neuropeptide y expression, localization and cellular transducing effects in huvec

Biol. Cell (2005) 97, 457–467 (Printed in Great Britain) Research article

Neuropeptide Y expression,localization and cellular transducingeffects in HUVECAntonio P. Silva*1, Jocelyne E. Kaufmann†, Cecile Vivancos‡, Stanislav Fakan‡, Claudia Cavadas*, Phillip Shaw§,Hans R. Brunner*, Ulrich Vischer† and Eric Grouzmann‖2

*Division d’Hypertension et de Medecine Vasculaire, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, †Division de Biochimie

Clinique, Centre Medical Universitaire, Geneva, Switzerland, ‡Centre de Microscopie Electronique de l’Universite de Lausanne, Lausanne,

Switzerland, §Unite de Pathologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, and ‖Division de Pharmacologie et

Toxicologie Cliniques, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

Background information. NPY (neuropeptide Y) may have an effect on the properties of vascular endothelial cellssuch as pro-angiogenic effects and potentiation of noradrenaline-induced vasoconstriction. In HUVEC (humanumbilical-vein endothelial cells), immunoreactive neuropeptide Y has been detected, but NPY synthesis, storageand secretion have not been studied. The aim of the present study was to establish NPY expression, storage andcellular transducing effects in HUVEC.

Results. HUVEC contain 0.19 fmol of NPY/µg of protein and 0.46 fmol of pro-NPY/µg of protein, as measured byELISA. RT (reverse transcriptase)–PCR confirmed the expression of NPY in HUVEC. Immunofluorescence revealedthe presence of NPY in small punctate structures, with a fluorescence pattern different from that observed for vonWillebrand factor, indicating distinct storage compartments. Double labelling for NPY and Rab3A demonstratedsimilar granular patterns, with at least partial co-localization. Electron microscopy showed NPY immunoreactivityin vesicle-like cytoplasmic structures, of a fine fibrillar texture, as well as in mitochondria and in the nucleus. Asimilar general distribution pattern was also obtained for Rab3A. Y1 and Y2 receptors were expressed in HUVECas assessed by RT–PCR, and they were functional since NPY induced a 42 nM intracellular calcium increase within100 s, representing 22% of the histamine-induced response. In contrast with histamine, NPY did not induce acutevon Willebrand factor secretion.

Conclusions. HUVEC produce, store and respond to NPY, suggesting an autocrine regulatory role for NPY in theendothelium.

IntroductionNPY (neuropeptide Y) is a 36-amino-acid peptide in-volved in the regulation of the cardiovascular system.

1Present address: Novartis Institutes for Biomedical Research, Basel,Switzerland.2To whom correspondence should be addressed ([email protected]).Key words: calcium, cytoplasmic vesicle, endothelial cell, Rab3A, WeibelPalade bodies.Abbreviations used: EC, endothelial cell; EEA1, early endosome-associatedprotein-1; HMVEC-L, human lung microvascular endothelial cells; HUVEC,human umbilical-vein endothelial cells; IBMX, isobutylmethylxanthine; NPY,neuropeptide Y; PSS, physiological saline solution; RT, reverse transcriptase;SMC, smooth muscle cell; SP, substance P; vWF, von Willebrand factor; WP,Weibel–Palade.

It has vasopressor effects and potentiates the effect ofother vasoconstrictor molecules such as noradrenalineor histamine (Dumont et al., 1992; Franco-Cerecedaand Liska, 1998; Schuerch et al., 1998). When used atlow, non-vasoconstrictive doses on cultured vascularSMCs (smooth muscle cells), NPY stimulates SMCproliferation, an effect potentiated by noradrenaline(Erlinge et al., 1994; Zukowska-Grojec et al., 1998a).NPY also acts on vascular ECs (endothelial cells). Thepotentiating effect of NPY on noradrenaline-inducedvasoconstriction has been shown to be endothelium-dependent on human saphenous veins (Fabi et al.,1998). NPY is capable of promoting EC proliferation,

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A.P. Silva and others

migration and adhesion on the extracellular matrix.It also stimulates capillary tube formation in vitro andangiogenesis in vivo (Zukowska-Grojec et al., 1998b).Similar to other secreted peptides, NPY is producedas a pre-pro-peptide. After removal of the signal pep-tide in the endoplasmic reticulum, pro-NPY is fur-ther cleaved by successive enzymes to generate thebiologically active amidated NPY. In neuroendocrinecells, mature NPY is localized in secretory gran-ules, e.g. in neurons (Adrian et al., 1983), chromaffincells (Cavadas et al., 2001) or in the pituitary (Morelet al., 1985). Immunoreactive NPY has been detectedin HUVEC (human umbilical-vein endothelial cells;Cai et al., 1993a, b), but NPY synthesis, storage andsecretion have not been studied.

