PF4+ Monocyte-derived macrophages protect the blood brain barrier after stroke - Current stroke therapies aim to restore blood flow and protect the post-mitotic cells in the brain, but few existing approaches influence the long-lasting immune responses that are responsible for tissue repair. In homeostasis, the brain parenchyma is protected by the blood-brain barrier (BBB), which prevents the passage of large molecules, pathogens, and leukocytes. Cellular distress unleashed by ischemia leads to BBB breakdown, which exacerbates neuroinflammation and brain injury. Angiogenesis is an important regenerative process after stroke. However, quick generation of new vessels without mature barrier function necessitates further BBB leakage, so the timing of angiogenesis must be controlled to avoid vascular leakage and runaway inflammation. Monocytes infiltrate the brain parenchyma early after stroke, which can cause harmful edema and worsen outcomes in the short term. However, the recruitment of blood monocytes is also essential for stroke recovery, as monocyte-derived macrophages (MDM) prevent hemorrhagic transformation and regulate angiogenesis and wound repair in mouse models of cerebrovascular injury. Blocking MDM recruitment has been reported to exacerbate behavioral deficits in stroke models. Using scRNAseq, we identified a population of C-C chemokine receptor type 2 negative (CCR2-) MDM present in the mouse brain 3 days after stroke. These cells express mRNA transcripts consistent with an anti-angiogenic, immuno-suppressive phenotype. Platelet factor 4 (PF4) is specifically expressed in this population. Infiltrating MDM express PF4Cre upon brain infiltration, allowing us to visualize PF4+ cells by their expression of tdTomato (PF4-tdT). We used the PF4Cre mouse to specifically deplete PF4+ MDM. I will use this model to characterize the phenotype and function of these cells after transient middle cerebral artery occlusion (tMCAO). I hypothesize that PF4+CCR2- MDM preserve BBB integrity in the early stages of stroke recovery by limiting angiogenesis and inflammation, and that specific cues from the CNS trigger monocyte differentiation into this state. Aim 1: Confirm that PF4+CCR2- MDM produce antiangiogenic and immunosuppressive proteins that inhibit early angiogenesis after stroke. I will prove that selective depletion of PF4+ MDM dysregulates angiogenesis after stroke by comparing endothelial cell proliferation and by observing vessel growth in vivo using longitudinal two- photon microscopy. Aim 2: Determine if PF4+ MDM promote BBB integrity after stroke. PF4+ MDM depletion and adoptive cell transfer will be applied to interrogate their function in maintaining BBB integrity. I will observe BBB permeability over the first week of tMCAO recovery in vivo using two-photon microscopy.
