Urokinase Inhibitor Research Articles

Plasminogen influence on the PAI-1 release by human platelets

РАІ-1 (plasminogen activator inhibitor type 1), as a major physiological inhibitor of tissue plasminogen activator and urokinase, plays a key role in the regulation of fibrinolysis in vivo. Besides, PAI-1 suppresses plasmin formation and affects cell migration through interaction with vitronectin. РАІ-1 is secreted from α-granules of platelets upon stimulation of cells by agonists. The aim of our study was to explore the effects of Glu- and Lys-forms of plasminogen on PAI-1 secretion by platelets and to evaluate the possible role of plasminogen in modulation of agonist-induced PAI-1 release. The secretion of PAI-1 by platelets was investigated by the Western blot analysis. It has been established that depending on the agonist, PAI-1 can be released from platelets in a free form, in a complex with a tissue plasminogen activator, as well as in the form of high-molecular complexes that contain a tissue activator and vitronectin molecules. The revealed induction of PAI-1 secretion under the action of Gly- and Lys-forms of plasminogen indicates their ability to activate intracellular signaling pathways that regulate the release of platelet α-granules. Our findings may be of importance for elucidating the pathogenetic mechanisms of many diseases associated with abnormally enhanced platelet function and PAI-1-related disorders. Keywords: Glu- and Lys-plasminogen, PAI-1, platelets, vitronectin, α-granules

FAM3C/ILEI protein is elevated in psoriatic lesions and triggers psoriasiform hyperproliferation in mice

FAM3C/ILEI is an important cytokine for tumor progression and metastasis. However, its involvement in inflammation remains elusive. Here, we show that ILEI protein is highly expressed in psoriatic lesions. Inducible keratinocyte‐specific ILEI overexpression in mice (K5‐ILEIind) recapitulates many aspects of psoriasis following TPA challenge, primarily manifested by impaired epidermal differentiation and increased neutrophil recruitment. Mechanistically, ILEI triggers Erk and Akt signaling, which then activates STAT3 via Ser727 phosphorylation. Keratinocyte‐specific ILEI deletion ameliorates TPA‐induced skin inflammation. A transcriptomic ILEI signature obtained from the K5‐ILEIind model shows enrichment in several signaling pathways also found in psoriasis and identifies urokinase as a targetable enzyme to counteract ILEI activity. Pharmacological inhibition of urokinase in TPA‐induced K5‐ILEIind mice results in significant improvement of psoriasiform symptoms by reducing ILEI secretion. The ILEI signature distinguishes psoriasis from healthy skin with uPA ranking among the top “separator” genes. Our study identifies ILEI as a key driver in psoriasis, indicates the relevance of ILEI‐regulated genes for disease manifestation, and shows the clinical impact of ILEI and urokinase as novel potential therapeutic targets in psoriasis.

Moisturizing at a molecular level - The basis of Corneocare.

This review covers the last 20 years of research we and our collaborators have conducted on ethnic differences in facial skin moisturization placed in historical context with previous research. We have focussed particularly on the biochemical and cellular gradients of the stratum corneum (SC) with the aim of discovering new skin moisturization and SC maturation mechanisms, identifying new technologies and/or providing conceptual innovations for ingredients that will improve our understanding and treatment of dry skin. Specifically, we discuss gradients for corneodesmosomes and proteases, corneocyte phenotype-inducing enzymes, filaggrin and natural moisturizing factor (NMF), and barrier lipids. These gradients are interdependent and influence greatly corneocyte maturation. The interrelationship between corneodesmolysis and the covalent attachment of ω-hydroxy ceramides and ω-hydroxy fatty acids to the corneocyte protein envelope forming the corneocyte lipid envelope is especially relevant in our new understanding of mechanisms leading to dry skin. This process is initiated by a linoleoyl-ω-acyl ceramide transforming enzyme cascade including 12R lipoxygenase (12R-LOX), epidermal lipoxygenase-3 (eLOX3), epoxide hydrolase 3 (EPHX3), short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7), ceramidase and transglutaminase 1. Our research has opened the opportunity of using novel treatment systems for dry skin based on lipids, humectants, niacinamide and inhibitors of the plasminogen system. It is clear that skin moisturization is a more complex mechanism than simple skin hydration.

