Figure 3: ECs Mechanotransduction. The shear stress on the ECs is sensed on the luminal surface by different receptors and various ion channels that activate downstream effects.[32]
1.3.3 Shear stress models
In order to study the effect of shear stress on ECs different in vivo, ex vivo and in vitro models have been developed. In vivo mouse models can be used for their naturally flow disturbed regions such as curved and branched arterial regions, however this will give at best “comparative results” between different arterial regions but will not “provide causative insight” onto the relation between shear stress and the expression of certain genes [69]. In consequence, surgical models have been used to investigate shear stress effects on the ECs, like arteriovenous fistulas, constrictive perivascular cuffs and partial ligation of arteries.
Arteriovenous fistulas originally created for dialysis patients were the first interventions to cause acute changes in shear stress [70]. The blood flow is increased in the artery from where the fistula is created. Here, it was shown that the artery dilates (increased lumen) in such a manner that the final experienced shear stress was not significantly changed compared to the original shear stress
[71]. The mouse aorto-‐caval fistula has been used as a model for human arteriovenous fistula [72].
The constructive perivascular cuff model is used in hyperlipidemic animals to accelerate atherogenesis. (Figure 4) Cheng et al. showed with a flow-‐modifying cuff around the carotid artery of Apolipoprotein E-‐deficient mice (ApoE-‐/-‐ mice) that local changes in hemodynamic conditions initiate atherosclerosis.
Interestingly, they also described that plaque vulnerability was associated with low unidirectional (laminar) shear stress rather than with oscillatory shear stress. Indeed, the regions of low laminar shear stress displayed an atherosclerotic lesion with large lipid core, many macrophages, low collagen content and few VSMCs [73, 74].
Figure 4: Constrictive perivascular cuff model [75]. A: The conical shaped cast creates three regions of shear stress: a low laminar shear stress (LLSS) upstream of the cast, a region of increasingly high laminar shear stress (HLSS) inside the cast, and a region with oscillatory shear stress (OSS) downstream of the cast. B: Wall shear stress (WSS, left) and oscillatory shear index (OSI, right) determined by micro computer tomography (μCT) in carotid artery after 9weeks of cast placement.
Several partial carotid artery ligation models exist to elicit different degrees of flow alterations and arterial remodeling in mice. Ligation of three of the four caudal branches of the left carotid artery (the internal carotid, occipital and the external carotid) after the branching of the superior thyroid artery induced significantly reduced flow but also flow reversal patterns during diastole characteristic for areas of disturbed flow [76]. (Figure 5)
Figure 5: Schematic representation of partial ligation of the left common carotid artery (LCA). Three branches of the LCA (external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA) are ligated leaving the superior thyroid artery (STA) open. Adapted from [76] .
Ligation was shown to reduce flow in the surgically ligated artery and was resulting in shear stress-‐dependent vascular remodeling [77-‐79]. Atheroma develops here in the untouched left common carotid artery (LCA) that is not manipulated during the procedure and can be compared with the right common carotid artery (RCA). Furthermore, surgery did not affect shear rate in the right common carotid artery [80, 81]. A recent study showed that partial carotid ligation in combination with adeno-‐associated-‐virus-‐8 (AAV8)-‐mediated overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) (AAV8-‐
PCSK9) induced within 3 weeks hyperlipidemia and atherosclerosis [82].
Inhibitors of PCSK9 are a promising new class of cholesterol lowering drug
because of their interference with cholesterol metabolism by the means of LDL receptor recycling in hepatocytes [83, 84]. PCSK9 is involved in the degradation of the low density lipoprotein receptor (LDLR) and is found primarily in the liver, intestine, and kidney [85]. Evidence shows that PCSK9 binds to the LDLR and redirect the LDLR to the lysosome. Decreasing the available LDLRs on the cell surface and thus resulting in increase LDL in the serum. Indeed, clinical studies using monoclonal antibodies (alirocumab and evolocumab) that inhibit PCSK9 showed a reduction of approximately 50% in blood plasma LDL cholesterol levels [86-‐88].
In transgenic mice the overexpression of the PCSK9 protein leads to hypercholesterolemia and atherosclerosis [89-‐91]. Instead of using transgenic animals, Bjorklund et al. developed a gain of function mutant of PCSK9 in a recombinant AAV8. One injection of AAV8-‐PCSK9 into wild type C57BL6 mice resulted in significant hypercholesterolemia and atherosclerotic plaque formation within 3 months. Making it a good alternative for germline knockout ApoE or LDLR mice models [92]. Finally, ligation of the left external carotid artery branch in another model was shown to reduce significantly the arterial flow through the left common carotid artery and resulted in flow-‐mediated reduction of the lumen diameter and medial wall mass followed by decreased VSMC proliferation and elastin content compared with the right common carotid
artery [79, 93, 94].
