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Stroke. 2004;35:e338-e340
Published online before print June 10, 2004, doi: 10.1161/01.STR.0000135294.08862.5d
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(Stroke. 2004;35:e338.)
© 2004 American Heart Association, Inc.

Letters to the Editor

Cerebral Ischemia, Matrix Metalloproteinases, and TNF-{alpha}: MMP Inhibitors May Act Not Exclusively by Reducing MMP Activity

Michael Dittmar, MD

Department of Anesthesiology, University of Regensburg, Regensburg, Germany

Georgios Kiourkenidis, PhD Markus Horn, MD

Department of Neurology, University of Regensburg, Regensburg, Germany

Susanne Bollwein Günther Bernhardt, PhD

Institute of Pharmacy, University of Regensburg, Regensburg, Germany

To the Editor:

We thank Drs Pfefferkorn and Rosenberg for their enlightening paper1 on matrix metalloproteinases (MMPs) after ischemic stroke.

MMPs are attributed an important role in the pathophysiology of cerebral ischemia. The gelatinases MMP-2 and MMP-9 are of particular interest in this respect. In numerous experimental settings, the reduction of gelatinase activity has been demonstrated to be associated with improved outcome. Beside natural MMP inhibitors (tissue inhibitors of MMPs),2 monoclonal antibodies,3 and genetic approaches,4 the broad-spectrum MMP inhibitors BB-94, BB-1101, and KB-R7785 have proven to reduce ischemic damage.1,4–8

In addition to their inhibitory effects on MMPs, broad-spectrum inhibitors impede the activity of other metalloendopeptidases such as tumor necrosis factor {alpha} (TNF-{alpha}) converting enzyme (TACE), which cleaves membrane-bound pro–TNF-{alpha} to active soluble TNF-{alpha}.9,10 TACE is inhibited by BB-94,11,12,13 BB-1101,14,15 and KB-R7785.16 TNF-{alpha} has been proven to display negative effects after cerebral ischemia,17 and its neutralization ameliorates ischemic lesions.17,18,19,20 TNF-{alpha} contributes to the opening of the blood–brain barrier (BBB) by a mechanism involving soluble guanylyl cyclase and protein tyrosine kinase.21 Therefore, broad-spectrum MMP inhibitors could have contributed to BBB protection via reducing TNF-{alpha} activity and not exclusively via inhibition of MMPs. Unfortunately, the literature on the impact of MMP inhibitors after stroke does not provide any insight into the possible involvement of TNF-{alpha} in this context.

We investigated the specific MMP-2/MMP-9 inhibitor I, N-([1,1'-biphenyl]-4-ylsulfonyl)-D-phenylalanine (Calbiochem),22 in focal cerebral ischemia. Experimental procedures were carried out in accordance with guidelines of the German law governing animal care and the European Communities Council Directive (86/609/EEC). Protocols were approved by the Ethics Committee for Animal Research of the Bavarian government.

Wistar rats (250 to 300 g; Charles River, Sulzfeld, Germany) were subjected to 90 minutes middle cerebral artery occlusion (n=38) or sham surgery (n=6) as described by Longa et al,23 with modifications previously described in detail.24 After reperfusion, the MMP-2/MMP-9 inhibitor I was locally infused into the internal carotid artery over 30 minutes. Nine animals received a dose of 1 mg/kg body weight or vehicle, respectively. A second consecutive group received 10 mg/kg or solvent (n=8 each). Four additional animals were used for quantification of plasma levels. They received a dose of 1 or 5 mg/kg (n=2, respectively). Blood specimens were collected before, immediately after, 30 minutes, 90 minutes, and 12 (n=1 per dosage) or 24 hours (n=1 per dosage) after infusion. Plasma levels were determined by isocratic (54% CH3CN/46% TFA (0.1%), 1 mL/min) high-performance liquid chromatography on a 5 µm LiChrospher 100 RP-18 column (250x4 mm) by uv detection (254 nm) using {alpha}-[([1,1'-biphenyl]-4-ylsulfonyl)amino]-N-hydroxy-({alpha}R)-benzenepropanamide (MMP-2/MMP-9 inhibitor II; Calbiochem) as internal standard. Deproteinisation was carried out by addition of 2 aliquots of ice-cold CH3CN to the plasma samples.

