Hyperglycaemia Inhibits Thymidine Incorporation and Cell Growth via Protein Kinase C, Mitogen-Activated Protein Kinases and Nitric Oxide in Human Umbilical Vein Endothelium

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Thymidine incorporation into DNA and cell proliferationare reduced in human umbilical vein endothelial cells(HUVECs) from pregnancies affected by gestationaldiabetes (Sobrevia et al. 1995) or in HUVECs cultured in thepresence of high extracellular D-glucose levels (i.e. hyper-glycaemia; Sobrevia et al. 1996; Zanetti et al. 2001).D-Glucose increases the protein level (Montecinos et al.2000) and activity (Sobrevia et al. 1996) of the endothelialnitric oxide synthase (eNOS), an enzyme that convertsL-arginine into L-citrulline and nitric oxide (NO; Sobreviaet al. 1995, 1996; Montecinos et al. 2000; Shaul, 2002).

The potent vasodilator NO has been shown to inhibit cellproliferation and DNA synthesis in HUVECs (Lau & Ma,1996; Zanetti et al. 2001; Bussolati et al. 2001), and other

endothelial cell types (Heller et al. 1999; Wang et al. 2002).cGMP, a second messenger for NO (Shaul, 2002), andcAMP are increased by high levels of D-glucose (Sobrevia etal. 1996), and induce inhibition (Zanetti et al. 2001; Wu etal. 2001; Kim et al. 2001) or stimulation (Grant et al. 1999;Wu et al. 2001) of endothelial cell proliferation. D-Glucosealso activates protein kinase C, which is associated withlonger term increases in NO production in HUVECs(Pan et al. 1995; Montecinos et al. 2000) and inhibitionof endothelial cell proliferation (Wang et al. 2002;Spyridopoulus et al. 2002). Activation of PKC (L. W. Wuet al. 2000; K. Y. Wu et al. 2001; Cheng et al. 2001) andeNOS (Haneda et al. 1997; Parenti et al. 1998; Montecinoset al. 2000) are required for D-glucose-induced stimulation

Hyperglycaemia inhibits thymidine incorporation and cellgrowth via protein kinase C, mitogen-activated protein kinases

and nitric oxide in human umbilical vein endothelium

Susana Rojas *, Romina Rojas *, Liliana Lamperti *, Paola Casanello *and Luis Sobrevia *

* Cellular and Molecular Physiology Laboratory (CMPL), Department of Physiology, Faculty of

Biological Sciences, Department of Clinical Biochemistry, Faculty of Pharmacy and

Department of Obstetrics and Gynaecology, Faculty of Medicine, University of Concepcin,

PO Box 160-C, Concepcin, Chile

(Manuscript received 21 October 2002; accepted 28 January 2003)

An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation andincorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells(HUVECs). Cells in their log-phase of growth (3.7 0.3 days, n = 27) incubated for 30 min with 25 mM D-glucose,but not with equimolar concentrations of L-glucose or D-mannitol, exhibited reduced [3H]thymidineincorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cellvolume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42and p44 mitogen-activated protein kinases (p42/44mapk), but inhibited superoxide dismutase (SOD).Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by thePKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitorL-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. Inthe presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKCactivator phorbol 12-myristate 13-acetate (PMA), the NO donor S-nitroso-N-acetyl-L,D-penicillamine (SNAP),dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A-23187. The effect of A-23187 was blocked bycalphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferationis associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellularlevels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cellgrowth in pathological conditions such as in diabetes mellitus or hyperglycaemia. Experimental Physiology(2003) 88.2, 209219.

2515

Publication of The Physiological Society Corresponding author: [email protected]

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of p42 (42 kDa) and p44 (44 kDa) mitogen-activatedprotein kinases (p42/p44mapk), which are also activated bycGMP and cAMP (Young et al. 1994; Frodin et al. 1994;Pan et al. 1995; Vossler et al. 1997; Parenti et al. 1998). Inaddition, D-glucose has also been shown to inhibitsuperoxide dismutase (SOD) in endothelial cells, suggestinga role for oxygen-derived free radicals in the alterations incell proliferation associated with hyperglycaemia (Zanettiet al. 2001).

We have studied the effect of hyperglycaemia on thymidineincorporation and proliferation of HUVECs. High levels ofD-glucose inhibited thymidine incorporation and cellproliferation associated with activation of PKC, eNOS andp42/44mapk, but with inhibition of SOD activity. In addition,the effect of D-glucose also involved cGMP, cAMP, Ca2+,and PKG and PKA activity.

Part of this study has been published in abstract form(Rojas et al. 2000).

