Elevated Expression of Urokinase Plasminogen Activator in Rodent Models and Patients With Cerebral Amyloid Angiopathy

Objective: To study the association of urokinase plasminogen activator (uPA) with development and progression of cerebral amyloid angiopathy (CAA) in a rat model for CAA type-I (rTg-DI) and in patients with sporadic (sCAA) and Dutch-type hereditary CAA (D-CAA). Additionally, to investigate the potential of uPA to serve as a diagnostic biomarker of CAA in cerebrospinal fluid (CSF) from patients with sCAA and D-CAA.Methods: We studied the expression of uPA mRNA by qPCR and co-localization of uPA with amyloid-β (Aβ) using immunohistochemistry in the cerebral vasculature of rTg-DI rats compared to wild type (WT) rats and in an sCAA patient and control subject using immunohistochemistry. CSF levels of uPA were measured in rTg-DI and WT rats and in two separate cohorts of sCAA and D-CAA patients and controls, using enzyme-linked immunosorbent assays (ELISA).Results: The presence of uPA was clearly detected in the cerebral vasculature of rTg-DI rats and an sCAA patient, but not in WT rats or a non-CAA human control. uPA expression was highly co-localized with microvascular Aβ deposits. In rTg-DI rats, uPA mRNA expression was highly elevated at 3 months of age (coinciding with the emergence of microvascular CAA deposition) and sustained up to 12 months of age (with severe microvascular CAA deposition) compared to WT rats. CSF uPA levels were elevated in rTg-DI rats compared to WT rats (p=0.03), and in two separate, independent groups of sCAA patients compared to controls (after adjustment for age of subjects, p=0.05 and p=0.03). No differences in CSF uPA levels were found between asymptomatic and symptomatic D-CAA patients and their respective controls (after age-adjustment, p=0.09 and p=0.44).Conclusion: uPA expression appears linked to the onset of CAA in rTg-DI rats. Increased cerebrovascular expression of uPA in CAA correlates with increased quantities of CSF uPA in rTg-DI rats and human CAA patients, suggesting that uPA could serve as a biomarker for CAA. No differential expression levels of uPA in CSF of (a)symptomatic D-CAA patients were discovered. These studies also further support the use of rTg-DI rat models as a useful preclinical model for the study of human CAA type-I.

Association of Tissue mRNA and Serum Antigen Levels of Members of the Urokinase-Type Plasminogen Activator System with Clinical and Prognostic Parameters in Prostate Cancer

The objective was to determine the mRNA expression and protein levels of uPA system components in tissue specimens and serum samples, respectively, from prostate cancer (PCa) patients and to assess their association with clinicopathological parameters and overall survival (OS). The mRNA expression levels of uPA, its receptor (uPAR), and its inhibitor type 1 (PAI-1) were analyzed in corresponding malignant and adjacent nonmalignant tissue specimens from 132 PCa patients by quantitative PCR. Preoperative serum samples from 81 PCa patients were analyzed for antigen levels of uPA system members by ELISA. RNA levels of uPA system components displayed significant correlations with each other in the tumor tissues. A significantly decreased uPA mRNA expression in PCa compared to the corresponding nonmalignant tissue was detected. High uPA mRNA level was significantly associated with a high Gleason score. Elevated concentration of soluble uPAR (suPAR) in serum was significantly associated with a poor OS of PCa patients (P=0.022). PCa patients with high suPAR levels have a significantly higher risk of death (multivariate Cox’s regression analysis;HR=7.12,P=0.027). The association of high suPAR levels with poor survival of PCa patients suggests a prognostic impact of suPAR levels in serum of cancer patients.

Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes

Quebec platelet disorder (QPD) is an autosomal dominant disorder with high penetrance that is associated with increased risks for bleeding. The hallmark of QPD is a gain-of-function defect in fibrinolysis due to increased platelet content of urokinase plasminogen activator (uPA) without systemic fibrinolysis. We hypothesized that increased expression of uPA by differentiating QPD megakaryocytes is linked to PLAU. Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing PLAU with a maximum multipoint logarithm of the odds (LOD) score of +11 between markers D10S1432 and D10S1136. Analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation. These findings implicate a mutation in an uncharacterized cis element near PLAU as the cause of QPD.

Increased Expression of Urokinase-Type Plasminogen Activator mRNA Determines Adverse Prognosis in ErbB2-Positive Primary Breast Cancer

Purpose To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival. Patients and Methods Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively. Results Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA–positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA–negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status. Conclusion After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.

