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Leo Hofstra

Publications and source records attributed to Leo Hofstra.

18 recordsLinked to original sources
PubMed2006

cNGR: a novel homing sequence for CD13/APN targeted molecular imaging of murine cardiac angiogenesis in vivo.

OBJECTIVE: Previously, the peptide sequence cNGR has been shown to home specifically to CD13/APN (aminopeptidase N) on tumor endothelium. Here, we investigated the feasibility of selective imaging of cardiac angiogenesis using the cNGR-CD13/APN system. METHODS AND RESULTS: CD13/APN induction and cNGR homing were studied in the murine myocardial infarction (MI) model. By real-time polymerase chain reaction (PCR) at 7 days after MI, CD13/APN expression was 10- to 20-fold higher in the angiogenic infarct border zone and the MI area than in non-MI areas. In vivo fluorescence microscopy confirmed specific homing of fluorophore-tagged cNGR to the border zone and MI territory at 4 and 7 days after MI with a local advantage of 2.3, but not at 1 or 14 days after MI. Tissue residence half-life was 9.1+/-0.3 hours, whereas the half-life in plasma was 15.4+/-3.4 minutes. Pulse chase experiments confirmed reversible binding of cNGR in the infarct area. Fluorescent labeled cNGR conjugates or antibodies were injected in vivo, and their distribution was studied ex vivo by 2-photon laser scanning microscopy (TPLSM). cNGR co-localized exclusively with CD13/APN and the endothelial marker CD31 on vessels. CONCLUSIONS: In cardiac angiogenesis endothelial CD13/APN is upregulated. It can be targeted specifically with cNGR conjugates. In the heart cNGR binds its endothelial target only in angiogenic areas.

Animals↗
PubMed2005

Annexin A5 inhibits engulfment through internalization of PS-expressing cell membrane patches.

Apoptosis and subsequent clearance of apoptotic cells are important for the prevention of diseases. Therefore, it is essential to understand the mechanisms underlying the biology of phagocytic clearance of apoptotic cells. The best characterized "eat me" signal on the surface of apoptotic cells is phosphatidylserine (PS). Recently, we demonstrated that annexin A5 mediates the internalization of PS-expressing membrane patches and down regulates surface expression of tissue factor. Here, we investigated the role of PS in the phagocytosis of apoptotic cells using annexin A5. Using a novel flow cytometric-based phagocytosis assay, we observed that engulfment was inhibited with 20% if annexin A5 was added to PS-expressing cells that had completed apoptosis. The inhibition increased to more than 50% if annexin A5 was added during the apoptotic process. This inhibition is specific for annexin A5, since the mutant M23 and annexin A1 did not further increase the inhibition of phagocytosis when added during the apoptotic process. Interestingly, cells with internalized annexin A5 still express PS at their surface. We conclude that other ligands within the PS-expressing membrane patch act together with PS as an "eat me" signal.

Animals↗
PubMed2005

Partial cell fusion: a newly recognized type of communication between dedifferentiating cardiomyocytes and fibroblasts.

OBJECTIVE: Fibroblasts have been shown to couple to neonatal cardiomyocytes in heterocellular cultures through functional gap junctions. Our objective was to provide evidence for an additional type of heterocellular communication between fibroblasts and adult cardiomyocytes in vitro and in vivo. METHODS: The contact areas in heterocellular co-cultures were evaluated by specific labeling and the intercellular communication was studied using preloading of fibroblasts with tracer molecules. Heterocellular fibroblast-cardiomyocyte contacts present in the in vitro setting and in the border zone of a rabbit myocardial infarction in vivo were further examined by electron microscopy. RESULTS: Addition of fibroblasts preloaded with the fluorescent low molecular weight tracer calcein-AM to cultured myocytes indicated early dye transfer via connexin 43 functional gap junctions. At a later time-period after co-culturing, dye transfer of fibroblasts preloaded with the high molecular weight tracer dextran 10,000 suggested partial cell fusion. The membrane continuity giving rise to this partial cell fusion was confirmed by electron microscopy, clearly showing areas of intercytoplasmic contacts between fibroblasts and phenotypically adapted (dedifferentiated) cardiomyocytes. Fluorescein-labeled annexin V affinity studies revealed transient exposure of phosphatidylserine at the contact sites, suggesting that phosphatidylserine mediates the fusion process. Close contacts between cardiac fibroblasts and dedifferentiated cardiomyocytes accompanied by disruption of the basal lamina were observed in the border zone of a rabbit myocardial infarction in vivo. CONCLUSION: Our results suggest that the partial cell fusion-type of heterocellular communication in our co-culture model and the contacts observed in vivo may lead to new insights in cardiovascular disease.

Animals↗
PubMed2005

Myocyte apoptosis in heart failure.

Human heart failure is preceded by a process termed cardiac remodeling in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Unlike necrosis, apoptosis is an orderly regulated process and, by inference, a logical therapeutic target if intervention occurs at an early stage. To identify potential therapeutic targets, it is imperative to have a full understanding of the apoptotic pathways that are functional in the cardiac muscle. Accordingly, the present review summarizes the apoptotic pathways operative in cardiac muscle and discusses therapeutic options related to apoptosis for the future treatment of human heart failure.

