Hans Hölschermann

Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection.

Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.

In Situ Detection of Tissue Factor within the Coronary Intima in Rat Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy is a major cause of morbidity and mortality of cardiac transplant recipients. The underlying cause of this disease remains unclear. Histological studies have implicated accelerated hemostasis and intravascular fibrin deposition in its pathogenesis. In the present study a defined model of this disease in the rat was used to elucidate the implication of tissue factor in the production of the hypercoagulable state observed in cardiac allograft vessels. Tissue factor protein and mRNA expression were studied in rat heart allografts developing allograft vasculopathy resembling human disease. Immunohistochemistry demonstrated tissue-factor-positive cells present in the allograft coronary intima and adventitia. Significant staining for tissue factor was detected in the endothelium lining coronary lesions in cardiac allografts and in interstitial mononuclear cells, respectively. Both transplant coronary endothelial cells and mononuclear cells contained tissue factor mRNA as indicated by oligo-cell reverse transcription polymerase chain reaction after laser-assisted cell picking. In contrast, tissue factor mRNA and protein were not or negligibly detectable within the coronary intima of nontransplanted control hearts. Thus, the present study clearly demonstrates that aberrant tissue factor expression occurs within the coronary intima after cardiac transplantation. Tissue factor, activating downstream coagulation mechanisms, may account for the intravascular clotting abnormalities observed in cardiac allografts and may represent a key factor in transplant atherogenesis.

The expression of angiotensin-I converting enzyme in human atherosclerotic plaques is not related to the deletion/insertion polymorphism but to the risk of restenosis after coronary interventions

Plasma and tissue concentrations of the angiotensin-I converting enzyme (ACE) have been shown to be associated with the ACE insertion/deletion (I/D) polymorphism. The purpose of this study was to examine the relation of ACE levels in atherosclerotic plaques to the ACE I/D polymorphism and to restenosis after balloon angioplasty and directional atherectomy (DCA). The study included 104 patients who underwent DCA and received angiographic follow-up at 12 to 18 months. The amount of ACE protein in various morphologically defined plaque components (fibrous, atheromatous, and complicated lesions) of the atherectomy specimens was determined by qualitative and semiquantitative immunohistochemistry. ACE levels were related to the ACE genotype, to plaque morphology and to the risk of restenosis. Sequential staining revealed that pathologic ACE overexpression of the atherosclerotic lesions occurred in intimal smooth muscle cells, fibrocytes/fibroblasts and macrophage/foam cells. The ACE content of the whole plaques and of the single plaque components was not associated with the I/D polymorphism, but with restenosis after coronary interventions. In addition, ACE levels in the atherosclerotic lesions correlated with the severity of vessel wall damage. The ACE phenotype might serve as an indicator for the risk of restenosis after coronary interventions.

Hirudin Reduces Tissue Factor Expression and Attenuates Graft Arteriosclerosis in Rat Cardiac Allografts

BACKGROUND-Intravascular clotting has been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV). We previously identified the expression of tissue factor (TF), the primary cellular initiator of blood coagulation, within the coronary intima, which was associated with neointimal thickening. In the present study, the effect of recombinant hirudin on CAV was assessed in Lewis to Fisher rat heterotopic cardiac allografts. METHODS AND RESULTS-Transplant recipients were randomized to a control group (n=10) and a hirudin-treated group (n=12; 2 mg. kg(-1). d(-1) SC). Histological evaluations of rejection, CAV, and TF staining were performed 120 days after transplantation. No significant differences were observed between the 2 groups with respect to the degree of rejection. Hirudin significantly (P<0.05) suppressed the development of CAV in the graft microvessels, but it was less effective in large coronary arteries. Graft intimal cells, isolated by laser-assisted cell picking, showed a marked upregulation of TF gene transcription, which was prevented by hirudin (P<0.01). As demonstrated by immunohistochemistry and quantitative analyses of TF mRNA levels by real-time polymerase chain reaction, hirudin treatment resulted in a significant reduction of TF protein and mRNA expression (P<0.001). CONCLUSIONS-Treatment with hirudin in this rat cardiac transplant model inhibited TF expression and decreased neointimal hyperplasia. These results suggest that TF inhibition by hirudin, in addition to its direct effect on thrombin, may attenuate the hypercoagulable state and prevent the development of CAV at least in restricted sites of the graft coronary vasculature.

