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Clinical Hemorheology and Microcirculation xx (20xx) x–xxDOI 10.3233/CH-151976IOS Press

1

Wall shear stress in the human eye1

microcirculation in vivo, segmental2

heterogeneity and performance of in vitro3

cerebrovascular models4

Aristotle G. Koutsiaris∗5

Department of Medical Laboratories, School of Health Sciences, Technological Educational Institute6

(TEI) of Thessaly, Larissa, Greece7

Abstract. Wall shear stress (WSS) is a very important hemodynamic parameter implicated in many physiological and pathological8

phenomena. In order to study these phenomena, it is more convenient to use in vitro models before testing on animals and humans.9

Dynamic in vitro cerebrovascular models are considered capable of simulating the in vivo hemodynamic conditions, but only10

few of them seem to meet the criteria for this task. It is now clear that in the human eye microcirculation a significant pulsation11

exists at the pre-capillary arterioles with average WSS values more than twice those in the venular side, for the same diameters.12

WSS heterogeneity is in support of segmental heterogeneity i.e. the endothelial phenotypic and functional difference among13

arterioles, capillaries and venules. In this review paper, the importance of WSS is described in detail and two more microvascular14

segments are proposed: a pre-capillary arteriolar and a post-capillary venular segment. The accurate hemodynamic simulation15

in all microvascular segments seems to be a prerequisite step in the development of dynamic in vitro blood brain barrier (BBB)16

models and microfluidic platforms on chip. Endothelial cells in the cardiovascular system seem to have sophisticated role acting17

like cardiovascular processing sensors (CPSs).18

Keywords: Wall shear stress, hemodynamics, human microcirculation in vivo, segmental heterogeneity, in vitro cerebrovascular19

models, blood-brain barrier, microfluidics20

Acronyms20

ACM Astrocyte-Conditioned MediumATP Adenosine TriPhosphateBBB Blood Brain BarrierCCM Cerebral Cavernous MalformationCD31 Cluster of Differentiation 31CD54 Cluster of Differentiation 54CD106 Cluster of Differentiation 106CPFC Cone-Plate Flow ChamberCSF CerebroSpinal Fluid

∗Corresponding author: Aristotle G. Koutsiaris, Department of Medical Laboratories, School of Health Sciences,Technological Educational Institute (TEI) of Thessaly, Larissa 41 110, Greece. E-mail: [email protected].

1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved

mailto:[email protected]

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2 A.G. Koutsiaris / Wall shear stress in the human eye microcirculation

CPS Cardiovascular Processing SensorD DiameterDa Arteriolar DiameterDv Venular DiameterDIV Dynamic in vitroDMEM Dulbecco’s modified Eagle’s MediumEBM Endothelial Basal MediumEDN1 EnDotheliN-1EMEM Eagle’s Minimal Essential MediumE-selectin Endothelial selectinG protein Guanine nucleotide-binding proteinHIV Human Immunodeficiency VirusHTS High-Throughput ScreeningIAM Immobilized Artificial MembraneICAM-1 InterCellular Adhesion Molecule - 1MDCK Madin-Darby canine kidneyMPC Microfluidic Platform on Chip�PIV micro Particle Image VelocimetryNO Nitric OxideNVU NeuroVascular UnitPAMPA Parallel Artificial Membrane Permeation AssayPDGF Platelet Derived Growth FactorPGF2a ProstaGlandin F2a

PGI2 ProstaGlandin I2

PC-1 PolyCystin-1PC-2 PolyCystin-2PECAM-1 Platelet Endothelial Cell Adhesion Molecule – 1P-selectin Platelet selectinPPFC Parallel-Plate Flow ChamberQ Volume flowRas-ERK Rat sarcoma - Extracellular signal Regulated KinasesRBC Red Blood CellRI Resistive IndexRNA RiboNucleic AcidSBP Systemic Blood PressureTEER Trans-Endothelial Electrical ResistanceTRP Transient Receptor PotentialVax Axial blood VelocityVs Average cross-sectional blood VelocityVCAM-1 Vascular Cell Adhesion Molecule – 1VEGF Vascular Endothelium Growth FactorVEGFR2 Vascular Endothelial Growth Factor Receptor 2WSR Wall Shear RateWSS Wall Shear StressZO Zonula Occludens

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A.G. Koutsiaris / Wall shear stress in the human eye microcirculation 3

1. Introduction21

Wall Shear Stress (WSS) is a very important hemodynamic parameter affecting a multitude of car-22

diovascular functions and diseases. An accurate quantification of WSS in the human microcirculation is23

important for better understanding of the physiological cardiovascular mechanisms and the pathological24

deviations from normal function.25

In this work, a detailed overview of the importance of WSS in various aspects of cardiovascular patho-26

physiology is presented. In addition, a description of the current knowledge about the WSS distribution27

in parts of the human eye microvasculature is given.28

Generally, it is more convenient to use in vitro models before testing directly on animals and humans29

and the arising question is how well these models simulate the real in vivo WSS situation. Therefore, in30

this review the focus was on how well the newest and most promising types of cell-based dynamic models31

