A role for P-selectin and complement in the pathological sequelae of germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. METHODS: We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal C57BL/6 J mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. RESULTS: Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. In addition, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. CONCLUSIONS: GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.

Complement Drives Chronic Inflammation and Progressive Hydrocephalus in Murine Neonatal Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is a pathology that occurs in infancy, with often devastating long-term consequences. Posthemorrhagic hydrocephalus (PHH) can develop acutely, while periventricular leukomalacia (PVL) is a chronic sequala. There are no pharmacological therapies to treat PHH and PVL. We investigated different aspects of the complement pathway in acute and chronic outcomes after murine neonatal GMH induced at postnatal day 4 (P4). Following GMH-induction, the cytolytic complement membrane attack complex (MAC) colocalized with infiltrating red blood cells (RBCs) acutely but not in animals treated with the complement inhibitor CR2-Crry. Acute MAC deposition on RBCs was associated with heme oxygenase-1 expression and heme and iron deposition, which was reduced with CR2-Crry treatment. Complement inhibition also reduced hydrocephalus and improved survival. Following GMH, there were structural alterations in specific brain regions linked to motor and cognitive functions, and these changes were ameliorated by CR2-Crry, as measured at various timepoints through P90. Astrocytosis was reduced in CR2-Crry-treated animals at chronic, but not acute, timepoints. At P90, myelin basic protein and LAMP-1 colocalized, indicating chronic ongoing phagocytosis of white matter, which was reduced by CR2-Crry treatment. Data indicate acute MAC-mediated iron-related toxicity and inflammation exacerbated the chronic effects of GMH.

A Role for P-selectin and Complement in the Pathological Sequelae of Germinal Matrix Hemorrhage.

Background Germinal Matrix Hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. Methods We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. Results Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. Also, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. Conclusion GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.