A model for shear stress sensing and transmission in vascular endothelial cells

Arterial endothelial cell (EC) responsiveness to flow is essential for normal vascular function and plays a role in the development of atherosclerosis. EC flow responses may involve sensing of the mechanical stimulus at the cell surface with subsequent transmission via cytoskeleton to intracellular transduction sites. We had previously modeled flow-induced deformation of EC-surface flow sensors represented as viscoelastic materials with standard linear solid behavior (Kelvin bodies). In the present article, we extend the analysis to arbitrary networks of viscoelastic structures connected in series and/or parallel. Application of the model to a system of two Kelvin bodies in parallel reveals that flow induces an instantaneous deformation followed by creeping to the asymptotic response. The force divides equally between the two bodies when they have identical viscoelastic properties. When one body is stiffer than the other, a larger fraction of the applied force is directed to the stiffer body. We have also probed the impact of steady and oscillatory flow on simple sensor-cytoskeleton-nucleus networks. The results demonstrated that, consistent with the experimentally observed temporal chronology of EC flow responses, the flow sensor attains its peak deformation faster than intracellular structures and the nucleus deforms more rapidly than cytoskeletal elements. The results have also revealed that a 1-Hz oscillatory flow induces significantly smaller deformations than steady flow. These results may provide insight into the mechanisms behind the experimental observations that a number of EC responses induced by steady flow are not induced by oscillatory flow.     

Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.     

Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.     

Resting smooth muscle cells as a model for studying vascular cell activation

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also re-acquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimic in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.     

Improved arterial wall model by coculturing vascular endothelial and smooth muscle cells

We have constructed an in vitro arterial wall model by coculturing bovine arterial endothelial cells (ECs) and smooth muscle cells (SMCs). When ECs were seeded directly over SMCs and cocultured in an ordinary culture medium, ECs grew sparsely and did not form a confluent monolayer. Addition of ascorbic acid to the culture medium at concentrations greater than 50 μg/ml increased the production of type IV collagen by the SMCs, and ECs formed a confluent monolayer covering the entire surface of SMCs. Histological studies showed that the thickness of the cell layer composed of ECs and SMCs increased with increasing duration of coculture. This arterial wall model, prepared by our method, may serve as a simple and good in vitro model to study the effects of factors such as biological chemicals and shear stress on cell proliferation and other physiological functions of arterial walls.     

Communication between endothelial and smooth muscle cells

Vascular endothelial cells play a fundamental role in the control of vascular tone, and therefore in the control of local blood flow, by releasing various contracting (endothelin, prostaglandins) and relaxing (prostacycline, NO) factors. An additional mechanism involving the hyperpolarization of the vascular smooth muscle cells is observed mainly in the coronary vascular bed and in the periphery. This phenomenon was attributed to an elusive endothelial factor called endothelium-derived hyperpolarizing factor (EDHF). This mechanism is now better understood. It involves first an increase in the endothelial intracellular concentration of calcium, the activation of endothelial potassium channels and the resulting hyperpolarization of the endothelial cells. The hyperpolarization of the endothelial cells is transmitted to the smooth muscle cells by different pathways. This hyperpolarization propagates along the vessels not only via the smooth muscle cells but also via the endothelial cells. Therefore, the endothelial layer can also be considered as a conducting tissue. The discovery of specific inhibitors of the endothelial cell hyperpolarization allows the assessment of the contribution of EDHF-mediated responses in the control of vascular tone.     

Differential effect of atorvastatin and tacrolimus on proliferation of vascular smooth muscle and endothelial cells

Although considered promising for use in drug-eluting stents (DES), tacrolimus failed clinically. Tacrolimus inhibits growth factor production but can also act as a growth factor on vascular smooth muscle cells (VSMC). This unexpected proliferative stimulus could reverse the beneficial effects of the drug on restenosis. We hypothesized that tacrolimus' association with statins, which lower cholesterol and impair cell proliferation, could restore tacrolimus' beneficial effect by abrogating the aberrant proliferative stimulus. Additionally, since maintenance of endothelial function represents a challenge for new-generation DES, we investigated the combined effect of tacrolimus and atorvastatin on endothelial cells. Human VSMC and umbilical vein endothelial cells (HUVEC) were incubated with 100 nM tacrolimus and increasing doses of atorvastatin (0-3.0 μM). Atorvastatin plus tacrolimus dose-dependently inhibited VSMC proliferation. The percentage of cells incorporating 5-bromo-2'-deoxyuridine (BrdU) in their DNA was 49 ± 14% under basal conditions, 62 ± 15% (P = 0.01) with tacrolimus, 40 ± 22% with 3 μM atorvastatin, and 30 ± 7% (P < 0.05) with 3 μM atorvastatin plus tacrolimus. Atorvastatin downregulated β-catenin, Erk1 and Erk2, and cyclin B in tacrolimus-stimulated VSMC. In contrast, atorvastatin plus tacrolimus did not affect proliferation of endothelial cells. The percentage of HUVEC incorporating BrdU in their DNA was 47 ± 8% under basal conditions, 58 ± 6% (P = 0.01) with tacrolimus, 45 ± 4% with 3 μM atorvastatin, and 49 ± 1% with 3 μM atorvastatin plus tacrolimus. Both agents stimulated endoglin production by HUVEC. Taken together, these results suggest that, when combined with tacrolimus, atorvastatin exerts a dose-dependent antiproliferative effect on VSMC. In contrast, atorvastatin acts in concert with tacrolimus in HUVEC to stimulate production of endoglin, a factor that has an important role in endothelial repair. Our study supports the conclusion that prevention of postcoronary in-stent restenosis and late thrombosis may benefit of concomitant association of tacrolimus and high doses of atorvastatin.     

A steady-state electrochemical model of vascular smooth muscle cells

A model of the steady-state electrochemical response of vascular smooth muscle cells to external stimuli is presented, which accounts for K, Na, and Ca fluxes. The results of the model are broadly in accordance with experimental data 1), at various transmural pressures; 2), with channel and pump blockade; and 3), under manipulation of external ionic concentrations. The model exhibits dual stable states which sometimes coexist, and abrupt transitions between these states may account for nongraded responses in arteries as external potassium or pressure is varied. The simulations suggest that changes in the intracellular sodium concentration ([Na]i) often accompany smooth muscle responses. For example, [Na]i values vary threefold over the range of pressures from 10 to 100 mmHg.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Molecular basis of the effects of shear stress on vascular endothelial cells

Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells (ECs) are subjected to the shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular functions, e.g., proliferation, apoptosis, migration, permeability, and remodeling, as well as gene expression. The ECs use multiple sensing mechanisms to detect changes in mechanical forces, leading to the activation of signaling networks. The cytoskeleton provides a structural framework for the EC to transmit mechanical forces between its luminal, abluminal and junctional surfaces and its interior, including the cytoplasm, the nucleus, and focal adhesion sites. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows. In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.     

A simple adhesion assay for studying interactions between platelets and endothelial cells in vitro

Cell adhesion plays a key role during various physiological and pathological processes. Many studies have been performed to understand the interaction of platelets with endothelial cells (ECs) during the past decades. Modulation of their interaction has been shown to be therapeutically useful in thrombotic diseases. Some methods of labeling platelets such as counting and radiolabeling have been applied in the study of the platelets-ECs interaction, but these methods did not obtain full approval. A rapid, simple and sensitive assay for platelets-ECs interaction was developed in this paper. Platelets were labeled with Sudan Black B (SBB) before adding to confluent ECs monolayer. Non-adherent platelets were removed by washing with PBS. The adherent platelets were lysed with dimethylsulfoxide (DMSO) and the absorbance was recorded at 595 nm by spectrophotometer. A linear correlation was observed between the absorbance of SBB and the number of platelets. By employing the SBB method, the influence of heparin on platelets-ECs interactions was observed. Heparin (3–100 units/mL) obviously reduced platelets adhering to ECs in a concentration-dependent manner.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

Protein tyrosine phosphatase LMW-PTP exhibits distinct roles between vascular endothelial and smooth muscle cells

The present study examined the cellular functions of low-molecular-weight protein tyrosine phosphatase (LMW-PTP), which consists of two active isoforms IF-1 and IF-2, in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), focusing on cell growth and migration. We transduced recombinant IF-1 and IF-2, and ribozyme targeting both isoforms using an adenovirus vector in these cells. We detected the expression of IF-1 and IF-2 in both types of cells. IF-1 as well as IF-2 inhibited PDGF-induced DNA synthesis and migration in VSMCs. In contrast, both isoforms enhanced lysophosphatidic acid-stimulated cell migration without change in DNA synthesis in ECs. Whereas there is a report indicating that reactive oxygen species-dependent inactivation of LMW-PTP regulates actin cytoskeleton reorganization during cell spreading and migration, the isoforms conversely suppressed the PDGF-induced H2O2 generation with subsequent decrease in the p38 activity in VSMCs. Catalytically inactive LMW-PTP exerted the opposite and similar effects to the wild type in ECs and in VSMCs, respectively, suggesting that substrates for the phosphatase differ between these cells. Moreover, high concentrations of glucose suppressed the expression of LMW-PTP in both cells. These data suggest that LMW-PTP negatively regulates the pathogenesis of atherosclerosis and that glucose-dependent suppression of LMW-PTP expression may promote the development of atherosclerosis in diabetics.     

Negative regulation of vascular smooth muscle cell migration by blood shear stress

Urocortin (UCN) protects hearts against ischemia and reperfusion injury whether given before ischemia or at reperfusion. Here we investigate the roles of PKC, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute UCN treatment improved hemodynamic recovery during reperfusion after 30 min of global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of 2-[(3)H]deoxyglucose). UCN pretreatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that UCN decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 h) treatment with UCN reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells that had been pretreated with UCN, suggesting that this is the mechanism by which UCN desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKC-epsilon or PKC-alpha translocate to the mitochondria following acute UCN treatment, inhibition of PKC with chelerythrine eliminated the effect of UCN on oxidative stress. Our data suggest that acute UCN treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.     

