CD44 mediates shear stress mechanotransduction in an in vitro blood‐brain barrier model through small GTPases RhoA and Rac1

Abstract

Fluid shear stress is an important mediator of vascular permeability, yet the molecular mechanisms underlying the effect of shear on the blood‐brain barrier (BBB) have yet to be clarified in cerebral vasculature despite its importance for brain homeostasis. The goal of this study is to probe components of shear mechanotransduction within the BBB to gain a better understanding of pathologies associated with changes in cerebral perfusion including ischemic stroke. Interrogating the effects of shear stress in vivo is complicated by the complexity of factors in the brain parenchyma and the difficulty associated with modulating blood flow regimes. The in vitro model used in this study is compatible with real‐time measurement of barrier function using a transendothelial electrical resistance as well as immunocytochemistry and dextran permeability assays. These experiments reveal that there is a threshold level of shear stress required for barrier formation and that the composition of the extracellular matrix, specifically the presence of high molecular weight hyaluronan, dictates the flow response. Gene editing to modulate the expression of CD44, a mechanosensitive receptor for hyaluronan, demonstrates that the receptor is required for the endothelial response to shear stress. Manipulation of small GTPase activity reveals CD44 activates Rac1 while inhibiting RhoA activation. Additionally, adducin‐γ localizes to tight junctions in response to shear stress and RhoA inhibition and is required to maintain the barrier. This study identifies specific components of the mechanosensing complex associated with the BBB response to fluid shear stress and, therefore, illuminates potential targets for barrier manipulation in vivo.

Exposure of human cerebral microvascular endothelial cells hCMEC/D3 to laminar shear stress induces vascular protective responses

“…RhoA inhibition was already reported in bovine primary BMEC cultured one day at 10 dyn.cm −2 [ 80 ]. More recently, DeOro et al reported in a pre-print an inhibition of RhoA in hCMEC/D3 cells (4 days at 2.1 dyn.cm −2 ) [ 83 ], consistent with our results at both 5 and 10 dyn.cm. −2 .…”

“…Brown et al showed that ZO-1 and Claudin-5 intensities at the cell membrane were increased after 2.73 dyn.cm −2 exposure for 18 h in a parallel-plate flow dual chamber [ 45 ]. DeOre et al confirmed the increase in ZO-1 expression by immunofluorescence [ 83 ]. They cultivated hCMEC/D3 cells with astrocytes in artificial capillaries with SS from 0.18 to 2.1 dyn.cm −2 for 4 days.…”

ECM-derived biophysical cues mediate interstitial flow-induced sprouting angiogenesis

“…In the absence of interstitial flow, we observe that ECM HA inhibits endothelial sprouting. This result is consistent with previous reports that under in vitro static conditions, addition of HA to collagen ECM inhibits capillary tube formation [37] and enhances vascular barrier function [35] . Indeed, we previously reported that the addition of HA to collagen-based matrices suppresses both vessel sprouting and permeability under static in vitro conditions and in the absence of CXCL12 treatment [21] .…”

“…This outcome is surprising because the CD44 receptor is known to be mechanosensitive. For example, a recent study showed that CD44 strengthens monoculture brain endothelial cell barrier function in the presence of intravascular fluid shear stress in vitro [35] . Moreover, multiple studies have reported that interstitial flow promotes cancer cell invasion via CD44-mediated mechanisms [36] .…”

Classical epithelial-mesenchymal transition (EMT) and alternative cell death process-driven blebbishield metastatic-witch (BMW) pathways to cancer metastasis

“…For example, CD44 regulates adhesion through Rac1 activation. 67,88 The metastatic foci, in general, have enhanced glycolytic capacity. 89 This reflects the underlying cellular signaling context of metastatic cells.…”