Alterations of Tear Mediators in Patients with Keratoconus after Corneal Crosslinking Associate with Corneal Changes

Keratoconus (KC) is the most common primary corneal ectatic disease which has considerable importance in public health. Corneal collagen crosslinking (CXL) is a procedure to mitigate progression of KC and reduce demand for corneal transplantation. Although studies have proven the efficacy of CXL regarding corneal shape, none have investigated the effects of CXL on tear biomarkers which are useful tools to understand molecular mechanisms behind CXL. Our purpose was to determine the effect of CXL on tear mediators in patients with KC and analyze associations with corneal changes. Tear samples were collected pre-CXL from 26 eyes of 23 patients and during a 12-month follow-up. The mediators’ concentration was measured by Cytometric Bead Array technology. Corneal topography parameters measured by Scheimpflug Camera included: Thinnest-corneal-thickness (ThCT), keratometry values (K1, K2), Radii-Minimum (Rmin), Keratoconus-Index (KI), Center-KI (CKI), Index-of-Height Asymmetry (IHA) and Index-of-Surface Variance (ISV). At baseline, KI was correlated negatively with chemokine (C-C motif) ligand 5 (CCL5) (p=0.015) and matrix metalloproteinase (MMP)-13 (p=0.007). At day 4, interleukin (IL)-6 and IL-8 increased, while IL-13, IL-17A, interferon (IFN)-γ, CCL5, MMP-13, epidermal growth factor (EGF), nerve growth factor (NGF) and plasminogen activator inhibitor (PAI-1) decreased significantly compared to pre-CXL concentrations (p≤0.02). At 6 months tissue plasminogen activator (t-PA) increased (p=0.02), while at 12 months Rmin increased (p≤0.004), and IL-6 and CXCL8 (p=0.005 and p=0.047) as well as K1, ISV and KI decreased. After 6 months CKI and ISV showed significant associations with IL-17A; CKI with IL-13 and ThCT with IL-13 (p≤0.02), while at 12 months there were reverse associations between ThCT and IL-6, IL-13, INFγ, CCL5 and PAI-1 (p≤0.02). Alterations of mediators in tear fluid after CXL associate with topographic changes highlight the fact that many mediators are involved in the complex mechanisms after CXL. Further studies on biomarkers to investigate the efficacy of CXL are needed.     

Evaluation of Intereye Corneal Asymmetry in Patients with Keratoconus. A Scheimpflug Imaging Study

Purpose

To assess the correlation between keratoconus severity and intereye asymmetry of pachymetric data and posterior elevation values and to evaluate their combined accuracy in discriminating normal corneas from those with keratoconus.

Methods

This study included 97 patients: 65 subjects with bilateral normal corneas (NC) and 32 with keratoconus (KC). Central corneal thickness (CCT), thinnest corneal thickness (ThCT) and posterior elevation (PE) at the thinnest point of the cornea were measured in both eyes using Scheimpflug imaging. Intereye asymmetry and its correlation with keratoconus severity were calculated for each variable. The area under the receiver operating characteristic curve (AUROC) was used to compare predictive accuracy of different variables for keratoconus.

Results

In normal eyes, intereye differences were significantly lower compared with the keratoconus eyes (p<0.001, for CCT, ThCT and PE). There was a significant exponential correlation between disease severity and intereye asymmetry of steep keratometry (r² = 0.55, p<0.001), CCT (r² = 0.39, p<0.001), ThCT (r² = 0.48, p<0.001) and PE (r² = 0.64, p<0.001). After adjustment for keratoconus severity, asymmetry in thinnest pachymetry proved to be the best parameter to characterize intereye corneal asymmetry in keratoconus. This variable had high accuracy and significantly better discriminating ability (AUROC: 0.99) for KC than posterior elevation (AUROC: 0.96), ThCT (AUROC: 0.94) or CCT (AUROC: 0.92) alone.

Conclusions

There is an increased intereye asymmetry in keratometry, pachymetry and posterior corneal elevation values in keratoconic patients compared to subjects with normal corneas. Keratoconus patients with more severe disease are also more asymmetric in their disease status which should be taken into account during clinical care.     

