Vappu Sirén

Tissue plasminogen activator and neuroserpin are widely expressed in the human central nervous system

Tissue plasminogen activator (tPA) is increasingly recognized to play important roles in various physiological and pathological processes in the central nervous system (CNS). Much of the data on the involvement of plasminogen activators in neurophysiology and -pathology have been derived from studies on experimental animals. We have now performed a systematic characterization of the expression of tPA and its inhibitor, neuroserpin, in normal human CNS. Brain and spinal cord samples from 30-36 anatomic locations covering all major brain regions were collected at 9 autopsies of donors with no neurological disease. Tissues were embedded in paraffin and tissue arrays were constructed. In two cases parallel samples were snap-frozen for biochemical analysis. Expression and activity profiling of tPA and neuroserpin were performed by immunohistochemistry, in situ hybridization, immunocapture and zymography assays. In the adult CNS, tPA was expressed at the mRNA and protein levels in many types of neurons, in particular in thalamus, cortex of cerebellum, pontine nuclei, neocortex, limbic system, and medulla oblongata. Interestingly, tPA was often co-expressed with its CNS inhibitor, neuroserpin. Despite overlapping expression of tPA and neuroserpin, zymography and immunocapture assays demonstrated that human neural tissue is a rich source of active tPA. Our analysis documents a detailed map of expression of tPA and its inhibitor in the human CNS and is compatible with the view that tPA is a key player in CNS physiology and pathology.

Plasminogen Activator Inhibitor-1 in Neointima of Vein Grafts Its Role in Reduced Fibrinolytic Potential and Graft Failure

BACKGROUND Intimal smooth muscle cell proliferation is an underlying pathogenetic mechanism for neointimal hyperplasia and consequent vein graft failure. This study characterizes the expression of tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor-1 (PAI-1) in hyperplastic vein grafts and normal venous tissue. METHODS AND RESULTS Failing graft and control vein specimens from 14 donors were homogenized, and TPA and PAI-1 were quantified with ELISA. The amount of PAI-1 was seven times higher (4.2+/-2.1 versus 0.6+/-0.6 ng/mg protein, P<.005), but the TPA antigen content was markedly lower (3.1+/-2.1 versus 8.1+/-3.7 ng/mg protein, P<.005) in the stenosed grafts compared with the control veins. Strong immunohistochemical PAI-1 reactivity and in situ hybridization signals for PAI-1 and UPA mRNA were associated with the smooth muscle cells of the thickened intima of the grafts. Functional assays of the graft specimens showed an increased UPA/TPA ratio and a decreased total fibrinolytic activity in comparison with normal veins. CONCLUSIONS Upregulation of PAI-1 mRNA expression and markedly increased amounts of PAI-1 antigen were detected in the vein grafts after the development of neointima. Furthermore, augmented UPA activity was found in the graft wall, but TPA was clearly depleted. Altogether, our findings imply decreased fibrinolytic potential in the stenosed graft, which may contribute to the graft occlusion.

Formation and activation of fibroblast spheroids depend on fibronectin-integrin interaction

Clustering of fibroblasts into spheroids induces a massive proinflammatory, proteolytic and growth-factor response, named nemosis, which promotes tumor cell invasiveness and differentiation of leukemia cells. We have now sought to investigate mechanisms leading to the formation of multicellular spheroids and subsequent activation of fibroblasts (nemosis). Cell lines either lacking fibronectin expression (FN-/-) or expressing FN with a mutated integrin-binding site (FNRGE/RGE) were unable to form compact spheroids. Furthermore, inhibition of FN synthesis by siRNA or functional inhibition of FN or its integrins impaired spheroid formation (alpha5, beta1) and quenched fibroblast activation (alphaV). The integrin ligand GRGDSP hexapeptide interfered with spheroid formation and induced activation of fibroblasts. Surprisingly, a 70 kDa FN fragment, which prevents deposition of FN matrix but does not interfere with FN-integrin interaction, prevented spheroid formation only marginally and did not block the activation. Our results present a new mechanism of fibroblast activation, which is initiated by interaction of FN with its integrin receptors.

