Glycocalyx is critical for blood‐brain barrier integrity by suppressing caveolin1‐dependent endothelial transcytosis following ischemic stroke

Abstract

The breakdown of the blood-brain barrier (BBB) is related to the occurrence and deterioration of neurological dysfunction in ischemic stroke, which leads to the extravasation of blood-borne substances, resulting in vasogenic edema and increased mortality. However, a limited understanding of the molecular mechanisms that control the restrictive properties of the BBB hinders the manipulation of the BBB in disease and treatment. Here, we found that the glycocalyx (GCX) is a critical factor in the regulation of brain endothelial barrier integrity.First, endothelial GCX displayed a biphasic change pattern, of which the timescale matched well with the biphasic evolution of BBB permeability to tracers within the first week after t-MCAO. Moreover, GCX destruction with hyaluronidase increased BBB permeability in healthy mice and aggravated BBB leakage in transient middle cerebral artery occlusion (t-MCAO) mice. Surprisingly, ultrastructural observation showed that GCX destruction was accompanied by increased endothelial transcytosis at the ischemic BBB, while the tight junctions remained morphologically and functionally intact. Knockdown of ca-veolin1 (Cav1) suppressed endothelial transcytosis, leading to reduced BBB permeability, and brain edema. Lastly, a coimmunoprecipitation assay showed that GCX degradation enhanced the interaction between syndecan1 and Src by promoting the binding of phosphorylated syndecan1 to the Src SH2 domain, which led to rapid modulation of cytoskeletal proteins to promote caveolaemediated endocytosis. Overall, these findings demonstrate that the dynamic degradation and reconstruction of GCX may account for the biphasic changes in BBB permeability in ischemic stroke, and reveal an essential role of GCX in suppressing transcellular transport in brain endothelial cells to maintain BBB integrity. Targeting GCX may provide a novel strategy for managing BBB dysfunction and central nervous system drug delivery. | M AT ER I A L S A N D M ET HOD S | AnimalsMale C57BL/6J background mice were obtained from the Experimental Animal Center of Southern Medical University and housed under a 12-h light/dark cycle in a pathogen-free facility with free access to water and blood-brain barrier, brain edema, caveolin1, glycocalyx, ischemia stroke, transcytosis

Two-photon microscopy in vivo reveals brain vessel type-specific loss of glycocalyx caused by apoM/S1P signaling impairment

“…Increased shedding of gcx accompanies aging [55,56] and pathologies affecting vascular function, e.g., stroke [10,57], sepsis [58,59] and diabetes [60]. These conditions have a common denominator, impaired S1P signaling [61][62][63][64][65] and all feature an increase in AMT [8,9,[11][12][13]66].…”

“…In vitro assays show that upon shedding, the remaining cytosolic fragment of syndecan-1 promotes Src-mediated phosphorylation of caveolin-1, and endocytosis [68-70]. Extrapolating to in vivo , this mechanism has been proposed to drive the fluctuations in transcytosis after stroke, where AMT increases simultaneously with gcx shedding and decreases synchronously with the restoration of syndecan-1 [10]. However, our results do not support syndecan-1 involvement in the recovery to physiological levels of AMT in Apom -/- mice.…”

“…In disease states, AMT is no longer suppressed, causing a progressive build-up of toxic levels of albumin and other plasma constituents in the brain parenchyma before evident changes in the BBB structure. This has been evidenced in aging [8], stroke [9, 10] and may precede and facilitate neurodegeneration in chronic brain disorders [11-13].…”

Vascularizing the brain in vitro

“…It is composed of proteoglycan polymers and glycosaminoglycan chains, including heparan sulfate, chondroitin sulfate, and hyaluronan ( Jin et al., 2021 ). Recent studies proved the importance of the presence of this substrate since it has a vital role in preventing coagulation and regulating the BBB permeability and the occurrence and development of inflammation ( Ando et al., 2018 ; Santa-Maria et al., 2019 ; Zhu et al., 2018 , 2021a ).…”

Data_Sheet_1.docx

“…The destruction of the blood-brain barrier is related to the occurrence and deterioration of neurological dysfunction in ischemic stroke; it causes edema in the brain, despite the fact that the tight junction formed is normal, and has a negative effect on mortality. GCX plays an essential role in brain endothelial cell transport system and central nervous system in maintaining the integrity of the blood-brain barrier (18). Regarding the pathogenesis of obesity paradox, to gain a deeper understanding of the mechanism of obesity paradox, it may be useful to measure the degree of GCX destruction and the thickness of GCX in the brain.…”