Corneal Sensitivity and Dry Eye Symptoms in Patients with Keratoconus

Purpose

To investigate corneal sensitivity to selective mechanical, chemical, and thermal stimulation and to evaluate their relation to dry eye symptoms in patients with keratoconus.

Methods

Corneal sensitivity to mechanical, chemical, and thermal thresholds were determined using a gas esthesiometer in 19 patients with keratoconus (KC group) and in 20 age-matched healthy subjects (control group). Tear film dynamics was assessed by Schirmer I test and by the non-invasive tear film breakup time (NI-BUT). All eyes were examined with a rotating Scheimpflug camera to assess keratoconus severity.

Results

KC patients had significatly decreased tear secretion and significantly higher ocular surface disease index (OSDI) scores compared to controls (5.3±2.2 vs. 13.2±2.0 mm and 26.8±15.8 vs. 8.1±2.3; p<0.001). There was no significant difference in NI-BUT between the two groups (KC: 9.8±4.8 vs. control: 10.7±3.8; p>0.05). The mean threshold for selective mechanical (KC: 139.2±25.8 vs. control: 109.1±24.0 ml/min), chemical (KC: 39.4±3.9 vs. control: 35.2±1.9%CO₂), heat (KC: 0.91±0.32 vs. control: 0.54±0.26 Δ°C) and cold (KC: 1.28±0.27 vs. control: 0.98±0.25 Δ°C) stimulation in the KC patients were significantly higher than in the control subjects (p<0.001, for all parameters). No correlation was found between age and mechanical, chemical, heat or cold thresholds in the patients with KC (p>0.05), whereas in the control subjects both mechanical (r = 0.52, p = 0.02), chemical (r = 0.47, p = 0.04), heat (r = 0.26, p = 0.04) and cold threshold (r = 0.40, p = 0.03) increased with age. In the KC group, neither corneal thickness nor tear flow, NI-BUT or OSDI correlated significantly with mechanical, chemical, heat or cold thresholds (p>0.05 for all variables).

Conclusions

Corneal sensitivity to different types of stimuli is decreased in patients with keratoconus independently of age and disease severity. The reduction of the sensory input from corneal nerves may contribute to the onset of unpleasant sensations in these patients and might lead to the impaired tear film dynamics.     

Subnormal cytokine profile in the tear fluid of keratoconus patients

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.     

杞菊地黄汤联合玻璃酸钠滴眼液对肝肾阴虚型干眼症患者眼表功能和泪液MMP-2、MMP-9水平的影响

摘要 目的：观察杞菊地黄汤联合玻璃酸钠滴眼液治疗肝肾阴虚型干眼症的疗效及对眼表功能和泪液基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）的影响。方法：病例来源于2018年6月~2021年3月期间就诊于湖南省直中医医院眼科门诊的干眼症患者,共98例。采用随机数字表法分为对照组和实验组,两组均49例。对照组给予玻璃酸钠滴眼液治疗,实验组在对照组基础上接受杞菊地黄汤治疗,两组均治疗4周。对比两组疗效、中医证候积分、眼表功能和泪液MMP-2、MMP-9水平变化,观察两组治疗期间不良反应发生率。结果：实验组的临床总有效率高于对照组（P<0.05）。实验组治疗4周后眼部干涩不爽、畏光、双目频眨、口干少津、白眼泛红、舌红、苔薄、脉细证候积分均低于对照组（P<0.05）。实验组治疗4周后角膜荧光素染色（FL）评分低于对照组,泪膜破裂时间（BUT）、泪液分泌试验（SIT）水平高于对照组（P<0.05）。实验组治疗4周后泪液MMP-2、MMP-9水平低于对照组（P<0.05）。两组间不良反应发生率无统计学差异（P＞0.05）。结论：杞菊地黄汤联合玻璃酸钠滴眼液治疗肝肾阴虚型干眼症,可促进患者症状缓解,改善眼表功能,降低泪液MMP-2、MMP-9水平,安全可靠。     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

Application of high resolution OCT to evaluate irregularity of Bowman's layer in asymmetric keratoconus

