Gelatinase Inhibitor Research Articles

Involvement of Fibronectin Type II Repeats in the Efficient Inhibition of Gelatinases A and B by Long-chain Unsaturated Fatty Acids

The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis- and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K(i) values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by long-chain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of long-chain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds.

Inhibition of human gelatinases by metals released from dental amalgam

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in pathologic oral processes such as periodontal tissue destruction, root caries, tumor invasion and temporomandibular joint disorders. The aim of this study was to test the effect of metal ions released from dental amalgam on the major gingival gelatinolytic MMPs. Gingival human explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers conditioned with dispersed phase or concentional phase dental amalgams. The major enzymes present in conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. The proteolytic activities of MMP-2 and MMP-9 were strongly inhibited by dispersed phase amalgams conditioned buffers. Inhibition of MMP-2 and MMP-9 activities was partly prevented by the addition of 1,10 phenanthroline, a divalent metal chelator, to the amalgam conditioned buffers. Dental amalgam conditioned buffer also inhibited the degradation of denatured type I collagen by purified MMP-2 on liquid phase assays. These findings suggest that the activity of oral tissue MMPs may be modulated by metal ions released from dental amalgam.

Tumor gelatinases and invasion inhibited by the green tea flavanol epigallocatechin-3-gallate.

Given the association of consumption of green tea with prevention of cancer development, metastasis, and angiogenesis, the effect of the main flavanol present, epigallocatechin-3-gallate (EGCG), on two gelatinases most frequently overexpressed in cancer and angiogenesis (MMP-2 and MMP-9) and on tumor cell invasion and chemotaxis were examined. Zymography, Western blotting, and enzyme linked immuoadsorbent assay were used to analyze the effect of EGCG on MMP-2 and MMP-9 activity, whereas its effect on tumor cell invasion and chemotaxis was examined using modified Boyden chamber assays. A Zn2+ chelation-independent, dose-dependent, noncompetitive inhibition by EGCG of both gelatinases was found at concentrations 500 times lower than that reported to inhibit urokinase. Tumor cell invasion of a reconstituted basement membrane matrix, but not chemotaxis, was reduced by 50% with EGCG concentrations equivalent to that in the plasma of moderate green tea drinkers, and 2 orders of magnitude below those of tissue inhibitors of MMPs. Although higher concentrations of EGCG were associated with increased levels of both cell-associated gelatinases and their activator MT1-MMP, no increased gelatinase activation was found, and TIMP-1 and TIMP-2 inhibitors were up-regulated. Finally, concentrations of EGCG active in restraining proliferation and inducing apoptosis of transformed cells were more than 100 times lower than those reported for normal cells. Epigallocatechin-3-gallate is a potent inhibitor of gelatinases and an orally available pharmacologic agent that may confer the antiangiogenic and antimetastatic activity associated with green tea.

Conformationally constrained analogues of endogenous tripeptide inhibitors of zinc metalloproteinases

Two diastereomeric furan-2-carbonylamino-3-oxohexahydroindolizino[8,7- b]indole carboxylates, highly constrained analogues of endogenous pyroglutamyl tripeptide inhibitors of snake venom endopeptidases, have been prepared as potential inhibitors of adamalysin II and matrix metalloproteinases. They proved to be inactive against adamalysin II and weak inhibitors of gelatinase A, gelatinase B, stromelysin 1 and human neutrophyl collagenase. Evaluation of the mode of binding of the (2 R,5 S,11b R) isomer in the active site of adamalysin II suggests that the decrease of potency may be due to the reorientation of the acylamino chain in three of the heterocyclic nucleus, to a short contact at the entrance of the S′ 1 hydrophobic cleft and to the loss of flexibility of the tetracyclic nucleus in the P′ 1, P′ 2 region of the inhibitor, which prevents optimal arrangement in the S′ 1 specificity subsite.

Potent and Selective Mechanism-Based Inhibition of Gelatinases

Potent and Selective Mechanism-Based Inhibition of Gelatinases

Glutathione, lactobionate, and histidine: cryptic inhibitors of matrix metalloproteinases contained in University of Wisconsin and histidine/tryptophan/ketoglutarate liver preservation solutions.