(capillary-like and chip-based microfluidic models) simulate WSS in comparison to the current state of32

knowledge of WSS distribution in the human microcirculation. Since WSS is considered a primary stim-33

ulus for the endothelium response, it is important for the microfluidic model to simulate WSS accurately34

and according to the principle of segmental heterogeneity. The concept of segmental heterogeneity, i.e.35

the endothelial phenotypic and functional differences among different vascular segments, is in strong36

support of differentiating the vascular segments in simulations in vitro.37

In the following sections, the issues described are: in Section 2 a brief description of the in vitro cere-38

brovascular models, in Section 3 an overview of the importance of WSS on various research disciplines39

ranging from molecular biology and angiogenesis, to biomechanics, pharmacokinetics and neuroscience,40

in Section 4 a full description of the current knowledge of WSS distribution in various diameters of the41

human eye microcirculation and a comparison to in vitro cerebrovascular model performance, in Section42

5 a description of segmental heterogeneity with a suggestion for two more vascular segments and in43

Section 6 the conclusions.44

2. In vitro cerebrovascular models45

Recently, some very interesting review papers [3, 34, 74, 84, 110] were published, investigating the pros46

and cons of the in-vitro blood-brain barrier (BBB) models, which are considered to be valuable tools that47

can precede and complement animal and human studies [74]. Using in general terms the categorization of48

Palmiotti et al. [84], in vitro cerebrovascular models can be classified (Fig. 1) into 3 broad categories: 1)49

non-cell based models, 2) isolated microvessel models and 3) cell based models. The same classification50

could in principle be used for every in vitro model constructed for studying the microvascular wall51

interface between blood and other tissues different from the nervous tissue, the difference being that in52

other tissues fewer cell types are involved than those in the BBB.53

Non-cell based models can be further subdivided into IAM (Immobilized Artificial Membrane) Chro-54

matography models and PAMPAs (Parallel Artificial Membrane Permeation Assays). The greatest55

advantage of non-cell based models is that they are low cost and High-Throughput Screening (HTS)56

devices. Their greatest disadvantage is their inability to simulate effectively the BBB function.57

Isolated microvessel models use functionally intact and purified microvessels from the brain or pia58

mater. Their greatest disadvantage is the difficulty associated with the isolation and purification procedure.59

Cell based models use cell cultures on an artificially designed platform. They can be further subdivided60

into static and dynamic models depending on weather or not they simulate the exposure of endothelium to61

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IN VITROCEREBROVASCULAR MODELS

NON-CELLBASED

MODELS

ISOLATEDMICROVESSEL

MODELS

CELL BASED MODELS

STATIC DYNAMICIAMCHROMATOGRAPHY

PAMPA

MONOCULTURE CPFC

BICULTURE

TRICULTURE CAPILLARY-LIKE

MPC(CHIPS)

PPFC

Fig. 1. In vitro cerebrovascular model classification was based on the work of Palmiotti et al. [84]. Dynamic models shownin double outline try to simulate the blood flow. The two most promising kinds of dynamic models, the capillary-like and themicrofluidic platforms on chip are shown in triple outline. IAM: Immobilized Artificial Membrane, PAMPA: Parallel ArtificialMembrane Permeation Assay, CPFC: Cone-Plate Flow Chamber, PPFC: Parallel-Plate Flow Chamber, MPC: MicrofluidicPlatform on Chip.

the mechanical forces induced by the flow of blood (Fig. 1). Static monoculture models use a monolayer62

of endothelial cells oversimplifying the structure and function of BBB, however, due to this simplicity63

they have been used extensively. Biculture and triculture static models simulate more efficiently the in64

vivo BBB, using astrocytes and pericytes, but they still lack WSS effects. Therefore it has been suggested65

that all static model generated results should be further validated by dynamic models [34].66

Based on the increasing evidence regarding the influence of blood flow on endothelium and on BBB67

structure and function (see next section) dynamic models are considered better simulators of the in vivo68

physiology. Dynamic cell based models can be further subdivided to Cone-Plate Flow Chambers (CPFC),69

Parallel-Plate Flow Chambers (PPFCs), capillary-like models and Microfluidic Platforms on Chip (MPC).70

CPFCs were the first dynamic cell based models [13, 22] but much later, more sophisticated dynamic71

models appeared namely, the capillary-like models [96], PPFCs [48, 111] and MPCs [10, 112]. One of72

the advantages of PPFCs and MPCs over the other dynamic models is the imaging compatible design and73

therefore the potential use of automatic 2D velocity field quantification techniques like �PIV [52, 53].74

In capillary-like models, endothelial cells are cultured in the lumen of artificial hollow fiber constructs75

(artificial capillaries) and glial cells are distributed on the ablumen side of the capillaries [20, 21, 96].76

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Usually, capillaries are suspended inside a sealed chamber with appropriate inlet and outlet ports. The77

culture medium is pumped through the system to create WSS effects similar to that of blood flow in vivo.78

The pressure waveform and other flow characteristics of some of these models [84] are very close to79

those observed in vivo. However, there are disadvantages [34] such as the thick layer of 150 �m between80

endothelial cells and astrocytes, the lack of direct visualization of the endothelium layer and the long81

time to reach steady-state TEER (Trans-Endothelial Electrical Resistance, 9–12 days).82

Microfluidic platforms on chip are low cost, HTS models that mimic BBB physiological structure83

and function on a miniaturized scale. The layer between endothelial cells and astrocytes can be less84

than 10 �m allowing trafficking studies [34]. Other advantages of chip based microfluidic systems are85

non-destructive imaging capabilities and dynamic monitoring of various variables such as TEER and86

permeability. On the downside, MPCs usually employ only 1 or 2 cell types and TEER is very low (up87

to 300 �·cm2) in comparison to in vivo values of the order of 1000 �·cm2.88

The majority of the latest dynamic models use some kind of culture medium to create WSS effects89

similar to that of blood flow in vivo. Some of the most common culture mediums reported are solutions90

with their composition based on Eagle’s Minimal Essential Medium (EMEM), Dulbecco’s modified91

