Regulation Of Neuroinflammation After Ischemic Stroke By Astroglial Endothelin Receptor Type-B Signaling
Date of Award
Doctor of Philosophy (PhD)
Jeffrey L. Spees
A large body of neuroscientific research has focused on reactive gliosis and glial scar formation because these are among the most prominent features of the cellular response to central nervous system (CNS) injury. Despite much progress in our understanding, controversy remains regarding the relative balance between the protective nature of the astroglial scar and its anti-regenerative features. Recent work suggests that astrocytes are heterogeneous in their resting state and in their reactivity. In traumatic injuries such as stroke and spinal cord injury, proliferative reactive astrocytes protect CNS tissue. By contrast, under neuroinflammatory and/or neurodegenerative conditions, neurotoxic astrocyte phenotypes may contribute to disease progression.
Our previously published work showed that endothelin receptor type-B (ETBR) signaling was required for reactive astrocyte proliferation in a distal middle cerebral artery occlusion (dMCAO) model of stroke. In the studies for this dissertation, we utilized stem cells derived from the peri-infarct area after stroke to model astroglial ETBR signaling and identified the array of paracrine factors released in response to endothelin signaling. The secreted factor with gene expression most decreased by astroglial ETBR-knockout was Angiopoietin-2 (Ang-2), a pro-angiogenic paracrine factor that affects stroke recovery. We found that astrocytes expressed significantly more Ang-2 after stroke. Ang-2 expression was specifically regulated by ETBR and downstream NFkB and MEK/ERK signaling. After stroke, we discovered that adult microglia, the sentinel immune cells of the brain, increased their expression of Tie-2, the receptor for Ang-2. To evaluate whether paracrine activity controlled by ETBR functioned in neuroinflammation, we performed astrocyte-specific conditional knockout of ETBR using GFAP-CreERTM-EDRNB-fl/fl mice. Notably, peri-infarct CD11b+ cells, corresponding to microglia/macrophages/monocytes, proliferated significantly less after dMCAO in astrocyte-specific ETBR-knockout mice as compared with controls. Taken together, our results indicated that astroglial ETBR signaling may promote Ang-2 release to regulate the proliferation, survival, and/or the inflammatory status of microglia after stroke.
In addition to Ang-2, we demonstrated that astroglial ETBR regulates the expression of CD109, a TGFb co-receptor and C3 alpha-2-macroglubulin family member. CD109 is the target of multiple proteases such as furin, and retains many of its functions as a released, soluble protein. In reactive astrocytes, increased expression of CD109 mRNA was reported to indicate a “neuroprotective” phenotype. We show that ETBR signaling increased astroglial expression of several forms of CD109. Astroglial CD109 expression increased following stroke, and ETBR-knockout prevented stroke-induced increases in CD109 expression. Consistent with a function for CD109 in altering astroglial neuroinflammatory status, in the presence of TNFα and an ETBR specific agonist, CD109-knockdown blocked ETB¬R-dependent effects on C3d expression. To test the disease modifying potential of soluble (free) CD109, we injected mice with recombinant human CD109 after stroke. At 7 days after dMCAO, CD109 treatment tended to increase the size of cortical infarcts and areas of secondary reactive astrocyte thalamic reactivity. Further research will be required to determine potential differences between the membrane-bound and protease- cleaved forms of CD109 and their effects on neuroinflammation, injury progression and resolution. Improved understanding of paracrine, juxtacrine, and/or autocrine cell signals regulated by astroglial ETBR may provide valuable targets to treat CNS injury and disease.
Number of Pages
McInnis, John, "Regulation Of Neuroinflammation After Ischemic Stroke By Astroglial Endothelin Receptor Type-B Signaling" (2020). Graduate College Dissertations and Theses. 1309.