Gender differences in the outcome of cardiac interventions.

AbstractI. The actual data base on the decision–making process of indication for revascularization reveals that angiographic severity of coronary artery disease (CAD) is the primary determinant of referral to coronary interventional procedures. Several recent studies demonstrated that after an acute myocardial infarction, women undergo cardiac catheterization to a lesser extent than men. Data of the MITI study and of the Cooperative Cardiovascular Project suggested that during acute treatment of myocardial infarction a somewhat less aggressive therapy is performed in women as compared to men.II. With respect to sex–related differences in the early and late outcome after elective PCI, the main problem is the small, limited amount of data due to the lack of randomized clinical studies including a larger number of women. The vast majority of data was obtained in patients with PTCA and stents. All the older studies and registers until 1993 revealed a three times higher periprocedural complication rate and in–hospital mortality in women. In recent studies such as BARI, after successful PCI women have an excellent long–term prognosis comparable or even better than in men. III.1. Several studies on the effect of interventional strategies in patients with unstable angina or non–ST elevation myocardial infarction NSTEMI) revealed superiority of an early invasive versus a more conservative, noninvasive approach. However, the data of the FRISC II and RITA–3 trials indicated that an early intervention strategy resulted in a beneficial effect only in men which was not seen in women. On the other hand, two studies (e.g., the TACTICS–TIMI– 18 study) showed an improved outcome of women with acute coronary syndrome after early invasive therapy.III.2. In numerous investigations, a higher early mortality after acute ST elevation myocardial infarction (STEMI) has been observed in women compared to men.Although placebo–controlled randomized trials of thrombolytic therapy have demonstrated a 25–30% reduction in early mortality, in–hospital survival has remained consistently lower for women than men after thrombolytic reperfusion.– In our clinic, prospective studies on clinical events during the early phase (30 days) and during long–term follow–up for 4 years after direct (primary) PTCA for acute STEMI were performed in women. Data were obtained in 204 consecutive and unselected women; results in women were compared with those of 577 consecutive and unselected men who had undergone direct angiography/primary PTCA for acute STEMI in the same time span. PTCA of the infarct–related artery was equally successful in both sexes (women 95%, men 94%). In the group of patients with acute STEMI who had been treated with primary infarct PTCA, no difference of early (30 days) mortality was detected in women versus men. Total cumulative mortality during 4 years of follow–up was 12.5%, 14.5%, 18% and 23% in women, respectively, versus 9%, 10.5%, 12% and 15%, respectively, in men. The general trend for a higher postdischarge mortality in women became apparent after 3 years and reached significance after 4 years. After multivariate analysis, female