MATERIALS AND METHODSMATERIALS AND METHODS Study Design This study was performed on the common carotid arteries of 87 male Sabra rats fed a normal diet (400±30g). The animals were divided, at random, into 4 treatment groups of angioplastied arteries (Figure 1). MMP activity in control and treated animals was determined in the carotid arteries of 20 additional rats sacrificed 3 days after balloon angioplasty (Figure 1).Balloon Angioplasty A 2F Fogarty balloon catheter was introduced through the left external carotid into the common carotid artery. The catheter was passed four times through a distance of 10-11 mm with the balloon inflated sufficiently to generate slight resistance. A 5-6 mm segment distal to the latter served as the non-injured control, reference segment for each vessel. (Figure 2)

Luminal NarrowingLuminal Narrowing was assessed as percent cross-sectional-area narrowing of the lumen by neointima %CSAN-N=IEL AREA-LA x 100/IEL AREA (LA - absolute luminal cross-sectional-area; IEL AREA -Internal Elastic Lamina area of reference). In order to avoid errors due to vessel deformation, %CSAN-N was calculated assuming the vessel to describe a perfect circle using the formula Area=C2/4.

RemodelingThe degree of remodeling was estimated by determining the ratio of the total vessel wall area (EEL area [calculated from perimeter using the formula Area=C2/4]) of the balloon-injured segment to the EEL area of the adjacent, non-injured reference segment [Remodeling Ratio (RR)]. Constrictive (negative) remodeling was defined as RR < 0.95, and expansive (positive) remodeling as RR > 1.05. Since reference segments were not obtained in 2 of the 87 animals (one Batimastat only [25D], one control [25D]), these 2 were excluded from the remodeling analyses.

Bromodeoxyuridine (BrdU) stainingIn order to identify proliferating cells, sections adjacent to those for histomorphometry were processed for BrdU staining. BrdU was injected intraperitoneally (50 mg/kg) 12 and 1 hour before sacrifice. Sections were deparaffinized at 60 degrees and rehydrated with xylene, 100% alchohol, 95% alcohol, and phosphate buffered saline (PBS) (3min, 2 times each). Sections were blocked with 0.3% hydrogen peroxide in absolute methanol for 15 min (37 degrees) and washed in PBS. After trypsinization (T-7168 SIGMA, 0.1% in deionized distilled water ,15 min, 37° C degrees, additional PBS wash (3min, 2 times), 2N HCl (30 min, 37° C), and another PBS wash (3min., 2 times), the sections were blocked with horse serum diluted 1:10 in PBS for 20-30 min (Biological Industries, Israel, CAT # 02-023-5A, 37 ° C). After blocking, the primary antibody, mouse-anti-rat BrdU (monoclonal Ab, clone BMC 9318, Boehringer), dissolved in 0.1% bovine serum albumin (BSA) in PBS (Ab dilution, 1: 20), was applied for 60 min. in a humified chamber (37° C). Following PBS wash (3min., 2 times), the secondary biotinylated, rat-anti-mouse absorbed antibody (Cat# BA-2001, Vector Laboratories, USA) in 0.1% bovine serum albumin (BSA) in PBS (Ab dilution,1:100), was applied for 60 minutes (humified chamber, 37°C). Amplification and developing solutions were applied in accordance with the protocol of the ABC kit (Vector laboratories, Cat # PK-4002) with exposure time of 30 min. in a humidified chamber (37° C). After PBS wash (3min., 2 times), the specimens were stained with diaminobenzidine (DAB, Sigma Fast DAB tablets, Cat #D4418) prepared by dissolving 2 tablets of DAB in deionized water (4 min.). Sections were counterstained with Mayer’s Haematoxylin (1 minute).

ZymographyFrozen segments of arteries from 20 additional rats sacrificed 3 days after balloon angioplasty were homogenized in a buffer containing 50 mM tris, 0.2M NaCl, 5 mM CaCl2, and 0.02% Brij 35 (w/v) adjusted to pH 7.6. Following extraction, the homogenate was centrifuged at 10,000 g for 5 min. at 4° C. The protein content was measured, and an equal amount of protein was separated on gelatin-impregnated (1mg/ml, Difco, Detroit, MI), 8% SDS acrylamide gel under non-reducing conditions Collagenolytic activity was determined blindly, the intensity of the various bands (Total enzymatic activity of MMP-2) was determined using a computerized densitometer (type 300A, Molecular Dynamics, using the Image Gauge V3 image analysis program) with activity presented in relative units with respect to non-treated control .