Several molecules released from ECs [e.g. vWF(von Willebrand factor), interleukin-8 and tissue-type plasminogen activator] are stored in theWP (Weibel–Palade) bodies (Wagner et al., 1982;Bonfanti et al., 1989; Rosnoblet et al., 1999). Thecontent of these granules is released in responseto an increase in intracellular calcium or cAMP.There is circumstantial evidence to suggest an ad-ditional protein storage compartment in ECs. In-deed, vasoactive substances such as atrial natriureticpeptide, calcitonin gene-related peptide, vasoactiveintestinal peptide, SP (substance P), endothelin-1and NPY have been shown to be present in ECs(Loesch et al., 1992; Cai et al., 1993a, b; Sextonet al., 1996), suggesting the production and/or endo-cytosis of these peptides by ECs. ATP, SP and acetyl-choline can be rapidly released from HUVEC byincreased shear stress (Milner et al., 1990). At leastsome of these proteins may be synthesized, stored inand released from ECs. However, their synthesis andsubcellular localization have not been investigated indetail.

The aim of the present study was to establishclearly the expression of NPY at the mRNA and pro-tein levels, as well as its subcellular localization byimmunocytochemistry in HUVEC. NPY action onHUVEC was also studied by measuring the increasein intracellular calcium ([Ca2+]i) and vWF secretionafter stimulation of the cells with NPY. Our resultsshow that NPY is expressed by HUVEC and thatNPY is localized in the nucleus, cytoplasm, mito-chondria and in a vesicle-like structure in HUVEC.Furthermore, exogenous NPY is capable of inducingan increase in intracellular calcium in HUVEC.

ResultsNPY is produced by ECsIntracellular NPY and pro-NPY contents were mea-sured by ELISA. HUVEC contained 0.19 fmol ofNPY/µg of protein and 0.46 fmol of pro-NPY/µgof protein. To determine whether the NPY was pro-duced by HUVEC, RT (reverse transcriptase)–PCRwas performed on total RNA. NPY mRNA was de-tected in all the HUVEC cultures tested as well as inHMVEC-L (human lung microvascular endothelialcells; Figures 1A and 1B). Labelling of NPY and pro-NPY with the highly specific monoclonal antibodyNPY02 revealed the presence of NPY and pro-NPYin HUVEC, appearing as small punctate structuresdisseminated in the whole cell (Figure 1C). As a nega-tive control, cells incubated only with the secondaryantibody showed only background fluorescence (Fig-ure 1D). Preincubation of NPY02 overnight with2 × 10−5 M NPY abolished specific labelling (resultsnot shown). NPY was also visualized in HUVEC afterincubation of the cells for 24 h in serum-free medium,arguing against the possibility that endothelial NPYis taken up from the serum (Figure 1E). A similarvesicular pattern was also observed on NPY labellingwith NPY05 antibody, another monoclonal antibody,which binds the amidated C-terminal part of NPY(results not shown).

NPY subcellular localizationTo investigate further the intracellular localization ofNPY immunoreactivity in HUVEC, WP bodies werevisualized by vWF labelling (Figure 2D). The stain-ing appeared distinct from that observed for NPY(Figure 2B), since the WP bodies appeared as largeelongated granules. Thus NPY is not stored in theWP bodies. To confirm that NPY was not taken upfrom the extracellular medium, we visualized earlyendosomes by EEA1 (early endosome-associatedprotein-1) staining. Figure 2(E) shows clearly thatEEA1 labelling is significantly different from that ob-served for NPY, indicating that NPY is not con-tained in early endosomes. To determine whetherNPY could be stored in other secretory granules,different from WP bodies, double labelling experi-ments with NPY and Rab3A, a marker for neuroen-docrine secretory granules (Lin and Scheller, 2000),were performed (Figures 2A and 2B). Both proteinsexhibited a granular labelling, with at least partialco-localization, as shown on the overlay picture