Rats were weighed, neurologically examined according to,25 and underwent in vivo magnet resonance imaging 24 hours, 2 days, and 8 days after surgery. Measurements were performed on a 1.5T MR scanner (Magnetom Symphony, Siemens). A T2-weighted turbo spin-echo sequence and a heavily T1-weighted inversion recovery sequence were applied in the axial and coronal orientations. Lesion volumes were determined from printed MR images. Midline shift was expressed as ratios of ischemic and nonischemic hemisphere diameters at the bottom of the 3rd ventricle.

Results revealed no differences in physiological parameters. Plasma concentrations of MMP-2/MMP-9 inhibitor I are displayed in the Table. There were no significant differences in lesion volumes, midline shift, body weight, and neurological examination between inhibitor-treated animals and controls.


View this table:
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  		Mean Plasma Concentrations of MMP-2/MMP-9 Inhibitor I [µmol] Over Time

In conclusion, broad-spectrum MMP inhibitors have been repeatedly reported to have beneficial effects after cerebral ischemia. In our study, an MMP-2/MMP-9 inhibitor failed to influence the effects of transient focal cerebral ischemia. Neither a pharmacodynamic nor a pharmacokinetic malfunction of this inhibitor can be ruled out. Nevertheless, these data, together with the present literature, suggest that therapeutic success due to synthetic MMP inhibitors may possibly not be attributed exclusively to its effects on MMP-2 and MMP-9. This hypothesis concurs with the finding of Pfefferkorn and Rosenberg that BB-94 reduced BBB opening without influencing zymographically determined MMP-2 and MMP-9 levels.1 Since broad-spectrum MMP inhibitors affect other enzymes like TACE as well, the effects on TNF-{alpha} activity should not be underestimated in the discussion of these drugs.

