Urokinase-like Plasminogen Activator Research Articles

PLAUR splicing pattern in hereditary angioedema patients’ monocytes and macrophages

The PLAUR gene encodes the urokinase-like plasminogen activator receptor (uPAR) and may undergo alternative splicing. Excluding cassette exons 3, 5 and 6 from the transcript results in truncated protein variants whose precise functions have not been elucidated yet. The PLAUR gene is one of several expressed in myeloid cells, where uPAR participates in different cellular processes, including the contact activation system and kallikrein-kinin system, which play an important role in hereditary angioedema (HAE) pathogenesis. A hypothesis about the PLAUR splicing pattern impact on HAE severity was tested. The RT-PCR quantified by capillary electrophoresis was used. Although no significant difference in alternative transcript frequency was observed between healthy volunteers and HAE patients, a significant increase in all cassette exon inclusion variants was revealed during monocyte-to-macrophage differentiation. PLAUR alternative splicing in monocytes and macrophages neither was different between HAE patients and healthy controls, nor reflected disease severity. However, the results showed an PLAUR splicing pattern was changing during monocyte-to-macrophage differentiation, but the significance of these changes is unknown and awaits future clarification.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.

MICROMICETES ASPERGILLUS RAPERI AND SAROCLADIUM STRICTUM AS PRODUCERS OF ENZYMES WITH UROKINASE-LIKE PLASMINOGEN ACTIVATOR ACTIVITY

Data were obtained on the general proteolytic activity of Aspergillus raperi and Sarocladium strictum and on proteolytic activity in relation to the main proteins of the hemostasis system (specific activity). It has been shown that Sarocladium strictum is a more promising strain producing enzymes with urokinase-like plasminogen activator activity due to the high specificity of this activity.

UPAR-targeted optical near-infrared (NIR) fluorescence imaging and PET for image-guided surgery in head and neck cancer: proof-of-concept in orthotopic xenograft model.

PurposeUrokinase-like Plasminogen Activator Receptor (uPAR) is overexpressed in a variety of carcinoma types, and therefore represents an attractive imaging target. The aim of this study was to assess the feasibility of two uPAR-targeted probes for PET and fluorescence tumor imaging in a human xenograft tongue cancer model.Experimental design and resultsTumor growth of tongue cancer was monitored by bioluminescence imaging (BLI) and MRI. Either ICG-Glu-Glu-AE105 (fluorescent agent) or 64Cu-DOTA-AE105 (PET agent) was injected systemically, and fluorescence imaging or PET/CT imaging was performed. Tissue was collected for micro-fluorescence imaging and histology. A clear fluorescent signal was detected in the primary tumor with a mean in vivo tumor-to-background ratio of 2.5. Real-time fluorescence-guided tumor resection was possible, and sub-millimeter tumor deposits could be localized. Histological analysis showed co-localization of the fluorescent signal, uPAR expression and tumor deposits. In addition, the feasibility of uPAR-guided robotic cancer surgery was demonstrated. Also, uPAR-PET imaging showed a clear and localized signal in the tongue tumors.ConclusionsThis study demonstrated the feasibility of combining two uPAR-targeted probes in a preclinical head and neck cancer model. The PET modality provided preoperative non-invasive tumor imaging and the optical modality allowed for real-time fluorescence-guided tumor detection and resection. Clinical translation of this platform seems promising.

Long-Lived αMUPA Mice Show Reduced Sexual Dimorphism in Lifespan, and in Energy and Circadian Homeostasis-Related Parameters.

Female αMUPA (alpha murine urokinase-like plasminogen activator) transgenic mice show increased lifespan, reduced body weight and food intake, and high-amplitude circadian rhythms with an endogenous period length (tau) of 24h, versus their wild types (WT) showing a 23.7-h tau. Our goal was to characterize αMUPA and WT male mice, and their in-strain sexual dimorphism, and to further understand the mechanisms underlying αMUPA's longevity. Male αMUPA mice showed increased lifespan, reduced body weight and food intake, and aligned endogenous rhythm with a tau of 24.0h versus a tau <24h in WT. However, no differences were found when intake was corrected for metabolic mass in male αMUPA mice. αMUPA's sexual dimorphism was damped or lacking in all studied traits, while WTs were sexually dimorphic, concluding that αMUPA's transgene overrides sex-dependent mechanisms involved in lifespan and in energy and circadian homeostasis. As enhanced resonance between tau and external circadian cycle correlates with increased lifespan and reduced body weight in other species, including humans, αMUPA's 24-h tau could contribute to their longevity. Focusing future research on the mechanistic interconnections between energy homeostasis, circadian homeostasis, sexual dimorphism, and aging, using αMUPA mice, may reveal mechanisms promoting reduced body weight and increased lifespan.

