Vascular permeability regulation by fluid shear stress

Abstract

Blood vessels deliver oxygen, nutrients and immune cells to all the tissues in our body. Once they reach target tissues, tissue-specific regulation of vascular permeability is critical for the controlled transport of macromolecules and immune cells across endothelial monolayers. Dysregulation of vascular integrity is involved in a number of vascular as well as non-vascular diseases. Blood flow patterns and flow rates vary depending on vessel type and geometry and regulate vascular barrier function by modulating endothelial mechanotransduction. In this review, we will focus on how shear stress influences various types of vascular permeability by acting on cell-cell adhesion, cell-ECM interaction, cytoskeletal changes and investigate related pathways. Flow-dependent vascular permeability diseases will be discussed in detail with updated therapeutic perspectives.

Introduction

Endothelial cells form monolayers lining the innermost layer of blood vessels and play a key role in vascular homeostasis and adaptation to stress. Cells or molecules pass through endothelial monolayers by transcytosis (endocytosis at apical side and exocytosis at basal side) or intercellular gaps in the disrupted cell-cell junction (paracellular transport). ¹ LDL is transported and accumulated in the subendothelial intima via caveolin-dependent transcytosis pathway. ² Monocytes employ both pathways but depend more on paracellular transport (70-90%) when they transmigrate to intima. ³ Vascular permeability is regulated for physiological remodeling but endothelial barrier function can be impaired in many diseases causing fatal organ dysfunction. ⁴

Endothelial cells sense fluid shear stress, tangential forces generated from the blood flow, using multiple mechanosensors including Pecam1/VE-cadherin/VEGFR junctional complex, PlexinD1, Latrophillin2, Piezo1, Serotonin receptor 1B and so on.^5–8 Applying tension directly on the mechanosensory molecules using antibody-coupled magnetic beads lead to induction of distinct sets of mechanotransduction pathways. And expression of these mechanosensors in non-endothelial cells enables them to induce flow-responses such as cell alignment to direction of flow. ⁵ Shear stress influences multiple endothelial phenotypes including endothelial permeability. ⁹ Atheroprone regions of arteries where blood flow with irregular direction, oscillation and low average shear stress value show increased vascular permeability to macromolecules such as LDL and monocyte. ¹⁰ Shear stress induces cytoskeletal changes leading to endothelial junction assembly or tyrosine kinase signaling modifying junctional proteins. Flow also modulates transcytosis pathway.

In this review, we summarize recent advances in endothelial flow signaling and its influence on vascular barrier function. Furthermore, relevant vascular permeability diseases will be discussed in terms of disease mechanism and possible therapeutic approaches.

Endothelial cells and vascular permeability

As cells forming the inner most layers of blood vessels, endothelial cells play a key role as a barrier and a transport gate for macromolecules and cells. Endothelial cells mediate transendothelial transport via transcellular route or paracellular route.^11,12

Paracellular route

Endothelial cells have cell-cell adhesion mediated by adherens junction and tight junction. Endothelial specific adherens junctions made by homotypic interaction between VE-cadherin. Tight junctions more apically located in endothelial cells are mediated by multiple tight junction proteins claudin, occluding and JAM. ¹³

Adherens junctions

VE-cadherins generally possess a modular structure consisting of five Ig-like extracellular cadherin domains, a single-pass transmembrane domain and a short cytoplasmic tail. ¹⁴ The tandem endothelial cell domains are responsible for Ca2⁺-dependent trans homotypic interaction between neighboring cells. VE-cadherin cytoplasmic tail has multiple tyrosine residues whose phosphorylation mediates interaction with β-catenin, p120-catenin and plakoglobin. β-catenin or plakoglobin binding to VE-cad stabilizes adherens junction by linking with actin cytoskeleton and p120-catenin regulates VE-cadherin internalization. Dynamic regulation of VE-cadherin cytoplasmic tail phosphorylation is important for vascular permeability for monocyte transmigration. VEGF induces Src-dependent phosphorylation of VE-cadherins and cadherins to promote vascular permeability. ¹⁵

