Subclinical involvement of the liver is associated with prognosis in treatment naïve cancer patients.

BACKGROUND: Routinely tested liver biomarkers as alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), butyryl-cholinesterase (BChE), albumin and bilirubin are altered in distinct malignancies and hepatic metastases. This study aimed to investigate whether all liver parameters have the ability to predict long-term mortality in treatment naïve cancer patients but without a malignant hepatic involvement. METHODS: We prospectively enrolled 555 consecutive patients with primary diagnosis of cancer without prior anticancer therapy. BChE, albumin, AST, ALT, GGT and bilirubin as well as the inflammatory makers C-reactive protein (CRP), serum amyloid A (SAA) and interleukin-6 (IL-6) were determined. All-cause mortality was defined as primary endpoint. RESULTS: During a median follow-up of 25 (IQR16-31) months 186 (34%) patients died. All liver parameters were significantly associated with all-cause mortality (p < 0.001 for all). However, for patients without a malignant primary or secondary hepatic involvement (82%) only the functional parameters BChE and albumin remained significantly associated with the primary endpoint (crude HR per 1-IQR increase 0.61, 95%CI:0.49-0.77; p < 0.001 for BChE and 0.58, 95%CI:0.47-0.70; p < 0.001 for albumin). This e ect was persistent after multivariate adjustment (adj.HR per 1-IQR increase 0.65, 95%CI:0.50-0.86; p = 0.002 for BChE and 0.63, 95%CI:0.50-0.79; p < 0.001 for albumin). BChE and albumin correlated inversely with CRP (r = -0.21, p < 0.001 and r = -0.36, p < 0.001), SAA (r = -0.19, p < 0.001 and r = -0.33, p < 0.001) and IL-6 (r = -0.13, p = 0.009 and r = -0.17, p = 0.001). CONCLUSIONS: Decreased serum BChE and albumin levels are associated with increased all-cause mortality in treatment-naïve cancer patients without a manifest malignant hepatic involvement irrespective of tumor entity or stage. This association may reflect progressing systemic inflammation and metabolic derangement with subclinical involvement of the liver.

GDF-15 Is Associated with Cancer Incidence in Patients with Type 2 Diabetes.

BACKGROUND: Diabetes has been linked epidemiologically to increased cancer incidence and mortality. Growth differentiation factor 15 (GDF-15) is increased in patients with diabetes and has recently been linked to the occurrence of cancer. We investigated whether circulating GDF-15 concentrations can predict the incidence of malignant diseases in a diabetic patient cohort already facing increased risk for cancer. METHODS: We prospectively enrolled a total of 919 patients with type 2 diabetes and no history of malignant disease, who were clinically followed up for 60 months. GDF-15, N-terminal pro-B-type natriuretic peptide and troponin T were measured at baseline; an additional 4 cardiovascular biomarkers were determined for a subpopulation (n = 259). Study end point was defined as the first diagnosis of any type of cancer during the follow-up period. RESULTS: During a median follow-up of 60 months, 66 patients (7.2%) were diagnosed with cancer. Baseline circulating GDF-15 concentrations were higher in patients that developed cancer over the follow-up period when compared to cancer-free patients. Increased GDF-15 concentrations were significantly associated with cancer incidence [crude hazard ratio (HR) per 1-IQR (interquartile range) increase 2.13, 95% CI 1.53-2.97, P < 0.001]. This effect persisted after multivariate adjustment with an adjusted HR of 1.86 (95% CI 1.22-2.84; P = 0.004). Among the 4 additionally tested cardiovascular markers in the subpopulation, only troponin T and C-terminal proendothelin-1 showed a significant association with future cancer incidence with unadjusted HRs of 1.71 (95% CI 1.28-2.28, P < 0.001) and 1.68 (95% CI 1.02-2.76, P = 0.042), respectively. CONCLUSIONS: Increased circulating concentrations of GDF-15 are associated with increased cancer incidence in patients with type 2 diabetes.

Cardiovascular safety of metformin and sulfonylureas in patients with different cardiac risk profiles.

