Multi-scale multi-physics model of brain interstitial water flux by transcranial Direct Current Stimulation.

Objective. Transcranial direct current stimulation (tDCS) generates sustained electric fields in the brain, that may be amplified when crossing capillary walls (across blood-brain barrier, BBB). Electric fields across the BBB may generate fluid flow by electroosmosis. We consider that tDCS may thus enhance interstitial fluid flow.Approach. We developed a modeling pipeline novel in both (1) spanning the mm (head),µm (capillary network), and then nm (down to BBB tight junction (TJ)) scales; and (2) coupling electric current flow to fluid current flow across these scales. Electroosmotic coupling was parametrized based on prior measures of fluid flow across isolated BBB layers. Electric field amplification across the BBB in a realistic capillary network was converted to volumetric fluid exchange.Main results. The ultrastructure of the BBB results in peak electric fields (per mA of applied current) of 32-63Vm-1across capillary wall and >1150Vm-1in TJs (contrasted with 0.3Vm-1in parenchyma). Based on an electroosmotic coupling of 1.0 × 10-9- 5.6 × 10-10m3s-1m2perVm-1, peak water fluxes across the BBB are 2.44 × 10-10- 6.94 × 10-10m3s-1m2, with a peak 1.5 × 10-4- 5.6 × 10-4m3min-1m3interstitial water exchange (per mA).Significance. Using this pipeline, the fluid exchange rate per each brain voxel can be predicted for any tDCS dose (electrode montage, current) or anatomy. Under experimentally constrained tissue properties, we predicted tDCS produces a fluid exchange rate comparable to endogenous flow, so doubling fluid exchange with further local flow rate hot spots ('jets'). The validation and implication of such tDCS brain 'flushing' is important to establish.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation.

OBJECTIVE: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. METHODS AND RESULTS: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. CONCLUSIONS: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

Direct current stimulation modulates gene expression in isolated astrocytes with implications for glia-mediated plasticity.

While the applications of transcranial direct current stimulation (tDCS) across brain disease and cognition are diverse, they rely on changes in brain function outlasting stimulation. The cellular mechanisms of DCS leading to brain plasticity have been studied, but the role of astrocytes remains unaddressed. We previously predicted that during tDCS current is concentrated across the blood brain-barrier. This will amplify exposure of endothelial cells (ECs) that form blood vessels and of astrocytes that wrap around them. The objective of this study was to investigate the effect of tDCS on the gene expression by astrocytes or ECs. DCS (0.1 or 1 mA, 10 min) was applied to monolayers of mouse brain ECs or human astrocytes. Gene expression of a set of neuroactive genes were measured using RT-qPCR. Expression was assessed immediately or 1 h after DCS. Because we previously showed that DCS can produce electroosmotic flow and fluid shear stress known to influence EC and astrocyte function, we compared three interventions: pressure-driven flow across the monolayer alone, pressure-driven flow plus DCS, and DCS alone with flow blocked. We show that DCS can directly modulate gene expression in astrocytes (notably FOS and BDNF), independent of but synergistic with pressure-driven flow gene expression. In ECs, pressure-driven flow activates genes expression with no evidence of further contribution from DCS. In ECs, DCS alone produced mixed effects including an upregulation of FGF9 and downregulation of NTF3. We propose a new adjunct mechanism for tDCS based on glial meditated plasticity.

Glycocalyx mechanotransduction mechanisms are involved in renal cancer metastasis.

