Urokinase-type plasminogen activator promotes corneal epithelial migration and nerve regeneration.

Urokinase-type plasminogen activator (uPA) is a serine protease that plays a central role in the pericellular fibrinolytic system, mediates the degradation of extracellular matrix proteins and activation of growth factors, and contributes to the regulation of various cellular processes including cell migration and adhesion, chemotaxis, and angiogenesis. The corneal epithelium responds rapidly to injury by initiating a wound healing process that involves cell migration, cell proliferation, and tissue remodeling. It is innervated by sensory nerve endings that play an important role in the maintenance of corneal epithelial homeostasis and in the wound healing response. We here investigated the role of uPA in corneal nerve regeneration and epithelial resurfacing after corneal injury with the use of uPA-deficient mice. Both the structure of the corneal epithelium and the pattern of corneal innervation in uPA-/- mice appeared indistinguishable from those in uPA+/+ mice. Whereas the cornea was completely resurfaced by 36-48 h after epithelial scraping in uPA+/+ mice, however, such resurfacing required at least 72 h in uPA-/- mice. Restoration of epithelial stratification was also impaired in the mutant mice. Fibrin zymography revealed that the expression of uPA increased after corneal epithelial scraping and returned to basal levels in association with completion of re-epithelialization in wild-type animals. Staining of corneal whole-mount preparations for ßIII-tubulin also revealed that the regeneration of corneal nerves after injury was markedly delayed in uPA-/- mice compared with uPA+/+ mice. Our results thus demonstrate an important role for uPA in both corneal nerve regeneration and epithelial migration after epithelial debridement, and they may provide a basis for the development of new treatments for neurotrophic keratopathy.

Discovery of a novel inhibitor against urokinase-type plasminogen activator, a potential enzyme with a role in atherosclerotic plaque instability.

The buildup of lipids, cholesterol, and other substances in and on the walls of the arteries is known as atherosclerosis and deposition is known as atherosclerotic plaque. Urokinase-type plasminogen activator (uPA) has multiple roles in the atherosclerotic plaque formation and even work simultaneously in making the atherosclerotic plaque unstable. Extracellular matrix plays a major role in the plaque remodeling and rapture. In this study, we have accessed that a higher interaction was observed in the molecular interaction score for uPA with ZINC380065722 having a GOLD fitness score of about 67.60, which is much higher as compared to the known standard inhibitor UK 122 which has reported an interaction score of 59.14. Ser217 and Asp192 are found to be the key amino acid residues in almost all the interactions. Protein frustration analysis has shown that these amino acid residues play a crucial role in the retention of the active pocket conformation and any mutation of these two residues can causes serious decrease in the overall function of the protein. It was observed that the molecule ZINC380065722 remained bound to the protein till 100 ns of simulation time. The average SASA for the apo-uPA and uPA-ligand complex was found to be stable. The network of hydrogen bonds for the intramolecular protein secondary structure and with the solvent system for the apo-protein and the uPA-ligand complex was found to be consistent.Communicated by Ramaswamy H. Sarma.

Urokinase-type plasminogen activator negatively regulates α-smooth muscle actin expression via Endo180 and the uPA receptor in corneal fibroblasts.

Corneal fibroblasts are embedded within an extracellular matrix composed largely of collagen type 1, proteoglycans, and other proteins in the corneal stroma, and their morphology and function are subject to continuous regulation by collagen. During wound healing and in various pathological conditions, corneal fibroblasts differentiate into myofibroblasts characterized by the expression of α-smooth muscle actin (α-SMA). Endo180, also known as urokinase-type plasminogen activator (uPA) receptor-associated protein (uPARAP), is a collagen receptor. Here we investigated whether targeting of Endo180 and the uPA receptor (uPAR) by uPA might play a role in the regulation of α-SMA expression by culturing corneal fibroblasts derived from uPA-deficient (uPA-/-) or wild-type (uPA+/+) mice in a collagen gel or on plastic. The expression of α-SMA was upregulated, the amounts of full-length Endo180 and uPAR were increased, and the levels of both transforming growth factor-ß (TGF-ß) expression and Smad3 phosphorylation were higher in uPA-/- corneal fibroblasts compared with uPA+/+ cells under the collagen gel culture condition. Antibodies to Endo180 inhibited these effects of uPA deficiency on α-SMA and TGF-ß expression, whereas a TGF-ß signaling inhibitor blocked the effects on Smad3 phosphorylation and α-SMA expression. Our results suggest that uPA deficiency might promote the interaction between collagen and Endo180 and thereby increase α-SMA expression in a manner dependent on TGF-ß signaling. Expression of α-SMA is thus negatively regulated by uPA through targeting of Endo180 and uPAR.

Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR).

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by mutations in the dystrophin gene. Transplantation of myogenic stem cells holds great promise for treating muscular dystrophies. However, poor engraftment of myogenic stem cells limits the therapeutic effects of cell therapy. Mesenchymal stem cells (MSCs) have been reported to secrete soluble factors necessary for skeletal muscle growth and regeneration. METHODS: We induced MSC-like cells (iMSCs) from induced pluripotent stem cells (iPSCs) and examined the effects of iMSCs on the proliferation and differentiation of human myogenic cells and on the engraftment of human myogenic cells in the tibialis anterior (TA) muscle of NSG-mdx4Cv mice, an immunodeficient dystrophin-deficient DMD model. We also examined the cytokines secreted by iMSCs and tested their effects on the engraftment of human myogenic cells. RESULTS: iMSCs promoted the proliferation and differentiation of human myogenic cells to the same extent as bone marrow-derived (BM)-MSCs in coculture experiments. In cell transplantation experiments, iMSCs significantly improved the engraftment of human myogenic cells injected into the TA muscle of NSG-mdx4Cv mice. Cytokine array analysis revealed that iMSCs produced insulin-like growth factor-binding protein 2 (IGFBP2), urokinase-type plasminogen activator receptor (uPAR), and brain-derived neurotrophic factor (BDNF) at higher levels than did BM-MSCs. We further found that uPAR stimulates the migration of human myogenic cells in vitro and promotes their engraftment into the TA muscles of immunodeficient NOD/Scid mice. CONCLUSIONS: Our results indicate that iMSCs are a new tool to improve the engraftment of myogenic progenitors in dystrophic muscle.

Plasminogen Activators in Neurovascular and Neurodegenerative Disorders.

The neurovascular unit (NVU) is a dynamic structure assembled by endothelial cells surrounded by a basement membrane, pericytes, astrocytes, microglia and neurons. A carefully coordinated interplay between these cellular and non-cellular components is required to maintain normal neuronal function, and in line with these observations, a growing body of evidence has linked NVU dysfunction to neurodegeneration. Plasminogen activators catalyze the conversion of the zymogen plasminogen into the two-chain protease plasmin, which in turn triggers a plethora of physiological events including wound healing, angiogenesis, cell migration and inflammation. The last four decades of research have revealed that the two mammalian plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), are pivotal regulators of NVU function during physiological and pathological conditions. Here, we will review the most relevant data on their expression and function in the NVU and their role in neurovascular and neurodegenerative disorders.

Superabsorbent Polymer Network Degradable by a Human Urinary Enzyme.

Owing to its superior water absorption capacity, superabsorbent polymer (SAP) based on a poly (acrylic acid) network is extensively used in industrial products such as diapers, wound dressing, or surgical pads. However, because SAP does not degrade naturally, a massive amount of non-degradable waste is discarded daily, posing serious environmental problems. Considering that diapers are the most widely used end-product of SAP, we created one that is degradable by a human urinary enzyme. We chose three enzyme candidates, all of which have substrates that were modified with polymerizable groups to be examined for cleavable crosslinkers of SAP. We found that the urokinase-type plasminogen activator (uPA) substrate, end-modified with acrylamide groups at sufficient distances from the enzymatic cleavage site, can be successfully used as a cleavable crosslinker of SAP. The resulting SAP slowly degraded over several days in the aqueous solution containing uPA at a physiological concentration found in human urine and became shapeless in ~30 days.

Urokinase-type Plasminogen Activator Induces Neurorepair in the Ischemic Brain.

Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to its receptor (uPAR) catalyzes the conversion of plasminogen into plasmin on the cell surface. Recent studies indicate that neurons but not astrocytes release uPA during the recovery phase from an ischemic injury, and that binding of uPA to uPAR promotes neurorepair in the ischemic brain by a mechanism that does not require plasmin generation. A combined approach of in vitro and in vivo studies has shown that uPA binding to uPAR induces the reorganization of the actin cytoskeleton in dendritic spines and axons that have suffered an ischemic injury. Furthermore, recent data indicate that uPA-uPAR binding induces astrocytic activation and a crosstalk between activated astrocytes and the injured neuron that triggers a sequence of biochemical events that promote the repair of synapses injured by the ischemic insult. The translational relevance of these observations is noteworthy because following its intravenous administrations recombinant uPA (ruPA) reaches the ischemic tissue, thus raising the question of whether treatment with ruPA is an effective therapeutic strategy to promote neurorepair functional recovery among ischemic stroke survivors.

