Nerve growth factor control of neuronal expressionof angiogenetic and vasoactive factorsLaura Calza*†‡, Luciana Giardino†‡, Alessandro Giuliani†, Luigi Aloe§, and Rita Levi-Montalcini§

†Department of Veterinary Morphophysiology and Animal Production, University of Bologna, 40064 Ozzano Emilia, Bologna, Italy; ‡Pathophysiology Centerfor the Nervous System, Hesperia Hospital, 41100 Modena, Italy; and §Institute of Neurobiology, Consiglio Nazionale delle Ricerche, 00137 Rome, Italy

Contributed by Rita Levi-Montalcini, December 29, 2000

In postnatal tissues, angiogenesis occurs in nontumoral conditionson appropriate stimuli. In the nervous tissue, hypoxia, neural graft,increased neural function, and synaptic activity are associated withneoangiogenesis. We have investigated the occurrence of neoan-giogenesis in the superior cervical ganglia (scg) of newborn ratstreated for 8–21 days with 6-hydroxy-dopamine (6-OHDA), nervegrowth factor (NGF), or 6-OHDA 1 NGF. The two latter treatmentsinduced a significant increase in scg size. However, the increaseafter combined treatment far exceeded that of NGF alone. Simi-larly, histological and histochemical analysis revealed neuronalhypertrophy and endothelial cell hyperplasia associated with stro-mal hypertrophy (as described by laminin immunostaining) andincreased vascular bed (as revealed by plateletyendothelial celladhesion molecule-1 immunostaining) in 6-OHDA 1 NGF-treatedpups. NGF, either alone or associated with 6-OHDA, also induced asignificant up-regulation of NADPH diaphorase, neuronal nitricoxide synthase, and vascular endothelial growth factor expressionin scg neurons. The present investigation suggests that the in-crease of scg size induced by NGF and 6-OHDA 1 NGF is associatedwith neoangiogenesis, and that the induction of vasoactive andangiogenic factors in neurons represents a further and previouslyundisclosed effect of NGF.

Nerve growth factor (NGF) is a neurotrophin that plays acrucial role in promoting growth, differentiation, and

function in sympathetic nerve cells (1). In recent years, NGFhas been described as a ‘‘pleiotropic’’ molecule (2), involved ina variety of peripheral actions such as tissue inf lammation (3),neuropeptide expression regulation (4), skin physiology (5),and peripheral tissue regeneration (6, 7). Furthermore, target-derived NGF, which binds to its low- and high-affinity recep-tors on nerve terminals and is retrogradely transported toneural sensory and sympathetic perikarya, is known to preventnatural cell death and to promote and modulate peripheralinnervation (8–10).

Previous findings by Levi-Montalcini et al. (11) showed thatNGF administration prevents nerve cell degeneration inducedby 6-hydroxy-dopamine (6-OHDA), a neurotoxic compoundthat causes a selective but reversible destruction of sympatheticnerve endings in adult animals and widespread lesions in the cellbody and neuronal cell death in newborn animals (12). Thecombined administration of 6-OHDA and NGF treatment indeveloping rodents results in an impressive growth of sympa-thetic ganglia because of an increase in the number and size ofpostganglionic neurites as well as glial and satellite cells (11, 13).These experiments led also to the still untested hypothesis thatNGF might influence endothelial cell proliferation and thatcombined treatment would magnify this effect.

Recent evidence indeed suggests that NGF alone or in com-bination with other biologically active endogenous molecules canexert its action on endothelial cells and most probably onangiogenic activity (14, 15). The known effects of NGF (16) oncells of the immune system [macrophages, mast cells (3)], onconnective tissue [fibroblast (17, 18) and osteoblasts (19)], onendothelial cells (20), and in tissue homeostasis and the wide-spread distribution as well of NGF receptors in peripheral tissues

(21) support this hypothesis. It has also been suggested that inperipheral sensory neurons, NGF stimulates the production ofvascular endothelial growth factor (VEGF), the most powerfulmitogen for endothelial cells (15).