Statistical AnalysisData are reported as mean number of arteries±standard deviation. The effect of the different treatments on individual histomorphometric or zymographic parameters was assessed by Analysis of Variance (ANOVA) with Fisher Protected Least Significant Difference (FPLSD) as the post hoc test. Two-factor, repeated measurements ANOVA was not used in order to retain the statistical and biological individuality of each time point. Correlation test was used for the correlation assessment. Categorical data were analyzed by the coded chi-square contingency analysis. The Statview II statistical package (Brain Power, Inc.) was was used for these calculations. This work was performed in in the Hebrew University of Jerusalem. (see also Atherosclerosis 163: 269-277)

RESULTSRESULTSThe absolute number of the BrDU stained cells was significantly lower in the neointima 14, 25, and 75 days after the injury in the group which received combined treatment vs the control group (p=0.0001, 0.001 and 0.01 respectively) see Figure 3 and 4. The mean relative intensity of the MMP-2 bands of control arteries excised 3 days after angioplasty (100±13), measured by computerized densitometry, was significantly greater than animals treated with Batimastat alone (72±10, p=0.001), avb3 receptor-inhibiting RGD peptide alone (69±12, p=0.0001), Batimastat plus peptide (angioplastied)(35±5, p=0.0001), or Batimastat plus peptide (no angioplasty)(25±3, p=0.0001 by ANOVA with FPLSD) see Figure 6. The %CSAN-N ratio was significantly greater, 14, 25, and 75 days after balloon injury in the carotid arteries of rats treated with the MMP inhibitor Batimastat alone, the avb3 inhibiting RGD peptide alone, or the two together compared to corresponding, non-treated, angioplastied controls; with these differences being most marked at 75 days (Figure 5). Strong positive correlation between the number of the BrdU stained cells in the intima and %CSAN-N was found r = 0.5 (14D), r = 0.7 (25D), r = 0.72 (75D). The number of the BrdU stained cells in the intima also strongly correlated with the MMP-2 activity measurements r = 0.73(14D), r = 0.99 (25D), r = 0.72 (75D). Constrictive vessel wall remodeling, most marked 75d after balloon injury, was completely prevented at this time point in treated animals (Figure 7). Augmentative effect of the vitronectin inhibitor peptide on cell proliferation was very significant 25 D after the injury: combined therapy versus RGD peptide (p=0.0001), combined therapy versus Batimastat (p=0.0001).

DISCUSSIONDISCUSSIONRheumatoid arthritis (RA) and osteoarthritis are chronic diseases that result in cartilage degradation and loss of joint function. Currently available drugs are predominantly directed towards the control of pain and/or the inflammation associated with joint synovitis but they do little to reduce joint destruction. Currently no disease modifying agents are used clinically for OA. In the future, it will be important to have drugs that prevent the structural damage caused by bone and cartilage breakdown.  MMPs are able to cleave all components of the cartilage matrix.Regulation of MMPs is aberrant in osteoarthritis and RA, and MMPs have been implicated in the collagen breakdown that contributes to joint destruction in these diseases.    We found that MMP inhibition resulted in a significant decrease in the cell proliferation and the remodeling of the extracellular matrix up to 2½ months after the MMP injection versus untreated control group and that these effects were associated with a marked reduction in MMP-2 activity.  The long term effect of the inhibitor may be related to it accumulation in the peritoneal cavity with a slow release effect as we described in our previous work. The long term effect of MMP inhibition on the extracellular matrix remodeling and cell proliferation supports the potential role of MMP inhibitors as disease modifying agents for the treatment of chronic pain induced by osteoarthritis and disk degeneration . However, results from clinical trials of MMP inhibitors have been equivocal, with some studies being terminated because of side effects or safety concerns.   The side effects of the MMP inhibitors that were tested in the clinical studiescould be related to the fact that the existing MMP inhibitors (including Batimastat as well as its oral analogue Marimastat ) are non-tissue specific.  We can suggest that local administration via facet joint injections or IDET may improve the efficacy and decrease the side effects.  The impressive long term effectiveness of the inhibitor in our study may indicate that smaller oral doses of the MMP inhibitors for longer time periods should be assessed in the arthritis and chronic pain trials in comparison to the high doses tested in the resent cancer studies in order to improve the side profile. We strongly advocate testing of matrix metalloproteinase inhibitors in larger animals and in the clinical setting as a potential disease modifying agent for non-cancer and cancer chronic pain syndromes.

