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Microglial P2RY12 mediates migration to and protection of cerebral microvasculature after ischemia–reperfusion via Caveolin-1
Introduction
Stroke, a leading global cause of both mortality and neurological disability, is predominantly characterized by the ischemic subtype, which accounts for approximately 87% of cases (Martin et al., 2025). The associated disease burden continues to escalate (Radak et al., 2017; Rathore et al., 2002). Disruption of the structural integrity and function of the blood-brain barrier (BBB) constitutes a pivotal event in the pathological progression of ischemic brain injury (del Zoppo et al., 2007). While vascular recanalization remains the cornerstone of clinical intervention, reperfusion-induced BBB damage contributes to vascular leakage and secondary brain injury, thus posing a significant limitation to patient prognosis (Krueger et al., 2015). Furthermore, the BBB directly regulates the trans-barrier delivery efficiency of neuroprotective agents (Wang et al., 2024; Yang et al., 2024). Consequently, maintaining BBB integrity carries substantial therapeutic implications, both for preventing further injury and for optimizing the efficacy of neuroprotective treatments.
Microglia, the resident immune cells of the brain, established connections with the vascular wall via morphological extensions, contributing to vascular homeostasis under physiological conditions (Bisht et al., 2021). Together with neurons and blood vessels, microglia constitute the neurovascular unit and play a critical role in maintaining BBB integrity (Thion et al., 2018; Prinz et al., 2019; Halder and Milner, 2020; Halder and Milner, 2019). P2RY12, a G protein-coupled purinergic receptor first identified in platelets, is a widely studied clinical target in cardiovascular and cerebrovascular diseases (Hollopeter et al., 2001). Its function is cell-type dependent: in platelets, it promotes thrombosis via Gi/o signaling, whereas in microglia, it mediates chemotactic migration (Davalos et al., 2005). By specifically recognizing purinergic signaling molecules (e.g., ATP/ADP) released at injury sites (Davalos et al., 2005). P2RY12-mediated chemotaxis of microglial processes is required for the rapid closure of the BBB, and it protects neurons from damage by forming microglia-neuron connections and plays a significant role in cerebral blood flow regulation (Cserép et al., 2020; Lou et al., 2016). Our preliminary investigations utilizing single-cell scRNA-seq datasets, the R package Cell Chat, and confirmatory experiments suggest that P2RY12 may function as a key trigger molecule through which microglia influence the cerebral microvasculature in ischemic brain injury (Wang et al., 2023). Nevertheless, its precise role and underlying mechanisms remain unclear.
As a key member of the caveolin protein family, Cav-1 functions as both a pivotal architectural protein for the formation of caveolae structures in the cell membrane and as an essential scaffold protein that maintains the structural integrity of specific functional domains within the cell membrane. It plays a crucial role in regulating cell membrane curvature and lipid raft assembly while integrating multiple transmembrane signaling pathways (Liu et al., 2002). In tumor biology, Cav-1 modulates cellular migration and invasion through molecular interactions, signaling pathways, and structural changes in cells (Singh et al., 2024; Du et al., 2009; Martínez et al., 2019; Chanvorachote et al., 2014; Lin et al., 2005; Li et al., 2018; Xiong et al., 2017; Sun et al., 2009). Research has demonstrated that the purinergic receptor P2Y family and Cav-1 regulate the structural and functional coupling between nucleotide receptors on the cell membrane and their downstream effectors, thereby affecting the efficiency of extracellular nucleotide signal transduction (Martinez et al., 2016). Notably, the lipid raft microdomain, serving as the functional platform for caveolae, the ability of Gαi (downstream of the P2Y12 receptor) to potentiate ADP-mediated platelet aggregation is highly dependent upon its localization to lipid rafts (Liu et al., 2022; Quinton et al., 2005). Furthermore, Cav-1 knockdown has been shown to reduce overall microglial migration, suggesting its potential involvement in microglial dynamics relevant to injury responses (Niesman et al., 2013). However, the specific role of Cav-1 in regulating P2RY12-mediated microglial function, particularly its chemotactic migration towards injured vasculature and microvascular protection within the central nervous system (CNS) during ischemic injury, remains unexplored.
This study employs an integrated approach to explore the regulatory mechanisms of the P2RY12/Cav-1 signaling axis in ischemic brain injury. Initially, the functional role of P2RY12 will be investigated through pharmacological inhibition with the antagonist PSB0739. This approach will assess the impact of P2RY12 blockade on cerebral I/R injury outcomes, evaluating its effects on microvascular integrity. Complementary in vitro studies using Bv2 microglial cells will further define the contribution of P2RY12 to microglial migration. Subsequently, both in vivo and in vitro models will be used to elucidate the mechanisms governing microglial chemotactic migration, employing the P2RY12 agonist 2MeSADP and the lipid raft disruptor methyl-β-cyclodextrin (MβCD).Finally, the study will experimentally determine the interaction between Cav-1 and P2RY12.
As a purinergic receptor uniquely expressed in microglia, the signaling network mediated by P2RY12 and its molecular regulatory mechanisms remain incompletely elucidated. Cav-1 may act as a critical molecular hub, potentially involved in P2RY12-induced microglial chemotactic migration. This research elucidates the molecular mechanisms by which microglial P2RY12 regulates the microcirculatory system during stroke pathology. The findings not only expand the theoretical framework of ischemic brain injury but also provide experimental evidence for developing potential therapeutic strategies focused on neurovascular unit protection.
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