Despite this extensive research, the role of NGF in theregulation of endothelial cells remains hypothetical. The aim ofthis research was to explore the influence of NGF on angiogenicmechanisms; we have therefore analyzed the effect of treatmentwith NGF and NGF 1 6-OHDA on the distribution of thebiological markers differentially involved in neoangiogenesisregulation in newborn rats. Here we report that vessel densityincreases in superior cervical ganglia of newborn rats treatedwith 6-OHDA 1 NGF, associated with increased expression ofthe angiogenic peptide VEGF and neuronal nitric oxide synthase(nNOS) in neurons. The result suggests that angiogenesis may beregulated through activation of angiogenic and vasodilatationagents directly produced by neurons on request, and NGFindirectly stimulates this process.

Materials and MethodsNGF Preparation and Animal Treatment. NGF was prepared frommouse submaxillary glands according to the method of Bocchiniand Angeletti (22). Fresh 6-OHDA (Sigma) solution in distilledwater containing 0.1 mgyml of ascorbic acid to prevent oxidationwas prepared shortly before use. Pregnant rats were purchasedfrom Charles River Italia (Calco, Varese, Italy) and housed insingle cages on arrival. A total of 40 newborn rats were treated.As soon as delivered, each litter was culled to eight pups, whichwere distributed into four groups of two rats each. The groupswere injected daily as follows: group 1 with NGF 10 mgyg of bodyweight (b.w.) in the morning and 6-OHDA 100 mgyg of b.w. inthe evening; group 2 with NGF 10 mgyg b.w. in the morning;group 3 with 6-OHDA 100 mgyg of b.w. in the evening; and group4 with saline 0.1 ml in the morning.

Animals from the four experimental groups were killed 8 or21 days after the beginning of treatment. According to pro-cedures for light microscopy studies, the animals were killed bycervical translocation; superior cervical ganglia (scg) waseither dissected under a stereomicroscope, fixed in alcoholicBouin and mounted in toto, or embedded in Paraplast (CarloErba, Milano, Italy), cut into 8-mm thick sections, and stainedwith toluidine blue. Few animals received a single adminis-tration of the above-mentioned treatments.

Immunocytochemistry. At the 8th postnatal day, pups from allexperimental groups were anaesthetized and perfused via theascending aorta with saline solution followed by 4% parafor-

Abbreviations: NGF, nerve growth factor; 6-OHDA, 6-hydroxy-dopamine; VEGF, vascularendothelial growth factor; nNOS, neuronal nitric oxide synthase; scg, superior cervicalganglia; PECAM-1, plateletyendothelial cell adhesion molecule-1; IR, immunoreactivity.

*To whom reprint requests should be addressed at: Department of Veterinary Morpho-physiology and Animal Production, University of Bologna, Via Tolara di Sopra 50, 40064Ozzano Emilia, Bologna, Italy. E-mail lcalza@vet.unibo.it.

The publication costs of this article were defrayed in part by page charge payment. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.§1734 solely to indicate this fact.

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maldehyde. scg was dissected under a stereomicroscope, im-mersed in ice-cold 4% paraformaldehyde fixative for 24 h, andthen rinsed in ice-cold 0.1 M Sorensen’s buffer 0.2 M, pH 7.5containing 5% sucrose for 48 h. After rinsing, scg was quicklyfrozen in CO2 and cut by using a Leitz cryostat 1720 (sections14-mm thick; 220°C). Sections were then collected on glass slidescoated with chrome alum and gelatin and immediately processedfor the immunofluorescence procedure. Slides from all animalswere run in the same assay. The sections were first incubated in0.1 M PBS at room temperature for 10–30 min, followed byincubation at 4°C overnight in humid atmosphere with theprimary antisera [mouse anti-NGF low-affinity receptor p75antibody, clone IgG192, generously supplied by E. M. Johnson(Washington University, St. Louis), diluted 1:250; rabbit anti-laminin (Sigma), 1:25; goat antiplateletyendothelial cell adhe-sion molecule-1 (PECAM-1) against a peptide mapping at thecarboxy terminus of mouse PECAM-1 (Santa Cruz Biotechnol-ogy), 1:300]. The staining specificity was assessed by overnightpreincubation in vitro of the antiserum with the respectiveantigen (100 mg of antigenyml diluted antiserum). This treat-ment prevented staining. The primary antibodies were diluted inPBS containing 0.3% Triton X-100, volyvol. The sections werethen rinsed in PBS for 30 min (3 3 10 min) and incubated at 37°Cfor 120 min in a humid atmosphere with fluorescein isothiocya-nate-conjugated anti-mouse, anti-rabbit, or anti-goat Ig (Dako)containing 0.3% Triton X-100. They were again rinsed in PBS asabove, mounted in glycerol and PBS (3:1, volyvol) containing0.1% 1,4-phenylenediamine, and finally examined by using aNikon Microphot FXA microscope equipped with a 100-Wmercury lamp and B-2A filter combination.