UW solution and HTK solution are both used for cold preservation of liver allografts. Although they are about equally effective, their compositions are very different, and they were formulated using different rationales. The authors recently showed an important role for MMPs in liver preservation injury and consequently postulated that these preservation solutions contain cryptic inhibitors of MMP activity. To determine this possibility, the ability of these solutions to inhibit MMP activity was studied. The source of MMP2 and MMP9 was human liver effluents obtained at the time of liver transplantation or commercially available human recombinant MMP2 and MMP9. MMP2 and MMP9 showed gelatinolytic activity at 37 degrees C and also at 4 degrees C, although activity at 4 degrees C was reduced. Activity was inhibited by University of Wisconsin (UW) and Histidine/Tryptophan/ Ketoglutarate (HTK) solutions. Examination of individual ingredients disclosed that reduced glutathione (GSH) and lactobionate in UW solution and histidine in HTK solution were the cryptic inhibitors. HTK solution was a more effective inhibitor than UW solution. GSH inhibited the activity of both enzymes, but was a much more effective inhibitor of MMP9 than MMP2. Oxidized glutathione(GSSG) was a much less effective inhibitor of the enzymes. The inhibitor constants (K(i)) of GSH for MMP2 and MMP9 were 34 micromol/L and 3 micromol/L, respectively. The authors conclude that MMP inhibition is a cryptic property of both commonly used liver preservation solutions and contributes importantly to their action. Furthermore, GSH appears to be an effective inhibitor of gelatinases at concentrations at which it is normally present in extracellular fluid.

Regulation of Membrane-type-1 Matrix Metalloproteinase Activity by Its Cytoplasmic Domain

Membrane-type-1 matrix metalloproteinase (MT1-MMP) has transmembrane and cytoplasmic domains, which target it to invasive fronts. We analyzed the role of the cytoplasmic tail by expressing wild type MT1-MMP and three mutants with progressively truncated C termini in human Bowes melanoma cells. We examined gelatinase A activation and the localization and processing of recombinant proteins in stable cell clones using gelatin zymography, immunoblotting, and immunofluorescence. Cell invasion was analyzed in vitro by Matrigel invasion assays. Gelatinase A was activated in all cell clones. However, the localization of MT1-MMP to the leading edge of migrating cells and cell invasion through Matrigel were strongly enhanced only in cells expressing either wild type or truncated MT1-MMP lacking 6 C-terminal amino acid residues (Delta577). Truncations of 10 or 16 amino acid residues in the cytoplasmic domain (Delta567 and Delta573, respectively) disturbed MT1-MMP localization. The expression of wild type and Delta577 MT1-MMPs induced also their cleavage to 43-kDa cell surface forms and the release of soluble, approximately 20-kDa N-terminal fragments containing the catalytic center. A synthetic MMP inhibitor but not a gelatinase inhibitor prevented the processing, suggesting that autocatalytic cleavage occurs. Purified soluble MT1-MMP was also autoproteolytically processed to 43- and 20-kDa forms in vitro. Our results indicate that the cytoplasmic domain has an important role in cell invasion by controlling both the targeting and degradation/turnover of MT1-MMP.

Curcuminoids Inhibit the Angiogenic Response Stimulated by Fibroblast Growth Factor-2, Including Expression of Matrix Metalloproteinase Gelatinase B

We have studied mechanisms controlling activation of the gelatinase B gene (matrix metalloproteinase-9) by fibroblast growth factor-2 (FGF-2) during angiogenesis, and the effects of the natural product curcuminoids on this process. Using a transgenic mouse (line 3445) harboring a gelatinase B promoter/lacZ fusion gene, we demonstrate FGF-2 stimulation of reporter gene expression in endothelial cells of invading neocapillaries in the corneal micropocket assay. Using cultured corneal cells, we show that FGF-2 stimulates DNA binding activity of transcription factor AP-1 but not NF-kappaB and that AP-1 stimulation is inhibited by curcuminoids. We further show that induction of gelatinase B transcriptional promoter activity in response to FGF-2 is dependent on AP-1 but not NF-kappaB response elements and that promoter activity is also inhibited by curcuminoids. In rabbit corneas, the angiogenic response induced by implantation of an FGF-2 pellet is inhibited by the co-implantation of a curcuminoid pellet, and this correlates with inhibition of endogenous gelatinase B expression induced by FGF-2. Angiostatic efficacy in the cornea is also observed when curcuminoids are provided to mice in the diet. Our findings provide evidence that curcuminoids target the FGF-2 angiogenic signaling pathway and inhibit expression of gelatinase B in the angiogenic process.

