Effects of AT1- and β-adrenergic receptor antagonists on TGF-β1-induced fibrosis in transgenic mice

Background Transforming growth factor‐β1 (TGF‐β1) is involved in interstitial remodelling promoting collagen synthesis and suppressing collagen degradation by inhibition of collagenases. TGF‐β1 mediates angiotensin II‐dependent effects and modulates β1‐adrenergic signalling. To study the effect of neuroendocrine antagonism on TGF‐β‐induced hypertrophic and fibrotic phenotype, we treated TGF‐β1 (Cys223,225Ser) transgenic mice (TGF‐β1‐TG) with either the β1‐receptor blocker metoprolol (MET), the angiotensin II type I (AT1)‐receptor antagonist telmisartan (TEL) or an antibody blocking TGF‐β1 signalling (TGFβ1‐sR‐Ab). Material and Methods Transforming growth factor‐β1‐TG mice (8 weeks) overexpressing TGF‐β1 were treated with either TEL (10 mg kg^?1), MET (350 mg kg^?1) or a soluble TGF‐β1 receptor antibody (1 mg kg^?1) for 6 weeks. Morphological analyses of interstitium and cardiomyocytes were related to expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by immunoblotting and zymography. Results In TGF‐β1‐TG mice, myocardial interstitial total collagen content was fourfold elevated compared to that of controls (P < 0·05) and was lowered under the treatment with TEL (P < 0·05). Protein expression of TIMP‐1 and ‐4 was increased in TGF‐β1‐TG but inhibited by TEL (TIMP‐1 and TIMP‐4) and MET (TIMP‐1), while collagenase activity was decreased in TGF‐β1‐TG and normalized by treatment with TEL (MMP‐1 and MMP‐13) and MET (MMP‐1) (P < 0·05). Morphometric measurements of cardiomyocyte diameter and area demonstrated similar antihypertrophic effects for all treatment groups. Conclusion The AT1‐antagonist TEL reduced myocardial hypertrophy and interstitial fibrosis in TGF‐β1‐TG mice by normalizing MMP/TIMP ratio. β1‐Adrenergic inhibition by MET as well as TGF‐β1 antagonism induced antihypertrophic rather than antifibrotic effects. Inhibition of both renin‐angiotensin system and β1‐adrenergic system may exert different but synergistic effects to reduce myocardial remodelling.     

Relationship between matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases systems and autoantibody patterns in systemic sclerosis

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis that reflects an imbalance between collagen production and degradation. Matrix metalloproteinases (MMPs) are a family of endopeptidases involved in the remodelling of extracellular matrix (ECM). This activity is controlled by tissue inhibitors of MMP (TIMPs). Aim of this study was the evaluation of MMP-9/TIMP-1 and MMP-2/TIMP-2 systems in patients with SSc. DESIGN AND METHODS: SearchLight Human MMP Array 1 was used to measure MMPs and TIMPs in 32 SSc patients and 32 matched healthy controls. RESULTS: SSc patients showed higher values of both MMP-9 and TIMP-1 in comparison with controls. The patients with anticentromere antibodies (ACA) positivity showed higher values of MMPs and TIMPs in comparison with either controls or the patients with anti-Scl70-positive antibodies. CONCLUSION: Results of this investigation suggest that SSc patients with ACA positivity, after a primary fibrogenetic noxa, react with a more abundant release of MMP/TIMP, whereas patients with anti-Scl70 antibody show a normal response.     

The effect of stress-deprivation and cyclic loading on the TIMP/MMP ratio in tendon cells: An in vitro experimental study

Purpose. To determine the effect of stress deprivation and cyclic loading on TIMP-1/MMP-13 mRNA expression ratio in rat tail tendon (RTT) cells.

Method. Adult RTTs were stress-deprived for 0, 24, 48, or 72 hours in the presence or absence of a MMP inhibitor (ilomastat), or subjected to 1%, 3%, or 6% strain for 24 h under tissue culture conditions. TIMP-1 and MMP-13 (rat interstitial collagenase) mRNA expression were measured using quantitative PCR and TIMP/MMP ratios were calculated for each group.

Results. The ratio of TIMP-1 to MMP-13 in control RTTs was 3.73:1 ± 0.73. Stress deprivation for 24 h significantly decreased the TIMP-1/MMP-13 ratio (0.25:1 ± 0.04) and MMP-13 expression continued to increase significantly with time of stress deprivation. Inhibition of MMP-13 mRNA expression with ilomastat in stress-deprived samples did not alter TIMP-1 expression when compared to normal controls. Cyclic loading significantly increased TIMP-1/MMP-13 expression at all strain levels examined.

