Teresa Padró

Overexpression of vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia

Vascular endothelial growth factor (VEGF) and its cellular receptor VEGFR-2 have been implicated as the main endothelial pathway required for tumor neovascularization. However, the importance of the VEGF/VEGFR-2 system for angiogenesis in hematologic malignancies such as AML remains to be elucidated. In 32 patients with newly diagnosed untreated AML, we observed by immunohistochemical analysis of bone marrow biopsies significantly higher levels of VEGF and VEGFR-2 expression than in 10 control patients (P <0.001). In contrast, VEGFR-1 staining levels in AML patients were in the same range as in the controls. Expression of VEGF and VEGFR-2 was significantly higher in patients with a high degree of microvessel density compared to those with a low degree (VEGF: P =0.024; VEGFR-2: P =0.040) and correlated well with bone marrow microvessel density (rs=0.566 and 0.609, respectively; P <0.001). Furthermore, in patients who achieved a complete remission following induction chemotherapy VEGFR-2 staining levels decreased into the normal range. In conclusion, our results provide evidence for increased expression of VEGF/VEGFR-2 of leukemic blasts and correlation with angiogenesis in the bone marrow of AML patients. Thus, VEGF/VEGFR-2 might constitute promising targets for antiangiogenic and antileukemic treatment strategies in AML.

Quantification of Plasminogen Activators and Their Inhibitors in the Aortic Vessel Wall in Relation to the Presence and Severity of Atherosclerotic Disease

Increased expression of plasminogen activator inhibitor-1 (PAI-1) has been demonstrated in the human atherosclerotic vessel wall and may contribute to the impaired plasma fibrinolytic capacity in patients at high risk of atherothrombotic events. In addition, the mural PA/plasmin system may have important pathobiologic functions during atherogenesis. We quantitatively analyzed PAs of the tissue type (TPA) and urokinase type (UPA), PAIs, and plasminogen in protein extracts from different layers of human aorta in relation to the presence and severity of atherosclerotic lesions. In comparison with normal control vessels, intimal and neointimal TPA concentrations were reduced in atherosclerotic aortas except in the necrotic core areas of advanced plaques, where TPA was mainly complexed to PAI-1 in extracellular matrix deposits. In the media, TPA antigen was higher in lesional segments and closely associated with smooth muscle cells. UPA antigen was increased in the intima of atherosclerotic lesions and colocalized with tissue-infiltrating macrophages and neointimal smooth muscle cells. By spectrophotometric assay, neither TPA nor UPA activity could be detected in intimal or medial extracts. PAI-1 concentrations increased significantly in the intima of atherosclerotic segments compared with adjacent uninvolved areas or control aortas. The immunohistochemical distribution of PAI-1 was similar to that observed for TPA. A large excess of PAI-1 over PA concentrations, particularly in the intimal layer, characterizes atherosclerotic lesions of the human aorta and suggests that PA action is locally confined and counterbalanced by enhanced PAI expression and accumulation.

Overexpression of tissue-type plasminogen activator in atherosclerotic human coronary arteries

UNLABELLED The plasminogen activator (PA)/plasmin system is involved in various pathological processes that are considered important features of atherogenesis and atherothrombosis. These include the proteolysis of fibrin deposits and extracellular matrix components as well as the induction of cell migration and mitogenesis. Tissue-type PA (TPA) is a key enzyme mediating plasminogen to plasmin conversion. TPA plasma concentrations are elevated in patients with advanced atherosclerosis and correlate with an increased risk for myocardial infarction and stroke. In this study, we have analysed the content and expression of TPA in human coronary arteries and their relation to the presence and severity of atherosclerotic lesions. METHODS Segments of coronary arteries obtained from heart explants (n = 15) were classified by the presence and types of atherosclerotic lesions. TPA was quantitatively determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. RESULTS PA activity entirely attributable to the presence of active TPA was consistently detected in the protein extracts. Extractable TPA antigen and activity showed a significant graded increase in relation to the presence and severity of atherosclerotic lesions. The ratios of active over total TPA were increased several-fold in extracts of advanced lesions despite a concomitant threefold increase in TPA complexed to its inhibitor PA-1. In macroscopically normal arterial segments and in early lesions, TPA was expressed in the endothelium and in colocalization with vascular smooth muscle cells (VSMCs). In advanced plaques, TPA mRNA was mainly detected in the lateral regions of the fibrous caps in association with migrating VSMCs and in the vicinity of the core areas infiltrated by CD68-positive macrophages. CONCLUSIONS TPA content and expression is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. This may contribute to plaque destabilization and disruption. Conversely, the increased intramural TPA activity may counteract mural fibrin deposition.