METHODS Umbilical cords and cell culture

Umbilical cords from full-term normal pregnancies with vaginaldeliveries (Regional Clinical Hospital of Concepcin, Chile) werecollected immediately after birth in a phosphate-buffered solution(PBS, 4 C) and stored until use. Approval of the Ethical Committeeof the University of Concepcin-DIUC and informed writtenconsent of the patients were obtained. Endothelium isolated bycollagenase (0.25 mg ml_1) digestion from human umbilicalveins was cultured (37 C, 5 % CO2) in medium 199 (M199)containing 5 mM D-glucose, 10 % newborn calf serum, 10 %fetal calf serum, 3.2 mM L-glutamine, 100 mM L-arginine and100 i.u. ml_1 penicillinstreptomycin. The incubation mediumwas changed 24 h prior to an experiment to serum-free M199.Experiments were performed in passage 2, subconfluent (5060 %)cells in the log-phase of growth (3.7 0.3 days, n = 27) (Sobreviaet al. 1994; Parodi et al. 2002; Casanello & Sobrevia, 2002; Floreset al. 2003).

Protein and DNA content, and cell volume

Cell protein was determined by addition of 100 ml Coomassieblue protein reagent (1:10 dilution), with bovine serum albumin(BSA) standards, and absorbances at 620 nm were measured in aMultiskan plate reader (Flow Laboratories, Irvine, UK) (Sobreviaet al. 1995). DNA was determined in cells extracted with sodiumdodecylsulphate (10 ml, 10 % in water) and mixed for 30 minwith 100 ml dye reagent (Hoechst 33258), and fluorescence at260 nm was measured in a Fluoroskan II (Labsystems, UK)microplate reader (Sobrevia et al. 1996). Cell volume wasdetermined from the distribution ratio of [3H2O] at equilibriumusing D-[14C]mannitol as an extracellular marker (Sobrevia et al.1994).

Cell number and viability

Cells cultured in the presence of 5 mM D-glucose (5060 %confluence) were incubated for periods of between 5 min and24 h with Krebs solution containing (mM): NaCl 131, KCl 5.6,NaHCO3 25, NaH2PO4 1, Hepes 20, CaCl2 2.5 and MgCl2 1;pH 7.4 at 37 C, and with D-glucose at concentrations rangingfrom 5 to 25 mM. Cell number was reduced (P < 0.05, n = 27) byD-glucose (half-maximal inhibitory concentration (K,c), 12.8 1.1 mM; time (K,t), 15 4 min; n = 27). Further experiments

were performed using 25 mM D-glucose for 30 min. Cells werecounted using a haemocytometer 1 h before and immediatelyafter the 30 min incubation period with 25 mM D-glucose. Krebssolution containing 25 mM D-glucose was changed to sera-freeM199 containing 5 mM D-glucose, and cells were counted after 1,2, 4, 6, 12, 18, 24 and 48 h of culture. Equimolar concentrationsof L-glucose or D-mannitol were used as osmotic controls(Sobrevia et al. 1994). Cell viability was determined by TrypanBlue exclusion (Sobrevia et al. 1995), and cell growth rates wereexpressed as number of cells per square centimetre of cell culturesurface per hour (Sobrevia et al. 1994, 1995).

Cells were co-incubated (30 min) with 5 or 25 mM D-glucose andphorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator),4a-phorbol 12,13-didecanoate (4a-PDD, 100 nM, less activePMA analogue), calphostin C (100 nM) or Ro-320432 (50 nM)(PKC inhibitors) (Kobayashi et al. 1989; Radallah et al. 1999),PD-98059 (10 mM, MAP kinase kinase 1/2 (MEK1/2) inhibitor)(Crews & Eriksson, 1992; Lazar et al. 1995), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, 100 mM, NO donor), NG-nitro-L-argininemethyl ester (L-NAME, 100 mM, NO synthase inhibitor), dibutyrylcGMP (dbcGMP, 100 nM) or dibutyryl cAMP (dbcAMP, 1 mM)(membrane-permeable forms of cGMP and cAMP), KT-5823(1 mM, PKG inhibitor) (Grider, 1993), KT-5720 (100 nM, PKAinhibitor) (Cabell & Audesirk, 1993), A-23187 (1 mM, calciumionophore) (Ziemianin et al. 1999) or superoxide dismutase(SOD, 50 U ml_1) (Misra, 1989). Drug concentrations wereselected from dose-dependence curves (not shown).

[3H]Thymidine incorporation

Cells were incubated with 10 mCi ml_1 [3H]thymidine (30 min,37 C), rinsed with Krebs solution and exposed to 5 % trichloro-acetic acid (TCA, 200 ml, 10 min). TCA was removed and themonolayers rinsed with 99 % methanol (200 ml) and digestedwith 25 mM formic acid for the determination of radioactivity(Sobrevia et al. 1994). The effect of D-glucose on [3H]thymidineincorporation (K,c, 11.7 0.9 mM D-glucose; K,t, 16 3 min;n = 27) was determined under the same conditions as for cellgrowth.

Protein kinase C activity

PKC activity was determined by measuring 32P incorporationfrom [g-32P]ATP into a synthetic PKC substrate peptide analoguecorresponding to a fragment of glycogen synthase (GS, Montecinoset al. 2000). PKC activity was determined in cytosolic andmembrane fractions from cells in 5 or 25 mM D-glucose (30 min),in the absence or presence of PMA (100 nM), 4a-PDD (100 nM),or calphostin C (100 nM). Results are expressed in pmol 32P (mgprotein)_1 min_1 (Montecinos et al. 2000).

