An antisense of protein kinase C-zeta inhibits proliferation of human airway smooth muscle cells

We hypothesized that an atypical isoform of protein kinase (PK) C, PKC-zeta, is essential for proliferation of human airway smooth muscle (HASM) cells in primary culture. Recombinant replication-deficient E1-deleted adenoviruses (100 plaque-forming units [pfu]/cell) expressing the antisense of PKC-zeta and the wild-type PKC-zeta (Ad-CMV-PKC-zeta) were added to actively growing cells that were subsequently incubated for 48 h in platelet-derived growth factor (PDGF) 40 ng/mL or 10% fetal bovine serum (FBS). Expression of the antisense at a virus concentration of 100 pfu/cell produced a significant (n = 3, P<0.05) decrease in the mean manual cell count in the presence of PDGF to 37+/-5% relative to that in cells with no virus (100%), whereas in cells infected with virus containing no construct, this figure was 102+/-13%. The increase in cell number in response to FBS, however, was not affected by the presence of the antisense. Corresponding values for cells in 10% FBS were 100+/-22%, 85+/-22%, and 122+/-18%. Western blotting revealed decreased levels of PKC-zeta protein, but not PKC-alpha or PKC-epsilon protein, in cells infected with the antisense when compared with levels in control cells. Thus, in HASM cells, PKC-zeta is involved in proliferation in response to PDGF, but not in response to FBS, for which alternate signal transduction pathways independent of PKC-zeta must exist.     

Levalbuterol inhibits human airway smooth muscle cell proliferation: therapeutic implications in the management of asthma

BACKGROUND: Racemic albuterol is a mixture of (R)- and (S)-enantiomers of albuterol. Its pharmacological activity and clinical efficacy reside in the (R)-enantiomer (levalbuterol), but the (S)-enantiomer exacerbates airway reactivity in nonclinical models. The role of albuterols in airway smooth muscle cell (SMC) proliferation is not well understood. METHODS: The effect of levalbuterol on human bronchial SMC growth was compared with the effects of racemic albuterol and (S)-albuterol. Cells were fed albuterols and 3H-thymidine in 5% FBS and incubated for 24 h. The effect of (S)-albuterol on levalbuterol actions was also studied and so were the effects of cAMP/PKA, PI-3 kinase, NK-kappaB, and retinoblastoma (Rb) proteins on albuterols and human bronchial SMC proliferation. RESULTS: Levalbuterol inhibited cell proliferation at low concentrations. The growth-inhibitory effect of levalbuterol occurs via activation of the cAMP/PKA pathway. Addition of (S)-albuterol to levalbuterol decreased the growth-inhibitory effect of levalbuterol, and (S)-albuterol attenuated levalbuterol-induced cAMP release by 65%. Levalbuterol inhibited NF-kappaB and Rb protein expressions. ICI-118551 abrogated the inhibitory properties of levalbuterol. The PAF receptor antagonist CV-3988 inhibited (S)-albuterol-induced cell growth, with no effect on levalbuterol. CONCLUSIONS: Levalbuterol inhibits cell growth by activating the cAMP/PKA pathway and inhibiting PI-3 kinase, NF-kappaB and Rb protein expression, and (S)-albuterol induces cell growth by activating PAF-receptor-mediated cell signaling.     

The PTEN tumor suppressor inhibits human airway smooth muscle cell migration

Airway remodeling in asthma is characterized by increased airway smooth muscle (ASM) mass, accompanied by cell migration. It is well known that the proliferation and migration of ASM cells (ASMCs) play a key role in airway remodeling, but the precise mechanism modulating these cellular events remains unclear. One of the genes most likely to be involved in this process is the phosphatase and tensin homolog (PTEN) gene, whose deletion from chromosome 10 can inhibit the proliferation and migration of many cell types. In this study, we investigated the effects of PTEN on human ASMCs. The cells were infected with recombinant adenovirus containing wild-type PTEN cDNA (Ad-PTEN), and the results were compared with those from the uninfected cells and those infected with the GFP-labeled adenovirus vector. Cell proliferation was measured using the MTT method. Cell migration was determined by wound-healing and transwell assays. The expressions of PTEN, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-focal adhesion kinase (FAK) and FAK, were examined by Western blot analysis. The results show that PTEN is expressed endogenously in ASMCs, and that Ad-PTEN inhibits the proliferation and migration of these cells. In addition, the Ad-PTEN treatment decreased the phosphorylation of Akt and FAK but not that of ERK1/2. In conclusion, this study demonstrates that PTEN overexpression inhibits the proliferation and migration of human ASMCs by down-regulating the activity of the Akt and FAK signaling pathways.     

