Mobilization of bone marrow-origin CD34+ cells was investigated 3 days (3d) after acute myocardial infarction (AMI) with/without ischemic preconditioning (IP) in relation to stromal-derived factor-1 ...(SDF-1α)/ chemokine receptor type 4 (CXCR4) axis, to search for possible mechanisms behind insufficient cardiac repair in the first days post-AMI.
Closed-chest reperfused AMI was performed by percutaneous balloon occlusion of the mid-left anterior descending (LAD) coronary artery for 90min, followed by reperfusion in pigs. Animals were randomized to receive either IP initiated by 3x5min cycles of re-occlusion/re-flow prior to AMI (n=6) or control AMI (n=12). Blood samples were collected at baseline, 3d post-AMI, and at 1-month follow-up to analyse chemokines and mobilized CD34+ cells. To investigate the effect of acute hypoxia, SDF-1α and matrix metalloproteinase (MMP)-2
were assessed, and a migration assay of CD34+ cells toward cardiomyocytes was performed.
Reperfused AMI induced significant mobilisation of CD34+ cells (baseline: 260±75 vs. 3d: 668±180; P<0.001) and secretion of MMP-2 (baseline: 291.83±53.40 vs. 3d: 369.64±72.89; P=0.011) into plasma, without affecting the SDF-1α concentration. IP led to the inhibition of MMP-2 (IP: 165.67±47.99 vs. AMI: 369.64±72.89; P=0.004) 3d post-AMI, accompanied by increased release of SDF-1α (baseline: 23.80±12.36 vs. 3d: 45.29±11.31; P=0.05) and CXCR4 (baseline: 0.59±0.16 vs. 3d: 2.06±1.42; P=0.034), with a parallel higher level of mobilisation of CD34+ cells (IP: 881±126 vs. AMI: 668±180; P=0.026), compared to non-conditioned AMI.
, CD34+ cell migration toward cardiomyocytes was enhanced by SDF-1α, which was completely abolished by 90min hypoxia and co-incubation with MMP-2.
. Non-conditioned AMI induces MMP-2 release, hampering the ischemia-induced increase in SDF-1α and CXCR4 by cleaving the SDF-1α/CXCR4 axis, with diminished mobilization of the angiogenic CD34+ cells. IP might influence CD34+ cell mobilization via inhibition of MMP-2.
Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. ...Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs.
Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 10
) or intramyocardially (group IM, 9.88 ± 1.44 × 10
). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery.
bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic fibroblast growth factor 2 (FGF2) and VEGF were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image.
AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min,
= 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm
,
= 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml,
= 0.006), with significant exponential decay between MMP-2 and CXCR4 (
= 0.679,
< 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%,
= 0.046) at the 1-month follow up.
Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.
The BCR/ABL1 inhibitor nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). However, nilotinib apparently induces metabolic changes, including an increase in the ...fasting glucose level. In addition, vascular adverse events, including progressive atherosclerosis with peripheral arterial occlusive disease (AOD) have been reported in nilotinib-treated CML patients. We reviewed and updated AOD events in our CML patients receiving nilotinib (n=34) and initiated preclinical in vitro and in vivo studies in order to dissect potential targets and mechanisms. After a median observation time (MOT) of 24 months, the frequency of AOD (26.5%) and severe AOD requiring surgical intervention and/or prolonged hospitalization (17.6%) was higher in nilotinib-treated patients compared to risk factor-, observation time-, and age-matched controls (34 imatinib-treated patients with CML, 34 with myelodysplastic syndromes, 34 with JAK2-mutated MPN and 34 with lymphoid neoplasms; <5% AOD, p<0.05). After a MOT of 36 months, the frequency of AOD amounted to 36.1% and the frequency of severe AOD was 19.4%. We next examined the in vitro effects of nilotinib on cultured human umbilical vein endothelial cells (HUVEC), human coronary artery-derived endothelial cells (HCAEC), and the human microvascular endothelial cell line HMEC-1. As determined by 3H-thymidine incorporation, nilotinib was found