Annulated pyrazole derivatives as a novel class of urokinase (uPA) inhibitors: Green synthesis, anticancer activity, DNA-damage evaluation, and molecular modelling study

Different series of annulated pyrazole derivatives were designed, synthesized via both green and traditional methods, and structurally characterized. In vitro uPA evaluation, antiproliferative activities and DNA binding damage was studied in this work. Thus, all the synthesized compounds were evaluated against three types of cancer cell lines; HepG-2, HCT-116, and MCF-7 cancer cell lines in addition to normal cell line WI38. Compounds 11, 20, 21, 23 and 24 displayed the most significant antiproliferative activity with IC50 ranging between 4.42 ± 0.59 μM to 11.05 ± 0.95 μM against HepG-2, HCT-116, and MCF-7 cancer cell lines compared to the reference drug, doxorubicin. Thus compound 11 exhibited cytotoxic activity with IC50 8.58 μM, 9.22 μM and 7.53 μM, compound 20 showed IC50 9.99 μM, 6.72 μM and 6.87 μM, analogue 21 displayed IC50 10.80 μM, 7.90 μM and 9.16 μM, compound 23 showed IC50 4.82 μM, 11.05 μM and 4.42 μM and derivative 24 exhibited potent cytotoxic activity with IC50 7.44 μM, 5.18 μM and 8.22 μM against HepG-2, HCT-116, and MCF-7 cancer cell lines, respectively. Additionally, compounds 11, 21, 23 and 24 showed significant uPA inhibitory activity with IC50 27.28 μM, 29.36 μM, 11.73 μM, and 7.96 μM respectively. Moreover, HCT-116 cell lines were treated with both compounds 23 and 24 that remarkably showed a high score of DNA binding damage. Mechanistic studies demonstrated the apoptotic activity of the most active tricyclic heteroaromatic analogue 24 on HCT-116 cancer cells by inducing a strong S phase cell cycle arrest suggesting that the mechanism of its antiproliferative activity may be through uPA inhibition. Finally, deeper insight illustrated that the hit compounds exhibited characteristic binding interactions in the active site of uPA that are required in the S pocket, which are important for activity Arg 217, Gly 219, and Ser 190.

SERPINE1 Overexpression Promotes Malignant Progression and Poor Prognosis of Gastric Cancer.

The serine protease inhibitor clade E member 1 (SERPINE1) is a major inhibitor of tissue plasminogen activator and urokinase, and has been implicated in the development and progression of a variety of tumors. In this study, mRNA microarray and TCGA database were used to comprehensively analyze the upregulation of SERPINE1 in gastric cancer (GC) tissues compared with the normal stomach tissues. Kaplan-Meier results confirmed that patients with high SERPINE1 expression exhibited worse overall survival and disease-free survival. In addition, cell proliferation, cell scratches, transwell migration and invasion assay showed that SERPINE1 knockdown inhibited the proliferation, migration and invasion of GC ells. Western blot showed that the expression of VEGF and IL-6 was significantly upregulated after overexpression of SERPINE1. Meanwhile, SERPINE1 was positively correlated with the level of immune infiltration using the online analysis tools TISIDB and TIMER. And SERPINE1 expression increased with the increase of malignancy of GC which were detected by Immunohistochemistry. Finally, tumorigenesis experiments in nude mice further demonstrated that SERPINE1 could promote the occurrence and development of GC, while deletion of SERPINE1 inhibited the progression of GC. In summary, SERPINE1 was highly expressed in GC tissues, and SERPINE1 was helpful for differential diagnosis of pathological grade of gastric mucosal lesions. SERPINE1 might regulate the expression of VEGF and IL-6 through the VEGF signaling pathway and JAK-STAT3 inflammatory signaling pathway, thus ultimately affecting the invasion and migration of GC cells.

