CD47 knockout mice exhibit improved recovery from spinal cord injury.

Recent data have implicated thrombospondin-1 (TSP-1) signaling in the acute neuropathological events that occur in microvascular endothelial cells (ECs) following spinal cord injury (SCI) (Benton et al., 2008b). We hypothesized that deletion of TSP-1 or its receptor CD47 would reduce these pathological events following SCI. CD47 is expressed in a variety of tissues, including vascular ECs and neutrophils. CD47 binds to TSP-1 and inhibits angiogenesis. CD47 also binds to the signal regulatory protein (SIRP)α and facilitates neutrophil diapedesis across ECs to sites of injury. After contusive SCI, TSP-1(-/-) mice did not show functional improvement compared to wildtype (WT) mice. CD47(-/-) mice, however, exhibited functional locomotor improvements and greater white matter sparing. Whereas targeted deletion of either CD47 or TSP-1 improved acute epicenter vascularity in contused mice, only CD47 deletion reduced neutrophil diapedesis and increased microvascular perfusion. An ex vivo model of the CNS microvasculature revealed that CD47(-/-)-derived microvessels (MVs) prominently exhibit adherent WT or CD47(-/-) neutrophils on the endothelial lumen, whereas WT-derived MVs do not. This implicates a defect in diapedesis mediated by the loss of CD47 expression on ECs. In vitro transmigration assays confirmed the role of SIRPα in neutrophil diapedesis through EC monolayers. We conclude that CD47 deletion modestly, but significantly, improves functional recovery from SCI via an increase in vascular patency and a reduction of SIRPα-mediated neutrophil diapedesis, rather than the abrogation of TSP-1-mediated anti-angiogenic signaling.

Sildenafil improves epicenter vascular perfusion but not hindlimb functional recovery after contusive spinal cord injury in mice.

Nitric oxide (NO) is an important regulator of vasodilation and angiogenesis in the central nervous system (CNS). Signaling initiated by the membrane receptor CD47 antagonizes vasodilation and angiogenesis by inhibiting synthesis of cyclic guanosine monophosphate (cGMP). We recently found that deletion of CD47 led to significant functional locomotor improvements, enhanced angiogenesis, and increased epicenter microvascular perfusion in mice after moderate contusive spinal cord injury (SCI). We tested the hypothesis that improving NO/cGMP signaling within the spinal cord immediately after injury would increase microvascular perfusion, angiogenesis, and functional recovery, with an acute, 7-day administration of the cGMP phosphodiesterase 5 (PDE5) inhibitor sildenafil. PDE5 expression is localized within spinal cord microvascular endothelial cells and smooth muscle cells. While PDE5 antagonism has been shown to increase angiogenesis in a rat embolic stroke model, sildenafil had no significant effect on angiogenesis at 7 days post-injury after murine contusive SCI. Sildenafil treatment increased cGMP concentrations within the spinal cord and improved epicenter microvascular perfusion. Basso Mouse Scale (BMS) and Treadscan analyses revealed that sildenafil treatment had no functional consequence on hindlimb locomotor recovery. These data support the hypothesis that acutely improving microvascular perfusion within the injury epicenter by itself is an insufficient strategy for improving functional deficits following contusive SCI.

Deletion of endoplasmic reticulum stress-induced CHOP protects microvasculature post-spinal cord injury.

Trauma introduces damaging stressors that compromise protein, lipid, and nucleic acid integrity. Aggregates of unfolded and misfolded proteins in the endoplasmic reticulum (ER) triggers the ER stress response (ERSR)/unfolded protein response (UPR) leading to activation of three signaling pathways mediated by PERK, ATF6, and IRE1. Initially, the ERSR/UPR is pro-homeostatic as it globally slows translation while increasing translation of chaperone proteins and inducing ER-associated degradation. If the cellular stress is not controlled, apoptosis is subsequently induced through several mechanisms, of which the most well-described is CHOP. Following spinal cord injury (SCI), mice deficient in CHOP signaling show increased spared white matter and enhanced locomotor recovery by 6 weeks. At 24 hours after SCI, ATF4 and CHOP are upregulated in under perfused microvessels. We observed vascular protection 3 days post-SCI and a significant decrease in macrophage infiltration by the end of the first week. These results suggest that modulating ER-stress signaling in endothelial cells and macrophages may protect against vascular injury and attenuate inflammation post-SCI.

Neutralizing endogenous VEGF following traumatic spinal cord injury modulates microvascular plasticity but not tissue sparing or functional recovery.

Acute loss of spinal cord vascularity followed by an endogenous adaptive angiogenic response with concomitant microvascular dysfunction is a hallmark of traumatic spinal cord injury (SCI). Recently, the potent vasoactive factor vascular endothelial growth factor (VEGF) has received much attention as a putative therapeutic for the treatment of various neurodegenerative disorders, including SCI. Exogenous VEGF exerts both protective and destabilizing effects on microvascular elements and tissue following SCI but the role of endogenous VEGF is unclear. In the present study, we systemically applied a potent and well characterized soluble VEGF antagonist to adult C57Bl/6 mice post-SCI to elucidate the relative contribution of VEGF on the acute evolving microvascular response and its impact on functional recovery. While the VEGF Trap did not alter vascular density in the injury epicenter or penumbra, an overall increase in the number of Griffonia simplicifolia isolectin-B4 bound microvessels was observed, suggesting a VEGF-dependency to more subtle aspects of endothelial plasticity post-SCI. Neutralizing endogenous VEGF neither attenuated nor exacerbated chronic histopathology or functional recovery. These results support the idea that overall, endogenous VEGF is not neuroprotective or detrimental following traumatic SCI. Furthermore, they suggest that angiogenesis in traumatically injured spinal tissue is regulated by multiple effectors and is not limited by endogenous VEGF activation of affected spinal microvessels.