Irregularity of the Bowman's layer (BL) in keratoconus (KC, sample size (n) = 28) and forme fruste keratoconus (FFKC, n = 18) eyes was evaluated. Subjects underwent high resolution OCT imaging (Bioptigen Inc., USA) and corneal tomography (Pentacam v1.20r41, OCULUS Optikgeräte GmbH, Germany). Anterior edge of the BL was segmented. A Bowman's roughness index (BRI) was defined as the sum of the enclosed areas between segmented edge and a smooth 3^rd order polynomial fit to the edge. BRI was compared with corneal aberrations, Keratoconus percentage index (KISA), Cone location magnitude index (CLMI) and Belin–Ambrosio enhanced ectasia display overall deviation index (BAD‐D). Area under the curve (AUC) was determined with logistic regression (LR). Mean BRI (×10^–3) was 2.12 mm², 1.81 mm² and 1.7 mm² in normal (n = 26), FFKC and KC eyes, respectively (p < 0.001). BAD‐D (0.79) and BRI (0.74) had the best AUC for FFKC. By combining BAD‐D and BRI, the AUC improved to 0.85 (p = 0.01). For KC eyes, KISA (0.94), CLMI (0.88), BAD‐D (0.96) and aberrations had comparable AUC (p > 0.05). However, LR with BRI and other indices didn't improve the AUC in KC eyes (p > 0.05). BRI was significantly reduced in FFKC and KC eyes. It improved the detection of FFKC but not KC eyes.

     

GnRH I and II up-regulate MMP-26 expression through the JNK pathway in human cytotrophoblasts

Background  

Matrix metalloproteinase-26 (MMP-26), one of the main mediators of extracellular matrix (ECM) degradation, has been shown to exist in trophoblasts of human placenta and to play a role in trophoblast cell invasion. However, little is known about the regulation of MMP-26 expression in human trophoblasts. Recently, gonadotropin-releasing hormone I (GnRH I) and GnRH II have been shown to regulate the expression of MMP-2, MMP-9/tissue inhibitor of metalloproteinases 1 (TIMP-1), and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) in human trophoblasts, suggesting that these two hormones may work as paracrine and/or autocrine regulators in modulating the activities of various protease systems at the feto-maternal interface. In this study, we determined the regulatory effects of GnRH I and GnRH II on the expression of MMP-26 in human immortalized cytotrophoblast-like cell line, B6Tert-1.     

Unique corneal tomography features of allergic eye disease identified by OCT imaging and artificial intelligence

The purpose of this study was to assess unique corneal tomographic parameters of allergic eye disease (AED) using optical coherence tomography (OCT) and artificial intelligence (AI). A total of 57 eyes diagnosed with AED were included. The curvature and aberrations of the air‐epithelium (A‐E) and epithelium‐Bowman's layer (E‐B) interfaces were calculated. Random forest AI models were built combing this data with the parameters of healthy, forme fruste keratoconus (FFKC) and KC eyes. The AI models were cross‐validated with 3‐fold random sampling. Each model was limited to 10 trees. The AI model incorporating both A‐E and E‐B parameters provided the best classification of AED eyes (area under the curve = 0.958, sensitivity = 80.7%, specificity = 98.5%, precision = 88.2%). Further, the E‐B interface parameters provided the highest information gain in the AI model. A few AED eyes (n = 9) had tomography parameters similar to FFKC and KC eyes and may be at risk of progression to KC.     

Genetic aspects of keratoconus development

Keratoconus (KC) is the most common form of keratoectasia characterized by changes in corneal topography and its thinning, stretching, and protrusion. The hereditary or genetic theory of keratoconus development is widely recognized. To date, a large number of candidate genes have been investigated in patients with KC. One of the most important of them are the gene encoding a homeodomain-containing protein that belongs to the subfamily of paired-like homeodomain proteins (VSX1), superoxidedismutase 1 (SOD1) gene, and the gene of lysyloxidase (LOX). The linkage analysis reveals over 17 chromosomal regions mutations in which can lead to the development of KC. In families with a hereditary form of keratoconus by GWAS analysis, the association of central corneal thickness (CCT) with a number of genetic loci is revealed. Thus, diverse results of genetic studies and a large number of identified chromosomal regions associated with keratoconus, firstly, show marked genetic heterogeneity of the disease and, secondly, are associated with challenges in DNA diagnosis of this disease. However, there are prerequisites that keratoconus belongs to both hereditary and genetically caused diseases and identified genetic variants are specific both to individual populations and to certain ethnic groups in general.     

