Haoqian Zhang

Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord.

In the injured spinal cord, a glial scar forms and becomes a major obstacle to axonal regeneration. Formation of the glial scar involves migration of astrocytes toward the lesion. Matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, govern cell migration through their ability to degrade constituents of the extracellular matrix. Although MMP-9 is expressed in reactive astrocytes, its involvement in astrocyte migration and formation of a glial scar is unknown. Here we found that spinal cord injured, wild-type mice expressing MMPs developed a more severe glial scar and enhanced expression of chondroitin sulfate proteoglycans, indicative of a more inhibitory environment for axonal regeneration/plasticity, than MMP-9 null mice. To determine whether MMP-9 mediates astrocyte migration, we conducted a scratch wound assay using astrocytes cultured from MMP-9 null, MMP-2 null, and wild-type mice. Gelatin zymography confirmed the expression of MMP-9 and MMP-2 in wild-type cultures. MMP-9 null astrocytes and wild-type astrocytes, treated with an MMP-9 inhibitor, exhibited impaired migration relative to untreated wild-type controls. MMP-9 null astrocytes showed abnormalities in the actin cytoskeletal organization and function but no detectable untoward effects on proliferation, cellular viability, or adhesion. Interestingly, MMP-2 null astrocytes showed increased migration, which could be attenuated in the presence of an MMP-9 inhibitor. Collectively, our studies provide explicit evidence that MMP-9 is integral to the formation of an inhibitory glial scar and cytoskeleton-mediated astrocyte migration. MMP-9 may thus be a promising therapeutic target to reduce glial scarring during wound healing after spinal cord injury.

Role of Matrix Metalloproteinases and Therapeutic Benefits of Their Inhibition in Spinal Cord Injury

SummaryThis review will focus on matrix metalloproteinases (MMPs) and their inhibitors in the context of spinal cord injury (SCI). MMPs have a specific cellular and temporal pattern of expression in the injured spinal cord. Here we consider their diverse functions in the acutely injured cord and during wound healing. Excessive activity of MMPs, and in particular gelatinase B (MMP-9), in the acutely injured cord contributes to disruption of the blood-spinal cord barrier, and the influx of leukocytes into the injured cord, as well as apoptosis. MMP-9 and MMP-2 regulate inflammation and neuropathic pain after peripheral nerve injury and may contribute to SCI-induced pain. Early pharmacologic inhibition of MMPs or the gelatinases (MMP-2 and MMP-9) results in an improvement in long-term neurological recovery and is associated with reduced glial scarring and neuropathic pain. During wound healing, gelatinase A (MMP-2) plays a critical role in limiting the formation of an inhibitory glial scar, and mice that are genetically deficient in this protease showed impaired recovery. Together, these findings illustrate complex, temporally distinct roles of MMPs in SCIs. As early gelatinase activity is detrimental, there is an emerging interest in developing gelatinase-targeted therapeutics that would be specifically tailored to the acute injured spinal cord. Thus, we focus this review on the development of selective gelatinase inhibitors.

Matrix Metalloproteinases and Neurotrauma: Evolving Roles in Injury and Reparative Processes

Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their participation in events that support recovery processes. Here the authors provide a brief overview of MMPs and their regulation, address their complex roles following traumatic injuries to the adult and developing CNS, and consider their time- and context-dependent signatures that influence both injury and reparative processes.

Keratan Sulfate Restricts Neural Plasticity after Spinal Cord Injury

Chondroitin sulfate (CS) proteoglycans are strong inhibitors of structural rearrangement after injuries of the adult CNS. In addition to CS chains, keratan sulfate (KS) chains are also covalently attached to some proteoglycans. CS and KS sometimes share the same core protein, but exist as independent sugar chains. However, the biological significance of KS remains elusive. Here, we addressed the question of whether KS is involved in plasticity after spinal cord injury. Keratanase II (K-II) specifically degraded KS, i.e., not CS, in vivo. This enzyme digestion promoted the recovery of motor and sensory function after spinal cord injury in rats. Consistent with this, axonal regeneration/sprouting was enhanced in K-II-treated rats. K-II and the CS-degrading enzyme chondroitinase ABC exerted comparable effects in vivo and in vitro. However, these two enzymes worked neither additively nor synergistically. These data and further in vitro studies involving artificial proteoglycans (KS/CS-albumin) and heat-denatured or reduced/alkylated proteoglycans suggested that all three components of the proteoglycan moiety, i.e., the core protein, CS chains, and KS chains, were required for the inhibitory activity of proteoglycans. We conclude that KS is essential for, and has an impact comparable to that of CS on, postinjury plasticity. Our study also established that KS and CS are independent requirements for the proteoglycan-mediated inhibition of axonal regeneration/sprouting.