Tight junctions

Three types of transmembrane proteins are involved in tight junction assembly. These three proteins Claudins, JAMs and TAMPs (TJ-associated MARVEL domain-containing proteins, occluding, tricellulin, MarvelD3) are associated and organized by cytosolic scaffold ZO proteins (Zonula occludens). ¹⁶ Genetic deletion of ZO proteins, claudins or occludin led to the impaired tight junction assembly and increased vascular permeability. Cytokines or shear stress induces serine/threonine phosphorylation of occludin to provoke vascular permeability. ¹⁷

Transcytosis

Transcytosis is the vectorial transfer of macromolecular cargo within the plasmalemmal vesicles from the circulation across capillary endothelial cells to the interstitium of tissues. The transport of macromolecules, including lipoproteins, across the endothelium is actively controlled by endothelial cells via the transcellular route. ¹⁸ Transcytosis involves either clathrin-mediated endocytosis or caveolae-mediated endocytosis or fluid-phase endocytosis. Blood brain barrier (BBB) controls transcytosis by Mfsd2a-mediated suppression of caveolae-mediated transcytosis. ¹⁹ Transcytosis of insulin through capillaries is the rate-limiting step in insulin action on skeletal muscle. ¹¹

Shear stress and endothelial cells

Endothelial cells are exposed to tissue-specific blood flow patterns and aberrant flows can promote disease progression. Shear stress, tangential force derived from blood flow, controls endothelial phenotypes and vascular homeostasis and diseases. For example, in PAH (pulmonary arterial hypertension) associated with congenital heart disease, increased pulmonary blood flow due to systemic-to-pulmonary shunting and resulting disturbed flow at branch points is an essential trigger for the neointimal lesions and PAH development. ²⁰ Decreased cerebral blood flow years before diagnosis has been implicated in onset of Alzheimer’s disease and suggested as a useful preclinical marker of AD. ²¹ Laminar shear stress refers to a unidirectional flow pattern with stable directional characteristics, whereas disturbed flow exhibits multidirectional oscillatory patterns with low mean shear stress, reflecting frequent flow reversal (Figure 1).

Figure 1.

Shear stress profiles define endothelial cell phenotypes. Laminar shear stress induces an aligned and barrier-stabilized endothelial phenotype, whereas disturbed shear stress promotes a dysfunctional state characterized by inflammation and increased vascular permeability.

Artery

Arteries are exposed to laminar shear stress with average value of 10-50 dyn/cm² with pulsatility in straight regions of arteries. However, disturbed flow pattern appears in regions of curvature or branching. These disturbed flow profiles are characterized by flow separation, recirculation zones, and increased oscillatory shear index, which mechanically destabilize endothelial alignment and junctional integrity. Laminar flow maintains endothelial homeostasis, whereas disturbed flow activates endothelial cells to promote endothelial cell turnover, permeability, angiogenesis and inflammation. ²² Atherosclerosis is an arterial disease strongly associated with endothelial responses to disturbed flow. Atherosclerotic plaques start to appear in the disturbed flow region of arteries and suppression of endothelial inflammation prevents atherosclerosis in vivo. ²²

Vein

Venous endothelial cells are also subjected to relatively low-level shear stress (1-5 dyn/cm²) but still under flow regulation of homeostasis and remodeling. Abnormal blood flow, such as stasis and turbulence, and resulting endothelial dysfunction are important pathogenic factors for venous thrombosis. ²³ The incidence of deep vein thrombosis (DVT) is higher in the paralysed leg compared to the mobile leg in stroke patients. ²³ Especially, valve pockets of large veins are more susceptible to low blood flow and contain more thrombi.