Superoxide dismutase activity

Cells were homogenized in buffer containing (mM): Tris 50, KCl100, sodium pyrophosphate 100, NaF 100 and 0.02 % TritonX-100 (pH 7.4) supplemented with trypsin inhibitors (aprotinin,4 mg ml_1; bezamidine, 1 mg ml_1; leupeptine, 5 mg ml_1) and200 mM sodium orthovanadate, an inhibitor of protein tyrosinephosphatases. Aliquots (1 mg protein ml_1) were incubated at25 C for 2 min with a potassium phosphate-buffered solution(50 mM, pH 10.2) containing adrenochrome (200 mM) andadrenaline (epinephrine; 10 mM), and absorbance was measuredat 480 nm (Misra, 1989; Flores et al. 2003). SOD activity wascalculated from the inhibition curve for adrenaline auto-oxidation versus protein concentration. Basal absorbance (100 %activity) was taken as the reaction in the absence of cell extracts.Results are expressed in U ml_1.

S. Rojas and others210 Exp Physiol 88.2

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Determination of cGMP and cAMP

Cells were incubated for 30 min in Krebs solution containing100 mM L-arginine, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), and 5 or 25 mM D-glucose or100 mM L-NAME at 37 C (Sobrevia et al. 1995, Casanello &Sobrevia, 2002). Cells were placed on ice and incubated with0.1 N HCl (1 ml per well, 60 min) and cGMP and cAMP levelswere determined by radioimmunoassay in 800 ml of cellsextracted in HCl (Sobrevia et al. 1995; Casanello & Sobrevia,2002).

L-[3H]Citrulline assay

Cells incubated with 100 mM L-[3H]arginine (4 mCi ml_1) and 5or 25 mM D-glucose (30 min, 37 C), in the absence or presenceof 100 mM L-NAME (30 min), were digested in 95 % formic acid.A sodium ion form of the cation ion-exchange resin Dowex-50W(50X8-200) was calibrated, and 200 ml of digested cells were passedthrough the column. The concentration of L-[3H]citrulline wasdetermined in the H2O eluate (Casanello & Sobrevia, 2002).

Determination of cell calcium concentration

Cells on glass coverslips were loaded (30 min, 23 C) with theacetoxymethyl derivative of fluo-3 (5 mM, Molecular Probes,Eugene, USA) and incubated (30 min) with 200 ml M199containing 5 or 25 mM D-glucose. Ca2+ was imaged using a ZeissLSM 410 confocal microscope (w 60 oil immersion lens;numerical aperture, 1.4 (Sobrevia et al. 1996; Flores et al. 2003).

Measurement of extracellular ATP concentration

ATP concentration was determined in M199 in cells incubatedfor 30 min in 5 or 25 mM D-glucose (Parodi et al. 2002). Aliquots

of 100 ml were mixed with 100 ml luciferase reagent (pH 7.7) andthe reaction was processed using the ATP bioluminescence assaykit CLS II (Roche, Germany), and monitored at 562 nm (10 s,22 C) in a luminometer (Lumat LB 9501, Berthold, Germany).The detection limit was 1 fmol of ATP.

Western blots

Cells preincubated (30 min) with PD-98059 (10 mM), KT-5823(1 mM), KT-5720 (100 nM) or SNAP (100 mM) were exposed(30 min) to Krebs solution containing 5 or 25 mM D-glucose.Lysed cells and proteins were separated by polyacrylamide gel(8 %) electrophoresis, transferred to Immobilon-P polyvinylidenedifluoride membranes and probed with a primary polyclonalmouse anti-phosphorylated p44/p42mapk (1:1000), rabbit anti-eNOS (1:2500) or rabbit anti-serine1177-phosphorylated eNOS(1:2500) antibody. Membranes were washed (w 6) in Tris-bufferedsaline Tween (TBST, 50 mM Tris-HCl, 150 mM NaCl, 0.02 % v/vTween 20; pH 7.4), incubated for 1 h in TBST with 0.2 % BSAcontaining horseradish peroxidase-conjugated goat anti-rabbitor anti-mouse antibodies, and proteins were detected byenhanced chemiluminescence (ECL; Casanello & Sobrevia, 2002;Flores et al. 2003).

