Serum matrix metalloproteinases MMP-2 and MMP-9 and metalloproteinase tissue inhibitors TIMP-1 and TIMP-2 in diabetic nephropathy

BACKGROUND: The regulation of mesangial extracellular matrix (ECM) turnover engages a number of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). High glucose concentration affects ECM degradation and the activities of MMPs and TIMPs. ECM accumulation is involved in the pathogenesis of diabetic nephropathy. METHODS: Serum MMP-9, MMP-2, TIMP-2 and TIMP-1 were measured with ELISA in patients with either chronic renal failure (CRF, n=20), type 2 diabetes mellitus (DM2, n=16) or diabetic nephropathy (DM2+CRF, n=14), and healthy controls (n=20). RESULTS: Diabetic nephropathy was related with profound decrease of serum TIMP-2 (122.2 +/- 47.2 vs. 263.0 +/- 89.2 ng/mL), TIMP-1 (242.5 +/- 96.9 vs. 347.4 +/- 87.2 ng/mL) and MMP-2 (385.4 +/- 42.6 vs. 517.2 +/- 75.4 ng/mL) (p<0.001). Both TIMP-1 and TIMP-2 were reduced in diabetic nephropathy in comparison with either diabetes alone (p<0.01 and p<0.001; respectively) or CRF alone (p<0.001 for both). An approximately 2-fold increase of MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio was found in diabetic nephropathy when compared with diabetes with normal renal function (p<0.01). Further, in DM2 patients, TIMP-2 was decreased when compared with CRF alone (219.2 +/- 71.8 vs. 296.8 +/- 58.4 ng/mL). MMP-2 was lowered in both groups of DM2 and CRF patients (413.8 +/- 59.0 ng/mL and 409.7 +/- 93.1 ng/mL, vs. normal control value of 517.2 +/- 75.4 ng/mL; p<0.001). CONCLUSIONS: These data indicate that circulating TIMP-1, TIMP-2 and MMP-2 are decreased in patients with diabetic nephropathy when compared with either CRF or diabetes.     

Sputum levels of metalloproteinase-9 and tissue inhibitor of metalloproteinase-1, and their ratio correlate with airway obstruction in lung transplant recipients: relation to tumor necrosis factor-alpha and interleukin-10.

BACKGROUND: Chronic transplant rejection is characterized by progressive narrowing of small airways caused by matrix remodeling and fibrosis. Matrix-metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are involved in the turnover of extracellular matrix. METHODS: To clarify the contribution of MMPs and TIMPs to airway inflammation in patients after lung transplantation (LTx), we used enzyme immunoassays to measure induced sputum concentrations of MMP-9, TIMP-1, and controlling cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 of 30 LTx patients and 15 control subjects. RESULTS: Sputum concentrations of MMP-9, TIMP-1, the MMP-9:TIMP-1 ratio, and TNF-alpha were higher in LTx patients than in control subjects (p < 0.04, all comparisons). The MMP-9, MMP-9:TIMP-1, and TNF-alpha levels were also significantly higher in LTx patients with chronic rejection compared with those with stable organ function (p < 0.03, all comparisons), whereas IL-10 levels were higher in the latter group (p = 0.05). In all LTx patients, MMP-9 and the MMP-9:TIMP-1 ratio were negatively correlated with forced expiratory volume in 1 second values (rho = -0.47, p = 0.01, and rho = -0.53, p = 0.003, respectively). We found that MMP-9 positively correlated with sputum neutrophils and TNF-alpha whereas MMP-9 and TIMP-1 did not correlate with IL-10. CONCLUSIONS: These data underline the possible contribution of proteases such as MMP-9 to chronic transplant rejection, and suggest that an imbalance of MMP-9 and TIMP-1 may be involved in the pathogenesis of airway obstruction after LTx. We found that MMP-mediated inflammation seems to be controlled by TNF-alpha whereas IL-10 might elicit anti-inflammatory effects through different pathways.     

