Connective tissue growth factor stimulates renal cortical myofibroblast-like cell proliferation and matrix protein production

Myofibroblasts primarily contribute to the pathogenesis of renal interstitial fibrosis by unregulated cell proliferation and synthesis of excessive amounts of extracellular matrix (ECM) proteins. We used cultured myofibroblast‐like cells obtained by outgrowth from explants of rat kidney cortex to study the effects and relevant signaling pathway of connective tissue growth factor (CTGF) on cell proliferation and ECM production. Exogenous CTGF stimulated proliferation of myofibroblast‐like cells in a dose‐ and time‐dependent manner. CTGF also increased the secretion of fibronectin and collagen I protein in the supernatant medium. Nevertheless, CTGF did not affect matrix‐degrading metalloproteinases‐2 and ‐9 activities in supernatant medium measured by gelatin zymography. CTGF induced activation of extracellular signal‐regulated protein kinase (ERK)1/2 mitogen‐activated protein kinase pathway as early as 5 minutes. Inhibition of ERK1/2 activation with PD98059 completely blocked CTGF‐induced cell proliferation as well as secretion of fibronectin and collagen I protein. The above results indicate that CTGF triggers cell proliferation and production of ECM proteins in cultured myofibroblast‐like cells through the ERK1/2 mitogen‐activated protein kinase pathway.     

罗格列酮抑制环孢素诱导的大鼠肾脏成纤维细胞基质金属蛋白酶9和金属蛋白酶1组织抑制剂表达

目的 观察罗格列酮(RGZ)对环孢素A(CsA)作用下大鼠肾脏成纤维细胞(NRK)过氧化物酶体增生物激活受体γ(PPARγ)和基质金属蛋白酶-9(MMP-9)表达的影响,探讨罗格列酮对环孢素A肾毒性保护作用的分子机制.方法 构建、筛选和扩增PPARγ基因的siRNA载体,并将载体转染至体外培养的NRK细胞.体外培养NRK细胞,随机分组.(1)对照组:不加处理;(2)RGZ组:加RGZ( 10 μmol/L);(3)CsA组:加CsA( 1.0 mg/L);(4)CsA+RGZ 组:同时加CsA( 1.0 mg/L)及RGZ(10 μmol/L);(5)CsA+RGZ+siRNA组:质粒pRNAT- U6.2/LentiPPARγ-236转染NRK细胞,然后同时加入CsA( 1.0 mg/L)及RGZ(10 μmol/L).培养24 h后,用实时荧光定量和RT-PCR法检测各组细胞中PPARγ、MMP-9、金属蛋白酶1组织抑制剂(TIMP-1) mRNA表达,用Western印迹法检测纤连蛋白(FN)的蛋白表达.结果 CsA明显上调PPARγ、MMP-9和TIMP-1 mRNA的表达(均P＜0.05);RGZ与CsA合用后,PPARγ、MMP-9和TIMP-1 mRNA表达下降(均P＜0.05);应用PPARγ siRNA后,与RGZ+ CsA组相比,PPARγ mRNA表达显著下降(P＜0.05),MMP-9 mRNA和TIMP-1 mRNA表达有所增加(均P＜ 0.05).CsA明显上调FN蛋白表达(P＜0.05);RGZ与CsA合用后,FN蛋白表达下降(P＜0.05);应用含PPARγ siRNA后,FN蛋白表达有所增加(P＜0.05).结论 罗格列酮可以显著减轻CsA诱导NRK细胞分泌的FN蛋白及MMP-9、TIMP-1 mRNA的表达,构建的siRNA质粒转染NRK细胞,能够有效地阻断NRK中PPARγ mRNA的表达,部分阻断罗格列酮对CsA毒性的改善作用.     