458 C© Portland Press 2005 | www.biolcell.org

Neuropeptide Y in HUVEC Research article

Figure 1 ECs express NPY(A) RT–PCR for NPY performed on HUVEC total RNA. Lane 1,

molecular-mass standard V; lane 2, negative control (H2O);

lane 3, positive control (human placenta RNA); lanes 4–6, RNA

from three HUVEC cultures obtained from three distinct umbil-

ical cords. (B) RT–PCR for NPY performed on HMVEC-L total

RNA. Lane 1, molecular-mass standard V; lane 2, negative

control (H2O); lane 3, positive control; lane 4, HMVEC-L RNA

incubated without RT; lane 5, HMVEC-L RNA with RT. (C) NPY

immunofluorescence in HUVEC, using the NPY02 antibody

coupled with Texas Red followed by a Texas Red-conjugated

goat anti-mouse antibody. (D) Negative control by omission

of NPY02 antibody. (E) NPY immunofluorescence in HUVEC

cultured in serum-free medium. Scale bar, 50 µm. Results of

immunocytochemistry are representative of three independ-

ent experiments performed in duplicate.

(Figure 2C). Rab3A expression in ECs was verified byRT–PCR. Both HUVEC and HMVEC-L expressedRab3A (Figure 3A). Rab3A PCR product identitywas verified by digestion with KpnI, leading to theexpected 252 and 404 bp fragments (Figure 3B) andsequence analysis of digestion products. To study therelease of NPY, we stimulated HUVEC with IBMX

(isobutylmethylxanthine)/forskolin (for 30 min) orhistamine, thrombin or PMA (for 20 min). Stimu-lations with IBMX/forskolin together with histam-ine and stimulation with IBMX/forskolin togetherwith PMA were also performed. Immunocytochem-istry did not reveal any difference in the signal ob-served for NPY between control cells and stimu-lated cells (results not shown). Electron microscopicanalyses revealed occasional labelling on vesicle-likecytoplasmic structures, consisting of finely fibrillartexture (Figures 4A and 4B). The cryopreparativetechnique used in our assays favours the immuno-cytochemical signal and allows one to obtain a goodstructural preservation, while membranes are poorlyvisualized. The cytoplasmic signal was also observedon mitochondria and in the free cytoplasm. Labellingwas frequently detected in the nucleus, where it oc-curred especially in association with the nucleoplas-mic ribonucleoprotein-containing perichromatin fib-rils (Figure 4C). Some signals could also be foundin nucleoli. Labelling experiments with anti-Rab3Aantibody revealed a similar general distribution pat-tern, with Rab3A being also present in occasionalvesicle-like cytoplasmic structures (Figure 5). The la-bel in the control grids, where primary antibody hasbeen omitted, was negligible.

NPY action on HUVECSince NPY affects endothelial cell function, we in-vestigated the presence of its receptors by RT–PCR.We found that cultured HUVEC express Y1 andY2 receptor mRNAs. However, neither Y4 nor Y5receptor mRNA was detected (Figure 6A). NPYeither induces intracellular calcium increase or in-hibits forskolin-induced intracellular cAMP increase(Olasmaa and Terenius, 1986; Motulsky and Michel,1988; Perney and Miller, 1989), and vWF secretiondepends on intracellular calcium increase or intracel-lular cAMP increase (Vischer and Wollheim, 1997).To determine whether NPY receptors were func-tional, the effect of NPY on vWF secretion was stud-ied (Figure 6B). Incubation of HUVEC with 100 nMNPY for 30 min did not induce the secretion of vWF(112 +− 8% of control, P = 0.36). NPY did not al-ter forskolin- and IBMX-induced release of vWF(345 +− 119% of control for NPY with IBMX andforskolin versus 375 +− 122% of control for IBMXand forskolin, P = 0.87), and it did not potenti-ate histamine-induced vWF secretion (313 +− 81% of



Figure 2 NPY is stored in vesicles associated with Rab3A and distinct from WP bodies(A) Rab3A was visualized in HUVEC with an anti-Rab3A polyclonal antibody followed by incubation with a goat anti-rabbit

antibody coupled with biotin and Alexa 488-coupled streptavidin. (B) NPY was visualized in HUVEC (same as in A) with the

NPY02 monoclonal antibody coupled with Texas Red followed by incubation with a goat anti-mouse antibody coupled with Texas