Serum von Willebrand factor,matrix metalloproteinase-9, and vascular endothelial growth factor levels predict the onset of cerebral vasospasm after aneurysmal subarachnoid hemorrhage

OBJECTIVE: Endothelial damage and intimal proliferation occur in vasospastic cerebral arteries after subarachnoid hemorrhage (SAH). In the peripheral vasculature, endothelial damage increases intimal matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) levels, causing neointimal proliferation. We hypothesized that serum von Willebrand factor (vWF) (a marker of endothelial cell death), MMP-9, and VEGF levels could serve as prognostic markers in predicting the occurrence of cerebral vasospasm. METHODS: Venous serum vWF, MMP-9, and VEGF levels were prospectively measured daily, for 12 days or until the onset of vasospasm, for 45 consecutive patients admitted with SAH (n = 38) or admitted for elective aneurysm clipping (control subjects, n = 7). The development of transcranial Doppler flow velocities of more than 180 cm/s and/or new focal neurological deficits with angiographically confirmed vasospasm was considered the onset of vasospasm. To establish whether these markers were specific for vasospasm versus ischemia, blood samples were obtained from a concurrent group of 42 patients within 24 hours after stroke onset unrelated to SAH. RESULTS: Fifty-seven percent of patients (22 of 38 patients) developed vasospasm, 4 to 11 days after SAH (median, 7 d). Mean peak serum vWF, MMP-9, and VEGF levels were increased in the SAH prevasospasm cohort, compared with the SAH nonvasospasm cohort (vWF, 5526 +/- 929 versus 4934 +/- 599 ng/ml, P = 0.01; MMP-9, 705 +/- 338 versus 438 +/- 154 ng/ml, P = 0.006; VEGF, 0.12 +/- 0.06 versus 0.06 +/- 0.06 ng/ml, P = 0.023). Mean peak vWF, MMP-9, and VEGF levels for the focal ischemia cohort (vWF, 4645 +/- 875 ng/ml, P = 0.01; MMP-9, 250 +/- 308 ng/ml, P = 0.001; VEGF, 0.03 +/- 0.04 ng/ml, P = 0.001) were markedly lower in comparison with the SAH prevasospasm cohort and were unchanged in comparison with the control cohort. vWF levels of more than 5500 ng/ml, VEGF levels of more than 0.12 ng/ml, and MMP levels of more than 700 ng/ml each independently increased the odds of subsequent vasospasm (18-, 20-, and 25-fold, respectively). CONCLUSION: The development of cerebral vasospasm after SAH was preceded by increases in serum vWF, MMP-9, and VEGF levels. Increased serum vWF, MMP-9, and VEGF levels could accurately predict the onset of cerebral vasospasm after SAH. These factors were not elevated by SAH alone or in a separate cohort of patients with ischemic stroke, suggesting that these factors might play a role in the pathogenesis of human cerebral vasospasm.     

Role of matrix metalloproteinase-9 in the basement membrane destruction of superficial urothelial carcinomas

PURPOSE: This study was conducted to clarify which matrix metalloproteinases (MMPs) play a key role in destruction of the underlying basement membrane (BM) of superficial urothelial carcinomas. Urine concentrations of MMP-9 and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) were also measured. MATERIALS AND METHODS: Overexpression of MMP-1, MMP-2 and MMP-9 was analyzed immunohistochemically in 60 patients with transitional cell carcinomas of the urothelium (41 were pTa or pis, 19 were pT1-4), and compared them with type IV collagen expression in tumor BM. In 33 of them, urine concentrations of MMP-9 and TIMP-1 were measured by one-step sandwich enzyme immunoassay. RESULTS: Positive expression of MMP-1, MMP-2 and MMP-9 was found in 53%, 17%, and 65% of tumors, respectively. Only MMP-9 expression rates were increased with grades and stages (p = 0.03). In pTa and pis tumors, type IV collagen expression was reduced in 17 of 26 (65.4%), and it was associated with positive MMP-9 expression (p = 0.0283). MMP-9 was detected in all urine samples of urothelial cancer patients, while urine TIMP-1 was detectable in 18 of 33 patients. In 16 healthy volunteers, both of them were below detectable levels. Balance between urinary MMP-9 and TIMP-1 were particularly kept in superficial urothelial carcinomas with intact tumor BM. Tumor BM status, however, was not associated with urinary MMP-9 or TIMP-1 levels. CONCLUSIONS: These results suggest that MMP-9 plays a key role in the invasion step of superficial urothelial carcinomas. Detection of urinary MMP-9 may become a new, non-invasive mean for the diagnosis of urothelial carcinomas.     

TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in renal proximal tubular cells

BACKGROUND: Tubulointerstitial fibrosis is a morphologic hallmark of chronic kidney disease and is a key factor in the prediction of progression to end-stage renal failure. Disruption of tubular basement membrane and interstitial extracellular matrix (ECM) via cytokine-induced alterations in matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) may be an important mechanism in this process. The presence of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and their effects on proximal tubular cells may be critical in this process. METHODS: Human proximal tubular cells were cultured in hormonally defined medium. Cells at 80% confluency were exposed to TNF-alpha (0.1 to 100 ng/mL) or IL-1beta (0.1 to 100 ng/mL) or a combination of both for 48 hours. Activity and expression of MMP-9 was examined by gelatin zymography and Western blot analysis. TIMP-1 expression was analyzed by Western blotting. Signaling through cytokine receptors, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways was investigated. RESULTS: TNF-alpha but not IL-1beta resulted in a dose-dependent increase in the latent form of MMP-9. TIMP-1 was decreased by treatment with either TNF-alpha or IL-1beta. Cotreatment with IL-1beta abolished the induction of MMP-9 but augmented the inhibition of TIMP-1 in the presence of TNF-alpha. Inhibition of PKC provided evidence of the importance of this pathway in mediating the cytokine-induced suppression of TIMP-1 in human kidney (HK-2) cells. Activation of the extracellular signal-regulated protein kinase (ERK1/2) MAPK mediated the up-regulation of MMP-9 by TNF-alpha whereas p38 was found to be involved in the IL-1beta-mediated inhibition of TNF-alpha-stimulated MMP-9. CONCLUSION: The differential effects of TNF-alpha and IL-1beta on proximal tubular MMP-9 and TIMP-1 expression are mediated through the TNF-RI, the IL-1-RI and the different signaling pathways of PKC, ERK1/2, and p38 MAPK. These findings may provide new insights into the role of proinflammatory cytokines TNF-alpha and IL-1beta in the development and possible therapeutic intervention in tubulointerstitial fibrosis.     

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     

Thrombin stimulates synthesis of type IV collagen and tissue inhibitor of metalloproteinases-1 by cultured human mesangial cells.

Glomerular accumulation of extracellular matrix (ECM) is the common pathologic feature following glomerular injury, and the alteration in the synthesis and degradation of ECM may be involved in the glomerular accumulation of ECM. Glomerular fibrin formation occurs in various forms of human and experimental glomerulonephritis, and it may play an important role in progressive glomerular injury. Thrombin, a multifunctional serine proteinase that is generated at the site of vascular injury, has central functions in hemostasis and it also shows various biologic effects. In this study, it is hypothesized that thrombin may alter the production and the degradation of type IV collagen, which is an important component of ECM in the glomeruli. Human mesangial cells (HMC) were cultured, and the levels of type IV collagen, tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-2 (MMP-2) in the culture supernatants were measured by enzyme immunoassay using specific antibodies. MMP-2 activity was also evaluated by zymography using polyacrylamide/ sodium dodecyl sulfate gel-containing gelatin. Thrombin increased the production of type IV collagen and TIMP-1 in a dose-and time-dependent manner, but it did not increase MMP-2. Thrombin also stimulated the gene expressions of the type IV collagen and TIMP-1 in HMC in a dose- and time-dependent manner. Thrombin treated with diisopropylfluorophosphate, a serine proteinase inhibitor, did not show any of these effects. Hirudin, a natural thrombin inhibitor, and anti-transforming growth factor-beta-neutralizing antibody inhibited the stimulating effect of thrombin. These findings suggest that thrombin may contribute to the excessive accumulation of ECM and progression of glomerulosclerosis through an increase of type IV collagen production and a decreased matrix degradation presumably via a transforming growth factor-beta-dependent mechanism.     

