Hirotaka Haro

Matrix metalloproteinase-7–dependent release of tumor necrosis factor-α in a model of herniated disc resorption

Herniated disc (HD), one of the major causes of low back pain, is often resolved spontaneously without surgical intervention. Resorption is associated with a marked increase in infiltrating macrophages, and the matrix metalloproteinases (MMP) MMP-3 and MMP-7 have been implicated in this phenomenon. We developed a murine organ culture model in which intact intervertebral discs were cocultured with peritoneal macrophages to investigate the role of MMPs in HD resorption. Using macrophages isolated from MMP-null mice, we report that macrophage-produced MMP-7 was required for proteoglycan degradation, loss of wet weight, and macrophage infiltration of cocultured discs. The inability of MMP-7-deficient macrophages to infiltrate discs could not be attributed to a defect in macrophage migration. MMP-7 was required for the release of the cytokine TNF-alpha from peritoneal macrophages. The generation of soluble TNF-alpha was essential for the induction of MMP-3 in disc cocultures, which in turn is required for the generation of a macrophage chemoattractant and subsequent macrophage infiltration. TNF-alpha release from macrophages was necessary but insufficient for disc resorption, which required macrophage infiltration. We conclude that there is extensive communication between macrophages and chondrocytes in HD resorption and that an essential component of this communication is the requirement for MMPs to release soluble bioactive factors.

The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene

It has been suggested that matrix metalloproteinase-3 (MMP-3, stromelysin-1) has an important role in the degeneration of intervertebral discs (IVDs). A human MMP-3 promoter 5A/6A polymorphism was reported to be involved in the regulation of MMP-3 gene expression. We suggest that IVD degeneration is associated with 5A/6A polymorphism. We studied 54 young and 49 elderly Japanese subjects. Degeneration of the lumbar discs was graded using MRI in the younger group and by radiography in the elderly. 5A/6A polymorphism was determined by polymerase-chain reaction-based assays. We found that the 5A5A and 5A6A genotype in the elderly was associated with a significantly larger number of degenerative IVDs than the 6A6A (p < 0.05), but there was no significant difference in the young. In the elderly, the IVD degenerative scores were also distributed more highly in the 5A5A and 5A6A genotypes (p = 0.0029). Our findings indicate that the 5A allele is a possible risk factor for the acceleration of degenerative changes in the lumbar disc in the elderly.

Matrix metalloproteinase-3–dependent generation of a macrophage chemoattractant in a model of herniated disc resorption

Herniated disc (HD) is a common health problem that is resolved by surgery unless spontaneous resorption occurs. HD tissue contains abundant macrophage infiltration and high levels of matrix metalloproteinases (MMPs) MMP-3 and MMP-7. We developed a model system in which disc tissue or isolated chondrocytes from wild-type or MMP-null mice were cocultured with peritoneal macrophages and used this system to investigate the role of MMPs and chondrocyte/macrophage interactions in disc resorption. We observed a marked enhancement of MMP-3 protein and mRNA in chondrocytes after exposure to macrophages. Chondrocytic MMP-3, but not MMP-7, was required for disc resorption, as determined by assaying for a reduction in wet weight and proteoglycan content after 3 days of coculture. Surprisingly, chondrocyte MMP-3 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by proteolytically active macrophages. We conclude that macrophage induction of chondrocyte MMP-3 plays a major role in disc resorption by mechanisms that include the generation of a bioactive macrophage chemoattractant.

Vascular endothelial growth factor (VEGF)-induced angiogenesis in herniated disc resorption.

Intervertebral disc herniation is a major cause of low back pain and sciatica. Spontaneous resorption of herniated disc (HD) is frequently detected by magnetic resonance imaging (MRI). Marked infiltration by macrophages and neo‐vascularization are observed upon histogical examination of HD. In addition, enhanced MRI studies suggest that HD resorption occurs more frequently in those completely exposed to the epidural space and that this correlates with their degree of vascularization. We have postulated that the angiogenic factor, vascular endothelial growth factor (VEGF), may be implicated in the neo‐vascularization of HD tissues. Here we demonstrate that VEGF and its receptors VEGFR‐1 and VEGFR‐2 are expressed in human surgical samples of HD. Using a co‐culture system comprised of murine peritoneal macrophages and intervertebral disc tissue as a model of the acute phase of HD developed previously, an increase in macrophage VEGF protein and mRNA expression was observed upon exposure to disc tissue. Tumor necrosis factor alpha (TNF‐α) was required for this induction of VEGF. Use of a novel angiogenesis assay revealed that addition of the conditioned media from the co‐culture system resulted in an increase of vascular tubule formation. This effect was strongly inhibited by anti‐VEGF antibody, but augmented by recombinant VEGF. We conclude that VEGF induction, under the co‐culture conditions tested can result in neo‐vascularization of intervertebral disc tissue and may thus play a role in the resorption of HD.