Connective tissue growth factor and igf-I are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose

Tubulointerstitial fibrosis is an important component in the development of diabetic nephropathy. Various renal cell types, including fibroblasts, contribute to the excessive matrix deposition in the kidney. Although transforming growth factor-beta (TGF-beta) has been thought to play a major role during fibrosis, other growth factors are also involved. Here we examined the effects of connective tissue growth factor (CTGF) and IGF-I on collagen type I and III production by human renal fibroblasts and their involvement in glucose-induced matrix accumulation. We have demonstrated that both CTGF and IGF-I expressions were increased in renal fibroblasts under hyperglycemic conditions, also in the absence of TGF-beta signaling. Although CTGF alone had no effect on collagen secretion, combined stimulation with IGF-I enhanced collagen accumulation. Furthermore, IGF-I also had a synergistic effect with glucose on the induction of collagens. Moreover, we observed a partial inhibition in glucose-induced collagen secretion with neutralizing anti-CTGF antibodies, thereby demonstrating for the first time the involvement of endogenous CTGF in glucose-induced effects in human renal fibroblasts. Therefore, the cooperation between CTGF and IGF-I might be involved in glucose-induced matrix accumulation in tubulointerstitial fibrosis and might contribute to the pathogenesis of diabetic nephropathy.     

血清基质金属蛋白酶2和抑制因子与绝经后骨质疏松症的相关性

目的探讨绝经后妇女血清基质金属蛋白酶2（MMP-2）和抑制因子（TIMP-2）水平及其与绝经骨质疏松症指标的关系。方法将202名48～65岁绝经后妇女分为正常组、低骨量组和骨质疏松组，用酶联免疫吸附试验（EIJSA）测定的血清MMP-2、TIMP-2以及骨保护蛋白（OPG）、骨保护蛋白配体（OPGL），计算MMP-2／TIMP-2和OPG／OPGL比值，用双能X线吸收法（DEXA）测定腰椎正位、股骨颈、华氏区和大粗隆的骨密度（BMD）。结果①骨质疏松组中血清MMP-2的数值（1392±121）μg／L高于正常组（1123±141）μg／L（P〈0．05），而TIMP-2的数值（44．3±36．2）ng/ml低于正常组（47．8±30．2）ng/ml。②骨质疏松组中血清MMP-2和MMP-2／TIMP-2比值与骨密度、血精OPGL数值存在明显负相关性（P〈0．05），和OPG和OPG／OPGL比值存在明显正相关性（P〈0．05），TIMP-2和华氏区骨密度和OPG存在明显正相关性（P〈0．05）。结论血清MMP-2和MMP-2／TIMP-2比值与绝经后骨质疏松症妇女骨密度和骨代谢指标OPG、OPGL和OPG／OPGL比值具有关联性。血清MMP-2水平升高和MMP-2／TIMP-2比值降低可能为绝经后骨质疏松症伴随骨代谢转换过程增快的表现。     

Platelet-derived growth factor stimulates matrix metalloproteinase-2 secretion in cultured human mesangial cells

Background. Platelet-derived growth factor (PDGF) is an important mediator of mesangial proliferative glomerulonephritis. Little is known about the role of PDGF in the regulation of intraglomerular extracellular matrix turnover. Method. Effects of PDGF on the secretion of matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), and type IV collagen by cultured human mesangial cells (HMCs) were examined in the present study. Secretion of MMP-2, TIMP-2, and type IV collagen by HMCs was quantified with an enzyme immunoassay. Collagenase activity of HMCs was evaluated by gelatin zymography. Results. Recombinant human PDGF (10–20 ng/ml) stimulated MMP-2 secretion by HMCs in a dose-dependent fashion. PDGF (20 ng/ml) increased TIMP-2 secretion by HMCs to a lesser extent. Enhanced activity of 72-kDa collagenase derived from HMCs incubated with PDGF was demonstrated by zymography. Although PDGF alone did not affect type IV collagen secretion by HMCs, PDGF increased type IV collagen secretion in the presence of TIMP. Conclusions. PDGF may contribute to intraglomerular matrix turnover by up-regulating secretion and activation of MMP-2 by HMCs. Received: January 24, 2001 / Accepted: September 13, 2002 Correspondence to:H. Osawa     

Expression of matrix metalloproteinase-7 and tissue inhibitor of metalloproteinase-2 in human endometrial carcinoma