References

  1. Pfefferkorn T, Rosenberg GA. Closure of the blood-brain barrier by matrix metalloproteinase inhibition reduces rtPA-mediated mortality in cerebral ischemia with delayed reperfusion. Stroke. 2003; 34: 2025–2030.[Abstract/Free Full Text]
  2. Rosenberg GA, Kornfeld M, Estrada E, Kelley RO, Liotta LA, Stetler-Stevenson WG. TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase. Brain Res. 1992; 576: 203–207.[CrossRef][Medline] [Order article via Infotrieve]
  3. Romanic AM, White RF, Arleth AJ, Ohlstein EH, Barone FC. Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke. 1998; 29: 1020–1030.[Abstract/Free Full Text]
  4. Asahi M, Asahi K, Jung J-C, del Zoppo GJ, Fini ME, Lo EH. Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab. 2000; 20: 1681–1689.[CrossRef][Medline] [Order article via Infotrieve]
  5. Sumii T, Lo EH. Involvement of matrix metalloproteinase in thrombolysis-associated hemorrhagic transformation after embolic focal ischemia in rats. Stroke. 2002; 33: 831–836.[Abstract/Free Full Text]
  6. Lee S-R, Tsuji K, Lee S-R, Lo EH. Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia. J Neurosci. 2004; 24: 671–678.[Abstract/Free Full Text]
  7. Rosenberg GA, Estrada EY, Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain. Stroke. 1998; 29: 2189–2195.[Abstract/Free Full Text]
  8. Jiang X-F, Namura S, Nagata I. Matrix metalloproteinase inhibitor KB-R7785 attenuates brain damage resulting from permanent focal cerebral ischemia in mice. Neurosci Lett. 2001; 305: 41–44.[CrossRef][Medline] [Order article via Infotrieve]
  9. Gearing AJ, Beckett P, Christodoulou M, Churchill M, Clements J, Davidson AH, Drummond AH, Galloway WA, Gilbert R, Gordon JL, Leber TM, Mangan M, Miller K, Nayee P, Owen K, Patel S, Thomas W, Wells G, Wood LM, Woolley K. Processing of tumor necrosis factor-alpha precursor by metalloproteinases. Nature. 1994; 370: 555–557.[CrossRef][Medline] [Order article via Infotrieve]
  10. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 1997; 385: 729–733.[CrossRef][Medline] [Order article via Infotrieve]
  11. Parvathy S, Karran EH, Turner AJ, Hooper NM. The secretases that cleave angiotensin converting enzyme and the amyloid precursor protein are distinct from tumour necrosis factor-K convertase. FEBS Letters. 1998; 431: 63–65.[CrossRef][Medline] [Order article via Infotrieve]
  12. Corbel M, Lanchou J, Germain N, Malledant Y, Boichot E, Lagente V. Modulation of airway remodeling-associated mediators by the antifibrotic compound, pirfenidone, and the matrix metalloproteinase inhibitor, batimastat, during acute lung injury in mice. Eur J Pharmacol. 2001; 426: 113–121.[CrossRef][Medline] [Order article via Infotrieve]
  13. Hernandez-Pando R, Orozoco H, Arriaga K, Pavön L, Rook G. Treatment with BB-94, a broad spectrum inhibitor of zinc-dependent metalloproteinases, causes deviation of the cytokine profile towards type-2 in experimental pulmonary tuberculosis in Balb/c mice. Int J Exp Path. 2000; 81: 199–209.[CrossRef][Medline] [Order article via Infotrieve]
  14. Clements JM, Cossins JA, Wells GM, Corkill DJ, Helfrich K, Wood LM, Pigott R, Stabler G, Ward GA, Gearing AJ, Miller KM. Matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumor necrosis factor-alpha inhibitor. J Neuroimmunol. 1997; 74: 85–94.[CrossRef][Medline] [Order article via Infotrieve]
  15. Barlaam B, Bird TG, Lambert-van der Brempt C, Campbell D, Foster SJ, Maciewicz R. New alpha-substituted succinate-based hydroxamic acids as TNFalpha convertase inhibitors. J Med Chem. 1999; 42: 4890–4908.[CrossRef][Medline] [Order article via Infotrieve]
  16. Morimoto Y, Nishikawa K, Ohashi M. KB-R7785, a novel matrix metalloproteinase inhibitor, exerts its antidiabetic effect by inhibiting tumor necrosis factor-alpha production. Life Sci. 1997; 61: 795–803.[CrossRef][Medline] [Order article via Infotrieve]
  17. Barone FC, Arvin B, White RF, Miller A, Webb CL, Willette RN, Lysko PG, Feuerstein GZ. Tumor necrosis factor-alpha: a mediator of focal ischemic brain injury. Stroke. 1997; 28: 1233–1244.[Abstract/Free Full Text]
  18. Nawashiro H, Martin D, Hallenbeck JM. Inhibition of tumor necrosis factor and amelioration of brain infarction in mice. J Cereb Blood Flow Metab. 1997; 17: 229–232.[Medline] [Order article via Infotrieve]
  19. Yang GY, Gong C, Qin Z, Ye W, Mao Y, Bertz AL. Inhibition of TNFalpha attenuates infarct volume and ICAM-1 expression in ischemic mouse brain. Neuroreport. 1998; 9: 2131–2134.[Medline] [Order article via Infotrieve]
  20. Martin-Villalba A, Hahne M, Kleber S, Vogel J, Falk W, Schenkel J, Krammer PH. Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ. 2001; 8: 679–686.[CrossRef][Medline] [Order article via Infotrieve]
  21. Mayhan WG. Cellular mechanisms by which tumor necrosis factor-alpha produces disruption of the blood-brain barrier. Brain Res. 2002; 927: 144–152.[CrossRef][Medline] [Order article via Infotrieve]
  22. Tamura Y, Watanabe F, Nakatani T, Yasui K, Fuji M, Komurasaki T, Tsuzuki H, Maekawa R, Yoshioka T, Kawada K, Sugita K, Ohtani M. Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivates. J Med Chem. 1998; 41: 640–649.[CrossRef][Medline] [Order article via Infotrieve]
  23. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989; 20: 84–91.[Abstract/Free Full Text]
  24. Dittmar M, Spruss T, Schuierer G, Horn M. External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats. Stroke. 2003; 34: 2252–2257.[Abstract/Free Full Text]
  25. Menzies SA, Hoff JT, Betz AL. Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model. Neurosurgery. 1992; 31: 100–107.[Medline] [Order article via Infotrieve]

