Physiological Levels Of Shear Stress Research Articles

Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Changes in Energy Metabolism and Cell Damage in RPTEC‐TERT1 Cells Exposed to Hypoxia or Ischemia after Shear Stress Conditioning

Study Objective Renal proximal tubules are exposed in vivo to physiological levels of shear stress. We aim to determine how preconditioning human proximal tubule cells with shear stress affects their sensibility to hypoxia or ischemia. Hypothesis Physiological levels of shear stress would induce further differentiation and metabolic specialization resulting in changes in their response to low oxygen or nutrient conditions. Methods Immortalized human proximal tubule cells (RPTEC-TERT1 from Evercyte) were grown to confluence in Ibidi µSlides and then exposed for three days to two flow regimes providing negligible (0.01 dyn/cm2) or physiological shear stress (0.3 dyn/cm2). Hypoxia or ischemic conditions were applied for 16h, by keeping cells under 1%O2 alone or together with glucose deprivation. Results RPTEC-TERT1 cells were first adapted to a culturing regime under 5 mM glucose culture medium, instead of 17 mM glucose present in the medium recommended by this cell line vendor. Proliferation rates and expression of phenotypic markers were unchanged. RPTEC-TERT1 cells switched from proliferative-glycolytic metabolism to non-proliferative-oxidative metabolism upon reaching confluency, approximately in 6-7 days. Seven days confluent, not-perfused RPTEC-TERT1 cells exposed to hypoxia or ischemia for 16 hours exhibited 58% and 75% mortality, respectively. In cells perfused for 3 days, preconditioning with shear stress reduced their sensitivity to hypoxia (mortality = 51% no-SS vs. 35% SS) and ischemia (mortality = 83% no-SS vs. 75% SS). Shear stress conditioning increased expression of genes related to energy metabolism and cells stress (AMPK alpha-2, HIF-1alpha, KIM-1) Conclusions Preconditioning of RPTEC-TERT1 by exposing them to physiological shear-stress modifies expression of genes related to mechanisms used to cope with cellular stress and this may explain the reduction in cell mortality when exposed to hypoxia or oxygen-glucose deprivation.

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Hemodynamic loads distinctively impact the secretory profile of biomaterial-activated macrophages - implications for in situ vascular tissue engineering.

Biomaterials are increasingly used for in situ vascular tissue engineering, wherein resorbable fibrous scaffolds are implanted as temporary carriers to locally initiate vascular regeneration. Upon implantation, macrophages infiltrate and start degrading the scaffold, while simultaneously driving a healing cascade via the secretion of paracrine factors that direct the behavior of tissue-producing cells. This balance between neotissue formation and scaffold degradation must be maintained at all times to ensure graft functionality. However, the grafts are continuously exposed to hemodynamic loads, which can influence macrophage response in a hitherto unknown manner and thereby tilt this delicate balance. Here we aimed to unravel the effects of physiological levels of shear stress and cyclic stretch on biomaterial-activated macrophages, in terms of polarization, scaffold degradation and paracrine signaling to tissue-producing cells (i.e. (myo)fibroblasts). Human THP-1-derived macrophages were seeded in electrospun polycaprolactone bis-urea scaffolds and exposed to shear stress (∼1 Pa), cyclic stretch (∼1.04), or a combination thereof for 8 days. The results showed that macrophage polarization distinctly depended on the specific loading regime applied. In particular, hemodynamic loading decreased macrophage degradative activity, especially in conditions of cyclic stretch. Macrophage activation was enhanced upon exposure to shear stress, as evidenced from the upregulation of both pro- and anti-inflammatory cytokines. Exposure to the supernatant of these dynamically cultured macrophages was found to amplify the expression of tissue formation- and remodeling-related genes in (myo)fibroblasts statically cultured in comparable electrospun scaffolds. These results emphasize the importance of macrophage mechano-responsiveness in biomaterial-driven vascular regeneration.

Staphylococcus aureus adhesion in endovascular infections is controlled by the ArlRS-MgrA signaling cascade.

Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS—MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS—MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS—MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS—MgrA regulatory system.