<正>Objective:To investigate the expression of matrix metalloproteinase-7(MMP-7) and its tissue inhibitor (TIMP-2) in endometrial carcinoma and analyze their significance in endometrial cancer's invasion and metastasis. Methods:Endometrial tissues were collected from 64 patients with endometrial carcinoma,20 patients with endometrial hyperplasia and 20 normal women.The expressions of MMP-7,TIMP-2 in endometrium were measured by immuohistochemistry. Results;Expressions of MMP-7,TIMP-2 in endometrium of patients with endometrial carcinoma were significantly higher than those in normal endometrium(P0.05).MMP-7 expression increased with surgical-pathological staging,depth of myometrial invasion,histologic grades and lymph node metastasis(P0.05),while TIMP-2 expression was related to lymph node metastasis(P0.05).TIMP-2 expression in endometrial cancer was significantly higher than that in hyperplastic endometrium(P0.05).Expressions of TIMP-2 and MMP-7 in endometrium of patients with endometrial carcinoma were positively correlated(r=0.654,P0.001). Conclusion:Highly expressed MMP-7 and TIMP-2 in endometrium may be related to development,invasion and metastasis of endometrial cancers.     

MMP-1、TIMP-1及PDGF在病理性瘢痕中的表达

目的研究基质金属蛋白酶-1（MMP-1）、金属蛋白酶组织抑制剂-1（TIMP-1）、血小板源性生长因子（PDGF）在病理性瘢痕中的表达及意义。方法对58例病理性瘢痕手术切除标本采用免疫组化方法。结果MMP-1、TIMP-1、PDGF在病理性瘢痕中呈阳性表达（62．07％，63．71％，55．17％），在正常皮肤与普通瘢痕中几乎无阳性表达，病理性瘢痕组与两对照组差异均有显著意义（P〈0．05）。病理性瘢痕中TIMP-1与PDGF呈正相关（r=0．331，P〈0．05）。结论TIMP-1、PDGF促进病理性瘢痕形成；在病理性瘢痕形成过程中PDGF可促进TIMP-1的表达；病理性瘢痕的形成与MMP-1、TIMP-1、PDGF相互作用失衡有关。     

MMP-1、TIMP-1及PDGF在病理性瘢痕中的表达

目的研究基质金属蛋白酶-1(MMP-1)、金属蛋白酶组织抑制剂-1(TIMP-1)、血小板源性生长因子(PDGF)在病理性瘢痕中的表达及意义。方法对58例病理性瘢痕手术切除标本采用免疫组化方法。结果MMP-1、TIMP-1、PDGF在病理性瘢痕中呈阳性表达(62.07%,63.71%,55.17%),在正常皮肤与普通瘢痕中几乎无阳性表达,病理性瘢痕组与两对照组差异均有显著意义(P<0.05)。病理性瘢痕中TIMP-1与PDGF呈正相关(r=0.331,P<0.05)。结论TIMP-1、PDGF促进病理性瘢痕形成;在病理性瘢痕形成过程中PDGF可促进TIMP-1的表达;病理性瘢痕的形成与MMP-1、TIMP-1、PDGF相互作用失衡有关。     

结缔组织生长因子与病理性瘢痕

病理性瘢痕是以成纤维细胞为主的细胞成分过度增殖和以胶原为主的细胞外基质过度沉积的皮肤纤维化疾病。近年来，越来越多的研究发现病理性瘢痕的纤维化过程与多种生长因子有关。目前普遍认为转化生长因子β1（Transforming Growth Factor，TGF-β1）是促进纤维化发展的最重要的生长因子之一，在随后的研究中，     

结缔组织生长因子诱导瘢痕成纤维细胞增殖的信号转导机制研究

目的：探讨结缔组织生长因子（connective tissue growth factor,CTGF）诱导增生性瘢痕（hypertrophics car,HS）成纤维细胞增殖的信号转导通路。方法：采用3H一胸腺嘧啶核苷（3H-TdR）掺入法观察不同浓度CTGF促Hs成纤维细胞增殖的效应,以Western Blot法检测CTGF刺激HS成纤维细胞0、5、10、15、30、60min后磷酸化及总细胞外信号调节激酶（extracellular-signal regulated kinase,ERK1／2）的表达,以二者的比值AI衡量信号通路活化程度;应用特异性阻断剂PD98059阻断ERK通路,MTT法检测CTGF诱导细胞增殖的变化。结果：CTGF在一定浓度范围内呈浓度依赖性促HS成纤维细胞增殖,ERK1／2在无CTGF刺激时AI为0．0131±0．00361,CTGF刺激HS成纤维细胞5、10、15、30、60min后的AI值分别为0．0221±0．00329,0．131±0．0361,0．209±0．0201,0．171±0．0379,0．0413±0．00361,在15min达高峰;采用P098059选择性阻断ERK1／2通路后（OD值=0．42±0．046）与CTGF刺激纽比较（OD=0．66±O．035）,细胞增殖显著受抑（P〈0．01）。结论：ERK1／2是CTGF诱导Hs戍纤维细胞增殖的主要信号通路。     