OBJECTIVES/BACKGROUND: Based on previous experiences, the Food and Drug Administration and the European Medicines Agency recommend that clinical trials for novel antidiabetic drugs are powered to detect increased cardiovascular risk. In this context, data concerning licensed drugs such as metformin and sulfonylureas are conflicting. The influence of baseline cardiovascular risk on any treatment effect appears obvious but has not been formally proven. We therefore evaluated association of metformin and sulfonylureas with cardiovascular events in patients with different cardiovascular risk profiles indicated by N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) levels. METHODS: 2024 patients with diabetes mellitus were included in this observational study. The primary endpoint was defined as a combination of cardiovascular events and death. Association of metformin and sulfonylureas was assessed using Cox regression models. Possible differences of these associations in patients with different NT-proBNP levels were studied by stratifying and through interaction analysis. RESULTS: During a median follow-up of 60 months, the primary endpoint occurred in 522 (26%) of patients. The median age was 63 years. A Cox regression analysis was adjusted for site of treatment, concomitant medication, age, gender, body mass index, glycated haemoglobin, duration of diabetes, glomerular filtration rate, cholesterol, and history of smoking and cardiac disease. Metformin was associated with a decreased risk in the cohort with elevated NT-proBNP ≥300 pg/mL (HR 0.70, p=0.014) and a similar association was found for the interaction between metformin and NT-proBNP (p=0.001). There was neither an association for sulfonylureas nor a significant interaction between sulfonylureas and NT-proBNP. CONCLUSIONS: Metformin is associated with beneficial cardiovascular outcomes in patients with diabetes only when (sub)clinical cardiovascular risk defined by NT-proBNP levels is present.

Cardiovascular biomarkers in patients with cancer and their association with all-cause mortality.

OBJECTIVE: Patients with cancer may display elevated levels of B-type natriuretic peptide (BNP) and high-sensitive troponin T (hsTnT) without clinical manifestation of cardiac disease. This study aimed to evaluate circulating cardiovascular hormones and hsTnT and their association with mortality in cancer. METHODS: We prospectively enrolled 555 consecutive patients with a primary diagnosis of cancer and without prior cardiotoxic anticancer therapy. N-terminal pro BNP (NT-proBNP), mid-regional pro-atrial natriuretic peptide (MR-proANP), mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-endothelin-1 (CT-proET-1), copeptin, hsTnT, proinflammatory markers interleukin 6 (IL-6) and C reactive protein (CRP), and cytokines serum amyloid A (SAA), haptoglobin and fibronectin were measured. All-cause mortality was defined as primary endpoint. RESULTS: During a median follow-up of 25 (IQR 16-31) months, 186 (34%) patients died. All cardiovascular hormones and hsTnT levels rose with tumour stage progression. All markers were significant predictors of mortality with HRs per IQR of 1.54 (95% CI 1.24 to 1.90, p<0.001) for NT-proBNP, 1.40 (95% CI 1.10 to 1.79, p<0.01) for MR-proANP, 1.31 (95% CI 1.19 to 1.44, p<0.001) for MR-proADM, 1.21 (95% CI 1.14 to 1.30, p<0.001) for CT-proET-1, 1.22 (95% CI 1.04 to 1.42, p=0.014) for copeptin and 1.21 (95% CI 1.13 to 1.32, p<0.001) for hsTnT, independent of age, gender, tumour entity and stage, and presence of cardiac comorbidities. NT-proBNP, MR-proANP, MR-proADM and hsTnT displayed a significant correlation with IL-6 and CRP. CONCLUSIONS: Circulating levels of cardiovascular peptides like NT-proBNP, MR-proANP, MR-proADM, CT-pro-ET-1 and hsTnT were elevated in an unselected population of patients with cancer prior to induction of any cardiotoxic anticancer therapy. The aforementioned markers and copeptin were strongly related to all-cause mortality, suggesting the presence of subclinical functional and morphological myocardial damage directly linked to disease progression.

Association of endostatin with mortality in patients with chronic heart failure.