A study of the effect of splenectomy on hepatic functional reserve and structural damage in patients with chronic hepatitis C virus infection by non-invasive serum markers. A prospective study

Several beneficial effects of splenectomy on the liver integrity have been recently reported by both experimental and clinical studies. However, the effects of splenectomy on hepatic functional reserve and structural damage in patients with chronic hepatitis C (CHC) were not studied by objective evidence. The aim of this study was to assess the effect of splenectomy on hepatic functional reserve and structural damage in patients with CHC by non-invasive serum markers. The study involved 22 patients with histopathological diagnosis of CHC undergoing splenectomy for treatment of associated hypersplenism. The hepatic functional reserve and structural damage markers were assessed before and after splenectomy surgery on the 2nd and 60th postoperative days by aspartate aminotransferase to alanine aminotransferase (AST/ALT ratio), AST to platelet ratio index (APRI) and serum levels of gamma-glutamyl transferase (GGT), hyaluronic acid (HA), type IV collagen (CIV) and tissue inhibitor of metalloproteinase-1 (TIMP-1). After splenectomy, the levels of serum HA showed a significant decrease in relation to the preoperative values both in PO-1 (mean pre-splenectomy: 272+/-88.6 versus 185+/-77.4 ng/ml; P=0.01) and PO-2 (169+/-58.1 ng/ml; 0.017). The levels of type IV collagen showed a significant decrease in relation to the preoperative values both in PO-1 (mean pre-splenectomy: 208+/-134 versus 125+/-100 ng/ml; P=0.01) and PO-2 (121+/-74.7 ng/ml; P=0.02). Serum levels of TIMP-1 also showed a significant decrease in relation to the preoperative values both in PO-1 (mean pre-splenectomy: 764+/-571 versus 261+/-195 ng/ml; P=0.006) and PO-2 (149+/-110.1 ng/ml; P=0.004). There was no significant difference between PO-1 and PO-2 mean values for each of those serum markers. This study found that splenectomy induced a reduction of biochemical markers of liver functional reserve and fibrosis in patients with chronic hepatitis C which reflect a change in the processes involved in of liver fibrosis. However, it cannot be concluded whether this reflects a change in the rate of its progression or a prevention of further fibrosis.     

Effect of a specific endothelin receptor A antagonist and an angiotensin-converting enzyme inhibitor on glomerular mRNA levels for extracellular matrix components, metalloproteinases (MMP) and a tissue inhibitor of MMP in aminonucleoside nephrosis

Background. We previously reported that mRNA levels for extracellular matrix (ECM) components and endothelin (ET)-1 are upregulated in glomeruli of puromycin aminonucleoside (PAN) nephrosis. Angiotensin-converting enzyme (ACE) inhibitors are effective in experimental models of renal injury, including PAN nephrosis. This study was designed to assess whether the glomerular expression of mRNA for ECM components, ET-1, metalloproteinases (MMP), and a tissue inhibitor of metalloproteinases (TIMP) is modulated by a specific endothelin receptor A antagonist (FR139317) or angiotensin-converting enzyme inhibitor (enalapril) in PAN-injected rats. Methods. Animals were divided into six groups. Group 1 consisted of PAN-injected rats given no treatment. In group 2, PAN-injected rats were given enalapril 35 mg/l. In group 3, PAN-injected rats were given an intraperitoneal injection of FR139317. Group 4 consisted of saline-injected rats given no treatment. In group 5, saline-injected rats were given enalapril. In group 6, saline-injected rats were given FR139317. We prepared glomerular RNA and performed Northern blot analysis in all groups. Results. In PAN nephrosis, glomerula mRNA levels for &agr;1 (IV) collagen chain, laminin B1 and B2 chains, ET-1, MMP-2 and TIMP-1 increased at the peak of proteinuria on day 8 and then decreased to the control level by day 20, whereas those for &agr;1 (I) and &agr;1 (III) collagen chains, MMP-1, MMP-3 and GAPDH showed little change in PAN nephrosis throughout the experimental periods. In contrast, mRNA levels for heparan sulphate proteoglycan (HSPG) decreased on day 8 and then increased to the control level by day 20. Both enalapril and FR139317 attenuated the increases in mRNA levels for &agr;1 (IV) collagen chain (P <0.01), laminin chains (P < 0.01), and ET-1 (P <0.01), and attenuated the decreases in mRNA levels for HSPG (P < 0.01) in glomeruli of PAN-injected rats. Enalapril had little effect on increased glomerular mRNA levels for MMP-2 and TIMP-1 in PAN nephrosis, whereas FR139317 attenuated the increases in glomerular mRNA levels for MMP-2 (P < 0.01) and TIMP-1 (P < 0.01). Conclusions. These data suggest that the beneficial effects of enalapril and FR139317 may be related to modulation of glomerular mRNA expression of ECM components and ET-1 and that these agents may follow a different mechanism in regulating the glomerular mRNA expression for MMP-2 and TIMP-1 in PAN nephrosis.     