to inhibit the proliferation of endothelial cells in a dose-dependent manner, with pharmacologically relevant IC50 values obtained in HUVEC (1.0 µM), HCAEC (100 nM), and HMEC-1 (1.0 µM), whereas imatinib showed little effect up to 5 µM. Moreover, nilotinib was found to inhibit the migration of HUVEC in a wound-scratch assay as well as angiogenesis in a tube-formation assay (relative capillary tubes: VEGF+control: 1.8±0.1, VEGF+nilotinib (100 nM): 1.3±0.1, VEGF+imatinib (100 nM): 1.7±0.05; n=3, p<0.01 for VEGF alone vs VEGF+nilotinib). In a mouse model of hindlimb ischemia, nilotinib (75 mg/kg/day p.o. for 28 days) was found to slow blood flow-recovery after induction of ischemia whereas imatinib (100 mg/kg/day p.o. for 28 days) showed no comparable effect (laser Doppler perfusion imaging ratio ischemic/control leg: control mice: 0.81±0.03, imatinib-treated mice: 0.79±0.04, nilotinib-treated mice: 0.68±0.04; n=13/group; p<0.05 for nilotinib vs control and for nilotinib vs imatinib). The decreased blood perfusion was accompanied by an increased rate of limb necrosis (necrosis score: control: 1.15±0.08, imatinib: 1.17±0.05, nilotinib: 1.54±0.18; p<0.05 for nilotinib vs control and nilotinib vs imatinib). Moreover, microvessel density was significantly lower in the affected hind limb in nilotinib-treated mice compared to imatinib-treated mice or control-mice (p<0.05). In addition, we found that nilotinib (between 1-10 µM), but not imatinib (1-10 µM) promotes the expression of pro-atherogenic cytoadhesion molecules (CAM) on HUVEC, including ICAM-1 (CD54), VCAM-1 (CD106) and E-Selectin (CD62E). By contrast, nilotinib (up to 10 µM) showed no effects on expression of plasminogen activators or uPA receptor (CD87) in cultured endothelial cells. As assessed by chemical proteomics profiling and phospho-array analysis, several angiogenesis-related and other endothelial antigens, including Tie-2/TEK, JAK1, BRAF and EPHB2 were identified as molecular targets of nilotinib, whereas imatinib did not bind to these vascular targets in endothelial cells. As assessed by immunohistochemistry using antibodies against KIT and mast cell tryptase, we also found that in our CML patients, nilotinib induces an almost complete depletion of KIT+ mast cells, a cell type that serves as unique source of heparin and uncomplexed tPA and has been implicated as a major repair cell in vascular disorders. However, imatinib was also found to induce mast cell depletion in our patients with CML. Neither nilotinib nor imatinib showed in vitro or in vivo effects on platelet adhesion or platelet aggregation. In conclusion, nilotinib exerts multiple effects on vascular endothelial cells and other perivascular cells such as mast cells, presumably through multiple mechanisms and targets. We hypothesize that these effects may contribute to nilotinib-induced vasculopathy in CML.
Wolf:Bristol-Meyers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding.
Macrophages produce the cytokine oncostatin M (OSM), which beside other functions is also involved in inflammation. The complement component C5a mobilizes and activates these cells at inflammatory ...sites. We examined the effect of C5a on OSM production in human monocytes and in human monocyte-derived macrophages.
For macrophage transformation peripheral blood monocytes were cultivated for 8 to 10 days in the presence of human serum. C5a significantly increased in these cells OSM antigen as determined by specific ELISA and mRNA as quantitated by real-time polymerase chain reaction in these cells as well as in plaque macrophages. This effect was blocked by antibodies against the receptor C5aR/CD88 and by pertussis toxin. The C5a-induced phosphorylation of p38 and JNK and the C5a-induced increase in OSM production in macrophages was abolished by 2 p38 inhibitors and by a JNK inhibitor. Furthermore C5a increased the nuclear translocation of c-fos and c-jun. Using different OSM promoter deletion mutant constructs we show that the putative AP-1 element is responsible for activation of OSM promoter activity by C5a.
Our data establish a link between the complement system and the gp130 receptor cytokine family with possible implications for the pathology of inflammatory diseases.
