Introduction

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Inflammation is often accompanied and characterized by an unwanted excess of cell proliferation that may ultimately lead to fibrosis and sclerosis. Sclerosis of renal tissues, such as the glomerulus, is an important cause of end-stage kidney disease in humans. Therapy of end-stage kidney disease requires demanding and expensive methods, such as dialysis and renal transplantation.

In the kidney, there exists a whole spectrum of proliferative forms of glomerular inflammatory disorders. Mesangial proliferative glomerulonephritis is a particularly prominent example of a difficult to treat inflammatory renal disease (Couser, 1999). Increased mesangial cell proliferation also plays an important role in other glomerular diseases, such as diabetic nephropathy. Therefore, we have chosen the mesangial cell and experimental mesangial proliferative glomerulonephritis as a model for the investigation of a new anti-inflammatory therapy.

Induction of cell cycle arrest and apoptosis represents an established method to treat malignant disorders (Shapiro et al., 1999). A similar approach may also be useful for the therapy of inflammation (Gao et al., 1998). We used matrix metalloproteinases (MMP) and their synthetic inhibitors to evaluate such a strategy. In mesangial cells, inhibition of MMP expression and activity has profound effects beyond the degradation of extracellular matrix (ECM) proteins. The constitutive synthesis of mesangial cell MMP-2 was greatly reduced with antisense RNA produced by an episomally replicating vector or with specific anti-MMP-2 ribozymes expressed by a retroviral transducing vector (Turck et al., 1996). The transfected or retrovirally infected cells reverted from a proliferative, inflammatory phenotype to the quiescent state that occurs in the normal renal glomerulus. The distinct differences included changes in synthesis of ECM proteins, loss of activation markers, and an almost total stop of proliferation (Turck et al., 1996).

These studies were extended by the use of a synthetic MMP inhibitor. Exposure of mesangial cells to Ro 31-9790 in vitro resulted in a dose-dependent and reversible inhibition of cell proliferation, associated with a reduction in the expression of -smooth muscle actin (Steinmann-Niggli et al., 1997).

Elevated MMP expression and activity occur in inflammatory disorders of the kidney. After induction of anti-Thy 1.1 nephritis in rats, mesangiolysis is followed by an increase in activation and proliferation of mesangial cells, associated with augmented expression of MMP-2 (Lovett et al., 1992; Marti et al., 1994). Therefore, we treated these nephritic rats with a synthetic MMP inhibitor. Total glomerular cell count reflecting the degree of mesangial cell proliferation and ECM accumulation were significantly reduced (Steinmann-Niggli et al., 1998). Consequently, glomerular histology was markedly improved and proteinuria showed a clear tendency toward a decrease.

It is known from studies with tumor cells that synthetic MMP inhibitors may induce cell cycle arrest or even promote apoptosis (Burke et al., 1997; Erba et al., 1999). Therefore, we hypothesized that these compounds may exert similar effects in nontumor cells also. In the present study, we used BB-1101 to demonstrate the induction of MMP inhibitor-related mesangial cell apoptosis in experimental mesangial proliferative glomerulonephritis. Subsequently, we studied the mechanisms of this phenomenon in cultured mesangial cells by the use of RO111-3456, a closely related compound.

	Materials and Methods

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Animals and Antibodies. Male Wistar rats were obtained from the local animal facilities at our hospital. Approval for rat studies was attained from the commission for animal studies, a local government agency. Anti-Thy1.1 IgG (OX-7) was prepared as described previously (Steinmann-Niggli et al., 1998). A mouse monoclonal anti-statin IgM was provided by Dr. Eugenia Wang, McGill University, Quebec, Canada (Wang and Pandey, 1995). Anti-c-myc antibody has been described previously (Brunner et al., 2000).

MMP-Inhibiting Agents. The MMP inhibitor BB-1101 (M_r = 389.5 g/mol) was obtained from British Biotech Pharmaceuticals, Oxford, UK. According to the instructions by the manufacturer, BB-1101 is designed for in vivo studies and is only for limited use in in vitro experiments because of low aqueous solubility in cell culture medium. BB-1101 was suspended in PBS/0.1% Tween 80 (v/v) and sonicated for 5 min before injection. BB-1101 was used in a dose of 30 mg/kg of body weight per day, given by single daily intraperitoneal injections, as described (Steinmann-Niggli et al., 1998).