[The urokinase-type plasminogen activator system and its role in tumor progression]. / Rol' sistemy aktivatora plazminogena urokinaznogo tipa v progressii opukholei.

In the multistage process of carcinogenesis, the key link in the growth and progression of the tumor is the invasion of malignant cells into normal tissue and their distribution and the degree of destruction of tissues. The most important role in the development of these processes is played by the system of urokinase-type plasminogen activator (uPA system), which consists of several components: serine proteinase - uPA, its receptor - uPAR and its two endogenous inhibitors - PAI-1 and PAI-2. The components of the uPA system are expressed by cancer cells to a greater extent than normal tissue cells. uPA converts plasminogen into broad spectrum, polyfunctional protease plasmin, which, in addition to the regulation of fibrinolysis, can hydrolyze a number of components of the connective tissue matrix (СTM), as well as activate the zymogens of secreted matrix metalloproteinases (MMР) - pro-MMР. MMРs together can hydrolyze all the main components of the СTM, and thus play a key role in the development of invasive processes, as well as to perform regulatory functions by activating and releasing from STM a number of biologically active molecules that are involved in the regulation of the main processes of carcinogenesis. The uPA system promotes tumor progression not only through the proteolytic cascade, but also through uPAR, PAI-1 and PAI-2, which are involved in both the regulation of uPA/uPAR activity and are involved in proliferation, apoptosis, chemotaxis, adhesion, migration and activation of epithelial-mesenchymal transition pathways. All of the above processes are aimed at regulating invasion, metastasis and angiogenesis. The components of the uPA system are used as prognostic and diagnostic markers of many cancers, as well as serve as targets for anticancer therapy.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) promote neurorepair in the ischemic brain.

Despite the fact that ischemic stroke has been considered a leading cause of mortality in the world, recent advances in our understanding of the pathophysiological mechanisms underlying the ischemic injury and the treatment of acute ischemic stroke patients have led to a sharp decrease in the number of stroke deaths. However, this decrease in stroke mortality has also led to an increase in the number of patients that survive the acute ischemic injury with different degrees of disability. Unfortunately, to this date we do not have an effective therapeutic strategy to promote neurological recovery in these growing population of stroke survivors. Cerebral ischemia not only causes the destruction of a large number of axons and synapses but also activates endogenous mechanisms that promote the recovery of those neurons that survive its harmful effects. Here we review experimental evidence indicating that one of these mechanisms of repair is the binding of the serine proteinase urokinase-type plasminogen activator (uPA) to its receptor (uPAR) in the growth cones of injured axons. Indeed, the binding of uPA to uPAR in the periphery of growth cones of injured axons induces the recruitment of ß1-integrin to the plasma membrane, ß1-integrin-mediated activation of the small Rho GTPase Rac1, and Rac1-induced axonal regeneration. Furthermore, we found that this process is modulated by the low density lipoprotein receptor-related protein (LRP1). The data reviewed here indicate that the uPA-uPAR-LRP1 system is a potential target for the development of therapeutic strategies to promote neurological recovery in acute ischemic stroke patients.

Hydroxytyrosol targets extracellular matrix remodeling by endothelial cells and inhibits both ex vivo and in vivo angiogenesis.

The health benefits of olive oil are attributed to their bioactive compounds, such as hydroxytyrosol. Previously, we demonstrated that hydroxytyrosol inhibits angiogenesis in vitro. The present study aimed to: i) get further insight into the effects of hydroxytyrosol on extracellular matrix remodeling; and ii) test whether hydroxytyrosol is able to inhibit angiogenesis ex vivo and in vivo. Hydroxytyrosol induced a shift toward inhibition of proteolysis in endothelial cells, with decreased expression of extracellular matrix remodeling-enzyme coding genes and increased levels of some of their inhibitors. Furthermore, this work demonstrated that hydroxytyrosol, at concentrations within the range of its content in virgin olive oil that can be absorbed from moderate and sustained virgin olive oil consumption, is a strong inhibitor of angiogenesis ex vivo and in vivo. These results suggest the need for translational studies to evaluate the potential use of hydroxytyrosol for angio-prevention and angiogenesis inhibition in clinical setting.

Clinical significance of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) for metastatic sentinel lymph node involvement in breast cancer.