We have recently shown that resting human mast cells (MCs) produce tissue-type plasminogen activator (t-PA) without simultaneously expressing plasminogen activator inhibitor 1 (PAI-1). In the present ...study we have identified the anaphylatoxin rhC5a as a potent inducer of PAI-1 expression in human MCs and basophils. In primary human skin MCs and primary blood basophils, exposure to rhC5a was followed by an increase from undetectable to significant levels of PAI-1. In addition, rhC5a induced a concentration- and time-dependent increase in PAI-1 antigen in the MC line HMC-1 and the basophil cell line KU-812 and increased the expression of PAI-1 mRNA in HMC-1. In conditioned media of HMC-1 treated with rhC5a, active PAI-1 could be detected. A simultaneous loss of t-PA activity in conditioned media from the same cells indicated that rhC5a-induced PAI-1 was capable of inhibiting the enzymatic activity of coproduced t-PA. Correspondingly, the levels of t-PA–PAI-1 complexes increased in rhC5a-treated cells. When HMC-1 cells were incubated with pertussis toxin or anti-C5a receptor antibodies, the effect of rhC5a on PAI-1 production was completely abolished. Treatment of C5a with plasmin resulted in loss of its ability to induce PAI-1 production in MCs. Considering the suggested role for MCs and components of the complement system in the development of cardiovascular diseases, we hypothesize that MCs, by producing t-PA in a resting state and by expressing PAI-1 when activated by C5a, might participate in the modulation of the balance between proteases and protease inhibitors regulating tissue injury and repair in such disease processes.
Overweight and obesity in many countries have developed into a serious health problem by themselves and by their impact on other pathologies such as insulin resistance, type 2 diabetes, hypertension, ...heart disease and cancer. The modulation of these diseases by adipose tissue-derived biomolecules, so-called adipokines, could be the key to differentiate between metabolically healthy and unhealthy obesity. This review will discuss the pathophysiological role of selected adipokines, primarily focusing on cardiovascular diseases. Furthermore, we will highlight possible therapeutic approaches, which target these biomolecules.
The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of ...progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.
Plaque destabilization leading to myocardial infarction is observed after surgery even if the intervention is of noncardiovascular nature. Mediators of peri‐ or postoperative stress responsible for ...such events could include catecholamines and lipopolysaccharide (LPS). Monocytes may be involved in destabilization of atherosclerotic plaques by production of matrix metalloproteinases (MMP). We examined whether catecholamines could affect the expression of MMPs in human monocytes/macrophages and whether catecholamines could modulate LPS‐stimulated expression of particular MMPs in these cells. Epinephrine and norepinephrine up‐regulated MMP‐1 and potentiated LPS‐induced expression of MMP‐1 in peripheral blood monocytes and monocyte‐ derived macrophages. We further characterized this effect employing the monocytic cell line U937 and showed that catecholamines potentiate LPS‐induced effects on MMP‐1 and MMP‐9 antigen and activity. mRNA levels of the respective MMPs also increased. These effects did not result from higher mRNA stability but rather from increased transcription possibly induced by enhanced DNA binding of AP‐1 and were mediated by either β1‐ or β2‐receptors. If this mechanism is also effective in vivo, our findings might, at least in part, help to explain the observation that cardiac events are important causes of morbidity and mortality after noncardiac surgery and support the findings that peri‐operative β‐blockade has been shown to reduce postoperative mortality from cardiac events.
Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the regulation of the fibrinolytic system and in the modulation of extracellular proteolysis. Increased PAI-1 was found in ...atherosclerotic lesions, and high PAI-1 plasma levels were associated with coronary heart disease. Smooth muscle cells (SMC) are a major source of PAI-1 within the vascular wall, and PAI-1 was implicated in SMC migration, proliferation, and apoptosis. We treated human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) with the glycoprotein 130 (gp130) ligands cardiotrophin-1, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), or oncostatin M (OSM). Only OSM increased PAI-1 antigen and activity production significantly in these cells up to 20-fold. OSM upregulated mRNA specific for PAI-1 up to 4.5-fold in these cells. HCASMC and HASMC express gp130, OSM receptor, IL-6 receptor, and LIF receptor. OSM induced extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylations in HASMC. A phosphatidylinositol 3-kinase inhibitor and a mitogen-activated protein/extracellular signal-regulated kinase inhibitor reduced Akt and ERK1/2 phosphorylation, respectively, and abolished OSM-induced PAI-1 upregulation. A janus kinase/signal transducer and activator of transcription inhibitor, a p38 mitogen-activated protein kinase inhibitor, or c-Jun NH(2)-terminal kinase inhibitor I did not inhibit the OSM-dependent PAI-1 induction. OSM enhanced proliferation of both HCASMC and HASMC by 77 and 90%, respectively. We hypothesize that, if the effect of OSM on PAI-1 expression in smooth muscle cells is operative in vivo, it could, via modulation of fibrinolysis and extracellular proteolysis, be involved in the development of vascular pathologies such as plaque progression, destabilization and subsequent thrombus formation, and restenosis and neointima formation.
PDF