Histopathology. Staining techniques for histological studies in-cluded toluidine blue, cresyl violet for Nissl staining, Mallory–Azan reaction, and hematoxylin-eosin staining. Visualization ofNADPH–diaphorase activity was obtained by incubation ofcryostat sections in 0.1 M phosphate buffer, pH 8.0, containing1 mM NADPH (Sigma), 2 mM nitro blue tetrazolium (Sigma),and 0.3% Triton X-100 at 37°C (17). Duration of the incubation(60–120 min) was determined by staining intensity. The reactionwas terminated by washing in 0.1 M PBS solution. The sectionswere then thoroughly washed in distilled water, air-dried, andfinally coverslipped with Eukitt (Kindler, Freiburg, Germany).

Image Analysis. The AIS Analytical Imaging Station ImagingComputer Research system (St. Catherines, ON, Canada) wasused for image analysis. Images of the processed sections wereloaded via a VarioCam PCO Computer Optics, Kelheim, Ger-many, charge-coupled device (CCD) camera. For quantitativeanalysis of the histochemical composition of ganglia (lamininand PECAM-1 immunoreactivity (IR) and Nissl-stained neu-rons) 320 objective was applied; digitalized images were thencontrast-enhanced to clearly differentiate positivity from back-ground, and a thresholding procedure was established to deter-mine the proportion of immunoreactive area in a constant areaof each section (512 3 512 pixels). Sections with anti-VEGF andNADPH-diaphorase staining were also loaded via a VarioCamCCD camera. The system was calibrated for density values, withdensity level 255 corresponding to 0.017 density relative opticaldensity (ROD) and density level 0 corresponding to 2.4 densityROD.

Statistical Methods. The animal sample was divided into fourexperimental groups, into which newborn rats from all litterswere randomly allocated and evenly distributed. Caring, housing,and feeding as well as treatment and experimental procedureswere identical for all animals studied. Allocation to eithercategory of treatment duration also occurred randomly. Scg sizewas expressed as neuronal area. The quantitative histochemical

analysis (laminin and PECAM-1 IR and Nissl-stained neurons)was carried out on three nonconsecutive sections from five areasof each ganglia from at least five pups in each experimentalgroup. Mean values of the proportion of immunoreactive area ina constant area of each section were used for statistical analysis.Twenty to twenty-five cells were measured in each ganglion forstaining intensity evaluation.

Descriptive analysis (mean values and standard error) of allrelevant variables was performed. Statistical analysis for com-parison of treatments was performed by one-way ANOVAfollowed by Dunnett’s test.

ResultsThe effect of treatment on growth of scg is illustrated in Fig. 1.In accordance with what is already described (11, 13), chemicalsympathectomy by 6-OHDA dramatically reduced scg size,whereas NGF treatment prevented reduction. Moreover, sgc wasfound to be larger in NGF-treated rats compared with controlanimals. scg grew even larger when combined treatment wasadministered. Mean neuronal area (mm2) measured 202.5 1 38.2(SD) in control rats and 247.1 1 47.1 in NGF-treated animals(P , 0.01). Coadministration of NGF and 6-OHDA not onlyprevented shrinkage of neurons but also further induced hyper-plasia (mean 1 SD, mm2: 6-OHDA 134.6 1 47.4; 6-OHDA 1NGF 280.4 1 40.3, P , 0.001). Neuron size, but not generalmorphology, was affected by treatment, as indicated by formfactor measurements (data not shown).