BACKGROUND: Experimental data imply that in decompensated heart failure (HF), the anti-angiogenic factor endostatin is increased. This study aimed to investigate whether the angiogenesis inhibitor endostatin is related to the risk of all-cause mortality in a prospective cohort study of chronic HF patients. METHODS: In this prospective observational cohort study, endostatin serum concentrations were determined in patients with chronic HF. Mortality data were recorded during a median follow-up of 31 months. RESULTS: One fifty one patients were included. The overall mortality rate was 20%. Baseline endostatin concentrations > 245 ng/mL were associated with higher risk of all-cause mortality [HR 8·7 (95% CI 2·5-30·0); P = 0·001] in the multivariate analysis as compared to endostatin concentrations ≤ 245 ng/mL. When both endostatin and NT-proBNP were above the calculated cut-off of 245 ng/mL and 2386 pg/mL, respectively, the prognostic utility of both biomarkers increased [HR 40·8 (95% CI 4·7-354·6); P = 0·001] compared with values lower than the cut-offs. CONCLUSIONS: Serum endostatin concentrations are independently associated with all-cause mortality. Furthermore, combination of endostatin and NT-proBNP discriminates patients at high risk.

Estimated glomerular filtration rate and albuminuria: true predictors of cardiovascular events in obese patients with type 2 diabetes?

BACKGROUND: The widely used MDRD formula underestimates kidney function in obese patients with diabetes mellitus. Therefore we aimed to evaluate the predictive value of estimated glomerular filtration rate (eGFR) for cardiovascular events in a typical cohort of patients with diabetes mellitus. METHODS: A total of 988 patients were analyzed. Cox regression models including the variables HbA1c, age, duration of diabetes, eGFR and urinary albumin to creatinine ratio (UACR) were run. First the whole collective was analyzed, in a second step the cohort was split into four different groups according to body mass index (BMI) and eGFR (Group 1, 475 Pts: eGFR > 60 ml/min; BMI < 30 kg/m(2), Group 2, 274 Pts: eGFR > 60 ml/min;BMI > 30 kg/m(2), Group 3, 110 Pts,: eGFR < 60 ml/min; BMI > 30 kg/m(2) and Group 4, 129 Pts.: eGFR < 60 ml/min;BMI < 30 kg/m(2)). eGFR was calculated using MDRD, Cockroft-Gault, and CKD-EPI formula. The endpoint was defined as unplanned cardiovascular hospitalization. RESULTS: Patients (571 male, 417 female) were 61 ± 22 years of age, mean duration of diabetes was 14.3 ± 12.3 years. After a median follow-up of 29 months 95 (9.6 %) patients reached the defined endpoint. The first model, including all patients showed that UACR (HR 1.001, p < 0.001) and eGFR (HR 0.957, p < 0.001) were significant predictors of the composite endpoint. In obese patients eGFR completely lost its predictive value for cardiovascular events. The prevalence of normoalbuminuria in patients with an eGFR below 60 ml/min was 59.4 %. CONCLUSION: In obese patients eGFR is not predictive for cardiovascular events.

A multi-biomarker risk score improves prediction of long-term mortality in patients with advanced heart failure.

BACKGROUND: Accurate risk prediction is important for an adequate management of heart failure (HF) patients. We assessed the incremental prognostic ability of a multi-biomarker approach in advanced HF. METHODS: In 349 patients with advanced HF (median 75 years, 66% male) we investigated the incremental prognostic value of 12 novel biomarkers involved in different pathophysiological pathways including inflammation, immunological activation, oxidative stress, cell growth, remodeling, angiogenesis and apoptosis. RESULTS: During a median follow-up of 4.9 years 55.9% of patients died. Using multivariable Cox regression and bootstrap variable-selection age, chronic obstructive pulmonary disease, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and the following 5 novel biomarkers were retained in the best mortality prediction model: the chemokine fractalkine, the angiogenic and mitogenic hepatocyte growth factor (HGF), the growth differentiation factor 15 (GDF-15) influencing cardiac remodeling and apoptosis, and the 2 pro-apoptotic molecules soluble apoptosis-stimulating fragment (sFAS) and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). This multi-biomarker score had strong discriminatory power for 5-year mortality (area under the Receiver Operating Characteristic curve [AUC]=0.81) and improved risk prediction beyond the prognostic power of a comprehensive conventional risk score including known clinical predictors and NT-proBNP (AUC=0.77). Net reclassification confirmed a significant improvement of individual risk prediction (p=0.003). CONCLUSIONS: Risk prediction by a multi-biomarker score is superior to a conventional risk score including clinical parameters and NT-proBNP. Additional predictive information from different biological pathways reflects the multisystemic character of HF.