Tabel 1: Advantages and disadvantages of in vivo shear modifying models in EC physiology
relationship between shear stress and oxidative stress used ex vivo porcine carotid arteries exposed to LLSS and OSS. Here, they showed that these explants reduced nitric oxide synthase 3 (eNOS) expression in low and oscillatory shear stress regions [103]. These ex vivo shear models have in time been refined to study the effects of other mechanical forces in the vascular environment in addition to shear stress. Here, parallel to the shear forces circumferential cyclic stretch can be controlled in addition to flow dynamics [104, 105]. The reduction of arterial compliance was shown to increase the risk of arterial disease through the interruption of the eNOS activation pathway and increasing vascular levels of oxidative stress [105]. Together, this ex vivo model makes it possible to dissect complex interactions of mechanical stresses in the vascular environment between shear and cyclic stretch [105].
The pressure myograph can be used to measure physiological functions and properties of small arteries, veins and other vessels with a maximal diameter of 6mm [106]. Here, a small segment of a vessel is mounted onto small glass cannula where they can be pressurized to a specific transmural pressure [107, 108]. In contrast to wire myograph where the constriction and dilation of the vessel is measured through a force transducer in high sensitivity isometric conditions, the pressure myograph uses a digital video edge-‐detection under isobaric conditions [107, 109, 110]. Therefore, the natural vessel diameter can be studied at a wide range of shear stresses and pressures applied to the lumen of the vessel [111]. The pressure myograph is primarily used for small vessels that have substantial vasoreactivity [112].
Tabel 2: Advantages and disadvantages of ex vivo models in EC physiology
cone above a stationary place containing ECs cultured on cover slips. This device was subsequently modified by other groups to integrate an optical system, which allowed the direct observation of EC in response to shear stress [118]. Next, Blackman et al. developed a shearing device based on the cone-‐and-‐plate using a micro-‐stepper motor technology to independently control the dynamics and steady components of the shear stress environment. Furthermore, this system was also fitted with a fluorescence microscope [119]. Finally, Tarbell and colleagues introduced the parallel disk viscometer [120]. (Figure 6C) Here, following the model of the cone-‐and-‐plate device the cone was replaced by a disk that was linked to a drive motor to produce a defined shear stress on the ECs and used to assess the effect of shear stress [121, 122].
Figure 6: Shear stress devices: A) parallel-‐plate flow chamber; B) cone-‐and-‐plate viscometer; C) parallel disk viscometer; D) orbital shaker; E) capillary tube. Adapted from [123]
Parallel-‐plate flow chamber systems have been used to analyze changes in the EC metabolism and morphology in response to shear stress [124-‐126]. Originally, Frangos, McIntire, and colleagues developed a flow chamber consisting of a polycarbonate plate, a rectangular silastic gasket and a glass slide with the EC monolayer [127, 128]. (Figure 7) The different parts of the device were held together by a vacuum at the periphery of the slide, forming a channel. At the time
flow was applied to the channel by a hydrostatic pressure head between the two media reservoirs to produce steady flow or via cam-‐driven clamps upstream of the chamber to achieve pulsatile flow.
Figure 7: The parallel plate flow chamber. Cover slips were covered with confluent ECs. A silastic gasket was applied to separate the cover slip from the deck of the flow chamber. Vacuum was applied to hold the device together. Adapted from [129]
Several modified designs have been used to date. Firstly, to assess the EC monolayer permeability the flow chamber was attached to a circulating luminal loop and basal non-‐circulating loop [130]. Next, using a flow chamber with at the center a series of arrow shaped channels allowed for variable shear stresses within the same flow chamber. Thus, by changing the geometry of the center channels changes in shear stress were introduced without altering the gap width or overall flow rate [131]. With this device the effect of shear rates on platelet adhesion onto immobilized fibrinogen and von Willebrand factor (vWF) matrices was studied [132]. The sudden-‐expansion flow chamber and the backward-‐
facing step flow chamber were designed to mimic the spatial and temporal gradients in shear stress that overlap in atherosclerosis prone regions [133, 134]. The sudden-‐expansion flow chamber leads to a flow separation due to the asymmetric expansion of the flow path. Here, the fluid flows from a narrow channel directly to a wider channel. At the location of the step the flow recirculates with the direction against the main flow to finally reattach to the
main unidirectional parabolic flow [134]. Finally, to study the effect of upstroke slopes of pulsatile flow (shear stress slew rates) the inlet and outlet of a parallel plate flow device was connected to symmetrical contractions and diffusers. Here, through precisely monitoring and controlling the frequency, amplitude and time-‐
average shear stress of pulsatile flow allowed the independent study of slew