gender was no independent risk factor of increased mortality.Thus, direct (primary) coronary angiography and PCI eliminate significant gender–specific differences in survival early after acute myocardial infarction. Long–term follow–up (4 years) also revealed no sex–related differences in mortality and cardiac morbidity after direct (primary) PCI for acute ST elevation myocardial infarction.ZusammenfassungI. Aus einigen Studien der letzten Jahre wurde der Schluss gezogen, dass diagnostische Untersuchungen bei Verdacht auf koronare Herzkrankheit und die nachfolgenden Behandlungsstrategien bei Frauen weniger aggressiv durchgeführt würden als bei Männern. Dies lässt sich anhand neuer Untersuchungen nicht in vollem Umfang aufrechterhalten:1. Der Schweregrad der koronaren Herzkrankheit stellt die Hauptdeterminante der Indikationsstellung dar; das Geschlecht spielt in der Regel keine wesentliche Rolle im Entscheidungsprozess der Indikation einer Koronarangiographie.2. Bei Frauen, die einer PTCA zugeführt werden, ist der Krankheitsprozess nicht weiter fortgeschritten als bei Männern.3. Hinsichtlich der Indikationsstellung zur Koronarangiographie bei Patientinnen nach akutem Myokardinfarkt finden sich in der Literatur widersprüchliche Daten: Frauen werden in geringerem Ausmaß als Männer nach abgelaufenem akuten ST–Elevations–Myokardinfarkt einer Herzkatheteruntersuchung unterzogen. Allerdings kann die niedrigere Anzahl an Herzkatheteruntersuchungen und interventionellen Eingriffen (PTCA, Stentimplantation etc.) bei Frauen nach abgelaufenem Myokardinfarkt auf Altersdifferenzen zurückgeführt werden.4. Daten der MITI–Studie und des Cooperative Cardiovascular Project legen nahe, dass der Geschlechtsunterschied bei der Frühletalität des akuten Myokardinfarkts auf einer niedrigeren Wahrscheinlichkeit einer akuten Koronarintervention bei Frauen beruht bzw. dass bei Frauen eine etwas weniger aggressive Therapie im Vergleich zu männlichen Patienten durchgeführt wird. Jedoch sind die Unterschiede bei den Behandlungsmaßnahmen sehr gering.II. Im Hinblick auf die Geschlechtsunterschiede bei elektiven interventionellen kardiologischen Eingriffen (bei stabiler Angina pectoris) ist das Hauptproblem das limitierte Datenmaterial, weil es an randomisierten klinischen Studien mangelt, die eine große Anzahl Frauen einschließen. Es zeigt sich:1. Bisher liegen nur Akut– und Langzeitergebnisse der PTCA vor. Repräsentative Daten größerer klinischer Studien über Geschlechtsunterschiede bei den Erfolgsraten neuerer interventioneller Verfahren sind kaum vorhanden.2. Alle älteren Studien und Register bis zum Jahre 1993 ergaben eine dreifach höhere periinterventionelle Komplikationsrate und Frühletaliät bei Frauen.3. Bei gleichzeitiger Berücksichtigung der Körperoberfläche jedoch ist das periinterventionelle Risiko der Frau nicht höher als dasjenige der Männer.4. Nach erfolgreicher elektiver PTCA/Stentimplantation besitzen Frauen eine exzellente Langzeitprognose, die mit derjenigen der männlichen Patienten vergleichbar oder sogar besser ist.III.1. In zahlreichen großen, prospektiven, multizentrischen Studien hinsichtlich des Effekts interventioneller Strategien bei Patienten mit tropo – nin–positivem