增生性瘢痕中基质金属蛋白酶及其抑制因子基因的表达

目的 了解基质金属蛋白酶2(MMP-2,又称明胶酶A)、MMP-9(又称明胶酶B)及其金属蛋白酶1组织抑制因子(TIMP-1)在增生性瘢痕不同形成时期的基因表达.方法 提取16例人体不同时期增生性瘢痕样本和8例正常皮肤样本总RNA,分离mRNA,用反转录-聚合酶链反应法检测MMP-2、MMP-9和TIMP-1基因在不同样本中的表达.结果 在正常皮肤中MMP-2、MMP-9和TIMP-1基因转录产物的灰度比值分别为(3.8±0.7)%、(5.8±4.4)%、(30.3±3.0)%,在增殖期瘢痕中分别为(13.5±4.5)%、(18.4±4.7)%、(37.7±4.3)%,明显高于正常皮肤(P＜0.05).成熟期瘢痕MMP-2和MMP-9基因表达量已恢复至正常水平,但TIMP-1基因较正常皮肤仍持续高表达(P＜0.05).结论 MMP-2、MMP-9和TIMP-1基因表达增强可能是增生性瘢痕形成的机制之一,MMP-2和MMP-9基因表达降低可能与增生性瘢痕达到相对稳定的成熟状态有关.     

基质金属蛋白酶9、3和金属蛋白酶组织抑制物1在原发性膜性肾病肾小球内的表达及意义

目的探讨基质金属蛋白酶9（MMP-9）、基质金属蛋白酶3（MMP-3）和金属蛋白酶组织抑制物1（TIMP-1）在原发性膜性肾病（IMN）患者肾小球内的表达变化及其与蛋白尿、Scr的关系。方法用免疫组织化学技术分别检测44例IMN患者（IMN组）与6例正常对照者（对照组）肾小球内MMP-9、MMP-3和TIMP-1的表达。结果MMP-9及MMP-3在对照组正常肾组织的肾小管上皮细胞、肾间质和肾小球足细胞有少量表达；在IMN组肾小球足细胞、系膜细胞、肾小球基底膜及肾小管上皮细胞有表达。IMN组肾小球内MMP-9及MMP-3的表达均较对照组显著增强（P〈0．05）。TIMP-1在对照组正常肾组织的肾小管上皮细胞有少量表达，肾小球内无表达；在IMN组肾小球足细胞、肾小管上皮细胞有表达。IMN组肾小球内TIMP-1的表达较对照组显著增强（P〈0．01）。IMN组24h尿蛋白定量显著高于对照组（P〈0．01）。Ⅱ期MN组肾小球MMP-9／TIMP-1及MMP-3／TIMP-1比值较Ⅰ期MN组明显下降（P〈0．01）。IMN组肾小球内MMP-9的表达与Scr呈负相关（r=-0．02，P〈0．05）；TIMP-1的表达与Scr呈正相关（r=0．34，P〈0．05）。结论MMP-9与TIMP-1从正反两方面影响IMN患者肾功能的改变。MMP-9、MMP-3的异常表达与IMN患者蛋白尿之间可能相关；IMN患者肾小球MMP／TIMP比值失衡可能与IMN患者肾小球基底膜增厚相关。     

