Effects of glucosamine and chondroitin sulfate on mediators of osteoarthritis in cultured equine chondrocytes stimulated by use of recombinant equine interleukin-1beta

OBJECTIVE: To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. SAMPLE POPULATION: Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. PROCEDURE: Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1beta. Effects of prior incubation with glucosamine (2.5 to 10.0 microg/mL) and CS (5.0 to 50.0 microg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor kappaB; and c-Jun-N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay. RESULTS: Glucosamine at a concentration of 10 microg/mL significantly reduced reIL-1beta-induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage-sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking.     

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

OBJECTIVE: To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. SAMPLE POPULATION: Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. PROCEDURES: Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. RESULTS: Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.     

Effects of methylprednisolone acetate and glucosamine on proteoglycan production by equine chondrocytes in vitro

OBJECTIVE: To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations. SAMPLE POPULATION: Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age). PROCEDURES: In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 microg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content. RESULTS: Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied. CONCLUSIONS AND CLINICAL RELEVANCE: Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.     

Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate

Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.     

Comparison of inhibitory effects of glucosamine and mannosamine on bovine articular cartilage degradation in vitro

OBJECTIVE: To compare the inhibitory effects of glucosamine and mannosamine on articular cartilage degradation and the effects on chondrocyte viability in vitro. SAMPLE POPULATION: Bovine articular cartilage explants. PROCEDURES: Explants were cultured in commercial medium for 48 hours. Cartilage was exposed to medium containing 10% fetal bovine serum, 10 microg of lipopolysaccharide/mL, and 0.5, 1.0, 2.5, 5.0, and 10.0 mg of glucosamine or mannosamine/mL for 24 hours. Nitric oxide (NO) production (nitrite concentration) and proteoglycan (PG) release (PG concentration) in media were measured. Cartilage extracts were analyzed via zymography to detect gelatinolytic activity. At the end of the experiment, explants were assessed for chondrocyte viability. RESULTS: Addition of lipopolysaccharide resulted in increased NO production and PG release, but no increase in gelatinolytic activity, compared with controls. Glucosamine and mannosamine at concentrations as low as 0.5 mg/mL inhibited NO production. Glucosamine inhibited PG release at a minimum concentration of 1.0 mg/mL, whereas mannosamine inhibited PG release at a concentration of 0.5 mg/mL. Concentrations of glucosamine < or = 5.0 mg/mL did not adversely affect chondrocyte viability; however, at a concentration of 10.0 mg/mL, cell death was evident. Mannosamine had a toxic effect at a concentration of 5.0 mg/mL and was associated with pronounced chondrocyte death at a concentration of 10.0 mg/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Glucosamine and mannosamine inhibit selected indices of bovine articular cartilage degradation at concentrations that do not affect chondrocyte viability. The potential for cytotoxic effects at higher concentrations underscores the importance of establishing appropriate dosage regimens for these aminomonosaccharides.     

Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system. SAMPLE POPULATION: Humeral head articular cartilage chondrocytes obtained from 6 adult dogs. PROCEDURE: Chondrocytes were cultured in a 3-D system for < or = 12 days in serum-free medium with IL 1alpha, IL-1beta, or TNF-alpha at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines. RESULTS: In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1beta (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-alpha-treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1beta (50 ng/mL) and IL-1beta (100 ng/mL) groups at days 1 and 3, in the IL-1beta (100 ng/mL) group at day 6, and in all TNF-alpha groups at days 1, 3, and 6. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that TNF-alpha more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1beta. In vitro, IL-1alpha appeared to have a minimal effect on canine chondrocytes.     

Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures

OBJECTIVE: To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. SAMPLE POPULATION: Cultured equine chondrocytes. PROCEDURE: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1beta and exogenous PGE2 (5 mg/ml) with appropriate controls. RESULTS: Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1beta-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10(-5) M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to relL-1beta and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1. CONCLUSIONS AND CLINICAL RELEVANCE: The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies.     

Matrix metalloproteinase expression and localization in turkey (Meleagris gallopavo) during the endochondral ossification process

Vertebrate long bones are formed by endochondral ossification, a process accompanied by changes in extracellular matrix synthesis and remodeling, performed mainly by the matrix metalloproteinases (MMP). The temporal/spatial expression patterns of 5 members of the MMP family known to be important for endochondral ossification were studied, for the first time, in the turkey growth plate during embryonic and juvenile stages. The expression of MMP-2 was detected in the proliferative zone, MMP-3, MMP-9, and MMP-13 in cells lining the blood vessels; MMP-13 was also detected in hypertrophic chondrocytes. The MMP-16 expression was detected in the reserve zone of the growth plate. These results present a detailed survey of turkey MMP, serving as a data source (atlas) for further studies in this subject.     

Evaluation of collagenase activity, matrix metalloproteinase-8, and matrix metalloproteinase-13 in horses with chronic obstructive pulmonary disease.

OBJECTIVES: To determine collagenase activity and evaluate matrix metalloproteinase (MMP)-8 and MMP-13 in horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 12 horses with COPD and 12 healthy control horses. PROCEDURE: Collagenase activity was determined by use of an assay for degradation of type-I collagen. Western immunoblot analysis was used to identify interstitial collagenases MMP-8 and MMP-13 in tracheal epithelial lining fluid (TELF). Immunocytochemistry and in situ hybridization were used to determine cellular expression of these 2 collagenases in cells in bronchoalveolar lavage fluid (BALF). RESULTS: Collagenase activity was approximately 7 times higher in samples obtained from horses with COPD, compared with control horses. During stabling, horses with COPD had significantly higher collagenase activity than after being maintained on summer pasture, when activity was similar to that of control horses. Immunoreactivity of MMP-8 and MMP-13 was significantly increased in TELF of horses with COPD, compared with healthy horses. In TELF, a positive correlation was detected between immunoreactivity of MMP-8 and MMP-13 and the amount of degradation of type-I collagen. Macrophages and epithelial cells were the major cellular sources of MMP-8 and MMP-13. CONCLUSIONS AND CLINICAL RELEVANCE: Increased collagenase activity in TELF indicates active ongoing disease and, thus, may reflect lung tissue changes in horses with COPD. Measurements of collagenase activity and MMP immunoreactivity may provide additional diagnostic tools to identify the active phase of chronic lung disease.     