Microvasculature

Microvasculature includes arterioles, venules and capillaries with diameters that range from 5 μm to 100 μm. Microvascular endothelial cells are typically exposed to relatively low shear stress compared to arterial endothelial cells. Flow through microcirculation shows non-Newtonian behavior due to a small vessel diameter and red blood cell behaviors such as aggregation and deformation. ²⁴ It also has the plasma-rich, low-viscosity layer near endothelium called cell-free layer (CFL). The thinning of CFL from inflammation or glycocalyx loss is linked to endothelial activation. ²⁵ Recent study reported data supporting critical role of shear stress in the restoration of pulmonary microvascular barrier function after ischemia reperfusion injury via ex-vivo experiment utilizing perfused isolated ventilated mouse lungs. ²⁶

Mechanism of flow regulation of vascular permeability

Fluid shear stress induces physical deformation of the endothelial glycocalyx and plasma membrane, which generates tensile forces across cell-cell junctions and focal adhesion complexes. This mechanical loading promotes conformational changes in mechanosensitive proteins such as PECAM-1 and integrins, enabling force transmission from the luminal surface to the actin cytoskeleton, ultimately leading to integrin-mediated activation of cytoskeletal contractility via RhoA signaling.

Paracellular route regulator: VE-cadherin phosphorylation and actomyosin contractility

VE-cadherin-mediated cell-cell junctions are dynamically controlled by mechanisms that involve protein phosphorylation and reorganization of the actin cytoskeleton. Src kinases phosphorylate multiple tyrosine residues on VE-cadherin which recruit cytoplasmic binding partners such as β-catenin, p120-catenin and plakoglobin. Knock-in animal studies revealed that site-specific mutations lead to differential effects on catenin bindings, VE-cadherin internalization and leukocyte transendothelial migration. ²⁷ VE-cadherin trans-interactions are stabilized by linkage via β-catenin and cortical actin bundles. Cytokines induce actin stress fiber promoting actomyosin contraction to break VE-cadherin junctions. ¹²

Rho family GTPase

RhoA activity is important for the endothelial barrier function and greatly influenced by fluid shear stress. ²⁸ Laminar flow elicits initial drop of RhoA activity transiently to block endothelial polarity to induce cell alignment and maintain relatively low level of RhoA activity.^29,30 However, disturbed flow elicits greater RhoA activation and sustains it. ³¹ Dynamic flow-dependent regulation of RhoA is mediated by mechanisms involving diverse RhoGEF and RhoGAP proteins. Direct force application on Pecam-1, which is a member of junctional mechanoreceptor complex, RhoA activation and cytoskeletal changes via activation of guanine nucleotide exchange factors (GEFs) GEF-H1 and LARG. ³² A recent study showed that spatial RhoA activation can contribute to endothelial barrier stabilization. In the study, physiological laminar shear stress induces Yes tyrosine kinase-dependent phosphorylation and activation of ARHGEF18. ARGFEF18 activates RhoA leading to p38-dependent tight junction stabilization. ³³ Yang et al. showed that p190RhoGAP, one of GTPase-activating proteins (GAPs), is recruited to caveolae and activated by integrin β1 and promotes actin stress fiber under laminar shear stress. ³⁴ ARHGAP18 is another flow-responsive Rho GAP and required for endothelial cell alignment, endothelial cell junction integrity. Its deletion has been shown to potentiate endothelial inflammatory responses and atherogenesis. ³⁵ In contrast to RhoA, Rac1 usually strengthens endothelial barrier function. Laminar shear stress induces polarized Rac1 activation in the downstream edge of cells. Rac1 GEF, Vav2 mediates flow-dependent Rac1 activation and another GEF, Tiam1 promotes polarized Rac1 activation with GEF activity independent manner. ³⁶ Kroon et al. revealed that RhoGEF Trio as a scaffold protein to induce polarized Rac1 activation and endothelial cell alignment. ³⁷ (Figure 2(a)).

Figure 2.

