, for some cell lines, a marked decrease human plasma (9) or medium conditioned by baby hamster at concentrations >1 ng/ml; e.g., arterial endothelial cells kidney cells genetically altered to express angiogenin (10). respond weakly to angiogenin concentrations comparable to The responses to plasma-derived and expressed angiogenin that in normal human plasma (-400 ng/ml). Chemical mod- were indistinguishable in all experiments. Stock solutions of ification of the active site of angiogenin or inhibition with angiogenin were free of endotoxins (<1 ng/ml in mg/ml placental ribonuclease inhibitor abolishes its activation of stocks) by the Limulus amoebocyte assay (11) using the endothelial cell phospholipase C; this correlates with the Sigma E-TOXATE kit. Low concentration solutions of concomitant loss of both intrinsic ribonucleolytic and angio- angiogenin were prepared from mg/ml stocks by dilution into genic activity. The response to low concentrations ofangiogenin Hanks' balanced salt solution (without bicarbonate and is consistent with its potency of inducing vascularization in phenol red) immediately before use. Caution in this regard is classical angiogenesis assays. In vivo, endothelial cells are essential since angiogenin is rapidly lost from dilute (<,g/ml) exposed to concentrations of angiogenin higher than that solutions by adsorption to the walls of containers (W. F. required to elicit a cellular response; it seems likely, therefore, Heath and M. D. Bond, personal communication). Chemi- that expression of a surface receptor or some other process cally modified derivatives of angiogenin were prepared as must be rate limiting in the cellular response. described (12). [inositol-2-3H]Phosphatidylinositol 4,5-bisphosphate (Ptd- Angiogenin, secreted by HT-29 human colon adenocarci- InsP2) (4 Ci/mmol; 1 Ci = 37 GBq), L-a-[myo-inositol-2-3H] noma cells (1), is a potent inducer of vascular development in phosphatidylinositol (PtdIns) (10 Ci/mmol), and D-[inositol- the chicken chorioallantoic membrane (1, 2). The primary 2-3H]inositol 1,4,5-trisphosphate (InsP3) (3.3 Ci/mmol) were structure of this 14-kDa protein exhibits significant sequence from New England Nuclear, [5,6,8,9,11,12,14,15-3H]arachi- homology to pancreatic ribonuclease (3). Moreover, it has donic acid was from either New England Nuclear (190-240 intrinsic ribonucleolytic activity distinct from that of Ci/mmol) or Research Products International, Mt. Prospect, "'classic" ribonucleases (4). Angiogenin is a very effective IL (130-180 Ci/mmol). myo-[2-3H]Inositol was either from inhibitor of cell-free protein synthesis in the rabbit reticulo- New England Nuclear (12.8 Ci/mmol) or Amersham (16.6 cyte lysate. Inhibition is a direct result of specific angiogenin Ci/mmol). Dulbecco's modified Eagle's medium (DMEM), cleavage of ribosomal RNA and consequent inactivation of fetal bovine serum, glutamine, fungizone, and gentamicin the protein synthesis machinery (5). To date there is no sulfate were from M. A. Bioproducts, Walkersville, MD; evidence that angiogenin either inhibits protein synthesis in bradykinin was from Vega Chemical, Tucson, AZ; and vivo or enters undamaged cells; hence, the physiological L-a-1,2-dioleoylglycerol, Ptdlns, and lysophosphatidylinosi- significance of these findings remains to be clarified. tol (lyso-PtdIns) from Avanti Polar Lipids, Birmingham, AL. Several other structurally characterized proteins-nota- Endothelial cell growth supplement from bovine neural tissue bly, tumor necrosis factor, transforming growth factors a and was from Sigma. ,3, epidermal growth factor, as well as acidic and basic Cell Culture. Human umbilical vein endothelial (HUVE) fibroblast growth factor-initially identified on the basis of CRL 1730 and calf pulmonary artery endothelial (CPAE) other biological activities, have also been shown to be CCL 209 cells were obtained from the American Type angiogenic (6-8). All of these proteins have been the subject Culture Collection. Bovine adrenal capillary endothelial of extensive cell biological research and the details of their (BACE) cells isolated by published procedures (13) were a interactions with various cell types have been carefully generous gift of William Heath. CPAE cells were cultured in documented. In contrast, little is known about the interaction DMEM supplemented with 20% fetal bovine serum/2 mM of angiogenin with cells, including the specificity of this glutamine/fungizone (1 ,g/ml)/gentamicin sulfate (5 ,ug/ml). interaction, the nature of the cellular response, how this Cells between passages 16 and 23 inclusive were seeded into relates to the ribonucleolytic activity of angiogenin, and how 35-mm wells and used at confluence (4-6 days). HUVE and this ultimately leads to vascularization. In particular, it was BACE cells were cultured on gelatin-coated Petri dishes in not known whether angiogenin interacts directly or indirectly DMEM supplemented with 10% fetal bovine serum/4 mM with the capillary endothelium. The results presented here glutamine/heparin (90 ,ug/ml)/gentamicin sulfate (5 ktg/ml)/ demonstrate that angiogenin activates endothelial cell phospholipase C at concentrations well below that in normal Abbreviations: CPAE, calf pulmonary artery endothelial; BACE, bovine adrenal capillary endothelial; HUVE, human umbilical vein endothelial; PtdIns, phosphatidylinositol; PtdInsP, phosphatidylino- The publication costs of this article were defrayed in part by page charge sitol 4-phosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; payment. This article must therefore be hereby marked "advertisement" Iyso-PtdIns, lysophosphatidylinositol; InsP3, inositol trisphosphate; in accordance with 18 U.S.C. §1734 solely to indicate this fact. InsP2, inositol bisphosphate; InsP, inositol monophosphate. 5961 Downloaded by guest on September 27, 2021 5962 Cell Biology: Bicknell and Vallee Proc. Natl. Acad. Sci. USA 85 (1988)

fungizone (0.5 pug/ml)/endothelial cell growth supplement A (30 gg/ml). All cells were passaged at confluence by har- vesting with trypsin/EDTA and seeding at a 1:3 ratio. Phospholipid Labeling, Extraction, and Isolation. Cell cultures were labeled at confluence by incubation with either 0 [3H]arachidonic acid (5 ,uCi/ml) in 10% fetal bovine serum supplemented basic medium for 48 hr or with myo-[3H]- -i inositol (40 puCi/ml) in standard growth medium for 72 hr. In 0 wcm the presence of growth supplement, BACE cells were mark- 0 edly elongated; however, after 48 hr in the absence of growth O supplement they formed a characteristic endothelial cell B "cobblestone" monolayer. All experiments were performed 0 with monolayers of the cobblestone morphology. Unincor- 0 porated label was removed by washing cultures two times w with unsupplemented DMEM, followed by incubation in this z medium in 5% C02/95% air for 3 hr prior to exposure to agonist. For InsP3 determinations, the cells were incubated for a further 30 min in Hanks' balanced salt solution containing 50 mM lithium chloride. The cells were further 10 15 20 washed with Hanks' balanced salt solution immediately TIME, min before exposure to agonist in the same medium. The reaction me- FIG. 1. Time course of angiogenin-stimulated diacylglycerol was terminated by aspiration of the agonist-containing increase in CPAE cells at 37TC. The angiogenin concentration was dium, followed by immediate addition of 1 ml of organic either 1 ng/ml (A) or 500 ng/ml (B). Each point represents the mean extraction medium. Neutral lipids were extracted with ± SEM (n = 4). The amount of diacylglycerol radioactivity in chloroform/methanol (1:1) and phospholipids were extracted controls was 4250 ± 220 cpm (A) and 2750 ± 30 cpm (B). with chloroform/methanol/conc. HCl (1:2:0.05). Extracts were treated as described (14). ng/ml. Although the response decreases somewhat at higher Phospholipids were resolved by thin-layer chromatogra- concentrations of angiogenin, it remains substantial, with a phy on Whatman LK5D linear-K silica gel plates dipped in concentration of 100 ng/ml in this instance. In contrast, 1% potassium oxalate/2 mM EDTA with a chloroform/ higher concentrations of angiogenin (100-1000 ng/ml) are