Nicht–ST–Hebungs–Myokardinfarkt (NSTEMI; FRISC–II–, TACTICS–TIMI 18–, RITA–3–Studie) konnte die Überlegenheit einer frühinvasiven Strategie (frühzeitige Koronarangiographie und PCI innerhalb von 24–48 h) im Vergleich zu einem konservativen nicht–invasiven Vorgehen nachgewiesen werden. In der FRISC–II– und der RITA–3–Studie profitierte jedoch das weibliche Geschlecht nicht von einer frühinvasiven Strategie im Gegensatz zu männlichen Patienten. Andererseits konnte in der TACTICS–TIMI 18–Studie und in der prospektiven Beobachtungsstudie in Bad Krozingen auch für Frauen eine Überlegenheit der frühinvasiven, interventionellen Strategie bei akutem Nicht–ST–Hebungs– Myokardinfarkt dokumentiert werden.III.2. Zahlreiche Untersuchungen der 90er Jahre haben ergeben, dass Frauen nach akutem Myokardinfarkt eine höhere Frühletalität als Männer aufweisen. Unterschiede hinsichtlich Lebensalter, Begleiterkrankungen, Zeitintervall zwischen Symptombeginn und Klinikaufnahme sowie Therapiemodalitäten können z.T. die schlechtere Prognose der Patientinnen mit akutem ST–Elevations– Myokardinfarkt erklären. Im Alter < 50 Jahre überstieg die Frühletalität der Frauen diejenige der Männer um mehr als das Zweifache. Dieser Unterschied der Frühletalität wurde mit steigendem Alter geringer und war in der Altersklasse > 74 Jahre nicht mehr nachweisbar. Der wesentliche Nachteil früherer Studien über geschlechtsbezogene Unterschiede des klinischen Verlaufs während und nach akutem ST–Hebungs– Myokardinfarkt liegt in dem Fehlen einer spezifischen Revaskularisationstherapie bzw. in der Anwendung lediglich nichtinvasiver Maßnahmen zur Wiedereröffnung der verschlossenen Infarktarterie.– In zwei in unserer Klinik durchgeführten prospektiven Studien wurden klinische Ereignisse während der Frühphase (30 Tage) und im Langzeitverlauf (bis zu 4 Jahren) nach primärer Infarkt– PTCA bei insgesamt 204 konsekutiven und unselektierten Frauen sowie 577 Männern mit akutem ST–Hebungs–Myokardinfarkt untersucht. Die PTCA des Infarktgefäßes war bei 95% der Frauen und 94% der Männer erfolgreich. Bei den insgesamt 691 Patienten mit akuter Infarkt–PTCA fand sich kein Unterschied der Frühletalität zwischen beiden Geschlechtern. Nach Adjustierung für Alter, Gefäßerkrankung, Infarktlokalisation und linksventrikuläre Auswurffraktion fand sich sogar ein Trend zu einer besseren Prognose der in der Akutphase des Myokardinfarkts mittels direkter (primärer) PCI behandelten Frauen. Die kumulative Gesamtmortalität während der ersten 4 Jahre nach primärer Infarkt–PTCA war 12,5%, 14,5%, 18% und 23% bei Frauen bzw. 9%, 10,5%, 12% und 15% bei Männern (nichtsignifikante Unterschiede bis zum 3. Jahr, ab dem 4. Jahr signifikant höhere Spätmortalität bei Frauen; p < 0,05). Bei Multivarianzanalyse war das Geschlecht kein unabhängiger Risikofaktor.Schlussfolgerung: Mit Hilfe der direkten (primären), systematisch durchgeführten Koronarangiographie und PTCA/Stentimplantation können geschlechtsspezifische Unterschiede in der Kurzzeitprognose nach akutem ST–Elevations–Myokardinfarkt eliminiert werden. Auch im Langzeitverlauf bis zu 4 Jahren konnten keine geschlechtsspezifischen Unterschiede bezüglich der Mortalität und kardialen Morbidität dokumentiert werden.