Eagle’s Medium (DMEM) or on some other kind of Endothelial Basal Medium (EBM) [11, 32, 86, 112].92

Some researchers use Astrocyte-Conditioned Medium (ACM) in order to invoke endothelial expression of93

important BBB proteins, such as tight junction proteins [86, 112]. In capillary-like models the recirculation94

of plasma cells is feasible [84] and experiments with blood leucocytes present in the luminal perfusate95

have been reported [60].96

However, experiments with whole blood or Red Blood Cell (RBC) suspensions as luminal perfusate97

in BBB in vitro models have not been reported yet. This has the following limitations.98

First, important solutes and cells in blood tissue are missing from culture media with unknown99

consequences, since all these blood components may trigger specific endothelial responses.100

Second, the reported dynamic viscosity of cultrure media ranges between 0.8 and 1.2 mPa.s [11, 112]101

which is at least three to four times less than human blood viscosity. This means that in order to achieve102

the same WSS values, flow rates must be increased accordingly, reducing the available diffusion time for103

important substances to be transported across the BBB.104

A more detailed description and analysis of the history, advantages and disadvantages of the cerebrovas-105

cular in vitro models can be found elsewhere [34, 84]. The ultimate goal seems to be the construction106

of a complete neurovascular unit on a chip [3] (NVU on chip) comprising separate compartments for107

the central nervous system, the blood and the cerebrospinal fluid (CSF) simulating three different brain108

barriers: blood-brain, blood-CSF and brain-CSF. This NVU on a chip should incorporate appropriate109

microfluidic devices and the full spectrum of cells implicated in the BBB: endothelial, astrocytes, peri-110

cytes, neurons and microglia. In addition, there are compatibility plans [3] with other “organs-on-a-chip”111

currently under construction.112

3. Importance of WSS113

The pressure exerted by a moving fluid on the inner surface of a tube, along the direction of the flow,114

is called Wall Shear Stress (WSS) and depends on the dynamic viscosity of the fluid and on the Wall115

Shear Rate (WSR) i.e. the spatial variation of velocity in a direction perpendicular to the vessel wall. In116

the case of the living vasculature the place of fluid is taken by blood.117

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AXONEME

GLYCOCALYXNECKLACE

MOTHERCENTRIOLE

PC1 and PC2

TIGHT JUNCTION

NUCLEUS

ADHERENCEJUNCTION

Fig. 2. A schematic of the endothelial primary cilium. The axoneme is composed of 9 doublet microtubules distributed cylin-drically (only 4 single microtubules are shown here). In contrast to the motile cilia there is no central pair of microtubules. PC1:PolyCystin-1, PC2: PolyCyctin-2. A real phase contrast image of an endothelial primary cilium can be seen at Nauli et al. [75].

WSS has been implicated in many physiological as well as pathological phenomena in the cardiovas-118

cular system and indirectly in other systems. All these phenomena start from the WSS influence on the119

endothelium, the innermost cellular layer of the blood vessel wall. In more detail, WSS affects endothe-120

lial mechanosensors and after elapsing a sufficient period of time, significant changes are observed121

in a plethora of endothelial functions and properties. The identity and function of all the endothelial122

mechanosensors has not been clarified yet. Candidates are the cytoskeleton (tensegrity theory), mem-123

brane mechanosensors (various channels and protein structures), membrane microviscosity, nonmotile124

primary cilia and the cell nucleus [41, 65, 75, 100, 101, 109]. It has been shown [47] that gene expression125

of 18 to 30-ion channel subunits was affected by WSS magnitude. In total, approximately 600 genes are126

shear-stress responsive [65, 80].127

The phenomenon of mechanotransduction is so complicated that it is possible more than one sensory128

pathway to be activated under different flow conditions or situations. For example it could be argued that129

in the low shear venular side the primary cilia mechanosensor is mainly used but in the high shear arterial130

side other mechanisms prevail.131

The primary cilium is an extraordinary and delicate organelle [88]. It is at least several microns long132

and has a skeleton of nine doublet microtubules (axoneme) on a basal body stemming from the mother133

centriole [93] (Fig. 2). The flow sensing capability of primary cilia was first demonstrated on Madin-134

Darby canine kidney (MDCK) cultured cells [87] where increasing the flow rate caused increasing of the135

intracellular calcium (as a second messenger) and cell hyperpolarization. Later, similar observations were136

made on cultured human endothelial cells [1, 39, 76] and soon, two hypothesis regarding the functional137

properties of the primary cilium were substantiated [93]: 1) it provides a means of isolating the centriole138

thus inhibiting cell division and 2) in many tissues, receptors on the ciliary membrane detect specific139

environmental signals (mechanical, chemical and others).140

Human endothelial primary cilia are fluid shear sensors regulating nitric oxide (NO) production through141

PolyCystin-1 (PC1) [76] and PolyCystin-2 (PC2) [1]. PC1 is a G-protein coupled transmembrane protein142

and PC2 is a mechanosensitive calcium channel. PC2 belongs to the transient receptor potential (TRP)143

ion channel superfamily and responds specifically to shear stress and not to mechanical stretch [1].144

In order to study the mechanosensory function of cultured cells two basic conditions must be ensured145

[75]: first that they are fully differentiated and second that they possess an optimal length. In addition,146