Memantine Reduces Hematoma Expansion in Experimental Intracerebral Hemorrhage, Resulting in Functional Improvement

Glutamate is accumulated in abundance during the early period of experimental hematoma, and the activation of N-methyl-D-aspartate (NMDA) receptors by glutamate can result in an influx of calcium and neuronal death in cases of intracerebral hemorrhage (ICH). Memantine, which is known to be a moderate-affinity, uncompetitive, NMDA receptor antagonist, was investigated with regard to its ability to block the glutamate overstimulation and tissue plasminogen activator (tPA)/urokinase plasminogen activator (uPA)/matrix metalloproteinase (MMP)-9 modulation in experimental ICH. Intracerebral hemorrhage was induced via the infusion of collagenase into the left basal ganglia of adult rats. Either memantine (20 mg/kg/day) or PBS was intraperitoneally administered 30 min after the induction of ICH, and, at daily intervals afterwards, for either 3 or 14 days. Hemorrhage volume decreased by 47% in the memantine group, as compared with the ICH-only group. In the memantine group, the numbers of TUNEL+, myeloperoxidase (MPO)+, and OX42+ cells decreased in the periphery of the hematoma. Memantine resulted in an upregulation of bcl-2 expression and an inhibition of caspase-3 activation. Memantine also exerted a profound inhibitory effect on the upregulation of tPA/uPA mRNA, and finally decreased the MMP-9 level in the hemorrhagic brain. In modified limbplacing test, the memantine-treated rats exhibited lower scores initially, and recovered more quickly and thoroughly throughout the 35 days of the study. Here, we show that memantine causes a reduction of hematoma expansion, coupled with an inhibitory effect on the tPA/uPA and MMP-9 level. Subsequently, memantine was found to reduce inflammatory infiltration and apoptosis, and was also determined to induce functional recovery after ICH.

Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells

Atherosclerotic plaques preferentially localize at arterial regions exposed to turbulent low-shear flow. Urokinase-type plasminogen activator (uPA) plays a role in vascular remodeling by facilitating smooth muscle cell migration and proliferation in addition to the proteolysis of extracellular matrix, and the expression of uPA is elevated in atherosclerotic lesions. In this study, we analyzed the effects of laminar and turbulent shear stress on uPA expression in cultured human coronary artery endothelial cells. The application of laminar shear stress (1.5 or 15 dyn/cm2) significantly decreased the amount of uPA mRNA as well as the secretion of uPA protein. In contrast, turbulent shear stress (average intensity, 1.5 dyn/cm2) markedly increased uPA gene expression and protein secretion. Laminar shear stress downregulated uPA gene expression transcriptionally and posttranscriptionally; laminar shear stress activated transcription factor GATA6, which binds to a GATA consensus element located between −692 and −687 bp in the uPA promoter, thereby inhibiting uPA gene transcription. Laminar shear stress also accelerated the degradation of uPA mRNA; the half-life of uPA mRNA decreased to about half of the static control's half-life. Although turbulent shear stress had no effect on the transcription of uPA, it significantly increased uPA mRNA stability; the half-life of uPA mRNA increased by about two times the static control's half-life. Our results suggest that endothelial uPA expression is flow sensitive and differentially regulated by laminar and turbulent shear stress in vitro. We speculate that this effect may contribute to the local nature of atherosclerosis.

Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells

The effects of flow-induced mechanical strains on the phenotype of proximal tubular cells were addressed in vivo and in vitro by subjecting LLC-PK1and mouse proximal tubular cells to different levels of flow. Laminar flow (1 ml/min) induced a reorganization of the actin cytoskeleton and significantly inhibited the expression of plasminogen activators [tissue-type (tPA) activity: 25% of control cells; tPA mRNA: 70% of control cells; urokinase (uPA) mRNA: 56% of control LLC-PK1cells]. In vivo, subtotal nephrectomy (Nx) decreased renal fibrinolytic activity and uPA mRNA content detectable in proximal tubules. Nx also induced a reinforcement of the apical domain of the actin cytoskeleton analyzed by immunofluorescence. These effects of flow on tPA and uPA mRNA were prevented in vitro when reorganization of the actin cytoskeleton was blocked by cytochalasin D and were associated, in vitro and in vivo, with an increase in shear stress-responsive element binding activity detected by an electrophoretic mobility shift assay in proximal cell nuclear extracts. These results demonstrate that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.

Prognostic Significance of the Combined Expression of Matrix Metalloproteinase-9, Urokinase Type Plasminogen Activator and its Receptor in Breast Cancer as Measured by Northern Blot Analysis

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.