Combination of flow and micropattern alignment affecting flow-resistant endothelial cell adhesion

Assuring cell adhesion to an underlying biomaterial surface under blood flow is vital to functional vascular grafts design. In vivo endothelial cells (ECs) are located under the microenvironment of both surface topography of the basement membrane and the mechanical loading resulting from blood flow. Both topographical and mechanical factors should thus be considered when designing vascular grafts to enhance the flow-resistant EC adhesion. This study aims to investigate effects of integrating biomaterial surface topography and flow on EC adhesion, which was a deficit in previous studies. Human umbilical vein endothelial cells (HUVECs) were cultured on different fibronectin (FN) micropatterns parallel or perpendicular to the flow direction and exposed to sustained flow with physiological levels of shear stress (15 dyne/cm2). We demonstrated that micropattern alignment parallel to the flow direction enhanced flow-resistant EC adhesion, while micropattern alignment perpendicular to the flow direction attenuated it. Experimental and numeric modeling analysis underlined that the flow-induced mechanic distribution on the surface of cells that were aligned on the micropatterned surfaces and the subsequent cytoskeleton rearrangement were responsible for the significant difference in EC adhesion. Furthermore, pressure on the surface of cells that were aligned on the micropatterned surfaces induced by flow provided a more critical role in EC adhesion than shear stress. These findings highlight the importance of proper combination of topographical and flow cues in enhancement of EC adhesion and may suggest new strategies for designing functional vascular grafts.

Shear stress with appropriate time-step and amplification enhances endothelial cell retention on vascular grafts.

Endothelial cells (ECs) are sensitive to changes in shear stress. The application of shear stress to ECs has been well documented to improve cell retention when placed into a haemodynamically active environment. However, the relationship between the time-step and amplification of shear stress on EC functions remains elusive. In the present study, human umbilical cord veins endothelial cells (HUVECs) were seeded on silk fibroin nanofibrous scaffolds and were preconditioned by shear stress at different time-steps and amplifications. It is shown that gradually increasing shear stress with appropriate time-steps and amplification could improve EC retention, yielding a complete endothelial-like monolayer both in vitro and in vivo. The mechanism of this improvement is mediated, at least in part, by an upregulation of integrin β1 and focal adhesion kinase (FAK) expression, which contributed to fibronectin (FN) assembly enhancement in ECs in response to the shear stress. A modest gradual increase in shear stress was essential to allow additional time for ECs to gradually acclimatize to the changing environment, with the goal of withstanding the physiological levels of shear stress. This study recognized that the time-steps and amplifications of shear stress could regulate EC tolerance to shear stress and the anti-thrombogenicity function of engineered vascular grafts via an extracellular cell matrix-specific, mechanosensitive signalling pathway and might prevent thrombus formation in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress.

Shear stress is known to stimulate an intracellular free calcium concentration ([Ca(2+)]i) response in vascular endothelial cells (ECs). [Ca(2+)]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca(2+)]i response is, in part, due to Ca(2+) release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca(2+) store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca(2+)]i in sheared ECs. Cultured ECs, loaded with a Ca(2+)-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca(2+)]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca(2+)]i transients/oscillations were present when experiments were conducted in Ca(2+)-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca(2+)]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca(2+) uniporter also inhibited the shear-induced [Ca(2+)]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca(2+) uptake/release, regulate the temporal profile of shear-induced ER Ca(2+) release. [Ca(2+)]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca(2+)]i response.

Abstract 11593: Thrombus Formation Under Flow is Inhibited by the Mechanosensitive Cation Channel Blockers GsMTx-4 Peptide and Gadolinium Chloride

Introduction: Ion channels contribute significantly to platelet physiology, including control of Ca 2+ as a second messenger. Although there is a clear link between platelet activation and shear stress, no study has examined the functional relevance of mechanosensitive (MS) ion channels in platelets or their precursors, megakaryocytes. Hypothesis: Human platelets express MS cation channels such as Piezo1, which contribute to platelet function and the development of thrombosis. Methods: Thrombus formation and [Ca 2+ ] i (intracellular Ca 2+ ) responses were studied by confocal microscopy using the indicators DiOC 6 and Fluo-3, respectively, under physiological levels of shear stress in a parallel-plate flow chamber. Aggregation and [Ca 2+ ] i responses to collagen and thrombin in washed human platelets were assessed by light transmission aggregometry and Fura-2 ratiometric measurements. GsMTx-4 (2.5μM) and gadolinium chloride (GdCl 3 ) (30μM) were used as MS cation channel inhibitors, targeting Piezo1 and TRPC6 in platelets. Piezo1 mRNA and protein expression were studied in platelets and the megakaryocytic cell line Meg-01. Results: Both inhibitors significantly inhibited thrombus formation on a collagen surface under normal arterial shear rate (average thrombus volume was reduced from 48132.7±7739.4μm 3 to 13478.9±2744.8μm 3 by GdCl 3 (n=6, P<0.01) and to 25125.9±6433.5μm 3 (n=6, P<0.05) by GsMTx-4). GsMTx-4 also caused a reduction in thrombin-induced platelet aggregation (to 53.6±14.0% of control, n=4, P<0.05), however GdCl 3 did not affect platelet shape change or aggregation under static conditions. Pre-treatment with either GsMTx-4 or GdCl 3 did not reduce calcium influx in response to TRPC6 stimulation with 60μM OAG, suggesting a MS role for Piezo1, which was found at the mRNA transcript and protein level in platelets and Meg-01 cells. Meg-01 cells showed shear rate-dependent increases in [Ca 2+ ] i levels; at a shear rate of 3989.2s -1 , F/F 0 increased to 1.38±0.05 from 1.00±0.00 baseline (n=53 cells, P<0.0001), a response which was diminished by GsMTx-4, and by chelation of extracellular Ca 2+ by EGTA. Conclusions: We provide evidence for the first time for a functional contribution of MS cation channels in platelet physiology and thrombosis.

Free thiol groups in von Willebrand factor (VWF) are required for its full function under physiological flow conditions

Introductionvon Willebrand factor (VWF) is rich in cysteine; next to important structural disulfide bonds, free thiol groups are present. Free thiols on the surface of plasmatic VWF have been shown to play a role in VWF self-association and in platelet binding under pathologically high levels of shear stress. The present study explores the role of VWF free thiol groups under physiological levels of shear stress and in interactions with collagen and platelet-GPIbα receptor. Materials and methodsFree and accessible thiol groups were blocked with N-ethylmaleimide (NEM) and the derivatized molecule was evaluated in functional assays. Reduced cysteine residues were identified using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation and by analysis of derivatized tryptic peptides by mass spectrometry. ResultsBlockade of free thiol groups significantly reduced VWF-mediated platelet recruitment to collagen under physiological flow conditions. This resulted from inhibition of VWF binding to both collagen and the platelet GPIb receptor. Evaluation of derivatization sites revealed a high level of derivatization in the cysteine-rich N- and C-termini of VWF. 19 MPB-derivatized peptides, 13 of which are described here for the first time, were identified by mass spectrometry. ConclusionsThis study shows a significant contribution of free thiol groups in VWF to the mediation of platelet adhesion under physiological shear stress conditions. The free thiol groups are shown to be involved in VWF binding to both collagen III and platelet GP1b receptor.

CMV infection of human sinusoidal endothelium regulates hepatic T cell recruitment and activation

Human cytomegalovirus infection (HCMV) is associated with an increased morbidity after liver transplantation, by facilitating allograft rejection and accelerating underlying hepatic inflammation. We hypothesized that human hepatic sinusoidal endothelial cells infected with HCMV possess the capacity to modulate allogeneic T cell recruitment and activation, thereby providing a plausible mechanism of how HCMV infection is able to enhance hepatic immune activation. Human hepatic sinusoidal endothelial cells were isolated from explanted livers and infected with recombinant endotheliotropic HCMV. We used static and flow-based models to quantify adhesion and transendothelial migration of allogeneic T cell subsets and determine their post-migratory phenotype and function. HCMV infection of primary human hepatic sinusoidal endothelial cells facilitated ICAM-1 and CXCL10-dependent CD4 T cell transendothelial migration under physiological levels of shear stress. Recruited T cells were primarily non-virus-specific CXCR3(hi) effector memory T cells, which demonstrated features of LFA3-dependent Th1 activation after migration, and activated regulatory T cells, which retained a suppressive phenotype following transmigration. The ability of infected hepatic endothelium to recruit distinct functional CD4 T cell subsets shows how HCMV facilitates hepatic inflammation and immune activation and may simultaneously favor virus persistence.

