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SummaryLittle is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development.

SummaryKallikrein-related peptidases (KLK), which represent a major tissue-associated proteolytic system, stand for a rich source of biomarkers that may allow molecular classification, early diagnosis and prognosis of human malignancies as well as prediction of response or failure to cancer-directed drugs. International research points to an important role of certain KLKs in female and male urogenital tract malignancies, in addition to cancers of the lung, brain, skin, head and neck, and the gastrointestinal tract. Regarding the female/male urogenital tract, remarkably, all of the KLKs are expressed in the normal prostate, testis, and kidney whereas the uterus, the ovary, and the urinary bladder are expressing a limited number of KLKs only. Most of the information regarding KLK expression in tumour-affected organs is available for ovarian cancer; all of the 12 KLKs tested so far were found to be elevated in the malignant state, depicting them as valuable biomarkers to distinguish between the normal and the cancerous phenotype. In contrast, for kidney cancer, a series of KLKs was found to be downregulated, while other KLKs were not expressed. Evidently, depending on the type of cancer or cancer stage, individual KLKs may show characteristics of a Janus-faced behaviour, by either expanding or inhibiting cancer progression and metastasis.

SummaryUrokinase-type plasminogen activator (uPA) and its inhibitor, PAI-1, play a key role in tumor invasion and metastasis. They were the first novel tumor biological factors to be validated at the highest level of evidence (LOE I) regarding their clinical utility in breast cancer. Their antigen levels are determined in tumor tissue extracts by standardized, quality-assured immunometric assays (ELISA). Since the late 1980s, numerous independent studies have demonstrated that patients with low levels of uPA and PAI-1 in their primary tumor tissue have a significantly better survival than patients with high levels of either factor. These prognostic data have recently been validated by an EORTC (European Organization for Research and Treatment of Cancer) pooled analysis comprising more than 8,000 breast cancer patients. In addition, results from a multicenter prospective randomized therapy trial in node-negative breast cancer (“Chemo N0”) showed that node-negative breast cancer patients with low levels of uPA and PAI-1 in their primary tumor have a very good prognosis, and may thus be candidates for being spared the burden of adjuvant chemotherapy. In contrast, node-negative patients with high uPA/PAI-1 are at substantially increased risk of disease recurrence, comparable to that of patients with three or more tumor cell positive axillary lymph nodes. The “Chemo N0” trial as well as retrospective data also indicate that these high-risk patients benefit from adjuvant chemotherapy. In conclusion, over a period of about 15 years sufficient evidence has been put forward to demonstrate that determination of uPA and PAI-1 in primary breast cancer patients supports risk-adapted individualized therapy decisions, particularily in patients with node-negative disease.

SummaryThe cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-specific monoclonal antibodies against uPAR. For quantification of uPAR mRNA variants, we established two highly sensitive real-time RT-PCR assays based on LightCycler technology. Study of their expression in a representative set of breast cancer tissues indicated that the novel mRNA variant uPAR-del4/5 was expressed very frequently and independently of uPAR mRNA variants covering exon 4 (uPAR-wt and uPAR-del5). Higher uPAR-del4/5 expression was significantly associated with shorter disease-free survival (p = 0.0004) of breast cancer patients. These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.