Retinoic acid inhibits airway smooth muscle cell migration

Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-alpha, -beta, -gamma, and retinoid X receptor (RXR)-alpha, -beta, but not RXR-gamma. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2-2 microM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein-protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.     

转染Kv1.5基因抑制人气道平滑肌细胞的增殖

 目的：转染Kv1.5基因对人气道平滑肌细胞(HASMCs)增殖及凋亡的影响。方法:通过脂质体介导瞬时转染Kv1.5基因于培养的HASMCs中，以转染空载体pRc/CMV的细胞及未转染质粒的细胞为对照；用Western blotting 检测平滑肌细胞Kv1.5蛋白表达；用荧光光度法检测HASMCs胞内钙浓度；用流式细胞术观察细胞周期；用MTT法检测HASMCs 增殖及DNA 末端转移酶介导的原位缺口末端标记技术(TUNEL)检测细胞的凋亡。 结果: (1) 转染质粒组Kv1.5蛋白质的表达明显高于未转染组及空载体转染组(P<0.01); (2) 转染质粒组细胞胞内钙浓度明显低于未转染组及空载体转染组(P<0.05)，且其细胞周期中的G₀/G₁期细胞比例明显高于、细胞增殖率显著低于未转染组及空载体转染组(P<0.01)；同时，转染质粒组细胞的凋亡率明显高于未转染组及空载体转染组 (P<0.01)。 结论: 转染Kv1.5基因能抑制HASMCs的增殖、促进其凋亡，为进一步探讨哮喘气道重塑的机制及其治疗提供实验依据。     

Adventitia-derived hydrogen peroxide impairs relaxation of the rat carotid artery via smooth muscle cell p38 mitogen-activated protein kinase

The role of adventitia-derived reactive oxygen species (ROS) in vascular disease and impaired vascular relaxation is not clear. Based on robust adventitial ROS generation and effects on MAPK involvement in vascular dysfunction, we hypothesized that adventitia-derived ROS hydrogen peroxide (H(2)O(2)) impairs vascular relaxation through activation of medial smooth muscle p38 MAPK. By using a novel in vivo model, the adventitial surface of rat carotid arteries was bathed in situ for 90 min with vehicle, angiotensin II (AngII; 500 nM), AngII+H(2)O(2)-scavenger catalase (3,000 U/ml), AngII+p38 MAPK inhibitor SB203580 (10 μM), or AngII+superoxide dismutase (SOD; 150 U/ml). After these in vivo treatments, ex vivo tone measurements on isolated vessels revealed that periadventitial application of AngII impaired both acetylcholine-induced (endothelium-dependent) and sodium nitroprusside-induced (endothelium-independent) relaxations. In vivo coincubation with catalase or SB203580 significantly improved, but SOD exacerbated AngII-induced impairment of in vitro endothelium-dependent and -independent vascular relaxations. Western blots of vascular media, separated from the adventitia, demonstrated increased medial p38 MAPK activation and decreased medial phosphatase SHP-2 activity in AngII-treated vessels. These effects were reversed by in vivo periadventitial addition of catalase. These findings provide the first evidence that adventitia-derived H(2)O(2) participates in vascular dysfunction through p38 MAPK activation and SHP-2 inhibition.     

Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism

BACKGROUND: Airway remodeling is a key feature of persistent asthma and includes alterations in the extracellular matrix protein profile around the airway smooth muscle (ASM) and hyperplasia of the ASM. We have previously shown that nonasthmatic ASM cells in culture produce a range of extracellular matrix protein proteins and that asthmatic ASM cells proliferate faster than cells from nonasthmatic patients. OBJECTIVE: In this study, we compared the profile of extracellular matrix proteins produced by nonasthmatic and asthmatic ASM cells. We also examined the influence of these extracellular matrix protein proteins and conditioned medium derived from nonasthmatic or asthmatic ASM cells on the proliferation of nonasthmatic and asthmatic ASM cells. METHODS: Extracellular matrix proteins were measured by ELISA; proliferation of ASM cells was measured by tritiated thymidine incorporation. RESULTS: Production of perlecan and collagen I by the cells from asthmatic patients were significantly increased. In contrast, laminin alpha1 and collagen IV were decreased. Chondroitin sulfate was detectable only in the cells from nonasthmatic patients. Compared with nonasthmatic extracellular matrix proteins, proteins from asthmatic cells enhanced ASM cell proliferation. Conditioned medium from asthmatic ASM cells did not induce greater proliferation compared with conditioned medium from nonasthmatic cells. CONCLUSIONS: The data show that the profile of extracellular matrix protein components is altered in asthmatic cells and that this altered profile and not soluble mediators secreted from the ASM cells has the potential to influence the proliferation of these cells. These changes are likely to contribute to the airway wall remodeling that occurs in asthma.     

