Macrophages Mediate The Repair Of Brain Vascular Rupture Through Direct Physical Adhesion And Mechanical Traction

Pathological bleeding in the brain includes hemorrhagic stroke and microbleeds. Hemorrhagic stroke, one of the leading causes of death and adult disability worldwide, is primarily caused by hypertensive atherosclerosis followed by the rupture of cerebral arterioles. Microbleeds result from ruptures in microvessels in the brain. Recently, improvements in imaging techniques have enabled the detection of previously invisible microbleeds. Damage to endothelial integrity is the key reason for cerebrovascular ruptures. Once these cerebrovascular ruptures have occurred, fast repair of the ruptures is clinically necessary. But the cellular mechanisms underlying the repair of cerebrovascular ruptures remain to be elucidated.To investigate the dynamic cellular events that occur during cerebrovascular repair, we developed an injury system in zebrafish. A high-energy multi-photon laser was used to focus on and irradiate brain vascular ECs. This endothelial lesion led to cerebral hemorrhage. By using this technique, a brain blood vessel was ruptured, and a lesion with two endothelial ends was generated. Our zebrafish model recapitulates both cerebral hemorrhage and microbleeds.Macrophages are associated with brain injury, hemorrhage, and several aspects of vascular development and remodeling. Although macrophages play multiple roles in vascular biology and pathology, whether they play a part in the repair of cerebrovascular ruptures, other than their role in phagocytosis, remains unknown. In vivo time-lapse imaging showed the arrival of a macrophage at the lesion site about thirty minutes after laser irradiation. This macrophage extended filopodia/lamellipodia that contacted both endothelial ends, then pulled out protrusions from the endothelial ends and mediated the endothelial ligation. Once the endothelial ends were completely ligated and the lesion repair was accomplished, the macrophage migrated away.To explore whether the migration of macrophages toward cerebrovascular lesions is mediated by extracellular ATP, we injected apyrase and ATP receptor inhibitor. Macrophages failed to arrive at the cerebrovascular lesion after apyrase injection. We also observed a dramatic increase in the cytoplasmic Ca2+ concentration in endothelial ends shortly after laser irradiation. All of these data indicate that extracellular ATP is the chemokine signal that attracts macrophages to the cerebrovascular rupture。In some rare cases, after cerebrovascular rupture the two endothelial ends were adhered by different macrophages. The lesion was not repaired in these cases. Cerebrovascular rupture was successfully repaired only if the two endothelial ends were in direct contact with the same macrophage. These results exclude factor secretion as the major mechanism through which macrophages mediate the repair of cerebrovascular ruptures.Once the macrophage was in direct contact with the endothelial ends, Adhesion protein accumulated at the macrophage-endothelial adhesion sites was observed. These data demonstrate that a stable physical adhesion forms between the macrophage and endothelial ends during the repair of cerebrovascular rupture.To investigate the role of mechanic forces in the repair of cerebrovascular ruptures a myosin II inhibitor was used. After inhibitor was applied in the middle, the macrophages exhibited arrested traction, and the rupture remained unrepaired. The main backbone of filopodia/lamellipodia is microfilaments,. To study the roles of microfilaments during this repair process, Using transgenic line we found an accumulation of F-actin became detectable at the adhesion site once the macrophage had physically adhered. We inhibited microfilament assembly with inhibitor. When a macrophage arrived at the lesion site, it would fail to extend filopodia/lamellipodia. The lesion remained unrepaired. When inhibitor was administered after the establishment of macrophage-endothelial physical adhesions, the macrophage appeared to lose its filopodia/lamellipodia, detach from and finally leave the endothelial ends. Lesion repair consequently failed. These results indicate that the macrophage-mediated repair of cerebrovascular rupture is dependent on microfilaments.To explore the regulatory molecules and signaling pathways involved in macrophage-mediated cerebrovascular repair, macrophages and endothelial end cells at three different lesion stages were photoconverted and sorted. Transcriptional profile analysis revealed several potentially important molecules that exhibited changes in transcriptional levels at different lesion stages. These molecules are involved in tight junctions, leukocyte transendothelial migration, and cell adhesion.Transcriptional profile analyses identified up-regulation of PI3 K and Rac in macrophages upon their arrival at the lesion site. To study the functions of PI3 K in the macrophage-mediated repair of cerebrovascular ruptures, Treatment with PI3 K inhibitor after macrophage-endothelial adhesion the lesion can not being repaired. And the repair could not be accomplished after inhibit endogenous Rac activity.If macrophages completely lost their motility to migrate toward the lesion. The lesion was slowly repaired. It imply that a macrophage-independent mechanism can repair the lesion. We found the indispensable role of microtubules in the endothelial self-extension-mediated, macrophage-independent repair of cerebrovascular ruptures. The macrophage-dependent mechanism of repair was significantly faster and more efficient.Real-time observations physical contact between the endothelial ends and the macrophage blocked the endothelial self-extension process. In the absence of a macrophage, Rac1 activity was reduced at the tip of the endothelial ends during self-extension. When inhibit Rac1 activity in the, endothelial self-extension was accelerated. These data indicate that endothelial self-extension after cerebrovascular rupture requires the repression of Rac1 activity.We used real-time live imaging, transcriptional profiles and functional studies to reveal the dynamic cellular events and regulatory molecules involved in the repair of cerebrovascular ruptures, providing important information that will be useful for the development of translatable post-microbleeding therapies. Thus, this study may be of both scientific and clinical interest.