Bowman's topography for improved detection of early ectasia

The aim of this study was to evaluate whether OCT topography of the Bowman's layer and artificial intelligence (AI) can result in better diagnosis of forme fruste (FFKC) and clinical keratoconus (KC). Normal (n = 221), FFKC (n = 72) and KC (n = 116) corneas were included. Some of the FFKC and KC patients had the fellow eye (VAE‐NT) with normal topography (n = 30). The Scheimpflug and OCT scans of the cornea were analyzed. The curvature and surface aberrations (ray tracing) of the anterior corneal surface [air‐epithelium (A‐E) interface in OCT] and epithelium‐Bowman's layer (E‐B) interface (in OCT only) were calculated. Four random forest models were constructed: (1) Scheimpflug only; (2) OCT A‐E only; (3) OCT E‐B only; (4) OCT A‐E and E‐B combined. For normal eyes, both Scheimpflug and OCT (A‐E and E‐B combined) performed equally in identifying these eyes (P = .23). However, OCT A‐E and E‐B showed that most VAE‐NT eyes were topographically similar to normal eyes and did not warrant a separate classification based on topography alone. For identifying FFKC eyes, OCT A‐E and E‐B combined performed significantly better than Scheimpflug (P = .006). For KC eyes, both Scheimpflug and OCT performed equally (P = 1.0). Thus, OCT Topography of Bowman's layer significantly improved the detection of FFKC eyes.     

Neuroprotective effect of neuroserpin in rat primary cortical cultures after oxygen and glucose deprivation and tPA

Besides its role as a thrombolytic agent, tissue plasminogen activator (tPA) triggers harmful effects in the brain parenchyma after stroke, such as inflammation, excitotoxicity and basal lamina degradation. Neuroserpin, a natural inhibitor of tPA, has shown neuroprotective effects in animal models of brain infarct. However, the molecular mechanisms of neuroserpin-mediated neuroprotection after brain ischemia remain to be well characterized. Then, our aim was to investigate such mechanisms in primary mixed cortical cell cultures after oxygen and glucose deprivation (OGD). Primary rat mixed cortical cultures containing both astrocytes and neurons were subjected to OGD for 150min and subsequently treated with either tPA (5μg/mL), neuroserpin (0.125, 0.25, 0.5 or 1μM), and tPA together with neuroserpin at the mentioned doses. Twenty-four hours after treatment, LDH release, caspase-3 activity, MCP-1, MIP-2, active MMP-9, GRO/KC and COX-2 were measured. Statistical differences were analyzed using Student's t-test or one-way ANOVA as appropriate. Treatment with tPA after OGD increased LDH release, active MMP-9, MCP-1 and MIP-2 (all p≤0.05), but not caspase-3, GRO/KC or COX-2 compared to control. Treatment with neuroserpin after OGD decreased LDH release and active MMP-9 (all p≤0.05). It had no effect on caspase-3 activity, or on MCP-1, MIP-2, GRO/KC or COX-2 expression compared to control. Administration of tPA together with neuroserpin decreased LDH release, active MMP-9 and MIP-2 (all p≤0.05) and showed no effect on MCP-1, GRO/KC or COX-2 compared to control. Our results suggest that neuroprotective activity of neuroserpin involves attenuation on tPA-mediated mechanisms of inflammation and BBB disruption after brain ischemia.     

Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen.

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.     