Materials

Sera, agarose and buffers were from Gibco Life Technologies.Collagenase type II (Clostridium histolyticum) was from BoehringerMannheim (FRG) and Bradford protein reagent was fromBioRad Laboratories (Herts, UK). Superoxide dismutase,ethidium bromide and Dowex-50W (50X8-200) were fromSigma. L-NAME and SNAP were from Calbiochem (La Jolla,USA). [6-3H]-thymidine (17.9 Ci mmol_1), L-[2,3-3H]-arginine

Modulation of cell growth by hyperglycaemiaExp Physiol 88.2 211

Table 1. Effect of D-glucose and PMA on thymidine incorporation and PKC activity in humanumbilical vein endothelial cells

5 mM D-glucose 25 mM D-glucose

Conditions Cytosol Membrane Cytosol Membrane

PKC activity

Control 187 54 27 12 * 45 13 * 169 11 Ro-320432 164 25 32 21 * 56 23 * 139 25 Ro-320432 + calphostin C 143 17 39 15 * 166 13 28 12 *PMA 37 45 129 21 * 45 13 * 171 10 PMA + Ro-320432 64 25 132 21 * 56 23 * 129 25 PMA + Ro-320432 + calphostin C 143 17 32 15 * 166 13 28 12 *

[3H]Thymidine incorporation

Control 1150 102 354 59 *Ro-320432 1285 120 596 205 *Ro-320432 + calphostin C 1235 152 1250 17 PMA 325 95 * 296 120 *PMA + Ro-320432 264 105 * 329 75 *PMA + Ro-320432 + calphostin C 1125 78 1284 129

PKC activity in cytosol and membrane fractions prepared from human umbilical vein endothelial cellsexposed for 30 min to Krebs solution containing 5 or 25 mM D-glucose (see Methods). [3H]Thymidineincorporation (10 mM, 30 min, 22 C) was determined as described in Methods. Experiments wereperformed in absence (Control) or presence (30 min) of Ro-320432 (50 nM), calphostin C (100 nM) orphorbol 12-myristate 13-acetate (PMA, 100 nM). PKC activity is given in pmol (mg protein)_1 min_1

(mean S.E.M., n = 12). * P < 0.05 versus Control for cytosolic fraction in the presence of 5 mMD-glucose; P < 0.05 versus values in membrane fraction in 5 mM D-glucose; P < 0.05 versus Controland Ro-320432 for membrane fraction in 25 mM D-glucose. [3H]Thymidine incorporation is expressedin d.p.m. (mg protein)_1 (30 min)_1 (n = 21). * P < 0.05 versus Control or corresponding values in 5 mMD-glucose; P < 0.05 versus Control in 25 mM D-glucose.

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(36.1 Ci mmol_1), and D-[1-14C]-mannitol (49.3 mCi mmol_1)were from NEN, Dreieich, and 3,5-cyclic GMP-TME (tyrosine-125I) was from ICN (UK). Anti-MAPK antibody was from SantaCruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-eNOSantibodies from Transduction Laboratories (USA).

Statistics

Values are mean S.E.M., where n indicates different cell cultureswith four to eight measurements. Statistical analyses were carriedout on raw data using the Peritz F multiple means comparisontest (Harper, 1984). Students t test was applied for unpaired dataand P < 0.05 was considered statistically significant.

RESULTSEffect of D-glucose on thymidine incorporation and cellgrowth

Cells exposed to 25 mM D-glucose for 30 min exhibitedreduced thymidine incorporation, which was reversed by


Figure 1

The effect of D-glucose on thymidine incorporationand cell growth in human umbilical vein endothelialcells. A, [3H]thymidine incorporation (10 mM,10 mCi ml_1, 30 min, 22 C) before (_1 h) and after(048 h) the 30 min incubation period with Krebssolution containing 5 (1) or 25 mM (0) D-glucose(see Methods). Values (mean S.E.M.) between 0.5and 18 h are significantly different (P < 0.05, n = 22).B, cell growth rates determined as in A (seeMethods). Values between 0.5 and 24 h aresignificantly different from values at time 0 (P < 0.05,n = 22). C, [3H]thymidine incorporation (5) andcell growth (4) in 5 or 25 mM D-glucose, or 5 mMD-glucose + 20 mM L-glucose or D-mannitol.* P < 0.03 versus all other values (n = 22).

Table 2. Effect of D-glucose and signalling molecules on cellgrowth in human umbilical vein endothelial cells

No. of cells (cm2 culture surface)_1 h_1

Conditions 5 mM D-glucose 25 mM D-glucose

Control 4011 102 655 48 *PMA 1466 315 * 461 92 *Calphostin C 4322 251 3987 221 PMA + calphostin C 3997 176 4248 241 Ro-320432 4189 201 430 67 *

PMA + Ro-320432 1316 101 * 577 74 *PD-98059 4226 298 4512 401 L-NAME 5766 319 * 5911 340 *SNAP 645 77 * 631 51 *SNAP + L-NAME 899 122 * 761 159 *

dbcGMP 542 98 * 331 76 *KT-5823 4109 301 3977 121 KT-5823 + dbcGMP 4226 211 3788 341 dbcAMP 355 61 * 478 101 *KT-5720 4228 204 3599 255

KT-5720 + dbcAMP 4261 233 4233 256 A-23187 1655 411 * 798 243 *A-23187 + L-NAME 1751 306 * 798 243 *A-23187 + calphostin C 4317 401 3755 311 A-23187 + PD-98059 3991 277 4701 644