NT-proBNP as a means of triage for the risk of hospitalisation in primary care.

BACKGROUND: In primary care, identification of patients who are at risk of major adverse events is of great importance. At the same time identifying individuals who are at very low risk and do not need further diagnostic workup and therapy is also important and may help to correctly allocate scarce healthcare resources. AIM: This study evaluated amino-terminal pro B-natriuretic peptide (NT-proBNP) as a risk marker in primary care patients with hypertension, diabetes, clinically suspected heart failure (HF), history of coronary artery disease or myocardial infarction. METHODS AND RESULTS: A prospective observational study was conducted in 1203 primary care patients. The primary endpoint, time to all-cause hospitalisation, was reached in 282 (24%) individuals within 12 months. Of all variables analysed, only NT-proBNP (HR 1.001 [1.000-1.001], p < 0.001) and age (HR 1.018 [1.007-1.028], p = 0.001) were of independent predictive value in a stepwise Cox regression analysis regarding all-cause hospitalisation. Neither systolic dysfunction nor signs and symptoms of HF added independent information to predict outcome. The negative predictive value (NPV) increased depending on the specificity of the endpoint (NPV was 86% for all-cause, 98% for cardiac and 100% for HF-related hospitalisation for 125 pg/ml). Positive predictive value and NPV were superior for NT-proBNP compared to clinical signs and symptoms of HF at every cut-point between 100 and 500 pg/ml. CONCLUSION: NT-proBNP levels predicted clinical events in primary care patients at risk. NPVs were excellent in this high risk population, proving NT-proBNP measurement a safe diagnostic tool.

Differences in the predictive value of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in advanced ischemic and non-ischemic heart failure.

OBJECTIVE: To assess the prognostic value of the pro-apoptotic, but also cell growth-inducing molecule soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) in heart failure (HF). METHODS: We assayed sTWEAK levels in 351 patients with advanced HF (non-ischemic: 130, ischemic: 221). During a median follow-up of 4.9 years, 195 patients (56%) died. RESULTS: sTWEAK concentrations were associated with extended survival in patients with non-ischemic (P=0.022), but not with ischemic HF (P=0.82). The inverse association in non-ischemic HF remained significant in a multivariable Cox regression model (P=0.025) with a hazard ratio of 0.40 (95% confidence interval: 0.21-0.77) comparing the third to the first tertile (P=0.007). CONCLUSION: Low sTWEAK levels independently predict mortality in advanced non-ischemic HF. sTWEAK-induced proliferation of cardiomyocytes may explain its impact on suvival. The different prognostic value of sTWEAK in ischemic and non-ischemic HF may point towards distinct pathogenic pathways determining the course of disease.

Prognostic value of pigment epithelium-derived factor in patients with advanced heart failure.

OBJECTIVE: Whereas angiogenesis, the formation of new blood vessels from preexisting vessels, may be beneficial in restoring failing myocardium, apoptosis may contribute to the progression of heart failure (HF). We investigated the role of pigment epithelium-derived factor (PEDF), a recently discovered antiangiogenic factor with additional proapoptotic effects, in patients with advanced HF. METHODS: We assayed PEDF levels in 351 patients with advanced HF at baseline. During the median follow-up time of 16 months, 50% of patients experienced the composite end point of rehospitalization and/or death. RESULTS: The risk of a clinical event increased with concentrations of the antiangiogenic marker PEDF, with a 1.94-fold higher risk in the third tertile compared with the first tertile (95% CI, 1.33-2.84). This association remained significant after adjustment for B-type natriuretic peptide (BNP) and other risk factors in a Cox regression model (P = .015). Experimental data revealed that PEDF may contribute to the progression of HF by inducing apoptosis in human cardiac myocytes and fibroblasts via activation of caspase 3. CONCLUSIONS: We suggest a role of PEDF in the progression of HF by inducing apoptosis of human cardiac myocytes and fibroblasts. Our clinical data suggest that PEDF concentrations may have the potential to become a valuable marker of the prognosis of HF, in addition to BNP.