Hydrogen sulfide inhibits proliferation and release of IL-8 from human airway smooth muscle cells

Hydrogen sulfide (H(2)S) is synthesized intracellularly by the enzymes cystathionine-γ-lyase and cystathionine-β-synthase (CBS), and is proposed to be a gasotransmitter with effects in modulating inflammation and cellular proliferation. We determined a role of H(2)S in airway smooth muscle (ASM) function. ASM were removed from resection or transplant donor lungs and were placed in culture. Proliferation of ASM was induced by FCS and the proinflammatory cytokine, IL-1β. Proliferation of ASM and IL-8 release were measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H(2)S "donors" inhibited this proliferation and IL-8 release. Methemoglobin, a scavenger of endogenous H(2)S, increased DNA synthesis induced by FCS and IL-1β. In addition, methemoglobin increased IL-8 release induced by FCS, but not by IL-1β, indicating a role for endogenous H(2)S in these systems. Inhibition of CBS, but not cystathionine-γ-lyase, reversed the inhibitory effect of H(2)S on proliferation and IL-8 release, indicating that this is dependent on CBS. CBS mRNA and protein expression were inhibited by H(2)S donors, and were increased by methemoglobin, indicating that CBS is the main enzyme responsible for endogenous H(2)S production. Finally, we found that exogenous H(2)S inhibited the phosphorylation of extracellular signal-regulated kinase-1/2 and p38, which could represent a mechanism by which H(2)S inhibited cellular proliferation and IL-8 release. In summary, H(2)S production provides a novel mechanism for regulation of ASM proliferation and IL-8 release. Therefore, regulation of H(2)S may represent a novel approach to controlling ASM proliferation and cytokine release that is found in patients with asthma.     

U46619对培养的人气道平滑肌细胞增殖的作用

目的和方法：研究Ｕ４６６１９（９,１１－ｄｉｄｅｏｘｙ－１１α,９α－ｅｐｏｘｙｍｅｔｈａｎｏ－ｐｒｏｓｔａｇｌａｎｄｉｎＦ２α）对培养的人气管平滑肌细胞增殖的作用。分离人的气管平滑肌细胞并且进行传代培养。在培养基中加入各种浓度的Ｕ４６６１９,计数细胞并且测定［３Ｈ］－胸腺嘧啶核苷（［３Ｈ］－ＴｄＲ）掺入量和三磷酸肌醇［Ｉｎｓ（１,４,５）Ｐ３］累积量。结果：Ｕ４６６１９在一定范围内（１ｎｍｏｌ／Ｌ～１００ｎｍｏｌ／Ｌ）以浓度依赖的方式增加人气管平滑肌的细胞数（Ｐ＜００１）。Ｕ４６６１９也增加［３Ｈ］－ＴｄＲ的掺入量和Ｉｎｓ（１,４,５）Ｐ３的累积量（Ｐ＜００１）。磷脂酶Ｃ抑制剂新霉素阻止Ｉｎｓ（１,４,５）Ｐ３累积量的增加（Ｐ＜００１）,但对Ｕ４６６１９的有丝分裂原的活性没有作用。结论：显示Ｕ４６６１９刺激培养的人气管平滑肌细胞增殖。     

Neutrophil-derived elastase induces TGF-beta1 secretion in human airway smooth muscle via NF-kappaB pathway