Gross Histology. Coadministration of NGF and 6-OHDA deter-mined an impressive growth of the stromal component in scg.Large nests of connective tissue were observed in pups treatedwith 6-OHDA 1 NGF for 21 days (Fig. 1D), whereas only veryfine trabeculae of connective tissue intermingled with neuronswere observed in control rats (Fig. 1B). The vascular componentof double-treated rats included mature elements and also vesselscharacterized by very high cellularity of the wall, with roundnuclei and buds indicating growth of neo-formed vessels (Fig. 1D and H–M). In pups treated with NGF, numerous mast cellswere observed around vessels. It is important to point out that,as already reported (11, 13), all cellular elements had a com-pletely normal individual morphology, and no signs of malignantproliferation were apparent.

Quantitative Immunohistochemistry. To investigate histologicalcomposition of scg after NGF and NGF 1 6-OHDA treatment,proteins of the extracellular matrix were analyzed. The experi-ments were performed after 8 days treatment, when the histo-logical effects were less pronounced but already present. Lami-nin immunostaining, which identifies basal membrane infibroblasts, neurons, and endothelial cells, labeled all areas of theganglia not occupied by neurons (Fig. 2). Quantitative evaluationof Nissl staining compared with laminin immunoreactivity (IR)confirmed that the cellular component did not completelyaccount for the ganglionic hypertrophy observed (Fig. 2, histo-grams), suggesting therefore an increase of the stromal compo-nent too. The vascular bed was also investigated by usingPECAM-1 as a marker (Fig. 2). In animals undergoing doubletreatment and in discrete areas of the scg, the percent areacovered by the PECAM-IR signal increased, indicating a higherdensity of the vascular component (Fig. 2, histograms). Labelingfor the NGF low-affinity receptor p75 was also up-regulated byNGF and double treatment as well, as shown in Fig. 2.

We investigated also the expression of angiogenetic andvasodilator agents to test for a regulatory effect on neoangio-genesis; in particular, the expression of VEGF and the neuralisoform of NO synthase were analyzed. We observed that neuralproduction of VEGF was up-regulated by NGF and 6-OHDA 1NGF after 8 days of treatment, whereas one single NGF

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administration produced no detectable change (Fig. 3 Upper andgraph bars). The intracellular level of enzymes associated withNO synthesis was also strongly up-regulated by NGF, as indi-cated by the level of nNOS expression (data not shown) andNADPH–diaphorase staining (Fig. 3 Lower and graph bars). Asingle NGF administration exerted a more powerful effect thana protracted 8-day administration; 6-OHDA coadministrationfurther potentiated NGF effect.

DiscussionIt has been previously shown that combined NGF and 6-OHDAtreatment in neonatal rats induces a paradoxical increase of scgsize as a result of enormous growth of postganglionic neuritesand, to a much lesser extent, of glial and satellite cells. In the

present study, we investigated the effect of NGF, alone or incombination with the neurotoxin 6-OHDA, on neoangiogenesismarkers. The results of this study show that NGF treatmentinduces impressive growth of the stromal component and de-termines vascular neoformation in sympathetic ganglia. Theseeffects are reinforced by 6-OHDA coadministration, thus sug-gesting that NGF effects are activated by lesioning. Concomi-tantly, NGF increases VEGF and nNOS expression andNADPH–diaphorase staining in neurons within the scg.