Differential gene expression in activated monocyte-derived macrophages following binding of factor VIIa to tissue factor

Factor VIIa/tissue factor (FVIIa/TF) interaction has been reported to induce intracellular signalling in cells constitutively expressing TF, independently of downstream activation of the coagulation cascade. It is unknown, however, whether binding of FVII to its cofactor TF alters the gene expression profile in cells which inducible express TF under inflammatory conditions. To address this issue, gene expression patterns in cultured LPS-stimulated monocyte-derived macrophages with or without exposure to FVIIa were compared by cDNA macro-array analysis. Of the 1176 genes examined on the array, a small set of six genes (IL-6, IL-8,TNF-a, GRO-beta alpha-thymosin, cathepsin H) were consistently up-regulated and one gene suppressed (alpha-antitrypsin) in response to FVIIa in activated monocyte-derived macrophages. Among the seven genes identified by array analysis, five genes were finally confirmed by real-time RT-PCR. Interestingly, all of these genes differentially regulated in response to FVIIa (GRO-beta, IL-6, IL-8, TNF-alpha and alpha-antitrypsin) are critical in inflammation. The changes in gene expression were reflected by corresponding changes in the protein concentrations of IL-6 and IL-8 as demonstrated by ELISA. Active site-inhibited FVIIa had no effect on gene expression indicating that FVIIa-induced gene alteration is dependent on the proteolytic activity of FVIIa. The FVIIa-induced alterations in gene expression were found to be TF-dependent but independent of downstream coagulation proteins like thrombin and FXa. In summary, this study demonstrates that binding of FVIIa to its cofactor TF enhances restricted pro-inflammatory genes in activated monocyte-derived macrophages. By up-regulation of chemokines critical for leukocyte recruitment, FVIIa/TF interaction on activated monocyte-derived macrophages could be relevant to prepare monocytes/macrophages for extravasation and may represent a novel amplification loop of leukocyte recruitment.

Cyclosporin A Inhibits Monocyte Tissue Factor Activation in Cardiac Transplant Recipients

BACKGROUND Fibrin deposition and thrombosis have been implicated in both allograft rejection and vasculopathy after cardiac transplantation. Because monocytes play a pivotal role in the pathophysiology of intravascular coagulation activation through their ability to synthesize tissue factor (TF), we asked (1) whether monocyte TF activation occurs in cardiac transplant recipients and (2) whether monocyte TF expression is affected by treatment with cyclosporin A (CsA). METHODS AND RESULTS We measured levels of TF activity in peripheral blood mononuclear cells and highly purified monocytes/macrophages from 10 consecutive cardiac transplant recipients and 10 healthy control subjects. TF activity generated by both unstimulated and endotoxin-stimulated cells was significantly higher in transplant recipients than in control subjects (P<.05). Increased monocyte TF expression in transplant recipients was shown to be adversely affected by treatment with CsA: TF induction was markedly reduced by CsA serum concentrations reaching peak CsA drug levels. Inhibition of TF induction in the presence of high CsA blood concentrations was also observed when stimulation of cells was performed with interferon-gamma or interleukin-1beta. As shown by reverse transcription-polymerase chain reaction and electrophoretic mobility shift assay, respectively, treatment with CsA leads to decreased TF mRNA expression and reduced activation of the NF-kappaB transcription factor, which is known to contribute to the induction of the TF promotor in human monocytes. CONCLUSIONS This study demonstrates that TF activation, occurring in mononuclear cells of cardiac transplant recipients, is inhibited by treatment with CsA. Inhibition of monocyte TF induction by CsA may contribute to its successful use in cardiac transplant medicine and might be useful in managing further settings of vascular pathology also known to involve TF expression and NF-kappaB activation.

Opposite Regulation of Tissue Factor Expression by Calcineurin in Monocytes and Endothelial Cells

Tissue factor (TF), the primary initiator of blood coagulation with structural homology to the cytokine receptor family, has been implicated in various vascular processes including metastasis, angiogenesis, and atherosclerosis. Within the vasculature, monocytes and endothelial cells (EC) can be activated to synthesize TF depending on the induction of NF-κB. Despite the undisputed value of cyclosporin A (CsA) as an immunosuppressant, problems have emerged due to induction of vascular changes by a poorly understood mechanism. We demonstrate that CsA has opposite effects on TF gene expression, inhibiting NF-κB-mediated TF gene transcription in monocytes but enhancing it in EC. To test whether CsA binding proteins (cyclophilins) can mediate these CsA effects we used a nonimmunosuppressant analog of CsA that binds to cyclophilins but does not inhibit the Ca2+/calmodulin-dependent phosphatase calcineurin (Cn). This drug lacked regulatory function for NF-κB and TF expression suggesting that Cn is responsible for the inverse gene regulation. The key function of Cn was supported by experiments demonstrating that other phosphatase inhibitors also either positively or negatively regulated NF-κB in monocytes and EC. Calcineurin was demonstrated to regulate NF-κB activation at the level of IκBα degradation, because agonist-induced phosphorylation and subsequent degradation of IκBα is prevented by Cn inhibitors in monocytes but enhanced in EC. These data identify Cn as an opposite regulator in generating transcriptionally active NF-κB, and they confirm the presumption that the ability of Cn to participate in NF-κB transactivation is not T cell specific.

