Levels Of Active Matrix Metalloproteinase-9 Research Articles

Pharmacological inhibition of receptor-interacting protein kinase 2 (RIPK2) elicits neuroprotective effects following experimental ischemic stroke

Ischemic stroke induces a debilitating neurological insult, where inflammatory processes contribute greatly to the expansion and growth of the injury. Receptor-interacting protein kinase 2 (RIPK2) is most well-known for its role as the obligate kinase for NOD1/2 pattern recognition receptor signaling and is implicated in the pathology of various inflammatory conditions. Compared to a sham-operated control, ischemic stroke resulted in a dramatic increase in the active, phosphorylated form of RIPK2, indicating that RIPK2 may be implicated in the response to stroke injury. Here, we assessed the effects of pharmacological inhibition of RIPK2 to improve post-stroke outcomes in mice subjected to experimental ischemic stroke. We found that treatment at the onset of reperfusion with a RIPK2 inhibitor, which inhibits the phosphorylation and activation of RIPK2, resulted in marked improvements in post-stroke behavioral outcomes compared to the vehicle-administered group assessed 24 h after stroke. RIPK2 inhibitor-treated mice exhibited dramatic reductions in infarct volume, concurrent with reduced damage to the blood-brain barrier, as evidenced by reduced levels of active matrix metalloproteinase-9 (MMP-9) and leakage of blood-borne albumin in the ipsilateral cortex. To explore the protective mechanism of RIPK2 inhibition, we next pretreated mice with RIPK2 inhibitor or vehicle and examined transcriptomic alterations occurring in the ischemic brain 6 h after stroke. We observed a dramatic reduction in neuroinflammatory markers in the ipsilateral cortex of the inhibitor-treated group while also attaining a comprehensive view of the vast transcriptomic alterations occurring in the brain with inhibitor treatment through bulk RNA-sequencing of the injured cortex. Overall, we provide significant novel evidence that RIPK2 may represent a viable target for post-stroke pharmacotherapy and potentially other neuroinflammatory conditions.

Neuroprotection by the cannabidiol aminoquinone VCE-004.8 in experimental ischemic stroke in mice

Synthetic cannabidiol (CBD) derivative VCE-004.8 is a peroxisome proliferator-activated receptor gamma (PPARγ) and cannabinoid receptor type 2 (CB2) dual agonist with hypoxia mimetic activity. The oral formulation of VCE-004.8, termed EHP-101, possesses anti-inflammatory properties and is currently in phase 2 clinical trials for relapsing forms of multiple sclerosis. The activation of PPARγ or CB2 receptors exerts neuroprotective effects by dampening neuroinflammation in ischemic stroke models. However, the effect of a dual PPARγ/CB2 agonist in ischemic stroke models is not known. Here, we demonstrate that treatment with VCE-004.8 confers neuroprotection in young mice subjected to cerebral ischemia. Male C57BL/6J mice, aged 3–4 months, were subjected to 30-min transient middle cerebral artery occlusion (MCAO). We evaluated the effect of intraperitoneal VCE-004.8 treatment (10 or 20 mg/kg) either at the onset of reperfusion or 4h or 6h after the reperfusion. Seventy-two hours after ischemia, animals were subjected to behavioral tests. Immediately after the tests, animals were perfused, and brains were collected for histology and PCR analysis. Treatment with VCE-004.8 either at the onset or 4h after reperfusion significantly reduced infarct volume and improved behavioral outcomes. A trend toward reduction in stroke injury was observed in animals receiving the drug starting 6h after recirculation. VCE-004.8 significantly reduced the expression of pro-inflammatory cytokines and chemokines involved in BBB breakdown. Mice receiving VCE-004.8 had significantly lower levels of extravasated IgG in the brain parenchyma, indicating protection against stroke-induced BBB disruption. Lower levels of active matrix metalloproteinase-9 were found in the brain of drug-treated animals. Our data show that VCE-004.8 is a promising drug candidate for treating ischemic brain injury. Since VCE-004.8 has been shown to be safe in the clinical setting, the possibility of repurposing its use as a delayed treatment option for ischemic stroke adds substantial translational value to our findings.

