The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion.

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

Olive oil polyphenol oleuropein inhibits smooth muscle cell proliferation.

OBJECTIVES: The Mediterranean diet, with a high content of olive oil, is associated with a reduced risk of coronary artery disease. The aim of this study was to determine the effect of oleuropein, one of the polyphenols in olive oil, on vascular smooth muscle cell (SMC) proliferation in vitro. DESIGN: This was an experimental study. MATERIALS AND METHODS: Bovine vascular SMCs were cultured in the presence of 100 µM of oleuropein. On days 1, 3 and 5, cell number was counted. Cell cycle analysis was performed by flow cytometry. Cell cycle regulators were assessed by immunoblotting. RESULTS: Cell proliferation in the presence of oleuropein was significantly inhibited by 92%. Cell cycle analysis revealed that oleuropein treatment blocked cells in the G1-S phase compared with the non-treated group. Among G1 phase regulators, retinoblastoma protein, cyclinD, p21 and p27 were not affected by oleuropein, but extracellular signal-regulated kinase 1/2 (ERK1/2) activation was inhibited. Growth of SMC treated with 100 µM of the mitogen-activated protein (MAP) kinase/ERK kinase 1 (MEK1) inhibitor PD98059 was also significantly inhibited by 70%. CONCLUSIONS: Oleuropein inhibited SMC proliferation through a cell cycle block between the G1 and the S phases, which may be regulated by ERK1/2. These results suggest a mechanism by which olive oil consumption may have beneficial effects on cardiovascular mortality by inhibiting SMC proliferation.

Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells.

In vivo, endothelial cells (EC) are subjected to hemodynamic forces which may influence the production of nitric oxide. This study was designed to examine the effect of cyclic strain on the expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic EC. EC were grown on flexible membranes which were subjected to deformation at 60 cycles/min with -5 or -20 kPa of vacuum. This results in an average strain of 6 and 10%, respectively, which is transmitted to the attached cells. Northern blot analysis of total cytosolic RNA demonstrated an increase in eNOS gene expression with both strain regimens but the increase with 10% average strain was greater than that at 6%. Nuclear runoff transcription assays confirmed the induction of eNOS transcripts. Western blot analysis showed an increase in eNOS level after 24 h of cyclic 10% average strain compared with controls or 6% average strain. Immunohistochemical staining of EC for eNOS was increased in the high strain periphery (7-24% strain) of membranes deformed with -20 kPa vacuum. These results demonstrate that cyclic strain upregulates the expression of eNOS transcripts and protein levels in bovine aortic EC thus emphasizing the importance of hemodynamic forces in the regulation of eNOS in vivo.

Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress.

Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress-induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress-induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate-responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress-induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

Shear Stress Induces Change in Extracellular Signal-Regulated Kinase 5 Levels with Sustained Activation under Disturbed and Continuous Laminar Flow.

Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction under laminar flow. Previously reported research indicates that under laminar flow ERK5 is activated with production of atheroprotective molecules. However, the characterization of ERK5 activation and levels under different flow patterns has not been investigated. Confluent HUVECs were serum-starved then seeded on glass slides. HUVECs incubated in 1% FBS were exposed to continuous laminar flow (CLF), to-and-fro flow (TFF), or pulsatile forward flow (PFF) in a parallel plate flow chamber. At the end of experimentation, cell lysates were immunoblotted with antibodies to phospho-ERK5 and total ERK5. ERK5 activation was assessed by the levels of phosphorylated ERK5. The densitometric mean ± SEM is calculated and analyzed by ANOVA. p < 0.05 is considered significant. Levels of ERK5 decreased with all flow conditions with the largest decrease in TFF flow condition. TFF and CLF exhibited sustained ERK5 phosphorylation in HUVECs stimulated for up to 4 hours. PFF had transient phosphorylation of ERK5 at 2 hours, which then became undetectable at 4 hours of exposure to flow. Also, TFF and CLF both showed decreased levels at 4 hours, suggesting a decrease in activation for these flow conditions. Exposure of HUVEC to different types of shear stress results in varying patterns of activation of ERK5. Activation of ERK5 with TFF suggests a role in the pathogenesis of atherosclerosis and vascular remodeling under disturbed flow conditions.

Urinary excretion of interferon, albumin, and beta 2-microglobulin during interferon treatment.