methanol/4 M NH40H (9:7:2) solvent (15). Neutral lipids required to induce a significant increase (75% ± 4% above were resolved on silica gel G plates (14) and were visualized controls) in the diacylglycerol level of the HUVE cell line by iodine staining, with L-a-1,2-dioleoylglycerol as standard. (Fig. 2C), and there is no evidence of a decrease in the Phospholipids were visualized by autoradiography on Kodak response at concentrations of angiogenin up to 1 u&g/ml. X-OMAT AR film after treatment with EN3HANCE (New Effect of Chemical Modification of Angiogenin on the Acti- England Nuclear). Tritiated PtdIns and PtdInsP2 (New En- gland Nuclear) and unlabeled lyso-PtdIns (Avanti Polar vation of Phospholipase C. Angiogenin was chemically mod- Lipids) (visualized by iodine stain) were used as standards. ified to abolish its ribonucleolytic activity by bromoacetate Lipids were quantitated by liquid scintillation counting. alkylation of one or both of the active site histidine residues Aqueous extracts containing labeled inositol phosphates (His-13 and His-114) (4, 12) to determine whether this were resolved by ion-exchange chromatography (16). enzymatic activity is needed to elicit a diacylglycerol response. Table 1 shows that carboxymethylation of His-13 and/or His-114 abolishes the capacity ofangiogenin to induce RESULTS an increase in CPAE cellular diacylglycerol. Angiogenin not Time Course and Dose Response of Angiogenin-Stimulated 100- A Diacylglycerol Formation in Cultured CPAE Cells. Stimula- 80 - tion of CPAE cell cultures by angiogenin (1 ng/ml) at 37°C 60- induces an increase in diacylglycerol that is detectable after 40- 30 s, peaks in 2.5 min at 59% ± 13% above controls, and 20 - returns to the resting level within 20 min (Fig. LA). The time w course for diacylglycerol formation on stimulation with much CD 100 higher concentrations of angiogenin (500 ng/ml) is similar 80 -B although the extent of the response is 10 times less (Fig. 1B). er 60 Fig. 2A illustrates the dose dependence of the diacylglycerol < 40 increase. With this cell line, the threshold for angiogenin- 20 induced diacylglycerol formation is 0.01 ng/ml, and the sX20 L-// I response peaks at 1 ng/ml and decreases sharply at higher * 100 c concentrations of angiogenin, such that only a small increase 80 in diacylglycerol levels is seen with angiogenin concentra- 60 tions of .100 ng/ml. In these experiments, bradykinin (1 40I ,ug/ml) served as a positive control agonist (17). The magni- 20I 20 / tude of the induced increases in diacylglycerol for angiogenin 0.001 0.01 0.1 1 10 100 1000 (1 ng/ml) (1140 ± 60 cpm above controls) and bradykinin (1 ANGIOGENIN, ng/ml ,ug/ml) (830 ± 70 cpm above controls) is similar. FIG. 2. Dose response of angiogenin-stimulated diacylglycerol Angiogenin-Stimulated Diacylglycerol Formation in Other increase in cultured endothelial cells at 37TC. (A) CPAE cells, 2.5 min Endothelial Cell Lines. Angiogenin also induces diacylglyce- exposure; peak (1 ng of angiogenin per ml), 2440 ± 60; control, 1300 rol formation in other endothelial cell lines. Fig. 2B shows a + 110 cpm. (B) BACE cells, 1 min exposure; peak (0.1 ng ofangiogenin dose-response curve with a capillary (BACE) cell line. per ml), 10700 ± 350; control, 7440 ± 1080 cpm. (C) HUVE cells, 2 min Again, low concentrations of angiogenin elicit a cellular exposure; peak (1 .ug ofangiogenin per ml), 3970 ± 80; control, 2270 + response, which in this case peaks with angiogenin at 0.1 290 cpm. Each point represents the mean ± SEM (n = 4). Downloaded by guest on September 27, 2021 Cell Biology: Bicknell and Vallee Proc. Natl. Acad. Sci. USA 85 (1988) 5963 Table 1. Effect of chemical modification and inhibition of decrease in PtdIns is seen over a period of at least 5 min after angiogenin on its induction of diacylglycerol in cultured exposure of CPAE cells to angiogenin (1 ng/ml) (data not CPAE cells shown). Thus, it appears that angiogenin stimulates a burst of Induced diacylglycerol (peak at 1 min, 1 ng of angiogenin per ml) by diacylglycerol, cpm phospholipase C catalyzed hydrolysis of PtdInsP2 