Kondrat'eva ligation: Diels-Alder-based irreversible reaction for bioconjugation.

Diversification of existing chemoselective ligations is required to efficiently access complex and well-defined biomolecular assemblies with unique and valuable properties. The development and bioconjugation applications of a novel Diels-Alder-based irreversible site-specific ligation are reported. The strategy is based on a Kondrat'eva cycloaddition between bioinert and readily functionalizable 5-alkoxyoxazoles and maleimides that readily react together under mild and easily tunable reaction conditions to afford a fully stable pyridine scaffold. The potential of this novel bioconjugation is demonstrated through the preparation of fluorescent conjugates of biomolecules and a novel Förster resonance energy transfer (FRET)-based probe suitable for the in vivo detection and imaging of urokinase-like plasminogen activator (uPA), which is a key protease involved in cancer invasion and metastasis.

Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency.

Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation, etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na(+) flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane-permeabilized cells. A decrease in the Na(+)-K(+)-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid reabsorption by MTEs. ERK1/2, but not Akt, phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs. those of wild-type controls. Expression of caspase 8, however, did not differ between wild-type and uPA(-/-) mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na(+)-K(+)-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function.

Effect of lysine antifibrinolytics and cyclooxygenase inhibitors on the proteolytic profile of breast cancer cells interacting with macrophages or endothelial cells

Extracellular matrix (ECM) proteases play a key role in the regulation of tumour invasion, growth, and transendothelial migration. The expression of ECM proteases and their endogenous inhibitors by cancer cells is regulated by stromal cells. We investigated the effect of commonly used perioperative medications on this regulation. Breast cancer cells (4T1) were cultured alone or with endothelial cells (H5V) or macrophages (RAW264.7). Cell grown alone or in cocultures were treated with clinically relevant concentrations of cyclooxygenase (COX) inhibitors, aspirin (ASA), ketorolac, celecoxib, or lysine antifibrinolytics, ɛ-aminocaproic acid (EACA) and tranexamic acid (TXA). We determined the level of the ECM proteases urokinase-like plasminogen activator (uPA), matrix metalloproteinase (MMP)-2 and MMP-9, and endogenous MMP inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in the conditioned media. Antifibrinolytics and COX inhibitors exerted a complex effect on cells grown alone and in cocultures. EACA increased the activity of MMP-9 and TIMP-1 in cocultures of 4T1 and RAW264.7. TXA increased TIMP-1 in the coculture without affecting MMP-9. EACA and TXA both attenuated MMP-2 detected in 4T1 and H5V cocultures. ASA and ketorolac both decreased the activity of MMP-2, MMP-9, and uPA. Celecoxib increased the activity of TIMP-1 in cocultures of 4T1 with both macrophages and endothelial cells. Antifibrinolytics and COX inhibitors can affect the proteolytic profile of the tumour microenvironment. Animal and clinical investigations are warranted to assess the effect of these proteolytic changes on the outcome of cancer surgery.

Morphine and breast tumor metastasis: the role of matrix-degrading enzymes

Opioids including morphine are commonly used in pain management during and after cancer surgery but have been linked to a variety of pro- and anti-tumor effects. In the present study the effect of morphine administration on the localization and growth of breast tumor cells in lungs and the level of extracellular matrix (ECM) proteases were investigated. In a mouse syngeneic model of intravenously inoculated breast cancer cells, morphine administration led to a reduction in the localization and growth of tumors in the lungs and a reduction in circulating matrix metalloproteinase-9 (MMP-9) and urokinase-like plasminogen activator (uPA). To model the involvement of non-malignant cells of the tumor microenvironment in the changes we observed in the level of proteases, we co-cultured breast cancer cells with macrophages, endothelial cells and fibroblasts. We found a significant elevation of matrix proteases as well as matrix protease inhibitors in co-cultures of breast cancer cells with macrophages or endothelial cells. Interestingly, morphine treatment of these co-cultures reduced the level of MMP-9 and increased its endogenous inhibitor, TIMP-1, thereby altering the proteolytic profile. Morphine affected the level of enzymes in co-cultures but not in cells grown individually. This suggests that anti-tumor effects of morphine observed in our in vivo model could be mediated at least in part through modulation of paracrine communication between cancer cells and non-malignant cells in the tumor microenvironment.