Azithromycin Treatment vs Placebo in Children With Respiratory Syncytial Virus–Induced Respiratory Failure

Despite a high disease burden, there is no effective treatment for respiratory syncytial virus (RSV) infection. To determine whether administration of azithromycin (AZM) to children with RSV-induced respiratory failure is safe and to define the effect of AZM therapy on nasal matrix metalloproteinase 9 (MMP-9) levels. This randomized, double-blind, placebo-controlled phase 2 trial was conducted at a single tertiary pediatric intensive care unit from February 2016 to February 2019. The study included children with RSV infection who were admitted to the pediatric intensive care unit and required respiratory support via positive pressure ventilation (invasive and noninvasive). A total of 147 children were screened; 90 were excluded for not meeting inclusion criteria, having an absent legal guardian, lacking pharmacy support, or having a language barrier and 9 declined participation, resulting in 48 patients enrolled in the study. Receipt of standard dose AZM (10 mg/kg/d), high-dose AZM (20 mg/kg/d), or a matching placebo of normal saline intravenously for 3 days. Nasal and endotracheal samples were collected at baseline as well as at 24 hours and 48 hours after start of treatment. The secondary outcome was to determine treatment effect on clinical outcome measures, including days of positive pressure ventilation and length of hospital stay. A total of 48 patients were enrolled in the trial, with a median (range) age at randomization of 12 (1 to 125) months; 36 participants (75.0%) were younger than 2 years. Overall, 26 participants (54.2%) were boys, and 29 (60.4%) had a comorbidity. A total of 16 patients were randomized into each trial group (ie, placebo, standard-dose AZM, and high-dose AZM). Baseline demographic characteristics were comparable among the 3 groups. Both doses of AZM were safe, with no adverse events observed. No difference in nasal MMP-9 levels were observed between treatment groups. Among those who required mechanical ventilation and received high-dose AZM, endotracheal active and total MMP-9 levels were lower on day 3. Compared with baseline, active and total MMP-9 levels in endotracheal aspirates were 1.0 log lower in the high-dose AZM group (active MMP-9: 99.8% CI, -1.28 to -0.64; P < .001; total MMP-9: 99.8% CI, -1.37 to -0.57; P < .001). Patients who received high-dose AZM had fewer median (interquartile range) hospital days compared with those receiving the placebo (8 [6-14] days vs 11 [8-20] days; mean ratio estimate, 0.57; 95% CI, 0.38-0.87; P = .01). In this phase 2 randomized clinical trial, both doses of AZM were safe. While nasal MMP-9 levels were unchanged among treatment groups, endotracheal MMP-9 levels were lower among those who received high-dose AZM. The positive secondary clinical outcome, while exploratory, provides insight for end points in a multicenter randomized trial. ClinicalTrials.gov Identifier: NCT02707523.

Hyperbaric oxygen therapy accelerates wound healing in diabetic mice by decreasing active matrix metalloproteinase-9.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

Coexpressed High Levels of VEGF-C and Active MMP-9 Are Associated With Lymphatic Spreading and Local Invasiveness of Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) usually has a good prognosis, but some patients develop an aggressive course of the disease, leading to a poor outcome. Vascular endothelial growth factor C (VEGF-C) and matrix metalloproteinase 9 (MMP-9) have been shown to play roles in tumor progression in various human malignancies. We analyzed VEGF-C and active MMP-9 expression profiles in PTC samples using immunohistochemistry and Western blotting. Immunohistochemistry showed positive staining for VEGF-C and active MMP-9 in 83% and 57% of PTCs, respectively (n = 60), with a positive correlation between their expression levels (Spearman, P < .001). Concomitant high expression of VEGF-C and active MMP-9 correlated with the presence of lymph node metastasis (P = .005), pT status (P = .004), pTNM tumor stage (P = .005), and particularly the degree of tumor infiltration (P < .001, Fisher exact test). Densitometric analysis of Western blot bands confirmed correlation between VEGF-C and active MMP-9 expression (Wilcoxon and Spearman tests) and significant association with the clinicopathologic parameters (Mann-Whitney and Kruskal-Wallis tests). Association of coexpressed high levels of VEGF-C and active MMP-9 with lymphatic spreading and local invasiveness of PTC suggests their potential usefulness as predictive biomarkers of aggressive PTC behavior.