Anti-Thy1.1 Nephritis: Experimental Design. Four groups of male Wistar rats (150 g of body weight at day 0) were studied (n = 54): group A, healthy rats (n = 9); group B, pretreated healthy rats (n = 9); group C, nephritic rats (n = 18); group D, pretreated nephritic rats (n = 18). MMP inhibitor BB-1101 (groups B and D) or the respective amount of solvent (groups A and C) was given intraperitoneally once daily from day 2 to +14, as described (Steinmann-Niggli et al., 1998). Anti-Thy1.1 nephritis was induced at day 0 (groups C and D) as described previously (Steinmann-Niggli et al., 1998); healthy control rats (groups A and B) received the respective amount of PBS only. Nephrectomy for histological analyses was performed at days +4, +8, +11, and +14 after induction of disease. Blood levels of BB-1101 at day +11 were analyzed by British Biotech Pharmaceuticals as reported (Steinmann-Niggli et al., 1998).

Histological Analyses. Renal tissues were fixed for 24 h in 5% buffered formalin, dehydrated, and embedded in paraffin. Subsequently, kidneys were cut longitudinally into 2-µm sections for periodic acid Schiff reaction, TUNEL, and -smooth muscle actin staining, as described previously (Baker et al., 1994; Ziswiler et al., 1998).

Mesangial Cell Cultures. Rat mesangial cells were isolated and propagated as described previously (Lovett et al., 1992; Steinmann-Niggli et al., 1997, 1998). Cell synchronization occurred in culture medium containing 1% FCS for 24 h, if required. Subsequently, culture medium containing 10% FCS was supplemented with either 5 to 100 µM RO111-3456 in 0.03% DMSO, 0.03% DMSO only, or 1 to 7 mM captopril (Trocme et al., 1998), and was added to cell cultures for periods of up to 24 h. Mesangial cell proliferation was assessed by manual cell counting.

Mesangial Cell Necrosis. The effect of RO111-3456 and captopril on mesangial cell viability and necrosis was assessed by light microscopy, trypan blue exclusion, and release of lactate dehydrogenase (LDH) in cell culture supernatant (Ziswiler et al., 1998).

Gelatin Zymography. SDS-polyacrylamide gel electrophoresis (PAGE) was performed on a 10-well, 10% polyacrylamide minigel containing 0.1% gelatin (w/v), as described by us in detail (Lovett et al., 1992; Steinmann-Niggli et al., 1997, 1998). For inhibition studies, the developing buffer was supplemented with 1, 50, 100, or 500 nM RO111-3456 in 0.003% DMSO, or with 0.003% DMSO only. In a separate series of experiments, the buffer was complemented by the addition of captopril in final concentrations of 0, 15, and 30 mM (Sorbi et al., 1993), respectively.

TUNEL- and Acridine Orange/Ethidium Bromide (AO/EB) Staining. Mesangial cells were grown on glass coverslides to reach subconfluency. TUNEL assays of cells exposed to RO111-3456 were performed using an in situ cell detection kit (catalog number 1684817; Boehringer-Mannheim, Mannheim, Germany). For AO/EB staining of RO111-3456- and captopril-treated cells, 5 µl of freshly prepared AO/EB solution (100 µg/ml AO and 100 µg/ml EB in PBS) was added, and apoptosis was assessed immediately using an inverted fluorescence microscope (Baker et al., 1994; Amarante-Mendes et al., 1998).

Assessment of DNA Content by Flow Cytometry. Cell cycle analyses and quantification of apoptosis in mesangial cells exposed to RO111-3456 and captopril were investigated by FACS analyses using propidium iodide (PI) staining of nuclear DNA as published (Nicoletti et al., 1991; Healy et al., 1998).

FACS Analyses of Annexin V Binding. After exposure to RO111-3456, apoptotic mesangial cell death reflected by phosphatidylserine externalization was assessed by analyses of annexin V binding (annexin V-FITC kit, catalog number BMS306FI; Bender MedSystems, Boehringer Ingelheim Bioproducts, Heidelberg, Germany) (Amarante-Mendes et al., 1998). Cells were counterstained with PI to distinguish between apoptotic and late apoptotic/necrotic cells.