Enhanced Prostate Cancer Targeting by Modified Protease Sensitive Photosensitizer Prodrugs

Prodrugs combining macromolecular delivery systems with site-selective drug release represent a powerful strategy to increase selectivity of anticancer agents. We have adapted this strategy to develop new polymeric photosensitizer prodrugs (PPP) sensitive to urokinase-like plasminogen activator (uPA). In these compounds (to be referred to as uPA-PPPs) multiple copies of pheophorbide a are attached to a polymeric carrier via peptide linkers that can be cleaved by uPA, a protease overexpressed in prostate cancer (PCa). uPA-PPPs are non-phototoxic in their native state but become fluorescent and produce singlet oxygen after uPA-mediated activation. In the present work, we studied the influence of side-chain modifications, molecular weight, and overall charge on the photoactivity and pharmacokinetics of uPA-PPPs. An in vitro promising candidate with convertible phototoxicity was then further investigated in vivo. Systemic administration resulted in a selective accumulation and activation of the prodrug in luciferase transfected PC-3 xenografts, resulting in a 4-fold increase in fluorescence emission over time. Irradiation of fluorescent tumors induced immediate tumor cell eradication as shown by whole animal bioluminescence imaging. PDT with uPA-PPP could therefore provide a more selective treatment of localized PCa and reduce side effects associated with current radical treatments.

Transcriptional Profiling after Lipid Raft Disruption in Keratinocytes Identifies Critical Mediators of Atopic Dermatitis Pathways

Lipid rafts are cholesterol-rich cell signaling platforms, and their physiological role can be explored by cholesterol depletion. To characterize transcriptional changes ongoing after lipid raft disruption in epidermal keratinocytes, a cell type that synthesizes its cholesterol in situ, we performed whole-genome expression profiling. Microarray results show that over 3,000 genes are differentially regulated. In particular, IL-8, urokinase-like plasminogen activator receptor, and metalloproteinases are highly upregulated after cholesterol extraction. Quantitative reverse transcriptase PCR validation and protein release measurements demonstrate the physiological relevance of microarray data. Major enriched terms and functions, determined by Ingenuity Pathways Analysis, identify cholesterol biosynthesis as a major function, illustrating the specificity of keratinocyte response toward cholesterol depletion. Moreover, the inflammatory skin disorder atopic dermatitis (AD) is identified as the disease most closely associated with the profile of lipid raft-disrupted keratinocytes. This finding is confirmed in skin of AD patients, in whom transcript levels of major lipid raft target genes are similarly regulated in lesional atopic skin, compared with non-lesional and normal skin. Thus, lipid raft disruption evokes typical features of AD, thereby suggesting that lipid raft organization and signaling could be perturbed in atopic keratinocytes.

Levonorgestrel‐intrauterine system effects on hemostasis and menstrual blood loss in women seeking contraception

The purpose of the study is to determine the effects of levonorgestrel use on hemostasis, and menstrual blood loss over 24 months in a cohort of women seeking contraception. Data from 30 women (median age 36 years) were analyzed. Samplings at pre-insertion, 1 and 3 months for blood and additional endometrial aspirates at 2 and 6 months were performed. Systemic determination for hemoglobin, hematocrit, computerized thromboelastography, tissue-type plasminogen activator, urokinase-like plasminogen activator and receptor, plasminogen activator inhibitor-1/2, D-dimer and von Willebrand Factor was performed. In endometrial extracts, tissue-type plasminogen activator, urokinase-like plasminogen activator and receptor and plasminogen activator inhibitor-1/2 were assayed. The Menstrual Blood Loss Pictorial Chart Score for measurement of menstrual blood loss was carried out for 24 months. There were no significant changes in systemic hemostasis within 3 months of device use. In the endometrium, there was a significant increase in tissue-type plasminogen activator antigen, plasminogen activator inhibitor-1/2 and urokinase-like plasminogen activator receptor levels from pre-insertion state, with the highest level seen by 6 months. Amenorrhea was seen in 20% of women by 6 months and 50% by 24 months. Enhanced expression of plasminogen activator inhibitor-1/2 in the presence of increased urokinase-like plasminogen activator receptor and tissue-type plasminogen activator antigen was seen in the endometrium. The effects on hemostasis appear to be localized in the endometrium. Systemic hemostasis was not duly affected and menstrual blood loss was reduced. No women in the study had a pregnancy or expulsion of the levonorgestrel-intrauterine system device in the 24 months of the study period.