Nauli et al. [76] reported an optimal shear stress of 7 dynes/cm2 for the greatest increase in cytosolic147

calcium and NO production. However, Iomini et al. [39] reported that at 15 dynes/cm2, primary cilia148

disassembled. It would be very useful to clarify if there is a WSS threshold, above which, cultured149

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endothelial cells disassemble their primary cilia, as it was argued recently [30, 88, 100]. It would also be150

very interesting to clarify if such a threshold exists in the human circulation in vivo. Goetz et al. [30] were151

the first to report endothelial ciliary flow response in vivo using non-invasive high-resolution imaging of152

live zebrafish embryos in a low blood flow environment. Since it is evident that ciliary sensitivity depends153

on its length would it be possible that primary cilium changed its length depending on the shear stress154

magnitude? Are primary cilia fully functional at every vascular segment (arteries, arterioles, capillaries,155

venules, veins) of the human circulation in vivo?156

Disruption of normal primary cilium morphology and function can lead to known abnormalities such157

as diabetes, obesity, cancer, atherosclerosis and various syndromes [93].158

Well known endothelial properties and functions affected by WSS [5, 8, 19, 84, 94] are the con-159

trol of vascular diameter, coagulation of blood, adhesion, transmigration of leukocytes, regulation of160

permeability between blood and surrounding tissues, TEER, energy metabolism, cytoskeletal structure-161

polarization-morphology, proliferation and angiogenesis. In the case of brain, the permeability between162

blood and surrounding tissue takes a more sophisticated and specialized form described by the term:163

blood brain barrier (BBB). A more detailed description of the WSS influence on the above functions and164

properties is given below.165

3.1. Vascular diameter control166

One of the first functions reported to be affected by the presence of WSS is the release of vasoactive167

substances, potent vasodilators or vasoconstrictors. Some examples of such substances, not limited to168

the brain, are prostacyclin or prostaglandin I2 (PGI2) [24, 31], nitric oxide (NO) [12, 25, 38, 48, 83],169

platelet derived growth factor (PDGF) [7], endothelin-1 (EDN1) [113], prostaglandin F2a (PGF2a) [78]170

and several neurotransmitters [70] such as histamine, acetylcholine, ATP and substance P. Some of the171

aforementioned substances are known vasodilators (PGI2, NO, ATP, acetylcholine, substance P) and172

others are known vasoconstrictors (PDGF, EDN1, PGF2a).173

3.2. Cell adhesion174

Cell adhesion is an endothelial property that is improved after WSS trigger [73, 82]. Integrins and175

cadherins are transmembrane proteins responsible for cell-cell and cell-matrix adhesions. When WSS176

was applied to integrins, an activation of the Ras-ERK pathways [40] and a signal transmission to the177

cytoskeleton [105] was reported. It was also reported [101] that vascular endothelial cadherin together178

with the platelet endothelial cell adhesion molecule-1 and the vascular endothelial growth factor receptor179

2 (VEGFR2) form a mechanosensory complex mediating the endothelial cell response to WSS.180

Leukocyte adhesion to the endothelium is considered an important step in the immune response during181

inflammation and in infectious diseases such as cerebral malaria [2, 71]. Leucocyte recruitment is a multi-182

step process that involves leucocyte rolling, adhesion and transendothelial migration and there are still183

significant gaps in our understanding of these steps in different microvascular beds [2]. WSS is important184

in leukocyte recruitment and in the design of vascular targeted drug carriers [14, 33] and differentially185

regulates endothelial uptake of nanocarriers targeted to distinct epitopes of Platelet Endothelial Cell186

Adhesion Molecule-1 (PECAM-1 or CD31) [33]. Cucullo et al. [19] reported a significant RNA level187

increase of several T lymphocyte adhesion molecules (InterCellular Adhesion Molecule ICAM-1 or188

CD54, Vascular Cell Adhesion Molecule VCAM-1 or CD106 and PECAM-1 or CD31) after exposure189

to WSS. In hematogenous metastasis, tumor cells adhere to the endothelium and this process is mediated190

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by adhesive factors such as E and P-selectin which are only effective under shear stress conditions in191

contrast to other adhesive factors [92].192

In addition, red blood cell adhesion to the endothelium is essential in inflammatory, infectious and193

thrombotic situations such as sickle cell disease, malaria, diabetes mellitus, polycythemia vera and central194

retinal vein occlusion [107]. Molecular mechanisms responsible for these situations perhaps are affected195

by hemodynamic forces such as WSS.196

3.3. Permeability197

Another important endothelial function is the regulation of permeability between blood and the sur-198

rounding tissue. More than 15 years ago, it was shown that bovine endothelial permeability [42] and199

endothelial adherence junctions [79] are affected by WSS. Then more evidence was accumulated over200

the years [99]. The degree of permeability depends on the organ, and as it is well known for the brain,201

the permeability is so low that the term “barrier” was used (“blood brain barrier”). To form this barrier,202

in the brain, the endothelium blocks paracellular molecular movement with tight junctions (or zonula203

occludens) and regulates intracellular molecular movement with specialized vesicular transporters. The204

main tight junction protein components are occludins, claudins and zonula occludens (ZO-1, ZO-2 and205

ZO-3). Vesicular transporters can be either bidirectional or unidirectional and can be expressed on both206

the luminal and abluminal sides of the endothelium or in either side [34].207

For BBB to function properly, endothelium combines forces with at least two other cell types, including208

astrocytes and pericytes of the neural system. Astrocytic endfeet cover more than 99% of the vascular209

surface and pericytes are embedded in the endothelial basement membrane between endothelial cells and210

astrocytes [34]. Pericytes cover around 30% of the abluminal surface of the brain microvasculature [110].211

