Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations

Bone marrow mesenchymal stem cells (MSCs) have multi-differentiation capability. Their endothelial cell (EC) oriented differentiation is the key to vasculogenesis, in which both mechanical and chemical stimulations play important roles. Most previous studies reported individual effects of VEGF or fluid shear stress (SS), when MSCs were subjected to shear stress of 10–15 dyn/cm² over 24 hr. In this paper, we investigated responses of MSCs from young Sprague Dawley rats to shear stress, VEGF and the combination of the two stimuli. Our study showed that the combined stimulation of shear stress and VEGF resulted in more profound EC oriented differentiation of MSCs in comparison to any individual stimulation. Furthermore, we subjected MSCs to prolonged period of fluid shear stimulation, i.e. 48 hr rather than 24 hr, and increased the magnitude of the shear stress from 10 dyn/cm² to 15, 20 and 25 dyn/cm². We found that without VEGF, the endothelium oriented differentiation of MSCs that was seen following 24 hr of shear stimulation was largely abolished if we extended the shear stimulation to 48 hr. A similar sharp decrease in MSC differentiation was also observed when the magnitude of the shear stress was increased from 10–15 dyn/cm² to 20–25 dyn/cm² in 24 hr shear stimulation studies. However, with combined VEGF and fluid shear stimulation, most of the endothelial differentiation was retained following an extended period, i.e. at 48 hr, of shear stimulation. Our study demonstrates that chemical and mechanical stimulations work together in determining MSC differentiation dynamics.     

Laminar shear stress induces the expression of aquaporin 1 in endothelial cells involved in wound healing

Laminar shear stress (LSS) due to blood flow contributes to the maintenance of endothelial health by multiple mechanisms including promotion of wound healing. The present study examined the hypothesis that the induction of water channel aquaporin 1 (AQP1) expression by LSS might be functionally associated with endothelial wound healing. When human umbilical vein endothelial cells were exposed to LSS at 12 dyn cm^?2 for 24 h, significant increases in AQP1 expression were observed at the mRNA and protein levels as compared with static control. In the in vitro scratch wound healing assay, LSS treatments before and after wound creation enhanced endothelial wound healing and this effect was significantly attenuated by selective suppression of AQP1 expression using small interfering RNA. Ectopic expression of AQP1 enhanced wound healing in the absence of LSS. This study demonstrated that LSS stimulates the endothelial expression of AQP1 that plays a role in wound healing.     

Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions

AimsTo determine how statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) affect endothelial cell (EC) shape and F-actin cytoskeleton arrangement in the presence of physiologically relevant wall shear stress (WSS) of 12.5 dyn/cm².Main methodsHuman abdominal aortic endothelial cells (HAAECs) were cultured to a confluent monolayer within three dimensional tissue culture models and presheared for 6 h at 12.5 dyn/cm² within a continuous flow loop. Statins were added to the perfusion media and the perfusion was continued for a further 24 h. ECs were then analyzed for morphology and F-actin cytoskeleton arrangement using light microscopy and laser scanning confocal microscopy.Key findingsECs became rounded with a significantly higher shape index with the addition of 10 μM simvastatin under both static and flow conditions. F-actin cytoskeleton structure was disorganized and fragmented with statin treatment under static and flow conditions. Neither of these findings were observed with the addition of both simvastatin and 200 μM mevalonate, confirming regulation through the cholesterol biosynthesis pathway.SignificanceEC morphology and F-actin cytoskeleton arrangement are regulated through the cholesterol biosynthesis pathway and are therefore impacted by statin treatment. ECs treated with statins became rounded, which is usually associated with unhealthy cells in regions of the vasculature prone to developing atherosclerotic plaques.     