Human umbilical vein endothelial cell (HUVECs) wereincubated (30 min) with Krebs solution containing 5 or 25 mMD-glucose in the absence (Control) or presence of phorbol 12-myristate 13-acetate (PMA, 100 nM), calphostin C (100 nM),Ro-320432 (50 nM), PD-98059 (10 mM), L-NAME (100 mM),SNAP (100 mM), dibutyryl cGMP (dbcGMP, 100 nM), dibutyrylcAMP (dbcAMP, 1 mM), KT-5823 (10 mM), KT-5720 (1 mM), orA-23187 (1 mM) (see Methods). Values are mean S.E.M.,n = 1223. * P < 0.05 versus Control in 5 mM D-glucose, P < 0.05 versus PMA in 5 mM D-glucose, P < 0.05 versusControl in 25 mM D-glucose, P < 0.05 versus correspondingdbcGMP values, P < 0.05 versus corresponding dbcAMPvalues, P < 0.05 versus corresponding A-23187 values.

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24 h (Fig. 1A). Cell growth rates were reduced 2 h after the30 min incubation period with 25 mM D-glucose, andreversed by 48 h (Fig. 1B). The effects of D-glucose werenot due to osmotic stress since equimolar concentrationsof L-glucose or D-mannitol did not significantly alterthymidine incorporation or cell growth (Fig. 1C).

Cell viability was > 98 % under all experimental conditions.Incubation of cells with 25 mM D-glucose did not alter(P > 0.05, n = 25) the cell volume (5 mM D-glucose,1.3 0.3; 25 mM D-glucose, 1.5 0.3 pl cell_1), DNA content(5 mM, 127 22; 25 mM, 98 23 mg (106 cells)_1) or proteincontent (5 mM, 205 35; 25 mM, 241 25 mg (106 cells)_1).Involvement of protein kinase C and MAP kinases

PMA inhibited thymidine incorporation in cells in thepresence of 5 mM D-glucose, but did not alter the degree ofinhibition induced by 25 mM D-glucose (Fig. 2A). PMAand 25 mM D-glucose induced a similar increase of PKCactivity in membrane fractions, and a decrease in cytosolicfractions from HUVECs (Fig. 2B). The effects of 25 mMD-glucose and PMA were blocked by calphostin C, but notby Ro-320432 (Table 1) or 4a-PDD (not shown). PMAalso inhibited cell growth in the presence of 5 mMD-glucose; however, inhibition of growth by 25 mMD-glucose was unaltered by PMA (Table 2). Inhibition ofcell growth by PMA and 25 mM D-glucose was blocked by

calphostin C, but unaltered by Ro-320432. Inhibition ofthymidine incorporation (Fig. 3A) and cell growth rate(Table 2) by 25 mM D-glucose was blocked by the MEK1/2inhibitor PD-98059. Parallel experiments showed that25 mM D-glucose also induced p42/44mapk phosphorylation(Fig. 3B), confirming previous observations in HUVECs(Montecinos et al. 2000; Flores et al. 2003).

Involvement of nitric oxide and calcium

Inhibition of thymidine incorporation (Fig. 4A) and cellgrowth (Table 2) induced by 25 mM D-glucose was blockedby L-NAME and mimicked by SNAP in cells in thepresence of 5 mM D-glucose. The effect of SNAP was notaltered by L-NAME, and SNAP did not alter the effect of25 mM D-glucose. Incubation of cells with 25 mM D-glucosealso increased L-citrulline production (Fig. 4B) and cGMPaccumulation (Fig. 4C), and SNAP increased cGMP levels,but only the effect of D-glucose was blocked by L-NAME.

Incubation of cells with 25 mM D-glucose induced Ser1177-eNOS phosphorylation (Fig. 5A), without altering the totaleNOS protein level. The basal Ca2+ concentration waselevated (P < 0.05, n = 57 cells in eight different cellcultures) by high levels of D-glucose (5 mM, 45 4 nM;25 mM, 172 12 nM), and the calcium ionophore A-23187(5 mM, 581 24; 25 mM, 541 46 nM). A-23187 inhibitedthymidine incorporation (Fig. 5B) only in the presence of


Figure 2

Protein kinase C involvement in the effect ofD-glucose on thymidine incorporation inhuman umbilical vein endothelial cells.A, [3H]thymidine incorporation (10 mM,10 mCi ml_1, 30 min, 22 C) in Krebs solutioncontaining 5 or 25 mM D-glucose, andcalphostin C and/or 12-myristate, 13-acetatephorbol ester (PMA). B, protein kinase Cactivity in membrane (5) or cytosolic (4)fractions (see Methods). Values aremean S.E.M. * P < 0.05 versus correspondingvalues (n = 12).

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5 mM D-glucose, an effect blocked by calphostin C,L-NAME and PD-98059. A-23187 also inhibited cellgrowth, and was blocked only by calphostin C andPD-98059 (Table 2).