Structure-based molecular insights into matrix metalloproteinase inhibitors in cancer treatments.

Protease inhibitors are of considerable interestas anticancer agents. Matrix metalloproteinases (MMPs) were the earliest type of proteases considered as anticancer targets. The developments of MMP inhibitors (MMPIs) by pharmaceutical companies can be dated from the early 1980s. Thus far, none of the over 50 MMPIs entering clinical trials have been approved. This work summarizes the reported studies on the structureof MMPs and complexes with ligands and inhibitors, based on which, the authorsanalyzed the clinical failures of MMPIs in a structural biological manner. Furthermore, MMPs were systematically comparedwith urokinase, a protease-generating plasmin, which plays similar pathological roles in cancer development;the reasons for the clinical successes of urokinase inhibitors andthe clinical failuresof MMPIs arediscussed.

Precision targeting of the plasminogen activator inhibitor-1 mechanism increases efficacy of fibrinolytic therapy in empyema.

Plasminogen activator inhibitor‐1 (PAI‐1) is an endogenous irreversible inhibitor of tissue‐type (tPA) and urokinase (uPA) plasminogen activators. PAI‐1‐targeted fibrinolytic therapy (PAI‐1‐TFT) is designed to decrease the therapeutic dose of tPA and uPA, attenuating the risk of bleeding and other complications. Docking site peptide (DSP) mimics the part of the PAI‐1 reactive center loop that interacts with plasminogen activators, thereby affecting the PAI‐1 mechanism. We used DSP for PAI‐1‐TFT in two rabbit models: chemically induced pleural injury and Streptococcus pneumoniae induced empyema. These models feature different levels of inflammation and PAI‐1 expression. PAI‐1‐TFT with DSP (2.0 mg/kg) converted ineffective doses of single chain (sc) tPA (72.5 µg/kg) and scuPA (62.5 µg/kg) into effective ones in chemically induced pleural injury. DSP (2.0 mg/kg) was ineffective in S. pneumoniae empyema, where the level of PAI‐1 is an order of magnitude higher. DSP dose escalation to 8.0 mg/kg resulted in effective PAI‐1‐TFT with 0.25 mg/kg sctPA (1/8th of the effective dose of sctPA alone) in empyema. There was no increase in the efficacy of scuPA. PAI‐1‐TFT with DSP increases the efficacy of fibrinolytic therapy up to 8‐fold in chemically induced (sctPA and scuPA) and infectious (sctPA) pleural injury in rabbits. PAI‐1 is a valid molecular target in our model of S. pneumoniae empyema in rabbits, which closely recapitulates key characteristics of empyema in humans. Low‐dose PAI‐1‐TFT is a novel interventional strategy that offers the potential to improve fibrinolytic therapy for empyema in clinical practice.

Analysis of Protein C Inhibitor/ SERPINA5

Protein C inhibitor is a non-specific serine protease inhibitor with extensive protease reactivity. Protein C inhibitor (SERPINA5, plasminogen activator inhibitor-3/PAI-3) is a secreted, extracellular clade A serpin. SERPINA5/PCI has originally been described as an inhibitor of activated protein C and independently as an inhibitor of the plasminogen activator urokinase. SERPINA5 binds glycosaminoglycans, phospholipids, and retinoic acid. Glycosaminoglycans and certain phospholipids can modulate its inhibitory activity and specificity. PCI plays role at thrombosis and fibrinolysis, regulation of fertilization, tissue regeneration, vascular permeability, tumor development, invasion, metastasis and angiogenesis. In this review; aimed to provide information about the functions of PCI and to provide guidance for studies.

Efforts towards an On-Target Version of the Groebke-Blackburn-Bienaymé (GBB) Reaction for Discovery of Druglike Urokinase (uPA) Inhibitors.

