IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

Effects of palmitoylation on the diffusional movement of BKCa channels in live cells

BK_Ca channels are palmitoylated at a cluster of cysteine residues within the cytosolic linker connecting the 1st and 2nd transmembrane domains, and this lipid modification affects their surface expression. To verify the effects of palmitoylation on the diffusional dynamics of BK_Ca channels, we investigated their lateral movement. Compared to wild-type channels, the movement of mutant palmitoylation-deficient channels was much less confined and close to random. The diffusion of the mutant channel was also much faster than that of the wild type. Thus, the lateral movement of BK_Ca channels is greatly influenced by palmitoylation.     

Palmitoylation of STREX domain confers cerebroside sensitivity to the BKCa channel

Our previous study reported that cerebrosides from traditional Chinese medicine Baifuzi directly interact with the STREX domain of BK_Ca channels, which in turn results in the therapeutic effect of Baifuzi on ischemic stroke. However, it is not known how cerebrosides in the plasma membrane could interact with the STREX domain that is in the cytoplasmic side. Using patch-clamp technique, effects of different cerebrosides on the BK_Ca channel were studied by measuring single channel currents in CHO cells expressing wild type or mutated BK_Ca channels. Palmitoylation of the STREX domain was removed either by site-directed mutagenesis or pharmacological inhibition. Removal of palmitoylation sites at C646 and C647 by mutating the residues to Ala abolished the ability of cerebrosides to activate the BK_Ca channel. In contrast, the mutation neither changed the single channel conductance nor voltage sensitivity of the channel. Both palmitoylation inhibitors tunicamycin and palmitic acid analog 2-bromopalmitate attenuated the activation of the BK_Ca channel by cerebrosides. Furthermore, confocal images on STREX-EGFP fragments demonstrated that STREX fragments no longer associated with the plasma membrane when the palmitoylation was removed or blocked. These findings suggest that palmitoylation of the STREX domain is necessary for cerebrosides to activate the BK_Ca channel and provide insight into the mechanism of how Baifuzi could exert therapeutic effect on ischemic stroke.     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells

We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p = 0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p < 0.05). Furthermore, OPG was detected by Western blotting in matrix vesicles (MV), nanoparticles that are released by VSMC with the capacity to nucleate mineral. In atherosclerotic arteries, OPG colocalized immunohistochemically with annexin VI, a calcium-dependent membrane and phospholipid binding protein found in MV. Thus, the calcification inhibitor OPG is contained in crystallizing MV and has a biphasic effect on VSMC: physiologic concentrations inhibit calcification, whereas high concentrations commonly seen in patients with vascular disease have no effect. Like other calcification inhibitors, OPG may be specifically loaded into these nanoparticles to be deposited at remote sites, where it acts to inhibit calcification.     

Enhancement of BKCa channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK_Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H₂O₂) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN_wt, PTEN_C124S, PTEN_G129E in HEK 293 cells. Typical BK_Ca channel currents could be recorded in cell-attached configurations. We found that PTEN_wt reduced the H₂O₂-induced BK_Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN_C124S/PTEN_G129E mutants that lack lipid phosphatase activity produced no regulation on the H₂O₂-induced BK_Ca channel activation. These results demonstrated that PTEN regulated the H₂O₂-induced BK_Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H₂O₂-induced BK_Ca channel activation was attenuated when cells were treated with H₂O₂ at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN_wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca²⁺]_i) induced by H₂O₂ was also inhibited. These findings may elucidate a new mechanism for H₂O₂-induced BK_Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H₂O₂.     