REGULATION OF THE PERICELLULAR ACTIVATION OF PLASMINOGEN AND ITS ROLE IN TISSUE‐DESTRUCTIVE PROCESSES

While it is generally recognized today that cells use specific adhesion proteins such as fibronectin and laminin, both in cell adhesion and migration, it is equally clear that pericelldar proteolysis is critically involved in the invasion of malignant and certain normal cells through the extracellular matrices, both interstitial connective tissue matrices and basement membranes. There is interesting new evidence suggesting that a key component in pericellular proteolysis, i.e. the urokinase-receptor complex, may have a role also in both cell adhesion and cell migration. In addition there is direct evidence that adhesion proteins of pericellular matrix can provide a molecular link between the membrane receptor proteins, known as integrins, and plasminogen.

Tissue plasminogen activator gene expression in multiple sclerosis brain tissue.

Recent studies have implicated tissue-type plasminogen activator (tPA) in neurodegeneration. We studied multiple sclerosis (MS) brain tissue for tPA gene and protein expression in comparison with reference tissue, by in situ hybridisation and immunohistochemistry. MS is characterised by demyelination in the central nervous system. In this study, neuronal cell bodies in MS brain showed high expression of tPA mRNA and protein, while in reference brains, staining for protein and mRNA expression were very low in neurons and mostly restricted to blood vessel walls. In MS, there was an additional staining of mononuclear cells within perivascular cuffs and foamy macrophages within demyelinating plaques. In view of evidence that the final process of demyelination in MS is thought to be enzyme-mediated, our work suggests the involvement of tPA and by inference plasmin, in the demyelinating process. Blocking tPA or plasmin activity may be a potentially beneficial therapeutic approach in MS.

Cell‐cell contact activation of fibroblasts increases the expression of matrix metalloproteinases

Background. We recently found that direct homotypic cell‐cell contacts between human dermal fibroblasts induce a novel form of cell activation leading to non‐apoptotic programmed cell death. As the major features of this process we identified massive induction of cyclo‐oxygenase‐2 and production of inflammatory prostaglandins. On the surface of the decomposing spheroids, activation of the major extracellular proteolytic cascade, plasminogen activation, associated with surface exposure of α‐enolase, took place. Aim. To further characterize pericellular proteolysis by cell‐cell contact‐activated fibroblasts we studied the role of the other major extracellular proteolytic system, matrix metalloproteinases (MMPs). Methods. MMP expression in fibroblast clusters and monolayers was compared using mRNA microarrays and immunoblot analyses. The activities of MMPs were confirmed using MMP inhibitors and caseinolysis. Results. In microarrays MMP‐1, ‐10, and ‐14 (MT1‐MMP) were induced 5.8‐, 106‐, and 5.6‐fold, respectively. These findings were confirmed by immunoblotting. Radial caseinolysis showed low level of proteolytic activity in spheroid‐conditioned media; ilomastat, a general inhibitor of MMPs, suppressed 50% of the proteolytic activity thus confirming it to be at least in part due to MMPs. A cocktail of tetracycline‐derived MMP inhibitors suppressed lactate dehydrogenase (LDH) release only 11%, and if combined with aprotinin 28%. Conclusions. Cell‐cell contact activation of fibroblasts induced MMP‐1, ‐10, and MT1‐MMP expression, suggesting similar signaling to that in inflammation and cancer.

Retinal Pigment Epithelial Cells Secrete Urokinase-Type Plasminogen Activator and Its Inhibitor PAI-1

Secretion of plasminogen activators and their inhibitors was examined in cultures of human retinal pigment epithelial (RPE) cells. The methods employed were zymography and reverse zymography, solid-phase immunocapture assay, metabolic labeling followed by immunoprecipitation, and immunofluorescence. The results showed that these cells produce urokinase-type plasminogen activator (u-PA) and a plasminogen activator inhibitor (PAI) which is immunologically and biochemically similar to PAI-1. Tissue-type plasminogen activator activity (t-PA) was not detected, but we detected small amounts of t-PA in an inactive complex with inhibitor in RPE cell-conditioned media. We conclude that RPE cells have the potential to utilize u-PA-catalyzed plasminogen activation which is subject to regulation by PAI-1. These results may have a bearing on the pathogenesis of proliferative retinal diseases.

