Vascular permeability regulation by fluid shear stress

Increased permeability and interstitial and pulmonary edema are prominent features of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) [1]. Vascular endothelial growth factor (VEGF) and its receptors have been implicated in the regulation of vascular permeability in many organ systems, including the lung. Data extrapolated from other organs and animal experiments have suggested that over-expression of VEGF would, therefore, be harmful within the lung. Recent data, from animal models as well as from patients with ALI, have shown decreased levels of VEGF in the lung. It is clear that the regulation of pulmonary vascular permeability and the roles of VEGF expression in the lung are complex. In this chapter, we explore the literature looking at the expression and function of VEGF in animal models of ALI and in patients with ALI. Novel evidence points to a potential role of VEGF in promoting repair of the alveolar-capillary membrane during recovery from ALI. Pro-VEGF therapy may therefore have potential as a rescue therapy for alveolar epithelial damage in ALI.

VEGFs and their receptors have been implicated in the regulation of vascular permeability in many organ systems, including the lung. Increased permeability and interstitial and pulmonary edema are prominent features of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Extrapolating data from other organ systems and animal experiments have suggested that overexpression of VEGF functions primarily as proinjurious molecules in the lung. Recent data, from animal models as well as from patients with ARDS, have shown decreased levels of VEGF in the lung. The role of VEGF and related molecules in ALI/ARDS is, therefore, controversial: what has become clear is that there are many unique features in the regulation of pulmonary vascular permeability and in VEGF expression in the lung. In this review, we explore a growing body of literature looking at the expression and function of VEGF and related molecules in different models of ALI and in patients with ALI/ARDS. Novel evidence points to a potential role of VEGF in promoting repair of the alveolar-capillary membrane during recovery from ALI/ARDS. Understanding the role of VEGF in this disease process is crucial for developing new therapeutic strategies for ALI/ARDS.

The endothelial monolayer is a signal transduction interface for blood-borne mechanical as well as chemical stimuli. The structural deformation arising from a mechanical stimulus, such as a change of hemodynamic shear stress, is conceptually different from the binding of a hormone or other agonist to its specific receptor, yet both elicit important endothelial signaling responses. Mechanical and chemical signaling appear to use common as well as unique pathways downstream of the initial stimulus. For example, signaling pathways arising from membrane deformation and hormone-receptor coupling may appear to converge through activation of phospholipases1 or nuclear factor κB transcription factor complex2 but later diverge at the level of DNA binding3 (Figure 1). However, simple interpretations of mechanotransduction are confounded by intracellular and pericellular force transfer, principally by the cytoskeleton,4,5⇓ that results in the generation of transduction pathways at multiple sites throughout the cell, ie, decentralized mechanotransduction.6,7⇓ Thus, although flow-induced shear stress acts at the luminal surface of the endothelial monolayer, the forces are transmitted to cell junctions, nuclear structures, basal adhesion sites, and organelles that are structurally interconnected. This spatial organization of intracellular signaling may result in the stimulation of multiple parallel, convergent, and/or divergent mechanotransduction pathways. The mechanism of transduction of a purely mechanical signal to second messenger pathways such as IP3-driven intracellular calcium mobilization8 or mitogen-activated protein kinases (MAPK) activation9 is unknown. Major efforts are therefore underway to understand endothelial mechanotransduction in terms of the initial stimulus, the generation of …

Podocytes are exposed to mechanical forces arising from glomerular capillary pressure and filtration. It has been shown that stretch affects podocyte biology in vitro and plays a significant role in the development of glomerulosclerosis in vivo. However, whether podocytes are sensitive to fluid shear stress is completely unknown. In the present study, we therefore exposed cells of a recently generated conditionally immortalized mouse podocyte cell line to defined fluid shear stress in a flow chamber, mimicking flow of the glomerular ultrafiltrate over the surface of podocytes in Bowman's space. Shear stress above 0.25 dyne/cm(2) resulted in dramatic loss of podocytes but not of proximal tubular epithelial cells (LLC-PK(1) cells) after 20 h. At 0.015-0.25 dyne/cm(2), lamellipodia formation in podocytes was enhanced and the actin nucleation protein cortactin was redistributed to the cell margins. Shear stress further diminished stress fibers and the presence of vinculin in focal adhesions. Linear zonula occludens-1 distribution at cell-cell contacts remained unaffected at low shear stress. At 0.25 dyne/cm(2), the monolayer was broken up and remaining cell-cell contacts were reinforced by F-actin and alpha-actinin. Because the cytoskeletal changes induced by shear stress suggested the involvement of tyrosine kinases (TKs), we tested several TK inhibitors that were all without effect on podocyte number under static conditions. At 0.25 dyne/cm(2), however, the TK inhibitors genistein and AG 82 were associated with marked podocyte loss. Our data demonstrate that podocytes are highly sensitive to fluid shear stress. Shear stress induces a reorganization of the actin cytoskeleton and activates specific tyrosine kinases that are required to withstand fluid shear stress.