Western Blot Analyses of Statin, c-Myc, Fas/FasL, Caspase-3, p53, p21, and bax. Subconfluent mesangial cells were exposed to 100 µM RO111-3456 for periods of 12 and 24 h, as indicated.

Assessment of Caspase-8 Activity. Caspase-8 activity of RO111-3456-exposed mesangial cells was determined using a fluorometric protease assay kit based on the detection of cleavage of the substrate IETD-AFC (Caspase-8/Flice Fluorometric Assay kit; BioVision, Palo Alto, CA). Thereafter, fluorescence was analyzed in a spectrofluorometer (SPECTRAmax GEMINI; Molecular Devices, Sunnyvale, CA) using a 400-nm excitation filter and a 505-nm emission filter.

Inhibition of Caspase Activity by Acetyl-Asp-Glu-Val-Asp-Aldehyde (DEVD-CHO). The caspase inhibitory peptide DEVD-CHO (Bachem, Bubendorf, Switzerland) was dissolved in water and used at concentrations of 0 to 200 µM, as reported previously (Ortiz et al., 1998). DEVD-CHO was added to cell culture medium together with 100 µM RO111-3456 or 0.03% DMSO only. Apoptosis was assessed 24 h later by FACS analyses using annexin V-FITC/PI staining, as described above. Positive control consisted of 50 ng/ml anti-Fas antibody (CH-11; Upstate Biotechnology, Lake Placid, NY)-treated Jurkat cells, as described (Zhou et al., 1999).

Immunocytology for Statin. Cells were harvested after exposure to 100 µM RO111-3456 or to 0.03% DMSO for 12 h. Thereafter, staining of cytocentrifuge cell samples was performed as described (Hirt et al., 1997).

Statistical Analyses. In vitro results were expressed as mean ± S.D., and comparison of means was done by the Student's t test. In vivo data are given as median (percentile P₂₅ to percentile P₇₅); analyses were performed by ANOVA (with the Bonferroni adjustment). For all experiments, probability of error (P values) <0.05 was regarded to be significant.

	Results

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Apoptosis of Mesangial Cells in Vivo

Increased Rates of Apoptosis in Experimental Glomerulonephritis. Kidney sections of healthy, healthy pretreated, nephritic, and pretreated nephritic rats were used for histological analyses. Therapy consisted of daily applications of BB-1101 resulting in blood levels of approximately 100 nM, equivalent to multiples of MMP IC₅₀ concentrations, as reported previously (Steinmann-Niggli et al., 1998). We selected this compound, since we have used it successfully in anti-Thy1.1 nephritis in the past (Steinmann-Niggli et al., 1998). Furthermore, there were no guidelines or published data available on the in vivo use of RO111-3456.

View larger version (87K): [in this window] [in a new window]   Fig. 1.   Induction of apoptosis over time in anti-Thy1.1 nephritis. A, TUNEL staining of a tissue section from a nephritic rat 11 days after induction of nephritis (arrow denotes TUNEL-positive cell). Original magnification, 400×. B, time course of apoptosis in nephritic and BB-1101-treated nephritic animals. Analyses of 50 glomeruli/animal were performed at days +4, +8, +11, and +14. The data are depicted in box plots with median values and range from percentile P25 to percentile P75. *P < 0.05 compared with untreated nephritic animals.

View larger version (112K): [in this window] [in a new window]   Fig. 2.   Mesangial cell apoptosis in anti-Thy1.1 nephritis. A, TUNEL and -smooth muscle actin staining (original magnification, 100×) identifies a double-positive apoptotic mesangial cell (arrow). B, electron micrograph (original magnification, 10,000×) shows the lumen (l) of a capillary loop. Immediately surrounding are fenestrated endothelial cells (E). In close apposition, between the endothelial cell(s) and the basement membrane is a mesangial cell (M) with an apoptotic nucleus indicated by condensed homogeneous chromatin (arrow).