Target-guided synthesis (TGS) has emerged as a promising strategy in drug discovery. Although reported examples of TGS generally involve two-component reactions, there is a strong case for developing target-guided versions of three-component reactions (3CRs) because of their potential to deliver highly diversified druglike molecules. To this end, the Groebke-Blackburn-Bienaymé reaction was selected as a model 3CR. We recently reported a series of druglike urokinase inhibitors, and these serve as reference compounds in the present study. Due to the limited number of literature reports on target-guided 3CRs, multiple experimental parameters were optimized here. Most challenging was the formation of imine intermediates under near-physiological conditions. This aspect was addressed by exploring chemical imine stabilization strategies. Notably, imines are also crucial intermediates of other 3CRs. Such systematic studies are strongly required for further development of the TGS domain but are largely absent in the literature. Hence, this work is intended as a reference for future multicomponent-based TGS studies.

Bifunctional inhibitors of urokinase and metalloproteinase-9 for cancer treatment - in silico evaluation

Matrix metalloproteinase-9 (MMP-9), and urokinase plasminogen activator (uPA) overexpression or/and increased activity are considered causative elements for cancer invasion and metastasis. These enzymes are degrading the extracellular matrix (ECM) providing space for cancer progression and cancer cell mobility. Process of angiogenesis, in which microvascular endothelial cells form blood vessels, requires local degradation of the underlying basal lamina to invade into the stroma proximal to cancer, and it strongly depends on the activity of MMP-9 and uPA as well. Malignant tumor invasion, cancer metastasis and angiogenesis have been documented as a fundamental factors in the morbidity and mortality among cancer patients, thus their inhibition can be exploit therapeutically. Numerous in vivo and in vitro studies have demonstrated that inhibition of proteolytic activity can reduce caner invasion, tumor size and limit angiogenesis. Consequently human clinical studies were designed inhibiting urokinase or MMPs, but these target specific inhibitors produce mix results. One of the possible explanation could be that cancers are overexpressing more than one enzyme simultaneously – for instance urokinase and MMPs. Thus upregulated net proteolytic activity should be normalized rather than trying to inhibit single proteolytic enzyme. Therefore, starting from specific inhibitors we have created - in silico - several hybrid molecules that could inhibit both uPA and MMP-9. The best hybrid (UI1xAGB) had theoretical affinities of Ki = 1.61-9 mol for MMP-9 and Ki = 1.36-9 mol for uPA. In the future each individual hybrid would need to be successfully synthesized and checked in the in vitro and in vivo analyses.

Abstract 4200: New potential therapeutic applications of WX-UK1, as a specific and potent inhibitor of human trypsin-like proteases

Abstract WX-UK1 (the active metabolite of upamostat) was originally developed as an inhibitor of the serine protease urokinase (uPA) with a Ki ~1 uM. To identify more sensitive targets, we performed a bioinformatic analysis of the ~200 human trypsin-like serine proteases, many of which play crucial roles in homeostasis and disease. Among these we selected a subset for biochemical analysis based on an inspection of modelled 3D structures of WX-UK1:protease complexes and sequence alignment of binding site residues. Samples of the selected proteases were prepared and characterized for their binding to WX-UK1; enzymatically with respect to inhibition constant (Ki) and by surface plasmon resonance with respect to dissociation constant (kd). We now report that WX-UK1 is a potent and specific inhibitor of five human serine proteases (trypsin-3, trypsin-2, trypsin-1 and matriptase-1 and trypsin-6), with Ki's down to the low nanomolar range, 19 nM for trypsin-3. Several of these serine proteases are known to be associated with cancer progression and metastasis. As a compound with an established clinical safety profile, targeted use of upamostat in oncology and extension to non-oncology indications may be assessed. Citation Format: Emil Oldenburg, Christine R. Schar, Eva Lange, Terry F. Plasse, Danielle T. Abramson, Reza Fathi, Eric M. Towler, Mark Levitt, Jan K. Jensen. New potential therapeutic applications of WX-UK1, as a specific and potent inhibitor of human trypsin-like proteases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4200.