Vascular endothelial growth factors (VEGFs) are key regulators of permeability. The principal evidence behind how they increase vascular permeability in vivo and in vitro and the consequences of that increase are addressed here. Detailed analysis of the published literature has shown that in vivo and in vitro VEGF-mediated permeability differs in its time course, but has common involvement of many specific signalling pathways, in particular VEGF receptor-2 activation, calcium influx through transient receptor potential channels, activation of phospholipase C gamma and downstream activation of nitric oxide synthase. Pathways downstream of endothelial nitric oxide synthase appear to involve the guanylyl cyclase-mediated activation of the Rho–Rac pathway and subsequent involvement of junctional signalling proteins such as vascular endothelial cadherin and the tight junctional proteins zona occludens and occludin linked to the actin cytoskeleton. The signalling appears to be co-ordinated through spatial organization of the cascade into a signalplex, and arguments for why this may be important are considered. Many proteins have been identified to be involved in the regulation of vascular permeability by VEGF, but still the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system, and a synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood.

Cell chirality is newly discovered as an intrinsic cell property characterized by the bias of cell polarizing along the left‐right axis. We have recently reported that the endothelial phenotype is right biased; however, little is known about its implications in vascular physiology. In this study, we measured chirality and permeability of human umbilical vein endothelial cells by utilizing micropatterning and transwell culture. Even with low‐level PKC activation, we observed a remarkable reversal of endothelial cell chirality, accompanied by a non‐monotonic change in the monolayer permeability. Using a co‐culture model and immunofluorescence‐based image analysis, we found that uniformity in cell chirality was important for endothelial barrier function such that chirality mismatch between adjacent cells resulted in disrupted cell‐cell junctions leading to increased permeability, which peaked when cell chirality was randomized in the endothelial monolayer. Pharmacological and genetic studies allowed us to determine that PKCα and PI3K/AKT mediated the switch of cell chirality, while other known vascular permeability factors such as VEGF, PAF, and histamine regulated permeability independent of cell chirality. Our study has demonstrated for the first time the role of endothelial cell chirality in vascular permeability regulation and provides unique opportunities for drug delivery across tightly connected cellular sheets.Support or Funding InformationThis work was supported by the National Institutes of Health (OD/NICHD DP2HD083961), National Science Foundation (CAREER CMMI‐1254656), American Heart Association (13SDG17230047), March of Dimes (MOD 5‐FY14‐111). Leo Q. Wan is a Pew Scholar in Biomedical Sciences (PEW 00026185), supported by the Pew Charitable Trusts.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Most of aneurysms in the cranial cavity occur at the bifurcation of anterior circulation system. A cerebral aneurysm is easily ruptured, and it is fatal for most patients. Generally it is known that aneurysm occurs when arterial wall is deformed by high pressure or high wall shear stress (WSS). A blood flow pattern and the geometry and the blood vessel are important factors for aneurysm formation and the location. The transient interaction between blood flow and the arterial wall affects for simulating deformation of the blood vessel. Thus, numerical analysis is performed for various bifurcation angles and flow rate ratio in bifurcation artery with different diameters to predict the location of aneurysm by hemodynamic characteristics of blood flow. A bifurcation angle between the internal carotid artery and the anterior cerebral artery (ACA) increased, a region of high pressure moved to the bifurcated artery with larger bifurcation angle when ratio of blood flow rate is constant case. When the ratio of blood flow increased, the region of high wall shear stress moved to the side of large flow rate ratio. Our results showed that the high WSS or high pressure region occur at the location of aneurysm as mentioned in the clinical research. Thus, this indicates that the geometry of blood vessel and blood flow rate affect the location of the anterior circulation aneurysm.