Cell Cycle Arrest and Apoptosis of Mesangial Cells in Vitro

Inhibition of MMP Activity. Conditioned medium of rat mesangial cells contained gelatinolytic activity exclusively derived from MMP-2. The zones of lysis in the zymogram were inhibited in a dose-dependent manner by exposure to RO111-3456, as demonstrated in Fig. 3A. Gelatinolytic activity in the gel strips clearly decreased following exposure to as little as 1 nM inhibitor and it almost completely disappeared after incubation with 500 nM RO111-3456. Similar inhibitor concentrations were used by us previously (Steinmann- Niggli et al., 1997). In addition, captopril lead to a dose-dependent and complete inhibition of MMP activity (Fig. 3B). All analyses were performed in duplicates with two identically treated samples on each gel strip.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   MMP-2 activity and proliferation of mesangial cells. A and B, zymography of MMP-2. Aliquots of conditioned medium from cell cultures were subjected to electrophoresis using 10% polyacrylamide gels impregnated with gelatin as described under Materials and Methods. A, gel was cut into five strips containing one lane each, and was developed by incubation in buffer containing 0.003% DMSO (vehicle) and RO111-3456 as follows: lane 1, 0.003% DMSO only; lane 2, 1 nM inhibitor; lane 3, 50 nM inhibitor; lane 4, 100 nM inhibitor; and lane 5, 500 nM inhibitor. B, gel was cut into three strips containing one lane each: lane 1, cell culture medium only; lane 2, 15 mM captopril; and lane 3, 30 mM captopril. C, analyses of cell proliferation. Subconfluent mesangial cells were exposed to RO111-3456 in concentrations up to 100 µM for 24 h. A concentration-response curve of RO111-3456 versus percentage inhibition of cell proliferation was obtained. Each point represents the mean ± S.D. of the assays of six culture wells.

Inhibition of Mesangial Cell Proliferation. Subconfluent mesangial cells were exposed to RO111-3456 in concentrations of 0 to 100 µM for 24 h. The addition of this MMP inhibitor to the culture led to a dose-dependent decrease in cell proliferation, as shown in Fig. 3C. An inhibition of 50% of cell proliferation occurred at a concentration of approximately 40 µM inhibitor and 61% inhibition at the maximal concentration of 100 µM.

Exclusion of Mesangial Cell Necrosis. Compared with untreated control cells, viability of mesangial cells exposed for 24 h to either 100 µM RO111-3456, 0.03% DMSO, or 7 mM captopril was not impaired, as analyzed by light microscopy and trypan blue exclusion (viability in all experiments >95%). Furthermore, LDH levels in mesangial cell culture supernatant expressed as percentage of total LDH release remained at constant levels: 6.1 ± 0.4% in control cells and 6.4 ± 0.4% in cells exposed to 100 µM RO111-3456 in 0.03% DMSO. LDH release was also not influenced by 0.03% DMSO alone, and by 7 mM captopril (data not shown). Therefore, both MMP-inhibiting agents caused no obvious signs of cell necrosis.

Cell Cycle Progression. For FACS analyses of nuclear DNA content by PI staining, subconfluent and synchronized mesangial cells were exposed to medium supplemented with 100 µM RO111-3456 or 0.03% DMSO only for time periods of 6, 12, and 24 h. Synchronization was realized by a previous 24-h incubation with culture medium containing 1% FCS. Subsequently, mesangial cells were stimulated with medium containing 10% FCS and the respective amount of inhibitors for further 24 h. All experiments were performed in triplicates.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Mesangial cell cycle progression. Effects of RO111-3456 treatment on the percentages of cells in G0/G1 phase (A) and in S phase (B) of the cell cycle. Mesangial cells were synchronized by serum starvation for 24 h. Subsequently, cells were exposed to 10% FCS containing medium in the absence (open circles) or presence of 100 µM RO111-3456 (solid circles). RO111-3456 treatment increasingly arrested cells in G0/G1 phase and reduced the percentage of cells in S phase accordingly. Each point represents the mean and vertical lines show S.E.M. (n = 3). *P < 0.01, compared with control cells without inhibitor exposure.