Tetrahydroindolocarbazoles (THICZs) as new class of urokinase (uPA) inhibitors: Synthesis, anticancer evaluation, DNA-damage determination, and molecular modelling study

Tetrahydroindolocarbazoles (THICZs) with versatile substituents, have been designed, synthesized, structure characterized, then investigated for their in-vitro anticancer screening, urokinase inhibition (uPA) evaluated, DNA-damage determination was further explored. Compounds 5, 8, 10 and 17 displayed the most promising antitumor activities against the breast cancer cell line as compared to the standard drug, doxorubicin with IC50 = 5.24 ± 0.37, 4.00 ± 0.52, 7.20 ± 0.90 and 9.60 ± 1.10 µg/ml (versus 3.30 ± 0.48 µg/ml for doxorubicin). Compounds 5, 8, 10 and 17 represents the most significant uPA inhibitors of our study with IC50 of 3.80, 2.70. 4.75, 10.80 (ng/ml) respectively. The expression levels of CDKN2A gene were decreased in 8, 10 and 17 cell lines as compared to those in positive control samples. Cell lines treated with 5, 8, 10 and 17 clearly observed a high score of damaged DNA cells. A deeper examination revealed that our hetroaromatics showed an extensive hydrogen bonding interactions that is required in the S pocket which is important for activity Arg 217, Gly 219, Gly 216, Lys 143 and Ser 190. So we present THICZs as promising uPA inhibitors expected as significant promise for further development as anti-invasiveness drugs.

Plasminogen activator inhibitor-1 in cancer research.

[Despite as a major inhibitor of urokinase (uPA), paradoxically,] Plasminogen activator inhibitor-1 (PAI-1) has been validated to be highly expressed in various types of tumor biopsy tissues or plasma compared with controls based on huge clinical data bases analysis, more importantly, PAI-1 alone or in conjunction with uPA have been identified as prognostic for disease progression and relapse in certain cancer types. particularly in breast cancer. In addition to play important roles in cell adhesion, migration and invasion, PAI-1 has been reported to induce tumor vascularization and thus promote cell dissemination and tumor metastasis. Furthermore, there are many tumor promoting factors involved in the modulation of PAI-1 expression and activity, which will strengthen the pro-tumorigenic roles of PAI-1. Undoubtedly, PAI-1 may be a promising target for therapeutic intervention of specific cancer treatment. In fact, some PAI-1 inhibitors are currently being evaluated in cancer therapy, which may be developed to new antitumor agents in the future.

Famotidine as Urokinase Inhibitor in Anaphylactic Shock

A QSAR study was performed on a series of naphthalene cyclohexane amines derivate with experimentally determine constant of inhibition (Ki ) in order to indentify a potent human urokinase inhibitor. A QSAR model was build using artificial neural networks (ANN). Model was used for screening of compounds with potent urokinase inhibitory properties and to compute Ki of resulted screening compounds. Compound with best Ki was chosen for discussion. An induced fit docking (protein - ligand) and a protein –protein docking were used in results discussion. A isoquinoline –diol base derivate (Famotidione) resulted to have urokinase inhibitory properties.

Abstract B055: New potential therapeutic applications of WX-UK1 as a specific and potent inhibitor of human trypsin-2 and human trypsin-3