Other cell types such as neurons and microglia have also been speculated to affect BBB permeability212

[34].213

The influence of WSS on BBB permeability has been studied mainly by tracer flux and TEER mea-214

surements. It was found that BBB permeability reduces [11, 19, 86] and TEER increases [11] as WSS215

increases. Griep et al. [32] reported a 3-fold increase of TEER after 18 hour application of WSS and216

Cucullo et al. [19] reported a 7-fold increase of TEER after 30 days application of WSS.217

From the above data it can be concluded as a general rule of thumb that cell adhesion and TEER218

increase as WSS increases, while BBB permeability reduces. This is a result of a profound change in the219

expression of numerous genes related to the BBB endothelial function namely, tight junction genes [11,220

17, 19, 32, 104] adherence junction genes [5] and membrane transporter genes [19, 69, 86]. Cucullo et al.221

[19] reported a statistically significant increase in the RNA levels of 13, 12, 10, 20 and 26 genes encoding222

for tight/adherens junction, multidrug resistance proteins, cytochrome P450, ion channels and membrane223

transporters respectively, after the exposure of endothelium at a WSS of 6.2 dynes/cm2. Booth et al. [11]224

quantified the increase in protein expression after the application of different WSS levels for 1 day and225

they reported a 5-fold and 6-fold increase in tight junction protein ZO-1 and efflux transporter protein226

P-gp expression respectively, at 58 dynes/cm2. In the absence of flow, despite the presence of abluminal227

astrocytes, the resulting BBB is much less stringent [20].228

3.4. Metabolic activity229

BBB endothelial cells manifest high metabolic activity since they are characterized by a very high230

density of mitochondria [18, 19]. As it was shown recently [19] WSS affects the energy metabolism231

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of endothelial cells by upregulating the RNA levels of enzymes involved in the Krebs cycle (aerobic232

respiration) and by downregulating the RNA levels of modulatory enzymes of the glycolytic pathway.233

3.5. Cytoskeletal structure and morphology234

A significant cytoskeletal gene expression increase has been reported [19], in parallel with a signif-235

icant decrease of the nuclear, cytosolic and membrane expression, after the application of WSS on the236

endothelium. The first observations of flow dependent endothelial orientation were reported more than237

30 years ago [23, 77]. Later, it was shown that morphological and cytoskeletal differentiation of endothe-238

lium depends on the time of exposure and magnitude of WSS [6, 26, 45, 63, 81, 108]. Franke et al. [26]239

suggested that the formation of endothelial actin filament stress fibers in response to critical levels of240

fluid shear stress is probably a functionally important mechanism that protects the endothelium from241

hydrodynamic injury and detachment. Garcia et al. [28] showed that gene expression and phenotype242

of the vascular endothelium is different between laminar and turbulent shear stress stimulation. Effects243

of oscillatory flow were reported by Helmlinger et al. [35]. Recent findings revealed the existence of244

a balance between a positive extracellular signal, i.e. WSS and a negative intracellular regulator, i.e.245

the Cerebral Cavernous Malformation (CCM) complex, for the control of �1 integrin activation and246

subsequent cytoskeletal shape and orientation remodeling [41].247

3.6. Cell membrane248

Endothelial membrane changes regarding pseudopodia formation have been reported [36] and a possible249

force-sensing mechanism was suggested [109] comprising first, membrane microviscosity changes linked250

to G proteins and second, cell-cell junction proteins.251

3.7. Angiogenesis252

Another important WSS effect is promoting endothelial sprouting and angiogenesis [4, 43]. Angio-253

genesis is necessary for development, menstruation, ovulation and wound healing and is also a key254

manifestation in important pathological events such as tumor growth, myocardial infarction and diabetic255

retinopathy [44, 46, 95]. It is now more than 15 years since the role of WSS in angiogenesis was reported256

[37, 114]. WSS activates the membrane Vascular Endothelium Growth Factor (VEGF) receptor in a257

different way from VEGF [106].258

In addition, a statistically significant difference on cell invasion and sprouting was reported at 5.3259

dynes/cm2 in comparison to lower or higher WSS values [43, 46]. Song and Munn [95] were the first to260

differentiate between luminal WSS exerted by the flowing blood tangentially on vessel wall and interstitial261

(transmural) WSS exerted perpendicularly through the vessel wall. Galie et al. [27] reported a luminal262

and transmural shear stress threshold of 10 dynes/cm2 for angiogenic sprouting. Regardless of the exact263

value and direction of shear stress it is now well accepted that it plays an important role in angiogenesis.264

Currently, the absence of flow related shear stresses in a proposed angiogenesis model is considered as265

a limitation [97].266

3.8. Atherosclerosis267

Even though this review was concentrated on microcirculation, the importance of WSS on large vessels268

and especially its correlation to atheromatic plaques in arteries are well known. It is generally accepted269

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that a low time-averaged WSS, caused by vessel geometry, vortices and/or oscillations, is a key factor for270

upregulating the expression of proatherosclerosis and prothrombosis genes and the development of an271

early atheromatic plaque [65], even though the evidence is less robust than is commonly assumed [85].272