Mammalian cells, including cancer cells, are covered by a surface layer containing cell bound proteoglycans, glycoproteins, associated glycosaminoglycans and bound proteins that is commonly referred to as the glycocalyx. Solid tumors also have a dynamic fluid microenvironment with elevated interstitial flow. In the present work we further investigate the hypothesis that interstitial flow is sensed by the tumor glycocalyx leading to activation of cell motility and metastasis. Using a highly metastatic renal carcinoma cell line (SN12L1) and its low metastatic counterpart (SN12C) we demonstrate in vitro that the small molecule Suberoylanilide Hydroxamic Acid (SAHA) inhibits the heparan sulfate synthesis enzyme N-deacetylase-N-sulfotransferase-1, reduces heparan sulfate in the glycocalyx and suppresses SN12L1 motility in response to interstitial flow. SN12L1 cells implanted in the kidney capsule of SCID mice formed large primary tumors and metastasized to distant organs, but when treated with SAHA metastases were not detected. In another set of experiments, the role of hyaluronic acid was investigated. Hyaluronan synthase 1, a critical enzyme in the synthetic pathway for hyaluronic acid, was knocked down in SN12L1 cells and in vitro experiments revealed inhibition of interstitial flow induced migration. Subsequently these cells were implanted in mouse kidneys and no distant metastases were detected. These findings suggest new therapeutic approaches to the treatment of kidney carcinoma metastasis.

Matrix Stiffness Affects Glycocalyx Expression in Cultured Endothelial Cells.

Rationale: The endothelial cell glycocalyx (GCX) is a mechanosensor that plays a key role in protecting against vascular diseases. We have previously shown that age/disease mediated matrix stiffness inhibits the glycocalyx glycosaminoglycan heparan sulfate and its core protein Glypican 1 in human umbilical vein endothelial cells, rat fat pad endothelial cells and in a mouse model of age-mediated stiffness. Glypican 1 inhibition resulted in enhanced endothelial cell dysfunction. Endothelial cell culture typically occurs on stiff matrices such as plastic or glass. For the study of the endothelial GCX specifically it is important to culture cells on soft matrices to preserve GCX expression. To test the generality of this statement, we hypothesized that stiff matrices inhibit GCX expression and consequently endothelial cell function in additional cell types: bovine aortic endothelial cells, mouse aortic endothelial cell and mouse brain endothelial cells. Methods and Results: All cell types cultured on glass showed reduced GCX heparan sulfate expression compared to cells cultured on either soft polyacrylamide (PA) gels of a substrate stiffness of 2.5 kPa (mimicking the stiffness of young, healthy arteries) or on either stiff gels 10 kPa (mimicking the stiffness of old, diseased arteries). Specific cell types showed reduced expression of GCX protein Glypican 1 (4 of 5 cell types) and hyaluronic acid (2 of 5 cell types) on glass vs soft gels. Conclusion: Matrix stiffness affects GCX expression in endothelial cells. Therefore, the study of the endothelial glycocalyx on stiff matrices (glass/plastic) is not recommended for specific cell types.

Heparan sulfate proteoglycan glypican-1 and PECAM-1 cooperate in shear-induced endothelial nitric oxide production.

This study aimed to clarify the role of glypican-1 and PECAM-1 in shear-induced nitric oxide production in endothelial cells. Atomic force microscopy pulling was used to apply force to glypican-1 and PECAM-1 on the surface of human umbilical vein endothelial cells and nitric oxide was measured using a fluorescent reporter dye. Glypican-1 pulling for 30 min stimulated nitric oxide production while PECAM-1 pulling did not. However, PECAM-1 downstream activation was necessary for the glypican-1 force-induced response. Glypican-1 knockout mice exhibited impaired flow-induced phosphorylation of eNOS without changes to PECAM-1 expression. A cooperation mechanism for the mechanotransduction of fluid shear stress to nitric oxide production was elucidated in which glypican-1 senses flow and phosphorylates PECAM-1 leading to endothelial nitric oxide synthase phosphorylation and nitric oxide production.

The Glycocalyx and Its Role in Vascular Physiology and Vascular Related Diseases.