Conclusions. RTTs normally have a positive TIMP-1/MMP-13 expression ratio. While cyclic loading increased the TIMP-1/MMP-13 ratio, loss of cellular homeostatic tension inversed this ratio through a significant increase in MMP-13 mRNA expression rather than a decrease in TIMP expression. A negative TIMP/MMP ratio has been implicated in the pathogenesis of tendinopathy. Increasing the TIMP/MMP ratios in these patients through exercise may be beneficial in the management of tendinopathy.     

慢性充血性心力衰竭患者血清基质金属蛋白酶与骨桥蛋白变化的相关性研究

目的探讨慢性充血性心力衰竭（CHF）患者血清基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）和骨桥蛋白（OPN）的变化及相关性。方法CHF患儿24例，正常对照组15例。采用ELISA法测定血清MMP-9和TIMP-1的含量,ELISA法测定血清OPN的含量，分析MMP-9／TIMP-1值与OPN和左室舒末内径（LVEDD）及射血分数（EF）的关系。结果CHF患者血清MMP-9、MMP-9／TIMP-1值和OPN含量明显增加（P均〈0．01），TIMP-1含量明显减少（P〈0．01），MMP-9／TIMP-1值与OPN含量和LVEDD呈正相关（P〈0．05），与EF呈负相关（P〈0．05）。结论CHF患者MMP-9／TIMP-1值与OPN含量呈明显的正相关；MMP-9／TIMP-1值与OPN含量的变化，可反映心衰的严重程度并加速了心功能的恶化。     

MMP-3、MMP-9和TIMP-1的动态平衡可作为心肌基质重构的重要标志

目的从基质金属蛋白酶（matrix metalloproteinase, MMPs）和基质金属蛋白酶组织抑制剂（tissue inhibitor of metalloproteinase, TIMps）的动态平衡着手探寻其对心肌基质的组织修复和再生作用,为心肌损伤后基质重构造成的心肌肥大、增厚等引起的心力衰竭提供的有效的生物检测方法及治疗。方法利用广州市红十字会医院（暨南大学第四附属医院）的心脏病患者作为研究对象,按照随机区组分组的方法根据进行的心脏手术不同,将研究对象分为3个组,第1组为先天性心脏病组（CHD组）;第2组为风湿性心脏病组（PHD组）;第3组为冠心病组（COR组）,由于其房室大小,心功能正常,设其为对照组。Elisa法对患者术前血液中的mmps和timps进行定量测定,寻求它们之间的动态平衡关系;术中切取心肌组织,用免疫组化方法测定心肌基质的胶原形态和分布;rt-PCR技术对心肌组织中mmps和timps的mRNA进行检测,查看其mRNA表达情况。结果CHD组、PHD组术前血液中的MMP-3、MMP-9、TIMP-1含量均较COR组高,MMP-9增多更为显著,差异有统计学意义（P < 0.05）,而CHD组和PHD组差异不显著,无统计学意义;免疫组化结果显示,CHD组、PHD组中MMP-3、MMP-9、TIMP-1均较COR组分布广泛,PCR结果显示CHD组、PHD组患者MMP-1、MMP-9、TIMP-1的mRNA表达较COR组升高,差异有统计学意义（P < 0.05）,同样CHD组和PHD组差异不显著,无统计学意义。结论MMP-1、MMP-9和TIMP-1的动态平衡可作为心肌基质重构的重要标志,调节MMPs和TIMPs的活性有望成为心脏病治疗的新方向。     

Expression and response to angiotensin-converting enzyme inhibition of matrix metalloproteinases 2 and 9 in renal glomerular damage in young transgenic rats with renin-dependent hypertension