De-N-acetyl GM3 Promotes Melanoma Cell Migration and Invasion through Urokinase Plasminogen Activator Receptor Signaling–Dependent MMP-2 Activation

We have recently discovered that de-N-acetyl GM3 [NeuNH(2)LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82-95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3-positive) and poorly invasive (d-GM3-negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases.

MTOR mediates human trophoblast invasion through regulation of matrix-remodeling enzymes and is associated with serine phosphorylation of STAT3

The intracellular signaling molecule mammalian target of rapamycin (mTOR) is essential for cell growth and proliferation. It is involved in mouse embryogenesis, murine trophoblast outgrowth and linked to tumor cell invasiveness. In order to assess the role of mTOR in human trophoblast invasion we analyzed the in vitro invasiveness of HTR-8/SVneo immortalized first-trimester trophoblast cells in conjunction with enzyme secretion upon mTOR inhibition and knockdown of mTOR protein expression. Additionally, we also tested the capability of mTOR to trigger signal transducer and activator of transcription (STAT)-3 by its phosphorylation status. Rapamycin inhibited mTOR kinase activity as demonstrated with a lower phosphorylation level of the mTOR substrate p70 S6 kinase (S6K). With the use of rapamycin and siRNA-mediated mTOR knockdown we could show that cell proliferation, invasion and secretion of matrix-metalloproteinases (MMP)-2 and -9, urokinase-like plasminogen activator (uPA) and its major physiological uPA inhibitor (PAI)-1 were inhibited. While tyrosine phosphorylation of STAT3 was unaffected by mTOR inhibition and knockdown, serine phosphorylation was diminished. We conclude that mTOR signaling is one major mechanism in a tightly regulated network of intracellular signal pathways including the JAK/STAT system to regulate invasion in human trophoblast cells by secretion of enzymes that remodel the extra-cellular matrix (ECM) such as MMP-2, -9, uPA and PAI-1. Dysregulation of mTOR may contribute to pregnancy-related pathologies caused through impaired trophoblast invasion.

Circadian changes in the hemostatic system in healthy men and patients with cardiovascular diseases

The highest risk of cardiovascular events is in the morning, which may be associated with circadian changes in hemostasis. There is a 10% circadian variability in von Willebrand factor (vWF). Circadian periodicity has been noted for both the number of platelets and their aggregability. The highest number of platelets was in the afternoon, while most studies observed an increase in platelet aggregation in the morning. Platelet activity has also been linked with physical activity after waking up. The highest values of coagulation system markers such as fragment 1+2, factor VIIa, and fibrinogen have been recorded in the late morning. Also, coagulation inhibitors such as protein C, antithrombin III, and a tissue factor pathway inhibitor are most active at this time of day. The levels of plasminogen and its activators (alpha 2-antiplasmin, urokinase-like plasminogen activator) do not undergo circadian periodicity, in contrast to changes in tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 levels (PAI-1). The most intensive activity of the fibrinolytic system inhibitor PAI-1 has been noted in the morning. The 24-h changes in the hemostatic system observed in healthy subjects often did not occur in individuals with health problems. The results of various studies suggest that circadian changes in the hemostatic system increase the incidence of cardiovascular events in the morning. This review considers the circadian rhythms of individual components involved in hemostasis (endothelium, platelets, coagulation, and fibrinolysis).

Membrane Regulation of EGFR Signaling by Gangliosides

Signal transduction is initiated at the cell membrane by the interaction of membrane-anchored receptors (such as growth factors or integrin) with extracellular stimuli (ligands). The availability of these receptors for their ligands and how the activated receptors triggers intracellular signaling cascades are regulated, at least in part, by cholesterol and sphingolipids of the plasma membrane. Gangliosides, sialylated glycosphingolipids, were first shown to regulate epidermal growth factor receptor (EGFR) signal transduction at the membrane level of epithelial cells in the mid-1980’s. Since that time gangliosides have been shown to influence the signaling of a variety of other growth factor receptors. Regulation of signaling by gangliosides involves ganglioside-induced shifts of signaling components at the membrane level that either promote or disrupt signaling complexes. To decrease phosphorylation of the EGFR, ganglioside GM3 has been shown to promote the formation of a complex in which the caveolin-1 and tetraspanin CD82-dependent association of PKC-α with EGFR leads to increased threonine phosphorylation of the EGFR and EGFR internalization, making it unavailable for ligand stimulation. The lack of available EGFR at the membrane may also impact EGFR crosstalk with other signaling pathways, such as those triggered by activation of integrins and the urokinase-like plasminogen activator receptor.