Shear stress upregulates IL‐1β secretion by Chlamydia pneumoniae‐ infected monocytes

Infectious agents are increasingly implicated in the development and progression of chronic inflammatory diseases. Several lines of evidence suggest that the common intracellular respiratory pathogen, Chlamydia pneumoniae contributes to the well-established risk factors of atherosclerosis but the exact mechanism is not well understood. It is believed that C. pneumoniae-infected monocytes travel from the lung to the atherosclerotic foci, during which the cells experience mechanical stimuli due to blood flow. In this work, we characterized the effect of physiological levels of shear stress on C. pneumoniae-infected human monocytes in an in vitro flow model. We found that a shear stress of 5 dyn/cm(2) enhanced the expression of pro-inflammatory cytokine IL-1β only in infected, but not in uninfected, monocytes. We also found that this enhancement is due to the upregulation of IL-1β gene expression due to shear stress. Our results demonstrate that mechanotransduction is an important, heretofore unaddressed, determinant of inflammatory response to an infection.

Characterization of Biomechanical Properties of Primary Endothelial Cells Exposed to Shear Stress

Mechanotransduction, the process where cells convert biophysical signals into chemical signals, directs the behavior and function of endothelial cells (ECs) because of their unique position on the luminal surface of blood vessels. ECs are in direct contact with blood and can sense changes in shear stress caused by alterations in flow rate. To investigate the biophysical implications of this response we recapitulated physiological levels of shear stress in vitro using two systems, imaged and measured the consequent cytoskeletal changes with immunofluorescence, and quantified cortical stiffness distributions. We developed a sampling method for Atomic Force Microscopy (AFM) stiffness measurements by which force-indentation data is collected in a uniform grid (force map) over multiple cells within a monolayer. In this way, the sampling bias resulting from the selection of force-indentation measurements was reduced. Hertzian contact modeling for AFM was used to specifically isolate the cortical stiffness of the cell using a pyramidal indenter and small indentation depths (100 nm). Nonparametric statistics were used to compare the distributions of moduli from a given experiment, as the data distribution was not Gaussian. Actin filament alignment was quantified using Hough Transforms on immunofluorescence images and increased when cells were exposed to shear stress. The medians of stiffness distributions within sheared EC monolayers increased in magnitude when compared to static monolayers, yielding a method-dependent increase in cortical stiffness of up to 60%. This demonstrates that physiological levels of shear stress dramatically affect ECs morphology in a way that cannot be simulated under static conditions. We concluded that the biophysical milieu of a cell's physiological microenvironment must be recapitulated in order to represent characteristic cellular mechanical properties in vitro.

Hydrogen sulfide impairs shear stress-induced vasodilation in mouse coronary arteries.

Hydrogen sulfide has emerged as an important endothelium-dependent vasodilator, but its role in shear stress-mediated dilation of coronary arteries is unclear. We examined the role of H2S on shear stress-mediated dilation of isolated mouse coronary arteries. In these vessels, Na2S produced concentration-dependent dilation, which was significantly inhibited by iberiotoxin and by 4-aminopyridine. In addition, BK and Kv currents in mouse coronary smooth muscle cells were directly activated by Na2S, suggesting that H2S produced vasodilation through BK and Kv channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm(2)) and produced graded dilatory responses, but such effects were diminished in the presence of 100 μM Na2S. Pre-incubation with the cystathionine γ-lyase inhibitor, D,L-propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress-mediated vasodilation. However, in the presence of L-NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress-mediated vasodilation, suggesting an interaction between NO and H2S signaling. Na2S inhibited eNOS activity in cultured mouse aortic endothelial cells and reduced the level of phospho-eNOS(serine 1177). These results suggest that both NO and H2S are important shear stress-mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H2S through inhibition of NO generation.