Thromb Haemost 2009; 101: 222–224 The scientist Emil-Karl Frey, a scholar of the famous surgeon ‘Geheimrat’ Ferdinand Sauerbruch, observed in 1925 a considerable reduction in arterial blood pressure when he injected human urine into dogs, an until then unknown cardioactive and vasoactive effect which he attributed to an unspecified substance with potential biological functions (1, 2). This then uncharacterized kininogenase was named kallikrein (Greek synonym for pancreas: kallikreas) by the three German scientists H. Kraut, E.-K. Frey and E. Werle, who in 1930 reported that the pancreas is a rich source of this endogenous hypotensive substance (3). A few years later, E.-K. Werle identified kallikrein as a proteolytic enzyme (KLK1 [EC 3.4.21.35] initially named tissue kallikrein, glandular kallikrein, kallikrein 1 or hk1) that liberates the biologically highly active, basic polypeptide ‘DK’ or kallidin (i.e. lys-bradykinin) from the blood plasma protein kallidinogen (now termed highand low-molecular-weight kininogen, HMWKand LMWK-kininogen) (4). The serine protease KLK1 is different from the later discovered plasma serine protease kallikrein (KLKB1 or Fletcher factor [EC 3.4.21.34], located on chromosome 4q34-q35) which is part of the plasma contact activation system and structurally related to coagulation factor XI (5), whereas KLK1 located on chromosome 19q13.4 is structurally related to the serine protease trypsin (5–7). Later on, two more glandular kallikreins were identified in humans: KLK2 (initially named human glandular kallikrein 1, hGK1, and later human kallikrein 2, hK2) (8) and KLK3 (also known as prostate-specific antigen [PSA]) (9, 10), both located on chromosome 19q13.4 as well (6). Some time later, before the turn of the millenium, 12 additional serine protease genes were independently discovered by three major research groups to be tandemly located on chromosome 19q13.4 and therefore assigned to the glandular kallikrein family as well, based on their localization to chromosome locus 19q13.4 and sequence/structural similarities with the three traditional glandular kallikrein genes (11–13), thus forming a new, “extended” kallikrein gene family (6, 7). Hence, a new comprehensive nomenclature was needed, and since 2006 of the 15 tissue-related kallikreins 14 are denoted kallikrein-related peptidases (KLK2 to KLK15) (14), except for kallikrein 1, since this is the only serine protease in the KLK-family with significant kininogenase activity (15). Now, with the comprehensive description of the human KLK gene locus available, one of the main bio-medical research endeavors is focused on the clarification of the (patho)physiological functions of the KLK (16). KLK are expressed in a wide range of tissues, although individual KLK do have quite restricted expression patterns suggesting a functional role in diverse (patho)physiological processes (17), e.g. skin desquamation and other skin diseases, tooth development and enamel defects, Alzheimer’s disease, Parkinson’s disease, and cancer (6, 15). Besides KLK3 (PSA), a well known biomarker for prostate cancer, other members of the KLK family are now considered as potential biomarkers in malignant disease states as well. Recently, several of the kallikrein-related peptidases have shown promise as prognostic and predictive cancer biomarkers, e.g. for cancer of the prostate, testis, kidney, breast, ovary, lung, colon, pancreas, and brain (6, 18). Interestingly, some of the KLK can auto-activate, while others activate each other, suggesting that the KLK may be part of an enzymatic cascade similar to the urokinase/plasminogen system or the matrix metalloprotease family, which are associated with the malignant potential of multiple malignant diseases (19, 20). Currently, three well-established technologies are in use to quantify kallikrein-related peptidase expression: RT-PCR (mRNA), ELISA (protein) and DNA-methylation (epigenetic modification of CpG islands in genomic DNA) (6, 21). From these assays, it can be assumed that certain members of the kallikrein-related peptidase gene family are differentially expressed in a wide variety of carcinomas. Although still under investigation, it has been shown that these factors can serve as new biomarkers for diagnosis, prognosis, and monitoring of cancer (6, 7, 15, 16). In Figure 1A the number of citations per year are displayed with reference to KLK3 (PSA) and cancer, encompassing investigations in human and animal tissues and cell lines, respectively. Between 1982 and 2008, 12,135 articles were published in MedLine including 1,631 reviews. Most of the experiments were conducted with human tumor tissues, tumor cell lines or blood components (12,047); 1,523 considering clinical trials. Only a small portion of the published articles dealing with the clinical impact of kallikrein-related peptidases (556, includ© 2009 Schattauer GmbH, Stuttgart

Extravasation and intravasation of solid malignant tumors is controlled by attachment of tumor cells to components of the basement membrane and the extracellular matrix, by local proteolysis and tumor cell migration. Strong clinical and experimental evidence has accumulated that the tumor-associated serine protease plasmin, its activator uPA (urokinase-type plasminogen activator), the receptor uPA-R (CD87), and the inhibitors PAI-1 and PAI-2 are linked to cancer invasion and metastasis. In cancer, increase of uPA, uPA-R, and/or PAI-1 is associated with tumor progression and with shortened disease-free and/or overall survival in patients afflicted with malignant solid tumors. uPA and/or its inhibitor PAI-1 appear to be one of the strongest prognostic markers so far described. Strong prognostic value to predict disease recurrence and overall survival has been documented for patients with cancer of the breast, ovary, cervix, endometrium, stomach, colon, lung, bladder, kidney, brain, and soft-tissue. Due to the strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the tumor invasion/ metastasis capacity of cancer cells, proteolytic factors have been selected as targets for therapy. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, enzyme inhibitors, and recombinant or synthetic uPA and uPA-R analogues.

Theme Issue: Update on the role of the fibrinolysis/ plasminogen activation system in a cellular context

Various parameters associated with fibrinolysis were examined in plasma and placenta tissue extract in 18 patients with HELLP syndrome (haemolysis, elevated liver enzymes, and low platelet count). A significant increase in plasma concentrations of tPA and PAl-1 was observed in the HELLP patients in comparison to 18 pregnant women of the control group (equal duration of pregnancy). In contrast, there was no difference in plasma concentrations of uPA, uPA-receptor and D-dimer. In placenta tissue extract, significant differences were found just as rarely for uPA, uPA-receptor and PAl-1 as for tPA and D-dimer. Our findings indicate, that endothelial damage with release of tPA may be involved in the pathophysiological pathway of HELLP syndrome. Increased plasma levels of PAl-1 may reflect deficient fibrinolysis resulting in impairment in microcirculation. Clinical relevance of tPA and PAl-1 plasma concentration as possible predictor of hypertensive pregnancy complications will have to be studied further.

Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.

A 31-year-old primigravida at 24 2/7 weeks of gestation presented with a 2-day history of diarrhoea and vomiting. On admission, her blood pressure was 160/100 mm Hg, increased for the first time during her pregnancy, but she had no further symptoms of preeclampsia such as proteinuria, oedema, headache, visual disturbance, hyperreflexia or right upper quadrant pain, and no personal or family history of chronic hypertension, renal disease or thromboembolic events. Laboratory findings were normal. Ultrasonography showed a reduced amniotic fluid index of 10 and a growth retarded fetus (below the tenth percentile for the expected weight). Doppler ultrasonography revealed an increased vascular resistance in the uterine arteries and . . .

F rom May 16-20, 2004, 60 scientists from 12 countries convened at the Certosa di Pontignano in Tuscany, Italy, for the Second Chianti Meeting on Proteases, “Cross-talk between Proteases and the Extracellular Environment”. Following the event, this issue of Thrombosis and Haemostasis presents a series of original contributions and review articles presented by some of the speakers at the Chianti Meeting. . . .