Prognostic value of apoptosis markers in advanced heart failure patients.

AIMS: Apoptosis plays an important role in the progression of heart failure (HF). The purpose of this study was to assess whether the pro-apoptotic molecules apoptosis-stimulating fragment (FAS, CD95/APO-1) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) predict event-free survival of HF patients. METHODS AND RESULTS: We assayed soluble (s)FAS and sTRAIL levels in 351 patients with advanced HF. During the median follow-up time of 16 months, 175 patients (50%) experienced the composite endpoints: rehospitalization and death. The hazard increased with sFAS concentrations, with a hazard ratio of 2.3 comparing fourth and first quartiles. This association remained significant after adjustment for B-type natriuretic peptide (BNP) and other risk factors in a Cox regression model (P = 0.014). Patients with high sFAS but low BNP had a comparable event-free survival rate with those with elevated BNP only (P = 0.78). Conversely, high sTRAIL concentrations were related to a better prognosis. Particularly, the risk of mortality dropped by 70% in the fourth quartile of sTRAIL (P = 0.001, multivariable Cox regression model). CONCLUSION: sFAS is an independent risk predictor in advanced HF patients. It may be of particular value for the identification of high-risk patients in addition to BNP. Conversely, sTRAIL appears to be protective and could be an interesting therapeutic agent.

NT-proBNP has a high negative predictive value to rule-out short-term cardiovascular events in patients with diabetes mellitus.

AIMS: This study evaluated the predictive value of NT-proBNP for patients with diabetes mellitus and compared the prognostic aptitude of this neurohumoral marker to traditional markers of cardiovascular events. METHODS AND RESULTS: A prospective observational study was conducted in 631 diabetic patients. The composite endpoint consisted of unplanned hospitalization for cardiovascular events or death within the observation period of 12 months. Of all variables analysed (age, gender, history of hypertension, ischaemic heart disease/any cardiac disease, smoking, duration of diabetes, body mass index, blood pressure, New York Heart Association-class, Dyspnoea score, Minnesota Living with Heart Failure Questionnaire, LDL-cholesterol, HbA(1c), creatinine, glomerular filtration rate), the logarithm of NT-proBNP gave the most potent information in a stepwise Cox regression analysis (P < 0.0001). Bootstrapping with 500 samples supports this result in 95% samples. The negative predictive value of a normal value (<125 pg/mL) of NT-proBNP for short-term cardiovascular events in diabetic patients is 98%. CONCLUSION: We have demonstrated a strong and independent correlation between NT-proBNP and short-term prognosis of cardiovascular events for patients with diabetes mellitus. With a high negative predictive value it can identify individuals who are not at intermediate risk for cardiovascular events. NT-proBNP proved to be of higher predictive value than traditional cardiovascular markers, in this unselected cohort.

Effect of single doses of SLV306, an inhibitor of both neutral endopeptidase and endothelin-converting enzyme, on pulmonary pressures in congestive heart failure.

SLV306, a potent neutral endopeptidase (NEP) inhibitor with additional endothelin-converting enzyme (ECE)-inhibitory activity, in doses of 200, 400, and 800 mg reduced pulmonary and right atrial pressures, although there was not a clear dose response. Systemic blood pressure, heart rate, and cardiac output were unaffected. SLV306 increased plasma natriuretic peptides and big endothelin-1 levels in a dose-dependent manner, confirming NEP and ECE inhibition. The combined inhibition of NEP and ECE may be useful in heart failure by reducing right and left cardiac filling pressures.

Interleukin-6 and B-type natriuretic peptide are independent predictors for worsening of heart failure in patients with progressive congestive heart failure.

BACKGROUND: Neurohormones and cytokines have been associated with poor prognosis in patients with congestive heart failure. However, direct comparisons between them are rare and the best predictor for worsening of heart failure remains to be elucidated. The aim of the study was to identify independent predictors for worsening of heart failure. METHODS: We studied 100 patients with congestive heart failure (LVEF

Natriuretic peptides predict symptom-free survival and postoperative outcome in severe aortic stenosis.