Serum and urinary levels of albumin, beta 2-microglobulin, and interferon were determined in ten patients undergoing interferon therapy. The pharmacokinetics during a phase I trial of interferon administration intramuscularly is presented. Only trace amounts of interferon activity are found in the urine, even during peak serum interferon activity. Serum beta 2-microglobulin levels increased after interferon treatment, especially at the higher dosing levels. Urinary excretion of beta 2-microglobulin increased due to the relatively low affinity of the transport system. Saturation, competition, or inhibition of the absorption process for beta 2-microglobulin was not attained. Measurement of the urinary albumin/urinary beta 2-microglobulin ratio reveals no glomerular or tubular lesion, and we conclude that interferon therapy does not result in a clinically significant nephrotoxicity.

Effects of ATP-MgCl2 and adenosine-MgCl2 administration on intracellular ATP levels in the kidney.

The effects of exogenous administration of 1 mM [8-14C]ATP-MgCl2 and adenosine-MgCl2 on intracellular accumulation of adenine nucleotides were examined in isolated, perfused rat kidneys. The kidneys were made filtering or non-filtering by increasing the colloid oncotic pressure of the perfusate solution in order to assess the relative contributions of the glomerular and peritubular routes in the uptake of the nucleotides. The results indicate that: although labeled ATP is undetectable in the perfusate after 20 min, there is a significant accumulation of labeled ATP in the tissue and although labeled adenosine-MgCl2 administration also leads to labeled intracellular ATP, the total intracellular ATP is much less than with ATP-MgCl2 administration.

Endothelin-stimulated monocyte supernatants enhance neutrophil superoxide production.

Endothelin-1 (ET-1) is a vasoconstrictive peptide released by ischemic/injured endothelium which increases intracellular ionized calcium [Ca2+]i in vascular smooth muscle. Previous work from this lab has shown that ET-1 also increases human peripheral blood monocyte [Ca2+]i, and that 24 h incubation of monocytes with 10(-9) M ET-1 causes production of prostaglandin E2 and interleukin-6. In these studies, ET-1-stimulated monocyte supernatants were evaluated for their effect on neutrophil superoxide production. While ET-1 alone had no direct effect, incubation of neutrophils for 20 min in ET-1-stimulated monocyte supernatants resulted in a 10-fold increase in superoxide production over basal levels, 44% as much superoxide production as induced by peptide N-formyl-methionyl-leucyl-phenylalanine (N = 6, p < .001). Monocyte supernatants were analyzed for interleukin-8 (IL-8 or neutrophil activation protein) content by radioimmunoassay. ET-1-stimulation resulted in production of 54% as much IL-8 as lipopolysaccharide controls (N = 6, p < .001). While a number of monokines can activate neutrophils, IL-8 has been shown to be a potent neutrophil activator as well as a superoxide primer. Therefore, ET-1-treated monocytes probably upregulate neutrophil superoxide production via a mechanism which includes IL-8.

Gene regulation by mechanical forces.

Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.

Cyclooxygenase expression in bovine aortic endothelial cells exposed to cyclic strain.

The aim of this study was to investigate the effect of cyclic strain on cyclooxygenase (COX)-1 and 2 expression in bovine aortic endothelial cells (EC). EC, subjected to 10% average strain at 60 cycle/min, were analyzed for induction of COX by Northern blot analysis and confirmed by analysis of promoter activity in transient transfection experiments. Exposure of EC to cyclic strain induced promoter activity and expression of COX-2 but not of COX-1. The extent of induction, however, was lower than that seen with stimulation of 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or lipopolysaccharide (LPS). These results demonstrate that, unlike shear stress, cyclic strain does not affect COX-1 expression and is a weak inducer of COX-2 promoter activity in bovine aortic EC with minimal effect on mRNA expression.

Cyclic stretch induces the expression of vascular endothelial growth factor in vascular smooth muscle cells.

OBJECTIVE: Accumulating evidence links the release of vascular endothelial growth factor (VEGF) by vascular smooth muscle cells (VSMC) to normal endothelial cell (EC) function, repair and maintenance. Using an in vitro model we investigate the role of cyclic stretch on both the release of VEGF by VSMC and the phosphorylation of a VEGF receptor on EC. METHODS: Bovine VSMC and EC were exposed to 10% cyclic strain for 4 hours. VEGF mRNA steady-state levels of VSMC were analysed by northern blot hybridisation. The presence of secreted VEGF from VSMC was determined by assaying the migration of EC. VEGF receptor phosphorylation on stretched EC was assayed by immunoblotting. RESULTS: The steady-state level of VEGF mRNA in stretched VSMC increased 3.3 (+/- 0.6) fold above that of unstretched VSMC (p < 0.005). Migration of EC was stimulated 8.3 (+/- 1.1) and 14.6 (+/- 1.3) fold by media from unstretched and stretched VSMC respectively, demonstrating a 1.8 fold increase due to stretch alone (p < 0.05). Cyclic stretch resulted in phosphorylation of the VEGF receptor KDR. CONCLUSION: Exposure of VSMC to physiological levels of stretch induces a biologically significant increase in VEGF secretion and may provide an arterial stimulus for maintenance of steady state levels of VEGF essential for EC survival.