Amniochorion Gelatinase-Gelatinase Inhibitor Imbalance In Vitro

In Brief Objective To estimate the effect of lipopolysaccharide on gelatinases and tissue inhibitors of matrix metalloproteinase 2 (gelatinase inhibitor) balance in human fetal membranes. Methods Amniochorionic membranes in organ explant were stimulated with 1000 ng/mL lipopolysaccharide for 24 hours after a 48-hour preincubation period. Quantitative competitive polymerase chain reaction (PCR) was done to quantitate messenger RNAs for gelatinase A and B (matrix metalloproteinase 2 and 9) and tissue inhibitor of metalloproteinase 2. Protein levels were assayed by enzyme-linked immunosorbant assay. The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 was calculated. Statistical evaluation was done by Mann-Whitney U test. Results Lipopolysaccharide stimulation produced 3.6 × 106 and 366 transcripts of gelatinase A and B, respectively, compared with only 5.9 × 104 (P = .009) and three transcripts (P = .006), respectively, in the controls. Lipopolysaccharide stimulation released 210 ng/mL compared with 7 ng/mL of gelatinase A and B proteins compared with 120 (P = .01) and 4.6 ng/mL (P = .3) in controls, respectively. Control amniochorion produced 5.7 × 105 transcripts of tissue inhibitor of metalloproteinase 2, whereas lipopolysaccharide stimulation produced 4.1 × 105 transcripts (P = .69). Lipopolysaccharide reduced the release of this inhibitor from 114 ng/mL to 68 ng/mL (P = .007). The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 increased from a balanced ratio of 1:1 to 3.1:1 after 1000 ng/mL of lipopolysaccharide. Conclusion Lipopolysaccharide increased the expression and release of gelatinases and decreased its inhibitor, which shifted the balance in favor of gelatinase activity leading to membrane degradation that predisposes to premature rupture of membranes. Lipopolysaccharide stimulation results in an imbalance in the molar ratio of gelatinases and tissue inhibitor of metalloproteinase 2 in human fetal membranes.

Amniochorion gelatinase-gelatinase inhibitor imbalance in vitro: a possible infectious pathway to rupture

Objective: To estimate the effect of lipopolysaccharide on gelatinases and tissue inhibitors of matrix metalloproteinase 2 (gelatinase inhibitor) balance in human fetal membranes. Methods: Amniochorionic membranes in organ explant were stimulated with 1000 ng/mL lipopolysaccharide for 24 hours after a 48-hour preincubation period. Quantitative competitive polymerase chain reaction (PCR) was done to quantitate messenger RNAs for gelatinase A and B (matrix metalloproteinase 2 and 9) and tissue inhibitor of metalloproteinase 2. Protein levels were assayed by enzyme-linked immunosorbant assay. The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 was calculated. Statistical evaluation was done by Mann-Whitney U test. Results: Lipopolysaccharide stimulation produced 3.6 × 10 6 and 366 transcripts of gelatinase A and B, respectively, compared with only 5.9 × 10 4 ( P = .009) and three transcripts ( P = .006), respectively, in the controls. Lipopolysaccharide stimulation released 210 ng/mL compared with 7 ng/mL of gelatinase A and B proteins compared with 120 ( P = .01) and 4.6 ng/mL ( P = .3) in controls, respectively. Control amniochorion produced 5.7 × 10 5 transcripts of tissue inhibitor of metalloproteinase 2, whereas lipopolysaccharide stimulation produced 4.1 × 10 5 transcripts ( P = .69). Lipopolysaccharide reduced the release of this inhibitor from 114 ng/mL to 68 ng/mL ( P = .007). The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 increased from a balanced ratio of 1:1 to 3.1:1 after 1000 ng/mL of lipopolysaccharide. Conclusion: Lipopolysaccharide increased the expression and release of gelatinases and decreased its inhibitor, which shifted the balance in favor of gelatinase activity leading to membrane degradation that predisposes to premature rupture of membranes.