Matrix metalloproteinase-9 index as a possible parameter for predicting acute coronary syndrome in diabetics.

Matrix metalloproteinase-9 (MMP-9) index is the ratio of active MMP-9 and total MMP-9 levels. It reflects the importance of MMP-9 in acute coronary syndrome (ACS). The study included 3 groups of patients (n = 87): the group 1 - non-diabetic subjects without ACS (control); the group 2 - diabetic patients with ACS [subgroups with unstable angina pectoris (UAP), myocardial infarction (MI) or reinfarction]; and the group 3 non-diabetics patients with ACS. Total and active MMP-9 were measured and used to create MIP-9 index. MMP-9 index, as a marker showed good sensitivity and specificity, of ACS in diabetics, with a cut-off value over 58.2. MMP-9 was higher in the study groups than in the control one. MMP-9 correlated with ACS occurrence and type of cardiovascular event. A statistically significant difference was found among the groups according to active MMP-9 (p < 0.001). The same was found with active MMP-9 between the control and the group with MI (p < 0.001). The control was highly statistically significantly different from the group of patients with UAP (p < 0.01). Statically significant differences in MMP-9 index was found between the control and the diabetics with ACS (P < 0.001). Statistically significant difference of MMP-9 index was also found in the controls compared to the value in non-diabetic patients with ACS (p < 0.01). MMP-9 index may be a possible marker of atheromatous plaque rupture in diabetics.

Intra-Individual Changes of Active Matrix Metalloproteinase-9 Are Associated with Clinical In-Stent Restenosis of Bare Metal Stents

Objectives: Increased chronic postprocedural levels of active matrix metalloproteinase-9 (MMP-9) have been associated retrospectively with a history of in-stent restenosis (ISR). This study aimed to determine whether index or post-percutaneous coronary intervention (PCI) plasma levels of active MMP-9 are a predictor of subsequent clinical ISR, in a standard population of patients treated with bare metal coronary stents. Methods: Four hundred thirty-two patients were prospectively recruited and sampled at index and 3 and 6 months after PCI. Those who developed symptomatic angiographically confirmed ISR were compared to randomly selected, asymptomatic controls, stratified by index presentation in a nested case-control design. Plasma samples were analyzed for the active form of MMP-9. Results: In all, 35 patients (8.1%) developed ISR, and these were compared to 98 controls. The increase in active MMP-9 over 3 months was significantly greater in the ISR group (p = 0.030) and independent of the established risk factors. Index clinical presentation was not associated with acute changes in active MMP-9; however, patients with ST-elevation myocardial infarction had greater increases in active MMP-9 at 3 months. Conclusions: The change in active MMP-9 over 3 months after bare metal coronary stent placement appears to be independently associated with the development of ISR in a standard PCI population.

Temporal profile of molecular signatures associated with circulating endothelial progenitor cells in human ischemic stroke