Influence of cytokines and growth factors on matrix metalloproteinase-2 production and invasion of human renal cancer

This study evaluated the influence of cytokines and growth factors on the production of matrix metalloproteinase-2 (MMP-2, 72-kDa type IV collagenase, gelatinase A) and invasion of the human renal cell carcinoma (HRCC) cell line KG-2. The cells were treated with cytokines and growth factors, and the gelatiolytic activity and in vitro invasion were examined. Basic fibroblast growth factor (bFGF) stimulated MMP-2 production by KG-2 cells to 2.0-, 4.84- and 4.53-fold that of the untreated group at 0.1, 1.0 and 10 ng/ml, respectively. Transforming growth factor-β1 (TGF-β1) at very low concentrations of 10 pg/ml and 100 pg/ml stimulated enzyme production in KG-2 cells by 1.74- and 2.83-fold, respectively. In contrast, interferon-γ (IFN-γ) decreased MMP-2 production by KG-2 cells at 10 and 100 U/ml to 69% and 41% of the level in the untreated group, respectively. At those concentrations, IFN-γ did not cause cytostasis in KG-2 cells. Moreover, bFGF and TGF-β1 (low concentrations) stimulated in␣vitro invasion of KG-2 cells, but IFN-γ decreased the invasive activity, which was well correlated with the levels of MMP-2. However, the expression of MMP-2 mRNA of KG-2 cells treated with 10 ng/ml bFGF, 100 pg/ml TGF-β1 and 100 U/ml IFN-γ was shown to be 3.8-, 3.4- and 0.7-fold, respectively, those in untreated groups. Thus the production of MMP-2 in HRCC was influenced by cytokines and growth factors, and MMP-2 plays an important role in the invasion and metastasis of certain types of HRCC. Received: 24 April 1997 / Accepted: 14 July 1997     

结缔组织生长因子通过ERK1/2途径刺激肾脏成肌纤维细胞增殖和Ⅰ型胶原生成

目的 观察结缔组织生长因子（CTGF）对大鼠肾脏成肌纤维细胞增殖及细胞外基质（ECM）成分Ⅰ型胶原生成的影响，并探讨ERK1/2途径在其中的作用。 方法 原代培养肾皮质成肌纤维细胞，应用5’-溴-2’-脱氧尿嘧啶（BrdU） 掺入法和细胞计数法检测细胞增殖；Western印迹法检测细胞上清液中Ⅰ型胶原的蛋白水平及细胞内ERK1/2的磷酸化水平。 结果 CTGF明显促进成肌纤维细胞的增殖，呈时间和剂量依赖性，100 μg/L组第4天时，细胞数为对照组的1.6倍（P < 0.05）。CTGF刺激可显著增加培养上清液中Ⅰ型胶原的蛋白水平 [为对照组的（2.6±1.2）倍， P < 0.05]。CTGF刺激细胞5 min后ERK1/2即发生磷酸化，持续至15 min， 30 min以后迅速降至基础水平。用ERK1/2阻断剂PD98059预处理30 min后，CTGF促进细胞增殖效应（7%±5%比85%±7%， P < 0.01）和促Ⅰ型胶原分泌作用（1.0±0.1比1.6±0.3, P < 0.05）被显著抑制。 结论 CTGF能够促进成肌纤维细胞的增殖和Ⅰ型胶原的分泌，其作用可能通过ERK1/2信号通路实现。     

Connective tissue growth factor stimulates cell proliferation and collagen type I secretion through ERK1/2 signaling pathway in cultured renal myofibroblasts