Abstract WX-UK1, a multi-serine protease inhibitor, was believed to function primarily as a synthetic small-molecule inhibitor of urokinase (uPA). We now reveal that WX-UK1 is a potent and rather specific inhibitor of human trypsin-2 and human trypsin-3, and propose that these new molecular targets offer potential for new indications in oncology and other diseases. Initially, we performed a bioinformatic analysis of the ~200 human trypsin-like serine proteases, most of which play crucial roles in homeostasis and disease. Among these we selected a subset for biochemical analysis based on an inspection of modeled 3D structures of WX-UK1:protease complexes and sequence alignment of binding site residues. Samples of the selected proteases were prepared and characterized biochemically with respect to inhibitory constant (Ki) of inhibition by WX-UK1-1 and dissociation constant (Kd). The Ki values were determined from enzymatic activity assays (37°C), and Kd values (affinity) by surface plasmon resonance (BIACORE) (25°C). Serine protease(human)Ki (nM)Mean ± SD (n)Kd (nM)Mean ± SD (n)Urokinase (uPA)874 ± 95 (3)720 ± 300 (3)Trypsin(-1)190 ± 10 (6)74 ± 18 (3)Trypsin-275 ± 3 (6)N.D.Trypsin-319 ± 4 (6)6 ± 4 (11) While previously described as an inhibitor of bovine trypsin, we now show WX-UK1 to be a specific competitive inhibitor of both human trypsin-2 and human trypsin-3 with Ki of 75 and 19 nM, respectively. With a Kd of 6 nM, WX-UK-1 binds human trypsin-3 with a >100-fold binding specificity relative to most other trypsin-like serine proteases tested so far, including uPA, and 10-fold over the classical human trypsin. WX-UK1 (and its oral prodrug, upamostat) was originally developed as an inhibitor of uPA and other members of the S1 family of trypsin-like serine proteases, essential to aspects of tissue remodeling associated with tumor invasion and metastasis. However, with the identification of human trypsin-3 and human trypsin-2 as high-affinity molecular targets, new disease indications on top of oncology indications may be feasible. As a low-nanomolar inhibitor of human trypsin-3, WX-UK1 may have potential utility in the treatment of inflammatory digestive diseases, including irritable bowel syndrome, inflammatory bowel disease, and pancreatitis, for the latter of which there are currently no approved therapies. Furthermore, WX-UK1, as a human trypsin-2 inhibitor, could potentially be used in the treatment of inflammatory lung diseases, including acute respiratory distress syndrome, acute lung injury, α-1 antitrypsin deficiency, and chronic obstructive pulmonary disease (COPD)–a major unmet medical need. Citation Format: Emil Oldenberg, Christine R. Schar, Eva L. Lange, Terry F. Plasse, Danielle T. Abramson, Mark L. Levitt, Reza Fathi, Jan K. Jensen. New potential therapeutic applications of WX-UK1 as a specific and potent inhibitor of human trypsin-2 and human trypsin-3 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B055.

3D descriptors calculation and conformational search to investigate potential bioactive conformations, with application in 3D-QSAR and virtual screening in drug design

The knowledge of the bioactive conformation for an active hit is relevant because of the easier interpretation and the general quality of the recognition models of protein and ligand. With the aim of investigating potential bioactive conformations without previous structural knowledge of the molecular target, we present herewith a ‘protocol’ that could be used which includes generation of low-energy conformations, calculations of tridimensional descriptors and investigation of structural similarity via principal component analysis. The protocol was used in the search for potential bioactive conformations. An initial selection of targets was made from a set of protein–ligand complexes with structure deposited in the Protein Data Bank, which was systematically filtered by lead-like rules, resulting in 45 ligands of 8 important therapeutic targets. After extensive optimization of the protocol and parameters of both OMEGA and Pentacle softwares, the best results were obtained for series of compounds such as the beta-trypsin and urokinase inhibitors, which are more structurally related among each other, inside the respective therapeutic class. Future improvements of the protocol, including a suitable choice and combination of robust 3D descriptors, could yield more reliable and less restrictive results, with general and diverse applications in drug design, in particular for improving the 3D-QSAR methodologies as well as virtual screening experiments for a more reliable selection of new lead compounds for different molecular targets.

The effects of benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide), a dual plasmin and urokinase inhibitor, on facial skin barrier function in subjects with sensitive skin.