Endothelial progenitor cells (EPC) have been associated with good functional outcome in ischemic stroke. From preclinical studies, it has been reported that EPC proliferation is mediated by several molecular markers, including vascular endothelial growth factor (VEGF), stromal cell-derived factor-1α (SDF-1α), and the activity of matrix metalloproteinase-9 (MMP-9). Therefore, our aim was to study the role of these molecular factors in EPC proliferation in human ischemic stroke. Forty-eight patients with first episode of nonlacunar ischemic stroke were prospectively included in the study within 12 hr of symptom onset. EPC colonies were classified as early-outgrowth colony forming unit-endothelial cell (CFU-EC) and quantified at admission, at 24 and 72 hr, at day 7, and at 3 months. At the same time, serum levels of VEGF, SDF-1α, and active MMP-9 were measured by ELISA. The primary endpoint was EPC increment during the first week, which was defined as the difference in the number of CFU-EC between day 7 and admission. We found that VEGF (r = 0.782), SDF-1α (r = 0.828), and active MMP-9 (r = 0.740) levels at 24 hr from stroke onset showed a strong correlation with EPC increment. Similar results were found for VEGF levels at 72 hr (r = 0.839) and at day 7 (r = 0.602) as well as for active MMP-9 levels at 72 hr (r = 0.442) and at day 7 (r = 0.474). In the multivariate analyses, serum levels of VEGF at 72 hr (B: 0.074, P < 0.0001) and SDF-1α at 24 hr (B: 0.049, P = 0.008) were independent factors for EPC increment during the first week of evolution. These findings suggest that VEGF and SDF-1α may mediate EPC proliferation in human ischemic stroke.

Elevated levels of active matrix metalloproteinase-9 cause hypertrophy in skeletal muscle of normal and dystrophin-deficient mdx mice

Matrix metalloproteinases (MMPs) are a group of extracellular proteases involved in tissue remodeling in several physiological and pathophysiological conditions. While increased expression of MMPs (especially MMP-9) has been observed in skeletal muscle in numerous conditions, their physiological significance remains less-well understood. By generating novel skeletal muscle-specific transgenic (Tg) mice expressing constitutively active mutant of MMP-9 (i.e. MMP-9G100L), in this study, we have investigated the effects of elevated levels of MMP-9 on skeletal muscle structure and function in vivo. Tg expression of enzymatically active MMP-9 protein significantly increased skeletal muscle fiber cross-section area, levels of contractile proteins and force production in isometric contractions. MMP-9 stimulated the activation of the Akt signaling pathway in Tg mice. Moreover, expression of active MMP-9 increased the proportion of fast-type fiber in soleus muscle of mice. Overexpression of MMP-9 also considerably reduced the deposition of collagens I and IV in skeletal muscle in vivo. In one-year-old mdx mice (a model for Duchenne muscular dystrophy, DMD), deletion of the Mmp9 gene reduced fiber hypertrophy and phosphorylation of Akt and p38 mitogen-activated protein kinase. Collectively, our study suggests that elevated levels of active MMP-9 protein cause hypertrophy in skeletal muscle and that the modulation of MMP-9 levels may have therapeutic value in various muscular disorders including DMD.

Microbleeds and free active MMP‐9 are independent risk factors for neurological deterioration in acute lacunar stroke

in this prospective study, we evaluated mutual relationships amongst microbleeds, matrix metalloproteinase-9 (MMP-9) and neurological deterioration in patients with their first acute lacunar stroke. based on diffusion-weighted image findings, we recruited 206 patients with their first acute lacunar stroke. Those without a MRI scan were excluded. Small (a maximum lesion diameter of 15 mm) areas of subcortical gray and white matter with increased signals were considered as lacunar infarctions. GRE images were obtained within 24 h of the onset of stroke symptoms. Venous blood was sampled at base line (within 24 h). Clinical, biochemical, rheological and inflammatory parameters, neurological scales and free, active MMP-9 levels were compared between patients with and without microbleeds. Neurological deterioration was defined as an increase in more than two points on the National Institutes of Health Stroke Scale Score baseline 14 days after the onset of lacunar stroke. of the patients, 79 (38.3%) had microbleeds and 48 (23.3%) showed neurological deterioration. Free, active MMP-9 and C-reactive protein (CRP) levels were significantly increased amongst patients with microbleeds (P < 0.001 and P = 0.047, respectively). Existence of microbleeds (RR = 2.47, 95% CI = 1.25-3.83) and increased free, active MMP-9 (RR = 1.10 per 10 ng/ml, 95% CI = 1.03-1.19) were identified as independent risk factors for neurological deterioration after adjusting for potential confounders. ncreased levels of active MMP-9 and the existence of microbleeds might be useful in predicting the deterioration following an initial acute lacunar stroke.