Objective To examine the effects of connective tissue growth factor (CTGF) on cell proliferation and production of collagen type Ⅰin cultured rat cortical myofibroblasts，and to investigate the role of ERK1/2 siganling pathway. Methods Myofibroblasts were obtained from normal rat renal cortex. 5’-bromodeoxyuridine (BrdU) incorporation assay and cell counting were used to detect cell proliferation. Western blot analysis was used to detect the levels of collagen typeⅠin the supernatant medium and the activation of ERK1/2 signaling pathway in cultured myofibroblasts. Results CTGF could induce the proliferation of myofibroblasts in a dose- and time-dependent manner. Cell number of 100 μg/L CTGF at day 4 was 1.6 folds of control group(P<0.05). Incubation with 100 μg/L CTGF also significantly increased secretion of collagen type I in the supernatant medium compared with control group (2.6±1.2 folds over control, P<0.01). ERK1/2 activation occurred as early as 5 minutes following 100 μg/L CTGF treatment, and persisted till 15 minutes later, and then declined back to the basal level after 30 minutes. Pretreatment with 50 μmol/L PD98059, a specific inhibitor of ERK1/2 pathway, abolished the effects of CTGF-induced cell proliferation (7%±5% vs 85%±7%, P<0.01) and CTGF-increased secretion of collagen type I (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusion CTGF promotes cell proliferation and secretion of collagen type I through ERK1/2 pathway in primary cultured rat myofibroblasts.     

An imbalance between matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 contributes to the development of early diabetic nephropathy.

BACKGROUND: High glucose and angiotensin-II (Ang-II) levels are the known important mediators of diabetic nephropathy. However, the effects of these mediators on matrix metalloproteinase-2 (MMP-2) and on tissue inhibitor of metalloproteinase-2 (TIMP-2) in proximal tubule cells have yet to be fully examined within the context of early stage diabetic nephropathy. METHODS: In this study, we attempted to characterize changes in MMP-2 and TIMP-2 in streptozotocin-induced diabetic rats. To further examine the molecular mechanisms involved, we evaluated the effects of high glucose (30 mM) or Ang-II on MMP-2, TIMP-2 and collagen synthesis in proximal tubule cells, and investigated whether MMP-2 and TIMP-2 are regulated via the TGF-beta1 pathway. RESULTS: In streptozotocin-induced diabetic rats, TIMP-2 mRNA and protein levels were significantly higher than in controls. Urinary protein excretion also showed a significant positive correlation with glomerular and tubular TIMP-2 protein expressions, and a negative correlation with MMP-2 expression. In cultured cells, both high glucose and Ang-II induced significant increases in TGF-beta1, TIMP-2, and in collagen synthesis, and significant decreases in MMP-2 gene expression and activity, and thus disrupted the balance between MMP-2 and TIMP-2. Moreover, treatment with a selective angiotensin type 1 (AT1) receptor antagonist significantly inhibited Ang-II mediated changes in TGF-beta1, MMP-2, TIMP-2, and in collagen production, suggesting the role of the AT1 receptor. The addition of exogenous TGF-beta1 produced an effect similar to those of high glucose and Ang-II. Furthermore, the inhibition of TGF-beta1 protein prevented Ang-II-induced MMP-2 and TIMP-2 alterations, suggesting the involvement of a TGF-beta1 pathway. CONCLUSIONS: High glucose or Ang-II treatment induce alterations in MMP-2 and TIMP-2 balance, which favour TIMP-2 over-activity. Moreover, Ang-II-mediated changes in the productions of MMP-2 and TIMP-2 occur via AT1 receptors and a TGF-beta1-dependent mechanism. These results suggest that an imbalance between the MMP-2 and TIMP-2, caused primarily by an increase in TIMP-2 activity, contributes to the pathogenesis of diabetic nephropathy.     