Involvement of PKG and PKA

Inhibition of thymidine incorporation (Fig. 6) and cellgrowth (Table 2) induced by 25 mM D-glucose was blockedby KT-5823 (Fig. 6A) and KT-5720 (Fig. 6B). IntracellularcAMP was increased (P < 0.05, n = 14) by 25 mM D-glucose(5 mM, 0.6 0.1; 25 mM, 3.2 0.2 pmol (mg protein)_1

(30 min)_1). Thymidine incorporation and cell growthwere also inhibited by dbcGMP or dbcAMP only in 5 mMD-glucose, effects blocked by KT-5823 or KT-5720,respectively, and by PD-98059 in both cases. Thesenucleotides also induced phosphorylation of p42/p44mapk

in the presence of 5 mM D-glucose, an effect that was alsoblocked by PD-98059 (Fig. 6C). However, p42/p44mapk

phosphorylation induced by 25 mM D-glucose was unalteredby these nucleotides (data not shown).


Figure 3

Involvement of p42/44mapk in the effect of D-glucoseon thymidine incorporation in human umbilical veinendothelial cells. A, [3H]thymidine incorporation(10 mM, 10 mCi ml_1, 30 min, 22 C) in 5 or 25 mMD-glucose, with or without PD-98059. Values aremean S.E.M. * P < 0.03 versus all other values(n = 618). B, Western blot of phosphorylatedp42/44mapk (p44~P, p42~P) under the sameconditions as A. Data are representative of similarresults in eight cell cultures.

Figure 4

Involvement of nitric oxide in the effect of D-glucoseon thymidine incorporation in human umbilical veinendothelial cells. Cells were incubated (30 min,22 C) with 5 or 25 mM D-glucose, L-NAME orSNAP, and [3H]thymidine incorporation (10 mM,10 mCi ml_1; A), L-[3H]citrulline formation fromL-[3H]arginine (B) and cGMP accumulation (C)were determined (see Methods). Values aremean S.E.M. * P < 0.05 versus all other values(n = 1719).

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nModulation of cell growth by hyperglycaemiaExp Physiol 88.2 215

Figure 5

eNOS phosphorylation and Ca2+ involvement in theeffect of D-glucose on thymidine incorporation inhuman umbilical vein endothelial cells. A, Westernblot (upper panel) and densitometry (lower panel) ofphosphorylated endothelial nitric oxide synthase(P~Ser1177 eNOS) or total eNOS in cells incubated(30 min, 22 C) with 5 or 25 mM D-glucose. Data arerepresentative of similar results in six cell cultures.B, [3H]thymidine incorporation (10 mM, 10 mCi ml_1,30 min, 22 C) in 5 mM (5) or 25 mM (4)D-glucose, in the absence or presence of A-23187,calphostin C, L-NAME or PD-98059 (see Methods).Values are mean S.E.M. * P < 0.05 versus all othervalues (n = 12).

Figure 6

The effect of cGMP and cAMP on thymidineincorporation and p42/44mapk phosphorylation inhuman umbilical vein endothelial cells. The effect of(A) dibutyryl cGMP (dbcGMP) or (B) dibutyrylcAMP (dbcAMP) on [3H]thymidine incorporation(10 mM, 10 mCi ml_1, 30 min, 22 C) was determinedin the presence of 5 mM (5) or 25 mM (4)D-glucose, in the absence or presence of KT-5823 orKT-5720 (see Methods). Values are mean S.E.M.* P < 0.05 versus all other values (n = 13). C, Westernblot of phosphorylated p42/44mapk in the presence of5 mM D-glucose, in the absence or presence ofdbcGMP, dbcAMP, or PD-98059. Data arerepresentative of similar results in nine cell cultures.

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Superoxide dismutase activity and ATP release

Basal SOD activity was reduced (P < 0.05, n = 8) by 25 mMD-glucose (5 mM, 12 1; 25 mM, 6 0.9 U ml_1). Thymidineincorporation (d.p.m. (mg protein)_1 (30 min)_1) in thepresence of 5 mM (1070 93) or 25 mM D-glucose (448 80)was unaltered (P < 0.05) by SOD (5 mM, 768 180;25 mM, 518 76). In addition, SOD did not alter the effectof 25 mM D-glucose on cell growth (5 mM, 3866 178;25 mM, 432 144 cells (cm2 culture surface)_1 h_1). BasalATP release (7.5 0.2 nmol (106 cells)_1) was unaltered(P > 0.05, n = 12) by 25 mM D-glucose (8.1 0.5 nmol(106 cells)_1).

DISCUSSIONIn this study we have shown that short-term incubationwith 25 mM D-glucose (i.e. hyperglycaemia) reducedthymidine incorporation and cell growth rates in HUVECs.The effects of hyperglycaemia involve the activity ofprotein kinases C, G and A, activation of eNOS andp42/44mapk, and increased intracellular Ca2+, cGMP andcAMP levels.