Neutrophils are infiltrated in airways of individuals with more severe and chronic asthma, with uncertain significance. Airway smooth muscle (ASM), apart from its contractile properties, is critically involved in the pathogenesis of asthma by producing inflammatory mediators. In the present study, we investigated the impact of neutrophil-derived elastase (NE) on ASM in terms of TGF-beta1 release, and we explored the underlying mechanisms. Primary ASM cells were serum starved for 24 h before stimulation with NE (0.01-0.5 microg/ml). TGF-beta1 in supernatant was determined by ELISA and mRNA quantified by real-time RT-QPCR. NF-kappaB nuclear translocation and activation was examined by Western blotting and kappaB-2 dEGFP reporter gene assay. Association of IL-1 receptor-associated kinase (IRAK) with MyD88 was studied by co-immunoprecipitation and Toll-like receptor 4 (TLR4) determined by FACS scan and Western blotting. We demonstrated that NE enhanced TGF-beta1 release in a time-dependent manner. This induction was inhibited by actinomycin D (5 mM), cycloheximide (5 mM), and NF-kappaB inhibitors, including pyrrolidine dithiocarbamate (PDTC, 1 mM), aspirin (2.5 mM), and sodium salyicylate (2.5 mM). Stimulation with NE was rapidly followed by association of IRAK with MyD88, phosphorylation of IkappaBalpha, and nuclear translocation of p65 with increased transactivation activity. We also found that TLR4 levels were reduced upon NE treatment. These data suggest that NE upregulates TGF-beta1 gene expression and release via My88/IRAK/NF-kappaB pathway, possibly through activation of TLR4, and shed light on a potential role of neutrophils in the pathogenesis of asthma.     

Tryptase inhibits motility of human spermatozoa mainly by activation of the mitogen-activated protein kinase pathway

BACKGROUND: We previously localized protease-activated receptor 2 (PAR-2) on human spermatozoa and demonstrated that activation of PAR-2 by the mast cell (MC) product tryptase inhibits sperm motility. Importantly, tryptase-secreting MCs are encountered in the male and female genital tract, implying that MC-spermatozoa interactions may be as yet unrecognized factors affecting sperm fertilizing ability. In order to elucidate how tryptase via activation of PAR-2 acts in human spermatozoa, we studied intracellular signal transduction events. METHODS AND RESULTS: Impairment of sperm motility by tryptase was not dependent on the presence of extracellular Ca2+ and tryptase did not alter intracellular Ca2+ levels. Pre-incubation with pertussis toxin (PTX) failed to prevent tryptase effects on sperm motility. Western blot analyses revealed that tryptase increased phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2, an effect which was blocked by the MAPK pathway inhibitor PD98059. Pre-treatment of spermatozoa with this inhibitor also blocked the inhibtion of sperm motility evoked by tryptase. CONCLUSIONS: These results indicate that tryptase acts via the ERK1/2 pathway to inhibit motility of human spermatozoa.     

CaN-NFAT信号通路参与苯肾上腺素介导的血管平滑肌细胞增殖

目的 研究钙调神经磷酸酶-活化T细胞核因子(CaN-NFAT)信号通路对苯肾上腺素诱导的血管平滑肌细胞(VSMCs)增殖的调节作用。方法：组织贴块法原代培养SD大鼠血管平滑肌细胞,MTT法和细胞计数法测定VSMCs增殖,间接免疫荧光法测定NFATc1细胞定位,Western blot测定CaN蛋白表达,定磷法检测CaN活性。结果：苯肾上腺素(PE,α1-受体激动剂)促进VSMCs增殖,哌唑嗪(prazosin,α1-受体抑制剂)和环胞霉素A(CsA,CaN抑制剂)降低PE诱发的VSMCs增殖,白屈菜红碱(chelerythrine,蛋白激酶C抑制剂)预处理VSMCs后,PE诱发的VSMCs吸光度和细胞数被抑制, 并且这种抑制作用可以被CsA进一步加强。CsA抑制PE诱发的CaN表达与活性。PE促进NFATc1从胞质易位入核,CsA抑制NFATc1核转位。结论：CaN-NFATc1信号通路参与调节苯肾上腺素诱导的VSMCs增生肥大。     

Rap1 promotes proliferation and migration of vascular smooth muscle cell via the ERK pathway

Background

Rap1 is involved in a multitude of cellular signal transduction pathways, which has extensively been linked to cell proliferation and migration. It has been shown to be important in the regulation of physiological and pathological processes. The present study aims to elucidate its detailed mechanistic in proliferation and migration.