PURPOSE: In 2007 the two senior authors wrote a review on the structure and function of the endothelial glycocalyx layer (Weinbaum in Annu Rev Biomed Eng 9:121-167, 2007). Since then there has been an explosion of interest in this hydrated gel-like structure that coats the luminal surface of endothelial cells that line our vasculature due to its important functions in (A) basic vascular physiology and (B) vascular related diseases. This review will highlight the major advances that have occurred since our 2007 paper. METHODS: A literature search mainly focusing on the role of the glycocalyx in the two major areas described above was performed using electronic databases. RESULTS: In part (A) of this review, the new formulation of the century old Starling principle, now referred to as the Michel-Weinbaum glycoclayx model or revised Starling hypothesis, is described including new subtleties and physiological ramifications. New insights into mechanotransduction and release of nitric oxide due to fluid shear stress sensed by the glycocalyx are elaborated. Major advances in understanding the organization and function of glycocalyx components, and new techniques for measuring both its thickness and spatio-chemical organization based on super resolution, stochastic optical reconstruction microscopy (STORM) are presented. As discussed in part (B) of this review, it is now recognized that artery wall stiffness associated with hypertension and aging induces glycocalyx degradation, endothelial dysfunction and vascular disease. In addition to atherosclerosis and cardiovascular diseases, the glycocalyx plays an important role in lifestyle related diseases (e.g., diabetes) and cancer. Infectious diseases including sepsis, Dengue, Zika and Corona viruses, and malaria also involve the glycocalyx. Because of increasing recognition of the role of the glycocalyx in a wide range of diseases, there has been a vigorous search for methods to protect the glycocalyx from degradation or to enhance its synthesis in disease environments. CONCLUSION: As we have seen in this review, many important developments in our basic understanding of GCX structure, function and role in diseases have been described since the 2007 paper. The future is wide open for continued GCX research.

Stenting-induced Vasa Vasorum compression and subsequent flow resistance: a finite element study.

Vascular stenting is a common intervention for the treatment for atherosclerotic plaques. However, stenting still has a significant rate of restenosis caused by intimal hyperplasia formation. In this study, we evaluate whether stent overexpansion leads to Vasa Vasorum (VV) compression, which may contribute to vascular wall hypoxia and restenosis. An idealized multilayered fibroatheroma model including Vasa Vasorum was expanded by three coronary stent designs up to a 1.3:1 stent/artery luminal diameter ratio (exp1.1, exp1.2, exp1.3) using a finite element analysis approach. Following Poiseuille's law for elliptical sections, the fold increase in flow resistance was calculated based on VV compression in the Intima (Int), Media (Med) and Adventitia (Adv). The VV beneath the plaque experiences the smallest degree of compression, while the opposite wall regions are highly affected by stent overexpansion. The highest compressions for Adv, Med and Int at exp1.1 are 60.7, 65.9, 72.3%, at exp1.2 are 62.1, 67.3, 73.5% and at expp1.3 are 63.2, 68.7, 74.8%. The consequent fold increase in resistance to flow for Adv, Med and Int at exp1.1 is 3.3, 4.4, 6.6, at exp1.2 is 3.5, 4.7, 7.2 and at exp1.3 is 3.8, 5.1, 7.9. Stent overexpansion induces significant VV compression, especially in the Intima and Media layers, in agreement with previously observed Media necrosis and loss in elasticity after stenting. The observed steep increase in flow resistance suggests the blood flow and associated oxygen delivery would drop up to five times in the Media and almost eight in the Intima, which may lead to intimal hyperplasia and restenosis.

The glycocalyx core protein Glypican 1 protects vessel wall endothelial cells from stiffness-mediated dysfunction and disease.