Optimization of intravascular shear stress assessment in vivo

The advent of microelectromechanical systems (MEMS) sensors has enabled real-time wall shear stress (WSS) measurements with high spatial and temporal resolution in a 3-D bifurcation model. To optimize intravascular shear stress assessment, we evaluated the feasibility of catheter/coaxial wire-based MEMS sensors in the abdominal aorta of the New Zealand white (NZW) rabbits. Theoretical and computational fluid dynamics (CFD) analyses were performed. Fluoroscope and angiogram provided the geometry of aorta, and the Doppler ultrasound system provided the pulsatile flow velocity for the boundary conditions. The physical parameters governing the shear stress assessment in NZW rabbits included (1) the position and distance from which the MEMS sensors were mounted to the terminal end of coaxial wire or the entrance length, (L_e), (2) diameter ratios of aorta to the coaxial wire (D_aorta /D_{coaxial wire}=1.5–9.5), and (3) the range of Reynolds numbers (116–1550). At an aortic diameter of 2.4 mm and a maximum Reynolds number of 212 (a mean Reynolds number of 64.2), the time-averaged shear stress (τ_ave) was computed to be 10.06 dyn cm^?2 with a systolic peak at 33.18 dyn cm^?2. In the presence of a coaxial wire (D_aorta /D_{coaxial wire}=6 and L_e=1.18 cm), the τ_ave value increased to 15.54 dyn cm^?2 with a systolic peak at 51.25 dyn cm^?2. Real-time intravascular shear stress assessment by the MEMS sensor revealed an τ_ave value of 11.92 dyn cm^?2 with a systolic peak at 47.04 dyn cm^?2. The difference between CFD and experimental τ_ave was 18.5%. These findings provided important insights into packaging the MEMS sensors to optimize in vivo shear stress assessment.     

Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca²⁺]_i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm² for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of Definity^TM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca²⁺]_i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca²⁺]_i compared to statically cultured cells. The maximum increases of PI uptake and [Ca²⁺]_i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca²⁺]_i waves in shear cultured HUVECs was reduced compared to the statically cultured cells.     

Shear stress regulates adhesion and rolling of CD44+ leukemic and hematopoietic progenitor cells on hyaluronan

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm² is exceeded. At shear stress ∼1 dyn/cm², the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow.     

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm²) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H₂O₂). Physiological concentrations of H₂O₂ (flux of 0.1 nM/min and 15 μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H₂O₂- and LSS-induced eNOS phosphorylation and reduced ^?NO levels. We propose a model in which LSS promotes the formation of signaling levels of H₂O₂, which in turn activate p38α MAPK and then stimulate eNOS, leading to increased ^?NO generation and protection of endothelial function.     

Acridin-3,6-dialkyldithiourea hydrochlorides as new photosensitizers for photodynamic therapy of mouse leukemia cells

Acridin-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been evaluated as a new group of photosensitizers (PSs) for photodynamic antitumor therapy (PDT). Mouse leukemia cells L1210 were used for testing of AcrDTUs as the new PSs. The irradiation (UV-A light (365 nm), 1.05 J/cm²) increased cytotoxicity of all derivatives against L1210 cells more than ten times. The highest photocytotoxicity was found for propyl-AcrDTU with IC₅₀ = 0.48 ± 0.03 μM after 48 h incubation. A generation of the superoxide radical anion upon UV-A irradiation of propyl-AcrDTU was confirmed by in situ photochemical EPR experiments. To explain a mechanism of photocytotoxic action of AcrDTUs, an intracellular distribution of propyl-AcrDTU has been studied. It was found that AcrDTU in non-irradiated cells was not present in their nucleus but in the lysosomes and partly in the mitochondria, and sequestration of propyl-AcrDTU was dependent on pH in lysosomes. After irradiation, the cell death was induced by oxidative damage of lysosomal and mitochondrial membranes. Concerning the cell cycle, flow cytometry after PDT with propyl-AcrDTU showed a significant increase of the cells in the subG₀ phase. Observed signs of necrosis, apoptosis, and autophagy indicate that PDT/AcrDTU leads to multiple cell death types (caspase independent apoptosis, necrosis, and autophagy).     