Extracellular matrix expansion in the glomerular mesangium contributes to the development of glomerulosclerosis and chronic renal disease in arterial hypertension. Transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) are involved in this process. Conflicting data are reported on the effects of angiotensin II (Ang II) and the response to angiotensin-converting enzyme inhibition on MMPs and TIMPs in early stages of hypertensive glomerular damage. We therefore investigated the effects of Ang II-dependent hypertension on MMP-2, MMP-9, TIMP-1, and TIMP-2 in isolated glomeruli of 8-week-old homozygous male rats overexpressing the mouse Ren2 gene [TGR(mRen2)27]. At this age, systolic blood pressure was already significantly elevated in Ren2 compared with Sprague-Dawley (SD) rats (197 +/- 38 versus 125 +/- 16 mm Hg, p < 0.01). Ren2 exhibited renal damage as determined by increased urinary albumin excretion, focal glomerulosclerosis, mesangial matrix expansion, and alpha-smooth muscle actin deposition. Quantification of mRNA levels in isolated glomeruli by real-time polymerase chain reaction showed a significant increase of TGF-beta1, a 2.3- and a 2.6-fold increase of MMP-2 and TIMP-1 in Ren2 compared with SD (p < 0.01, respectively) and no strain differences for TIMP-2. In contrast, MMP-9 mRNA expression was markedly suppressed to 10% of control levels in Ren2 (p < 0.01). Early treatment with ramipril completely prevented renal damage in Ren2 and restored mRNA expression of TGF-beta1, MMP-2, and TIMP-1 to SD control levels. Interestingly, down-regulation of MMP-9 mRNA, protein, and activity was not affected by ramipril, indicating that the protective effect of this compound is not attributable to restoration of MMP-9 in the glomerulus.     

Increase in the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1 in saliva from patients with primary Sjögren's syndrome

BACKGROUND: To investigate the ratio of matrix metalloproteinase (MMP) to tissue inhibitor of metalloproteinase (TIMP) in primary Sj?gren's syndrome (PSS), patients and healthy subjects MMP-2, 9 and TIMP-1, 2 levels were measured in saliva. METHODS: Stimulated whole-mixed saliva was collected from 32 patients and 26 healthy subjects. MMP-2, 9 and TIMP-1, 2 levels were measured using enzyme-linked immunosorbent assay (ELISA) and the sandwich enzyme immunoassay (sandwich EIA). Zymography and reverse zymography were used to identify MMPs and TIMPs. RESULTS: MMP-9 (gelatinase-B) level in saliva was significantly increased in the patients. MMP-9 (ng/ml): patients 231.02 +/- 151.77 (mean +/- S.D.), healthy subjects 145.87 +/- 111.65 (p < 0.05). MMP-2 levels were not detected with this system kit in either healthy subjects or patients. The differences in TIMPs were only trends and not statistically significant (p > 0.05). Accordingly, MMP-9/TIMP-1 was greatly increased in the patients (2.60 +/- 1.18) than in the healthy subjects (1.28 +/- 1.11) (p < 0.01). CONCLUSION: This study found that MMP-9/TIMP-1 and MMP-9 levels in the saliva were significantly higher in pSS patients than those in healthy subjects. Our results indicate that the increase in MMP-9/TIMP-1, rather than the increase in MMP-9, in pSS patients' saliva is strongly involved in destruction of glandular and salivary duct tissues.     

Effect of chemically modified tetracycline on transforming growth factor-beta1 and caspase-3 activation in liver of septic rats

OBJECTIVES: We have previously demonstrated that hepatic matrixmetalloproteinase (MMP)-9 and gelatinase activity increased significantly after sepsis, and pretreatment with chemically modified tetracycline (CMT-3) inhibited these expressions and improved survivability. It has been established that MMP-9 release from hepatic nonparenchymal cells activates transforming growth factor (TGF)-beta1, which in turn catalyzes the conversion of procaspase-8 into active caspase-8. Caspase-8 activates caspase-3, which in turn degrades fibronectin and focal adhesion kinase and leads to disruption of hepatic architecture and integrity. We have been interested in investigating the role of posttreatment with CMT-3 on hepatic MMP-9, TGF-beta1, and caspase-3 activity following sepsis. DESIGN: Laboratory experiment. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: In this study, sepsis was induced in rats by cecal ligation and puncture (CLP), and 2 hrs later, half of the rats received CMT-3 (25 mg/kg), whereas the other half received vehicle by gavage. Twenty-four and 48 hrs after sepsis induction, blood and liver samples were collected. MEASUREMENTS AND MAIN RESULTS: Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined by enzymatic method, and the activation states of hepatic MMP-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-beta1, and caspase-3 were determined by Western immunoblotting. Plasma GOT, GPT, and hepatic MMP-9 activity increased 2.5-fold, and TFG-beta1 and caspase-3 activity increased 1.5- to 2-fold at 24 hrs and 48 hrs post-CLP; CMT-3 treatment blocked these increases. Furthermore, CMT-3 treatment also led to increased TIMP-1 level, an in vivo inhibitor of MMP-9. MMP-2 level was unaffected by CLP. The 24-hr and 48-hr mortality rates for CLP rats were 29% and 50%, whereas posttreatment with CMT-3 resulted in 0% mortality. CONCLUSIONS: Our results are consistent with an MMP-9-induced caspase-3 activation in response to CLP. CMT-3 posttreatment increased TIMP-1 level and thereby inhibited MMP-9, which in turn decreased TGF-beta1 and caspase-3 signaling pathways and improved survivability in septic rats.     