AIMS: Arterial stiffness is an underlying risk factor and a hallmark of cardiovascular diseases. The endothelial cell (EC) glycocalyx is a glycan rich surface layer that plays a key role in protecting against EC dysfunction and vascular disease. However, the mechanisms by which arterial stiffness promotes EC dysfunction and vascular disease are not fully understood, and whether the mechanism involves the protective endothelial glycocalyx is yet to be determined. We hypothesized that endothelial glycocalyx protects the endothelial cells lining the vascular wall from dysfunction and disease in response to arterial stiffness. METHODS AND RESULTS: Cells cultured on polyacrylamide (PA) gels of substrate stiffness 10 kPa (mimicking the subendothelial stiffness of aged, unhealthy arteries) showed a significant inhibition of glycocalyx expression compared to cells cultured on softer PA gels (2.5 kPa, mimicking the subendothelial stiffness of young, healthy arteries). Specifically, gene and protein analyses revealed that a glycocalyx core protein Glypican 1 was inhibited in cells cultured on stiff PA gels. These cells had enhanced endothelial cell dysfunction as determined by enhanced cell inflammation (enhanced inflammatory gene expression, monocyte adhesion, and inhibited nitric oxide expression), proliferation, and EndMT. Removal of Glypican 1 using gene-specific silencing with siRNA or gene overexpression using a plasmid revealed that Glypican 1 is required to protect against stiffness-mediated endothelial cell dysfunction. Consistent with this, using a model of age-mediated stiffness, older mice exhibited a reduced expression of Glypican 1 and enhanced endothelial cell dysfunction compared to young mice. Glypican 1 gene deletion in knockout mice (GPC1-/-) exacerbated endothelial dysfunction in young mice, which normally had high endothelial expression, but not in old mice that normally expressed low levels. Endothelial cell dysfunction was exacerbated in young, but not aged, Glypican 1 knockout mice (GPC1-/-). CONCLUSION: Arterial stiffness promotes EC dysfunction and vascular disease at least partly through the suppression of the glycocalyx protein Glypican 1. Glypican 1 contributes to the protection against endothelial cell dysfunction and vascular disease in endothelial cells.

The role of oxygen transport in atherosclerosis and vascular disease.

Atherosclerosis and vascular disease of larger arteries are often associated with hypoxia within the layers of the vascular wall. In this review, we begin with a brief overview of the molecular changes in vascular cells associated with hypoxia and then emphasize the transport mechanisms that bring oxygen to cells within the vascular wall. We focus on fluid mechanical factors that control oxygen transport from lumenal blood flow to the intima and inner media layers of the artery, and solid mechanical factors that influence oxygen transport to the adventitia and outer media via the wall's microvascular system-the vasa vasorum (VV). Many cardiovascular risk factors are associated with VV compression that reduces VV perfusion and oxygenation. Dysfunctional VV neovascularization in response to hypoxia contributes to plaque inflammation and growth. Disturbed blood flow in vascular bifurcations and curvatures leads to reduced oxygen transport from blood to the inner layers of the wall and contributes to the development of atherosclerotic plaques in these regions. Recent studies have shown that hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor associated with hypoxia, is also activated in disturbed flow by a mechanism that is independent of hypoxia. A final section of the review emphasizes hypoxia in vascular stenting that is used to enlarge vessels occluded by plaques. Stenting can compress the VV leading to hypoxia and associated intimal hyperplasia. To enhance oxygen transport during stenting, new stent designs with helical centrelines have been developed to increase blood phase oxygen transport rates and reduce intimal hyperplasia. Further study of the mechanisms controlling hypoxia in the artery wall may contribute to the development of therapeutic strategies for vascular diseases.

Heparan sulfate proteoglycan, integrin, and syndecan-4 are mechanosensors mediating cyclic strain-modulated endothelial gene expression in mouse embryonic stem cell-derived endothelial cells.

It is widely believed that the differentiation of embryonic stem cells (ESCs) into viable endothelial cells (ECs) for use in vascular tissue engineering can be enhanced by mechanical forces. In our previous work, we reported that shear stress enhanced important EC functional genes on a CD31+ /CD45- cell population derived from mouse ESC committed to the EC lineage. In the present study, in contrast to the effects of shear stress on this cell population, we observed that cyclic strain significantly reduced the expression of EC-specific marker genes (vWF, VE-cadherin, and PECAM-1), tight junction protein genes (ZO-1, OCLD, and CLD5), and vasoactive genes (eNOS and ET1), while it did not alter the expression of COX2. Taken together, these studies indicate that only shear stress, not cyclic strain, is a useful mechanical stimulus for enhancing the properties of CD31+ /CD45- cells for use as EC in vascular tissue engineering. To begin examining the mechanisms controlling cyclic strain-induced suppression of gene expression in CD31+ /CD45- cells, we depleted the heparan sulfate (HS) component of the glycocalyx, blocked integrins, and silenced the HS proteoglycan syndecan-4 in separate experiments. All of these treatments resulted in the reversal of cyclic strain-induced gene suppression. The current study and our previous work provide a deeper understanding of the mechanisms that balance the influence of cyclic strain and shear stress in endothelial cells.