UNLABELLED: Urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) are key factors for tumor invasion and development of metastases in breast cancer. Prospective studies confirmed the prognostic significance of these factors for development of distant metastases. The predictive impact of uPA and PAI-1 for metastatic sentinel lymph node involvement is unclear. PATIENTS AND METHODS: Between 2006 and 2008 uPA and PAI-1 were measured in 184 out of 1,035 patients for primary breast cancer. uPA and PAI-1 were analyzed with an ELISA assay. Measured concentrations were considered as negative for uPA <3 ng/ml and for PAI-1 <14 ng/ml. RESULTS: In a retrospective analysis, 173/184 women had a negative sentinel lymph node and 11/184 women had a metastatic sentinel lymph node. From the 11 women with a positive sentinel lymph node 7 had elevated values for uPA and 4 had elevated values for PAI-1. Four and 7 women were uPA- and PAI-1-negative, respectively. Sensitivity, specificity, positive and negative predictive values for uPA were 63.3%, 50.9%, 7.6%, 95.6% and for PAI-1 36%, 52.6%, 4.7%, 92.9%. Even the combination of both uPA and PAI-1 values did not detect 3/11 women with metastatic lymph node involvement. CONCLUSION: uPA and PAI-1 alone or in combination did not identify all patients with metastatic lymph node involvement. Thus, uPA and PAI-1 cannot be considered as predictive selection parameters to avoid sentinel lymph node biopsy in case of negative values for uPA or PAI-1.

The effects of urokinase-type plasminogen activator (uPA) on cell proliferation and phenotypic transformation of rat mesangial cells induced by high glucose.

AIMS: To investigate the effects of urokinase-type plasminogen activator (uPA) on proliferation and phenotypic transformation of rat mesangial cells (MCs) under high glucose conditions and its possible signal transduction pathway. METHODS: Rat MC were divided into 4 groups: the control group, the high glucose group, the high glucose and wortmannin group, and the high glucose and uPA group. MC proliferation in all groups was detected by the 3-(4,5-dimethylthiazol-)-2,5-diphenyltetrazolium bromide (MTT) method. MC cell cycle was analyzed by flow cytometry. Expression of cyclin dependent kinase 2 (CDK2), and activity of the signaling protein Akt in MC were detected by Western blot. Expression pattern and quantity of α-smooth muscle actin (α-SMA) in MC were examined using laser confocal microscopy. The expression of plasminogen activator inhibitor-1 (PAI-1), and collagen IV in renal tissues in rats was tested with immunohistochemistry and Western blotting methods. RESULTS: Activation of Akt induced by high glucose can be reduced significantly by wortmannin and uPA. There was no obvious change in CDK2 protein expression in different groups (P>0.05). Expression of α-SMA in MC cytoplasm increased dramatically (P<0.01). Expression of α-SMA decreased significantly in the high glucose and wortmannin group and the high glucose and uPA group compared with that of the high glucose group (P<0.01). In diabetic rats, uPA down-regulated PAI-1 and collagen IV expression in mesangial matrix (P<0.05). CONCLUSION: uPA antagonizes cell proliferation and phenotypic transformation of MCs induced by high glucose through inhibiting Akt signaling pathway.

Targeted therapies in cancer - challenges and chances offered by newly developed techniques for protein analysis in clinical tissues.

In recent years, new anticancer therapies have accompanied the classical approaches of surgery and radio- and chemotherapy. These new forms of treatment aim to inhibit specific molecular targets namely altered or deregulated proteins, which offer the possibility of individualized therapies.The specificity and efficiency of these new approaches, however, bring about a number of challenges. First of all, it is essential to specifically identify and quantify protein targets in tumor tissues for the reasonable use of such targeted therapies. Additionally, it has become even more obvious in recent years that the presence of a target protein is not always sufficient to predict the outcome of targeted therapies. The deregulation of downstream signaling molecules might also play an important role in the success of such therapeutic approaches. For these reasons, the analysis of tumor-specific protein expression profiles prior to therapy has been suggested as the most effective way to predict possible therapeutic results. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow the rapid, precise, inexpensive and simultaneous analysis of many network components while requiring only a small amount of clinical material.Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling the quantitative measurement of proteins. Together with recently developed protocols for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues, RPPA may provide the means to quantify therapeutic targets and diagnostic markers in the near future and reliably screen for new protein targets.With the possibility to quantitatively analyze DNA, RNA and protein from a single FFPE tissue sample, the methods are available for integrated patient profiling at all levels of gene expression, thus allowing optimal patient stratification for individualized therapies.