R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells

Prostaglandins (PGs) and matrix metalloproteinases (MMP) have been implicated in lowering intraocular pressure (IOP) by facilitating aqueous humor outflow. A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing an impaired COX-2 expression in the nonpigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. Using human NPE cells, the present study therefore investigated the effect of the IOP-lowering cannabinoid R(+)-methanandamide [R(+)-MA] on the expression of COX-2 and different MMPs and tissue inhibitors of MMPs (TIMPs). R(+)-MA led to a concentration- and time-dependent increase of COX-2 mRNA expression. R(+)-MA-induced COX-2 expression was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK and was abrogated by inhibitors of both pathways. Moreover, R(+)-MA increased the mRNA and protein expression of MMP-1, MMP-3, MMP-9, and TIMP-1 but not that of MMP-2 and TIMP-2. Inhibition of COX-2 activity with NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide] was associated with a virtually complete suppression of R(+)-MA-induced MMP-9 and TIMP-1 expression. Consistent with these data, MMP-9 and TIMP-1 expression was also induced by PGE2, a major COX-2 product. Two other COX-2-inducing cannabinoids, anandamide and Delta9-tetrahydrocannabinol, caused the same pattern of MMP and TIMP expression as R(+)-MA both in the absence and presence of NS-398. Altogether, cannabinoids induce the production of several outflow-facilitating mediators in the human NPE. Our results further imply an involvement of COX-2-dependent PGs in MMP-9 and TIMP-1 expression. In conclusion, stimulation of intraocular COX-2 and MMP expression may represent a potential mechanism contributing to the IOP-lowering action of different cannabinoids.     

Evaluation of plasmatic MMP-8, MMP-9, TIMP-1 and MPO levels in obese and lean women

ObjectivesTo compare the plasma concentrations of matrix metalloproteinase (MMP)-9, tissue inhibitor of MMP (TIMP)-1, MMP-8, and myeloperoxidase (MPO) for obese and lean women.Design and methodsWe recruited 30 lean and 36 obese women without comorbidities. The MMP-9, TIMP-1, and MMP-8 levels were measured using enzyme-linked immunosorbent assay (ELISA). MPO activity was assessed by a colorimetric assay.ResultsObese women had higher MMP-9 levels and MMP-9:TIMP-1 ratios than lean women. Conversely, the MMP-8 levels and MMP-8:TIMP-1 ratios in the obese women were significantly lower than those in the lean women despite neutrophil activation, which was assessed by MPO activity.ConclusionWe observed that MMP-9 and MMP-8 had distinct profiles, which suggested that these 2 enzymes play different roles in obesity.     

Insulin-blood cardioplegia decreases matrix metalloproteinase activity in ischaemia-reperfusion injury during coronary artery bypass surgery

Reperfusion of myocardium during coronary bypass activates matrix metalloproteinases (MMPs) with changes occurring in the levels of tissue inhibitors of metalloproteinases (TIMPs) in the myocardium. This study investigated the effects of insulin-blood cardioplegia on MMP activity and TIMP levels during reperfusion. Non-diabetic patients undergoing coronary artery bypass graft with cardiopulmonary bypass were randomized into a control group (n = 12) or an insulin group (n = 12). Blood cardioplegia was used for both groups; insulin and glucose were added to the insulin group. Blood samples were obtained from the coronary sinus just before aortic cross clamping and after 1 and 30 min of reperfusion. Plasma proenzyme MMPs (proMMP-2 and -9) and TIMPs (TIMP-1 and TIMP-2) levels were measured. There were no differences between groups for MMP-2 and TIMP-2 levels. However, insulin diminished proMMP-9 activation, although some still occurred. TIMP-1 consumption lessened during reperfusion which, we conclude, was as a result of the diminished MMP activation. This is the first open heart surgery study in which diminished MMP activation was achieved via a metabolic change.     

Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts

Interleukin (IL)-13 has been implicated in the pathogenesis of various diseases characterized by fibrosis. We describe the effects of IL-13 on collagen homeostasis from normal (NF) and keloid (KF) fibroblasts and compare these effects with those of IL-4 and transforming growth factor (TGF)-beta(1). Total collagen generation was up-regulated in NF after 48 h of stimulation by IL-13; in KF, IL-13 stimulated a more rapid collagen response. The kinetics and magnitude of collagen generation induced by IL-13 were equivalent to those induced by similar concentrations of IL-4 and TGF-beta(1). Collagen type I production paralleled total collagen generation from both NF and KF; however, IL-4-induced collagen type I and total collagen production from KF was more transient than that induced by either IL-13 or TGF-beta(1). Procollagen 1alpha1 gene expression was induced in KF by stimulation with IL-13 for 24 h. Moreover, IL-13 was unique among these three cytokines in its ability to induce gene expression for procollagen 3alpha1. Finally, IL-13 inhibited IL-1beta-induced matrix metalloproteinase (MMP)-1 and MMP-3 production and enhanced tissue inhibitor of metalloproteinase (TIMP)-1 generation from NF; although similar effects were observed with IL-4, TGF-beta(1) transiently enhanced MMP-1 and MMP-3 generation without effecting TIMP-1. In KF, IL-13 and IL-4 inhibited MMP-3, whereas TGF-beta(1) enhanced MMP-3; TIMP-1 was unaffected by any of the three cytokines. These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.     

A farnesoid x receptor-small heterodimer partner regulatory cascade modulates tissue metalloproteinase inhibitor-1 and matrix metalloprotease expression in hepatic stellate cells and promotes resolution of liver fibrosis

The farnesoid X receptor (FXR) is expressed by and regulates hepatic stellate cells (HSCs). In the present study, we investigated whether 6-ethyl chenodeoxycholic acid (6-ECDCA or INT-747), a semisynthetic derivative of chenodeoxycholic acid (CDCA), modulates tissue metalloproteinase inhibitor (TIMP)-1 and matrix metalloprotease (MMP)-2 expression/activity in HSCs and in the liver of rats rendered cirrhotic by 4-week administration of CCl(4). Exposure of HSCs to FXR ligands increases small heterodimer partner (SHP) mRNA by 3-fold and reduces basal and thrombin-stimulated expression of alpha1(I)collagen, alpha-smooth muscle actin (alpha-SMA), TIMP-1, and TIMP-2 by approximately 60 to 70%, whereas it increased matrix metalloprotease (MMP)-2 activity by 2-fold. In coimmunoprecipitation, electromobility shift, and transactivation experiments, FXR activation/overexpression caused a SHP-dependent inhibition of JunD binding to its consensus element in the TIMP-1 promoter. Inhibition of TIMP-1 expression by SHP overexpression enhanced the sensitivity of HSCs to proapoptogenic stimuli. Administration of 3 mg/kg 6-ECDCA, but not 15 mg/kg ursodeoxycholic acid, resulted in early (3-5-day) induction of SHP and prevention of early up-regulation of TIMP-1 mRNA induced by CCl(4). In the prevention protocol, 4-week administration of 6-ECDCA reduced alpha1(I)collagen, alpha-SMA, and TIMP-1 mRNA by 60 to 80%, whereas it increased MMP-2 activity by 5-fold. In the resolution protocol, administration of 3 mg/kg 6-ECDCA promoted liver fibrosis resolution and increased the apoptosis of nonparenchyma liver cells. By demonstrating that a FXR-SHP regulatory cascade promotes the development of a quiescent phenotype and increases apoptosis of HSCs, this study establishes that FXR ligands may be beneficial in treatment of liver fibrosis.     