Activation of matrix metalloproteinase-9 is associated with mobilization of bone marrow-derived cells after coronary stent implantation

Background After stent-related vascular injury, an inflammatory response triggers the mobilization of bone marrow-derived stem cells, including both endothelial and smooth muscle progenitors, leading to re-endothelialization as well as restenosis. It has been postulated that neutrophil-released matrix metalloproteinase-9 (MMP-9) induces stem cell mobilization. Aim To elucidate the mechanistic link between inflammation and stem cell mobilization after coronary stenting. Methods In 31 patients undergoing coronary stenting, we serially measured activated Mac-1 on the surface of neutrophils and active MMP-9 levels in the coronary sinus blood plasma, and the number of circulating CD34-positive cells in the peripheral blood. Results After bare-metal stent implantation (n = 21), significant increases in the numbers of CD34-positive cells (maximum on post-procedure day 7, P < 0.001), activated Mac-1 (at 48 h, P < 0.001), and active MMP-9 levels (at 24 h, P < 0.001) were observed. However, these changes were absent after sirolimus-eluting stent implantation (n = 10). In overall patients, the numbers of CD34-positive cells on day 7 (R = 0.58, P < 0.01) and activated Mac-1 at 48 h (R = 0.58, P < 0.01) were both correlated with active MMP-9 levels at 24 h. Stimulation of activated Mac-1 on the surface of isolated human neutrophils produced active MMP-9 release in vitro. Conclusions These results suggest that stent-induced activation of Mac-1 on the surface of neutrophils might trigger their MMP-9 release, possibly leading to the mobilization of bone marrow-derived stem cells. These reactions were substantially inhibited by sirolimus-eluting stents.

Neutrophil MMP-9 Proenzyme, Unencumbered by TIMP-1, Undergoes Efficient Activation in Vivo and Catalytically Induces Angiogenesis via a Basic Fibroblast Growth Factor (FGF-2)/FGFR-2 Pathway

The structural and catalytic requirements for neutrophil MMP-9 proenzyme (proMMP-9) to induce angiogenesis were investigated using a quantitative angiogenesis model based on grafting of collagen onplants onto the chorioallantoic membrane of chick embryos. Both physiological activation of neutrophil proMMP-9 and proteolytic activity of the generated MMP-9 enzyme were critically dependent on the tissue inhibitor of metalloproteinase (TIMP)-free status of the zymogen. The presence of an intact active site and hemopexin domain were required for full angiogenesis-inducing activity of the MMP-9 enzyme. Timed additions of TIMP-1 to the onplants containing TIMP-free neutrophil proMMP-9 indicated that in vivo activation of the zymogen occurred during the first 24 h after grafting. Within the onplant tissue, MMP-9 activation was accompanied by proteolytic modifications of fibrillar collagen and an influx of host proteins, the rate of which depended on the TIMP-free status of the zymogen. By quantifying the levels of host angiogenic factors, we demonstrated that basic fibroblast growth factor (FGF-2) was a major cytokine becoming bioavailable in the onplant tissue undergoing a neutrophil proMMP-9-mediated angiogenic switch. Inhibition of angiogenesis with specific function-blocking antibodies further indicated an involvement of a FGF-2/FGFR-2 pathway in neutrophil proMMP-9-induced angiogenesis. The enhanced angiogenesis catalyzed by neutrophil MMP-9 appears to evoke also a localized, low threshold level vascular endothelial growth factor (VEGF)/VEGFR-2 pathway, likely functioning in the formation and/or stabilization of blood vessels. That neutrophil proMMP-9, unencumbered by TIMP-1, directly mediates FGF-2-dependent angiogenesis was also demonstrated in our quantitative mouse angiogenesis model employing subcutaneous collagen implants, thus implicating the novel TIMP-free MMP-9/FGF-2/FGFR-2 pathway in proMMP-9-induced angiogenesis in a mammalian setting.

