Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor

Degradation of the extracellular matrix plays an important role in a number of normal and pathological conditions involving active tissue remodeling such as postlactational mammary gland involution, wound healing and tumor invasion and metastasis. The expression of a high-affinity, glycolipid-anchored receptor for the urokinase-type plasminogen activator (uPAR) is often up-regulated during such tissue remodeling events. uPAR may, therefore, in cooperation with various matrix metalloproteases, serve to facilitate the proteolytic breakdown of the extracellular matrix via uPA catalyzed plasminogen activation at the foci where cellular invasion occurs. Consistent with such a role for uPAR in pericellular proteolysis is the observation that the membrane assembly of both plasminogen, via its lysine binding-sites, and of pro-uPA, via its tight binding to uPAR, is required to favor and confine plasminogen activation potential in proximity of the cell surface. This review will focus on molecular properties of uPAR including its membrane attachment by glycosyl-phosphatidylinositol, its multidomain structure and its relationship to the Ly-6/uPAR/alpha-neurotoxin protein domain family. Furthermore a mapping of the functional epitopes for uPA binding as well as a competitive peptide antagonist of the uPA-uPAR interaction will be discussed.     

Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: inhibitory effects on receptor-mediated uPA activity in vitro and in vivo

Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.     

Rational targeting of the urokinase receptor (uPAR): development of antagonists and non-invasive imaging probes

In the last two decades, the urokinase-type plasminogen activator receptor (uPAR) has been implicated in a number of human pathologies such as cancer, bacterial infections, and paroxysmal nocturnal hemoglobinuria. The primary function of this glycolipid-anchored receptor is to focalize uPA-mediated plasminogen activation at the cell surface, which is accomplished by its high-affinity interaction with the growth factor-like domain of uPA. Detailed insights into the molecular basis underlying the interactions between uPAR and its two bona fide ligands, uPA and vitronectin, have been obtained recently by X-ray crystallography and surface plasmon resonance studies. Importantly, these structural studies also define possible druggable target sites in uPAR for small molecules and provide guidelines for the development of reporter groups applicable for non-invasive molecular imaging of uPAR expression in vivo by positron emission tomography. In this review, we will discuss recent advances in our perception of the structure-function relationships of uPAR ligation and how these may assist translational research in preclinical intervention studies of uPAR function.     

Tumour cell expression of C4.4A, a structural homologue of the urokinase receptor, correlates with poor prognosis in non-small cell lung cancer

PURPOSE: C4.4A expression has been implicated in human cancer progression. This protein is a structural homologue of the urokinase receptor, uPAR, which constitutes a well-established prognostic marker in various human cancers. Nonetheless, little is known about the prognostic significance of C4.4A expression. In the present study, we therefore explored the possible association between C4.4A expression and prognosis in patients with non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Tissue sections from 108 NSCLC patients were subjected to immunohistochemical staining using a polyclonal antibody that specifically recognises human C4.4A. Staining frequency and intensity was scored semiquantitatively and grouped into cancers with high and low expression of C4.4A. Kaplan-Meier survival curves were generated to evaluate the significance of C4.4A expression in prognosis of NSCLC patients. RESULTS: High C4.4A expression was observed in 42% of the NSCLC specimens analysed, and this correlates with overall survival (p = 0.012). A remarkably strong correlation was noted between high expression of C4.4A in pulmonary adenocarcinoma and survival (p < 0.0001). Multivariate Cox regression analysis shows that high C4.4A expression is an independent predictor of poor disease outcome in NSCLC (risk ratio, 1.42; 95% confidence interval, 1.09-1.86; p = 0.009). Although histological type is not a predictor of outcome in NSCLC, high C4.4A expression in adenocarcinoma is nevertheless a very strong predictor of poor disease outcome (risk ratio, 1.62; 95% confidence interval, 1.24-2.09; p = 0.001). CONCLUSIONS: High tumour cell C4.4A expression is associated with shorter survival for NSCLC patients. Patients with pulmonary adenocarcinoma have a particularly poor prognosis if this histological type is combined with high tumour cell C4.4A expression.     

Evidence of accumulated stress in Achilles and anterior knee tendons in elite badminton players

