Occludin degradation makes brain microvascular endothelial cells more vulnerable to reperfusion injury in vitro

Abstract

Intracerebral hemorrhage is the most dangerous complication in tPA thrombolytic therapy for ischemic stroke, which occurs as a consequence of endothelial cell death at the blood–brain barrier (BBB) during thrombolytic reperfusion. We have previously shown that cerebral ischemia‐induced rapid occludin degradation and BBB disruption. Here we demonstrated an important role of occludin degradation in facilitating the evolution of ischemic endothelial cells toward death. Cultured brain microvascular endothelial cells (bEnd.3 cells) were exposed to oxygen‐glucose deprivation (OGD) or incubated with occludin siRNA or occludin AAV to achieve an occludin deficiency or over‐expression status before exposing to reoxygenation (R) or TNF‐α treatment. Cell death was assessed by measuring lactate dehydrogenase release, TUNEL staining, and flow cytometry analysis. Inhibition of OGD‐induced occludin degradation with SB‐3CT or over‐expression of occludin with occludin AAV both significantly attenuated OGD/R‐induced apoptosis and pyroptosis in bEnd.3 cells. Consistently, knockdown of occludin with siRNA potentiated TNF‐α‐induced apoptosis, supporting an important role of occludin integrity in endothelial cell survival. Similar results were observed for pyroptosis, in which occludin knockdown with siRNA led to a significant augmentation of cytokines secretion, inflammasome activation, and pyroptosis occurrence in TNF‐α‐treated bEnd.3 cells. Lastly, up‐regulation of c‐Yes, PI3K/AKT, and ERK concurrently occurred with occludin degradation after OGD/R or TNF‐α treatment, and the level of these proteins were further increased when inhibition of occludin degradation or over‐expression of occludin. These data indicate that occludin degradation inflicted during ischemia makes BBB endothelial cells more vulnerable to reperfusion‐associated stress stimuli.

Circular noncoding RNA circ_0007865, serves as a competing endogenous RNA, targeting the miR-214-3p/FKBP5 axis to regulate oxygen-glucose deprivation-induced injury in brain microvascular endothelial cells

“…Previous documents presented that AIS is responsible for HBMEC death, blood-brain barrier disruption, and neurological damage [35,36]. Moreover, it has been confirmed that the processes of neurogenesis and angiogenesis has a positive correlation with the survival rate in patients with stroke [37].…”

Chinese Herbal Preparation SaiLuoTong Alleviates Brain Ischemia Via Nrf2 Antioxidation Pathway Dependent Cerebral Microvascular Protection

“…Besides, these two proteins also have a protective effect on endothelial cells against hazardous stimuli. For example, knockdown of occludin potentiated cytokines secretion, in ammasome activation, and pyroptosis occurrence in TNF-α-treated bEnd.3 cells [28].…”

The Role of NLRP3 Inflammasome in Cerebrovascular Diseases Pathology and Possible Therapeutic Targets

“…Studies have shown that the NLRP3 inflammasome enhances the permeability of brain microvessel endothelial cells via IL-1b (Yang et al, 2014) and downregulates the expression of tight junction proteins, occludin and zona occludens-1, after ischaemia/reperfusion injury (Figure 4) (Cao et al, 2016;Qu et al, 2019). Occludin degradation renders the blood-brain barrier more vulnerable to reperfusion injury in vitro (Zhang, Li, et al, 2020). A recent study observed that the NLRP3 inflammasome influences the distribution of aquaporin-4 in the infarct area .…”