Abstract 1706: Elevation of Pericardial Fluid Active Matrix Metalloproteinase-9 (MMP-9) Level Is Closely Associated With Left Ventricular Remodeling

Background: The development of congestive heart failure (CHF) is associated with left ventricular (LV) remodeling. However, fundamental mechanisms that contribute to this remodeling process remain unclear. The matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been demonstrated to play a significant role in tissue remodeling through a number of pathological processes. Objectives: The purpose of this study was to investigate whether MMPs and TIMPs represent autocrine/paracrine factors and are accumulated in pericardial fluid. We previously reported that active MMP-2 levels in pericardial fluid served as more sensitive and accurate indicators of LV remodeling than did active MMP-2 levels in plasma. Methods: We measured the concentrations of the enzyme (active MMP-9) in both plasma and pericardial fluid in 20 patients during coronary artery bypass graft surgery. Results: The active MMP-9 level was significantly higher in pericardial fluid than in plasma (4.68 ± 1.17 vs. 2.58 ± 0.29 ng/ml, p&lt;0.0001). Interestingly, the pericardial fluid levels of active MMP-9 were significantly higher in patients with impaired LV function than in those with normal LV function (5.25 ± 0.57 vs. 2.96 ± 0.65 ng/ml, p&lt;0.0001). Pericardial fluid active MMP-9 levels had closer relations with LVEDVI (r=0.459, p=0.047) and LVESVI (r=0.490, p=0.0321) than did plasma active MMP-9 levels (LVEDVI: r=0.065, p=NS; LVESVI: r=0.254, p=NS). Active MMP-9 levels in pericardial fluid but not in plasma inversely correlated with LV ejection fraction (r=−0.626, p=0.0033). Conclusions: Active MMP-9 levels in pericardial fluid serve as more sensitive and accurate indicators of LV remodeling than did active MMP-9 levels in plasma. Thus, active form of MMP-9 may be also secreted from the heart into the pericardial space with the progression of CHF, and it may have a pathophysiologic role in LV remodeling process as an autocrine/paracrine factor.

Bronchoalveolar matrix metalloproteinase 9 relates to restrictive lung function impairment in systemic sclerosis

Systemic sclerosis (SSc) is frequently associated with interstitial lung disease (ILD) often leading to lung fibrosis. In this study we investigated whether matrix metalloproteinase 9 (MMP-9) and its natural inhibitor; the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), would be associated with remodelling in ILD in SSc. Levels of total MMP-9, pro-MMP-9 and TIMP-1 were measured in bronchoalveolar lavage (BAL) fluid from nine SSc patients with ILD, seven SSc patients without ILD and 16 age- and sex-matched healthy controls. Total MMP-9 and pro-MMP-9 levels were significantly elevated in SSc patients with ILD, compared to levels in SSc patients without ILD and healthy controls. In SSc patients with ILD calculated active MMP-9 levels were significantly higher than in SSc patients without ILD and tended to be higher than in healthy controls. TIMP-1 levels were elevated in both patient groups compared to healthy controls. Total-, pro- and active MMP-9 levels as well as pro-MMP-TIMP-1 and active MMP-9/TIMP-1 ratios were inversely associated with total lung capacity. The present study suggests that MMP-9 plays a pathophysiological role in the remodelling in ILD and lung fibrosis associated with SSc, and may represent a new therapeutic target in this condition.

Elevated Plasma Active Matrix Metalloproteinase-9 Level Is Associated With Coronary Artery In-Stent Restenosis

Objective— This study aimed to determine whether the plasma levels of matrix metalloproteinase-9 (MMP-9) or tissue inhibitor of metalloproteinases-1 (TIMP-1) were altered in patients with a history of symptomatic in-stent restenosis (ISR). Methods and Results— A group of 158 patients with a history of ISR were compared with 128 symptom-free patients. Plasma samples and a detailed risk factor history were collected. Plasma samples were analyzed for pro–MMP-9 and latent MMP-9 and active MMP-9, latent MMP-3, and TIMP-1. Several variables were associated with ISR, including index coronary disease extent and severity (number of diseased vessels and American College of Cardiology/American Heart Association lesion classification), number, diameter, and total length of stent(s) inserted, and plasma high-density lipoprotein cholesterol. Plasma active MMP-9 (odds ratio, 1.96; 95% CI, 1.43 to 2.69) showed independent risk association with ISR. Patients with multiple sites of ISR had significantly higher levels of active MMP-9 compared with patients with only a single ISR lesion or no ISR. Conclusion— Plasma active MMP-9 levels may be a useful independent predictor of bare metal stent ISR.

Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis

In this study, we employed a sensitive activity assay system to measure cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-9 (MMP-9) in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and nine primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also studied, as neurological controls, 48 patients with other inflammatory neurological disorders (OIND) and 48 with non-inflammatory neurological disorders (NIND). To assess active MMP-9/TIMP-1 circuit, CSF and serum levels of MMP-9 tissue inhibitor TIMP-1 were quantified by ELISA in the same patient population. CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P <0.05, <0.02 and <0.02, respectively), serum active MMP-9/TIMP-1 ratio was higher in MS (P<0.01) and OIND (P<0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P<0.05). More importantly, serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P<0.001, <0.001 and <0.05, respectively) and MRI (P<0.001, <0.001 and <0.02, respectively) disease activity, whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P<0.02 and <0.01, respectively). Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS immune dysregulation. In addition, our results indicate that CSF and serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity. In particular, serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

Elevated Plasma Active Matrix Metalloproteinase-9 Level Is Associated With Coronary Artery In-Stent Restenosis

This study aimed to determine whether the plasma levels of matrix metalloproteinase-9 (MMP-9) or tissue inhibitor of metalloproteinases-1 (TIMP-1) were altered in patients with a history of symptomatic in-stent restenosis (ISR). A group of 158 patients with a history of ISR were compared with 128 symptom-free patients. Plasma samples and a detailed risk factor history were collected. Plasma samples were analyzed for pro-MMP-9 and latent MMP-9 and active MMP-9, latent MMP-3, and TIMP-1. Several variables were associated with ISR, including index coronary disease extent and severity (number of diseased vessels and American College of Cardiology/American Heart Association lesion classification), number, diameter, and total length of stent(s) inserted, and plasma high-density lipoprotein cholesterol. Plasma active MMP-9 (odds ratio, 1.96; 95% CI, 1.43 to 2.69) showed independent risk association with ISR. Patients with multiple sites of ISR had significantly higher levels of active MMP-9 compared with patients with only a single ISR lesion or no ISR. Plasma active MMP-9 levels may be a useful independent predictor of bare metal stent ISR.

Downregulation of Angiogenic Factors in Ewing Tumor Xenografts by the Combination of Human Interferon‐α or Interferon‐β with Ifosfamide

Ewing sarcoma is the second most common bone tumor in childhood. Despite aggressive chemotherapy and radiotherapy, the prognosis of metastatic disease remains poor. In a nude mouse model of Ewing tumor xenografts, we recently showed that human type I interferons (IFNs) inhibit the growth of established xenografts. Combined therapy with human IFNs and ifosfamide (IFO), an alkylating agent widely used in high-dose chemotherapy of Ewing tumors, results in a strong synergistic antitumor effect. We have investigated the effect of IFNs/IFO treatment on the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP-9), and urokinase plasminogen activator receptor (uPAR), three key mediators of tumor growth and angiogenesis, in tumor xenografts generated either from a primary tumor (EW7) or from a metastatic tumor (COH). COH tumors expressed 5-fold higher levels of VEGF than EW7 tumors. IFNs/IFO treatment reduced by >70% the amount of VEGF in COH and EW7 tumors. We did not detect constitutive MMP-9 activity in EW7 tumors. In contrast, the metastasis-derived COH tumor expressed very high levels of active MMP-9. Although the total amount of MMP-9 remained unchanged, active MMP-9 was reduced by up to 75% in IFNs/IFO-treated COH tumors. IFNs/IFO treatment triggered in both COH and EW7 tumors the downregulation of uPAR expression, a molecule involved in vascularization and endothelial cell migration. Our results partly explain the mechanism of tumor growth inhibition by IFNs/IFO therapy and provide a rational foundation for the development of a new therapeutic approach to Ewing tumors resistant to conventional chemotherapy.

