Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21cip1 in vascular remodelling during vein arterialization in the rat

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while α-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs . the media layer (100 vs . 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27^Kip1 and p16^INKA remained unchanged, whereas p21^Cip1 was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21^Cip1 is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.     

自体静脉移植后管壁超微结构的变化

目的　探讨自体静脉移植后内皮形态特征和中膜SMC增生特点 ,为静脉搭桥后再狭窄的防治提供资料。方法　兔 18只 ,随机分 6组 ,均作模拟血管搭桥手术 ,即将颈外静脉移植于颈总动脉 ,术后取材 ,光电镜观察。结果　移植后 1～ 4周管壁渐增厚 ,4～ 6周时达最厚 ,随后管壁不再继续增厚。对照侧静脉无微绒毛梭形内皮被移植段具有丰富微绒毛呈不同形态的内皮细胞所替代。移植后 1～ 2周 ,SMC肌丝和致密体减少 ,与合成和分泌有关的细胞器异常丰富 ,SMC从收缩型转变成合成型 ,四周时分泌型细胞器略有减少 ,但细胞间的胶原纤维明显增多。第 6～ 12周 ,SMC又渐从分泌型向收缩型转变。结论　自体静脉移植后 4～ 6周管壁厚度达高峰 ,内皮微绒毛增多。中膜SMC在 1～ 2周转型增殖最明显。提示术后 2周内是药物抑制SMC增殖的良机     

Changes in the arrangement of actin bundles during heart looping in the chick embryo

Summary We assessed the arrangement of actin bundles in the looping chick heart. Actin filaments were stained with rhodamine-labeled phalloidin, and their total arrangement was observed in whole mount specimens. Before the straight heart tube was formed, actin bundles were in a net-like arrangement as if to indicate the cell borders. With progress of the heart tube formation, actin bundles were gradually arranged in a circumferential direction. In the looped heart, regional differences in actin arrangements were observed. In the truncus arteriosus, actin bundles ran in a net-like arrangement. In the bulbus cordis, actin bundles ran in random directions. In the ventricle, actin bundles were roughly arranged in a circumferential direction. Between these three regions, actin bundles ran in a circumferential direction especially on the concave side. Near the right contour on the ventral face, some actin bundles ran in a longitudinal direction along the axis of the tubular heart. In the bulbus cordis and the ventricle at the looped stage, there was another group of actin bundles in the inner layer of the myocardium which ran in a circumferential direction. We presume that the arrangement of actin bundles is related to heart looping.     

Arrangements of actin filaments in the cytoskeleton of human platelets

Assembly of actin molecules into filaments is closely associated with platelet shape change and exercise of contractile function. Since assembled actin filaments serve both as a framework for distortion of discoid shape and for generation of contractile force, it is important to determine the organizations of actin filaments capable of serving the two opposed functions of distortion and contraction. The present study has used negative staining alone and after combined glutaraldehyde fixation and detergent extraction to examine the arrangements of actin filaments in the cytoskeletons of surface activated platelets. Actin filament assembly developed as one of the earliest manifestations of platelet activation. Small protuberances containing random networks of actin filaments extending beyond the circumferential microtubules appeared to be an initial step in the response to stimulation. Transformation into dendritic forms was associated with development of parallel bundles of actin filaments organized into paracrystalline lattices with a periodicity of 5.5 nm at an angle of 60 degrees with the long axis of the pseudopod. Parallel bundles of actin filaments formed the concave borders of late dendritic forms and expanded to become the convex margin of most spread cells, suggesting a possible role in the spreading process. Other bundles of actin filaments resembled stress fibers radiating through the cytoplasm into pseudopods or organized in a variety of other apparently stable configurations. More loosely associated masses of actin filaments formed concentric layers around constricted rings of microtubules or a random network in the peripheral cytoplasm of spread cells. The arrangements of newly assembled actin filaments suggest their involvement in cell deformation, as well as contractile events.     