Red. (C) An overlay of (A) and (B), showing the co-localization of Rab3A and NPY. (D) HUVEC were stained with an anti-vWF

polyclonal antibody and a Texas Red-conjugated goat anti-rabbit antibody. (E) EEA1 visualized in HUVEC with an anti-EEA1

monoclonal antibody followed by incubation with a Texas Red-conjugated goat anti-mouse antibody. Scale bar, 50 µm. Results

are representative of three independent experiments performed in duplicate.

control for NPY with histamine versus 348 +− 95%of control for histamine alone, P = 0.79). Intracellu-lar cAMP measurements revealed that NPY did notalter forskolin-induced cAMP increase in HUVEC(Figure 6C). However, incubation of HUVEC with10 nM NPY induced a 42 +− 28 nM increase in in-tracellular calcium (n = 105), reaching a maximumlevel within approx. 100 s, followed by a long-lasting decrease (Figure 7A) compared with the rapiddecrease observed with histamine (Figure 7B). Themaximal effect was at a dose of 10 nM NPY since,at 1 nM, NPY-induced calcium response was weaker(18 +− 5 nM) and, at 100 nM, the calcium increase wasrather the same as that at 10 nM. The NPY (10 nM)-induced increase was 22% of the peak response tohistamine (1 µM; Figures 7B and 7C).

DiscussionThe present study shows the expression of NPY andits precursor pro-NPY in HUVEC at the mRNA

and protein levels as demonstrated by RT–PCR andELISA. NPY expression has previously been deter-mined in HUVEC (Zukowska-Grojec et al., 1998b)only in a small number of HUVEC cultures. In thepresent study, NPY expression was evidenced in allcell preparations tested, independent of the mediumused for cell culture (results not shown). The differ-ence between the results of these two studies may bedue to a different initial amount of RNA used forRT–PCR and the number of passages, since we usedcells at passage 1, whereas Zukowska et al. workedon HUVEC between passages 3 and 7.

The presence of NPY and pro-NPY in HUVECwas also assessed by immunofluorescence. NPY im-munoreactivity appeared as small punctate granularstructures disseminated in the cytoplasm. The anti-body NPY02 used in our study is directed againstan epitope borne by both NPY and pro-NPY, thusleading to the labelling of both NPY and its pre-cursor. The small punctate granular appearance ofNPY labelling looks similar to the labelling for



Figure 3 HUVEC and HMVEC-L express Rab3ALane 1, negative control; lanes 2–6, five distinct HUVEC cul-

tures (1 µg of total RNA); lane 7, HMVEC-L (160 ng of total

RNA). (A) PCR products (656 bp) before digestion with KpnI

and (B) PCR products (404 and 252 bp) after digestion with

KpnI.

endocytic vesicles as observed by Niles and Malik(1999). However, it is unlikely that intracellularNPY derives from cell-culture medium since NPYstaining is not lost or altered in HUVEC incubatedin the absence of serum. Furthermore, labelling ofEEA1, an early endosome marker, showed a stainingcompletely different from the immunolabelling ofNPY.

Since many molecules secreted from HUVEC arestored in the WP bodies, we investigated the possiblelocalization of NPY in these structures. Comparisonof NPY and vWF labellings did not reveal the samelocalization for both products, thus suggesting thatNPY vesicles are distinct from WP bodies. To de-termine whether NPY could be included in anothertype of secretory granules, not yet characterized, weperformed a double labelling for Rab3A and NPY.