Real-Time Reverse-Transcription PCR to Quantify a Panel of 19 Genes in Breast Cancer: Relationships with Sentinel Lymph Node Invasion

At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0-T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2-4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERalpha, ERbeta), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35-40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.

Real-time reverse-transcription PCR to quantify a panel of 19 genes in breast cancer: relationships with sentinel lymph node invasion.

At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0-T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2-4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERalpha, ERbeta), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35-40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.

Activators of Plasminogen and the Progression of Small Abdominal Aortic Aneurysms

The aim of this study was to examine the role of activating pathways of plasminogen in the natural history of abdominal aortic aneurysms (AAA). To fulfill this objective 70 male patients with small AAA (> 3 cm) were interviewed and examined. Their blood samples were taken at diagnosis. The patients were scanned annually for a minimum period of 1 year and a maximum of 5 years (mean 2.5 years), and referred for surgery if the AAA exceeded 5 cm in diameter. Plasma levels of urokinase-like plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen-activator-inhibitor-1 (PAI-1), macrophage-inhibiting factor (MIF), transforming-growth-factor-beta1 (TGF-beta1), homocysteine, and serum levels of IgA-antibodies against Chlamydia pneumoniae (IgA-CP) and cotinine (a nicotine metabolite) were measured. The annual expansion rate correlated positively with tPA, IgA-CP, and S-cotinine; rho = 0.37 (P = 0.004), 0.28 (P = 0.01), and 0.24 (P = 0.04), while PAI-1, uPA, TGF-beta1, homocysteine, and MIF did not. S-cotinine and PAI-1 also correlated positively with tPA, rho = 0.24 (P = 0.04), and 0.33 (P = 0.005). IgA-CP did not correlate with tPA. By receiver operating characteristics (ROC) curve analysis, tPA showed to be predictive of cases expanding to above 5 cm within the first 5 years with an optimal sensitivity and specificity of 0.73 and 0.71, respectively (P = 0.015). The aortic matrix degradation in AAA may be partly caused by an activation of plasminogen by tPA, but not by uPA, which usually dominates matrix degradation. Smoking seems to be an important factor for this pathway, while the pathway of IgA-CP seems different.

Long-lived αMUPA transgenic mice exhibit pronounced circadian rhythms

Robust biological rhythms have been shown to affect life span. Biological clocks can be entrained by two feeding regimens, restricted feeding (RF) and caloric restriction (CR). RF restricts the time of food availability, whereas CR restricts the amount of calories with temporal food consumption. CR is known to retard aging and extend life span of animals via yet-unknown pathways. We hypothesize that resetting the biological clock could be one possible mechanism by which CR extends life span. Because it is experimentally difficult to uncouple calorie reduction from temporal food consumption, we took advantage of the murine urokinase-like plasminogen activator (alphaMUPA) transgenic mice overexpressing a serine protease implicated in brain development and plasticity; they exhibit spontaneously reduced eating and increased life span. Quantitative real-time PCR analysis revealed that alphaMUPA mice exhibit robust expression of the clock genes mPer1, mPer2, mClock, and mCry1 but not mBmal1 in the liver. We also found changes in the circadian amplitude and/or phase of clock-controlled output systems, such as feeding behavior, body temperature, and enteric cryptdin expression. A change in the light-dark regimen led to modified clock gene expression and abrogated circadian patterns of food intake in wild-type (WT) and alphaMUPA mice. Consequently, food consumption of WT mice increased, whereas that of alphaMUPA mice remained the same, indicating that reduced food intake occurs upstream and independently of the biological clock. Thus we surmise that CR could lead to pronounced and synchronized biological rhythms. Because the biological clock controls mitochondrial, hormonal, and physiological parameters, system synchronicity could lead to extended life span.