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.     

Assessment of the catabolic effects of interleukin-1beta on proteoglycan metabolism in equine cartilage cocultured with synoviocytes

OBJECTIVE: To evaluate the effects of interleukin (IL)-1beta on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes. SAMPLE POPULATION: Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses. PROCEDURES: 3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1beta (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectrophotometric assay; RNA was isolated from synoviocytes and cartilage and analyzed for expression of matrix metalloproteinases (MMP)-3 and -13 (cartilage and synoviocytes), aggrecan (cartilage), collagen type IIB (cartilage), and 18S as a control (cartilage and synoviocytes) by use of quantitative PCR assays. Cartilage matrix metachromasia was assessed histochemically. RESULTS: IL-1beta-induced GAG loss from cartilage was significantly less in cocultures than in cartilage-only cultures. Cartilage aggrecan gene expression was also significantly less downregulated and synoviocyte MMP-3 expression was less upregulated by IL-1beta in cocultures, compared with cartilage- and synoviocyte only cultures. Histochemical findings supported the molecular and biochemical results and revealed maintenance of matrix metachromasia in cocultured cartilage treated with IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synoviocytes secrete 1 or more mediators that preferentially protect matrix GAG metabolism from the degradative effects of IL-1beta. Further studies involving proteomic and microarray approaches in similar coculture systems may elucidate novel therapeutic targets for the treatment of osteoarthritis.     

大肠杆菌及脂多糖对奶牛乳腺上皮细胞基质金属蛋白酶表达的影响

为研究大肠杆菌(E.coli)及脂多糖(LPS)对奶牛乳腺上皮细胞(BMECs)基质金属蛋白酶(MMPs)表达的影响,以及MMPs与基质金属蛋白酶组织抑制因子(TIMPs)、尿激酶型纤溶酶原激活物(uPA)系统在调控细胞外基质(ECM)代谢中的作用,分别以106 CF U/mL热灭活E.coli菌液、7.5μg/mL ...     

Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage

OBJECTIVE: To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage. SAMPLE POPULATION: Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers. PROCEDURE: Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency. Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured. Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores. Liquid culture media were collected every 4 days and GAG content measured. Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37 degrees, 45 degrees, or 55 degrees C. Cell viability was determined via 2 different assays immediately and 24 hours after treatment. RESULTS: Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining. Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period. Cell viability was significantly different between controls and cells treated at 55 degrees C immediately and 24 hours after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences. The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation.     

Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis

OBJECTIVE: To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. PROCEDURE: Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP-13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. RESULTS: Addition of IL-1beta caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Human recombinant IL-1beta resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis.     

In vitro effects of lactoferrin on lipopolysaccharide-induced proliferation, gene expression, and prostanoid production by bovine peripheral blood mononuclear cells

Objective-To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro. Sample Population-PBMCs isolated from 15 Holstein bull calves. Procedures-Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 mug/mL) in a checkerboard design. Incorporation of (3)H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E(2) production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays. Results-Lactoferrin supplementation significantly reduced LPS-induced incorporation of (3)H-thymidine and production of prostaglandin E(2) by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA. Conclusions and Clinical Relevance-Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E(2) production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.     

Effects of autologous stem cells on immunohistochemical patterns and gene expression of metalloproteinases and their tissue inhibitors in doxorubicin cardiomyopathy in a rabbit model

This study aims to investigate the expression of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in chronic doxorubicin cardiomyopathy in a rabbit model and to evaluate the effects of bone marrow-derived mesenchymal stem cell (MSC) transplantation in this disease. Thirty-nine 3-month-old New Zealand rabbits were divided into 4 groups: group 1 (n = 9) was the untreated control. Groups 2-4 were treated with 6 weeks of doxorubicin (3 mg/kg). Group 2 (n = 6) received no further treatment. In group 3 (n = 9), animals were treated with culture medium (CM) alone. In group 4 (n = 15), autologous MSCs (1.5-2.0 x 10(6)/ml) were injected in the left ventricular (LV) wall. Hearts were stained with HE and picrosirius red. MMP-1, -2, -3 and -9 and TIMP-2 and -3 were detected immunohistochemically. The mRNA levels were determined by real-time polymerase chain reaction. The results confirmed that doxorubicin treatment resulted in minimal myocardial fibrosis and showed that expression of MMPs increased and TIMP-3 decreased. The injection procedure resulted in increased myocardial fibrosis in groups 3 and 4. After MSC injection, MMP-1, MMP-2, and TIMP-3 expression was higher than that in group 2. CM injection led to more fibrosis, elevated TIMP-3, but diminished MMP-1 and MMP-2 expression compared with MSC injection. The mRNA levels of MMPs and TIMPs were not significantly different among all groups. In conclusion, chronic doxorubicin cardiomyopathy was characterized by increased MMP and decreased TIMP-3 expression. MSCs injection into the LV resulted in marked differences of collagen content and MMP/TIMP expression in the whole heart, although significant numbers of living MSCs were not detected after 4 weeks.