NGF and Histological Components. The increase of the stromalcomponent of sympathetic ganglia on NGF and NGF 16-OHDA administration is well illustrated by laminin immu-nostaining, a marker for the basal membrane in most cell types

Fig. 1. (A) Whole-mount preparation of superior cervical ganglia of littermate rats (26 days) treated with NGF and 6-OHDA, NGF, 6-OHDA, and saline. 6-OHDAadministration induces a well known degeneration of the sympathetic neurons, which, on the contrary, is prevented by NGF treatment. scg are larger inNGF-treated pups than in control pups; however, growth induced by double treatment is impressive. Toluidine-blue micrographs show general morphology incontrol (B), NGF (C), and 6-OHDA 1 NGF- (D) treated pups. High-magnification micrographs show intragangliar blood vessels in controls (E), NGF-treated (F andG), and 6-OHDA 1 NGF-treated (H–M) pups. In the latter group, within the vessel and capillary wall, high cellularity can be observed, and buds of newly formedvessels are clearly detectable (arrow, H). (Bars 5 B–D, 200 mm; E–M, 100 mm.)

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(23). NGF receptors have been described in stroma in differentorgans, such as human prostate tissue (24), cornea (25, 26),synovial tissue (17), and bone marrow (27). However, lamininalso stimulates growth of nerve process and favors NGF actionin vivo and in vitro. Indeed, the addition of laminin to in vitrosystems significantly enhances neurite extension and stronglystimulates NGF action in cultured dorsal root ganglia (28) andsuperior cervical ganglia (29). The finding that the expressionof p75 increased in both neurons and extracellular elementsafter NGF administration, alone or in association with6-OHDA, is particularly meaningful. This increase is notassociated with neuronal death; on the contrary, neuronsappear to be numerous and hypertrophic, with no histologicalmarks of degeneration.

NGF and Neuronal Component of scg. The effect of NGF alone andas double treatment on regulation of vasoactive and angioge-

netic factors was investigated by the assessment of VEGF andNO production in scg. VEGF is a secreted mitogen specific forthe vascular endothelium; it is known to exert a pivotal role innormal and pathological angiogenesis (30). NO is consideredthe link between neural activity and activity-dependent re-gional vasodilatation (31). We have observed that, whereasVEGF production by sympathetic neurons is very low incontrol animals, it is strongly stimulated by NGF administra-tion. Moreover, this effect is enhanced by the concomitantadministration of 6-OHDA. It has been shown that neuralproduction of VEGF in the developing nervous tissue corre-lates with vasculogenesis (32) and that at birth, VEGF pro-duction switches from neurons to astrocytes (33). VEGFexpression in the peripheral nervous system is also stimulatedby some pathological conditions, such as diabetic neuropathy(15) and, in mature central nervous systems, by hypoxia andischemia (34, 35). Moreover, when exogenously applied,VEGF proves to exert a neurotrophic effect on cultured adultmouse scg, measured as axonal outgrowth (36). NO productionis also up-regulated by NGF, the NGF effect being extremelypowerful and fast of onset.

NGF and Neoangiogenesis. The demonstration of induction ofpostnatal angiogenesis by NGF represents the most interestingresult of this research. This effect is well documented in prep-arations featuring toluidine blue staining and immunostainingfor PECAM-1. Angiogenesis, the sprouting of new capillariesfrom preexisting blood vessels, is complete around postnatal day20 (37) and is inhibited in mature tissues. However, severalnontumoral conditions can trigger neoangiogenesis in nervoustissue by tilting the balance between angiogenic and angiostaticfactors toward activation. Adaptation of brain circulation tosevere chronic hypoxia is achieved by both angiogenesis andmicrovascular hypertrophy (38, 39). Angiogenesis has beenshown to occur also in nerve cell grafts (40) and on spinal cordlesion (41), during peripheral nerve regeneration (42). Angio-genesis is also triggered by physiological tasks that increaseneural function and synaptic activity (43). Overall, these dataindicate that migration and proliferation of endothelial cells,remodeling of extracellular matrix, and functional maturation ofnewly assembled vessels can be triggered also in postnatalnontumoral nervous tissue.