Rab3 proteins are small GTP-binding proteins of theRas family. They are reported to be involved in the se-cretion of hormones and neurotransmitters and phys-ically associated with secretory vesicles (Darchen andGoud, 2000). The results showed at least partial co-localization of NPY and Rab3A in small vesicles.Therefore NPY is packaged within a new class ofgranules in ECs, associated with a secretory markernormally found in neuroendocrine cells. A previousreport has shown that HUVEC express Rab3B but notRab3A by immunoblot analysis (Karniguian et al.,1993). Despite this negative result, we found thepresence of Rab3A by direct staining of the cells aswell as its mRNA by RT–PCR. The polyclonal anti-body used in the present study to visualize Rab3Aby immunocytochemistry has been used in a previ-ous study, where the authors showed that this anti-body does not cross-react with either recombin-ant Rab3B or recombinant Rab3C (Regazzi et al.,1996). The discrepancies between immunocytochem-istry and Western-blot analysis are probably due tothe low abundance of Rab3A in HUVEC, which pre-vents visualization by a method having a low sensi-tivity such as immunoblot analysis. To determinewhether Rab3A was indeed expressed in HUVEC,we performed RT–PCR and the presence of Rab3AmRNA in HUVEC as well as HMVEC-L was re-vealed. During these experiments, the mRNA foranother Rab3 isoform was amplified together withRab3A mRNA. DNA sequence analysis before di-gestion with KpnI suggests that the second isoformmight be Rab3D. Furthermore, our electron micro-scope observation of the occurrence of NPY in vesicle-like cytoplasmic structures confirms the present im-munofluorescence findings. However, these struc-tures are relatively rare and their labelling is notabundant. Rab3A immunoreactivity was also ob-served in similar vesicle-like structures by immuno-electron microscopy. In contrast with fluorescenceimmunocytochemistry, immunoelectron microscopydid not reveal co-localization of NPY and Rab3A inthese structures. This might be explained by the factthat the immunological reaction takes place only atthe surface of ultrathin resin sections. Taking intoaccount the rather low amounts of NPY and Rab3Ain HUVEC, the number of epitopes available for an-tibody labelling will be relatively low. If, in addi-tion, two different epitopes occur in close proximity,their visualization by two different antibodies can be



Figure 4 Immunoelectron micrographs of HUVEC labelled with anti-NPY, visualized by means of 12 nm colloidal goldparticles(A) A vesicle-like cytoplasmic domain exhibiting a significant signal. Some label is also observed in the cytoplasm. (B) Significant

labelling is detected in a vesicle-like structure (black arrow). Some label also occurs in mitochondria (open arrows) and in the

surrounding cytoplasm. (C) Portion of the nucleoplasm showing preferential labelling on perichromatin fibrils (some indicated by

arrows). c, condensed chromatin regions. Scale bar, 0.25 µm.

prevented due to steric hindrance phenomena. Inter-estingly, NPY signal is also detected in mitochondriaand in the ground cytoplasm. The presence of NPY

in the nucleus has recently been suggested by animmunofluorescence study (Jacques et al., 2003). Inthe present study, we have been able to show that



Figure 5 Immunoelectron micrograph of HUVEC labelledwith anti-Rab3A, visualized by means of 15 nm goldparticlesA vesicle-like fibrillar structure similar to that shown in Fig-

ure 4(A) is also labelled for Rab3A. Scale bar, 0.25 µm.

NPY is mostly associated with nucleoplasmic peri-chromatin fibrils, previously shown as in situ formsof pre-mRNA transcripts (Fakan, 1994). Our re-

sults therefore suggest a possible involvement of theneuropeptide in the formation or regulation of somepre-mRNAs and/or in their processing.

The release of NPY was studied by immunocyto-chemistry, by stimulating cells with IBMX/forskolin(for 30 min) or with histamine, thrombin or PMA (for20 min). Stimulation with IBMX/forskolin togetherwith histamine and stimulation with IBMX/forskolintogether with PMA were also performed. No differ-ence could be observed in the signal for NPY betweencontrol cells and stimulated cells. This result indi-cates either that NPY secretion is not stimulated bythe agents tested (i.e. IBMX, forskolin, PMA, histam-ine and thrombin) or that the amount of NPY releasedon cell stimulation by these agents is not sufficient tobe observed. This latter hypothesis is consistent withthe observation made for NPY secretion by adrenalchromaffin cells after stimulation by 100 µM nico-tine which account for 0.28% of the total intracellularcontent (Cavadas et al., 2001). If such a release oc-curs for NPY in HUVEC, it means that the amountsecreted in the presence of these secretagogues wouldbe in the range of 0.08 pM, which is far below thesensitivity of the ELISA for NPY at 0.5 pM. The se-cretion of NPY from HUVEC was strongly suggestedin a study focused on the role of dipeptidyl peptidase

Figure 6 HUVEC express Y1 and Y2 receptor mRNALane 1, negative control; lane 2, positive control (Y1, SKNMC cell line; Y2, LN 319 cell line; Y4, human nasal mucosa; and Y5,

human placenta); lanes 3–5, three HUVEC cultures obtained from three distinct umbilical cords. (B) NPY does not induce vWF

secretion from HUVEC. NPY does not alter IBMX/forskolin-induced vWF secretion and does not potentiate histamine-induced

vWF secretion from HUVEC. Results are expressed as a percentage of the control condition and are the means +− S.D. for three

independent experiments. (C) NPY does not alter forskolin-induced cAMP accumulation in HUVEC. Results are expressed as a

percentage of the cAMP concentration in the presence of forskolin and are the means +− S.D. for three independent experiments.