Occludin modulates organization of perijunctional circumferential actin in rat endothelial cells

The tight junction is not a constitutional junctional apparatus in endothelial cells, but develops in a particular lineage of endothelia, such as the capillary endothelia in the brain and retina, and thus is considered to be pivotal for the maintenance of the blood-tissue barrier. Occludin is an integral membrane component of tight junctions, but the role of occludin in the endothelial cell function remains to be elucidated. We have cloned and transfected rat full-length occludin cDNA into a rat endothelial cell line (RLE) that expressed only a trace amount of occludin with no fine circumferential actin bundles at the cell border in native conditions. Occludin was expressed at the cell border of RLE cells, and circumferential fine actin bundles developed in close relation to the sites of occludin localization. Even under subconfluent culture conditions, fine circumferential actin bundles were formed at the sites where occludin-positive cell-cell contact was achieved. In immunoelectron microscopy, occludin was localized at distinct areas of the plasma membrane, always in association with the cytoplasmic actin filaments. On the other hand, actin bundles were not seen in occludin-negative juxtaposing plasma membranes. Collectively, these data strongly suggested a possible determinant function of occludin for the organization of actin in endothelial cells.     

Subnormal shear stress-induced intimal thickening requires medial smooth muscle cell proliferation and migration

Arterial intimal thickening is consisted of predominately smooth muscle cells (SMC). The source of these SMCs and mechanisms response for their changes have not been well cleared. Using a model of rabbit common carotid artery (CCA) shear induced intimal thickening, we sought to identify and describe the source of SMCs in intima. The enlarged CCA 28 days after arteriovenous fistula (AVF) creation was subjected to subnormal wall shear stress (WSS) for 1, 3, and 7 days by closure of the AVF. To determine SMC proliferation, BrdU pulse labeling of SMCs was performed. BrdU-labeled SMCs were tracked over time to further confirm SMC migration. In response to subnormal WSS intimal thickening developed progressively. BrdU-labeled SMCs localized in the subendothelial area. When the BrdU-labeled medial SMCs were tracked 1 day after AVF closure, progenies of these BrdU-incorporated SMCs increased by 4.8-fold with 75% of them in the intima. They were 12-fold increased with 83% in the intima 7 days after. En face examination showed an accumulation of SMCs in internal elastic lamina gap after AVF closure, which later migrated into subendothelial area. In situ hybridization revealed increased TGF-beta1 mRNA expression in intimal SMCs. This study demonstrates that the medial SMCs are the predominant cells in subnormal WSS-induced intimal thickening. Early expression of TGF-beta1 may play an important role in the process of intimal thickening.     

不同转移潜能肿瘤细胞胸腺素β10表达水平及微丝结构的差异性研究

目的 研究胸腺素β10(Tlaymosinβ10,Tβ10)表达水平与人类肿瘤转移潜能的相关性以及细胞内微丝骨架的变化差异。方法 利用Northern-blot和细胞爬片免疫组织化学的方法检测4组9种具有不同转移潜能的人类肿瘤细胞系中Tβ10 mRNA和蛋白的表达水平;通过组织化学的方法显示细胞内微丝骨架的变化。结果 Tβ10的mRNA和蛋白表达水平在高转移潜能的人类肺癌、黑色素瘤和乳腺癌细胞中高于相应的不／低转移肿瘤细胞;高转移性癌与不／低转移癌细胞比较,前者细胞内肌动蛋白多聚体减少,微丝骨架杂乱、模糊,而低转移癌系微丝骨架粗大、结构清晰、排列整齐。结论 在所研究的人类肿瘤中,Tβ10 mRNA和蛋白表达水平与肿瘤的转移能力呈正相关性;肿瘤转移潜能的增高伴有肌动蛋白多聚体的丢失和微丝结构的解聚,微丝骨架的这种变化与Tβ10的表达增高具有相关性。     

Neonatal pulmonary hypertension prevents reorganisation of the pulmonary arterial smooth muscle cytoskeleton after birth