Comparative analysis of plasminogen activator inhibitor-1 expression in different types of atherosclerotic lesions in coronary arteries from human heart explants

OBJECTIVES Clinical manifestations of coronary heart disease result primarily from the progressive development of atherosclerotic plaques and subsequent thrombus formation: processes which may be accelerated by an enhanced expression of plasminogen activator inhibitor (PAI-1) in the vessel wall. In the present study, content and expression of PAI-1 were comparatively analyzed in human coronary arteries in relation to the presence and severity of atherosclerotic lesions. METHODS Segments of coronary arteries obtained from heart explants (n = 15) were classified by the presence and types of atherosclerotic lesions. Antigen and activity levels of PAI-1 were determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. RESULTS Total PAI-1 antigen consistently increased from macroscopically normal areas (MNAs) to early lesions (ELs) and to maximal levels in fibrous (FPs) and calcified (CPs) plaques. No PAI activity was detected, although PAI-1 in its free form was present in all vascular specimens. Both free PAI-1 and PAI-1 complexed with plasminogen activators were significantly increased in extracts of advanced lesions. However, there was a 2-3 fold molar excess of free versus complexed PAI-1 in FPs and CPs. These findings suggest the presence of relevant amounts of PAI-1 in its substrate rather than in its inhibitor conformation in areas of advanced lesions. Compared with MNAs, PAI-1 mRNA was strongly expressed within the thickened intima of ELs. The highest PAI-1 expression was observed in FPs and CPs, being mainly localized in areas surrounding the necrotic cores in co-localization with infiltrating macrophages. CONCLUSIONS PAI-1 content is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. The concomitant elevation of PAI-1 mRNA suggests that the PAI-1 increase in regulated by local synthesis in the areas of atherosclerotic lesions.

Tissue plasminogen activator and plasminogen activator inhibitor in patients with acute ischemic stroke: Relation to stroke etiology

Recent studies suggest that high plasma levels of tissue-type plasminogen activator (tPA) and its inhibitor (plasminogen activator inhibitor-1, PAI-1) are markers of an increased risk of atherothrombotic ischemic events such as stroke and myocardial infarction. In this prospective study, we measured tPA antigen, PAI-1 antigen and activity, as well as tPA/PAI-1 complex in patients with acute stroke. Stroke subtypes were classified according to the TOAST criteria. From 132 consecutively screened patients, 89 (100%) were enrolled in this study, including 42 patients (47%) with large artery atherosclerosis (LAA), 32 (36%) with small vessel occlusion (SVO), and 15 (17%) with cardioembolism (CE). Nineteen age-matched neurologic patients without manifestations of cerebrovascular disease served as control subjects (CS). Patients with acute stroke had significantly higher plasma levels of tPA antigen (p < 0.001), PAI-1 antigen (p < 0.05) and PAI activity (p < 0.05) than patients in the control group. t-PA antigen, PAI activity and tPA/PAI-1 complex levels were similar regardless of stroke etiology. Only PAI-1 antigen was lower in patients with cardioembolic stroke than in stroke patients with LAA (p < 0.05). Plasma tPA antigen, PAI-1 antigen, and PAI activity are significantly increased in patients with acute ischemic stroke. Except for PAI-1 antigen, this increase appears not to be related to the underlying stroke etiology.

Thalidomide for the Treatment of Acute Myeloid Leukemia

Abstract In analogy to solid neoplasms, accumulating data suggest the requirement of angiogenesis also for the development and progression of hematopoietic malignancies including acute myeloid leukemia (AML). Inhibition of increased microvessel density in bone marrow (BM) might be a promising target for pharmacological interventions aimed at reducing disease activity. Among the putative inhibitors of angiogenesis, thalidomide has demonstrated a considerable efficacy in myelodysplastic syndromes (MDS) and AML with overall response rates up to 56% and 25%, respectively. Responders experienced hematologic improvements with increased hemoglobin and platelet counts resulting in temporary transfusion independence. In AML, partial responses—defined as reduction of the leukemic blast cell infiltration of at least 50% in BM—occurred in four of 20 patients after one month of thalidomide administration in a previous phase I/II study. Additionally, we observed a long-term response in one AML patient of more than 20 months, meanwhile fulfilling the criteria of complete remission. The decrease in leukemic blast infiltration in BM of responders was accompanied by a significant reduction of the microvessel density. Overall adverse events caused by the drug consisted mainly of fatigue, constipation, skin rash and polyneuropathy with a tolerable dose of 200–400 mg p.o. per day. In conclusion, thalidomide as a single agent has significant anti-leukemic activity with some evidence for anti-angiogenic effects in BM, although the precise mechanism of action remains to be elucidated.