Incorporation of thymidine into DNA has been used as anindex of cell proliferation in HUVECs (Sobrevia et al.1996; Lau & Ma, 1996; Zanetti et al. 2001; Bussolati et al.2001) and other endothelial cell types (Parenti et al. 1998;Heller et al. 1999; Grant et al. 1999; Wu et al. 2001; Kim etal. 2001; Wang et al. 2002; Spyridopoulus et al. 2002).Thymidine incorporation was reduced in HUVECsincubated with 25 mM D-glucose for 24 h (Sobrevia et al.1995). Our results show that short-term incubation(30 min) with a high level of D-glucose reduced cell growthand thymidine incorporation with no changes in totalDNA. Furthermore, protein content is not altered by highD-glucose. Thus, inhibition of thymidine incorporationand cell growth by hyperglycaemia may be due to reducedDNA turnover, rather than reduced protein synthesis. Inaddition, reduced thymidine incorporation is not due tochanges of intracellular space distribution for thymidinesince cell volume was unaltered in hyperglycaemia. Thelack of effect of L-glucose or D-mannitol suggests that theactions of D-glucose are not due to an osmotic effect.

Involvement of protein kinases

Inhibition of thymidine incorporation and cell growth inhyperglycaemia was blocked by calphostin C (an inhibitorof diacylglycerol- and phorbol ester-sensitive PKC)(Kobayashi et al. 1989). In our study, 25 mM D-glucoseincreases PKC activity, which modulates nucleosidetransport in HUVECs (Montecinos et al. 2000) and othercell types (Sen et al. 1993; Soler et al. 1998). Thus, PKCactivity is needed for D-glucose to affect thymidineincorporation and cell growth in HUVECs. This issupported by the results showing that the PKC activatorPMA also reduced thymidine incorporation and cellgrowth and that calphostin C blocked the effects of PMA.HUVECs express the PKC isoforms PKC-a (Morigi et al.1998), PKC-b1 (Deisher et al. 1993), PKC-e and PKC-z(Ross & Joyner, 1997). Since PKC-a and PKC-e are

phorbol ester sensitive and inhibited by calphostin C(Kobayashi et al. 1989), and are activated by 25 mMD-glucose in HUVECs (Morigi et al. 1998), it is likely thatone or both of these isoforms are involved in the effects ofPMA in HUVECs. However, the effects of D-glucose orPMA were unaltered by 50 nM Ro-320432, an inhibitorwith reported IC50 values of ~20 nM for PKC-a andPKC-b1, but ~110 nM for PKC-e (Wilkinson et al. 1993;Pedron et al. 2000). Since the effects of 25 mM D-glucoseand PMA were blocked in cells co-incubated withRo-320432 and calphostin C, PKC-e, rather than PKC-a or-b1, could play a key role in the modulation of thymidineincorporation and cell growth by hyperglycaemia or PMA.

It has been reported that PKC-e activation increases,instead of reducing, thymidine incorporation in responseto vascular endothelial growth factor (VEGF) in HUVECs(Wu et al. 2000). However, VEGF also down-regulatesPKC-a and PKC-z in this cell type (Wellner et al. 1999).Thus, VEGF-dependent increased thymidine incorporationcould be due not only to PKC-e activation, but also todown-regulation of PKC-a and PKC-z. This possibilityseems unlikely in our study since the inhibitory effects ofD-glucose were blocked by calphostin C. In addition,discrepancies between our results with 25 mM D-glucoseand reported results with VEGF (Wellner et al. 1999; Wu etal. 2000) could be due to different cell signallingmechanisms involved in the response to VEGF andD-glucose, for example activation of the cell surfacemembrane receptors KDR/Flk1 and Flt1 by VEGF(Millauer et al. 1993; De Vriese et al. 2000) compared witha metabolic effect of high D-glucose (Sobrevia et al. 1996;Lau & Ma, 1996; Bussolati et al. 2001; Parodi et al. 2002).The latter is supported by our results showing that cellpretreatment with the non-metabolizable/non-transportableD-glucose analogue L-glucose did not alter thymidineincorporation or cell growth in HUVECs.

Parallel experiments confirmed our previous observationsof increased p42/p44mapk phosphorylation induced byhyperglycaemia in HUVECs (Montecinos et al. 2000).Incubation of cells with 25 mM D-glucose reducedthymidine incorporation and cell growth, and p42/p44mapk

phosphorylation was blocked by PD-98059, suggesting theinvolvement of the MEK/ERKs signalling pathway in theeffect of D-glucose. Activation of PKC-e also increasesp42/44mapk phosphorylation in HUVECs (Wu et al. 2000).Thus, it is feasible that the effects of hyperglycaemia couldbe due to activation of p42/44mapk following activation ofPKC-e. This is supported by previous observationsshowing that calphostin C blocks D-glucose-inducedp42/44mapk phosphorylation in HUVECs (Montecinos et al.2000). Activation of p42/44mapk has been implicated in bothcytoprotection and cytotoxicity, leading to cell death inseveral cell types (for review see Kyriakis & Avruch, 2001).It has been reported that D-glucose-induced apoptosis inHUVECs occurs only after 36 h of incubation, andinvolves activation of c-Jun NH2-terminal kinase (JNK)instead of p42/44mapk or p38mapk (Ho et al. 2000). In ourstudy HUVECs were incubated with high D-glucose for