Regional variation of corneal stromal deformation measured by high-frequency ultrasound elastography

The cornea’s mechanical response to intraocular pressure elevations may alter in ectatic diseases such as keratoconus. Regional variations of mechanical deformation in normal and keratoconus eyes during intraocular pressure elevation have not been well-characterized. We applied a high-frequency ultrasound elastography technique to characterize the regional deformation of normal and keratoconus human corneas through the full thickness of corneal stroma. A cross-section centered at the corneal apex in 11 normal and 2 keratoconus human donor eyes was imaged with high-frequency ultrasound during whole globe inflation from 5 to 30 mmHg. An ultrasound speckle tracking algorithm was used to compute local tissue displacements. Radial, tangential, and shear strains were mapped across the imaged cross-section. Strains in the central (1 mm surrounding apex) and paracentral (1 to 4 mm from apex) regions were analyzed in both normal and keratoconus eyes. Additional regional analysis was performed in the eye with severe keratoconus presenting significant thinning and scarring. Our results showed that in normal corneas, the central region had significantly smaller tangential stretch than the paracentral region, and that within the central region, the magnitudes of radial and shear strains were significantly larger than that of tangential strain. The eye with mild keratoconus had similar shear strain but substantially larger radial strains than normal corneas, while the eye with severe keratoconus had similar overall strains as in normal eyes but marked regional heterogeneity and large strains in the cone region. These findings suggested regional variation of mechanical responses to intraocular pressure elevation in both normal and keratoconus corneas, and keratoconus appeared to be associated with mechanical weakening in the cone region, especially in resisting radial compression. Comprehensive characterization of radial, tangential, and shear strains through corneal stroma may provide new insights to understand the biomechanical alterations in keratoconus.     

Comparison of Early Changes in Ocular Surface and Inflammatory Mediators between Femtosecond Lenticule Extraction and Small-Incision Lenticule Extraction

Purpose

To evaluate the short-term changes in ocular surface measures and tear inflammatory mediators after femtosecond lenticule extraction (FLEx) and small-incision lenticule extraction (SMILE) procedures.

Methods

Eighteen subjects (18 eyes) underwent FLEx and 23 subjects (23 eyes) underwent SMILE in this single-center and prospective study. Central corneal sensitivity, Schirmer I test (SIT), noninvasive tear breakup time (NI-TBUT), tear meniscus height, corneal fluorescein (FL) staining, and ocular surface disease index (OSDI) were assessed in all patients. Concentrations of interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), nerve growth factor (NGF), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase-9 (MMP-9) in collected tears were measured by multiplex antibody microarray.

Results

Central corneal sensitivity was reduced in both groups, but the scores in the SMILE group were higher than those in the FLEx group at all time points postoperatively (P<0.01). Lower FL scores and longer NI-BUT were observed in the SMILE group 1 week after surgery (P<0.05). OSDI scores in both groups increased rapidly at 1 day and 1 week postoperatively, then returned to their preoperative levels within 1 month (P<0.05). There were no significant differences in SIT or tear meniscus height between the two groups. Lower and faster recovery of tear NGF, TGF-β1 and IL-1α concentration were found in the SMILE group compared to the FLEx group postoperatively. No significant difference was found in tear TNF-α, IFN-γ and MMP-9 for either group before or after surgery. Tear NGF, TGF-β1 and IL-1α show a correlation with ocular surface changes after FLEx or SMILE surgery.

Conclusion

SMILE has superiority over FLEx in early ocular surface changes and NGF, TGF-β1 and IL-1α may contribute to the process of ocular surface recovery.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02540785","term_id":"NCT02540785"}}NCT02540785     

Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3).

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.     

Urokinase directly activates matrix metalloproteinases-9: a potential role in glioblastoma invasion

Previous reports showed that urokinase plasminogen activator (uPA) converts plasminogen to plasmin which then activates matrix metalloproteinases (MMPs). Here, we report that uPA directly cleaved pro-MMP-9 in a time-dependent manner at both C- and N-terminus and generated two gelatinolytic bands. uPA-activated-MMP-9 efficiently degraded fibronectin and blocked by uPA inhibitor B428 and recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1). B428 inhibited basal and PMA-induced active MMP-9 in glioblastomas (GBM) U1242 cell media as well as cell invasion in vitro. A combination of MMP-9 and uPA antibodies more significantly inhibited U1242 cell invasion than uPA or MMP-9 antibody alone. Both uPA and MMP-9 were highly expressed in U1242 cell and GBM patient specimens. Furthermore, two active MMP-9 fragments with identical molecular weights to the uPA-activated MMP-9 products were detected in GBM patient specimens. These results suggest that uPA-mediated direct activation of MMP-9 may promote GBM cell invasion.     