抗Fas抗体诱导人肾间质成纤维细胞凋亡

目的检测人肾间质成纤维细胞(hRIFs)Fas表达并以抗Fas抗体诱导hRIFs凋亡。方法分离人肾肾乳头组织培养hRIFs,以细胞形态、免疫细胞化学染色和右旋缬氨酸选择性培养基培养作细胞鉴定。采用RT-PCR、免疫细胞化学染色检测正常hRIFsFas表达;不同γ干扰素浓度(γ-IFN,500U/ml、1000U/ml、l500U/ml和2000U/ml)与hRIFs孵育48h后,采用Northern杂交、Western印迹和流式细胞术检测Fas表达。经γ-IFN预处理(500U/ml,48h)的hRIFs,加人抗Fas抗体(IgM,O.5mg/m1)作用12h,以形态学、DNA电泳和流式细胞术观察细胞凋亡。结果培养细胞呈梭形,波形蛋白(+),上皮细胞膜抗原(-),选择性培养基内逐渐死亡。正常hRIFs有FasmRNA和蛋白表达,γ-IFN可明显上调其表达水平,直接免疫荧光流式细胞术测定未加γ-IFN的hRIFs中Fas表达阳性细胞占5.91％,上述浓度的γ-IFN作用后依次为59.44％、69.39％、70.06％和7547％,呈剂量依赖性增强。抗Fas抗体可引起高表达Fas的hRIFs核固缩、核断裂,DNA电泳呈现梯形结构,流式细胞术呈现亚二倍体凋亡细胞峰,对照组凋亡细胞百分数为7.99％,上述浓度抗Fas抗体作用后依次为24.35％、27.89％、29.38％和31.21％。结论Fas表达于hRIFs且可被γ-IFN显著上调,抗Fas抗体能够诱导高表达Fas的hRIFs凋亡。     

Localization and quantification of mRNA for matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in human benign and malignant prostatic tissue

BACKGROUND: The family of matrix metalloproteinases (MMPs) has been shown to be involved in proteolytic degradation of the extracellular matrix, which is an essential step in tumor invasion and metastasis. MMPs are tightly regulated by the levels of active enzymes and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). MMP-2 and its ratio to TIMP-2 have been associated with tumor recurrence and progression in a number of human malignancies. METHODS: We examined the relationship between MMP-2 and TIMP-2 mRNA expression in 42 men with malignant (n = 32) and benign (n = 10) prostates using nonisotopic in situ hybridization and Northern blot analysis. RESULTS: mRNA for MMP-2 and TIMP-2 was localized to the malignant epithelial cells of both high- and low-grade tumors in the periphery of the glands and in areas of extracapsular involvement, and to the glandular epithelium in the benign prostates. Using Northern blot analysis, the mean MMP-2 to TIMP-2 ratio was approximately one in the benign prostates and low-grade and -stage cancers. The MMP-2 to TIMP-2 ratio increased to 3.3 in the high-grade and 2.8 in the high-stage tumors. CONCLUSIONS: The results suggest a close association between MMP-2/TIMP-2 expression and local tumor invasion, with a disruption in expression of the two genes leading to disease progression. Future studies should focus on the activity of these enzymes and on the ratio of enzyme/inhibitor expression, which may become a useful prognostic marker in prostate cancer.     

Mitogenic bovine whey extract modulates matrix metalloproteinase-2, -9, and tissue inhibitor of matrix metalloproteinase-2 levels in chronic leg ulcers

Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the wound-healing process. An imbalance in the expression of these molecules is thought to contribute to the failure of chronic ulcers to heal. We investigated whether a mitogenic bovine whey extract enriched with growth factors modulated the expression and activity of MMP-2 and -9, and the tissue inhibitor of MMP-2 (TIMP-2) in chronic leg ulcers. Wound fluids and biopsies were collected from chronic leg ulcer patients whose ulcers were treated topically for 4 weeks with placebo or mitogenic bovine whey extract at concentrations of 2.5, 10, and 20 mg/mL. The levels of MMP-2 and -9 in wound fluid samples was assessed by gelatin zymography and showed a decrease in active MMP-2 in the 2.5 and 10.0 mg/mL mitogenic bovine whey extract-treated ulcers compared with placebo (p<0.05). Immunohistochemical analysis of ulcer biopsies for MMP-2, -9, and TIMP-2 expression showed a reduction in the number of MMP-2-positive dermal fibroblasts in the mitogenic bovine whey extract-treated ulcers compared with pretreatment biopsies (p<0.05) that persisted over the course of the study. In contrast, a transient increase in the number of MMP-9- and TIMP-2-positive cells was observed in mitogenic bovine whey extract treated ulcer biopsies compared with pretreatment levels (p<0.05). These results show that topical application of mitogenic bovine whey extract was able to modulate the expression of MMP-2, -9, and TIMP-2 in chronic leg ulcers and that its constituent growth factors may have the potential to redress the proteolytic imbalance observed in nonhealing chronic ulcers.