Fibroblast growth factor-2 binding to the endothelial basement membrane peaks at a physiologically relevant shear stress

Fibroblast growth factor-2 (FGF2) is produced and released by endothelial cells and binds to heparan sulfate proteoglycans in the endothelial basement membrane (BM), an important FGF2 storage reservoir. Experimental and computational models of FGF2 binding kinetics to both cells and BM under static conditions are well established in the literature but remain largely unexplored under flow. We now examine BM-FGF2 binding kinetics in fluid flow conditions. We hypothesized that FGF2 binding to the endothelial BM would decrease as fluid shear stress increased. To investigate this, BM-FGF2 equilibrium, associative, and dissociative bindings were measured at various shear stresses. Surprisingly, FGF2 binding increased up to a physiological arterial shear stress of 25 dynes/cm², after which it decreased to a level similar to the 1 dyne/cm² condition. Both BM-FGF2 dissociation and BM binding site availability increased with flow, while association remained constant. This suggests that force-dependent FGF2 equilibrium binding varies with shear stress due to a combination of an increase in binding site availability and FGF2 dissociation with flow. This improved understanding of BM-FGF2 binding with flow enriches current knowledge of FGF2 binding kinetics under physiologic conditions, which may contribute to improved growth factor therapy development.     

NOX4-dependent Hydrogen peroxide promotes shear stress-induced SHP2 sulfenylation and eNOS activation

Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H₂O₂) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12 dyn/cm²) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H₂O₂, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.     

Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro

Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm² to 6, 10, or 20 dyn/cm² elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability.     

Occurrence of molecular abnormalities of cell cycle in L132 cells after in vitro short-term exposure to air pollution PM2.5

To improve the knowledge of the underlying mechanisms implying in air pollution Particulate Matter (PM)-induced lung toxicity in humans, we were interested in the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation in the L132 target human lung epithelial cell model. The most toxicologically relevant physical and chemical characteristics of air pollution PM_2.5 collected in Dunkerque, a French highly-industrialized sea-side city, were determined. L132 cells were exposed during 24, 48 and 72 h to Dunkerque City's PM_2.5 (i.e. Lethal Concentration (LC)₁₀ = 18.84 μg PM/mL or 5.02 μg PM/cm²; LC₅₀ = 75.36 μg PM/mL or 20.10 μg PM/cm²), TiO₂ and desorbed PM (i.e. dPM; EqLC₁₀ = 15.42 μg/mL or 4.11 μg PM/cm²; EqLC₅₀ = 61.71 μg/mL or 16.46 μg PM/cm²), benzene (7 μM) or Benzo[a]Pyrene (B[a]P; 1 μM). Dunkerque City's PM_2.5 altered the gene expression and/or the protein concentration of several key cell cycle controllers from TP53-RB gene signaling pathway (i.e. P53; BCL2; P21; cyclin D1, cyclin-dependent kinase 1; retinoblastoma protein) in L132 cells, thereby leading to the occurrence of cell proliferation and apoptosis together. The activation of the critical cell cycle controllers under study might be related to PM-induced oxidative stress, through the possible involvement of covalent metals in redox systems, the metabolic activation of organic chemicals by enzyme-catalyzed reactions, and phagocytosis. Taken together, these results might ask the critical question whether there is a balance or, in contrast, rather an imbalance between the cell proliferation and the apoptosis occurring in PM-exposed L132 cells, with possible consequences in term of PM-induced lung tumorgenesis.     