Role of caveolae in shear stress-mediated endothelium-dependent dilation in coronary arteries

Caveolae are membrane microdomains where important signalling pathways are assembled and molecular effects transduced. In this study, we hypothesized that shear stress-mediated vasodilation (SSD) of mouse small coronary arteries (MCA) is caveolae-dependent. MCA (80-150 μm) isolated from wild-type (WT) and caveolin-1 null (Cav-1(-/-)) mice were subjected to physiological levels of shear stress (1-25 dynes/cm(2)) with and without pre-incubation of inhibitors of nitric oxide synthase (L-NAME), cyclooxygenase (indomethacin, INDO), or cytochrome P450 epoxygenase (SKF 525A). SSD was endothelium-dependent in WT and Cav-1(-/-) coronaries but that in Cav-1(-/-) was significantly diminished compared with WT. Pre-incubation with L-NAME, INDO, or SKF 525A significantly reduced SSD in WT but not in Cav-1(-/-) mice. Vessels from the soluble epoxide hydrolase null (Ephx2(-/-)) mice showed enhanced SSD, which was further augmented by the presence of arachidonic acid. In donor-detector-coupled vessel experiments, Cav-1(-/-) donor vessels produced diminished dilation in WT endothelium-denuded detector vessels compared with WT donor vessels. Shear stress elicited a robust intracellular Ca(2+) increase in vascular endothelial cells isolated from WT but not those from Cav-1(-/-) mice. Integrity of caveolae is critical for endothelium-dependent SSD in MCA. Cav-1(-/-) endothelium is deficient in shear stress-mediated generation of vasodilators including NO, prostaglandins, and epoxyeicosatrienoic acids. Caveolae plays a critical role in endothelial signal transduction from shear stress to vasodilator production and release.

Abstract 473: Fluid Shear Stress Specifically Exacerbates Chlamydia pneumoniae-mediated Pro-inflammatory Responses in Monocytes

Background and Significance The precise role of Chlamydia pneumoniae infection in various stages of the atherosclerosis is not well understood. During the transit from lung to the atherosclerotic sites, C. pneumoniae-infected monocytes travel through circulation and are subjected to shear stress due to blood flow. Elucidating the effects of these mechanical stimuli on infected monocytes is critical in our understanding of the link between C. pneumoniae infection and atherosclerosis. Objectives We hypothesized that fluid shear stress alters the inflammatory response of C. pneumoniae-infected monocytes. Using an in vitro model of blood flow, we determined the effect of physiological levels of shear stress on monocyte responses pertinent to atherosclerosis including cytokine secretion, adhesion molecule expression, intracellular signaling, and endothelial adhesion. Methods Primary human monocytes and THP-1 cells were infected with C. pneumoniae at an MOI-2 for 2 h at 35 °C with intermittent rocking, and cultured for 36 h. The infected cells were then subjected to a shear stress of 7.5 dyn/cm2 for 1 h, and the supernatants were analyzed for a panel of 17 cytokines, and the cells were analyzed for signaling proteins, surface receptors, and endothelial adhesion. Uninfected cells and static conditions were used as controls, and a P<0.05 (Student’s t-test) was considered significant. Results We observed that shear stress on infected cells resulted in a caspase-independent upregulation of pro-inflammatory IL-1β (3-fold), a modest increase in MIP-1α and MIP-1β, a decrease in anti-inflammatory IL-10, and no change in other cytokines including TNFα and IL-12. These changes were manifested as an increase in the adhesion of monocytes to endothelium activated with the infected supernatants. We also observed differences in the chemotaxis of monocytes exposed to sheared vs. static supernatants. Conclusions We observe that physiological shear stress drives Chlamydia infected monocytes towards a pro-inflammatory state in circulation suggesting a critical synergy between mechanical and chemical factors in atherogenesis.

Paradoxical vasoconstrictive effects of H2S on shear stress‐mediated vasodilation

We examined the role of H2S on shear stress‐mediated dilation of isolated mouse coronary arteries. Na2S produced concentration‐dependent dilation in these vessels, which was significantly inhibited by iberiotoxin. BK currents in mouse coronary smooth muscle cells were directly activated by Na2S, suggesting H2S produced vasodilation through BK channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm2), producing graded dilatory responses, but such effects were diminished in the presence of 100 μM Na2S. Pre‐incubation with the cystathionine γ‐lyase inhibitor, D,L‐propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress‐mediated vasodilation. However, in the presence of L‐NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress‐mediated vasodilation, suggesting an interaction between NO and H2S signaling. Using cultured bovine aortic endothelial cells, eNOS activity was inhibited by Na2S. These results suggest that both NO and H2S are important shear stress‐mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H2S through inhibition of NO generation. (Supported by grants from NIH)