Figure 1

STUDY DESIGNRAT CAROTID ARTERIES

GROUP I GROUP II GROUP III GROUP IV CONTROL BATIMISTAT* anti-v3 BATIMASTAT PEPTIDE† PLUS PEPTIDE

SURVIVAL TIMES AFTER ANGIOPLASTY FOR EACH GROUP3 DAYS (n = 6,4,6,5)

14 DAYS (n = 5,6,5,6)25 DAYS (n = 5,6,6,6)75 DAYS (n = 5,6,6,6)

ADDITIONAL CAROTID ARTERIES FOR METALLOPROTEINASE ACTIVITY3 DAY SURVIVAL ONLY

GROUP I (n = 4)GROUP II (n = 4)

GROUP III ( n = 4)GROUP IV (n = 4)

GROUP IV w/o BA (n = 4)*4-(N-Hydroxyamino)-2R-isobutyl-3S-(thienylthiomethyl)succinyl]-L-phenylalanine-N-methylamide †Gly-Penicillamine-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala

COMMON CAROTID A.

INJURED SEGMENT

REFERENCE SEGMENT

INTERNAL CAROTID A.

EXTERNAL CAROTID A.

10-11mm

5-6 mm

(catheter entry site)

Figure 2

Figure 3

BrDU and Movat Stained Sections of Rat Common Carotid Arteries 75 Days After Balloon Injury:

Note marked reduction in luminal narrowing by neointima in artery of animal treated with the MMP-inhibitor Batimastat plus the alpha-v beta-3 vitronectin receptor inhibiting peptide B versus non-treated angioplastied control A.

A. B.

C: BrdU stained section of small intestine (external control of the staining). Note very intensive and specific BrdU staining of the nuclei D and E: BrDU stained common carotid arteries 75 days after injury. D = control group. E = maximally treated group. Note the significant decrease of the BrDU staining in the neointima of the treated vs control artery. A-E Mayer’s Haematoxylin counterstained x 320.

C. D. E.

Note that the absolute number of the BrDU stained cells was significantly lower in the neointima 14, 25, and 75 days after the injury in the group which received combined treatment vs the control group.

Figure 4

BrdU Staining in Neointima

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Note marked reduction in d %CSAN-N and marked increase in luminal area ratio 14, 25, and 75 days after balloon angioplasty particularly in animals treated with Batimastat combined with the v3 receptor peptide compared to non-treated angioplastied control.

Figure 5

%CSAN-N

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Zymography:Mean relative intensity of collagenolytic activity of MMP-2 by computerized densitometry in rat carotid arteries 3 days after balloon injury. Note marked reduction in MMP-2 activity after Batimastat (p=0.001), peptide(p=0.001), or combined treatment (p=0.0001) compared to non-treated angioplastied control arteries. Both@ = combined treatment without angioplasty.

Figure 6

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Note significant constrictive remodeling (RR< 0.95) in non-treated control arteries 14, 25 and 75 days after balloon injury. For specific values of RR in each. Prevention of constrictive remodelling by Batimastat, peptide or combined treatment was most marked at 75 days.

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Figure 7

Remodelling ratio:

INTRODUCTIONINTRODUCTION Matrix metalloproteinases (MMP) are the key enzymes in the pathogenesis of osteoarthritis and inflammatory arthritis. Inhibition of the matrix metalloproteinases has been postulated to block metastasis invasion and to play an important role in the pathogenesis of both non-cancer and cancer chronic pain.  Gelatinase A (matrix metalloproteinase-2) participates in the degradation of the extracellular matrix components in a variety of arthritis. Gelatin zymography revealed that the activaty of Gelatinase A (matrix metalloproteinase-2) in synovial fluid joints of gouty arthritis was associated with the local arthritis activity scores (LAS) (p < 0.001) and correlated well with the neutrophil counts in the synovial fluid as well (p < 0.001).