Figure 7 NPY induces intracellular calcium increase inHUVEC(A) A typical trace of [Ca2+]i increase after stimulation with

10 nM NPY. The basal calcium concentration was 30 +−0.34 nM before and 29 +− 0.46 nM after the addition of vehicle.

Addition of NPY or buffer is indicated by an arrow. (B) Typical

trace of [Ca2+]i increase after stimulation with 1 µM histamine.

Addition of histamine is indicated by an arrow. (C) Compar-

ison of 10 nM NPY- and 1 µM histamine-induced peak values

of [Ca2+]i increase. Results are expressed as the means +− S.D.

IV in NPY-mediated HUVEC migration. The au-thors showed that blocking the cleavage of NPY intoNPY3-36 by DPP IV with a monoclonal anti-DPPIV antibody decreased the HUVEC migration belowthat obtained in the absence of exogenous NPY (in

serum-free medium), thus arguing for an autocrineNPY released from HUVEC (Ghersi et al., 2001).

Many studies report NPY effects on HUVEC, butlittle is known about the presence of the specific re-ceptors and the signal transduction events leading tothese cell responses. NPY was characterized previ-ously as a novel angiogenic factor due to its abilityto stimulate endothelial cell proliferation, migrationand morphological changes into a capillary tube net-work (Zukowska-Grojec et al., 1998b). These effectswere attributed to Y1 and Y2 receptors. One of theintracellular events known to mediate NPY effectson cells is the increase in cytosolic free calcium(Motulsky and Michel, 1988; Perney and Miller,1989). Calcium measurements revealed that NPY wascapable of raising the free cytosolic calcium concen-tration by approx. 42 nM within 100 s. This long-lasting response contrasted with the rapid, transientand large increase induced by histamine (Rotrosen andGallin, 1986). The plateau phase observed for NPY-induced calcium increase may be due to the ab-sence of NPY wash out in these experiments. SincevWF secretion can be induced by an increase ofcytosolic free calcium and an increase of intracellu-lar cAMP concentration (Hamilton and Sims, 1987),we investigated whether NPY can trigger vWF se-cretion, using histamine and IBMX/forskolin as pos-itive controls (Hamilton and Sims, 1987). As ex-pected, histamine and IBMX/forskolin markedlyincreased vWF secretion, but NPY alone had no ef-fect. Since potentiating effects between calcium- andcAMP-elevating agents have been reported (Vischerand Wollheim, 1997), we investigated the potentialeffects of NPY on IBMX/forskolin- and histamine-induced vWF secretion. NPY did not alter or po-tentiate either the effects of the calcium-elevatingagent histamine, or the effects of the cAMP-elevat-ing agents IBMX and forskolin. These results contrastwith the potentiating effects of calcium and cAMP-elevating agents described previously (Vischer andWollheim, 1997). These results can be explained bythe observation that vWF release occurs only after arapid and substantial free cytosolic calcium increase,similar to the one induced by histamine (Hamiltonand Sims, 1987).

We hypothesize that the previously reported effectsof NPY on EC function are related to signal trans-duction events shown in the present study. In addi-tion, the fact that NPY is produced and stored in



vesicle-like structures is in favour of an auto-crine/paracrine role for NPY at the endothelial celllevel. Moreover, the present study demonstrates a newstorage compartment in ECs.

Materials and methodsCell cultureHUVEC were isolated from freshly delivered umbilical cordsafter incubation at 37◦C for 15 min with 1 mg/ml collagenasetype IV (Worthington Biochemical, Lakewood, NJ, U.S.A.)enzyme solution and plated on to gelatin-coated T25 flasks.Cells were cultured in RPMI 1640 containing glutamax I (LifeTechnologies, Grand Island, NY, U.S.A.), supplemented with1000 units/ml penicillin, 1000 µg/ml streptomycin (Life Tech-nologies), 20 mM Hepes buffer (Biochrom, Berlin, Germany),4.5 units/ml heparin (Becton Dickinson, Franklin Lakes, NJ,U.S.A.), 10% (v/v) fetal bovine serum (Life Technologies)and 80 µg/ml endothelial cell growth supplement (BectonDickinson). Experiments were performed with cells at passage1 or 2.