This study suggests that angiogenesis is triggered by theincrease of scg size because of NGF and NGF 1 6-OHDAtreatment. We also suggest that in our experimental conditionsa ‘‘neural drive’’ for angiogenesis might occur in postnatalnervous tissue, correlated with increased metabolic request, andthat survival factors, like NGF, act to promote it. We are of theopinion that NGF promotes neuron-induced angiogenesis bystimulating VEGF production.

A number of stimuli may induce VEGF expression, includ-ing several growth factors and cytokines, hormones, phorbolesters, oncogenes, NO, and hypoxia through hypoxia-induciblefactor (44). In addition, neuronal VEGF expression has beenfound to correlate with angiogenesis (45). Moreover, brain-derived neurotrophic factor, a member of the neurotrophinfamily, also seems to exert a direct angiogenic action onendothelial cells in the heart (46), whereas local NGF admin-istration has proved to increase angiogenesis during sciaticnerve regeneration (47).

The effect of NGF on NO production also supports ourhypothesis, because a single NGF injection induces a strongup-regulation of the neuronal isoform of NO synthesis enzymeand of the associated histochemical NAPDH–diaphorase reac-tion. This is followed by VEGF-induced angiogenesis and con-sequently by a reduction of NO synthesis enzyme production. Weacknowledge that early in hypertrophy induction, a NO-mediated vasodilation allows metabolic supply to occur, whereas

Fig. 2. Histochemical composition of scg after treatment. The figure showslaminin, p75, and PECAM-1 IR in scg of controls (Left) and pups treated for 8days with 6-OHDA associated with NGF (Right). The graph shows the quanti-tative histochemical composition. Data are expressed as area covered by theimmunoreactive signal inside a standard sampling window (proportionalarea). As also seen in micrographs, in the 6-OHDA-treated group laminin IRoccupies the largest proportional area because of the very low presence ofneuronal component. In double treatment preparations, a true increase of thestromal component can be measured. p75 IR can be detected intra- andextracellularly: the percentage related to its proportional area increases inNGF and 6-OHDA 1 NGF-treated pups. PECAM-1 IR depicts nets of newlyformed vessels in double-treated pups. An overall increase of the percent ofarea covered by PECAM-1 IR can be measured. Statistical evaluation: ANOVAand Dunnett’s test; *, P , 0.05. [Bars 5 100 (laminin also for p75) and 50(PECAM-1) mm.]

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later, VEGF-mediated angiogenesis can provide adequate met-abolic supply. This pattern of coordinated actions between NOand VEGF has been demonstrated in different tissues (48, 49),under different physiological (50) and pathological nontumoral(51) conditions.

In conclusion, our data show an increased vessel density insuperior cervical ganglia of newborn rats treated with6-OHDA 1 NGF; this effect is associated with increasedexpression of the angiogenic peptide VEGF and nNOS inneurons. The results suggest that angiogenesis may be regu-lated through activation of angiogenic and vasodilation agents

directly produced by neurons on request, and that NGFindirectly stimulates this process.

The technical assistance of Nadia De Sordi (Department of VeterinaryMorphophysiology and Animal Production, University of Bologna) isgratefully acknowledged. This work was supported by the Pathophysi-ology Center for the Nervous System, Hesperia Hospital, Modena, Italy.The contribution of L.A. was supported by PF5 subproject 3 Biotec-nologie, Consiglio Nazionale delle Ricerche, to R.L.-M. and L.A. NGFwas prepared by Luigi Manni, a recipient of a grant from the Levi-Montalcini Foundation.

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Fig. 3. VEGF IR and NADPH–diaphorase staining in neurons in scg of controls and newborn rats treated with NGF (1 and 8 days) and 6-OHDA 1 NGF (8 days)and density evaluation. Eight days of NGF treatment induces a significant increase in VEGF expression; a greater increase occurs after concomitant 6-OHDA andNGF administration. Single NGF administration strongly up-regulates NADPH–diaphorase staining, but the increase induced by 8 days of combined 6-OHDA andNGF treatment far exceeds that of NGF single administration. Statistical analysis: ANOVA and Dunnett’s test; *, P , 0.05. (Bar 5 100 mm.)

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