Tendon-related injuries are a major problem, but the aetiology of tendinopathies is unknown. In tendinopathies as well as during unaccustomed loading, intra-tendinous flow can be detected indicating that extensive loading can provoke intra-tendinous flow. The aim of present study is to evaluate the vascular response as indicated by colour Doppler (CD) activity in both the Achilles and patella tendon after loading during high-level badminton matches. The Achilles tendon was subdivided into a mid-tendon, pre-insertional, and insertional region and the anterior knee tendons into a quadriceps-, patella- and tuberositas region. Intra-tendinous flow was measured using both a semi-quantitative grading system (CD grading) and a quantitative scoring system (CF) on colour Doppler. Intra-tendinous flow in the Achilles and anterior knee tendons was examined in fourteen single players before tournament and after 1st and 2nd match, respectively on both the dominant and non-dominant side. All players had abnormal intra-tendinous flow (Colour Doppler ≥ grade 2) in at least one tendon in at least one scan during the tournament. At baseline, only two of the 14 players had normal flow in all the tendons examined. After 1st match, tendencies to higher intra-tendinous flow were observed in both the dominant patella tendon and non-dominant quadriceps tendon (P-values n.s.). After 2nd match, intra-tendinous flow was significant increased in the dominant patella tendon (P = 0.009). In all other locations, there was a trend towards a stepwise increase in intra-tendinous flow. The preliminary results indicate that high amount of intra-tendinous flow was found in elite badminton players at baseline and was increased after repetitive loading, especially in the patella tendon (dominant leg). The colour Doppler measurement can be used to determine changes in intra-tendinous flow after repetitive loading.     

Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B

Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether beta-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a beta-cell-specific, 2-fold overexpression of the cAMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from advanced fasting hyperinsulinemia and early development of hyper-glycemia, in spite of hyperinsulinemia, as well as impaired capacity of insulin to suppress plasma glucose in an insulin tolerance test. In vitro analyses of insulin-stimulated lipogenesis in adipocytes and glucose uptake in skeletal muscle did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet perturbations, such as centrally located alpha-cells and reduced immunostaining for insulin and GLUT2 in islets from RIP-PDE3B/2 mice. Additionally, in vitro experiments revealed that the insulin secretory response to glucagon-like peptide-1 stimulation was markedly reduced in islets from high-fat-fed RIP-PDE3B/2 mice. We conclude that accurate regulation of beta-cell cAMP is necessary for adequate islet adaptation to a perturbed metabolic environment and protective for the development of glucose intolerance and insulin resistance.     

Small dense LDL particles - a predictor of coronary artery disease evaluated by invasive and CT-based techniques: a case-control study

Background  

Coronary angiography is the current standard method to evaluate coronary atherosclerosis in patients with suspected angina pectoris, but non-invasive CT scanning of the coronaries are increasingly used for the same purpose.     

Genetic control of eosinophilia in guinea pig strains inbred for high or low bronchial allergic reactivity 2. A genetic study of spontaneous and immunization-induced eosinophilia

Ploug Winthereik M, Spärck JV, Lundberg L, Sompolinsky D. Genetic control of eosinophilia in guinea pig strains inbred for high or low bronchial allergic reactivity. 2. A genetic study of spontaneous and immunization-induced eosinophilia.
In 4 inbred strains of guinea pig the tendency to develop peripheral high-eosinophilia was shown to be genetically controlled. The development of eosinophilia with age and following immunization was examined in high- and low-eosinophilic parental strains, in F₁-hybrids and in backcross offspring. The results show that probably only one or a very few segregating genes control eosinophilia, and they also indicate that different genes are involved in the determination of spontaneous (age-related) and immunization-induced eosinophilia.     

Effect of force development on contraction induced glucose transport in fast twitch rat muscle

A previous study has shown that in fast twitch frog sartorius muscle contraction stimulated glucose transport depends only on stimulation frequency and not on workload. In contrast, we have recently shown that in rat slow twitch muscle stimulated to contract at constant frequency, glucose transport varies directly with force development and, in turn, metabolism. The present study was carried out to clarify whether the discrepancy between the earlier studies reflected differences in physiological behaviour between fast and slow twitch muscle. We investigated the effect of force development on glucose transport in incubated fast twitch rat flexor digitorum brevis (rich in type 2a fibres) and epitrochlearis (rich in type 2b fibres) muscle. Muscles were electrically stimulated to perform repeated tetanic contractions at 1 Hz for 10 min. Resting length was adjusted to achieve either no force or maximum force. Glucose transport (2‐deoxyglucose uptake) increased when force was produced compared with when it was not (P < 0.05) in both flexor digitorum brevis (19 ± 7 (basal), 163 ± 14 (no force) and 242 ± 17 (max force) nmol × g^–1 × 5 min^–1) and epitrochlearis (60 ± 4 (basal), 100 ± 7 (no force) and 125 ± 6 (max force) nmol × g^–1 × 5 min^–1). In both muscles glucose transport increased in parallel with metabolic rate, as reflected by muscle lactate concentrations and 5′ AMP‐activated protein kinase activity, during contractions. In conclusion, as previously shown for rat soleus muscle, at a given stimulation frequency glucose transport varies directly with force development in rat flexor digitorum brevis and epitrochlearis muscle. Accordingly, force development enhances glucose transport in all mammalian muscle fibre types. The influence of force development probably reflects effects of enhanced 5′ AMP‐activated protein kinase activity resulting from reduced intra‐cellular energy status and pH.