AP-1 and STAT-1 decoy oligodeoxynucleotides attenuate transplant vasculopathy in rat cardiac allografts

AIMS Cardiac allograft vasculopathy (CAV) continues to be an unsolved clinical problem requiring the development of new therapeutic strategies. We have previously demonstrated that ex vivo donor allograft treatment with decoy oligodeoxynucleotides (ODN) targeting the transcription factors, activator protein-1 (AP-1) or signal transducer and activator of transcription-1 (STAT-1), delays acute rejection and prolongs cardiac allograft survival. Here, we investigated whether this treatment regime also prevents the occurrence of CAV in a fully allogeneic rat heart transplantation model. METHODS AND RESULTS Wistar-Furth rat cardiac allografts were perfused ex vivo with AP-1 decoy ODN, STAT-1 decoy ODN, or buffer solution and transplanted into the abdomen of Lewis rats immunosuppressed with cyclosporine. Treatment with both decoy ODNs but not vehicle significantly attenuated the incidence and severity of CAV. Laser-assisted microdissection/real-time polymerase chain reaction as well as immunohistochemistry analyses revealed a significant increase in CD40 abundance in the coronary endothelial cells and medial smooth muscle cells on day 1 post transplantation which was virtually abolished upon AP-1 or STAT-1 decoy ODN treatment. While the AP-1 decoy ODN primarily attenuated basal CD40 expression, the STAT-1 decoy ODN suppressed tumour necrosis factor-alpha-/interferon-gamma-stimulated expression of CD40 in rat native endothelial cells. CONCLUSION Treating donor hearts with decoy ODNs neutralizing AP-1 or STAT-1 at the time of transplantation prevents upregulation of CD40 expression in the graft coronary arteries and effectively inhibits CAV.

Factor Seven Activating Protease (FSAP) levels during normal pregnancy and in women using oral contraceptives

INTRODUCTION Factor seven activating protease (FSAP) is a plasma serine protease involved in haemostasis and remodeling processes. We have investigated whether pregnancy or the use of oral contraceptives (OCs) influences circulating FSAP levels. The effect of female sex hormones on FSAP expression in cultured cells was also determined. MATERIALS AND METHODS FSAP levels and activity was measured in plasma samples obtained at different gestation stages from healthy pregnant women (n=101), from non-pregnant women, pre-menopausal women who currently use OCs (n=48), and non-pregnant women who did not use OCs (n=69). RESULTS In late pregnancy the plasma FSAP antigen (median 2.28 PEU/ml [range 1.11 to 2.62 PEU/ml]; p<0.001 vs control group) and activity (median 2.98 PEU/ml [range 1.05 to 4.24 PEU/ml]; p<0.001 vs control group) was significantly higher compared with levels in non-pregnant women and remained elevated after delivery. Plasma FSAP levels in women using OCs was also significantly elevated compared to the control group. Ex vivo experiments demonstrated enhanced FSAP expression in monocytes isolated from women using OCs. In vitro experiments showed that FSAP mRNA levels were strongly induced by estradiol in monocytes but not in hepatocytes. CONCLUSIONS Increased levels of circulating FSAP in pregnancy and in women using OCs indicate that hormonal status critically influences FSAP expression. Hormonal influences could be observed in monocytes in vivo and ex-vivo but not in hepatocytes indicating cell-specific regulation. Future studies designed to investigate the role of FSAP in haemostasis and remodeling processes should consider the role of female sex hormones on FSAP expression.