The pulmonary arterial smooth muscle cell (SMC) cytoskeleton was studied in tissue from 36 piglets aged from within 5 min of birth to 21 d of age, and in 8 adults. An additional 16 piglets were made pulmonary hypertensive by exposure to hypobaric hypoxia (50.8 kPa) for 3 d. In conduit intrapulmonary elastic arteries α, β and γ actin, the 204, 200 and 196 kDa myosin heavy chain (MHC) isoforms and vinculin were localised by immunohistochemistry. The total actin content, the proportion of monomeric to filamentous α and γ actin and changes in the proportions of the MHC isoforms were determined biochemically. Dividing SMCs were localised and quantified using Ki-67. We found a transient reduction in immunohistochemical expression of γ actin, 204 kDa MHC isoform and vinculin at 3 and 6 d in the inner media, associated with a transient increase in Ki-67 labelling. The actin content also decreased at 3 and 6 d ( P < 0.05), but there was a postnatal, permanent increase in monomeric actin, first the α then the γ isoform. The relative proportions of the MHC isoforms did not change between birth and adulthood in elastic pulmonary arteries but in muscular arteries the 200 kDa isoform increased between 14 d and adulthood. Pulmonary hypertension prevented both the immunohistochemical changes and the postnatal burst of SMC replication and prevented the transient postnatal reduction in actin content. These findings suggest that rapid remodelling of the actin cytoskeleton is an essential prerequisite of a normal postnatal fall in pulmonary vascular resistance.     

Actin filament organization in aligned prefusion myoblasts

The organization of the actin cytoskeleton in prefusion aligning myoblasts is likely to be important for their shape and interaction. We investigated actin filament organization and polarity by transmission electron microscopy (TEM) in these cells. About 84% of the filaments counted were either found in a subplasmalemma sheet up to 0.5 microm thick that was aligned with the long axis of the cell, or in protrusions. The remaining filaments were found in the cytoplasm, where they were randomly orientated and not organized into bundles. The polarity of the subplasmalemma filaments changed progressively from one end of the cell to the other. At the ends of the cells and in protrusions, the majority of filaments were organized such that their barbed ends faced the tip of the protrusion. We did not find any actin filament bundles or stress fibres in these cells. Time-lapse phase microscopy demonstrated that aligned cells were still actively migrating at the time of our TEM observations, and their direction of movement was restricted to the long axis of the cell group. The ability of these cells to locomote actively in the absence of actin filament bundles suggests that in these cells the subplasmalemma actin sheet contributes not only to cell shape but also to cell locomotion.     

Development of an Endothelial–Smooth Muscle Cell Coculture Model Using Phenotype-Controlled Smooth Muscle Cells

A coculture of endothelial cells (ECs) and smooth muscle cells (SMCs), which mimics cellular interactions appearing in vivo, has been performed in studies on the relationship between atherogenesis and fluid shear stress conditions. Although healthy arteries in vivo consist of contractile phenotype SMCs, cultured cells used in many studies normally exhibit a synthetic phenotype. Here, we developed an EC–SMC coculture model to investigate the interactions between ECs and contractile SMCs, and examined the effect of shear stress applied to ECs on SMC phenotypes. Cultured human umbilical artery SMCs were differentiated into contractile states by arresting cell growth using a serum-free medium. Western blotting confirmed that SMC expression of contractile protein markers, α-smooth muscle actin (SMA) and calponin, increased to levels similar to those observed in arterial cells. After coculturing contractile SMCs with ECs separated by a collagen gel layer, the expression of α-SMA decreased under static conditions, indicating that the SMC phenotype tended to be synthetic by coculturing with ECs, but shear stress applied to cocultured ECs maintained the level of α-SMA expression in SMCs. The coculture model constructed in the present study will be a useful tool to investigate interactions between ECs and contractile SMCs under shear conditions.     

Evaluation of electrostatically endothelial cell seeded expanded polytetrafluoroethylene grafts in a canine femoral artery model