concomi- Agonist tantly with a slower release of diacylglycerol by phospholipase Angiogenin (1 ng/ml) 2300 ± 450 C hydrolysis of PtdIns. Bromoacetate modified Fig. 3C shows that angiogenin (0.1 ng/ml) also induces a (both histidines) (1 ng/ml)* 100 ± 370 time-dependent increase in the intracellular lyso-PtdIns level Bromoacetate modified of BACE cells. A similar increase in lyso-PtdIns occurs with (one histidine) (1 ng/ml)* 430 ± 415 CPAE cells (data not shown). These observations suggest Angiogenin that, in addition to its effect on phospholipase C, angiogenin (1 ng/ml) + PRI (10 ng/ml)t 10 ± 390 activates an endothelial cell phospholipase A2, as has been Conditions: Cells were exposed to agonist for 1 min. cpm are mean proposed for bradykinin (17, 18). (above control of 2250 ± 270 cpm) ± SEM (n = 5). Effect of Angiogenin on Inositol Phosphate Release. Inositol *See ref. 12. phosphates were quantitated by column chromatography (16) tAngiogenin and placental ribonuclease inhibitor (PRI) were mixed after exposure of BACE cells to angiogenin (0.1 ng/ml). before addition to the cells. Without preincubation with lithium chloride, there is little, if any, characterizable increase in the amount of inositol phos- exposed to the modification reagent but treated in an other- phates. In contrast, after incubation for 30 min with 50 mM wise identical way elicits the usual response. Placental lithium chloride, an inhibitor ofboth inositol monophosphate ribonuclease inhibitor (10 ng/ml) also completely abolishes phosphatase and inositol polyphosphate 1-phosphatase (19, the capacity of angiogenin (1 ng/ml) to elicit a response. 20), intracellular InsP3 increases 20% (Fig. 4A), peaks at 15 Ribonuclease A, although homologous with angiogenin (3), s, and returns to baseline by 2.5 min. does not stimulate diacylglycerol formation at concentrations The transient increase in InsP3 is followed by an increase ranging from 1 to 100 ng/ml (data not shown). in inositol bisphosphate (InsP2) (Fig. 4A, peak at 30 s) and Angiogenin-Stimulated Inositol Phospholipid Turnover. Inosi- subsequently inositol monophosphate (InsP) (Fig. 4B, peak tol-containing phospholipids of BACE cell membranes were at 1 min). InsP remains elevated, presumably reflecting labeled with myo-[3H]inositol and the cellular phospholipid inhibition of inositol monophosphate phosphatase (19) by content was determined before and afterexposure to angiogenin Li+ and prolonged hydrolysis of PtdIns. It is clear that the (0.1 ng/ml) forvarious periods oftime. BACE cells were chosen inositol-specific phospholipase C is activated because (i) the for detailed study because of their substantially greater incor- successive peaks of InsP3, InsP2, and InsP follow precedent, poration of myo-inositol label compared to that of CPAE and and (ii) there is a large increase in counts in the InsP fraction, HUVE cells (data not shown). Angiogenin promotes a decrease >4000 ± 350 cpm at 1 min. The latter is regarded as the most in cellular PtdInsP2, which reaches a minimum at 1 min (an 18% reliable indicator of inositol-specific phospholipase C activa- decrease) and returns to the resting value within 2.5 min. Over tion (21). The endothelial cell agonist bradykinin promotes a the same time period, intracellular phosphatidylinositol 4- 500% increase in InsP3 (18); by comparison, the 20% increase phosphate (PtdlnsP) does not change significantly (Fig. 3A). induced by angiogenin appears small. The decrease in PtdInsP2 is accompanied by a marked decrease in intracellular PtdIns: after 1 min ofexposure to angiogenin, the Ptdlns fraction had 30,000 cpm less than that of the controls (a DISCUSSION 21% decrease). However, in contrast to PtdInsP2, the PtdIns Activation of the Endothelial Cell Inositol-Specific Phospho- level does not rapidly return to that ofunstimulated cells in the lipase C by Angiogenin. Angiogenin has been found to interact continued presence of angiogenin (Fig. 3B). A similar 20% 1.8r A 0.8 r 1.4 I \ 0.6 P2 A 1.0 I'0 I' 0.4 0.6 11 - r 0.2 15 0 0 x X 13 B E 011 1~~~~~~~~~P 1.5 I'F I ' 1.0,k LPI ., C 0.5.

1 2 TIME, min TIME, min FIG. 4. Time course of angiogenin-induced inositol phosphate FIG. 3. Time course of angiogenin-induced inositol phospholipid release in cultured BACE cells. Cells were prelabeled with myo- metabolism in cultured BACE cells. Cells were prelabeled with [3H]inositol, incubated with lithium chloride (50 mM) for 30 min, and myo-[3H]inositol and exposed to angiogenin (0.1 ng/ml) at 37°C. then exposed to angiogenin (0.1 ng/ml) at 37°C. (A) InsP3 (o) Effect on intracellular (A) PtdInsP2 (o) and PtdlnsP (o), (B) Ptdlns, (control, 700 + 25 cpm) and InsP2 (a) (control, 1055 + 70). (B) InsP and (C) lyso-Ptdlns. (control, 12,200 ± 1650). Downloaded by guest on September 27, 2021 5964 Cell Biology: Bicknell and Vallee Proc. Natl. Acad. Sci. USA 85 (1988) with cultured endothelial cells to stimulate changes in the responses are well characterized for mitogen activation of intracellular concentrations of second messengers. It induces protein kinase C (33), stimulation ofdiacylglycerol formation a rapid 2-fold increase in the intracellular level of diacylglyc- by nonmitogens does not seem to evoke similar cellular erol, a putative cellular activator of protein kinase C (22). reactions. Thus, interferon inhibits cell growth and, despite This is accompanied by a concomitant decrease in intracel- a well characterized increase in diacylglycerol (24, 34), none lular inositol-containing phospholipids and a transient 20% of the aforementioned "classic" responses to protein kinase increase in InsP3. Hydrolysis of PtdInsP2 is an early event C activation has been observed (35). Nevertheless, interfer- that leads to a small burst ofInsP3. PtdInsP2 and InsP3 return on-dependent protein kinase activities have been noted with to the resting value within 2.5 min. In contrast, a decrease in mouse 3T3-F442A and 3T3-C2 fibroblasts. Interferon stimu- PtdIns and increases in both diacylglycerol and InsP last for lation promotes cAMP-independent phosphorylation of a at least 10 min. The decrease in PtdIns could be due to (i) protein that is similar to the double-stranded RNA-dependent hydrolysis by phospholipase C, (ii) hydrolysis by phospho- eukaryotic initiation factor 2a protein kinase (36), which lipase A2 to lyso-PtdIns, or (iii) phosphorylation to PtdInsP regulates protein synthesis in rabbit reticulocytes (37, 38). It and PtdInsP2. The threshold for diacylglycerol induction appears that with nonmitogens (such as angiogenin), protein (0.001-0.01 ng/ml) is similar in all three endothelial cell lines kinase C activation phosphorylates and activates proteins examined. In contrast, the maximal response is variable, that are crucial but as yet largely unidentified. peaking at 1 ng/ml in arterial and at 0.1 ng/ml in capillary Comparison of the Effect of Angiogenin and Phorbol Esters endothelial cells, but requiring higher concentrations of on Endothelial Cells. Although angiogenin stimulates endo- angiogenin in HUVE cells (100-1000 ng/ml). Reasons for this thelial cells to increase the intracellular level of diacylglyc- variability, apart from different species of origin of the cells, erol, the putative in vivo activator ofprotein kinase C, it does are not clear. With CPAE and BACE cells, the ability to not induce changes in cell morphology. This contrasts induce an increase in diacylglycerol falls sharply with higher sharply with the effect of the "artificial" activator of protein concentrations of angiogenin. Indeed, at concentrations of kinase C, phorbol 12-myristate 13-acetate, which promotes angiogenin comparable to those in normal human plasma, striking morphological changes in capillary endothelial cells angiogenin induces only a small increase in intracellular (39). Recent work has shown, however, that phorbol esters diacylglycerol in arterial endothelial cells. elicit other cellular responses not known to be mediated by Although InsP3 is postulated to release calcium from stores protein kinase C activation (40). within the endoplasmic reticulum (23), we have been unable Protein kinase C activators-e.g., A3-phorbol 12,13-dibu- to detect an angiogenin-induced calcium flux when monitor- tyrate-suppress the stimulation of capillary endothelial cell ing fura-2 fluorescence in cultured BACE monolayers (R.B., growth induced by an angiogenic endothelial mitogen (41). It unpublished observations). Interferon