Matrix Metalloproteinase-9 and Stromal Cell-Derived Factor-1 Act Synergistically to Support Migration of Blood-Borne Monocytes into the Injured Spinal Cord

The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28–30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

Brain keratan sulfate and glial scar formation.

Abstract:  In response to injury to the central nervous system (CNS), reactive astrocytes appear and accumulate in the wounded area, leading to glial scar formation. Glial scar is the physical barrier to axonal regeneration of injured neurons. Chondroitin sulfate proteoglycans are inhibitory to axon outgrowth and are upregulated in reactive astrocytes upon CNS injury. It is known that keratan sulfate proteoglycans (KSPGs) are also augmented after CNS injury and act as inhibitory cues. We give a brief overview of CNS injury and cover our recent data regarding the relationship between glial scar formation and KS. KS expression in the developing brain is detectable with 5D4, a KS‐specific monoclonal antibody. These 5D4 immunoreactivities are eliminated in mice deficient in N‐acetylglucosamine 6‐O‐sulfotransferase‐1. In adult mice, brain injury apparently upregulates mRNA expression of N‐acetylglucosamine 6‐O‐sulfotransferase‐1 as well as 5D4‐reactive KS in the wounded area. Intriguingly, the expression of 5D4‐reactive KS and reactive astrocyte accumulation in the wounded area are dramatically diminished in the sulfotransferase‐deficient mice. Consequently, the deficient mice exhibit a marked reduction in scar formation and enhancement of neuronal regeneration after brain injury. Thus, N‐acetylglucosamine 6‐O‐sulfotransferase‐1 plays indispensable roles in brain KS biosynthesis and glial scar formation after brain injury.

Elevated MMP-9 in the lumbar cord early after thoracic spinal cord injury impedes motor relearning in mice.

Spinal cord injury results in distant pathology around putative locomotor networks that may jeopardize the recovery of locomotion. We previously showed that activated microglia and increased cytokine expression extend at least 10 segments below the injury to influence sensory function. Matrix metalloproteinase-9 (MMP-9) is a potent regulator of acute neuroinflammation. Whether MMP-9 is produced remote to the injury or influences locomotor plasticity remains unexamined. Therefore, we characterized the lumbar enlargement after a T9 spinal cord injury in C57BL/6 (wild-type [WT]) and MMP-9-null (knock-out [KO]) mice. Within 24 h, resident microglia displayed an activated phenotype alongside increased expression of progelatinase MMP-3 in WT mice. By 7 d, increases in active MMP-9 around lumbar vasculature and production of proinflammatory TNF-α were evident. Deletion of MMP-9 attenuated remote microglial activation and restored TNF-α expression to homeostatic levels. To determine whether MMP-9 impedes locomotor plasticity, we delivered lumbar-focused treadmill training in WT and KO mice during early (2–9 d) or late (35–42 d) phases of recovery. Robust behavioral improvements were observed by 7 d, when only trained KO mice stepped in the open field. Locomotor improvements were retained for 4 weeks as identified using state of the art mouse kinematics. Neither training nor MMP-9 depletion alone promoted recovery. The same intervention delivered late was ineffective, suggesting that lesion site sparing is insufficient to facilitate activity-based training and recovery. Our work suggests that by attenuating remote mechanisms of inflammation, acute treadmill training can harness endogenous spinal plasticity to promote robust recovery.

Transforming growth factor-β1 upregulates keratan sulfate and chondroitin sulfate biosynthesis in microglias after brain injury

After injury to the adult central nervous system, levels of extracellular matrix molecules increase at the injury site and may inhibit the repair of injured axons. Among these molecules, the importance of proteoglycans, particularly their chondroitin sulfate chains, has been highlighted. We have recently reported that keratan sulfate-deficient mice show better axonal regeneration after injury. Here, we investigated the regulation of keratan sulfate and chondroitin sulfate biosynthesis after neuronal injuries. Several key enzymes required for glycosaminoglycan biosynthesis (beta3GlcNAcT-7 and GlcNAc6ST-1 for keratan sulfate; CS synthase-1 and C6ST-1 for chondroitin sulfate) were expressed at significantly higher levels in the lesion 7 days after a knife-cut injury was made to the cerebral cortex in adult mice. These increases were accompanied by increased expression of TGF-beta(1) and bFGF. Since microglias at the injury sites expressed both keratan sulfate and chondroitin sulfate, the effects of these cytokines were examined in microglias. TGF-beta(1) induced the expression of the above-named enzymes in microglias, and consequently induced keratan sulfate and chondroitin sulfate biosynthesis as well as the expression of the chondroitin/keratan sulfate proteoglycan aggrecan in these cells. TGF-beta(1) also induced bFGF expression in microglias. bFGF in turn induced TGF-beta(1) expression in astrocytes. Astrocyte-conditioned medium following bFGF stimulation indeed induced keratan sulfate and chondroitin sulfate production in microglias. This production was blocked by TGF-beta(1)-neutralizing antibody. Taken together, our data indicate that the biosyntheses of keratan sulfate and chondroitin sulfate are upregulated in common by TGF-beta(1) in microglias after neuronal injuries.

Deficiency in matrix metalloproteinase-2 results in long-term vascular instability and regression in the injured mouse spinal cord

Angiogenesis plays a critical role in wound healing after spinal cord injury. Therefore, understanding the events that regulate angiogenesis has considerable relevance from a therapeutic standpoint. We evaluated the contribution of matrix metalloproteinase (MMP)-2 to angiogenesis and vascular stability in spinal cord injured MMP-2 knockout and wildtype (WT) littermates. While MMP-2 deficiency resulted in reduced endothelial cell division within the lesioned epicenter, there were no genotypic differences in vascularity (vascular density, vascular area, and endothelial cell number) over the first two weeks post-injury. However, by 21days post-injury MMP-2 deficiency resulted in a sharp decline in vascularity, indicative of vascular regression. Complementary in vitro studies of brain capillary endothelial cells confirmed MMP-2 dependent proliferation and tube formation. As deficiency in MMP-2 led to prolonged MMP-9 expression in the injured spinal cord, we examined both short-term and long-term exposure to MMP-9 in vitro. While MMP-9 supported endothelial tube formation and proliferation, prolonged exposure resulted in loss of tubes, findings consistent with vascular regression. Vascular instability is frequently associated with pericyte dissociation and precedes vascular regression. Quantification of PDGFrβ+ pericyte coverage of mature vessels within the glial scar (the reactive gliosis zone), a known source of MMP-9, revealed reduced coverage in MMP-2 deficient animals. These findings suggest that acting in the absence of MMP-2, MMP-9 transiently supports angiogenesis during the early phase of wound healing while its prolonged expression leads to vascular instability and regression. These findings should be considered while developing therapeutic interventions that block MMPs.

Neutrophils as Determinants of Vascular Stability in the Injured Spinal Cord

While a number of studies have examined the complex roles of leukocytes in the acute and chronically injured cord, few have specifically focused on neutrophils, where we have only recently begun to appreciate their involvement in both vascular pathogenesis and early wound healing. Here we address the mechanisms underlying neutrophil-mediated endothelial destabilization, their synergism with monocytes in modulating permeability, and their putative role as initiators of angiogenesis in the acutely injured spinal cord. Neutrophils contain a variety of bioactive molecules that are stored in granules. Studies have shown that certain of these molecules, and most notably proteases, contribute to endothelial destabilization as neutrophils degranulate during their transmigration across this front. Neutrophils have historically been regarded as detrimental to the acutely injured cord. However, there is growing evidence that this may be an oversimplified view as it fails to take into account their ability to release proteases that degrade the extracellular matrix, releasing latent growth factors that may in turn support early angiogenesis.