Up-regulated expression of matrilysin and neutrophil collagenase in human herniated discs

Spontaneous resorption of herniated nucleus pulposus (HNP) is commonly observed when there is substantial contact of the disc with the spinal canal. We already demonstrated the expression of matrix metalloproteinase (MMP)-3 (stromelysin-1) in the granulation tissues of HNP, suggesting its role in the resorption process of HNP. Recent studies of osteoarthritic cartilages reported an up-regulated expression of metalloproteinases including MMP-7 (matrilysin) and MMP-8 (neutrophil collagenase), suggesting their roles in the matrix degradation. To clarify the expression of MMP-7 and MMP-8 in HNP, immunohistological analysis of various types of HNP was performed. We found MMP-7 was expressed in infiltrated mononuclear cells and chondrocytes, whereas MMP-8 was specifically expressed in chondrocytes. The positive rate for both MMP-7 and MMP-8 significantly increased when HNP was exposed to the epidural space (p < 0.01). Our data suggest that not only MMP-3 but also MMP-7 and MMP-8 may play a role in the resorption process of HNP.

Chemonucleolysis with human stromelysin-1.

Study Design. Immunohistologic analysis was performed on surgically removed samples of herniated nucleus pulposus to examine the expression of stromelysin‐1. We performed in vitro and in vivo experiments to determine whether recombinant human (rh) stromelysin‐1 is capable of degrading nucleus pulposus. Objective. To analyze the production of stromelysin‐1 in various types of herniated nucleus pulposus, and to examine the effects of this recombinant protein on nucleus pulposus tissues. Summary of Background Data. The authors previously demonstrated a progressive decrease in herniated nucleus pulposus size in some of the transligamentous and sequestration types of herniated nucleus pulposus using magnetic resonance imaging. An increased production of stromelysin‐1, a cartilage proteoglycan degrading enzyme, in herniated nucleus pulposus was reported recently. The authors speculated that if stromelysin‐1 is involved in the degradation of herniated nucleus pulposus, stromelysin‐1 itself may be used as a chemonucleolytic agent. Methods. Immunohistologic analysis using streptoavidin‐biotin method was performed on 20 herniated nucleus pulposus samples to investigate the expression of stromelysin‐1. Five herniated nucleus pulposus samples were incubated in a tissue culture medium in the presence or absence of rh stromelysin‐1. After 24 hours of incubation, their weight changes were measured, and the loss of proteoglycan was assessed by Safranin O staining. Rat nucleus pulposus tissues were obtained from coccygeal intervertebral discs, and autologous subcutaneous transplantation was performed. Rh stromelysin‐1 was injected into the grafted materials, and the reduction in size was followed by two‐dimensional measurements from the skin surface, using engineers calipers. Results. Immunohistologic analysis demonstrated the production of stromelysin‐1 in the granulation tissues of herniated nucleus pulposus. When stromelysin‐1 was injected into the murine nucleus pulposus tissues, they reduced in size more rapidly than the control group. In addition, human herniated nucleus pulposus materials obtained at surgery showed significant weight loss when treated with stromelysin‐1 in an organ culture system. Safranin O staining revealed extensive depletion of proteoglycan in these herniated nucleus pulposus samples. Conclusions. Stromelysin‐1 is a possible key enzyme in herniated nucleus pulposus resorption, and stromelysin‐1 may be a good candidate for use in chemonucleolysis. Administration of human stromelysin‐1 may physiologically facilitate the resorption process of herniated nucleus pulposus, increase the healing rate, and decrease complications after chemonucleolysis.

Sequential dynamics of matrix metalloproteinases, tumor necrosis factor–α, vascular endothelial growth factor and plasmin expressions in the resorption process of herniated disc