Endothelial cells appear polygonal in static conditions (atheroprone phenotype) but are transformed to273

elongated cells with their long axis oriented to the direction of flow (atheroresistant phenotype) when they274

are exposed to physical WSS [65, 102]. Lin et al. [66] reported inhibition of endothelial cell proliferation275

after exposure at a laminar WSS of 12 dynes/cm2 for more than 12 hours and proposed this as an276

atherogenesis preventing mechanism. In addition, it seems that flow conditions and WSS magnitude277

affect plaque phenotype [65] which can be of the vulnerable or the more stabilised type.278

3.9. Other forces on endothelial cells279

Endothelial cells are also exposed to other two mechanical forces namely, systemic blood pressure280

(SBP) which acts at a direction vertical to the vessel wall and a cyclic stretch which is a circumferential281

tensile stress due to the arterial dilatation caused by the cardiac pulse. It has been suggested that these282

forces are also taken into account in the endothelial cell responses [94].283

Recently, Vozzi et al. [103] considered for the first time hydrostatic pressure and WSS separately, using284

custom made bioreactors [68] and showed that both pressure forces affect endothelial cell function in285

different ways and that both should be taken into account when planning in vitro experiments.286

Considering that SBP is reduced as blood travels through the different vascular segments from the aorta287

to the vena cava, the above experimental results reinforce the need for more accurate fluid mechanical288

simulation in different vascular segments in developing dynamic in vitro models. Also, it would be289

interesting to see if SBP affects mechanosensor function as for example the function of the primary290

cilium.291

3.10. Summary292

As time went by, the importance of WSS in the function of endothelium and BBB, both in physiological293

and pathological conditions, was made clear. Today it seems logical to consider that the first step in294

implementing an accurate in vitro BBB model is the proper simulation of the physiological WSS values295

observed in the corresponding vascular segments. The development and maintenance of appropriate shear296

stress values at the endothelial barrier is considered as a critical factor in the development of a brain-on-297

a-chip [110]. Therefore, in the next section, a comparison is made between the latest in vivo and in vitro298

data, regarding physiological and simulated WSS values respectively.299

4. WSS in the human microvasculature and in vitro performance300

Even today, WSS can not be measured directly in the human microcirculation in vivo. This can be301

attributed to the lack of experimental apparatuses as well as to difficulties in persuading humans to302

participate in invasive experimental procedures. Perhaps the use of special nanosensors in the future will303

permit the direct measurement of WSS in the human microcirculation.304

In the present, what is usually done is to estimate WSS from other physical quantities such as axial305

blood velocity (Vax) and average cross sectional blood velocity (Vs) or the velocity profile along vessel306

diameter (D) [50, 53, 72, 90]. Even in this indirect way, and despite the critical importance of WSS in307

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-25 -20 -15 -10 -5 0 5 10 15 20 25Arteriolar Diameter Da (μm) Venular Diameter Dv (μm)

WSS

(D

ynes

/cm

2 )

15 subjects30 pre-capillary arterioles

Koutsiaris et al. [54]

50

17 subjects104 post-capillary venules

Koutsiaris et al. [56]

155

Average WSS

Systolic WSS

Diastolic WSS

Fig. 3. Wall shear stress is shown in relation to the pre-capillary arteriolar [54] and post-capillary venular [56] diameters ofthe human eye. The direction of flow is from left to right and for this reason arteriolar diameters have negative values. In thearteriolar (left) side, average and peak systolic wall shear stress points are shown as gray dots and triangles respectively. Theend diastolic points are not shown here for a better presentation. WSS trend lines are shown in black for systolic, average anddiastolic phase. The average WSS trend line is thicker. At a pre-capillary arteriolar diameter of 6 �m, the systolic, average anddiastolic WSS values are 152, 105 and 70 dynes/cm2 respectively. In the post-capillary venular (right) side, the WSS trend linevalues of 50, 15 and 5 dynes/cm2 correspond to diameters of 5, 10 and 20 �m, respectively. The peak systolic WSS trianglecorresponding to the highest average WSS value (211 dynes/cm2) is not shown for a better presentation.

unravelling important mysteries of the physiology and pathology, WSS quantification reports from direct,308

non-invasive, blood velocity measurements in vivo are limited. A more indirect way, usually employed309

in large vessels with more complex geometries, is to put blood pressure as input to numerical simulations310

using Navier Stokes equations.311

The microvessel diameter D is an important variable affecting many hemodynamic quantities namely,312

Vax [51], resistive index (RI) [57, 58], Vs [54, 56, 59], volume flow (Q) [54, 56, 59], wall shear rate313

(WSR) [54, 56] and WSS [54, 56]. Some of these quantities (Q, WSR) are strongly affected by diameter,314

in contrast to others (RI) which relate to D in a logarithmic way [58].315

Lately, a method was introduced [54, 56] for the estimation of WSS from Vax in small microvessels316

(<25 �m), taking into account the influence of D, on the estimation of Vs [54] and viscosity (in vivo317

viscosity law [89]). In this way, WSS was quantified in the post-capillary venules [56] and in the pre-318

capillary arterioles [54] of the human eye using a slit lamp based system [55], and now it is possible, for319

the first time, to have a more accurate picture of the WSS distribution in the smallest human microvessels320

(Fig. 3).321

As it is shown in Fig. 3, in every diameter of the arteriolar side, WSS varies during the cardiac cycle,322

from a peak systolic value down to an end diastolic value. The average WSS during the cardiac cycle is323

shown in a thicker black line. The WSS values increase hyperbolically as arteriolar diameter decreases. At324

pre-capillary arteriolar diameters of approximately 6 �m (–6 in Fig. 3), systolic WSS trend line values are325

of the order of 150 dynes/cm2. However, there are WSS measurement points higher than 200 dynes/cm2.326