PAI-1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA

Plasminogen activator inhibitor-1 (PAI-1) increases injury in several liver, lung and kidney disease models. The objective of this investigation was to assess the effect of PAI-1 deficiency on cholestatic liver fibrosis and determine PAI-1 influenced fibrogenic mechanisms. We found that PAI-1(-/-) mice had less fibrosis than wild type (WT) mice after bile duct ligation. This change correlated with increased tissue-type plasminogen activator (tPA) activity, and increased matrix metalloproteinase-9 (MMP-9), but not MMP-2 activity. Furthermore, there was increased activation of the tPA substrate hepatocyte growth factor (HGF), a known anti-fibrogenic protein. In contrast, there was no difference in hepatic urokinase plasminogen activator (uPA) or plasmin activities between PAI-1(-/-) and WT mice. There was also no difference in the level of transforming growth factor beta 1 (TGF-beta1), stellate cell activation or collagen production between WT and PAI-1(-/-) animals. In conclusion, PAI-1 deficiency reduces hepatic fibrosis after bile duct obstruction mainly through the activation of tPA and HGF.     

Efficacy of Corneal Collagen Cross-Linking for Treatment of Keratoconus: A Meta-Analysis of Randomized Controlled Trials

ObjectiveTo evaluate the efficacy of corneal collagen cross-linking (CXL) for the treatment of keratoconus.MethodsWe performed a literature search for randomized controlled trials that assessed the effect of CXL in slowing progression of keratoconus. The primary outcome measures included changes of topographic parameters, visual acuity, and refraction. Efficacy estimates were evaluated by weighted mean difference (WMD) and 95% confidence interval (CI) for absolute changes of the interested outcomes.ResultsSignificant decrease in mean keratometry value, maximum keratometry value and minimum keratometry value were demonstrated in the CXL group compared with the control group (WMD = -1.65; 95% CI: -2.51 to -0.80; P < 0.00001; WMD = -2.05; 95% CI: -3.10 to -1.00; P < 0.00001; WMD = -1.94; 95% CI: -2.63 to -1.26; P < 0.00001; respectively). Best spectacle-corrected visual acuity improved significantly in CXL group (WMD = -0.10; 95% CI: -0.15 to -0.05; P < 0.00001), whereas uncorrected visual acuity did not differ statistically. Manifest cylinder error decreased significantly in patients undergoing CXL procedure compared with control patients in sensitivity analysis (WMD = -0.388; 95% CI: -0.757 to -0. 019; P = 0.04). The changes in central corneal thickness and intraocular pressure were not statistically significant.ConclusionCXL may be an effective option in stabilizing keratoconus. Further long-term follow-up studies will be necessary to assess the persistence of CXL.     

Inactivation of plasminogen activator inhibitor-1 by specific proteolysis with stromelysin-1 (MMP-3)

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically hydrolyzes the Ser(337)-Ser(338) (P10-P9) and Val(341)-Ile(342) (P6-P5) peptide bonds in human plasminogen activator inhibitor-1 (PAI-1). Cleavage is completely abolished in the presence of the metal chelators EDTA or 1,10-phenanthroline. A stabilized active PAI-1 variant was also cleaved by MMP-3. At an enzyme/substrate ratio of 1/10 at 37 degrees C, PAI-1 protein cleavage occurred with half-lives of 27 or 14 min for active or stable PAI-1 and was associated with rapid loss of inhibitory activity toward tissue-type plasminogen activator with half-lives of 15 or 13 min, respectively. A substrate-like variant of PAI-1, lacking inhibitory activity but with exposed reactive site loop, was cleaved with a half-life of 23 min, whereas latent PAI-1 in which a major part of the reactive site loop is inserted into the molecule, was resistant to cleavage. Biospecific interaction analysis indicated comparable binding of active, stable, and substrate PAI-1 to both proMMP-3 and MMP-3 (K(A) of 12-22 x 10(6) m(-1)), whereas binding of latent PAI-1 occurred with lower affinity (1.7-2.3 x 10(6) m(-1)). Stable PAI-1 bound to vitronectin was cleaved and inactivated by MMP-3 in a manner comparable with that of free PAI-1; however, the cleaved protein did not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antiproteolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration.