Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Denmark.
Basic Clin Pharmacol Toxicol. 2011 Sep;109(3):208-16. doi: 10.1111/j.1742-7843.2011.00713.x. Epub 2011 Jun 3.
Increased expression of matrix metalloproteinase (MMP)-2, -3 and -9 has been demonstrated in Crohn's disease fistulas, but it is unknown whether these enzymes are biologically active and represent a therapeutic target. Therefore, we investigated the proteolytic activity of MMPs in fistula tissue and examined the effect of inhibitors, including clinically available drugs that beside their main action also suppress MMPs. Fistula specimens were obtained by surgical excision from 22 patients with Crohn's disease and from 10 patients with fistulas resulting from other causes. Colonic endoscopic biopsies from six controls were also included. Total functional MMP activity was measured by a high-pressure liquid chromatography (HPLC)-based, fluorogenic MMP-substrate cleavage assay, and the specific activity of MMP-2, -3 and -9 by the MMP Biotrak Activity Assay. The MMP inhibitors comprised ethylene-diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin-converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline. In Crohn's disease fistulas, about 50% of the total protease activity was attributable to MMP activity. The average total MMP activity was significantly higher (about 3.5-times) in Crohn's fistulas (471 FU/μg protein, range 49-2661) compared with non-Crohn's fistulas [134 FU/μg protein, range 0-495, (p < 0.05)] and normal colon [153 FU/μg protein, range 77-243, (p < 0.01)]. MMP-3 activity was increased in Crohn's fistulas (1.4 ng/ml, range 0-9.83) compared with non-Crohn's fistulas, [0.32 ng/ml, range 0-2.66, (p < 0.02)]. The same applied to MMP-9 activity [0.64 ng/ml, range 0-5.66 and 0.17 ng/ml, range 0-1.1, respectively (p < 0.04)]. Ramiprilate significantly decreased the average total MMP activity level by 42% and suppressed the specific MMP-3 activity by 72%, which is comparable to the effect of GM6001 (87%). Moreover, MMP-9 activity was completely blunted by ramiprilate. Doxycycline had no effect on MMP activity. Increased functional MMP activity, notably MMP-3 and -9, is present in Crohn's fistulas and may be inhibited by ramiprilate, a widely available ACE inhibitor.
在克罗恩病瘘管中已经证实基质金属蛋白酶（MMP）-2、-3 和 -9 的表达增加，但这些酶是否具有生物活性并代表治疗靶点尚不清楚。因此，我们研究了瘘管组织中 MMP 的蛋白水解活性，并检查了抑制剂的作用，包括临床可用的除主要作用外还能抑制 MMP 的药物。通过手术切除从 22 例克罗恩病患者和 10 例其他原因引起的瘘管患者获得瘘管标本，还包括 6 例对照的结肠内镜活检。通过基于高压液相色谱（HPLC）的荧光 MMP 底物裂解测定法测量总功能 MMP 活性，通过 MMP Biotrak 活性测定法测量 MMP-2、-3 和 -9 的特异性活性。MMP 抑制剂包括乙二胺四乙酸（EDTA）、合成的广谱抑制剂 GM6001、血管紧张素转换酶（ACE）抑制剂雷米普利和四环素、多西环素。在克罗恩病瘘管中，约 50%的总蛋白酶活性归因于 MMP 活性。克罗恩病瘘管的平均总 MMP 活性明显较高（约 3.5 倍）[471 FU/μg 蛋白，范围 49-2661]，而非克罗恩病瘘管[134 FU/μg 蛋白，范围 0-495，（p<0.05）]和正常结肠[153 FU/μg 蛋白，范围 77-243，（p<0.01）]。与非克罗恩病瘘管相比，克罗恩病瘘管中的 MMP-3 活性增加[1.4 ng/ml，范围 0-9.83]（p<0.02）。同样适用于 MMP-9 活性[0.64 ng/ml，范围 0-5.66 和 0.17 ng/ml，范围 0-1.1，分别（p<0.04）]。雷米普利可使平均总 MMP 活性水平降低 42%，并使特异性 MMP-3 活性降低 72%，与 GM6001（87%）的作用相当。此外，雷米普利完全阻断 MMP-9 活性。多西环素对 MMP 活性无影响。功能性 MMP 活性增加，特别是 MMP-3 和 -9，存在于克罗恩病瘘管中，可被广泛应用的 ACE 抑制剂雷米普利抑制。