BACKGROUND: The prognostic value of natriuretic peptides in aortic stenosis (AS) remains unknown. METHODS AND RESULTS: B-type natriuretic peptide (BNP), N-terminal BNP (NtBNP), and N-terminal atrial natriuretic peptide (NtANP) were determined in 130 patients with severe AS (mean age, 70+/-12 years; mean gradient, 64+/-21 mm Hg; valve area, 0.64+/-0.15 cm2) who were followed up for 377+/-150 days. Natriuretic peptides increased with NYHA class and with decreasing ejection fraction (EF). Even asymptomatic patients frequently had elevated neurohormones. Asymptomatic patients who developed symptoms during follow-up had higher BNP and NtBNP levels at entry compared with those remaining asymptomatic (median for NtBNP, 131 pmol/L [interquartile range, 50 to 202 pmol/L] versus 31 pmol/L [range, 19 to 56 pmol/L]; P<0.001). Symptom-free survival at 3, 6, 9, and 12 months for patients with NtBNP <80 versus > or =80 pmol/L was 100%, 88+/-7%, 88+/-7%, and 69+/-13% compared with 92+/-8%, 58+/-14%, 35+/-15%, and 18+/-15%, respectively (P<0.001). Seventy-nine patients eventually underwent surgery because of symptoms. Considering preoperative neurohormone levels, age, NYHA class, aortic valve area, EF, and presence of coronary artery disease, we found that neurohormones, EF, and NYHA class predicted survival; neurohormones predicted postoperative symptomatic status; and neurohormones and preoperative EF predicted postoperative EF. However, by multivariate analysis, NtBNP was the only independent predictor of outcome. CONCLUSIONS: In severe AS, natriuretic peptides provide important prognostic information beyond clinical and echocardiographic evaluation. NtBNP independently predicts symptom-free survival, and preoperative NtBNP independently predicts postoperative outcome with regard to survival, symptomatic status, and left ventricular function. Thus, neurohormones may gain particular importance for timing of surgery in asymptomatic severe AS.

Oncostatin-M in myocardial ischemia/reperfusion injury may regulate tissue repair.

AIM: Oncostatin-M, a member of the gp 130 family of cytokines, has been associated with inflammation, connective tissue production, and extracellular matrix turnover. Since the reperfused heart is associated with an intense inflammatory reaction followed by scar formation, we tested the hypothesis that oncostatin-M is upregulated in response to cardiac injury and may play a part in cardiac repair. METHODS: Using a canine model of myocardial ischemia/reperfusion injury, we examined the expression of oncostatin-M at various time points of reperfusion, ranging from 1 h to 7 days. In situ hybridization was used to determine oncostatin-M mRNA expression. Immunohistochemistry was performed to detect oncostatin-M protein. Stimulatory effects of oncostatin-M on isolated endothelial cells and fibroblasts were investigated. RESULTS: Oncostatin-M mRNA expression was detected in ischemic segments within the first 3 h of reperfusion and was persistent over the whole observation period. At the early time points, infiltrating and endothelial cells were the primary source of oncostatin-M mRNA expression. At later time points, macrophages and myofibroblasts were the main contributors. We previously demonstrated in vivo that monocyte-chemoattractant-protein (MCP-1) mRNA is induced early after reperfusion. We stimulated isolated endothelial cells with oncostatin-M and postischemic lymph, which have both upregulated MCP-1 expression in endothelial cells. In addition, isolated fibroblasts stimulated with oncostatin-M demonstrated a dose-dependent proliferative response. CONCLUSION: Oncostatin-M may contribute to the process of healing after myocardial infarction.

Prostaglandin E1 induces vascular endothelial growth factor-1 in human adult cardiac myocytes but not in human adult cardiac fibroblasts via a cAMP-dependent mechanism.