Hepatocyte growth factor (HGF) modulates matrix turnover in human glomeruli

BACKGROUND: The imbalance between synthesis and degradation of mesangial matrix causes glomerulosclerosis and leads to renal failure. Hepatocyte growth factor (HGF) has been shown to reduce the progression in murine models of chronic renal failure. The present study evaluated the effect of HGF on the extracellular matrix turnover and on c-met receptor in human glomeruli. METHODS: Human glomeruli microdissected from donor kidney biopsies before transplantation were incubated with culture media containing HGF (50 ng/mL). After 24 and 48 hours, the expression of c-met, (alpha2) IV collagen, transforming growth factor-beta (TGF-beta), metalloprotease (MMP) 2 and 9 and of the inhibitor of MMP-2, tissue inhibitors of metalloprotease-1 (TIMP-1), was evaluated by polymerase chain reaction (PCR). beta-actin was used as housekeeping gene. The production of collagen type IV and TGF-beta was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blotting and the activity of MMP by zymography. RESULTS: (alpha2) IV collagen, TGF-beta, and TIMP-1 mRNA levels were markedly decreased in glomeruli treated with HGF at 24 and 48 hours. The expression of c-met was up-regulated by HGF treatment. HGF reduced the production of collagen type IV and TGF-beta. MMP-2 but not MMP-9 mRNA level was increased in HGF-treated glomeruli, although the gelatinolytic activity of the supernatant was not changed. By light microscopic examination kidney biopsies neither showed glomerular hypercellularity nor mesangial expansion. CONCLUSION: HGF reduced expression and synthesis of TGF-beta and collagen type IV and increased MMP-2 mRNA level in normal human glomeruli. These results suggest an antifibrotic effect of HGF on glomerular cells and may explain its beneficial role in glomerulosclerosis.     

The no donor DETA-NONOate decreases MMP-9 expression and activity in abdominal aortic explants

Objective: The goal of this project is to examine the role of an exogenous NO donor, DETA-NONOate (DETA), on MMP-9, MMP-2, and TIMP-1 expression and activity in interleukin-1β (IL-1β) induced rat aortic explants (RAE). Methods: RAEs were incubated with IL-1β (2 ng/ml) and increasing concentrations of DETA (0, 5.0, 50, 100, and 500 μM) (n = 3 per group). Messenger RNA (mRNA) was extracted from cells after 24 hours and analyzed for MMP-9, MMP-2, and TIMP-1 expression levels by real time RT-PCR. Media at 48 hours was collected and assayed for NO₂ and NO₃ (NO_x) by the Saville Assay, MMP-9 and MMP-2 activity by gelatin zymography, and TIMP-1 activity by reverse zymography. All statistical analyses were performed by ANOVA and Pearson correlation. Results: DETA administration resulted in a dose-dependent increase in media NO_x concentration (0.001 +/- 0.0003 ng NO_x / mg protein to 0.062 +/- 0.004 ng NO_x / mg protein, p < 0.01). In RAE, MMP-9 expression and activity decreased significantly in a dose dependent fashion with increasing DETA concentrations (p < 0.01). At the maximal dose of 500 μM DETA, a 78% decrease in MMP-9 expression (p < 0.05) and a 72% decrease in pro-MMP-9 activity (p < 0.05) was demonstrated compared to RAE treated with IL-1β alone (0 μM DETA). There were no significant differences seen in MMP-2 and TIMP-1 expression or activity in response to DETA exposure. Conclusion: The NO donor DETA-NONOate decreased IL-1β induced MMP-9 expression and activity in RAE in a dose dependent fashion. These data suggest that NO donors may be beneficial in decreasing MMP-9 levels, an enzyme believed to be critical in vessel wall remodeling, and therefore may serve to inhibit MMP-9 dependent vessel wall degradation seen during AAA formation.     

High glucose decreases matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth factor-beta1.

Diabetic nephropathy is characterized by accumulation of mesangial matrix. Glucose-induced inhibition of matrix-degrading enzymes such as collagenases is believed to contribute to matrix accumulation. We have previously demonstrated that 72 kDa type IV collagenase activity is decreased in the rat mesangial cells cultured in high glucose media [Diabetes 1995;44:929-935]. The present studies were designed to investigate if the cytokine transforming growth factor-beta1 (TGF-beta1) mediates this effect of glucose. Type IV collagenases degrade type IV collagen as well as gelatin (denatured collagen) and are thus also called gelatinases. They belong to the family of matrix metalloproteinases (MMPs); MMP activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). The activity of 72 kDa type IV collagenase, also known as matrix metalloproteinase-2 (MMP-2), was assessed using three methods: (1) fluoresceinated gelatin degradation assay to detect free enzyme activity (activity which is present in excess of TIMP-inhibited activity); (2) zymography to measure total (free + TIMP-bound) enzyme activity; (3) ELISA using specific antibodies to measure MMP-2 levels. TGF-beta1 and TIMP-2 levels were also determined by ELISA. Incubation of primary cultures of rat mesangial cells for 5 days in 30 vs. 5 mM glucose resulted in a 3-fold increase in production of total TGF-beta1, a significant decrease in MMP-2 activity and immunoreactive MMP-2 levels, and an increase in TIMP-2 levels. Addition of exogenous TGF-beta1 to mesangial cells incubated in 5 mM glucose replicated the high glucose effect by producing a significant decrease in MMP-2 levels with a concurrent increase in TIMP-2 levels. Furthermore, glucose-induced inhibition of MMP-2 activity was completely blocked by neutralization of TGF-beta1 with anti-TGF-beta1 antibody. We conclude that the decrease in MMP-2 activity induced by glucose loading is mediated via TGF-beta1.     