Elevated levels of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during the acute phase of Kawasaki disease.

Kawasaki disease (KD) is an acute, self-limiting, multisystem vasculitis of unknown etiology affecting infants and young children. Unless treated promptly with high-dose intravenous gamma globulin and aspirin, patients frequently develop coronary aneurysms. Previously, matrix metalloproteinase 9 (MMP-9), which is secreted complexed to tissue inhibitor of metalloproteinase 1 (TIMP-1), has been implicated in abdominal aortic aneurysm formation. Since the clinical and pathological features of KD include inflammation and weakening of blood vessels, we analyzed acute- and convalescent-phase paired plasma or serum samples from 31 KD patients, 7 patients who did not completely meet the criteria for KD, and 26 non-KD controls (9 febrile and 17 afebrile patients) for pro-MMP-9 (92 kDa) enzyme activity by gelatin zymography and for active MMP-9 (83 kDa), pro-MMP-9, and TIMP-1 protein levels by enzyme-linked immunosorbent assay. Statistical analysis was performed by using Student t tests, linear regression, and the Wilcoxon rank-sum test. Markedly elevated pro-MMP-9 enzymatic activity, pro-MMP-9 protein levels, and TIMP-1 protein levels were found during the acute phase of illness in patients with clinically established KD and in patients who were suspected of having KD but did not meet all of the criteria. There was no significant difference in active MMP-9 levels. Furthermore, pro-MMP-9 and TIMP-1 protein levels were significantly elevated among KD patients, compared to those of febrile and afebrile non-KD controls. The significantly elevated pro-MMP-9 enzyme and protein levels during the acute phase of KD may reflect vascular remodeling or an inflammatory response to a microbial agent, suggesting a pathophysiological role for MMP-9 in coronary aneurysm formation.

Matrix metalloproteinase-9 activity in plasma of patients following surgical resection of endometrioma

Objective: It is well established that endometriomas are detected with ultrasound and laparoscopy with pathologic confirmation. In some cases, however, the ultrasound finding may not be definitive. It has been established that MMPs are involved in the development of endometriosis. The present pilot study was performed to elucidate whether matrix metalloproteinase-9 (MMP-9) levels can be used to screen for recurrence of endometriomas. Design: Prospective observational study in patients treated with oral contraceptives to allow accurate measurement of levels in the midfollicular phase. Materials/Methods: Five patients with endometriomas were studied. Three patients had unilateral endometrioma and the two patients had bilateral endometriomas. All patients had definite confirmation of these findings on laparoscopy and pathology. We tested the preoperative plasma MMP-9 activity after 7 days of oral contraceptives (OCP) and again post-operatively after another 7 days of OCP. This method of testing avoided any inclusion of samples from the luteal phase or during menses. A control group of five patients who are tubal ligated and free of endometriosis were included. In the control group, we demonstrated that MMP-9 levels peaked during menses and minimum levels were seen in midluteal phase. The mid-follicular phase levels however were constant. Thus, treating patient with OCP avoided any false drop in the levels secondary to this cycle variation. Results: The mean active preoperative plasma MMP-9 levels were 57.00+/−8.98, whereas the mean postoperative plasma MMP-9 levels after resection were 33.93+/−3.84. Analysis of these findings with a two tailed t-test showed a statistical significance in the drop in MMP-9 activity following surgical resection of the endometrioma (p <.04). While this is a pilot study consisting of five patients, it supports the potential diagnostic role of MMP-9 in endometriosis. Conclusions: Preliminary data suggest that active MMP-9 levels may be used to confirm a clinical diagnosis of recurrence of endometriomas. Further studies including additional patients are underway. Considering MMP-9 levels vary in the menstrual cycle, if MMP-9 levels are to be used for diagnostic or prognostic purposes, the samples have to be matched in the menstrual cycle. In this study we controlled the normal variability by placing patients on oral contraceptives. Supported by: Department of Obstetrics, Gynecology and Women’s Health at University of Louisville.