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only 30 min, a time period that did not alter cell viability,suggesting that activation of p42/44mapk could not resultin acute hyperglycaemia-induced cell death. p42/44mapk

activation has also been implicated in the proliferation ofHUVECs, but activation may have occurred to aninsufficient degree in this study to achieve proliferation (asreported for expression of matrix metalloproteinase-9 inthis cell type; Genersch et al. 2000). Also, inhibition of cellproliferation could result from the increased NO level inresponse to elevated D-glucose as reported to occur in thiscell type (Lau & Ma, 1996; Zanetti et al. 2001; Bussolatiet al. 2001). Thus, NO-dependent inhibition of cellproliferation could be the over-riding stimulus (Kyriakis& Avruch, 2001). However, the significance of ourapparently paradoxical findings of reduced proliferation ofHUVECs in the face of p42/44mapk activation underconditions of acute hyperglycaemia remains unclear.

Involvement of NO, cGMP and cAMP in the D-glucoseeffect

NO reduces proliferation of HUVECs (Lau & Ma, 1996;Zanetti et al. 2001; Bussolati et al. 2001). In our study,D-glucose increased L-citrulline formation from L-arginine,suggesting the activation of eNOS associated with Ser1177-phosphorylation. Since the NO synthase inhibitor L-NAMEabolishes D-glucose-dependent inhibition of thymidineincorporation and cell growth, the effects of D-glucosecould be due to eNOS activation. In addition, the NOdonor SNAP also reduced thymidine incorporation andcell growth. Our results also show that intracellular Ca2+

concentration was increased by D-glucose, suggesting thatCa2+ may be required for eNOS activation in response toD-glucose.

Hyperglycaemia is associated with the inhibition of cellproliferation and thymidine incorporation, effects associatedwith increased generation of anion superoxide (O2

_) andblocked by over-expression of SOD in HUVECs (Zanetti etal. 2001). In addition, a reduced NO level results fromlower SOD activity and accumulation of O2

_ (a scavengerfor NO) in diabetic vessels (Schnackenberg & Wilcox,2001). In our study SOD activity was reduced inhyperglycaemic conditions, and addition of SOD to theculture medium did not block the effects of D-glucose.Thus, SOD activity could not be required for modulationby D-glucose of HUVEC proliferation; however, thepossibility that exogenous SOD did not enter the cellscannot be ruled out. We recently found in HUVECs that25 mM D-glucose increased the release of ATP, a nucleotideknown to inhibit nucleoside transport (Parodi et al. 2002).Since 30 min incubation with 25 mM D-glucose did notchange ATP release from HUVECs, it is unlikely thatD-glucose-dependent inhibition of cell growth andthymidine incorporation was due to ATP.

Dibutyryl cGMP (dbcGMP) induced p42/44mapk phos-phorylation, confirming similar observations in coronaryvenular endothelium (Parenti et al. 1998). This resultsuggests that hyperglycaemia-induced inhibition ofthymidine incorporation and cell growth may involve

p42/44mapk activation via cGMP in HUVECs. In addition,the inhibitor of PKG, KT-5823 (Grider, 1993), blockedp42/44mapk activation by D-glucose suggesting that this wasdependent on PKG activity. It has been reported thathyperglycaemia also increases intracellular cAMP levels inendothelium (Zhang et al. 2000) and that cAMP activatesp42/44mapk (Young et al. 1994; Frodin et al. 1994; Pan et al.1995; Vossler et al. 1997). We have confirmed these resultsand found that cAMP-induced phosphorylation ofp42/44mapk is blocked by KT-5720, an inhibitor of PKA(Cabell & Audesirk, 1993), involving both cAMP and PKAactivity in the modulation by D-glucose of thymidineincorporation and cell growth in HUVECs.

We have established in this study that hyperglycaemiainhibits thymidine incorporation and cell proliferation inHUVECs, and that this is associated with increased PKCand eNOS activity, cGMP and cAMP levels, and p42/44mapk

phosphorylation. The effects of cGMP and cAMP could bemediated by PKG and PKA, respectively. The cellular eventsinhibiting cell proliferation in hyperglycaemia could beimportant mechanisms in pathological conditions such asdiabetes mellitus where endothelial cell growth is reduced(Sobrevia et al. 1995).

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Acknowledgements

This study was supported by Fondo Nacional de Ciencia yTecnologa (FONDECYT 1030781, 1030607, 1000354 and7000354) and Direccin de Investigacin-Universidad deConcepcin (DIUC 201.084.003-1 and 201.072.025-1) Chile, andThe Wellcome Trust, UK. P.C. and L.L. hold PhD fellowships(Beca Docente-University of Concepcin, Chile). We thank themidwives on the labour ward at the Hospital Regional-Concepcin, Chile for the supply of umbilical cords, and IsabelJara for secretarial assistance.


Documents

Hyperglycaemia Inhibits Thymidine Incorporation and Cell Growth via Protein Kinase C, Mitogen-Activated Protein Kinases and Nitric Oxide in Human Umbilical Vein Endothelium