Angiogenese bei hämatologischen Neoplasien

Angiogenesis in Patients with Hematologic Malignancies The importance of angiogenesis for the progressive growth and viability of solid tumors is well established. Emerging data suggest an involvement of angiogenesis in the pathophysiology of hematologic malignancies as well. Recently, we and others have reported increased angiogenesis in the bone marrow of patients with acute myeloid leukemia (AML) and normalization of bone marrow microvessel density when patients achieved a complete remission (CR) after induction chemotherapy. Tumor angiogenesis depends on the expression of specific mediators that initiate a cascade of events leading to the formation of new microvessels. Among these, VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor) and angiopoietins play a pivotal role in the induction of neovascularization in solid tumors. These cytokines stimulate migration and proliferation of endothelial cells and induce angiogenesis in vivo. Recent data suggest an important role for these mediators in hematologic malignancies as well. Isolated AML blasts overexpress VEGF and VEGF receptor 2. Thus, the VEGF/VEGFR-2 pathway can promote the growth of leukemic blasts in an autocrine and paracrine manner. Therefore, neovascularization and angiogenic mediators/receptors may be promising targets for anti-angiogenic and anti-leukemic treatment strategies. The immunomodulatory drug thalidomide inhibits angiogenesis in animal models. Moreover, it has significant activity in refractory multiple myeloma. In a current phase II study for patients with primary refractory or relapsed multiple myeloma using a combination of thalidomide with hyperfractionated cyclophosphamide and dexamethasone (Hyper-CDT), we observed a partial remission in 12 of 14 evaluable patients (86%). Thus, this combination seems to be very potent. Furthermore, we evaluated the safety and efficacy of thalidomide in patients with AML not qualifying for intensive cytotoxic chemotherapy. 20 patients aged 58–85 (median 69) years were recruited to this phase I/II study and were treated with a dose of 200–400 mg per os daily for a duration of 1–40 (median 6) weeks, dependent on the individual tolerability of the drug. In 4 patients we observed a partial response (PR – defined as more than 50% reduction in leukemic blast infiltration in the bone marrow). This was accompanied by an increase in platelet counts and hemoglobin values. One additional patient showed a significant improvement of peripheral blood counts without fulfilling the criteria of a PR. In parallel, we observed a significant decrease in microvessel density in these 5 patients during treatment with thalidomide. In conclusion, thalidomide seems to have anti-angiogenic as well as anti-leukemic activity in AML. The VEGF/VEGFR-2 pathway seems to play an important role in AML. Therefore, receptor tyrosine kinase inhibitors like SU5416 or SU6668 are currently evaluated in the context of phase II studies in AML. We could recently induce a stable remission in a patient with second relapse of her AML refractory towards chemotherapy by administration of SU5416 (compassionate use), a tyrosine kinase inhibitor of VEGFR-2 and ckit. Current and future studies will clarify the role of anti-angiogenic treatment strategies in AML and other hematologic malignancies.

Evidence of Angiogenesis in Acute Myeloid Leukemia

Angiogenesis plays a key role in the growth of solid tumors and in the development of metastases. An increased angiogenesis in bone marrow has been reported in children with acute lymphoblastic leukemia (Am J Pathol 150:815-821,1997). The purpose of the present study was to assess angiogenesis in bone marrow biopsies from 47 patients with newly diagnosed, untreated acute myeloid leukemia (AML). Control specimens (n=20) were obtained from patients with neo-plasic disorders without bone marrow involvement. The FAB distribution of the AML cases was as follows: 7 M1, 21 M2,1 M3,7 M4, 7 M5,2 M6 and 2 AMLs without defined subtype. The endothelial cells of microvessels were highlighted by immunohistochemical staining for thrombomodulin (TM) and von Willebrand factor (vWF). The 3 areas with the highest microvessel density in representative sections of each bone marrow core biopsy specimen were selected and the microvessels scored in x500 fields by two observers using light microscopy. A significant correlation was found between microvessel counts in bone marrow sections stained by vWF and TM antibodies (r = 0.828; p< 0.001). Using TM staining, AML marrows had (median [interquartile range]) 25.5 [22.1-29.3] microvessels / field while normal marrows had 13.2 [11.4-14.8] microvessels / field. Using vWF staining of the same specimens, AML marrows had 22.9 [16.1-26.2] microvessels / field while normal marrows had 9.8 [7.7-10.5] microvessels / field. The differences between the number of vessels / field in AML and controls were statistically significant for both TM (p= 0.0003) and vWF (p= 0.001) staining. When analyzed by FAB category, there was no difference in the average number of microvessels / field between the different subgroups of AML. In summary, we demonstrated that bone marrow in AML is associated with increased microvessel density. These findings suggest that antiangiogenic therapy might constitute a novel strategy for the treatment of acute myeloid leukemia.