High Concentrations of Plasminogen Activator Inhibitor-1 in Lungs of Preterm Infants With Respiratory Distress Syndrome

BACKGROUND. Among preterm infants, respiratory distress syndrome (RDS) is characterized by the presence of intraalveolar fibrin deposition. Fibrin monomers inhibit surfactant function effectively. However, little is known about potential disturbances of intraalveolar fibrinolysis in RDS. We studied levels of major plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA) in lungs of preterm infants with RDS. METHODS. The antigen levels of PAI-1, tPA, and uPA were measured in 262 samples of tracheal aspirate fluid collected from 37 intubated preterm infants during the first 2 postnatal weeks. To examine the expression of PAI-1, tPA, and uPA in lung tissue, immunohistochemical analyses were performed on autopsy specimens from 7 preterm infants with RDS and 6 newborn infants without pulmonary pathologic conditions. RESULTS. For infants with an immature surfactant profile, as indicated by lecithin/sphingomyelin ratios in tracheal aspirate fluid of <10, PAI-1 levels and ratios of PAI-1 to uPA and tPA were significantly higher during postnatal days 1 to 2, compared with infants with lecithin/sphingomyelin ratios of ≥10. Moreover, infants who subsequently developed bronchopulmonary dysplasia (BPD) (n = 15) had higher PAI-1 levels on days 3 to 4 and days 7 to 8 than did those who survived without BPD. For preterm infants with RDS, immunohistochemical analyses demonstrated increased expression of PAI-1, tPA, and uPA predominantly in alveolar epithelium. CONCLUSIONS. High concentrations of PAI-1 and an increased ratio of PAI-1 to uPA, with a concurrently less-increased ratio of PAI-1 to tPA, are associated with the severity of RDS among preterm infants during the first postnatal days. Pulmonary inhibition of fibrinolysis is a pathophysiologic feature of RDS and may play a role in the development of BPD.

Urokinase, tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in severely stenosed and occluded vein grafts with thrombosis.

Intimal hyperplasia and subsequent thrombotic occlusions limit the success of vascular reconstructive procedures. Plasminogen activation in situ may be an important factor affecting re-stenosis of the graft. Tissue specimens from eight patients with failing or failed infra-inguinal vein bypasses and three specimens from normal veins were harvested to study urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) by in situ hybridization and immunohistochemistry. The possible presence of thrombi was monitored by platelet and fibrin-specific stainings. In occluded grafts, platelet endothelial cell adhesion molecule (PECAM-1) antibody stained the thrombi but not the endothelial area, indicating the absence of endothelium. Platelet glycoprotein (GP) IIb/IIIa co-localized with PECAM-1 and, furthermore, GP IIb/IIIa staining was positive on the vein walls with thrombi and to some extent in the grafts without thrombi. PAI-1 and u-PA were uniformly upregulated in intimal thickening in grafts without thrombus. In organized thrombi, enhanced u-PA, t-PA and PAI-1 reactivity was detected in the ingrowing subendothelium. In non-occluded grafts with small thrombi, u-PA expression was enriched beneath microthrombi co-localizing with the graft wall injury, while PAI-1 was scattered in the (sub)endothelium. We conclude that fibrinolytic system is upregulated at sites of graft stenosis, and local proteolytic degradation of the graft wall associates with thrombus formation.

Proteinases in subretinal fluid

Abstract⊎ Background: Degradation of the extracellular matrix by secreted proteases is connected to cell migration and proliferation in invasive growth and in scar tissue formation. In retinal detachment, retinal pigment epithelium (RPE) cells loosened from their monolayer are often seen in the subretinal fluid (SRF) and the vitreous, where they may participate in the scar tissue formation of proliferative vitreoretinopathy. To evaluate the role of SRF constituents on the release of RPE cells, we analyzed SRF in patients with retinal detachment for the presence of enzymes able to degrade extracellular matrix. ⊎ Methods: SRF was collected altogether from 16 patients undergoing retinal reattachment surgery and analyzed for activities against some of the key enzymes in extracellular proteolysis, namely collagenases, gelatinases, elastase and cathepsin G. ⊎ Results: Seventy-two-kilodalton gelatinase was found in all SRF samples studied, whereas the neurophil-type 92-kDa gelatinase could not be detected. Low collagenase, elastase and cathepsin G activities could also be detected in some samples. ⊎ Conclusions: The predominant type of matrix metalloproteinase present in SRF is the 72-kDa MMP-2. The proteolytic activity in SRF may be connected to the release of RPE cells into SRF and to degradation of components of the vitreous exposed to SRF.