Abstract 18001: Shear Stress-Induced Endothelium-Dependent Dilation of Mouse Coronary Arteries is Caveolae-Dependent

BACKGROUND: Shear stress-induced dilation (SSD) is an important vascular endothelial function that is impaired in diseases such as diabetes mellitus. Caveolin-1 is the major structural protein required for the formation of endothelial caveolae, where signaling molecules and pathways are assembled. In this study, we hypothesized that SSD of mouse small coronary arteries (MCA) is caveolae-dependent. METHODS: MCA (80-130 μm) freshly isolated from WT and Cav-1 knockout (KO) mice were mounted in a vessel chamber, pressurized at 80 mmHg, pre-contracted to 60-80% baseline diameter with endothelin-1, and subjected to physiological levels of shear stress at 25 dyn/cm 2 using a microinjection pump coupled to a pressure servo controller. SSD was measured by videomicroscopy and expressed as % of maximum diameter measured in the absence of Ca 2+ . Donor (intact WT or KO) and detector (WT with no endothelium) vessels were mounted in separate chambers connected by a micropipette. Diabetes mellitus was produced by streptozotocin (STZ) injection. RESULTS: SSD of MCA is significantly diminished in KO mice. 25 dyn/cm 2 produced 92.9±1.2% SSD in WT but only 42.0±0.7% in KO (n=10 for both, *p<0.05). Removal of endothelium reduced SSD to 22.3±3.3% in WT and to 14.9±1.3% in KO (n=8 for both, * vs. intact vessels but not WT vs. KO). Inhibition of eNOS (L-NAME), PGIS (Indocin), and CYP monooxygenase (SKF525A) reduced endothelial-dependent SSD by 44%, 34%, and 21% respectively in WT (all * vs control) but had no effect in KO, suggesting deficiency in shear stress-induced generation of vasodilators in KO. This is confirmed by the significant reduction in SSD in detector vessels with KO serving as donors (*42.0±0.7%, n=6 vs. 64.3±6.4% n=4, WT donor). With diabetes, MCA from WT and KO lost the ability for SSD (WT 25.9±2.4%, n=6 and KO 24.4±2.3%, n=5), which was similar to the effects of endothelium denudation. CONCLUSION: Integrity of caveolae is critical for endothelium-dependent SSD in MCA. KO endothelium is deficient in shear stress-mediated generation of vasodilators including NO, PGI 2 , and EET. Diabetic MCA have impaired endothelial function with loss of SSD. Caveolae plays a critical role in endothelial signal transduction from shear stress to vasodilator production and release.

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.

Endothelial cells seeded on elastin–heparin matrices express normal EC markers and resist detachment on exposure to shear stress: a histological study

Endothelialization of vascular graft surfaces has been suggested as a method to prevent thrombosis-related failure. The purpose of this study was to examine the ability of endothelial cells (ECs) seeded on novel elastin–heparin scaffolds to resist detachment on exposure to physiological levels of shear stress and to evaluate whether these cells maintain a quiescent profile on these scaffolds by investigating the expression of thrombomodulin (TM) and adhesive molecules platelet endothelial cell adhesion molecule-1 (PECAM-1) and intercellular adhesion molecule-1 (ICAM-1). To evaluate the functional response of the cultured endothelium on injury, the cellular response (ICAM-1 expression) to physiological stimulatory factor tumor-necrosis factor (TNF)-alpha was analyzed. The ECs formed a confluent monolayer expressing PECAM-1 and TM very similar to native aortic ECs. ICAM-1 expression was slightly higher than that observed on tissue culture polystyrene (TCPS), but was similar to that seen on several other polymers. Furthermore, the cells responded to stimulation by TNF-alpha (expression of ICAM was elevated by ∼30%). These results indicate that the ECs seeded on elastin–heparin scaffolds express normal EC molecules and may provide an efficient anti-thrombogenic surface for these tissue-engineered vascular grafts.