RT–PCRTotal RNA extraction and cDNA synthesis were performed asdescribed previously (Cavadas et al., 2001). The resulting cDNAwas submitted to PCR using specific primer pairs for NPY, NPYreceptors (see Cavadas et al., 2001 for sequences) and Rab3A:forward, 5′-ATGGCATCCGCCACAGACTCG-3′, and reverse,5′-GCGCAGTCCTGGTGCGGTGGCAC-3′ (Smeland et al.,1994), and the product size was 663 bp. All amplifications wereperformed in a 2.5 mM MgCl2-containing buffer (except for Y2receptor, which was amplified in 1.5 mM MgCl2), with 200 µMdNTP, 0.5 pM of each primer and 25 units/ml of hot start TaqDNA polymerase (all provided in the PCR kit obtained fromQiagen, Valencia, CA, U.S.A.). Reactions were performed inDNA thermal cycler Hybaid® (Promega, Madison, WI, U.S.A.)under the following conditions: 15 min at 95◦C, 35 cycles at95◦C for 1 min, annealing temperature (Y1, 58◦C; Y2, 66◦C;Y4, 67◦C; Y5, 61◦C; NPY, 60◦C; and Rab3A, 64◦C) for 1 min,72◦C for 1 min and a final extension at 72◦C for 10 min. PCRproducts were analysed by electrophoresis on a 1.5% agarose gelcontaining ethidium bromide. Gels were visualized by UV ir-radiation and photographed. The identity of PCR products wasconfirmed by sequence analysis. For Rab3A, the PCR productwas first submitted to digestion with KpnI (Promega) to dis-tinguish Rab3A from the other Rab3 isoforms that was alsoamplified with the primer pair used. The resulting fragmentswere identified by sequence analysis.

ImmunofluorescenceHUVEC grown on gelatin-coated glass coverslips were fixedin 4% (w/v) paraformaldehyde and permeabilized with 0.1%saponin in PBS containing 0.5% BSA. NPY was visualizedby sequential incubation with an anti-NPY monoclonal anti-body NPY 02 (Grouzmann et al., 1992a) coupled with TexasRed (1/600) overnight at 4◦C and after exposure to Texas Red-conjugated goat anti-mouse antibodies diluted 1:400 (JacksonImmunoresearch Laboratories, West Grove, PA, U.S.A.). Rab3Awas visualized with a polyclonal antibody (dilution 1:200; a

gift from Dr A. Zahraoui, Curie Institute, Paris, France), fol-lowed by biotin-conjugated goat anti-rabbit antibody diluted1:200 (Jackson Immunoresearch Laboratories) and with Alexa488-conjugated streptavidin diluted 1:300 (Molecular Probes,Eugene, OR, U.S.A.). vWF was visualized by sequential incub-ation with an anti-vWF polyclonal antibody (2 µg/ml; Dako,Glostrup, Denmark) and with Texas Red-conjugated goat anti-rabbit antibodies diluted 1:200 (Jackson Immunoresearch Labor-atories). EEA1 was visualized by sequential incubation with ananti-EEA1 monoclonal antibody (BD Transduction Laborator-ies, Palo Alto, CA, U.S.A.) diluted 1:50 and with a Texas Red-conjugated goat anti-mouse antibody diluted 1:400 (JacksonImmunoresearch Laboratories). The slides were examined eitherwith a Zeiss-Axioskop 2 microscope or an LSM510 confocal mi-croscope (Carl Zeiss, Jena, Germany). Pictures were acquiredwith a Spot RT colour digital CCD camera (Diagnostic Instru-ments, Michigan, IN, U.S.A.) controlled by the Spot advanced3.1 software (Diagnostic Instruments).