Matrix metalloproteinase profile in patients with Creuztfeldt-Jakob disease

Preliminary findings suggest that abnormalities in matrix metalloproteinase (MMP) activity may be found in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). In this study of 16 subjects with CJD and 16 age-, and sex-matched controls, we determined the presence of MMP-2 and MMP-9 in their active and proenzyme forms, the relative levels of MMP-3 and four inhibitors of MMP activity (TIMP-1, TIMP-2, TIMP-3 and TIMP-4), and the concentration of 4-3-3 protein. The methodology used involved zymography and immunological techniques. The results indicate that, compared with controls, CJD patients have a significantly higher positive frequency of pro-MMP-9 and of the active form of MMP-2, along with significantly higher levels of TIMP-1 and TIMP-2, classical inhibitors of MMP-9 and MMP-2, respectively. We also found a positive correlation between 14-3-3 protein concentration and that of TIMP-1 and TIMP-2 levels (correlation coefficients of 0.793 and 0.798, respectively). These results suggest that abnormalities in MMP and TIMP profiles may be helpful in the biochemical characterisation of CJD.     

Comparative assessment of matrix metalloproteinase (MMP)-2 and MMP-9, and their inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in preeclampsia and gestational hypertension

ObjectivesTo compare the circulating levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and the MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios in preeclampsia and gestational hypertension with those found in normotensive pregnancies.Design and methodsWe studied 83 pregnant women (30 healthy pregnant women with uncomplicated pregnancies, 26 with gestational hypertension, and 27 with preeclampsia) and 30 healthy nonpregnant women in a cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively.ResultsWe found higher plasma pro-MMP-9 levels, and higher pro-MMP-9/TIMP-1 ratios in women with gestational hypertension (95%-CI: 1.031 to 2.357, and 0.012 to 0.031, respectively), but not with preeclampsia, compared with those found in normotensive pregnant women (95%-CI: 0.810 to 1.350, and 0.006 to 0.013, respectively; both P < 0.05). We found no significant differences in pro-MMP-2 levels (P > 0.05).ConclusionsThe higher net MMP-9 (but not MMP-2) activity in gestational hypertension compared with normotensive pregnancy suggests that MMP-9 plays a role in the pathophysiology of gestational hypertension. Conversely, the lack of such alterations in preeclampsia is consistent with the notion that different pathophysiological mechanisms are involved in these hypertensive disorders.     

Immunohistochemical expression of matrix metalloproteinases 1 and 2 (MMP-1 and MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in ruptured and non-ruptured tubal ectopic pregnancies

OBJECTIVE: Recent evidence suggests that matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are crucial for trophoblast implantation in normal pregnancy. To evaluate the expression of MMP-1, MMP2, and the tissue inhibitor of MMP-2 (TIMP-2) along the invasive pathway of trophoblast in ruptured and non-ruptured tubal ectopic pregnancies, we performed a retrospective immunohistochemical study. METHODS: In 15 tissue specimens of patients with ruptured (N = 7) and non-ruptured (N = 8) first trimester tubal ectopic pregnancies who underwent laparoscopic salpingectomy, immunohistochemical staining against MMP-1, MMP-2, and TIMP-2 was performed. Serial paraffin sections were photographed and digitized for a computerized quantitative image analysis. Mean percentages of positive stained areas by MMP-1, MMP-2, and TIMP-2 antibodies in the extravillous trophoblast were determined for ruptured and non-ruptured tubal ectopic pregnancies and compared. RESULTS: In our 15 tissue specimens of ectopic pregnancies MMP-1 and TIMP-2 were found to be more prominent in the immunohistochemical distribution pattern than MMP-2. However, no statistically significant difference could be detected between the mean percentages of positive stained area by MMP-1, MMP-2, and TIMP-2 antibodies in ruptured and non-ruptured tubal pregnancies. DISCUSSION: For the first time, we measured the comparative immunohistochemical expression of MMP-1, MMP-2, and TIMP-2 in ruptured and non-ruptured tubal ectopic pregnancies. Although our results did not show any statistically significant difference between ruptured and non-ruptured tubal ectopic pregnancies, we conclude that MMP-1, MMP-2, and TIMP-2 are functionally involved in the highly proliferative early first part of ectopic implantation.     