At much higher diameter microvessels of the human eye, Nagaoka and Yoshida [72] differentiated the327

systolic from the diastolic hemodynamic values in arterioles and reported an average arteriolar (throughout328

the cardiac cycle) WSS of 40 and 54 dynes/cm2 at average diameters of 100 and 110 �m, respectively.329

In the venular side, they reported an average WSS of the order of 23 dynes/cm2 for venular diameters330

ranging between 110 and 173 �m.331

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Α

Β

D0

20

40

60

80

100

120

140

160

-25 -20 -15 -10 -5 0 5 10 15 20 25

Arteriolar Diameter Da (μm) Venular Diameter Dv (μm)W

SS

(Dyn

es/c

m2 )

C

Fig. 4. A comparison of in vivo WSS values (in black trend-lines) to maximum WSS values (in black dotted horizontal lines)reached by in vitro models. A: 86 dynes/cm2 by Booth et al. [11], B: 23 dynes/cm2 by Cucullo et al. [20], C: 9 dynes/cm2 byYeon et al. [112] and D: 6 dynes/cm2 by Griep et al. [32].

In summary, regarding microvascular hemodynamics of the human eye, three points are now clear:332

1) a significant pulsation exists at the pre-capillary arteriolar side [57], in accordance with experimental333

data from other mammals [49], which disappears as blood continues its way towards the venular side, 2)334

average arteriolar WSS values are more than double than those in the venular side, for the same diameters;335

for example, as it is shown in Fig. 3, at a diameter of 10 �m, the cardiac cycle averaged arteriolar WSS336

of 32 dynes/cm2 is more than double the venular WSS of 15 dynes/cm2 and 3) at pre-capillary arteriolar337

diameters of the order of 6 �m, WSS values higher than 200 dynes/cm2 can be observed [54]. This is338

more than 3 times higher than it was anticipated as “physiological” in recent in vitro works [11].339

Taking into account the aforementioned data, it seems that the full in vivo WSS spectrum in the human340

eye microcirculation ranges approximately from 2 to 200 dynes/cm2. The suggested upper limit of this341

spectrum could be further increased in special cases of increased peripheral blood flow (heat, drugs,342

exercise) but there are no relative measurements. In addition, the lower limit of this spectrum could be343

further decreased in greater venular diameters.344

The in vitro cerebrovascular models constructed so far do not cover the full in vivo WSS spectrum345

(Fig. 4). The upper WSS limit of the order of 200 dynes/cm2 is about 9 times higher than the highest346

value observed at dynamic in vitro models (23 dynes/cm2, DIV-BBB model [20]) and about 20 to 35347

times higher than the highest value observed at microfluidic models on chip (9 dynes/cm2 at Yeon et al.348

[112] and 5.8 dynes/cm2 at Griep et al. [32]).349

Recently, Booth et al. [11] reported a maximum value of 86 dynes/cm2 but in order to reach 200350

dynes/cm2 they would have perhaps to add on their chip a fifth channel with a width smaller than351

0.73 mm. Their microfluidic platform has the advantage of simultaneously achieving a wide range (15×)352

of WSS values and allowing trans-monolayer assays such as permeability, TEER, cell morphometry353

and protein expression. In addition, their results showed impressive correlation of increasing TEER and354

protein expression to increasing WSS, supporting the importance of WSS in cerebrovascular model355

design. However, on the downside, TEER values were low (120–240 �·cm2) in comparison to in vivo356

values and there was no report on simulating the WSS pulsation observed at the arteriolar side in vivo.357

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Much higher TEER values (>700 �·cm2) were achieved by Cucculo et al. [20] who reported simulation358

of the pulsating flow in the arteriolar side. However, their maximum WSS values (23 dynes/cm2) are low359

compared to in vivo values (Fig. 4) and their system is not on a chip. Visualization of cells cultured within360

the artificial microvessel is difficult and there are no HTS capabilities [84].361

At the moment, it has not been reported if the dynamic in vitro on chip models can simulate pre-capillary362

pulsation [3, 10, 11, 32, 86] and most of them are far below (≈20 times, Fig. 4) the human physiological363

WSS values.364

5. Segmental heterogeneity365

The importance of more accurate in vitro simulation of the WSS difference among the vascular seg-366

ments is supported strongly by the observed endothelial heterogeneity in the brain [29, 67]. In general,367

the endothelial phenotypic and functional difference among different vascular segments is regarded as368

segmental heterogeneity [9]. It has been recognized and proved in vitro [15] that there are at least three,369

organ specific, vascular segments, with large differences in the endothelium genotype: 1) arterial, 2)370

microvascular and 3) venous.371

In addition, accumulating data [29, 67] support the argument that there are at least 5 vascular segments:372