Electron microscopyHUVEC seeded in plastic culture flasks were washed withserum-free medium, scraped off with a rubber policemanand immediately centrifuged to form a pellet. The concen-trated suspension of cells was introduced into cellulose tubes(Hohenberg et al., 1994), cryofixed by high-pressure freezing,cryosubstituted with pure acetone and embedded into LR Whiteresin as described previously (Von Schack and Fakan, 1993).Ultrathin sections were incubated with anti-NPY antibody di-luted 1:25, followed by a goat anti-mouse antibody coupledwith 12 nm colloidal gold particles (Jackson ImmunoresearchLaboratories) or with anti-Rab3A antibody (1:50) followedby a goat anti-rabbit colloidal gold complex (15 nm; Aurion,Wageningen, The Netherlands) according to a standard proced-ure (Cmarko et al., 2002). As a control, some grids were incub-ated in the absence of the primary antibody. The preparationswere stained with uranyl acetate and lead citrate and observedin a Philips CM10 transmission electron microscope at 80 kVusing a 30–40 µm objective aperture.

NPY and pro-NPY assaysConfluent cells were washed three times with PBS containing0.01% (v/v) Tween 20 (Pierce, Rockford, IL, U.S.A.) and har-vested by scraping in Krebs–Henseleit buffer (111 mM NaCl,4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 CaCl2,25 mM NaHCO3, 11 mM glucose and 15 mM Hepes, pH 7.4)containing 50 mM EDTA and 0.08% Tween 20. After sonica-tion and centrifugation, peptide concentrations were measuredin supernatants, and pellets were used for the protein assay. NPYand pro-NPY were measured by ELISA as described previously(Grouzmann et al., 1992b; Brakch et al., 2002).

Determination of [Ca2+]iHUVEC were seeded on to fibronectin-coated glass coverslips,and [Ca2+]i was determined using the fluorescent probe fluo3-AM (Molecular Probes) as described previously (Grouzmannet al., 1997, 1998). Briefly, cells were loaded with 2.5 µMfluo 3-AM in the presence of pluronic acid. Cells were washedthree times with PSS (physiological saline solution) containing140 mM NaCl, 2 mM CaCl2, 4.6 mM KCl, 1.0 mM MgCl2,10 mM glucose and 10 mM Hepes (pH 7.4; for measurementswithout extracellular calcium, a calcium-free buffer was used).



Finally, cells were placed in a chamber with 0.4 ml of PSS formeasurements. Basal calcium concentration was monitoredfor 30 s before the stimulation of cells by the addition of NPYor histamine (100 µl) to this 0.4 ml volume of buffer. Fluor-escence images were acquired every 10 s with a laser-scannedconfocal microscope (MRC 500 confocal imaging system; Bio-Rad Laboratories, Hemel Hempstead, Herts., U.K.) equippedwith an argon ion laser and a 488 nm fluorescein filter cart-ridge. Calibration of the Ca2+ signal was performed after eachexperiment using the non-fluorescent Ca2+ ionophore 4-bromo-A-23187 (2 × 10−5 M) in the presence of extracellular Ca2+to saturate the intracellular dye with Ca2+ and thereby obtainthe maximal fluorescence Fmax. The minimal fluorescence Fmin

was measured after the addition of an excess of EDTA (80 mM).Fluorescence intensities (F) are then translated into [Ca2+] usingthe equation:

[Ca2+] = K d(F − Fmin)

(Fmax − F )(1)

where Kd is the dissociation constant (320 nM for fluo 3-AM asindicated by the supplier).

vWF release studiesvWF levels were measured by ELISA as described previously(Vischer et al., 1995). A standard curve was constructed fromserial dilutions of normal pooled plasma by assuming a plasmaconcentration of 10 µg/ml. Results are expressed in terms ofng · well−1 · (unit time)−1.

cAMP assaycAMP concentrations were determined in HUVEC as describedpreviously (Grouzmann et al., 1997, 1998). The results are ex-pressed as a percentage of the control conditions.

Protein assayCell extracts were solubilized in 0.1 M NaOH supplementedwith 2% (w/v) Na2CO3 and 0.1% SDS. Proteins were quanti-fied with the commercially available BCA (bicinchoninic acid)protein assay kit (Pierce).

AcknowledgementsWe thank N. Aebischer for skilful technical assist-ance, J. Fakan, F. Voinesco and C. Bouchet-Marquisfor excellent assistance with electron microscopy andW. Blanchard for photographic work. This work is apart of the thesis work of A.P.S. at the University ofLausanne and was supported by the Swiss NationalScience Foundation (grant no. 3100-05325.98).

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Received 16 August 2004; accepted 13 September 2004

Published as Immediate Publication 25 April 2005, DOI 10.1042/BC20040102


neuropeptide y expression, localization and cellular transducing effects in huvec

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