Metastasis is the cause of more than 90% of cancer‐related deaths. An important step in metastatic process is the migration of the disseminating cancer cells through the endothelial monolayer during entrance or exit from the vasculature. Activation of endothelial RhoA‐ROCK pathway has been implicated in the regulation of vascular permeability in various pathophysiological conditions. In the present study, we have investigated the role of this pathway in trans‐endothelial migration and metastasis of breast cancer cells. RhoA activation was identified by RhoA pull‐down experiments of murine and human breast cancer cell supernatant on primary endothelial cells. Cancer cell trans‐endothelial migration efficiency was explored through an in vitro transwell‐based two‐cell co‐culture model. RhoA signaling pathway was blocked by both pharmacological inhibition and knockdown experiments. In vivo metastatic potential was identified by experimental metastasis models of murine and human breast cancer cell lines using endothelial‐specific RhoA‐deficient mice or clinically relevant ROCK inhibitors. We identified that the diverse metastatic potential of a panel of human breast cancer cell lines correlated with the RhoA activation efficiency of their supernatant on primary endothelial cells. Secretome analysis revealed that IL‐8 (over)expression was relevant to breast cancer cell aggressiveness and its functionality was tested with gain‐and loss‐of‐function experiments. In vivo, experimental metastasis experiments with the syngeneic breast cancer cell line E0771 showed that endothelial RhoA deficiency decreased the metastatic potential. Similar inhibition was obtained with treatment with the clinically‐relevant ROCK inhibitor, Fasudil, in experimental metastasis experiments of murine and human cell lines using immune‐competent and immune‐deficient mice respectively. Collectively, our in vitro and in vivo findings demonstrate that the endothelial RhoA‐ROCK pathway affects breast cancer trans‐endothelial migration and metastasis and propose endothelial RhoA inhibition as a novel therapy for breast cancer metastases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial cell (EC) morphology and functions can be highly impacted by the mechanical stresses that the cells experience in vivo. In most areas in the vasculature, ECs are continuously exposed to unsteady blood flow-induced shear stress and vasodilation-contraction-induced tensile stress/strain simultaneously. Investigations on how ECs respond to combined shear stress and tensile strain will help us to better understand how an altered mechanical environment affects EC mechanotransduction, dysfunction, and associated cardiovascular disease development. In the present study, a programmable shearing and stretching device that can apply dynamic fluid shear stress and cyclic tensile strain simultaneously to cultured ECs was developed. Flow and stress/strain conditions in the device were simulated using a fluid structure interaction (FSI) model. To characterize the performance of this device and the effect of combined shear stress-tensile strain on EC morphology, human coronary artery ECs (HCAECs) were exposed to concurrent shear stress and cyclic tensile strain in the device. Changes in EC morphology were evaluated through cell elongation, cell alignment, and cell junctional actin accumulation. Results obtained from the numerical simulation indicated that in the "in-plane" area of the device, both fluid shear stress and biaxial tensile strain were uniform. Results obtained from the in vitro experiments demonstrated that shear stress, alone or combined with cyclic tensile strain, induced significant cell elongation. While biaxial tensile strain alone did not induce any appreciable change in EC elongation. Fluid shear stress and cyclic tensile strain had different effects on EC actin filament alignment and accumulation. By combining various fluid shear stress and cyclic tensile strain conditions, this device can provide a physiologically relevant mechanical environment to study EC responses to physiological and pathological mechanical stimulation.