Gender differences in rat aortic smooth muscle cell matrix metalloproteinase-9

BACKGROUND: A predilection exists for men to develop abdominal aortic aneurysms (AAAs), but the reasons for this gender predisposition are not known. Matrix metalloproteinase-9 (MMP-9) has been implicated in both human and experimental AAAs. This investigation tested the hypothesis that male and female gender differences exist in the production of MMP-9 by rat aortic smooth muscle cells (RASMCs). STUDY DESIGN: In the first set of experiments, cultured male and female RASMCs were stimulated with interleukin-1 beta (IL-1beta) at 2 ng/mL. Messenger RNA was extracted from the RASMCs and gene expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1), an MMP-9 inhibitor, was measured by quantitative real-time polymerase chain reaction. Cell culture media were collected for measurement of MMP-9 protein levels and MMP-9 activity by Western blotting and gelatin zymography, respectively. In the second set of experiments, male RASMCs were treated with 17-beta-estradiol (10(-10) to 10(-6) mol/L) and MMP-9 activity was measured. In the third set of experiments, male rats were pretreated with estradiol, and MMP-9 activity was measured in the media from explanted aortas. RESULTS: MMP-9 gene expression was 10-fold higher in male versus female RASMCs (p=0.003). MMP-9 protein levels (p=0.005) and gelatinolytic activities (p=0.01) were also greater in male than female RASMCs. TIMP-1 expression was fourfold higher in male versus female RASMCs (p<0.001). Estradiol-treated male RASMCs did not exhibit a decrease in MMP-9 activity. But aortic explants from male rats pretreated with 17-beta-estradiol had 60% less MMP-9 activity than explants from male controls (p=0.03). CONCLUSIONS: MMP-9 and TIMP-1 are greater in male than in female RASMCs. These findings support the tenet that gender-related differences in MMP-9 may contribute to AAA formation.     

Proteolysis of the abdominal aortic aneurysm wall and the association with rupture.

PURPOSE: to investigate proteolysis of the abdominal aortic aneurysm (AAA) wall and the association with rupture. METHODS: levels of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) were measured in the walls of medium-sized (5-7 cm in diameter) ruptured AAA (rAAA) (n =30) and large (> or = 7 cm in diameter) asymptomatic AAA (aAAA) (n=30). RESULTS: MMP-2 levels (median, range) were significantly higher in the walls of large aAAA (165 ng/g AAA tissue, 50-840) than from medium-sized rAAA (110 ng/g AAA tissue, 47-547, p=0.007). MMP-9 levels were significantly higher in the walls of medium-sized rAAA (107 ng/g AAA tissue, 19-582) than from large aAAA (55 ng/g AAA tissue, 11-278, p=0.012). TIMP-1 and TIMP-2 levels were equivalent. There was a positive correlation between MMP-2 and the diameter of aAAA (r=0.54, p=0.002), but a negative correlation with MMP-9 (r= -0.44, p=0.017). No significant correlations were found between aAAA diameter and TIMP-1 or TIMP-2. CONCLUSION: AAA rupture is associated with higher levels of MMP-9. There is no association with TIMP-1 or TIMP-2 levels. MMP-2 levels are positively, whereas MMP-9 levels are negatively, correlated with aAAA size. MMP-9 may play a role in the progression towards rupture, whereas MMP-2 may play a role in expansion.