噻托溴铵抑制瘦素促大鼠气道平滑肌细胞的增殖

目的 探讨噻托溴铵对瘦素促大鼠气道平滑肌细胞(ASMCs)增殖的作用.方法 体外培养大鼠ASMCs,瘦素(100 μg/L)干预后,加入不同浓度噻托溴铵(μg/L)(15、30、60和120),CCK-8法测定ASMCs增殖,荧光实时定量RT-PCR及免疫印迹(Western blot)分别检测瘦素受体mRNA及蛋白含量水平表达.结果 与对照组比较,瘦素可明显促进大鼠ASMCs增殖(P＜0.05),加入噻托溴铵则抑制瘦素的促增殖作用(P＜0.05),并与浓度相关(P＜0.01).瘦素作用后,其受体mRNA表达增加,蛋白含量由对照组的0.327 ±0.011升至0.541 ±0.016(P＜0.05);噻托溴铵干预后,瘦素受体mRNA表达减少,蛋白含量分别回降至(0.514±0.017、0.468±0.025、0.331±0.023、0.313 ±0.017) (P ＜0.05);并与浓度相关(P＜0.01).结论 噻托溴铵可抑制瘦素的促ASMCs增殖作用,其机制可能与下调大鼠ASMCs表面的瘦素受体表达有关.     

Role of RhoA inactivation in reduced cell proliferation of human airway smooth muscle by simvastatin

Enhanced proliferation of smooth muscle cells contributes to airway remodeling of bronchial asthma. Recently, statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, have been shown to inhibit proliferation of both vascular and airway smooth muscle cells independently of lowering cholesterol. However, the mechanisms remain to be elucidated. The purpose of this study was to determine molecular processes by which statins inhibit proliferation of human bronchial smooth muscle cells. Simvastatin (0.1-1.0 muM) significantly inhibited cell proliferation and DNA synthesis induced by FBS in a concentration-dependent manner. The inhibitory effects of simvastatin were antagonized by mevalonate and geranylgeranylpyrophosphate, whereas the effects were not affected by squalene and farnesylpyrophosphate. The antiproliferative effects of simvastatin were mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, and Y-27632, an inhibitor of Rho-kinase, a target protein of RhoA. Western blot analysis showed that the level of membrane localization of RhoA (active Rho) was markedly increased by FBS, and that the level of active RhoA increased by FBS was reduced by simvastatin. Moreover, the inhibitory effect of simvastatin on FBS-induced RhoA activation was also antagonized by geranylgeranylpyrophosphate, but not by farnesylpyrophosphate. Because these isoprenoids are required for prenylation of small G proteins RhoA and Ras, respectively, the present results demonstrate that an inhibition in airway smooth muscle cell proliferation by simvastatin is due to prevention of geranylgeranylation of RhoA, not farnesylation of Ras. Therefore, statins may have therapeutic potential for prohibiting airway remodeling in bronchial asthma.     

Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype

Mature airway smooth muscle cells are characterized by a low proliferative index and expression of contractile marker proteins such as smooth muscle alpha-actin (sm-alpha-actin), calponin, and smooth muscle myosin heavy chain (sm-MHC). In the present study, defined extracellular matrix (ECM) components were examined on the proliferative and phenotypic status of mitogen-stimulated, cultured human airway smooth muscle cells. The results demonstrate that although cells adhered and spread on plates precoated with (1 to 100 microg/ml) of fibronectin (FN), collagen I (Col I), laminin (LN), or Matrigel, their subsequent proliferative response varied qualitatively. FN and Col I enhanced proliferation in response to either platelet-derived growth factor (PDGF)-BB or alpha-thrombin, compared with cells on plastic. LN, however, reduced mitogen-stimulated proliferation. A similar reduction was found in cells cultured on Matrigel. The effect of ECM substrates on contractile phenotype was determined by examining cellular expression of sm-alpha-actin, sm-MHC, and calponin using immunocytochemical and flow cytometric methods. Approximately 75% of PDGF-BB-stimulated cells, cultured on LN or Matrigel, expressed sm-alpha-actin, calponin, and sm-MHC, but only 8 to 10% stained for the Ki67 nuclear antigen proliferation marker. In contrast, more than 75% of cells cultured on FN or Col I were positive for Ki67 antigen, but only 20% were positive for contractile proteins. Flow cytometric analysis of sm-alpha-actin and DNA content confirmed the immunocytochemical findings and showed that the observed reduction in sm-alpha-actin content after culture on FN or Col I, compared with LN and Matrigel, occurred in the majority of the cell population, supporting bidirectional phenotype modulation. Overall, the data suggest that ECM substrates modulate both proliferation and phenotype of human airway smooth muscle cells in culture.     

Hyperglycaemia inhibits thymidine incorporation and cell growth via protein kinase C,mitogen-activated protein kinases and nitric oxide in human umbilical vein endothelium

An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation 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]thymidine incorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cell volume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen-activated protein kinases (p42/44(mapk)), but inhibited superoxide dismutase (SOD). Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitor L-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. In the presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator 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 by calphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.