Response:

Gary A. Rosenberg, MD

Department of Neurology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico

Thomas Pfefferkorn, MD

Department of Neurology, Ludwigs-Maximilian-Universität, Munich, Germany

We appreciate the comments of Dr Dittmar and colleagues on the role of selective MMP-2/MMP-9 inhibitors in cerebral ischemia. Most of the inhibitors used in the neurological studies have been broad-spectrum agents such as the one that we used.1 The finding that an MMP-2/MMP-9 inhibitor failed to affect the endpoints they tested suggests that other MMPs or possibly tumor necrosis factor-{alpha} converting enzyme (TACE) are also involved. The emphasis of the earlier studies on MMP-2 and MMP-9 (gelatinase A and B, respectively) was based on the availability of gelatin zymography, a highly sensitive quantitative method to detect gelatinases.2 It is not surprising to find that selective blockade of MMP-2 and MMP-9 is not sufficient to block ischemic damage since over 20 MMPs have been discovered.3 Other MMPs implicated in stroke include MMP-3 (stromelysin-1) and MMP-14 (membrane-bound MMP).4,5

The current MMP inhibitors, including the one tested by Dittmar and colleagues, are poorly soluble.6 Little is known about the penetration of these agents into the brain. Because they are poorly soluble, a diluent such as DSMO, which may be neuroprotective by itself, is used. The authors do not specify the diluent used. Another potential drawback of this study is that the selected endpoints did not address the effect on the blood-brain barrier (BBB), which is the major site of action of the MMP inhibitors. It would be interesting to know whether the MMP-2/MMP-9 inhibitor had any effect on the BBB, which may be separate from its effect on lesion size.

Computer-aided drug design has increased the number of selective MMP inhibitors.7 The challenge will be to compare the ones with promising drug profiles against the broad-spectrum inhibitors and other classes of agents that interfere with the expression or action of the MMPs, such as the tetracycline derivatives doxycycline and minocycline.8 Studies such as that described by Dittmar and colleagues will aid greatly in the selection of optimal agents to control the neuroinflammation associated with MMP expression in stroke.

References

  1. Pfefferkorn T, Rosenberg GA. Closure of the blood-brain barrier by matrix metalloproteinase inhibition reduces rtPA-mediated mortality in cerebral ischemia with delayed reperfusion. Stroke. 2003; 34: 2025–2030.[Abstract/Free Full Text]
  2. Kleiner DE, Stetler-Stevenson WG. Quantitative zymography: detection of picogram quantities of gelatinases. Anal Biochem. 1994; 218: 325–329.[CrossRef][Medline] [Order article via Infotrieve]
  3. Rosenberg GA. Matrix metalloproteinases in neuroinflammation. Glia. 2002; 39: 279–291.[CrossRef][Medline] [Order article via Infotrieve]
  4. Rosenberg GA, Cunningham LA, Wallace J, Alexander S, Estrada EY, Grossetete M, Razhagi A, Miller K, Gearing A. Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures. Brain Res. 2001; 893: 104–112.[CrossRef][Medline] [Order article via Infotrieve]
  5. Chang DI, Hosomi N, Lucero J, Heo JH, Abumiya T, Mazar AP, del Zoppo GJ. Activation systems for latent matrix metalloproteinase-2 are upregulated immediately after focal cerebral ischemia. J Cereb Blood Flow Metab. 2003; 23: 1408–1419.[CrossRef][Medline] [Order article via Infotrieve]
  6. Brown PD. Synthetic inhibitors of matrix metalloproteinases. In: Parks WC, Mecham RP, eds. Matrix Metalloproteinases. San Diego: Adademic Press, 1998: 243–262.
  7. Overall CM, Lopez-Otin C. Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer. 2002; 2: 657–672.[CrossRef][Medline] [Order article via Infotrieve]
  8. Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan, PH, Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci U S A. 1999; 96: 13496–13500.[Abstract/Free Full Text]




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