Prostaglandin E(1) (PGE(1)) has been used to treat pulmonary hypertension and peripheral artery occlusive disease and has been successfully employed for pharmacological bridging to transplantation in patients with chronic end-stage heart failure. In addition to its vasoactive effects PGE(1) was shown to stimulate angiogenesis in animal models. Recently we showed that PGE(1)-induced angiogenesis in hearts of patients with ischemic heart disease. We proposed that the angiogenic action of PGE(1) is mediated by vascular endothelial growth factor (VEGF). In the present paper we studied a possible effect of PGE(1) on the expression of VEGF-1 in cultured human adult cardiac myocytes (HACM) and cultured human adult cardiac fibroblasts (HACFB), respectively, to identify a cellular source of VEGF-1 in patients treated with PGE(1). We also aimed to delineate mechanisms involved in a possible regulation of VEGF-1 by PGE(1) in these cells. When HACM, isolated from human myocardial tissue, were treated with PGE(1), a significant up to 3-fold increase in VEGF-1 production could be observed. These results could be confirmed on the level of specific mRNA expression as determined by real-time polymerase chain reaction. The effect of PGE(1) on VEGF-1 expression could be blocked by H089, an inhibitor of cAMP-dependent protein kinase A. In HACFB, also isolated from human myocardial tissue, no effect of PGE(1) on VEGF-1 production was seen. If this effect of PGE(1) is also operative in the in vivo situation, one could speculate that cardiac myocytes could be a cellular source of PGE(1)-induced VEGF-1 expression in patients treated with this drug.

Glycoprotein 130 ligand oncostatin-M induces expression of vascular endothelial growth factor in human adult cardiac myocytes.

OBJECTIVE: In murine and rat cardiac myocytes the gp130 system transduces survival as well as hypertrophic signals and via induction of the expression of the potent angiogenic factor VEGF in these cells also indirectly contributes to cardiac repair processes through the development of new blood vessels. There are, however, species differences in receptor specificity and receptor crossreactivity in the gp130-gp130 ligand system. We asked whether gp130 signaling is also involved in the regulation of VEGF in human cardiac myocytes and if so which gp130 ligands are critical for such an effect. METHODS: Human adult cardiac myocytes (HACMs) were isolated from myocardial tissue and characterised by positive staining for myocardial actin, troponin-I and cardiotin. HACMs were treated with the gp130 ligands CT-1, IL-6, LIF or OSM and VEGF-1 was determined by a specific ELISA in the conditioned media of these cells. RT-PCR and Western blot analysis was used in order to detect gp130, IL-6-receptor, LIF-receptor or OSM-receptor specific protein and mRNA in human adult cardiac myocytes and for detection of VEGF-1 specific mRNA in cardiac myocytes after incubation with OSM. Pieces of myocardial tissue were incubated ex vivo in the presence and absence of OSM and VEGF was determined in supernatants of these cultures and immunohistochemistry was performed on the tissue using specific antibodies for VEGF-1. Immunohistochemistry was also employed to detect VEGF in sections from a healthy human heart and in a heart from a patient suffering from acute myocarditis. RESULTS: OSM, but not CT-1, IL-6 or LIF increased VEGF-1 production in human adult cardiac myocytes dose-dependently derived from five different donors. This selective stimulation of VEGF by gp130 ligands was also reflected by a specific receptor expression on these cells. We detected high levels of mRNA for gp130 and the OSM receptor in freshly isolated human cardiac myocytes but only low amounts of mRNA for the IL-6 receptor whereas mRNA for the LIF receptor was hardly detectable by RT-PCR. OSM receptor and IL-6 receptor were also detectable by Western blotting whereas LIF receptor was only present as a faint band. OSM also increased the expression of VEGF-1 mRNA in cardiac myocytes. When pieces of human myocardial tissue were incubated with the gp130 ligands in an ex vivo model only OSM resulted in an increase in VEGF-1 in the supernatants of these cultures. Furthermore, VEGF increased in tissue samples treated with OSM in cardiac myocytes as evidenced by immunohistochemistry. In addition, we found increased VEGF-1 expression in myocardial tissue from a patient suffering from acute myocarditis. CONCLUSION: The gp130-gp130 ligand system is also involved in VEGF regulation in human cardiac myocytes and OSM is the gp130 ligand responsible for this effect in the human system whereas LIF and CT-1 which had been shown to regulate VEGF expression in mouse and rat cardiac myocytes had no effect. Thus we have added OSM, which is produced by activated T lymphocytes and monocytes, to the list of regulatory molecules of VEGF production in the human heart. Our results lend further support to the notion that besides hypoxia, inflammation via induction of VEGF through autocrine or paracrine pathways plays a key role in (re)vascularisation of the myocardium.