The cancer cell glycocalyx proteoglycan Glypican-1 mediates interstitial flow mechanotransduction to enhance cell migration and metastasis.

BACKGROUND: Previous studies have demonstrated that the glycosaminoglycans (GAGs) heparan sulfate (HS) and hyaluronic acid (HA) are mechanosensors for interstitial flow on cancer cells. The proteins that link the GAGs to the cancer cell for mechanotransduction, however, are not known. OBJECTIVE: To assess whether the HS proteoglycan core proteins, Glypican-1 and Syndecan-1, or the HA receptor, CD44, provides the mechanical linkage to the cell. METHODS: The highly metastatic renal carcinoma cell line (SN12L1) and its companion low metastatic cell line (SN12C) were analyzed by Western blot, siRNA, and a 3-dimensional interstitial flow migration assay. RESULTS: There was significant elevation of Glypican-1 protein expression in the SN12L1 cells relative to the SN12C cells while there were no significant differences in Syndecan-1 or CD44. Knock down of Glypican-1 by siRNA completely blocked flow induced migration in SN12L1 cells. MAPK inhibitors also blocked flow induced migration in SN12L1 cells. CONCLUSIONS: Glypican-1 provides the mechanical linkage from HS (the flow sensor) to the SN12L1 cell where mechanotransduction leading to the enhancement of migration (metastasis) occurs. MAPKs downstream of Glypican-1 propagate the signal. The HS, Glypican-1, MAPK signaling axis suggests opportunities for pharmaceutical intervention.

Endothelial surface glycocalyx (ESG) components and ultra-structure revealed by stochastic optical reconstruction microscopy (STORM).

BACKGROUND: In order to play different roles in vascular functions as a mechanosensor to blood flows and as a barrier to transvascular exchange, the endothelial surface glycocalyx (ESG) should have an organized structure. Due to the limitations of optical and electron microscopy, the ultra-structure of ESG has not been revealed until the recent development of super-resolution optical microscopy, STORM. OBJECTIVES: To investigate the ESG components and their organization on bEnd3 (mouse brain microvascular endothelial cells) monolayer. METHODS: ESG was immunolabeled with anti-heparan sulfate (HS), followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated hyaluronic acid (HA) binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM. RESULTS: HA is a long molecule weaving into a network which covers the endothelial luminal surface. In contrast, HS is a shorter molecule, perpendicular to the cell surface. HA and HS are partially overlapped with each other at the endothelial luminal surface. We also quantified the length, diameter, orientation, and density of HS at the top, middle and bottom regions of the endothelial surface. CONCLUSIONS: Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve.

The Role of Endothelial Surface Glycocalyx in Mechanosensing and Transduction.

The endothelial cells (ECs) forming the inner wall of every blood vessel are constantly exposed to the mechanical forces generated by blood flow. The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system. A variety of mechanosensors and transducers, locating on the EC surface, intra- and trans-EC membrane, and within the EC cytoskeleton, have thus been identified to ensure proper functions of ECs. Among them, the most recent candidate is the endothelial surface glycocalyx (ESG), which is a matrix-like thin layer covering the luminal surface of the EC. It consists of various proteoglycans, glycosaminoglycans, and plasma proteins and is close to other prominent EC mechanosensors and transducers. This chapter summarizes the ESG composition, thickness, and structure observed by different labeling and visualization techniques and in different types of vessels. It also presents the literature in determining the ESG mechanical properties by atomic force microscopy and optical tweezers. The molecular mechanisms by which the ESG plays the role in EC mechanosensing and transduction are described as well as the ESG remodeling by shear stress, the actin cytoskeleton, the membrane rafts, the angiogenic factors, and the sphingosine-1-phosphate.

Direct current stimulation of endothelial monolayers induces a transient and reversible increase in transport due to the electroosmotic effect.