Mechanism of flow-induced vascular permeability (a) Disturbed shear stress induces sustained RhoA activation through PECAM-1–dependent mechanotransduction, promoting cytoskeletal tension and endothelial barrier destabilization. In contrast, laminar shear stress transiently suppresses and spatially restricts RhoA activity while inducing polarized Rac1 activation at the downstream edge, supporting cortical actin organization and junction stabilization. (b) Laminar flow enhances Ang1–Tie2 signaling by promoting VE-PTP internalization and sustained Tie2 phosphorylation, leading to CDC42-dependent cortical actin stabilization. Disturbed flow increases Ang2 release and maintains VE-PTP at the cell surface, resulting in Tie2 inactivation and junction destabilization. (c) Low shear stress and leukocyte adhesion activate Src family kinases through mechanical cues and Piezo1-mediated Ca²⁺ influx, leading to VE-cadherin phosphorylation and internalization, localized junctional remodeling, and leukocyte diapedesis. (d) Flow-induced activation of the PECAM-1–VE-cadherin–VEGFR2/3 mechanosensory complex promotes PI3K signaling and integrin activation. Under homeostatic conditions, integrins support endothelial barrier stability, whereas under pathological conditions, extracellular matrix remodeling together with Ang2 signaling drives pro-inflammatory integrin signaling and increased vascular permeability. (e) Caveolin-1–dependent caveolae mediate LDL transcytosis via luminal receptors. Laminar shear stress–induced follistatin-like 1 (FSTL1) suppresses caveolae formation and LDL transcytosis, thereby limiting endothelial inflammation.

Ang-Tie2

Ang1 (Angiopoietin-1) maintains stable vascular barrier by binding to Tie2 and inducing signaling leading to cortical actin structures. ³⁸ Ang2, an antagonist of Ang1-Tie2 signaling, is secreted from endothelial cells by inflammatory stimuli including disturbed flow. ³⁹ VE-PTP inhibits Tie2 signaling by dephosphorylation of cytoplasmic tyrosine residues of Tie2. ⁴⁰ A recent report suggests VE-PTP association with Tie2 is regulated by flow patterns. Laminar flow induces internalization of VE-PTP and strong phosphorylation and activation of Tie2, however, disturbed flows maintains VE-PTP on cell surface and promote Tie2 dephosphorylation leading to junction destabilization. ⁴¹ In addition to inducing Tie2 phsphorylation and activation, VE-PTP inhibition also influences phosphorylation of FGD5, CDC42 GEF, to promote cortical actin bundles to stabilize endothelial cell junctions. ⁴² (Figure 2(b)).

Src

Src family protein tyrosine kinases have been implicated in the endothelial hyperpermeability through both the paracellular and transcellular pathways. Src induces hyperpermeability via both working on VE-cadherin-mediated cell-cell junctions and actomyosin contractility. Src phosphorylation of VE-cadherin cytoplasmic tails on Y645, Y658, Y685, Y731 and leads to dissociation of catenins and adherens junction disassembly. ⁴³ Shear stress-induced junctional Src activation and Y658 and Y685 phosphorylation of VE-cadherin happens in veins but not in arteries, which was explained by in vitro results showing high shear stresses (in arteries) leads to decreased Src activation in junctions. ⁴⁴ Low shear stress and leukocyte adhesion-dependent membrane tension has been shown to mechanical opening of Piezo1 and Ca2 + influx leading to activation of Src/Pyk2 promoting leukocyte diapedesis. ⁴⁵ Jin et al. suggests that Yes is involved in the shear stress-dependent Y658, Y685, Y731 phosphorylation using knockout mouse studies and demonstrated Yes-dependent phosphorylation mediates VE-cadherin internalization and vascular permeability. ⁴⁶ (Figure 2(c)).