1) arterial, 2) arteriolar, 3) capillary, 4) venular and 5) venous. On surplus, given the high number373

of human genes responsive to WSS [80], with specializations on temporal responses (eleven different374

temporal profile clusters) and on flow type (laminar or turbulent) [80], and taking in to account our current375

knowledge of the vascular system and the WSS data shown in Fig. 3, it would seem logical to propose376

two more microvascular segments: a pre-capillary arteriolar and a post-capillary venular, raising the total377

number of vascular segments to seven. The post-capillary vascular segment is already recognized as the378

most preferable site for leukocyte extravasation [29, 67].379

It is known that pulsation is an important energy saving mechanism for the heart, but this pulsation,380

together with the high WSS observed at the pre-capillary arterioles, could lead to important morphological381

and functional endothelial differentiation and therefore, to different BBB restrictive properties and mech-382

anisms among the arteriolar, capillary and venular segments [67] and perhaps among the pre-capillary383

arteriolar, the post-capillary venular and the other vascular segments.384

From a hemodynamical and biomechanical point of view the 7 proposed vascular segments are certainly385

different (Table 1). In the arterial and venous segments, the blood can be considered as a “continuum” but386

this is not the case for the pre and post-capillary segments where the RBCs flow separately constituting a387

different liquid phase from plasma. In the pre and post-capillary segments, the biphasic nature of blood388

is manifested, with RBCs moving in groups, and the velocity profile can not be defined in the ordinary389

sense [53]. In the capillary segment, the flow is also biphasic, but in this case RBCs flow in file (one by390

one) with lengthy plasma gaps between them. More specializing features discriminating the 7 vascular391

segments regarding flow type, flow medium and vessel wall type are presented in Table 1. It is noted that392

each vascular segment has a unique combination of flow type, flow medium and vessel wall type.393

More details on the vessel wall structure and composition can be found in textbooks of anatomy and394

physiology. Rhodin [91] has subdivided arteries and veins into large and medium – sized segments taking395

into account differences in wall structure and composition. In addition, it is well known that many sizes396

(classes) of arterioles and venules exist. In Table 1, an abstract-kind segmentation of the vascular system397

is given without depicting the full complexity and specialization of the vascular system.398

Only few research groups have published measured functional differences among some of the vascular399

segments using in vitro cerebrovascular systems. Cucullo et al. [20] measured statistically significant400

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Table 1

The seven vascular segments

Number Segment Name Flow Type Flow Medium Vessel Wall Type

1 Arterial Unsteady – Periodic(Cardiac Cycle)

Continuum Thick - Muscular -Distensible

2 Arteriolar Unsteady – Periodic(Cardiac Cycle

Continuum with thinPeripheral PlasmaLayer

Thick - Muscular - Rigid

3 Pre-capillary arteriolar Unsteady – Periodic(Cardiac Cycle)

Biphasic (RBCs ingroups)

Rigid

4 Capillary Decaying Velocity Pulse Biphasic (RBCs passingone by one)

Rigid

5 Post-capillary venular Steady Biphasic (RBCs ingroups)

Rigid

6 Venular Steady Continuum with thinPeripheral PlasmaLayer

Thick - Rigid

7 Venous Unsteady – Periodic(Breathing Cycle)

Continuum Thick – Collapsible(mostly with valves)

differences in TEER, permeability and bioenergetic behavior, between the capillary and venular brain401

segments in vitro. More importantly, they showed that capillary segments under low, venular-level, WSS402

exhibited low TEER values comparable to that of the venular segments.403

Endothelial phenotypic and functional differences among vascular segments may be important not404

only to the BBB mechanism, but also to other brain microvascular manifestations in angiogenesis,405

inflammation, infection (HIV-associated neurocognitive disorders and other neurotropic viruses), stroke,406

trauma, toxic transformation of approved drugs, epilepsy, Alzheimer’s disease, schizophrenia, Parkinson’s407

disease, drug addiction, cancer and aging [3, 29, 34].408

Similarly, segmental heterogeneity should be taken into account in the design of dynamic in vitro chip409

models of other organs such as the liver [62]. In addition, segmental heterogeneity could be proved an410

essential variable in the study of the glycocalyx [61, 98, 99], the tissue fluid balance [64] and in red blood411

cell deformability [16].412

Kaunas et al. [46] using dermal, retinal and umbilical vein cells in culture, localized angiogenic sprout-413

ing at the post-capillary venular vascular segment, basing their argument on the relatively thin wall of this414

segment and on their statistically significant results of highest sprouting occurring at about 5 dynes/cm2.415

Observing Fig. 3, similar WSSs occur at human post-capillary venules of about 20 microns in diameter.416

Song and Munn [95] consider that distinct phenotypes are exhibited by the endothelium during sprouting,417

dilation and quiescence even within the same vessel segment depending on fluid forces and biochemical418

gradients.419

May be it is difficult to simulate in the same in vitro microvascular model three different microvascular420

segments (arteriolar, capillary and venular) and even more complex to add another two segments (pre-421

capillary arteriolar and post-capillary venular). However, given all the above data, it seems very important422

to accurately simulate the different vascular segments not only structurally but also hemodynamically.423

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6. Conclusions424

All the above lead to the idea that endothelial cells in the cardiovascular system have a very sophisticated425

role far more complex than it was though of in the last half of the previous century. Apart from their426

obvious function as the internal linning of all blood vessels, it seems that they are able of integrating427

a multitude of local signals to determine weather they remain quiescent or undergo morphogenesis,428

phenotypic differentiation, proliferation or activate specific signal pathways. It could be argued that they429

act as processing sensors specialized for the cardiovascular system or in other words as cardiovascular430

processing sensors (CPSs).431

In conclusion, it seems important that the dynamic in vitro BBB models should simulate more accurately432

the in vivo WSS values depicting at least the important differences between the arteriolar and venular433

segments. This is even more important for the microfluidic platforms on chip given their advantages of434

low cost, small size, dynamic monitoring, high throughput screening and high resolution imaging.435

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