Design, combinatorial chemical synthesis and in vitro characterization of novel urea based gelatinase inhibitors

A novel series of hydroxamate/urea-based inhibitors of gelatinases has been discovered via solid-phase combinatorial chemistry. SAR of P 1′, P 2′, and P 3′ has been exploited and structures different from traditional succinate-based MMP inhibitors have been found.

Tumor targeting with a selective gelatinase inhibitor.

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

Collagen breakdown in soft connective tissue explants is associated with the level of active gelatinase A (MMP-2) but not with collagenase.

Recent data suggest that gelatinase A (matrix metalloproteinase-2, MMP-2) plays an important role in the degradation of collagen of soft connective tissues. In an attempt to investigate its participation in more detail we assessed the digestion of collagen in cultured rabbit periosteal explants and compared this with the level of active MMP-2 and collagenases. The data demonstrated that both collagen degradation and MMP activity increased with time. Conditioned medium obtained from explants cultured for 72 h showed that the level of active MMP-2 correlated with collagen degradation (r = 0.80, d.f. = 23, P < 0.0001). Such a relationship was not found with collagenase activity (r = -0.08, d.f. = 21, NS). The possible involvement of MMP-2 in collagen degradation was investigated further by incubating explants with selective gelatinase inhibitors (CT1166, CT1399 and CT1746). In the presence of these compounds breakdown of collagen was almost completely abolished (approximately 80%). Finally we assessed whether periosteal fibroblasts had the capacity to degrade collagen type I that conferred resistance to collagenase activity. Breakdown of this collagen did not differ from degradation of normal collagen. Taken together, our data provide support for the view that MMP-2 plays a crucial role in collagen degradation of soft connective tissue.

Inhibition of gelatinase A by oleic acid.

Inhibition of gelatinase A by oleic acid.

Selective inhibition of collagenase-1, gelatinase A, and gelatinase B by chemotherapeutic agents.

Selective inhibition of collagenase-1, gelatinase A, and gelatinase B by chemotherapeutic agents.

Inhibition of gelatinase A (MMP-2) by batimastat and captopril reduces tumor growth and lung metastases in mice bearing Lewis lung carcinoma.

We have examined the effects of the synthetic matrix metalloproteinase inhibitor, batimastat (BB-94) and the angiotensin-converting enzyme inhibitor, captopril, on metalloproteinase activity of murine Lewis-lung-carcinoma cells (3LL) in vitro, and on local growth and lung metastasis of the same tumor implanted intramuscularly in syngeneic C57BL/6 mice. The effect of BB-94 and captopril on the survival of the 3LL-tumor-bearing mice was also examined. Here we report that captopril treatment resulted in decreased transcription and protein levels of gelatinase A by 3LL cells. Both BB-94 and captopril also prevented substrate degradation by gelatinase A and B released in conditioned medium by cultured cells. Treatment of tumor-bearing animals with BB-94 (i.p.) or captopril (in drinking water) resulted in significant inhibition of the mean tumor volume (25 and 33% respectively) and of the mean lung metastasis number (26 and 29% respectively). When both agents were given, they acted in synergy, resulting in 51 and 80% inhibition of tumor growth and metastasis. The survival time of the mice treated with both BB-94 and captopril was also significantly longer compared with the groups treated with each agent alone or with the vehicle. Our data support the hypothesis of an essential role of metalloproteinase(s) in the metastatic process. Moreover, blockade of invasion, angiogenesis and other processes mediated by metalloproteinases may underlie the anti-tumor and anti-metastatic effect of BB-94 and captopril and their combination. It is conceivable that this combination could be tested in selected clinical conditions as an adjuvant modality to cytotoxic therapy.

Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Gelatinase A and membrane-type 1 matrix metalloproteinase mRNA: expressed in adrenocortical cancers but not in adenomas.