BackgroundWith the implementation of transcatheter aortic valve replacement (TAVR) in lower-risk patients, evaluation of blood flow characteristics and the effect of TAVR on aortic dilatation becomes of considerable interest. We employed 4D flow MRI in the ascending aorta of patients after TAVR to assess wall shear stress (WSS) and compare blood flow patterns with surgical aortic valve replacement (SAVR) and age- and gender-matched controls.MethodsFourteen post-TAVR patients and ten age- and gender-matched controls underwent kt-PCA accelerated 4D flow MRI of the thoracic aorta at 3.0 Tesla. Velocity and wall shear stress was compared between the two groups. In addition, aortic flow eccentricity and displacement was assessed and compared between TAVR patients, controls and 14 SAVR patients recruited as part of an earlier study.ResultsCompared to controls, abnormally elevated WSS was present in 30±10% of the ascending aortic wall in TAVR patients. Increased WSS was present along the posterior mid-ascending aorta and the anterior distal-ascending aorta in all TAVR patients. TAVR results in eccentric and displaced flow in the mid- and distal-ascending aorta, whereas blood flow displacement in SAVR patients occurs only in the distal-ascending aorta.ConclusionThis study shows that TAVR results in increased blood flow velocity and WSS in the ascending aorta compared to age- and gender-matched elderly controls. This finding warrants longitudinal assessment of aortic dilatation after TAVR in the era of potential TAVR in lower-risk patients. Additionally, TAVR results in altered blood flow eccentricity and displacement in the mid- and distal-ascending aorta, whereas SAVR only results in altered blood flow eccentricity and displacement in the distal-ascending aorta.Key Points• TAVR results in increased blood flow velocity and WSS in the ascending aorta.• Longitudinal assessment of aortic dilatation after TAVR is warranted in the era of potential TAVR in lower-risk patients.• Both TAVR and SAVR result in altered blood flow patterns in the ascending aorta when compared to age-matched controls.

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.

The balance of RhoA activation is vital in maintaining endothelial function in part due to its role in cell contraction, a function which regulates vascular permeability. Previous studies have implicated PKCδ activity in the regulation of endothelial barrier function via modulation of RhoA activity. To understand the relationship between PKCδ and RhoA in the pulmonary endothelium, we used co‐precipitation experiments to identify Rho effectors that bind RhoA in the presence and absence of the PKCδ inhibitor, rottlerin.We noted PKCδ binding with both constitutively inactive, RhoAG17A, as well as the constitutively active RhoAQ63L, forms of RhoA. Chemical inhibition of PKCδ resulted in increased PKCδ binding to RhoAG17A but decreased PKCδ binding to RhoAQ63L. Altered binding of RhoAQ63L and RhoAG17A with p190RhoGAP and p115RhoGEF, respectively, was observed upon PKCδ inhibition. Using electric cell‐substrate impedance sensing (ECIS) system, we noted that similar to PKCδ inhibition, Rho kinase inhibition with Y27632 promoted endothelial barrier dysfunction.The data demonstrates that PKCδ influences RhoA activity in pulmonary endothelial cells and the decline in PKCδ or Rho kinase signaling correlated with dysfunction of the endothelial barrier. We propose that PKCδ functions as a regulator of vascular permeability and thus presents an interesting target in the regulation of the pulmonary endothelium.

Regulation of blood-retinal barrier cell-junctions in diabetic retinopathy

See related article, pages 988–995 One of the most widely recognized mechanisms to initiate an inflammatory response is calcium entry into endothelial cells.1–3 Recent investigations have demonstrated that there are multiple mechanisms which determine calcium flux into endothelial cells (including ligand gated calcium channels, store operated calcium channels and mechanosensitive channels), and that these different mechanisms are preferentially distributed between different endothelial cells. The functional consequences of these observations for regulation of vascular tone and remodeling are beginning to be understood4,5 but this is not the case for the regulation of endothelial barrier function in intact organs. In this issue Alvarez et al describe an important example of segmental vascular permeability regulation in lung.6 They demonstrate that activation of the vanilloid subset of the family of calcium channels known as transient receptor potential channels (in this case the TRPV4 channels) preferentially increased the permeability of the endothelial and epithelial layers of the primary gas exchanging septal regions of the lung microvasculature whereas other calcium channels, such as the store operated calcium channels, increased permeability outside the primary gas exchange regions (so called extra-alveolar regions). These are important observations because disruption of the endothelium at the septal barrier is more likely to cause alveolar flooding and impair gas exchange than disruption in extra-alveolar vessels. So far at least 28 mammalian TRP isoforms have been discovered. TRP channels have been subdivided into 3 main classes, TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) although more recently other classes have also been proposed (TRPP [polycystin], TRPML [mucolipin], and TRPA [ankyrin]).4,7 A general feature of the channels is that they pass only very small currents (calcium ion flux) and act as passive conductance pathways for calcium entry, ie, they are not voltage gated. Thus the calcium flux depends on the …

Increased vascular permeability after cardiopulmonary bypass in patients with diabetes is associated with increased expression of vascular endothelial growth factor and hepatocyte growth factor