The aim of this study was to optimize the synthesis of the plasmin and urokinase (uPA) inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide) (BSFAB), to characterize its activity and mechanism of action and to assess its use to improve stratum corneum (SC) barrier function. Peptide coupling methods were used to synthesize BSFAB, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) together with 1 H- and 13 C-nuclear magnetic resonance spectroscopy (NMR) were applied to clarify its structure and determine its purity. Its binding mode was determined by docking studies to the catalytic domains of plasmin and uPA. Inhibition constants (Ki ) were determined by enzyme kinetic studies, and the effect of BSFAB on plasmin, uPA and transglutaminase 1 expression was evaluated in non-cytokine and cytokine-stimulated keratinocytes. A vehicle-controlled clinical study on SC barrier function was conducted on facial skin of subjects with self-perceived sensitive skin. BSFAB was synthesized with high purity (97.3%). In silico studies indicated that the amidine moiety of BSFAB was anchored in the S1 pocket of both enzymes by binding to Asp189, Ser190 and Gly219, whereas the backbone of the D-Ser residue makes an anti-parallel β-sheet interaction with Gly216. BSFAB was shown to be an effective inhibitor of plasmin and uPA with Ki values of 29 and 25 nM, respectively. BSFAB also inhibited keratinocyte-secreted protease activities in basal (plasmin inhibition 37.7%, P < 0.05 and uPA inhibition 96.6%, P < 0.01) and cytokine-induced conditions (plasmin inhibition 41.1%, P < 0.05 and uPA inhibition 97.0%, P < 0.001) and stimulated the gene expression of transglutaminase 1 in cytokine-stimulated keratinocytes (approximately 4.5 times increased expression, P < 0.01). Clinically, BSFAB was shown to improve SC barrier integrity (P < 0.02 on day 29) and subjective improvements in the perception of healthy skin (P < 0.05 on day 28). BSFAB binds as a reversible competitive inhibitor to the active sites of plasmin and uPA. Additionally, BSFAB positively improved keratinocyte differentiation gene expression (transglutaminase 1). These effects were translated into improvements in SC barrier integrity clinically in subjects with dry and sensitive skin and improved their perception of having a healthy skin condition.

SerpinB2 Deficiency Results in a Stratum Corneum Defect and Increased Sensitivity to Topically Applied Inflammatory Agents

SerpinB2 (plasminogen activator inhibitor type 2) is constitutively expressed at high levels by differentiating keratinocytes in mice and humans; however, the physiological function of keratinocyte SerpinB2 remains unclear. Herein, we show that SerpinB2(-/-) mice are more susceptible to contact dermatitis after topical application of dinitrofluorobenzene, and show enhanced inflammatory lesions after topical applications of phorbol ester. Untreated SerpinB2(-/-) mice showed no overt changes in epithelial structure, and we were unable to find evidence for a role for keratinocyte SerpinB2 in regulating immunity, apoptosis, IL-1β production, proteasomal activity, or wound healing. Instead, the phenotype was associated with impaired skin barrier function and a defective stratum corneum, with SerpinB2(-/-) mice showing increased transepidermal water loss, increased overt loss of stratum corneum in inflammatory lesions, and impaired stratum corneum thickening after phorbol ester treatment. Immunoblotting suggested that SerpinB2 (cross-linked into the cornified envelope) is present in the stratum corneum and retains the ability to form covalent inhibitory complexes with urokinase. Data suggest that the function of keratinocyte SerpinB2 is protection ofthe stratum corneum from proteolysis via inhibition of urokinase, thereby maintaining the integrity and barrier function of the stratum corneum, particularly during times of skin inflammation. Implications for studies involving genetically modified mice treated with topical agents and humandermatological conditions, such as contact dermatitis, are discussed.

321 Improvement of barrier impairment by topical application of a dual plasmin and urokinase inhibitor

321 Improvement of barrier impairment by topical application of a dual plasmin and urokinase inhibitor

Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen–plasmin system

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood–brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen–plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.