Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3299-3303, May 1990 Neurobiology gene-related stimulates proliferation of human endothelial cells (sensory neurons//) A. HA-GERSTRAND*t, C.-J. DALSGAARD**, B. JONZON§, 0. LARSSON¶, AND J. NILSSONII Departments of *Anatomy and lPharmacology, Karolinska Institute, Stockholm, Sweden; §Department of Clinical Pharmacology, Huddinge University Hospital, Huddinge, Sweden; and Departments of tPlastic Surgery and IlMedicine, Karolinska Hospital, Stockholm, Sweden Communicated by Viktor Mutt, December 29, 1989 (receivedfor review November 2, 1989)

ABSTRACT The effects of the vasoactive perivascular increased phosphatidylinositol (PI) turnover (6). Further- calcitonin gene-related peptide (CGRP), neuro- more, vasoactive intestinal polypeptide (VIP) has been kinin A (NKA), Y (NPY), and vasoactive intes- shown to inhibit serum-induced proliferation of rat smooth tinal polypeptide (VIP) on proliferation of cultured human muscle cells and to stimulate proliferation of human kerati- umbilical vein endothelial cells (HUVECs) were investigated. nocytes, both events paralleled with adenosine 3',5'-cyclic CGRP was shown to increase both cell number and DNA monophosphate (cAMP) formation (7, 8). synthesis, whereas NKA, NPY, and VIP were ineffective. In this study, the proliferative effect on cultured human l I-labeled CGRP was shown to bind to HUVECs and this umbilical vein endothelial cells (HUVECs) of the vasoactive binding was displaced by addition of unlabeled CGRP, sug- neuropeptides calcitonin gene-related peptide (CGRP), gesting the existence of specific CGRP receptors. The effect of NKA, (NPY), and VIP was examined by CGRP on formation of adenosine 3',5'-cycdic monophosphate determining the increase in cell number and incorporation of (cAMP) and inositol phosphates (InsP), two intracellular mes- [3H]thymidine. For comparison bFGF, an endothelial cell sengers known to be involved in regulation ofcell proliferation, mitogen (9), was used. Since CGRP and bFGF both stimu- was investigated. CGRP stimulated cAMP formation but was lated proliferation, theireffects on cAMP formation and PI without effect on the formation of InsP. Proliferation, as well turnover, two intracellular signal systems that frequently as cAMP formation, was also stimulated by cholera toxin. Basic have been found to be involved in regulation of cell prolif- fibroblast stimulated growth without affecting eration (10, 11), were examined. The proliferative effects of cAMP or InsP formation, whereas thrombin, which increased cholera toxin (CT), a potent inducer ofcAMP formation (12), InsP formation, did not stimulate proliferation. We thus sug- and thrombin, which induces breakdown ofPI (13), were also gest that CGRP may act as a local factor stimulating prolifer- investigated. ation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physi- MATERIALS AND METHODS ological and pathophysiological events such as ischemia, in- Cell Culture. HUVECs were isolated and cultured mainly flammation, and wound healing. as described by Jaffe et al. (14). Briefly, veins of fresh umbilical cords were rinsed with 50-100 ml of phosphate- Several different factors have been shown to stimulate the buffered saline (PBS) and subsequently filled with a colla- formation of new vessels, angiogenesis, and/or to be mito- genase solution (0.1%; Worthington) and incubated at 370C genic for cultured endothelial cells. These factors include for 20 min. Harvested cells were centrifuged at 800 x g for polypeptide growth factors-i.e., basic and acidic fibroblast 5 min and resuspended in culture medium-i.e., medium 199 growth factor (bFGF, aFGF)-endothelial cell growth fac- (M199; GIBCO) with addition of 20% fetal calf serum (FCS; tor, which is a precursor form ofaFGF, transforming growth GIBCO) and antibiotics (penicillin, 50 pug/ml; streptomycin, factors a and /3, angiogenin, and a (1). 50 units/ml) and cultured in gelatin-coated (0.2% gelatin in Angiogenic factors may be important when released locally PBS at +40C for 30 min) culture flasks (Costar). by cells adjacent to the endothelium, possibly also produced Cell Proliferation Assays. Secondary and tertiary cultures by the endothelial cells themselves or as circulating factors in were used for experiments. For determinations ofincrease in plasma. cell number and [3H]thymidine incorporation, HUVECs Indirect evidence has suggested trophic effects of periph- were transferred to gelatin-coated 4-well plates (1.9 cm2, 104 eral neurons. In the newt, naturally occurring limb regener- cells per well; Nunc) and 96-well plates (3 x 103 cells per well; ation is prevented by damage to the peripheral nerve inner- Nunc), respectively. HUVECs were allowed to attach over- vating the limb (2). Intact sensory innervation has been night in culture medium and were subsequently stimulated shown to be important for corneal wound healing in the rat with CGRP, NKA, NPY, VIP (all 100 nM, Peninsula; CGRP (3), and in humans suffering from Parry-Romberg syndrome, was also kindly provided by I. MacIntyre), bFGF (1 ng/ml; a marked hemifacial dystrophia is observed in the area kindly provided by California Biotechnology), CT (0.1 nM; innervated by the trigeminal nerve (4). Although direct evi- Sigma), or thrombin (1 unit/ml; kindly provided by P. T. dence from in vivo studies is essentially lacking, recent Larsson, Karolinska Institute) in assay medium-i.e., M199 studies have shown that sensory neuropeptides regulate cell containing 5% FCS and antibiotics. Cells used for studies of proliferation in vitro. (NKA) and (SP), which are structurally related, have been shown to Abbreviations: HUVEC, human umbilical cord vein endothelial cell; induce proliferation of human fibroblasts and rat smooth CGRP, calcitonin gene-related peptide; SP, substance P; NKA, muscle cells (5). This effect was shown to be parallel with neurokinin A; VIP, vasoactive intestinal polypeptide; NPY, neu- ropeptide Y; bFGF, basic ; PI, phosphati- dylinositol; InsP, inositol phosphate; CT, cholera toxin. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Anatomy, Karolinska Institute, Box 60400, S-104 01 Stockholm, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Sweden. 3299 Downloaded by guest on September 30, 2021 3300 Neurobiology: Hagerstrand et al. Proc. Natl. Acad. Sci. USA 87 (1990) DNA synthesis were serum starved for 12 hr prior to 24 hr of cAMP Formation. Tertiary cultures were grown to subcon- stimulation in assay medium containing [3H]thymidine (3 fluence in 12-well plates (4 cm2 per well; Costar) in culture puCi/ml; 1 Ci = 37 GBq; Amersham). medium. Cells were rinsed with serum-free M199 and stim- A dose-response study on the proliferative effect ofCGRP ulated with CGRP (0.01-100 nM), bFGF (1 ng/ml), or CT (0.1 (0.001-100 nM) was performed. Stimulation of[3H]thymidine nM) in 0.33 ml of serum-free M199 containing a phosphodi- incorporation by CGRP-containing (0.01-1 nM) medium pre- esterase inhibitor (Rolipram, 20 ,uM; Schering) for 15 min at incubated with a polyclonal CGRP antibody (2 hr at room 37°C. The effect of forskolin (1 uM) or forskolin (1 ,uM) plus temperature; antibody dilution, 1:100; Peninsula) was also CGRP (100 nM) on cAMP formation was also examined. examined. Reactions were terminated by addition of ice-cold HCl04 to Cell number was determined after 4 days of stimulation a final concentration of0.4 M. After addition of HCl04, cells (medium was renewed every 2nd day) by detachment of cells were frozen, thawed, and scraped, and the material was with a trypsin/EDTA (0.05%/0.01%) solution. Cells were frozen until analysis. The insoluble material was spun down suspended into single cells and counted in a cell counter and aliquots from supernatants were neutralized with KOH (Analys Instrument, Stockholm). Incorporation of [3H]thy- and Tris base and used for cAMP analysis performed essen- midine was examined in a Beckman scintillation counter after tially as described by Brown et al. (16). lysis with Triton X-100 (0.1% in distilled water). PI Breakdown. Tertiary cultures of HUVECs were grown CGRP Receptor Binding Assay. Tertiary cultures were to subconfluence and incubated for 48 hr with myo-[3H]- grown to subconfluence in 12-well plates (4 cm2 per well; inositol (10 ACi/ml; specific activity, 14.3 Ci/mmol; NEN) in Costar) in culture medium. The binding assay was performed M199 with 5% FCS. Cells were washed with PBS containing mainly as described by Heldin et al. (15). Briefly, cells were Na2HPO4 (6.5 mM), KH2PO4 (1.5 mM), KCI (2.7 mM), NaCI rinsed twice with PBS containing 0.1% bovine serum albumin (137 mM), CaCl2 (1 mM), MgCI2 (1 mM), and Hepes (2 mM). (Sigma) and triplicate cultures were incubated for 2 hr at Cells were then incubated for 10 min with 0.6 ml of the same +40C in PBS containing 1251-labeled CGRP (125I-CGRP) (1 buffer containing 10 mM LiCl and were subsequently stim- nM; 70 Bq/fmol; kindly supplied by E. Theodorsson- ulated by the addition of 66-,A aliquots of the , Norheim, Karolinska Hospital) alone or with the addition of resulting in final concentrations of CGRP (0.1 ,uM), bFGF (1 unlabeled CGRP (0.001-1000 nM). Cells were rinsed repeat- ng/ml), CT (0.1 nM), or thrombin (1 unit/ml). The reaction edly with PBS and lysed with Triton X-100 (1%) in distilled was terminated by addition of ice-cold HC104 to a final water. Radioactivity in lysates was determined in a y-counter concentration of 0.27 M. The standard procedure for sepa- (LKB). ration of inositol phosphates (InsP) was, with minor modifi- b

100 I 100 90 _ 90 > _~~~~~~~~80 OI cis 80 $1-4 70 _ 70 .= LC- 60 *** ~ -60 0 ,0- 50 - 50 (U In 40 40 30 30 -4_ 20 20 o 10 10 °

0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 CGRP, nM CGRP, nM FIG. 1. Proliferation of HUVECs examined by means of increased cell number (a), and incorporation of [3H]thymidine (b), after stimulation with CGRP (e, 0.001-100 nM), bFGF (o, 1 ng/ml), or CT (i, 0.1 nM) in M199 with addition of 5% FCS. (a) Cell number was determined after 4 days of culture (medium was renewed twice) by detachment and counting of suspended single cells. (b) Cells were serum starved for 12 hr prior to 24 hr of stimulation with the addition of3 ,uCi of [3H]thymidine per ml to the medium. Cells were lysed and radioactivity was determined in a scintillation counter. Data points are expressed as % increase [means ± SEM of triplicate (a) and quadruplicate (b) cultures] from control cultures. It was shown that CGRP increased cell number and [3H]thymidine incorporation with a maximum of 45% ± 3.3% (P < 0.01) and 53% ± 6.1% (P < 0.002) at 10 nM and 1 nM, respectively. It was also shown that the CGRP-induced increase in DNA synthesis was reduced by preincubation with a CGRP antibody (o). bFGF (1 ng/ml) increased cell number by 79% ± 6.8% (P < 0.001) and DNA synthesis by 103% ± 8.8% (P < 0.001), whereas CT (0.1 nM) induced a 58% ± 3.2% (P < 0.002) and 75% ± 11.3% (P < 0.001) increase in cell number and DNA synthesis, respectively. For statistical analysis, Student's t test was used. *, P < 0.01; **, P < 0.002; ***, P < 0.001. Downloaded by guest on September 30, 2021 Neurobiology: Hxgerstrand et al. Proc. Natl. Acad. Sci. USA 87 (1990) 3301 cations, carried out as described by Bone et al. (17). Radio- activity was measured in a scintillation counter (LKB). 0r.°c RESULTS CZ c.) 2 Cell Proliferation. CGRP, but not NKA, NPY, or VIP (all 100 nM), increased the cell number after 4 days of stimula- tion. CGRP induced a concentration-dependent increase in u: cell number as compared to control cultures with a maximal 1 effect of 45% ± 3.3% (P < 0.01) at 10 nM (Fig. la). bFGF (1 ng/ml) and CT (0.1 nM) increased cell number by 79% ± 6.8% (P < 0.001) and 58% ± 3.2% (P < 0.002), respectively (Fig. la), whereas thrombin (1 unit/ml) was ineffective (data not shown). 0.01 0.1 1 10 100 Similar results were obtained when the cells were serum CGRP, nM starved for 12 hr and subsequently stimulated in the presence of [3H]thymidine. CGRP induced a concentration-dependent FIG. 3. Formation of cAMP by HUVECs after stimulation with stimulation of [3H]thymidine uptake with a maximum of53% CGRP (0.01-100 nM), bFGF (1 ng/ml), or CT (0.1 nM). Subconfluent HUVECs were stimulated in serum-free M199 for 15 min in the ± 6.1% (P < 0.002) at 1 nM (Fig. lb). NKA, VIP, and NPY presence of agonists and the phosphodiesterase inhibitor Rolipram (all 100 nM) were ineffective in stimulating [3H]thymidine (20 uM). cAMP formation was determined on supernatants from incorporation. It was also shown that CGRP-induced [3H]- lysed HUVECs and was assayed by protein saturation essentially as thymidine uptake could be reduced by preincubating the described by Brown et al. (16). Data are presented as means ± SEM CGRP-containing culture medium with a polyclonal CGRP ofquadruplicate cultures. It was shown that CGRP (o) concentration antibody (Fig. lb). bFGF (1 ng/ml) induced a 103% ± 8.8% dependently increased cAMP formation compared to control levels. (P < 0.001) increase in DNA synthesis and CT (0.1 nM) CT (n, 0.1 nM) increased cAMP formation by 330%o ± 13.2% (P < 0.01), whereas bFGF (o, 1 ng/ml) was ineffective. cAMP formation induced an increase of 75% ± 11.3% (P < 0.001; Fig. lb), in control cultures is indicated by the horizontal line. For statistical whereas thrombin (1 unit/ml) was ineffective in stimulating analysis, Student's t test was used. *, P < 0.01. DNA synthesis. CGRP Receptor Binding. 1251-CGRP (1 nM) was shown to change cAMP formation. cAMP formation in control cultures bind to cultured HUVECs (1408 ± 125 cpm per well, corre- was 0.55 ± 0.15 pmol per 105 cells. sponding to 0.19 fmol per 105 cells). It was also shown that PI Breakdown. CGRP (100 nM), CT (0.1 nM), and bFGF (1 this binding was reduced in a dose-dependent manner when ng/ml) were all unable to significantly change InsP levels in unlabeled CGRP (0.001-1000 nM) was added (Fig. 2). When HUVECs after 15 min of stimulation, whereas thrombin (1 a 1000-fold excess of unlabeled CGRP was added 125I-CGRP unit/ml) increased InsP levels by 256% ± 32.2% (P < 0.001; binding was reduced to 40% ± 5.1% of control binding. Fig. 4). InsP formation in control cultures corresponded to cAMP Formation. It was shown that CGRP stimulated 523 dpm per 105 cells. As previously shown (13), InsP2 and cAMP formation in a concentration-dependent manner (Fig. InsP3 levels were not changed from control levels after 15 min 2). CT (0.1 nM) increased cAMP formation by 330%o ± 13.2% of stimulation (data not shown). (P < 0.01), whereas bFGF was ineffective (Fig. 3). It was also shown that the effect of CGRP (10 nM), which increased cAMP formation by 168% ± 7.2% (P < 0.01), was potentiated DISCUSSION by addition of forskolin (1 ,uM), a compound that commonly CGRP was first identified as a polypeptide encoded by the enhances receptor-mediated cAMP formation, to 703% + calcitonin gene of the rat (18), and later a human counterpart 3.7% ofcontrol cultures. Forskolin alone did not significantly was isolated and sequenced (19). In addition to thyroid tissue, 100 0 _ 10

0 "I', 1)-.~I-- 50D x S

CGRP bFGF CT Thrombin FIG. 4. Formation ofInsP after stimulation with CGRP (100 nM), 0.001 0.01 0.1 1 10 100 1000 bFGF (1 ng/ml), CT (0.1 nM), or thrombin (1 unit/ml). Subconfluent CGRP, nM HUVECs were preincubated with LiCl (10 mM) in assay buffer for 10 min and were subsequently stimulated for 15 min after addition of FIG. 2. Binding of 1251-CGRP to cultured HUVECs. Subconflu- agonists. PIs were analyzed in supernatants from lysed cells and ent tertiary cultures of HUVECs were incubated (for 2 hr, at +4°C) were separated as described by Bone et al. (17). Data are presented with 1251-CGRP alone (1 nM) or with the addition of unlabeled CGRP as means ± SEM of quadruplicate cultures. It was shown that (0.001-1000 nM). Data are expressed as means SEM of triplicate thrombin (1 unit/ml) increased InsP formation by 256% ± 32.2% (P cultures. It was shown that binding of 1251-CGRP in the absence of < 0.001), whereas CGRP (0.1 AM), bFGF (1 ng/mI), and CT (0.1 nM) unlabeled CGRP (set as 100o) was 1408 + 125 cpm per well, did not significantly change InsP formation. InsP formation in corresponding to 0.19 fmol per 105 cells, and that this binding was control cultures is indicated by the horizontal line. For statistical dose dependently reduced by addition of unlabeled CGRP. analysis, Student's t test was used. ***, P < 0.001. Downloaded by guest on September 30, 2021 3302 Neurobiology: Hxgerstrand et al. Proc. Natl. Acad. Sci. USA 87 (1990) CGRP is widely distributed within nervous tissue (20). In the suggests a role in angiogenesis, including formation of new periphery, CGRP is present in sensory and autonomic nerve vessels in ischemia, , and wound healing. fibers, of which the sensory neurons show close relationship to blood vessels (21-23). CGRP-like immunoreactive nerve We thank Professor I. MacIntyre for providing human CGRP fibers have also been shown to be intimately related to (founded by Bartos Foundation) and for valuable discussions; Dr. E. capillary loops in the papilla of the dermis and also to be Theodorsson-Norheim for providing 125I-CGRP; Dr. P. T. Larsson for highly endometrial stroma (24). providing thrombin; Ms. H. Vrang, Ms. S. Mattisson, and Ms. M. abundant in the vascularized Lind for excellent technical assistance; and Ms. E. Melander and Degradation of the basal membrane, proliferation, and colleagues at the Department of Gynecology and Obstetrics at the migration of endothelial cells is required to complete the Karolinska Hospital for their help in collecting umbilical cords. This formation of new vessels, which is a crucial step in embryo- work was supported by grants from the Karolinska Institute, the genesis, tumor neovascularization, inflammation, and heal- Swedish Medical Research Council (Grants 7126 and 7464), SRA, ing of injured tissue (1). A role of sensory neuropeptides in Marcus and Amalia Wallenberg's Memorial Foundation, T. Nilsson's these events is implicated by several findings (25). Foundation, and Knut and Alice Wallenberg's Foundation. CGRP is a potent vasodilator (26) and is released from sensory nerve fibers during ischemia (27). Experimental 1. Folkman, J. & Klagsbrun, M (1987) Science 235, 442-447. that functionally intact sensory neurons 2. Singer, M. (1952) Q. Rev. Biol. 27, 169-200. studies have shown 3. Rook, A., Wilkinson, D. S. & Ebling, F. J. G. (1968) Textbook and treatment with CGRP increase survival of ischemic of Dermatology (Davis, Philadelphia), pp. 475-488. surgical flaps (28, 29). Thus, CGRP released from peripheral 4. Converse, J. M. (1977) Plastic and Reconstructive Surgery nerve endings during ischemia may play a dual role by (Saunders, Philadelphia), 2nd Ed., pp. 610-660. increasing the blood flow and by stimulating formation ofnew 5. Nilsson, J., von Euler, A. & Dalsgaard, C.-J. (1985) Nature vessels. (London) 315, 61-63. Sensory neurons have been shown to participate in both 6. Hultgirdh-Nilsson, A., Nilsson, J., Jonzon, B. & Dalsgaard, acute and chronic inflammatory events. Capsaicin pretreat- C.-J. (1988) J. Cell. Physiol. 137, 141-145. 7. Hultgirdh-Nilsson, A., Nilsson, J., Jonzon, B. & Dalsgaard, ment, which depletes the neuropeptide content in sensory C.-J. (1988) Regul. Pept. 22, 267-274. neurons, attenuates joint injury in experimental arthritis in 8. Hwgerstrand, A., Jonzon, B., Dalsgaard, C.-J. & Nilsson, J. the rat (30). The severity of adjuvant-induced arthritis in the (1989) Proc. NatI. Acad. Sci. USA 86, 5993-5996. rat is increased by SP, which is costored with CGRP in 9. Oospodarowicz, D., Brown, K. D., Birdwell, C. R. & Zetter, sensory neurons. The severity is also correlated to the B. R. (1978) J. Cell Biol. 77, 774-788. density of sensory innervation (31). Since inflammation is 10. Boynton, A. L. & Whitfield, J. F. (1983) Adv. Cyclic Nucleo- associated with both increased turnover of sensory neu- tide Res. 15, 193-294. 11. Berridge, M. (1986) in Oncogenes and Growth Control, eds. ropeptides and vessel formation, a release of CGRP may Kahn, P. & Graf, T. (Springer, Heidelberg), pp. 147-153. contribute to the vascularization of inflamed tissue. 12. O'Keefe, E. & Cuatrecasas, P. (1978) J. Membr. Biol. 42, 61-79. During wound healing, sprouting of sensory nerve fibers in 13. Jaffe, E. A., Grulich, J., Weksler, B. B., Hampel, G. & Wa- relation to blood vessels, hair follicles, and sweat gland ducts tanabe, K. (1987) J. Biol. Chem. 262, 8557-8565. toward the wound surface has been described (32). It was 14. Jaffe, E. A., Nachmar, R. L., Becker, C. G. & Minick, R. recently shown that SP stimulates monocytes-macrophages (1973) J. Clin. Invest. 52, 2745-2756. to produce tumor necrosis factor a (33), which has been 15. Heldin, C.-H., Backstrom, G., Ostman, A., Hammacher, A., Ronnstrand, L., Rubin, K., Nister, M. & Westermark, B. shown to be angiogenic in vivo but does not stimulate (1988) EMBO J. 4, 1387-1393. proliferation in vitro (34). It is thus an intriguing possibility 16. Brown, B. C., Albano, J. B. M., Ekins, R. P., Sgherzi, A. M. that sprouting sensory neurons may be important for both & Tampion, W. (1971) Biochem. J. 121, 561-562. directional guidance, via activation of macrophages, and 17. Bone, E. A., Fretten, P., Palmer, S., Kirk, C. J. & Michell, proliferation of endothelial cells. R. H. (1984) Biochem. J. 221, 803-811. The intracellular mechanisms involved in stimulation of 18. Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S. & HUVEC proliferation are at present not known. In human Evans, R. M. (1982) Nature (London) 298, 240-244. dermal capillary cells, CT and the phosphodiesterase inhib- 19. Morris, H. R., Panico, M., Etienne, T., Tippins, J., Girgis, S. T. & MacIntyre, I. (1984) Nature (London) 308, 746-748. itor isobutylmethylxanthine have been shown to stimulate 20. Rosenfeld, M. G., Mermod, J.-J., Amara, S. G., Swanson, cell proliferation (35). L. W., Sawchenko, P. E., Rivier, J., Vale, W. W. & Evans, Activation of protein kinase C in capillary cells has been R. M. (1983) Nature (London) 304, 129-135. shown to induce elongation of the cells and to reduce the 21. Hanko, J., Hardebo, J. E., Kahrstrom, J., Owman, C. & mitogenic response induced by growth factors (36). In this Sundler, F. (1985) Neurosci. Lett. 57, 91-95. context, it is interesting to note that CGRP has been shown 22. Lundberg, J. M., Franco-Cereceda, A., Hua, X., Hokfelt, T. & to activate adenylate cyclase in different cells, including Fischer, J. A. (1985) Eur. J. Pharmacol. 108, 315-319. HUVECs (37). In porcine aortic endothelial cells, however, 23. Gibbins, I. L., Furness, J. B., Costa, M., MacIntyre, I., Hill- endothelial cell growth factor and thrombin, which both yard, C.-J. & Girgis, S. (1985) Neurosci. Lett. 57, 125-130. 24. Kruger, L., Silverman, J. D., Mantyh, P. W., Sterini, C. & stimulate PI breakdown, also appear to be mitogenic (38, 39). Brecha, P. W. (1989) J. Comp. Neurol. 280, 291-302. The present findings suggest that cAMP formation rather 25. Dalsgaard, C.-J., Hultgardh-Nilsson, A., Haegerstrand, A. & than PI breakdown is associated with proliferation of HU- Nilsson, J. (1989) Regul. Pept. 25, 1-9. VECs. Whether increased cAMP formation per se stimulates 26. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. & proliferation or the effect is indirect by potentiating a stim- MacIntyre, I. (1985) Nature (London) 313, 54-56. ulatory effect offactors in serum is not known. However, the 27. Franco-Cereceda, A., Saria, A. & Lundberg, J. M. (1989) Acta presence of serum factors may be regarded as a natural Physiol. Scand. 135, 173-187. environment for endothelial cells both in vivo and in vitro. 28. Kjartansson, J. & Dalsgaard, C.-J. (1987) Eur. J. Pharmacol. has been shown to be for develop- 142, 355-358. CGRP recently trophic 29. Kjartansson, J., Dalsgaard, C.-J. & Jonsson, C.-E. (1986) Plast. ment of the nicotinic receptor at the motor end plate (40) and Reconstr. Surg. 79, 218-221. also to act as a differentiating factor by inducing a dopami- 30. Levine, J. D., Dardick, S. J., Roizen, M. F., Helms, C. & nergic phenotype in the mouse olfactory bulb (41). In addition Basbaum, A. I. (1986) J. Neurosci. 6, 3423-3429. to the previously shown effects of CGRP, our findings 31. Levine, J. D., Clark, R., Devor, M., Helms, C., Moskowitz, M. suggest that CGRP may also act as a local factor stimulating & Basbaum, A. 1. (1984) Science 226, 547-549. cell proliferation. The effect of CGRP on endothelial cells 32. 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