Clinical benefit of prostaglandin E1-treatment of patients with ischemic heart disease: stimulation of therapeutic angiogenesis in vital and infarcted myocardium.

New evidence suggests that Prostaglandin E1 (PGE-1) stimulates myocardial angiogenesis in human chronic ischemic myocardium. We sought to investigate whether PGE-1 may participate in the process of neoangiogenesis within the myocardial infarct scar. Neovascularization was investigated in 14 explanted hearts from patients with ischemic cardiomyopathy, who had been bridged to heart transplantation (HTX) with PGE-1 and compared with 14 hearts from patients who did not receive PGE-1 prior to HTX. In transmural sections obtained from the left ventricular wall and containing myocardial scar tissue, CD34 and vascular endothelial growth factor (VEGF) were quantified immunohistochemically to estimate capillary density and amount of angiogenesis. Additionally, to assess the hypoxic state of myocardium of the infarct border zone, hypoxia inducible factor 1-alpha (HIF-1alpha) was determined by immunohistochemistry and quantified by means of planimetric analysis. PGE-1-treated patients had significantly more CD34-and VEGF-positive cells in infarct areas as compared to nonPGE-1 group, respectively (CD34: 116.7 +/- 5.9 vs. 45.1 +/- 5.2 capillary profiles/mm(2), P < 0.001, and VEGF: 48.3 +/- 4.9 vs. 22.9 +/- 4.7 capillary profiles/mm(2)). HIF-1alpha enrichment (in %) as well as staining intensity (in estimated units (eU)) was significantly decreased in PGE-1-treated as compared to non-treated controls (enrichment: 11.3 +/- 2.5% vs. 19.4 +/- 4.36%; staining intensity: 0.95 +/- 0.3 vs. 1.97 +/- 0.44 eU). Our data demonstrate that PGE-1 stimulates neoangiogenesis in infarct areas adjacent to viable myocardium, via upregulation of VEGF expression. The induction of therapeutic angiogenesis along with the improved hypoxic state of chronic ischemic myocardial tissue might explain the favorable clinical outcome in PGE-1 treated patients.

Revascularization of myocardial scar tissue following prostaglandin E1-therapy in patients with ischemic heart disease.

Prostaglandin E1 (PGE-1) treatment has proved to stimulate angiogenesis in vital non-infarcted myocardium of patients with ischemic cardiomyopathy (ICMP). We investigated infarcted myocardial tissue for a possible angiogenic response to PGE-1. Neovascularization was investigated in infarcted areas of 12 hearts explanted from patients with ICMP who had been treated with PGE-1 before heart transplantation (HTX). In transmural sections containing myocardial scar tissue, CD34 and VEGF were immunohistochemically quantified to estimate capillary density and the extent of angiogenesis. To investigate a possible effect of PGE-1 on collagen turnover, the collagen content was determined in myocardial scar tissue by assessing the intensity of the area positively stained with sirius red. PGE-1-treated patients had significantly more CD34- and VEGF-positive cells in infarcted areas, and showed a significant reduction in collagen content as compared with the non-PGE-1 group (CD34: 120.3 +/- 6.1 vs. 47.7 +/- 6.1 capillary profiles/mm2; VEGF: 52.8 +/- 5.6 vs. 24.0 +/- 4.8 capillary profiles/mm2, and collagen content: 2.18 +/- 0.4 eU vs. 3.59 +/- 0.38 eU). Our data demonstrate that PGE-1 stimulates angiogenesis by upregulating VEGF expression, and reduces fibrosis in cardiac scar tissue of ischemic origin. The induction of therapeutic angiogenesis in vital and at sites of putative dead myocardial scar tissue, along with the hemodynamic improvement in patients with severe ICMP, might explain the favorable clinical outcome in PGE-1-treated patients before HTX.