The integrin-mediated cyclic strain-induced signaling pathway in vascular endothelial cells.

The irregular distribution of plaque in the vasculature results from the interaction of local hemodynamic forces with the vessel wall. One well-characterized force is cyclic circumferential strain, the repetitive pulsatile pressure distention on the arterial wall. This review summarizes current research, which has aimed to elicit the signal transduction pathway by which cyclic strain elicits functional and structural responses in endothelial cells; specifically, it summarizes the signaling pathway that begins with the reorganization of integrins. One method by which these extracellular matrix receptors affect signal transduction is through their ability to initiate the process of phosphorylation on tyrosine residues of cytoplasmic protein kinases, including focal adhesion kinase. The strain-induced pathway appears to also involve ras and the mitogen-activated protein kinase family of enzymes, and preliminary data suggests a role for src as well. Ultimately, it is the regulation of gene expression through the modulation of transcription factors that allows endothelial cells to respond to changes in local hemodynamics.

Prostacyclin synthetic activity in cultured aortic endothelial cells undergoing cyclic mechanical deformation.

The effect of mechanical stretching on prostacyclin (PGI2) synthesis was studied by growing bovine aortic endothelial cells on flexible-bottomed culture plates that could be deformed by vacuum. A stress unit was used to exert an elongation of 24% at maximum downward deflection of the culture plate bottom. The experimental group was subjected to cycles of 10 seconds of elongation, 10 seconds of relaxation for 1, 3, or 5 days. The control group was subjected to similar incubations but without cyclic stretch. Twenty-four hours before collection, the medium was replaced with new medium that was devoid of serum. On days 1, 3, and 5, the 24-hour culture medium was collected (basal state). Arachidonic acid (20 mumol/L) was then added to each culture and incubated for 5 minutes at 37 degrees C. The medium was then collected to assess prostacyclin synthetic activity (stimulated state). Media were assayed for PGI2 and thromboxane A2 by radioimmunoassays for 6-keto-PGF1 alpha and thromboxane B2, the respective stable hydrolysis product. The results indicate that cyclic stretching, while not altering the basal production of PGI2, increases PGI2 synthetic capacity in a time-dependent manner. These data suggest that it may be inappropriate to extrapolate the mechanisms of in vivo PGI2 release from studies of endothelial cells in stationary culture.

Protein kinase C is a mediator of the adaptation of vascular endothelial cells to cyclic strain in vitro.

BACKGROUND: The mechanism by which hemodynamic forces influence the function of the endothelium lining a blood vessel are unknown. The aim of this study was to determine the effect of in vitro cyclic strain on endothelial cell (EC) activation of protein kinase C (PKC). METHODS: Confluent bovine aortic ECs grown on flexible-bottomed culture plates were subjected to 24% maximum strain at a frequency of 60 cycles/min for 24 hours. Changes in PKC activity and evidence of translocation from cytosol to membrane fractions were assessed by immunocytochemical staining of ECs with antibodies specific to PKC and direct measurement of PKC activity in cytosol and membrane. To determine whether activation of PKC was responsible for some effects of cyclic stretch on ECs, a specific PKC inhibitor, calphostin C, was added to ECs subjected to cyclic stretch for 5 days and control ECs grown under static conditions. RESULTS: Immunocytochemical staining of ECs demonstrated translocation of PKC alpha- and beta-antibody fluorescence from the cytosol to the perinuclear and nuclear regions in ECs subjected to cyclic strain. This was confirmed by direct measurements of PKC activity, which demonstrated an early transient translocation of PKC activity from cytosol to membrane fraction at 10 seconds followed by a sustained elevation in PKC activity in the membrane at 100 seconds. Calphostin C abrogated the increase in EC proliferation that occurs in response to stretch. CONCLUSIONS: We conclude that cyclic stretch of ECs results in activation of PKC, which may be responsible for mediating the effects of cyclic stretch on EC growth.

Enhanced production of endothelium-derived contracting factor by endothelial cells subjected to pulsatile stretch.