Abstract Purpose of study: Granulation tissues of herniated disc (HD) are composed of marked infiltration macrophages and neovascularization that is not observed in the healthy intervertebral disc. Magnetic resonance imaging (MRI) study has shown that epidurally displaced HD tissues more commonly exhibited a gradual decrease in the size of HD. We previously showed that infiltrating macrophages, neovascularization induced by vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP)-3 and MMP-7 have been implicated in this resorption process with the study of an in vitro coculture model reproducing the acute HD phase. In this report, we examine the interaction of those molecules by investigating their sequential expressions of mRNA at an early time point in that coculture system with semiquantitative reverse transcriptase polymeric chain reaction (RT-PCR). Methods used: Activated murine macrophages were obtained by intraperitoneal administration of thioglycollate 3% medium. Cells were harvested by peritoneal lavage 4 days after, and coccygeal intervertebral disc tissues were obtained. Whole disc tissues or peritoneal macrophages were incubated alone or cocultured. Every 3 hours after cell culture started, total RNA was extracted, and by RT-PCR we examined the semiquantitative expression of MMP-3, -7, VEGF, TNF-α, plasminogen activator tissue type (t-PA) and urokinase type (u-PA), plasmin and GAPDH. Then we analyzed the change of mRNA expression and the interaction of those molecules. of findings: In both singly and cocultured macrophages, MMP-7 and VEGF were expressed constitutively. In cocultured macrophages, TNF-α was expressed at 3 hours and u-PA was expressed at 6 hours. They were changed to weak expression after 24 hours. Plasmin and MMP-3 were detected constitutively after 12 hours. In disc tissues, MMP-3 was expressed before culture and was strongly expressed in cocultured disc tissues at 3 hours; then both singly and cocultured the expression was detected constitutively. u-PA and plasmin were expressed at 3 hours and changed to weak expression after 24 hours. TNF-α was expressed at 6 hours and changed to weak expression after 18 hours. VEGF was not expressed in singly cultured disc tissues. However, VEGF was expressed at 3 hours after coculture, and after 12 hours the expression was detected constitutively. MMP-7 could not be detected in disc tissues. t-PA did not change expression at each time point in each condition. Relationship between findings and existing knowledge: Activated macrophages are known to generate proinflammatory cytokines, such as TNF-α, which is a potent inducer of various MMPs and also a potential regulator of angiogenesis by inducing of VEGF. VEGF also induces PAs, resulting in the generation of plasmin. In turn, plasmin activates MMPs. The activation of MMPs resulting from VEGF induction may result in macrophage infiltration of HD. These factors therefore affected each other and were upregulated in the mechanism of the resorption process of HD. In our cocultured macrophages, TNF-α was first upregulated and then u-PA was expressed. After that, both plasmin and MMP-3 were upregulated. We can conclude a putative mechanism that after the contact between disc tissues and macrophages, coculture condition initiated TNF-α upregulated expression. TNF-α could accelerate both angiogenesis and matrix degrading. Overall significance of findings: In our coculture system, TNF-α was first upregulated after the beginning of coculture. TNF-α could be the key molecule to accelerate the cascade of angiogenesis and disc matrix degradation. Disclosures: No disclosures. Conflict of interest: No conflicts.

The role of matrix metalloproteinases and vascular endothelial growth factor in the resorption process of herniated disc

Abstract Purpose of study: Spontaneous resorption of herniated disc (HD) is frequently detected by magnetic resonance imaging. The tendency of HD to spontaneously resorb is directly proportional to the degree of Gd-DTPA enhancement suggesting a vascularization-mediated process that also correlates with prominent macrophage infiltration. Both infiltrating macrophages and factor VIII–positive cells were abundant in epidurally displaced HD, suggesting neovascularization process in HD resorption. We developed an in vivo coculture system of macrophages and disc tissues, which could reproduce the acute phase of HD. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen and plays an essential role in the formation of new blood vessels and vascular tubule formation. Currently, we have used this coculture system to investigate the role of MMPs and VEGF in angiogenesis in herniated disc resorption. Methods used: Murine coccygeal intervertebral discs were isolated from mice. Murine macrophages were obtained after intraperitoneal administration of thioglycollate. The isolated murine discs and macrophages were then either cultured separately or in direct contact as cocultures. The conditioned media from the various cultures and cocultures were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) to reveal the cell source of MMP-3, MMP-7, and VEGF and western blotting analysis for determination of MMPs and VEGF expression. To assess the angiogenetic activity of coculture system, we used angiogenesis assay (TCS Biologicals), which can be used to study new blood formation and vascular tubule formation. of findings: Cocultures of disc tissue and macrophages produced elevated levels of MMP-3, MMP-7 and VEGF as determined by western blotting analysis. However, weak expression of them was observed in the single-culture of macrophage or disc tissue. RT-PCR revealed that the majority of the MMP-7 and VEGF were produced by the macrophages, and MMP-3 was by disc chondrocytes under coculture conditions, but both cell types produced detectable levels of all. CD31 (PECAM-1)–positive endothelial cells and formation of anastomosing tubule network were abundant in media derived from coculture compared with single culture. Relationship between findings and existing knowledge: Our study demonstrated that coculture condition resulted in disc degradation mediated by MMP upregulation. In addition, our results suggest that the interaction of macrophages with disc tissues leads to VEGF induction with a stimulation of new blood formation. VEGF is also known to promote monocyte chemotaxis. Thus, VEGF could be essential both for macrophage infiltration into HD tissues, subsequent angiogenesis and HD resorption. Overall significance of findings: In the resorption process of herniated disc, proteoglycan enzyme MMPs and a potent angiogenetic factor VEGF play a pivotal role in degrading disc matrix. These factors may be possible candidates for clinical use as a chemonucleolysis for patients with herniated disc. Disclosures: No disclosures. Conflict of interest: Hirotaka Haro, grant research support: Ministry of Education, Japan.