Integrin

Flow-induced integrin activation is one of the major pathways in endothelial mechanotransduction. Activation of the Pecam1-VEcadherin-VEGFR2/3 mechanosenor complex leads to activation of PI3K pathways and induces high affinity state of integrin heterodimers forming new adhesions. ⁵ Integrin signaling is involved in regulation of homeostasis and remodeling of vasculatures and different ECMs control endothelial cell behavior via controlling integrin specific signalings.^47,48 Function in vascular permeability varies depending on ECM types, integrin isoforms, whether endothelial cells are in resting condition or under pathological situation. Provisional ECM molecules such as fibronectin are upregulated in developing or reorganizing vasculatures and provoke endothelial pro-inflammatory signaling and vascular permeability. ¹⁰ Modulation of fibronectin receptors in endothelial cells seems to have pleiotropic outcome. Integrin α5β1 blockade, integrin α5 knockout or pharmacological inhibition maintains vessel barrier functions in ischemic stroke condition.^49–51 Integrin β1 promotes or inhibits vascular permeability depending on physiological contexts. Endothelial knockout of integrin β1 showed that integrin β1 is required for the formation of stable, non-leaky blood vessels during development. ⁵² However, under pathological conditions, integrin β1 promoted vascular permeability via Ang2-dependent cytoskeletal changes.^53,54 (Figure 2(d)).

Transcellular route regulator

Transcytosis is important for the delivery of various cargos from plasma into the organs on demand. Insulin transcytosis is the rate limiting step for insulin action in skeletal muscle for glucose utilization. Transcytosis is very tightly regulated in BBB since transcytosis is one of the very few ways for large molecule uptake. MFSD2A limits caveolin-mediated transcytosis by influx of DHA to change lipid composition to prevent caveolae formation. ⁵⁵ Caveolin1 (Cav1), a key membrane protein of caveolae, is important in LDL transcytosis. ⁵⁶ SR-B1 and Alk1 are two major endothelial LDL receptors binding circulating LDL and mediating caveolae-dependent transcytosis into subendothelial space.^2,57 Atheroprone region of arteries showed presence of caveolae-like vesicles in hyperlipidemic mice and LDL uptake into endothelial cells under disturbed flow was strongly reduced by Cav1 knockdown suggesting shear stress regulates caveolae-dependent LDL transcytosis. ⁵⁸ Recently, FSTL1 (follistatin-like 1 protein) secreted from endothelial cells stimulated with laminar flow reduced LDL transcytosis and endothelial inflammation (Figure 2(e)).

Flow-regulated vascular permeability diseases

Atherosclerosis

Focal initiation of atherosclerotic lesions is controlled by blood profiles, and endothelial inflammation and vascular permeability is increased in disturbed flow regions and contributes to lesion formation. These observations provide a mechanistic explanation for the focal distribution of atherosclerotic plaques at arterial bifurcations despite the systemic presence of circulating risk factors, as disturbed shear stress enhances endothelial dysfunction and promotes inflammatory signaling through mechanosensitive pathways. Various inflammatory stimuli including cytokines or shear stress induce vascular permeability increase along with endothelial inflammation. Increased vascular permeability allows transport of macromolecules (LDL, Lp(a), fibrinogen) and immune cells (monocytes, T cells) to subendothelial space to promote atherogenesis. A number of reports showed that inhibition of vascular permeability can be a therapeutic target for atherosclerosis prevention or treatment. VE-PTP inhibition using AKB-9785 which induces vascular permeability by Tie2 activation suppressed atherosclerosis in hyperlipidemic mice. Ang2 neutralizing antibody reduced fatty streak formation in mouse model. Vascular permeability is closely related to plaque stability. MRI study showed that endothelial permeability is significantly increased in vulnerable plaques and MRI-based vascular permeability measurement can be used to predict plaque ruptures. ⁵⁹

Pulmonary arterial hypertension (PAH)