基质金属蛋白酶-9及组织抑制因子-1在老年犬持续性心房颤动心房组织中表达

目的探讨基质金属蛋白酶（matrixmetalloproteinase,MMP）-9及组织抑制因子（tissueinhibitorsofmetalloproteinase,TIMP）-1在老年犬持续性心房颤动（房颤）心房肌组织中表达变化及其与心房颤动及纤维化发生的关系。方法健康比格犬18只随机分为成年、老年窦性心律组各5只、成年、老年持续性房颤组各4只。采用Masson三色法染色,计算胶原容积分数以测定纤维化程度,采用RT-PCR检测左心房MMP-9及TIMP-1的mRNA水平表达情况,采用蛋白质印迹法测定其蛋白水平表达情况。结果与成年窦性心律组比较,其余3组心房肌组织纤维化程度明显增高（P〈0．01）;TIMP-1mRNA表达水平下降（P〈0．05）,MMP-9蛋白表达水平升高（P〈0．01）,TIMP-1蛋白表达水平下降（P％0．01）;老年窦性心律组及老年持续性房颤组MMP-9mRNA表达水平较成年窦性心率组升高（P〈0．05）;与成年持续性房颤组比较,老年持续性房颤组纤维化程度增加,胶原容积分数明显增高（P％0．05）,TIMP-l蛋白表达水平下降（P〈0．01）。结论心房组织中MMP9和TIMP1基因表达失衡可能是影响胶原代谢、造成增龄性心房颤动时心房肌纤维化的分子机制之一,与增龄性心房颤动的发生和持续有关。     

Assessment of matrix metalloproteinase (MMP)-2, MMP-8, MMP-9, and their inhibitors,the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in obese children and adolescents

ObjectivesTo compare the circulating levels of matrix metalloproteinase (MMP)-8, pro-MMP-2, pro-MMP-9, and total MMP-9, their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2, and the MMP-8/TIMP-1, MMP-9/TIMP-1, and MMP-2/TIMP-2 ratios in normotensive obese children and adolescents with those found in non obese children and adolescents.Design and methodsWe studied 40 obese and 40 non obese (controls) children and adolescents in this cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA.ResultsObese children and adolescents had higher circulating MMP-8 concentrations, lower plasma TIMP-1 concentrations, and higher MMP-8/TIMP-1 ratios than non obese controls (P < 0.05). We found no differences in pro-MMP-9 or total MMP-9 levels, or in MMP-9/TIMP-1 ratios between groups (P > 0.05). While we found no significant differences in pro-MMP-2 levels (P > 0.05) obese subjects had higher TIMP-2 concentrations and lower pro-MMP-2/TIMP-2 ratios (P < 0.05) than non obese controls.ConclusionsIn conclusion, we found evidence indicating higher net MMP-8 (but not MMP-9 and MMP-2) activity in childhood obesity. The increased MMP-8 levels found in obese children suggest a possibly relevant pathophysiological mechanism that may be involved in the increase of cardiovascular risk associated with childhood obesity.     

Dilinoleoylphosphatidylcholine prevents transforming growth factor-beta1-mediated collagen accumulation in cultured rat hepatic stellate cells

Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines, protects against alcoholic and nonalcoholic liver fibrosis in baboons and rats, respectively. In this study, we assessed the antifibrogenic action of dilinoleoylphosphatidylcholine (DLPC), the main phosphatidylcholine species of PPC, against transforming growth factor-beta1-mediated expression of alpha1(I) procollagen, tissue inhibitor of metallopreoteinase-1 (TIMP-1) and matrix metalloproteinase-13 (MMP-13) in cultured rat hepatic stellate cells (HSCs). In primary culture-activated HSCs, TGF-beta1 up-regulated the alpha1(I) procollagen mRNA level with a concomitant increase in type I collagen accumulation in culture media. Whereas TIMP-1 mRNA levels and TIMP-1 accumulation in media were also increased by TGF-beta1, MMP-13 mRNA expression and MMP-13 concentration in media were not altered. DLPC fully blocked TGF-beta1-induced increase in alpha1(I) procollagen mRNA expression and decreased collagen accumulation in media. Whereas TIMP-1 mRNA level and TIMP-1 accumulation in media were decreased by DLPC, MMP-13 mRNA expression and MMP-13 concentration in media were not changed by this treatment. Palmitoyl-linoleoylphosphatidylcholine (PLPC), the second most abundant component of PPC, had no effect on the concentrations of collagen, TIMP-1, and MMP-13 in HSC culture. We conclude that DLPC prevents TGF-beta1-mediated HSC fibrogenesis through down-regulation of alpha1(I) procollagen and TIMP-1 mRNA expression. The latter effect leads to a decreased accumulation of TIMP-1 that, in the presence of unchanged MMP-13 mRNA expression and MMP-13 concentration, results in a larger ratio of MMP-13/TIMP-1 concentrations in the culture media, favoring collagen degradation and lesser collagen accumulation. This effect of DLPC may explain, at least in part, the antifibrogenic action of PPC against alcoholic and other fibrotic disorders of the liver.