Analyses of Statin and c-Myc. The effects of 100 µM RO111-3456 on mesangial cell cycle arrest in the G₀/G₁ phase were confirmed by the analyses of statin, a 57-kDa protein expressed by truly quiescent cells in the G₀ phase (Turck et al., 1996). MMP inhibitor-treated cells displayed a distinctively higher level of statin after an incubation period of 12 h, as shown by Western blot analyses (Fig. 5, A and B) and immunocytology (Fig. 5C). Correspondingly, the synthesis of c-myc, a G₁ phase-specific gene, somewhat decreased as a result of the inhibitor exposure (Fig. 5, A and B) (Wang and Pandey, 1995; Brunner et al., 2000). Therefore, the expected inverse regulation of statin and c-myc expression was observed due to cell cycle arrest before the initiation of apoptosis (Wang and Pandey, 1995).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Synthesis of statin and c-myc by mesangial cells. Mesangial cells were exposed for 12 h to 100 µM RO111-3456. A, Western blot analyses demonstrated higher level of statin but lower amount of c-myc in inhibitor-treated cells compared with control cells. B, Western blot densitometry demonstrated approximately a 4-fold increase of statin and a 35% decrease of c-myc in inhibitor-treated cells compared with controls. C, immunocytology confirmed higher expression of statin, stained in red, in inhibitor-exposed cells (right), compared with control cells (left). Photographs were taken under identical conditions. Original magnification, 160×.

TUNEL and AO/EB Staining. TUNEL-positive, apoptotic mesangial cells were not present in the untreated control group (Fig. 6A). Exposure of cells to even 50 µM RO111-3456 for 24 h resulted in a distinct increase of TUNEL-positive cells, as shown in Fig. 6B.

View larger version (95K): [in this window] [in a new window]   Fig. 6.   Apoptosis detection in mesangial cell cultures: TUNEL staining. TUNEL staining of mesangial cells treated with RO111-3456 or solvent for 24 h. A, solvent of RO111-3456 alone caused no visible apoptosis. B, already 50 µM RO111-3456 treatment resulted in a significant number of apoptotic cells.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Apoptosis detection in mesangial cell cultures: AO/EB staining. AO/EB staining of mesangial cells treated with RO111-3456, solvent or captopril for 24 h. A, solvent-treated cells showed virtually no signs of apoptosis or necrosis. B, exposure to 100 µM RO111-3456 resulted in mostly late apoptotic events featuring a high number of cells with condensed nuclei; only very few necrotic cells were visible. C, exposure to 7 mM captopril revealed signs of early apoptosis with bright green nuclear dots, but hardly any necrosis. Photographs were taken under identical conditions. Original magnification, 250×.

Annexin V Binding. An early event during apoptosis is the externalization of phosphatidylserine, a phospholipid normally restricted to the inner leaflet of the plasma membrane (Healy et al., 1998). This apoptotic event can be monitored using annexin V, a phosphatidylserine-specific binding protein.

View larger version (34K): [in this window] [in a new window]   Fig. 8.   FACS analyses of annexin V binding. Mesangial cells were treated for 24 h with solvent (A) or with 100 µM RO111-3456 (B). The externalization of phosphatidylserine was assessed by measuring FITC-annexin V binding using PI as a counterstain. MMP inhibitor treatment led to a substantial increase in annexin V binding of mesangial cells. One representative experiment of three is shown.

Dose-Dependent Induction of Apoptosis. As an estimate of apoptosis, we calculated the percentage of cells in the "sub-G₀/G₁ phase" (Erba et al., 1999). This sub-G₀/G₁ peak represents a population of cells with reduced DNA content due to DNA fragmentation (Healy et al., 1998). To avoid potential cytotoxicity, we did not use inhibitor concentrations higher than 100 µM and incubation times longer than 24 h.

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Quantification of apoptosis in mesangial cells by FACS analyses. A, apoptosis induced by 100 µM RO111-3456. Left, a DNA profile of solvent-exposed control cells is depicted. Right, cells treated with 100 µM RO111-3456 for 24 h showed a 25% increase in apoptotic cells in the sub-G0/G1 phase of the cell cycle. B, dose-dependent induction of apoptosis by 24-h exposures to increasing concentrations of up to 100 µM RO111-3456. Results are expressed as percentage of cells in the sub-G0/G1 phase of the cell cycle and represent mean ± S.E.M. of six independent experiments. *P < 0.01 compared with untreated control cells.

