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.
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
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.
Electrical impedance techniques have been used to characterize endothelium morphology, permeability, and motility in vitro. However, these impedance platforms have been limited to either static endothelium studies and/or induced laminar fluid flow at a constant, single shear stress value. In this work, we present a microfabricated impedance sensor for real-time, in vitro characterization of human umbilical vein endothelial cells (HUVECs) undergoing oscillatory hydrodynamic shear. Oscillatory shear was applied with an orbital shaker and the electrical impedance was measured by a microfabricated impedance chip with discrete electrodes positioned at radial locations of 0, 2.5, 5.0, 7.5, 10.0, and 12.5 mm from the center of the chip. Depending on their radial position within the circular orbital platform, HUVECs were exposed to shear values ranging between 0.6 and 6.71 dyne/cm(2) (according to numerical simulations) for 22 h. Impedance spectra were fit to an equivalent circuit model and the trans-endothelial resistance and monolayer's capacitance were extracted. Results demonstrated that, compared to measurements acquired before the onset of shear, cells at the center of the platform that experienced low steady shear stress (∼2.2 dyne/cm(2) ) had an average change in trans-endothelial resistance of 6.99 ± 4.06% and 1.78 ± 2.40% change in cell capacitance after 22 hours of shear exposure; cells near the periphery of the well (r = 12.5 mm) experienced transient shears (2.5-6.7 dyne/cm(2) ) and exhibited a greater change in trans-endothelial resistance (24.2 ± 10.8%) and cell capacitance (4.57 ± 5.39%). This study, demonstrates that the orbital shear platform provides a simple system that can capture and quantify the real-time cellular morphology as a result of induced shear stress. The orbital shear platform presented in this work, compared to traditional laminar platforms, subjects cells to more physiologically relevant oscillatory shear as well as exposes the sample to several shear values simultaneously. Biotechnol. Bioeng. 2016;113: 1336-1344. © 2015 Wiley Periodicals, Inc.
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.
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