Role of integrin-linked kinase in vascular smooth muscle cells: regulation by statins and angiotensin II

Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Tissue inhibitor of metalloproteinases-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis

In a previous study, we have demonstrated that overexpression of the tissue inhibitors of metalloproteinases-4 (TIMP-4) can inhibit the neointima formation in the rat carotid model. To define the functions of tissue inhibitor of metalloproteinases-4 (TIMP-4) in SMCs, we transduced human TIMP-4 cDNA into rat aortic SMCs by using adenoviral vector. Overexpression of TIMP-4 blocked the conversion of pro-MMP-2 to the active form and inhibited basic fibroblast growth factor-induced migration by 56.7% (p < 0.01). Overexpression of TIMP-4 markedly increased apoptotic cell death without changing their proliferation. Importantly, overexpression of human TIMP-4 in the wall of balloon-injured rat carotid artery also increased SMC apoptosis. The percentages of TUNEL-positive cells of total cells increased significantly in AdTIMP-4 infected group compared with AdGFP infected group. These findings demonstrate that TIMP-4 can inhibit SMCs migration and induce apoptosis in vitro and in vivo, which may generate new targets for prevention and treatment of vascular diseases.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Adiponectin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the AMPK/mTOR pathway

Vascular calcification is common in patients with peripheral artery diseases and coronary artery diseases. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) contributes significantly to vascular calcification. Adiponectin has been demonstrated to exert a protective effect in osteoblastic differentiation of VSMCs through regulating mTOR activity. However, the upstream and downstream signaling molecules of adiponectin-regulated mTOR signaling have not been identified in VSMCs with osteoblastic differentiation. In this study, the VSMC differentiation model was established by beta-glycerophosphate (β-GP) induction. The mineralization was identified by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot or immunofluorescence. Adiponectin attenuated osteoblastic differentiation and mineralization of β-GP-treated VSMCs. Adiponectin inhibited osteoblastic differentiation of VSMCs through increasing the level of p-AMPKα. Pretreatment of VSMCs with AMPK inhibitor blocked while AMPK activator enhanced the effect of adiponectin on osteoblastic differentiation of VSMCs. Adiponectin upregulated TSC2 expression and downregulated mTOR and S6K1 phosphorylation in β-GP-treated VSMCs. Adiponectin treatment significantly attenuates the osteoblastic differentiation and calcification of VSMCs through modulation of AMPK–TSC2–mTOR–S6K1 signal pathway.     

Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells

It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) modulate vascular smooth muscle cell functions. In the present study, we investigated the effect of simvastatin on vascular endothelial growth factor (VEGF) release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. Administration of simvastatin increased the VEGF level in rat plasma in vivo. In cultured cells, simvastatin significantly stimulated VEGF release in a dose-dependent manner. Simvastatin induced the phosphorylation of p44/p42 MAP kinase but not p38 MAP kinase or SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). PD98059 and U-0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly reduced the simvastatin-induced VEGF release in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by simvastatin was reduced by PD98059 or U-0126. Moreover, a bolus injection of PD98059 truly suppressed the simvastatin-increased VEGF level in rat plasma in vivo. These results strongly suggest that p44/p42 MAP kinase plays a role at least partly in the simvastatin-stimulated VEGF release in vascular smooth muscle cells.     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P < 0.05). Caspase-like activity was inhibited to a greater degree (77.2% P < 0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the α and β subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.     

高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道研究

目的:研究高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道(KCa)的功能活动。方法:应用膜片钳制技术内面向外式单通道记录方法。结果:①人肠系膜动脉平滑肌细胞KCa开放具有电压依赖性。KCa通道电导在高血压组、正常组分别为191.4pS、197.7pS。胞内侧应用TEA可阻断通道。②增加浴液中Ca2 浓度(从0增至10-8、10-7、5×10-7、10-6mol/L),各组KCa开放概率(Po)均呈浓度依赖性增加,高血压组Po从0.016增至0.023、0.031、0.053、0.094,正常组Po从0.004增至0.023、0.041、0.072、0.184。通道平均开放时间延长,平均关闭时间缩短。③Ca2 浓度为0时,高血压组KCa开放概率明显高于正常组,在其它Ca2 浓度下高血压组KCa开放概率等于或低于正常组。结论:高血压病患者肠系膜动脉平滑肌细胞KCa的Ca2 敏感性较低,可能促进高血压的发生。     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.