Pulmonary arterial hypertension (PAH) is a life-threatening disorder characterized by increased pulmonary pressure and right heart failure due to occlusive pulmonary artery remodeling. ⁶⁰ Endothelial dysfunction from hypoxia, toxins, inflammatory cytokines or pathological shear stress plays critical roles in the disease onset and progression. Abnormally high arterial shear stress from heart defect such as Left-to-Right shunt.^20,61 Increased shear stress over the course of vessel occlusion accelerate lesion formation and progression to plexiform lesion. Multiple evidences showed that vascular permeability is increased in PAH and contributes to the development of PAH. ⁶² Several reports suggest that TRPC4-induced calcium entry mediates hyperpermeability in PAH lungs. ⁶³ Rafikova et al. showed that plasma hemoglobin level is increased in PAH patients and inhibition of HCP-1 (heme carrier protein 1) restored RVPSP and Fulton index. ⁶⁴ Endothelial specific knockout of ROR2 (receptor tyrosine kinase-like orphan receptor 2) exacerbated PAH severity in mouse hypoxia model. It turned out that ROR2 knockout boosted integrin β1 activation and endothelial contractility inducing vascular permeability. ⁶⁵ Notch1 activation is reduced in PAH patients and animal model and blockade of Dll4 notch1 ligand led to increased endothelial permeability. Endothelial expression of Notch1 intracellular domain reduced pulmonary hypertension development. ⁶⁶

Cerebral cavernous malformation (CCM)

Cerebral cavernous malformations (CCMs) belongs to low-flow vascular malformations in the venous-capillary vascular beds of central nervous system (CNS). CCM lesions are raspberry-like clusters of enlarged endothelial channels surrounded by a thick, segmental layer of basal membrane. The affected vasculature has leaky junctions causing recurrent cerebral hemorrhages. Familial CCM study revealed that CCM 1-3 promotes endothelial cell-cell junction mainly via suppression of RhoA-Rho kinase pathway and also directly affecting tight junction or adherens junction proteins. Rho kinase inhibitor fasudil has been shown to CCM lesions in a mouse model. Zhou et al. showed that upregulation of Mekk3-KLF2/4 signaling in CCM patients and animal models is responsible for Rho/ROCK activation.^67,68 How laminar flow-responsive KLF2, KLF4 signaling leads to Rho A activation and vascular permeability is intriguing but not very clear yet. However, Li et al. showed that CCM develops in regions of vasculature with low shear stress and CCM2 knockdown aberrantly upregulated KLF2 and KLF4 expression and disease related transcriptome signatures only under low shear condition. ⁶⁹ Ginsberg group identified thrombospondin1 (TSP1) as a KLF2/4-dependent repression target and showed that the treatment of 3TSR, a recombinant TSP1 fragment, preserved tight junction and prevents lesion development in a mouse model of CCM1. ⁷⁰

Conclusion

Mechanical stimuli play a crucial role in maintaining homeostasis of our body and different kind of mechanical inputs are sensed and processed overlapping and distinct mechanotransduction pathways.^71–74 Blood vessels are also actively engaged in maintaining homeostasis by changing endothelial phenotypes to adapt to extrinsic stresses including biochemical or mechanical injuries. Endothelial cells sense blood flow profile (shear stress value and direction) and induce change of permeability via paracellular or transcellular route. Proper transport regulation of plasma ligands and metabolites across endothelium to underlying tissues should be ensured for normal organ functions. Migration of blood leukocytes to sites of injury or infection is crucial in the development of an inflammatory response and host defense. Endothelial cells accommodate this immune cell diapedesis by weakening cell-cell junctions and promoting transcytosis pathway. However sterile inflammation also causes same permeability increase, which can often lead to tissue damages and diseases. For instance, arteries are supposed to bend and branch, which disturbs laminar blood flow. This inherent disturbed flow generates a vulnerable endothelium that, when combined with additional insults such as aging, toxins, hyperglycemia, and hypercholesterolemia, is prone to initiating inflammation and atherosclerotic lesion formation. Shear stress controls vascular permeability by regulating various proteins involved in endothelial cell junction assembly, endothelial cell-ECM adhesion, actin cytoskeleton. Identification of these flow-dependent permeability regulating factors and underlying molecular mechanisms would give us chance to manipulate vascular barrier function to fight multiple permeability diseases such as atherosclerosis, PAH, CCM and cancers. Understanding how altered shear stress is transduced into biochemical signals regulating endothelial permeability may inform therapeutic strategies targeting mechanosensitive pathways, including integrin-mediated signaling, junctional mechanocomplexes, and downstream cytoskeletal regulators such as RhoA.