The purpose of this study was to assess the role of cyclic deformation in modulating the production by endothelial cells (ECs) in culture of a recently described endothelium-derived smooth muscle cell contracting factor, endothelin. We grew bovine aortic ECs to confluence on culture plates with flexible membrane bottoms. Vacuum (-20 kPa) was applied to deform the membrane to 24% strain at 60 cycles/min for 1, 3, or 5 days. Control ECs were grown on the same membrane but without vacuum deformation. The conditioned media were collected, centrifuged at 10,000 rpm for 10 minutes to remove cells and debris, and the supernatant fluid was subjected to radioimmunoassay for endothelin. The results demonstrate that bovine aortic ECs release a basal level of endothelin under stationary conditions (107.1 +/- 14.7 pg/10(5) cells), and this production increased fivefold to sixfold with cyclic stretch. Thus physical forces exerted on ECs in culture can influence the secretion of this vasoconstrictive molecule.

Treatment with verapamil and adenosine triphosphate-MgCl2 reduces cyclosporine nephrotoxicity.

Nephrotoxicity is a serious side effect that limits the use of cyclosporine (CyA) as an immunosuppressive agent. The purpose of this study was to develop a model of CyA nephrotoxicity in the isolated perfused rat kidney and to evaluate the effects of adenosine triphosphate (ATP)-MgCl2 and verapamil treatment on this model. Kidneys were perfused for 90 minutes in a 7.5% albumin-Krebs-HCO3 solution containing 3H-inulin (glomerular marker) and 5.0 micrograms/ml 14C-cytochrome C (cyt) (marker of tubular protein absorption). After 10-minute equilibration, perfusate and urine samples were collected with 10-minute clearance periods. After two control clearance periods, 500 ng/ml CyA was added to the perfusate. In some experiments, 1 micrograms/ml of verapamil was added 10 minutes before CyA and in others 2 mmol/l ATP-MgCl2 was added with CyA. Cyt and inulin radioactivity, [Na+] and [K+], were measured in perfusate and urine. Tissue ATP levels were also determined. The results demonstrate that CyA treatment leads to a marked depression of glomerular filtration rate (GFR), tubular absorption of protein, urine output, and renal flow. ATP-MgCl2 cotreatment improved GFR, tubular absorption, and renal perfusate flow but the increase in urine output was not dramatic. Verapamil pretreatment markedly improved GFR and urine and renal perfusate flow but not tubular function. The combination of verapamil and ATP-MgCl2 treatment with CyA returned GFR to control values, significantly improved tubular absorption, urine and renal perfusate flow, and enhanced renal tissue ATP of isolated kidneys to levels seen in vivo. These data lead us to conclude that ATP-MgCl2 cotreatment with CyA after verapamil pretreatment greatly reduces the nephrotoxic potential of this immunosuppressive agent.

Homocysteine stimulates MAP kinase in bovine aortic smooth muscle cells.

BACKGROUND: Hyperhomocysteinemia is recognized as a risk factor for atherosclerotic disease. However, the mechanism of homocysteine effects on smooth muscle cell proliferation, which is a hallmark of atherosclerosis, is unknown. The object of this study was to test the effects of homocysteine on smooth muscle cell proliferation, and to examine the mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase 1 and 2, that are known to be involved in cell proliferation. METHODS: For the proliferation study, bovine aortic smooth muscle cells (BASMC, 10, 000/well) were allowed to grow for 2 days before 2 mmol/L D,L -homocysteine was added for 2, 4, 6, and 8 days to simulate the clinical hyperhomocysteinemic condition. For the MAP kinase study, quiescent BASMC were exposed to 2 mmol/L D,L -homocysteine for 1.5, 5, 10, 20, 30, and 60 minutes, and the active forms of MAP kinase were detected with Western immunoblotting. The degree of phosphorylation of MAP kinase was determined by densitometry. RESULTS: D,L -homocysteine stimulated BASMC proliferation by 20% by day 8. MAP kinase phosphorylation was activated as much as six fold by D,L -homocysteine, with a peak at 30 minutes. PD98059, an inhibitor of MAP kinase phosphorylation, inhibited the homocysteine-induced MAP kinase phosphorylation and attenuated the increase in BASMC proliferation. CONCLUSIONS: These data are consistent with the hypothesis that D,L -homocysteine stimulation of BASMC proliferation involves MAP kinase activation.