We investigated the effects of direct current stimulation (DCS) on fluid and solute transport across endothelial cell (EC) monolayers in vitro. Our motivation was transcranial direct current stimulation (tDCS) that has been investigated for treatment of neuropsychiatric disorders, to enhance neurorehabilitation, and to change cognition in healthy subjects. The mechanisms underlying this diversity of applications remain under investigation. To address the possible role of blood-brain barrier (BBB) changes during tDCS, we applied direct current to cultured EC monolayers in a specially designed chamber that generated spatially uniform direct current. DCS induced fluid and solute movement across EC layers that persisted only for the duration of the stimulation suggesting an electroosmosis mechanism. The direction of induced transport reversed with DCS polarity - a hallmark of the electroosmotic effect. The magnitude of DCS-induced flow was linearly correlated to the magnitude of the applied current. A mathematical model based on a two-pore description of the endothelial transport barrier and a Helmholtz model of the electrical double layer describes the experimental data accurately and predicts enhanced significance of this mechanism in less permeable monolayers. This study demonstrates that DCS transiently alters the transport function of the BBB suggesting a new adjunct mechanism of tDCS.

Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling.

Nitric oxide (NO) is a regulatory molecule in the vascular system and its inhibition due to endothelial injury contributes to cardiovascular disease. The glycocalyx is a thin layer of glycolipids, glycoproteins, and proteoglycans on the surface of mammalian epithelial cells. Extracellular forces are transmitted through the glycocalyx to initiate intracellular signaling pathways. In endothelial cells (ECs), previous studies have shown the glycocalyx to be a significant mediator of NO production; degradation of the endothelial glycocalyx layer (EGL) drastically reduces EC production of NO in response to fluid shear stress. However, the specific EGL components involved in this process are not well established. Recent work using short-hairpin RNA approaches in vitro suggest that the proteoglycan glypican-1, not syndecan-1, is the dominant core protein mediating shear-induced NO production. We utilized atomic force microscopy (AFM) to apply force selectively to components of the EGL of confluent rat fat pad ECs (RFPECs), including proteoglycans and glycosaminoglycans, to observe how each component individually contributes to force-induced production of NO. 4,5-diaminofluorescein diacetate, a cell-permeable fluorescent molecule, was used to detect changes in intracellular NO production. Antibody-coated AFM probes exhibited strong surface binding to RFPEC monolayers, with 100-300 pN mean adhesion forces. AFM pulling on glypican-1 and heparan sulfate for 10 min caused significantly increased NO production, whereas pulling on syndecan-1, CD44, hyaluronic acid, and with control probes did not. We conclude that AFM pulling can be used to activate EGL-mediated NO production and that the heparan sulfate proteoglycan glypican-1 is a primary mechanosensor for shear-induced NO production.

Mechanotransmission in endothelial cells subjected to oscillatory and multi-directional shear flow.

Local haemodynamics are linked to the non-uniform distribution of atherosclerosic lesions in arteries. Low and oscillatory (reversing in the axial flow direction) wall shear stress (WSS) induce inflammatory responses in endothelial cells (ECs) mediating disease localization. The objective of this study is to investigate computationally how the flow direction (reflected in WSS variation on the EC surface over time) influences the forces experienced by structural components of ECs that are believed to play important roles in mechanotransduction. A three-dimensional, multi-scale, multi-component, viscoelastic model of focally adhered ECs is developed, in which oscillatory WSS (reversing or non-reversing) parallel to the principal flow direction, or multi-directional oscillatory WSS with reversing axial and transverse components are applied over the EC surface. The computational model includes the glycocalyx layer, actin cortical layer, nucleus, cytoskeleton, focal adhesions (FAs), stress fibres and adherens junctions (ADJs). We show the distinct effects of atherogenic flow profiles (reversing unidirectional flow and reversing multi-directional flow) on subcellular structures relative to non-atherogenic flow (non-reversing flow). Reversing flow lowers stresses and strains due to viscoelastic effects, and multi-directional flow alters stress on the ADJs perpendicular to the axial flow direction. The simulations predict forces on integrins, ADJ filaments and other substructures in the range that activate mechanotransduction.

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38.

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2 Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5ß1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5ß1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1-/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals.NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).