Shear stress promotes anoikis resistance of cancer cells via caveolin-1-dependent extrinsic and intrinsic apoptotic pathways

Circulating tumor cells (CTCs) need to acquire resistance to anoikis to survive after they experience fluid shear stress in the circulatory and lymphatic systems. However, the mechanism by which tumor cells resist anoikis under shear stress conditions remains unknown. Here, we found that the application of low shear stress (LSS; 2 dyn/cm²) to human breast carcinoma cells (MDA-MB-231) resulted in increased anoikis resistance when tumor cells were grown under anchorage-independent conditions. Caveolin-1 (Cav-1), the major component of plasma membrane caveolae, was overexpressed in LSS-treated cells and prevented tumor cells from anoikis, while depletion of Cav-1 restored sensitivity to anoikis. LSS-induced dissociation of Cav-1–Fas inhibited formation of the death-inducing signaling complex, caspase-8 activation, and rendered tumor cells resistant to anoikis. Likewise, LSS blocked the mitochondrial pathway through promotion of integrin β1–focal adhesion kinase-mediated multicellular aggregation and suppression of truncated BID translocation mediated crosstalk between the extrinsic and intrinsic apoptotic pathways. Our findings provide insights into the mechanisms by which LSS induces anoikis resistance in breast carcinoma cells through inhibition of Cav-1-dependent extrinsic and intrinsic apoptotic pathways, and serves as a potential therapeutic target for CTCs and metastatic breast cancer.     

Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1 mg/kg, i.p., 48 h). Echocardiographic, cardiomyocyte contractile and intracellular Ca^2 + properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca^2 + properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.     

Agitation,aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus

Factorial design and response surface analyses were used to optimize the production of inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) by Kluyveromyces marxianus ATCC 16045, using sucrose as carbon source. Effects of aeration, agitation and type of impeller (disk turbine, marine, pitched blade) were studied in a batch stirred reactor. Two factorial designs 2² were carried out. Agitation speed varied from 50 to 550 rpm (revolution per minute), aeration rate from 0.5 to 2.0 vvm (air volume/broth volume·minute). It has been shown that the enzyme production was strongly influenced by mixing conditions, while aeration rate was shown to be less significant. Additionally, the increase in the agitation speed is limited by the death rate, which increases drastically at high speeds, lowering the enzyme production. Also, the impeller type has significant influence in the production, the disk impeller at 450 rpm and aeration at 1.0 vvm led to an activity of 121 UI/mL, while the pitched blade was shown to be the best impeller for this process, leading to the best production, 176 UI/mL, at 450 rpm and 1.0 vvm. The maximum shear stress for inulinase production was about 0.22 Pa, since higher values cause higher cell death rates, affecting the enzyme production. The same results were confirmed with another microorganism, which was also sensible to shear stress. Therefore, it has been concluded that in some cases, mainly when the microorganism is sensible to shear stress, the interaction between mass transfer and mechanical stress should be considered in scale up processes.     

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 10⁶ cells ml⁻¹). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm⁻¹ could be measured without signal saturation of the Fogale Biomass system^®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC⁻¹ (0.15 × 10⁶ cells cm⁻²). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.     

Wall shear over high degree stenoses pertinent to atherothrombosis

Atherothrombosis can induce acute myocardial infarction and stroke by progressive stenosis of a blood vessel lumen to full occlusion. Since thrombus formation and embolization may be shear-dependent, we quantify the magnitude of shear rates in idealized severely stenotic coronary arteries (≥75% by diameter) using computational fluid dynamics to characterize the shear environment that may exist during atherothrombosis. Maximum shear rates in severe short stenoses were found to exceed 250,000 s^?1 (9500 dynes/cm²) and can reach a peak value of 425,000 s^?1 for a 98% stenosis. These high shear rates exceed typical shear used for in vitro blood flow experiments by an order of magnitude, indicating the need to examine thrombosis at very high shear rates. Pulsatility and stenosis eccentricity were found to have minor effects on the maximum wall shear rates in severe stenoses. In contrast, increases in the stenosis length reduced the maximum shear to 107,000 s^?1 (98% stenosis), while surface roughness could increase focal wall shear rates to a value reaching 610,000 s^?1 (90% stenosis). The “shear histories” of circulating platelets in these stenoses are far below reported activation thresholds. Platelets may be required to form bonds in 5 μs and resist shear forces reaching 8000 pN per platelet. Arterial thrombosis occurs in the face of pathological high shear stress, creating rapid and strong bonds without prior activation of circulating platelets.