M. Elizabeth Fini

Effects of Matrix Metalloproteinase-9 Gene Knock-Out on the Proteolysis of Blood–Brain Barrier and White Matter Components after Cerebral Ischemia

Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood–brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood–brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood–brain barrier-associated protein, occludin. Correspondingly, blood–brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood–brain barrier and white matter components.

Role for Matrix Metalloproteinase 9 after Focal Cerebral Ischemia: Effects of Gene Knockout and Enzyme Inhibition with BB-94

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches, Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size, However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that function to maintain and remodel tissue architecture. Their substrates represent an astounding variety of extracellular matrix components, secreted cytokines and cell surface molecules, and they have been implicated in a wide range of processes and diseases. To date MMPs have been found in virtually every tissue of the eye under conditions of health and disease. Although their functions in vivo remain poorly understood, it is clear they impact on essentially every aspect of eye physiology. This chapter reviews the expanding literature on MMPs in the eye and attempts to place it in the context of basic MMP biology. A general overview of MMP functions is presented first, and then the discussion moves to examples of possible MMP roles in two eye structures. For the cornea, we present recent work on the roles of MMPs during various aspects of wound healing. For the retina, we describe the activities of MMPs in specific disease states from which common principles may emerge.

Matrix Metalloproteinase-9 Knockout Confers Resistance to Corneal Epithelial Barrier Disruption in Experimental Dry Eye

Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin.

Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype.

The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-κB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eyes aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.

Mitogen-Activated Protein Kinase Inhibition in Traumatic Brain Injury: In Vitro and In Vivo Effects

The authors provide the first in vitro and in vivo evidence that perturbations in mitogen-activated protein kinase (MAPK) signal-transduction pathways are involved in the pathophysiology of traumatic brain injury. In primary rat cortical cultures, mechanical trauma induced a rapid and selective phosphorylation of the extracellular signal-regulated kinase (ERK) and p38 kinase, whereas there was no detectable change in the c-jun N-terminal kinase (JNK) pathway. Treatment with PD98059, which inhibits MAPK/ERK 1/2, the upstream activator of ERK, significantly increased cell survival in vitro. The p38 kinase and JNK inhibitor SB203580 had no protective effect. Similar results were obtained in vivo using a controlled cortical impact model of traumatic injury in mouse brain. Rapid and selective upregulation occurred in ERK and p38 pathways with no detectable changes in JNK. Confocal immunohistochemistry showed that phospho-ERK colocalized with the neuronal nuclei marker but not the astrocytic marker glial fibrillary acidic protein. Inhibition of the ERK pathway with PD98059 resulted in a significant reduction of cortical lesion volumes 7 days after trauma. The p38 kinase and JNK inhibitor SB203580 had no detectable beneficial effect. These data indicate that critical perturbations in MAPK pathways mediate cerebral damage after acute injury, and further suggest that ERK is a novel therapeutic target in traumatic brain injury.

Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia.

Matrix metalloproteinases (MMPs) may contribute to tissue damage after cerebral ischemia. In this study, wildtype and MMP-2 knockout mice were subjected to permanent and transient (2 h) occlusions of the middle cerebral artery. Gelatin zymography showed that MMP-9 levels were increased in all brains after ischemia. MMP-2 levels did not show a significant increase in wildtype mice, and were not detectable in knockout mice. Laser doppler flowmetry demonstrated equivalent ischemic reductions in perfusion in wildtype and knockout mice. In both permanent and transient occlusion paradigms, there were no statistically significant differences between wildtype and knockout mice in terms of 24 h ischemic lesion volumes. These data suggest that MMP-2 does not contribute to acute tissue damage in this model of focal ischemia.

Secretion of Matrix Metalloproteinase-2 and -9 after Mechanical Trauma Injury in Rat Cortical Cultures and Involvement of MAP Kinase

Matrix metalloproteinases (MMP) are involved in the pathophysiology of brain injury. We recently showed that knockout mice deficient in MMP-9 expression were protected against traumatic brain injury. However, the cellular sources of MMP activity after trauma remain to be fully defined. In this study, we investigated the hypothesis that resident brain cells secrete MMP after mechanical trauma injury in vitro, and mitogen-activated protein (MAP) kinase signal transduction pathways are involved in this response. Rat primary cortical neurons, astrocytes, and co-cultures were subjected to needle scratch mechanical injury, and levels of MMP-2 and MMP-9 in conditioned media were assayed by zymography. MMP-2 and MMP-9 were increased in cortical astrocytes and co-cultures, whereas only MMP-2 was increased in neurons. Western blots showed that phosphorylated extracellular signal regulated kinase (ERK1/2) and p38 were rapidly upregulated in co-cultures after mechanical injury. No change in phosphorylated c-jun N-terminal kinase (JNK) was observed. In-gel kinase assays confirmed this lack of response in the JNK pathway. Treatment with either 10 microM of U0126 (a MAP kinase/ERK1/2 kinase inhibitor) or 10 microM of SB203580 (a p38 inhibitor) had no detectable effect on MMP-2 and MMP-9 levels after mechanical injury. However, combination treatment with both inhibitors significantly reduced secretion of MMP-9. Herein, we demonstrate that (1) resident brain cells secrete MMP after mechanical injury, (2) astrocytes are the main source of MMP-9 activity, and (3) ERK and p38 MAP kinases are upregulated after mechanical injury, and mediate the secretion of MMP-9.

Matrix Metalloproteinase 9 Promoter Activity Is Induced Coincident with Invasion during Tumor Progression

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

Transcription Factors Pax6 and AP-2α Interact To Coordinate Corneal Epithelial Repair by Controlling Expression of Matrix Metalloproteinase Gelatinase B

ABSTRACT Pax6 is a paired box containing transcription factor that resides at the top of a genetic hierarchy controlling eye development. It continues to be expressed in tissues of the adult eye, but its role in this capacity is unclear. Pax6 is present in the adult corneal epithelium, and we showed that the amount of Pax6 is increased at the migrating front as the epithelium resurfaces the cornea after injury (J. M. Sivak, R. Mohan, W. B. Rinehart, P. X. Xu, R. L. Maas, and M. E. Fini, Dev. Biol. 222:41-54, 2000). We also showed that Pax6 controls activity of the transcriptional promoter for the matrix metalloproteinase, gelatinase B (gelB; MMP-9) in cell culture transfection studies. gelB expression is turned on at the migrating epithelial front in the cornea, and it coordinates and effects aspects of epithelial regeneration (R. Mohan, S. K. Chintala, J. C. Jung, W. V. Villar, F. McCabe, L. A. Russo, Y. Lee, B. E. McCarthy, K. R. Wollenberg, J. V. Jester, M. Wang, H. G. Welgus, J. M. Shipley, R. M. Senior, and M. E. Fini, J. Biol. Chem. 277:2065-2072). We define here two positively acting Pax6 response elements in the gelB promoter. Pax6 binds directly to one of these sites through the paired DNA-binding domain. It binds the second site indirectly by interaction with AP-2α, a transcription factor that also exerts control over eye development. Pax6 control of gelB expression was examined in vivo by using a corneal reepithelialization model in mice heterozygous for a Pax6 paired-domain mutation (Sey+/−). A reduced Pax6 dosage in these mice resulted in a loss of gelB expression at the migrating epithelial front. This effect was correlated with an increase in inflammation and the rate of reepithelialization, a finding consistent with the phenotype of gelB knockout mice. Together, these data indicate that Pax6 controls activity of the gelB promoter through cooperative interactions with AP-2α and support an active role for Pax6 in maintenance and repair of the adult corneal epithelium.