Bindarit

The integrity of the endothelial monolayer is critical for preventing life-threatening hemorrhaging and thrombosis. However, how severe endothelium-denuded injury is rapidly repaired remains unknown. Given the common biological properties between endothelial cells and circulating monocytes, we aimed to examine whether blood monocytes are involved in endothelium wound healing. The in vivo common carotid artery endothelium-denuded (CCAED) model was established through a wire-induced injury. Monocyte adhesion was assessed using immunofluorescence and a parallel plate flow chamber. We initially observed that the circulating monocyte-mediated endothelialization was better downstream of kinase 3 deficient mice (DOK3^-/-) than that of wild-type (WT) mice following induction of the CCAED model. Rapid endothelialization increased endothelial integrity, prevented coagulation, and decreased thrombosis. Mechanistically, following endothelium-denuded injury, monocyte chemoattractant protein 1 (MCP1) disassociated from DOK3 and C-C chemokine receptor type 2B (CCR2B), increased the intracellular Ca²⁺ concentration, and promoted adhesion in circulating monocytes. However, this process was inhibited by the CCR2B inhibitor INCB3344. Moreover, the adhesive functions of circulating monocytes isolated from DOK3^-/- mice were stronger than those from WT mice. Furthermore, adhered monocytes expressed endothelial-specific markers and compensated for endothelium-dependent vasorelaxation in WT mice. Similarly, these effects were enhanced in DOK3^-/- mice. Bindarit, a selective MCP1 inhibitor, suppressed endothelialization following CCAED surgery in WT mice but not in DOK3^-/- mice. In conclusion, endothelialization mediated by circulating monocytes repairs endothelium-denuded injury to compensate for endothelial functions through MCP1/DOK3/CCR2B/Ca²⁺ signaling. Our findings indicate that circulating monocyte adhesion is an important endothelial wound healing mechanism.

The poor prognosis of subarachnoid hemorrhage (SAH) is closely linked to neuroinflammation and neuronal apoptosis. The CCL2/CCR2 signaling axis, a cytoplasmic pathway responsible for recruiting immune cells, plays a significant role in regulating neuroinflammation in neurological diseases. Bindarit, an inhibitor of chemokine CC motif ligand 2(CCL2), has been shown to demonstrate neuroprotective effects in various central nervous system diseases. This study aimed to investigate the anti-inflammatory effects of Bindarit after SAH.

Pre-processed RNA-seq transcriptome datasets GSE79416 from the Gene Expression Omnibus (GEO) database were analyzed to identify genes differentially expressed between mice with SAH and control mice using bioinformatics methods. Bindarit, a CCL2 inhibitor, was administered intraperitoneally one hour after SAH. Recombinant CCL2 protein was administered via the lateral ventricle one hour before SAH induction. HT22 cells were cultured and stimulated by oxyhemoglobin to establish an in vitro model of SAH.

Analysis of GSE79416 datasets revealed upregulation of CCL2 expression, identifying it as a hub gene in SAH. Following SAH, CCL2 expression increased in rat brain tissue, reaching the highest level 24 h after SAH. Bindarit improved the short-term and long-term neurological deficits after SAH and also exhibited the anti-inflammatory effects following SAH. Conversely, administration of recombinant CCL2 protein attenuated the protective effects of Bindarit. In vitro, Bindarit significantly reduced neuronal inflammation.

Endogenous CCL2 expression was upregulated in the rat SAH model. Bindarit improved neurological functions and reduced neuroinflammation by regulating the CCL2/CCR2/NF-κB pathway in early brain injury after SAH.