(1) PURPOSE: The purpose of this study was to evaluate the extent (luminal coverage) of the endothelial cell (EC) lining/neointimal development and the thromboresistance of electrostatically EC seeded small diameter e-PTFE vascular grafts. (2) METHODS: This evaluation consisted of harvesting autologous, canine jugular vein ECs, electrostatically EC seeding the e-PTFE grafts (4 mm GORE-TEX, Length = 6 cm), implanting the grafts in a canine femoral artery model for six weeks, and excising the graft for histological and scanning electron microscopy evaluations. (3) RESULTS: The results of the histological evaluation (mid-graft region only) indicated that the electrostatic EC seeding significantly affected neointimal development (p < 0.01) and the degree of thrombus formation (p < 0.001) within the EC seeded grafts versus the untreated control grafts. Scanning electron microscopy examination demonstrated a mature, confluent endothelium with a "cobblestone" appearance on the EC seeded graft luminal surface. The control grafts demonstrated an equal distribution of SMCs through the graft wall while the electrostatically EC seeded graft sections exhibited an uneven SMC cellular distribution which was skewed toward the graft luminal surface. (4) CONCLUSIONS: The presence of electrostatic EC seeding significantly (p < 0.01) enhanced the development of a neointima and reduced the incidence of thrombosis in e-PTFE grafts implanted in a canine femoral artery model. Results of the mid-graft SMC migration measurements indicate that the electrostatic EC seeding had a significant (p < 0.001) impact on the acute healing of the standard wall e-PTFE vascular graft specimens.     

The roles of fascins in health and disease

Fascins are actin-binding proteins that cross-link filamentous actin into tightly packed parallel bundles. These bundles are important for the organization and morphology of an extremely diverse set of sub-cellular structures that include dynamic and stable cell-surface protrusions, stress fibres, and the specialized actin bundles of photoreceptor and stereocilia cells. In this review, we discuss the fascin gene family and its evolution, the actin-bundling activity of fascins and the molecular pathways by which it is regulated, and the role of the diverse actin/fascin structures in normal cellular processes. We discuss the mechanisms by which fascins contribute to disease pathologies, especially cancer, where fascin-1 is emerging as a novel therapeutic target in carcinoma metastasis.     

The modulation of smooth muscle cell phenotype is an early event in human aorto-coronary saphenous vein grafts

Summary The morphological changes in human vein grafts occurring in the first days after a coronary bypass operation (CBP) are rarely reported in the literature. Sections of aorto-coronary vein grafts from 11 patients who died during the first 10 days after a CBP were obtained at autopsy. The number of vein grafts per patient ranged from 1 to 4, yielding a total of 28 vein grafts. The early changes in the vein grafts have been studied by light microscopy, immunohistochemistry, transmission and scanning electron microscopy. The study demonstrates that soon after grafting, the vein wall is infiltrated by polymorphonuclear leucocytes (PMN). At 24 h the endothelium shows extensive desquamation. The massive migration of PMN through the venous wall occurs simultaneously with the endothelial damage. The circular layer of the media is severely damaged, resulting in a loss of smooth muscle cells (SMC). The remaining SMC in this layer show a change toward the synthetic phenotype and a reduced expression of α-smooth muscle actin. These early changes in the SMC function may initiate the process of fibrosis in the intima and the media of the vein grafts.     

The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells

Hydrogels based on poly(ethylene glycol) (PEG) are of increasing interest for regenerative medicine applications and are ideal materials to direct cell function due to the ability to confer key functionalities of native extracellular matrix (ECM) on PEG's otherwise inert backbone. Given extensive recent evidence that ECM compliance influences a variety of cell functions, PEG-based hydrogels are also attractive due to the ease with which their mechanical properties can be controlled. In these studies, we exploited the chemical and mechanical tunability of PEG-based gels to study the impact of ECM chemistry and mechanics on smooth muscle cells (SMCs) in both 2-D and 3-D model systems. First, by controlling the extent of crosslinking and therefore the mechanical properties of PEG-based hydrogels (tensile moduli from 13.7 to 423.9kPa), we report here that the assembly of F-actin stress fibers and focal adhesions, indicative of the state of actin contractility, were influenced by the compliance of 2-D PEG gels functionalized with either short adhesive peptides or full-length ECM proteins. Varying ECM ligand density and identity independent of gel compliance affected the physical properties of the focal adhesions, and also influenced SMC spreading in 2-D. Furthermore, SMCs proliferated to a greater extent as gel stiffness was increased. In contrast, the degree of SMC differentiation, which was qualitatively assessed by the extent of smooth muscle alpha-actin bundling and the association of calponin and caldesmon with the alpha-actin fibrils, was found to decrease with substrate stiffness in 2-D cultures. In 3-D, despite the fact that their viability and degree of spreading were greatly reduced, SMCs did express some contractile markers indicative of their differentiated phenotype when cultured within PEG-RGDS constructs. Combined, these data suggest that the mechanical and chemical properties of PEG hydrogels can be tuned to influence SMC phenotype in both 2-D and 3-D.     