Cell Cycle Arrest and Apoptosis over Time. RO111-3456 treatment in the maximal concentration of 100 µM for 6, 12, and 24 h in medium containing 10% FCS caused time-dependent increases in the percentage of the cells in the G₀/G₁ phase that was paralleled by decreases of cells in the G₂/M (mitotic/dividing) phases, as depicted in Fig. 10 and Table 1. These findings explain the dose-dependent antiproliferative effect of RO111-3456 in mesangial cells. Already after a 6-h exposure of MMP inhibitor, nonsynchronized mesangial cells started to accumulate in G₀/G₁ phase, representing cell cycle arrest. DNA fragmentation after 6 and 12 h remained minimal and only reached significant values after 24 h. Therefore, it can be concluded that cell cycle arrest occurs first and the induction of apoptosis is a subsequent event.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Temporal relation of cell cycle arrest and apoptosis. Mesangial cells were incubated in culture medium containing 10% FCS and 100 µM RO111-3456 for 0, 6, 12, and 24 h. Cell cycle arrest in the G0/G1 phase occurred first and apoptosis followed subsequently. One representative experiment of three is shown.

Fas/FasL and Caspases-3/-8. Alterations in Fas and FasL expression as a result of 100 µM RO111-3456 treatment were investigated following incubation periods of 12 and 24 h. At both time points, Western blot analysis failed to show an increase in the expression of 45-kDa Fas protein, 37-kDa FasL protein, and caspase-3 in the MMP inhibitor-exposed cells compared with untreated controls (data not shown). Furthermore, there was only a slight tendency toward an approximately 1.5-fold increase in caspase-8 activity in MMP inhibitor-treated cells at 24 h, as analyzed by fluorometric measurements (data not shown).

Inhibition of Caspases by DEVD-CHO. The peptide DEVD-CHO inhibits caspase-3 and related caspases (Ortiz et al., 1998). Apoptosis induced by RO111-3456 was attempted to be inhibited by this peptide. Whereas anti-Fas-induced apoptosis in Jurkat cells was dose dependently inhibited by DEVD-CHO as expected, DEVD-CHO in concentrations of 0 to 200 µM failed to attenuate 100 µM RO111-3456-induced apoptosis in mesangial cells. Results are summarized in Table 3. Therefore, we had no evidence that the Fas/FasL and probably also the TNF-/TNF-R pathways played a major role in the antiproliferative effect of the MMP inhibitor.

Analyses of p53, p21, and bax. Mesangial cells were exposed to 100 µM RO111-3456 for 12 h. At this time point distinct cell cycle arrest of mesangial cells was observed.

View larger version (28K): [in this window] [in a new window]   Fig. 11.   Synthesis of p53, bax, and p21 by mesangial cells. A, mesangial cells were exposed for 12 h to 100 µM RO111-3456. Scanning densitometry of Western blot analyses demonstrated approximately 4 times higher levels of p53, a 3-fold increase of bax, and almost 2 times higher amounts of p21 in inhibitor-treated cells compared with controls (representative results of one of three identical experiments are depicted). B, Western blot analysis of nuclear extracts obtained from glomeruli at day +8 demonstrated a distinct increase in p53 synthesis in nephritic animals as a result of BB-1101. Healthy rats showed little p53 expression (representative results of one of three animals are depicted). C, scanning densitometry of the Western blot demonstrated a 3- and 9-fold increase of p53 in BB-1101-treated nephritic rats compared with nephritic and healthy animals, respectively.

	Discussion

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The present study describes the profound effects of synthetic MMP inhibitors on cell cycle arrest and apoptosis in glomerular mesangial cells. MMPs are zinc-dependent metalloendopeptidases belonging to the collagenase supergene family. Primarily based on substrate specificity, they are classified into several groups, such as interstitial collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -7, -10, -11), and membrane type-MMPs (membrane type-MMP-1, -2, -3, -4) (Cuvelier et al., 1997). MMPs are secreted in a latent form as pro-MMP and extracellular activation occurs in a complex manner by conformational change and by proteolytic action of proteinases, such as plasmin and membrane-type matrix metalloproteinase (Davies et al., 1992). MMP activity is regulated by natural inhibitors, mainly, the tissue inhibitors of metalloproteinases (tissue inhibitor of metalloproteinase-1 to -4) (Nagase, 1997). The regulation of ECM metabolism is probably the main function of these proteolytic enzymes and their inhibitors. Recently, a broad spectrum of very effective, low-molecular-weight MMP inhibitors was developed by the addition of a zinc-chelator, mostly a hydroxamate, to a peptidyl moiety based on MMP substrates (Vincenti et al., 1994). Both compounds used in our studies belong to this category. Notably, certain synthetic low-molecular-weight MMP inhibitors also inhibit related metalloproteinases that process membrane-bound cytokines and growth factors, such as TNF- (McGeehan et al., 1994).