stimulates InsP3 for- was suggested that this response may operate when prolif- mation in diploid fibroblasts (24) but without induction of a erating endothelial cells differentiate and organize into non- detectable calcium flux. The angiogenin-induced increase in growing tubes. The induction of diacylglycerol by angiogenin InsP3 is small (=20%) compared to that reported for calcium could similarly potentiate an endothelial cell response to mobilizing agonists such as bradykinin (-500%) (18), and angiogenic mitogens, such as fibroblast growth factor. How- possibly insufficient to stimulate a detectable calcium flux in ever, mixtures of angiogenin and acidic fibroblast growth this cell line. This contrasts with the large increases in factor are neither additive nor synergistic in the chorioallan- diacylglycerol and InsP. After exposure of the endothelial toic membrane assay (42). cell to angiogenin, diacylglycerol appears to be the major Ribonucleolytic Activity of Angiogenin and the Cellular intracellular messenger (Fig. 5). Response. The present data show that the ribonucleolytic The associated increase in lyso-PtdIns suggests that angio- activity of angiogenin is essential to elicit a cellular response. genin activates an endothelial cell phospholipase A2. How- This is in agreement with earlier observations that placental ever, a recent report failed to detect phospholipase A2 ribonuclease inhibitor abolishes both the ribonucleolytic and activity in CPAE cell extracts (25) and cautions against taking angiogenic activities of angiogenin (43). In this respect, the lyso-PtdIns formation alone as evidence for activation of endothelial cell response to angiogenin is similar to its phospholipase A2. Nevertheless, the activity of agonist- response to thrombin, which promotes an increase in stimulated cell extracts was not examined, and it is possible diacylglycerol by activating the inositol-speciflic phospholi- that detectable phospholipase A2 activity is seen only after pase C in endothelial cells (44). In the latter case, the exposure of intact cells to agonist. catalytic-proteolytic as contrasted with ribonucleolytic- Angiogenin-Stimulated Diacylglycerol Formation and Acti- activity is an absolute requirement for thrombin activation of vation of the Diacylglycerol-Dependent Protein Kinase. endothelial cell phospholipase C (45). Diacylglycerol activates protein kinase C, which phospho- Angiogenin interacts with both RNA-e.g., ribosomal rylates critical proteins, potentiating the cellular response to RNA (5)-and proteins-e.g., tight binding to placental the original agonist. Well known targets of protein kinase C ribonuclease inhibitor (43). Both interactions require an are an 80-kDa protein of unknown function (26), the epider- intact active site (46). Attempts to identify specific angiogen- mal growth factor receptor (27, 28), and the Na+/H+ in receptors on endothelial and many other cell lines have pump-either directly or indirectly (29-32). Although these shown that angiogenin binds rapidly to the cell surface. However, extensive nonspecific binding (W. F. Heath, per- ANGIOGENIN sonal communication), presumably due to the exceptionally high pI (>9.5) of angiogenin (3) and the acidic nature of the ARACHIDONATE - 1 cell surface, has made it difficult to characterize the low level + LPI ,- INOSITOL - DG PLASMA 1_ PHOSPHOLIPIDS A J MEMBRANE of specific binding. It is hoped that further work will enable characterization of putative angiogenin receptors. CYTOSOL Relevance to in Vivo Angiogenesis. The relevance of the IP(+ IP3) present data to the in vivo angiogenic activity of angiogenin is still in need ofclarification. A simplistic explanation would FIG. 5. Proposed endothelial cell response to angiogenin. The propose that the principal reservoir of angiogenin is blood mechanism of transduction is not yet characterized, as indicated by plasma, although other (including abnormal) reservoirs may dashed arrows. LPI, lyso-PtdIns; DG, diacylglycerol; PLA2, phos- yet be identified. Release of low concentrations of angiogenin pholipase A2; PLC, phospholipase C; IP, InsP; IP3, InsP3. from plasma, its normal physiological reservoir, could lead to Downloaded by guest on September 27, 2021 Cell Biology: Bicknell and Vallee Proc. Natl. Acad. Sci. USA 85 (1988) 5965

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