Quick layer-by-layer assembly of aligned multilayers of vascular smooth muscle cells in deep microchannels

One of the main challenges in vascular tissue engineering has been mimicking the complex native three-dimensional (3D) architecture of smooth muscle cells (SMCs). In the current study, we performed layer-by-layer (LBL) seeding of SMCs in a microchanneled scaffold, with or without interleaving a thin layer of collagen type I hydrogel, toward fabricating the 3D microarchitecture. This LBL process avoids the "steric hindrance" effect observed in direct 3D culture of SMCs in collagen hydrogel. More importantly, the LBL process enables the building up of multilayers of aligned and confluent SMCs. Within each layer, the SMCs as well as the SMC F-actin and alpha-actin filaments align along the direction of the scaffold microchannels, which would potentially improve the tensile and contractile strength of the tissue engineered construct, desirable properties for an engineered vasculature. In addition, rapid two-dimensional (2D) patterning of SMCs is possible with high seeding density, which makes the LBL method feasible for fabrication of multilayered structures in a short time, rendering it useful in clinical therapeutic applications.     

Expression of matrix Gla protein and osteonectin mRNA by human aortic smooth muscle cells.

BACKGROUND: Recent data indicate that matrix proteins such as matrix Gla protein (MGP) and osteonectin (ON) influence not only mineralization of vasculature but smooth muscle cell (SMC) differentiation. METHODS: We examined whether MGP and ON are expressed by human aortic SMCs in vivo using Northern blotting, in situ hybridization and immunohistochemistry. RESULTS: MGP and ON mRNAs were strongly expressed in the aorta without atherosclerosis from newborn and four young subjects up to 10 years old. In the aorta from 15 adult cases, MGP and ON mRNAs were decreased as atherosclerosis developed. We determined cell type and distribution of the MGP- and ON mRNA-expressing cells by in situ hybridization and immunohistochemistry. In the aorta obtained from newborn and young subjects, SMCs in the media and thin intima expressed MGP mRNA and, to a lesser extent, ON mRNA. In the adult aorta with fibrous thickening, MGP mRNA was expressed by intimal SMCs and subpopulation of medial SMCs. Osteonectin mRNA was expressed mainly by intimal SMCs and few medial SMCs. Double immunohistochemical staining revealed that both MGP- and ON protein-expressing cells were positive for anti-alpha-smooth muscle actin antibody, aortic SMCs. CONCLUSIONS: These results suggested that MGP and ON expression by aortic SMCs might be regulated by the degree of atherosclerosis and SMC differentiation in human aorta.     

A system for the direct co-culture of endothelium on smooth muscle cells

The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/cm(2) for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.     

Structural relationships between the endothelial actin system and the underlying elastic layer in the distal interlobular artery of the rat kidney

Summary The structure of the intracellular actin filaments and the extracellular matrices was studied in the distal interlobular arteries in the rat kidney, employing three different morphological techniques, including rhodamin-phalloidin staining of cryosections, resorcin-fuchsin staining of paraffin sections, and a cold dehydration procedure for electron microscopy. The endothelial cells possess longitudinally running stress fibers. The inner elastic layer is composed of meshworks of elastic fibers encompassing numerous pores. The smooth muscle cells containing abundant actin filaments are arranged circumferentially around the vascular axis. The endothelial stress fibers are found mainly in the basal half of the endothelial cells, and anchor onto the basal cell membranes. The elastic meshworks send off longitudinal branch fibers to contact the endothelial cell membranes at the anchoring sites of stress fibers. In addition circumferential branches run toward the smooth muscle cells. The functional significance of the intracellular contractile apparatus and the extracellular tensile component in small arteries was discussed.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress.

The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.