Synthetic MMP inhibitors already demonstrated significant benefits in various animal models for inflammatory diseases, such as arthritis (Conway et al., 1995), experimental allergic encephalomyelitis (Hewson et al., 1995), asthma (Kumagai et al., 1999), and glomerulonephritis, as already mentioned (Steinmann-Niggli et al., 1998). However, the precise mechanism on the cellular level beyond MMP inhibition remains to be fully determined.

Unopposed mesangial cell proliferation associated with accumulation of ECM proteins is an important cause of glomerular scarring with loss of kidney function. However, if mesangial proliferation resolves in a timely manner, glomerular structure and function may revert back to normal (Baker et al., 1994). We speculated that resolution of excess mesangial cell proliferation may be facilitated by the induction of cell cycle arrest with subsequent apoptosis. Apoptosis plays a central role in maintaining homeostasis in tissues and organs such as the kidney by the deletion of "unwanted" cells without induction of an inflammatory reaction (Baker et al., 1994). Increased apoptotic cells were detected in glomeruli of humans with IgA nephropathy (Tashiro et al., 1998). In animals, apoptosis was shown to play a prominent role in the resolution of anti-Thy1.1 nephritis, a rat model of mesangial proliferative glomerulonephritis (Baker et al., 1994). In this disease, anti-Thy1.1 antibodies induce a self-limited mesangial cell proliferation associated with marked increases in cyclin A and cyclin-dependent kinase 2 (Shankland et al., 1996). Apoptosis occurred approximately 10-fold more frequently in glomeruli of nephritic rats compared with healthy controls with clear morphological evidence of mesangial apoptosis leading to phagocytosis by neighboring mesangial cells (Baker et al., 1994).

Since MMP inhibitors can induce cell cycle arrest or apoptosis in malignant cells (Burke et al., 1997; Erba et al., 1999), we examined the effect of BB-1101 in anti-Thy1.1 nephritis. The process of mesangial cell clearance by apoptosis was greatly enhanced by MMP inhibitor treatment. TUNEL staining was used to discover glomerular cells with fragmented DNA, a hallmark of apoptosis. The therapy with BB-1101 led to a 4.5- to 6-fold increase in TUNEL-positive apoptotic glomerular cells 11 and 14 days after induction of nephritis, respectively. In anti-Thy1.1 nephritis, the overwhelming majority of proliferating cells that may be arrested in their cell cycle by any type of intervention are mesangial cells (Steinmann-Niggli et al., 1998). Accordingly, in our study apoptotic cells were almost exclusively limited to the mesangium, as described (Baker et al., 1994). Importantly, mesangial cell apoptosis was confirmed by electron microscopy as well as by TUNEL and -smooth muscle actin double-staining.

Apoptosis remained negligible in the tubulo-interstitium and there were no signs of a concomitant inflammatory reaction as a result of an unlikely necrosis. Furthermore, the amounts of apoptotic cells, in relation to absolute numbers of glomerular cells, followed the time course of the proliferation rates of mesangial cells and were lower at day +14 than at day +11, despite the continuous application of the MMP inhibitor. In the case of nonspecific toxicity, one might have expected rather a more relentless rise in apoptosis.

Therefore, we selected the mesangial cells as a model to study the MMP inhibitor effect on the cellular level. RO111-3456 caused significant cell cycle arrest in G₀/G₁ phase in cultured mesangial cells associated with increased expression of statin. Cell cycle arrest was followed by apoptosis. Increased induction of apoptosis was demonstrated and confirmed by various morphological and biochemical tests. Importantly, the induction of statin may well exclude a nonspecific, toxic effect of the MMP inhibitor treatment.