Cyclic strain increases endothelial nitric oxide synthase activity.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) is an important enzyme that controls the production of a potent vascular smooth muscle relaxing factor, nitric oxide. However, the role of hemodynamic forces (blood pressure, cyclic strain, and shear stress) on the regulation of eNOS has not been fully elucidated. Recently, we showed that cyclic strain increases eNOS gene and protein in cultured bovine aortic endothelial cells (EC). Because an increase in gene transcription and protein synthesis may not necessarily translate into an increase in functional activity, the aim of this study was to determine the effects of cyclic strain on eNOS activity. METHODS: EC were seeded onto plates with flexible bottoms that can be deformed by vacuum and were then exposed to 60 cycles/minute of either 24% maximum strain (-20 kPa vacuum) or 10% maximum strain (-5 kPa vacuum) for 24 hours. eNOS activity was assessed, and nitric oxide production was determined (as nitrite) by the Greiss reaction. RESULTS: Twenty-four percent strain, at 60 cycles/min, but not 10% strain significantly increases eNOS activity compared with stationary controls. Both strain regimens increased nitric oxide (as nitrite) in culture media compared with stationary controls, although nitrite in media of EC exposed to high strain were significantly increased compared with the lower strain. CONCLUSIONS: Cyclic strain increases eNOS activity in cultured bovine aortic EC. These results may indicate the importance of hemodynamic forces in the regulation of eNOS in vivo.

Thrombospondin-1 regulation of smooth muscle cell chemotaxis is extracellular signal-regulated protein kinases 1/2 dependent.

BACKGROUND: Thrombospondin-1 (TSP-1), an extracellular matrix protein, induces vascular smooth muscle cell (VSMC) chemotaxis. We hypothesized that extracellular signal-regulated protein kinases 1/2 (ERK1/2), a pathway of the mitogen activated protein kinase (MAPK) family, is important in TSP-1-induced VSMC chemotaxis. METHODS: A modified Boyden chamber was used to assess chemotaxis. First, a concentration curve was performed to determine the level for optimal TSP-1-induced chemotaxis. Then quiescent VSMCs were preincubated (30 minutes) in serum-free medium, dimethyl sulfoxide (the inhibitor vehicle), or PD98059 (10 mumol/L, an upstream inhibitor of ERK1/2). VSMCs (50,000 cells/well) with the appropriate preincubation were placed in the top chamber. The bottom chamber contained TSP-1 (20 micrograms/mL) or serum-free medium. Results were recorded as cells/5 fields (400x). Then quiescent VSMCs were exposed to TSP-1 (20 micrograms/mL) for 0, 1, 5, 10, 30, 120, or 300 minutes. Platelet-derived growth factor (10 ng/mL) was the positive control for ERK1/2 activation. Western blot analysis was performed for activated ERK1/2. All comparisons were made by a paired t test (n = 3). RESULTS: TSP-1-induced chemotaxis peaks by a concentration of 20 micrograms/mL. PD98059 inhibited TSP-1-induced chemotaxis (P < .05). ERK1/2 was activated by TSP-1-stimulated VSMCs. CONCLUSIONS: TSP-1-stimulated VSMCs activated ERK1/2. An ERK1/2 inhibitor abolished chemotaxis, suggesting the functional importance of MAPK in TSP-1-induced VSMC chemotaxis.

Cyclic strain activates the pro-survival Akt protein kinase in bovine aortic smooth muscle cells.

BACKGROUND: Pulsatile pressure induced by the beating heart causes cyclic strain on arterial endothelial cells and smooth muscle cells (SMCs). This study examined whether Akt, a serine/threonine protein kinase known to promote cell survival by inhibiting apoptosis, is activated by cyclic strain in bovine aortic SMCs. METHODS: Bovine aortic SMCs were cultured on flexible-bottomed membranes and then serum-starved for 24 to 36 hours. The cells were then exposed to 150-mm Hg repetitive deformations, which created an average of 10% strain on the monolayer SMCs at a frequency of 60 cycles/minute for 0 (negative control) and 30 minutes. Platelet-derived growth factor (PDGF)--stimulated SMCs were used as positive controls. Phosphorylation of Akt was determined by means of Western blot analysis. An apoptosis assay (TUNEL) was also performed on SMCs exposed to cyclic strain. RESULTS: Akt phosphorylation was significantly increased over that of the negative control after 30 minutes of cyclic strain and in the PDGF group. Cyclic strain did not increase the prevalence of apoptosis in SMCs over the control. CONCLUSIONS: Cyclic strain activated the pro-survival Akt kinase. The pro-survival function was supported by the fact that cyclic strain did not increase apoptosis in bovine aortic SMCs. This experiment suggests that cyclic strain may induce arterial wall thickening by tipping the balance toward arterial SMC proliferation through the inhibition of apoptosis.