Co-cultivation of vascular endothelial and smooth muscle cells using microcarrier techniques

A system is described which uses microcarrier culture techniques for the co-cultivation of different cell types without direct contact between cell populations. In co-cultivation, arterial endothelial cells induced proliferation in > 90% of quiescent homologous arterial smooth muscle cells in the absence of serum-derived growth factors. The microcarrier coculture system allows investigation of potent local humoral interactions between vascular cells in vitro.     

Antiproliferative effect of L-NAME on rat vascular smooth muscle cells

The nitric oxide synthase (NOS) inhibitor L-NAME may have growth inhibitory effects in vivo. We investigated in vitro the potential growth inhibitory effects of three different NOS inhibitors: L-NAME (1 mM), LNMMA (1 mM) and aminoguanidine (0.5 mM), on fetal bovine serum (FBS) and platelet derived growth factor (PDGF-BB)-stimulated growth in cultured vascular smooth muscle cells (VSMCs). [3H]-thymidine incorporation into rat mesenteric VSMCs was measured as an index of VSMCs proliferation (DNA synthesis) and activation of extracellular signal regulated kinase (ERK1/2), a major signaling event in cell growth, was measured by western blot assay. PDGF-BB (0-5 ng/mL) and FBS (0-5%) increased [3H]-thymidine incorporation in a dose-dependent manner up to 6-10 fold. L-NAME significantly reduced PDGF-BB (5 ng/ml) and FBS (5%) stimulated DNA synthesis by 46% and 38% respectively. The increase of [3H]-thymidine incorporation induced by PDGF-BB and FBS was unaltered by L-NMMA. In contrast, aminoguanidine induced an increase in FBS and PDGF-BB-stimulated [3H]-thymidine incorporation of 64% and 34% respectively above cells not exposed to aminoguanidine. ERK1/2 phosphorylation induced by PDGF-BB and FBS was not affected by pre-treatment with L-NAME or aminoguanidine. In conclusion, NOS inhibitors differentially influence DNA synthesis in VSMCs: L-NAME inhibits FBS and PDGF-BB-stimulated cellular proliferation whereas aminoguanidine accentuates FBS and PDGF-BB-stimulated VSMCs proliferation. These phenomena are independent of the ERK1/2 pathway. The growth inhibitory effects of L-NAME may be related to differences in properties from other NOS inhibitors, and independent of its ability to inhibit NOS.     

A model for shear stress-induced deformation of a flow sensor on the surface of vascular endothelial cells

Fluid mechanical shear stress elicits humoral, metabolic, and structural responses in vascular endothelial cells (ECs); however, the mechanisms involved in shear stress sensing and transduction remain incompletely understood. Beyond being responsive to shear stress, ECs distinguish among and respond differently to different types of shear stress. Recent observations suggest that endothelial shear stress sensing may occur through direct interaction of the flow with cell-surface structures that act as primary flow sensors. This paper presents a mathematical model for the shear stress-induced deformation of a flow sensor on the EC surface. The sensor is modeled as a cytoskeleton-coupled viscoelastic structure exhibiting standard linear solid behavior. Since ECs respond differently to different types of flow, the deformation and resulting velocity of the sensor in response to steady, non-reversing pulsatile, and oscillatory flow have been studied. Furthermore, the sensitivity of the results to changes in various model parameters including the magnitude of applied shear stress, the constants that characterize the viscoelastic behavior, and the pulsatile flow frequency (f) has been investigated. The results have demonstrated that in response to a suddenly applied shear stress, the sensor exhibits a level of instantaneous deformation followed by gradual creeping to the long-term response. The peak deformation increases linearly with the magnitude of the applied shear stress and decreases for viscoelastic constants that correspond to stiffer sensors. While the sensor deformation depends on f for low f values, the deformation becomes f -independent above a critical threshold frequency. Finally, the peak sensor deformation is considerably larger for steady and non-reversing pulsatile flow than for oscillatory flow. If the extent of sensor deformation correlates with the intensity of flow-mediated endothelial signaling, then our results suggest possible mechanisms by which ECs distinguish among steady, non-reversing pulsatile, and oscillatory shear stress.     

Dynamic modeling for shear stress induced ATP release from vascular endothelial cells

A dynamic model is proposed for shear stress induced adenosine triphosphate (ATP) release from endothelial cells (ECs). The dynamic behavior of the ATP/ADP concentration at the endothelial surface by viscous shear flow is investigated through simulation studies based on the dynamic ATP release model. The numerical results demonstrate that the ATP/ADP concentration against time at endothelium-fluid interface predicted by the dynamic ATP release model is more consistent with the experimental observations than that predicted by previous static ATP release model.     

The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.