 Matrix metalloproteinases have been implicated in the excessive breakdown of extracellular matrix components during disc degeneration. The activity of Gelatinase A (matrix metalloproteinase-2) was increased in the ovine model of intervertebral disc degeneration one of the major causes of the low back pain in humans.

 Chronic constriction injury to peripheral nerve causes a painful neuropathy in association with a process of axonal degeneration and endoneural remodeling that involves macrophage recruitment and local increase in matrix metalloproteinases, including Gelatinase A (matrix metalloproteinase-2) activity with a possible relevance to the pathogenesis of neuropathic pain  Extracellular matrix remodeling is the major pathologic process in the pathogenesis of osteoarthritis.

Extracellular matrix cell proliferation is involved in the pathogenesis of inflammatory arthritis and metastasis progression in cancer pain syndromes. Our study determines the long term effect of metalloproteinase inhibition on extracellular matrix remodeling, cell proliferation and migration in rats. The study was performed on the common carotid artery model because of the good accessibility and reliable injury assessment techniques of this model in comparison to articular cartilage. It addresses the direct effect of the mechanical injury on matrix metalloproteinase 2 activity using the methods developed in the previous study . This is a long term prospective randomized study that studies the long term effect of of metalloproteinase inhibitor on cell proliferation and extracellular matrix remodeling in vivo

Matrix Metalloprteinase Inhibitors from Animal Model to Clinical StudyMatrix Metalloprteinase Inhibitors from Animal Model to Clinical Study

Leon Margolin, MD, PhD a; Reuven Reich, PhDb; Louise S. Perez, BSc b; S. David Gertz, MD, PhD b  Leon Margolin, MD, PhD a; Reuven Reich, PhDb; Louise S. Perez, BSc b; S. David Gertz, MD, PhD b  a Department of Pain Medicine, UPMC b Hebrew University of Jerusalem, Israel;a Department of Pain Medicine, UPMC b Hebrew University of Jerusalem, Israel;

ABSTRACTABSTRACT Introduction: Matrix metalloproteinases (MMP) are the key enzymes in the pathogenesis of osteoarthritis and inflammatory arthritis. Inhibition of the matrix metalloproteinases has been postulated to block metastasis invasion and to play an important role in the pathogenesis of both non-cancer and cancer chronic pain.

Our study determines the long term effect of metalloproteinase inhibition on extracellular matrix remodeling, cell proliferation and migration in rats. The study was performed on the common carotid artery model because of the good accessibility and reliable injury assessment techniques of this model in comparison to articular cartilage. It addresses the direct effect of the mechanical injury on matrix metalloproteinase 2 activity using the methods developed in the previous study. 

Methods: 91 slides from the the common carotid artery of Sabra rats treated with the metalloproteinase inhibitor underwent immunohistochemical staining and histomorphometric evaluation

 Results: The absolute number of the BrDU stained cells was significantly lower in the neointima 14, 25, and 75 days after the injury in the group which received combined treatment vs. the control group (p=0.0001, 0.001 and 0.01 respectively). The mean relative intensity of the MMP-2 bands (100±13), measured by computerized densitometry was significantly greater than in control group than in the treated animals.

Conclusion: We found that MMP inhibition resulted in a significant decrease in the cell proliferation and the remodeling of the extracellular matrix up to 2½ months after the MMP injection versus untreated control group and that these effects were associated with a marked reduction in MMP-2 activity. We strongly advocate testing of matrix metalloproteinase inhibitors in larger animals and in the clinical setting as a potential disease modifying agent for non-cancer and cancer chronic pain syndromes.

 Since Batimastat as well as its oral analogue Marimastat are non-tissue specific matrix metalloproteinase inhibitors we can suggest that local administration via facet joint injections may improve the efficacy and decrease the side effects.