In an attempt to understand the mechanism behind the invasion and metastasis in adrenocortical cancer we performed mRNA in situ hybridization on 30 tumors for three matrix metalloproteinases (MMPs): gelatinase A, membrane type 1 matrix metalloproteinase (MT1-MMP), and collagenase-3. All are known to participate in the invasion and metastasis of other tumor forms by degrading the extracellular matrix. Thirteen of sixteen cancers, but only one of fourteen benign lesions showed expression of gelatinase A, which was localized in stromal cells. MT1-MMP is thought to assist in tumor invasion and metastasis by activating the zymogen gelatinase A. Of 14 malignant tumors analyzed, 12 showed MT1-MMP mRNA expression, which in 7 cases was detected in both neoplastic and stromal cells. The benign tumors showed MT1-MMP expression in only 3 of 11 cases, and it was restricted to tumor cells. Fourteen tumors (11 cancers, 3 adenomas) were also analyzed for collagenase-3 mRNA, but no expression was detected. In conclusion, our data show that gelatinase A mRNA is expressed in most malignant adrenocortical tumors but not in the benign tumors. Gelatinase A mRNA expression is restricted to stromal cells, whereas its activator, MT1-MMP, is expressed in both stromal and neoplastic cells. Inhibition of gelatinase A and other proteinases may in the future become important as a form of cancer treatment.

Tetracycline-Based MMP Inhibitors Can Prevent Fibroblast-Mediated Collagen Gel Contraction In Vitro

Collagen gels in vitro can be contracted by fibroblasts. The role of matrix metalloproteinases (MMPs) in the contraction of collagen lattices by human neonatal foreskin fibroblasts (HuFFs) was investigated in tissue culture media supplemented by various doses of known gelatinase inhibitors. Fluorescent assays with model gelatinase substrates and media conditioned by fibroblasts apparently confirmed the ability of chemically modified tetracyclines (CMTs) to act as inhibitors of MMP2, and zymography demonstrated that this was the major cell-derived MMP activity. There were no observable effects on the rate of contraction of attached FPCLs containing 6 x 10(4) HuFFs (passages 18-25) with either CMT-5 or CMT-2 at all concentrations tested (0-100 micrograms/mL). However, at greater than 20 micrograms/mL doxycycline and greater than 5 micrograms/mL CMT-3, FPCL contraction was completely abolished. Quantitative assessment of cell viability by means of the MTT assay in monolayer and qualitatively within the FPCLs with CalceinAM suggested that differences were not due to cytotoxic effects. Seeding FPCLs with lower-passage fibroblasts produced identical trends. These results may implicate the involvement of MMPs in the process of gel contraction, although tetracyclines have effects additional to their ability to inhibit MMPs directly.

Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain.

Reperfusion disrupts cerebral capillaries, causing cerebral edema and hemorrhage. Middle cerebral artery occlusion (MCAO) induces the matrix-degrading metalloproteinases, but their role in capillary injury after reperfusion is unknown. Matrix metalloproteinases (MMPs) and tissue inhibitors to metalloproteinases (TIMPs) modulate capillary permeability. Therefore, we measured blood-brain barrier (BBB) permeability, brain water and electrolytes, MMPs, and TIMPs at multiple times after reperfusion. Adult rats underwent MCAO for 2 hours by the suture method. Brain uptake of 14C-sucrose was measured from 3 hours to 14 days after reperfusion. Levels of MMPs and TIMPs were measured by zymography and reverse zymography, respectively, in contiguous tissues. Other rats had water and electrolytes measured at 3, 24, or 48 hours after reperfusion. Treatment with a synthetic MMP inhibitor, BB-1101, on BBB permeability and cerebral edema was studied. Brain sucrose uptake increased after 3 and 48 hours of reperfusion, with maximal opening at 48 hours and return to normal by 14 days. There was a correlation between the levels of gelatinase A at 3 hours and the sucrose uptake (P<0.05). Gelatinase A (MMP-2) was maximally increased at 5 days, and TIMP-2 was highest at 5 days. Gelatinase B and TIMP-1 were maximally elevated at 48 hours. The inhibitor of gelatinase B, TIMP-1, was also increased at 48 hours. Treatment with BB-1101 reduced BBB opening at 3 hours and brain edema at 24 hours, but neither was affected at 48 hours. The initial opening at 3 hours correlated with gelatinase A levels and was blocked by a synthetic MMP inhibitor. The delayed opening, which was associated with elevated levels of gelatinase B, failed to respond to the MMP inhibitor, suggesting different mechanisms of injury for the biphasic BBB injury.