The MMP inhibitor concentrations used in vitro were much higher than its blood levels achieved in vivo. However, the application of BB-1101 in vivo occurred over a relatively prolonged period and achieved rates of apoptotic cells in the low range of pars pro mille. In contrast to these experiments, RO111-3456 was given in vitro over a very short time period and attained rates of apoptotic cells in the order of many percentages. The high amount of apoptosis in cultured mesangial cells facilitated its detection and confirmation by the various tests used.

Elimination of apoptotic cells is a rapid process with a clearance time of only a few hours (Baker et al., 1994). Therefore, even minute changes in absolute amounts of apoptotic cells over time may have significant effects on the cell content of a given tissue (Baker et al., 1994). In this respect, for future clinical studies, even lower amounts of MMP inhibitors may prove to be successful when given over a longer time period. To induce apoptosis in mesangial cells, MMP inhibitors may be particularly useful since these cells are believed to be very resistant to this type of cell death (Baker et al., 1994). Therefore, MMP inhibitors may be especially useful for the therapy of mesangial cell-mediated kidney inflammation.

To the best of our knowledge, this is the first report showing synthetic MMP inhibitor-induced cell cycle arrest followed by apoptosis in nontumor cells. Although MMP activity is inhibited by these compounds, it remains to be demonstrated to which extent precisely this effect is functionally linked to proapoptotic actions in mesangial cells.

Cell cycle arrest of mesangial cells as a result of RO111-3456 treatment is well explained by induction of p53 and p21 (Shaw, 1996; Amundson et al., 1998). Since captopril, a structurally very different agent inhibiting MMP activity, also influenced cell cycle of these cells to a similar extent, it is possible, although far from being proven, that inhibition of MMP in fact played a causative role. Future studies are needed to resolve this issue.

The subsequent apoptosis of mesangial cells is more complex, although well explained by the up-regulation of p53, also shown in vivo, and of bax (Shaw, 1996; Amundson et al., 1998). Furthermore, the observed down-regulation of c-myc, an important proliferation signal, probably is also the result of p53 induction (Amundson et al., 1998).

Hydroxamic acid-based MMP inhibitors, such as the compounds used in our study, were shown to inhibit the metalloproteinase responsible for processing of transmembrane FasL (Tanaka et al., 1998). Therefore, it might have been conceivable that RO111-3456 facilitated apoptosis by stabilization of the transmembrane form of FasL and hence prevented the down-regulation of FasL by its shedding (Tanaka et al., 1998). However, a significant participation of the Fas/FasL pathway, including the downstream caspases, was not detectable. Therefore, MMP inhibitor-related apoptosis appeared to follow a caspase-independent pathway (Brown et al., 2000). The investigation of the gene products responsible for p53-mediated apoptosis beyond bax is very extensive and involves a separate project.

The role of MMP and their inhibitors in the regulation of cell cycle and apoptosis is still emerging. MMPs themselves are involved in the regulation of cell proliferation (Turck et al., 1996; Steinmann-Niggli et al., 1997, 1998) and to a certain extent apoptosis (Vu et al., 1998). The influence of synthetic MMP inhibitors on cell cycle and apoptosis is well documented, especially for tumor cells. The MMP inhibitor batimastat (BB-94) was shown to enhance interferon--induced apoptosis in mice with ovarian cancer (Burke et al., 1997) and to block ovarian cancer cells in the G₀/G₁ phase of the cell cycle unrelated to p53 expression (Erba et al., 1999). AG3340 promoted apoptosis in human prostate and colon carcinoma models (Shalinski et al., 1999). Furthermore, GM-6001 was shown to induce apoptosis in smooth muscle cells (Cowan et al., 2000).

In summary, MMP inhibitors induce cell cycle arrest with subsequent apoptosis in mesangial cells in vitro and in vivo. These features may explain the beneficial anti-inflammatory features, such as reduction of excess mesangial cell proliferation and of ECM accumulation. Therefore, it may be concluded that the concept of induction of cell cycle arrest with subsequent apoptosis may contribute to the development of new perspectives in the therapy of inflammation even beyond the scope of kidney diseases.