Animals↗
PubMed2005

Abdominal aortic aneurysm screening during transthoracic echocardiography in an unselected population.

OBJECTIVE: We sought to investigate the echocardiographic prevalence of abdominal aortic aneurysm (AAA) in an unselected group of patients referred for regular transthoracic echocardiography (TTE). METHODS: Prospectively, during a 3-month period, a limited ultrasound examination of the infrarenal aorta was performed. AAA was defined as a diameter of 30 mm or more. RESULTS: The abdominal aorta could be visualized in 742 patients. The prevalence of AAA was 4.6%. AAA prevalence increased with age, especially in men. In 34 patients AAA was unknown and aortic diameters exceeded 50 mm in 4 patients. Two underwent elective but urgent operation. Patients with AAA were older and had an increased ascending aorta diameter, larger left ventricular dimensions, higher left ventricular mass index, and lower ejection fraction. CONCLUSION: AAA is prevalent in patients referred for regular TTE. Routine rapid screening of the abdominal aorta during TTE is beneficial and should, therefore, be part of a standard TTE examination for patients older then 50 years.

Adult↗
PubMed2005

Resolution of apoptosis in atherosclerotic plaque by dietary modification and statin therapy.

UNLABELLED: Although apoptosis within atherosclerotic plaques is associated with plaque vulnerability and rupture, the role of inhibition of the apoptotic process is not clear. We evaluated the impact of dietary modification and statin therapy (measures known to favorably influence outcomes in coronary disease) on the incidence of apoptosis in experimental atherosclerotic lesions. METHODS: A total of 30 animals were studied; 1 group of 6 animals served as the controls (group 1), and the remaining 24 animals were subjected to balloon de-endothelialization of the abdominal aorta and a high-cholesterol diet. These atherosclerotic animals were randomized as follows: high-cholesterol diet for 4 mo (n=6; untreated atherosclerotic group [group 2]), high-cholesterol diet for 3 mo and normal chow diet for 1 mo (n=6; diet withdrawal group [group 3]), and high-cholesterol diet for 4 mo and simvastatin orally every day of the last month (n=6; statin therapy group [group 4]). 99mTc-Annexin A5 was used for noninvasive detection of apoptosis in groups 1-4. The remaining 6 rabbits on a high-cholesterol diet for 4 mo were studied with radiolabeled mutant annexin A5 (n=6; nonspecific control group [group 5]). Quantitative annexin A5 uptake in the abdominal aorta was determined and compared with the histologic and immunohistochemical characteristics of the atherosclerotic lesions. RESULTS: Maximum annexin A5 uptake (mean+/-SD, 0.051+/-0.009 percentage injected dose per gram [%ID/g] of tissue) was observed in the untreated atherosclerotic animals. The uptake was substantially reduced in the diet withdrawal (0.03+/-0.006%ID/g; P<0.0001) and statin therapy (0.03+/-0.006%ID/g; P<0.0001) groups. The plaques in the untreated high-cholesterol group demonstrated advanced atherosclerotic lesions. On the other hand, the diet withdrawal and statin therapy groups showed histologic characteristics of stabilization, including the resolution of macrophage infiltration and an increase in smooth muscle cell content. There was a marked reduction in the apoptosis of macrophages. No significant uptake of annexin A5 or mutant annexin A5 was seen in rabbits on the normal chow diet or atherosclerotic rabbits, respectively. CONCLUSION: Dietary modification and statin therapy in atherosclerosis lead to a reduction in apoptosis and contribute to plaque stabilization. It can be hypothesized that a reduction in apoptosis is a favorable process in atherosclerotic disease.

Animal Feed↗
PubMed2004

Annexin A5 down-regulates surface expression of tissue factor: a novel mechanism of regulating the membrane receptor repertoir.

Phosphatidylserine (PtdSer) is exposed on the external leaflet of the plasma membrane during apoptosis. The protein annexin A5 (anxA5) shows high affinity for PtdSer. When anxA5 binds to the PtdSer-expressing membranes during apoptosis, it crystallizes as an extended two-dimensional network and activates thereby a novel portal of cell entry that results in the internalization of the PtdSer-expressing membrane patches. This novel pathway of cell entry is potentially involved in the regulation of the surface expression of membrane receptors. In this study we report the regulation of surface expression of the initiator of blood coagulation tissue factor (TF) by this novel pathway of cell entry. AnxA5 induces the internalization of tissue factor expressed on the surface of apoptotic THP-1 macrophages. This down-regulation depends on the abilities of anxA5 to bind to PtdSer and to form a two-dimensional crystal at the membrane. We furthermore show that THP-1 cells produce and externalize anxA5 that cause the internalization of TF in an autocrine type of mechanism. We extended our in vitro work to the in vivo situation and show in a mouse model that anxA5 causes the down-regulation of TF expression by smooth muscle cells of the media of the carotid artery that was mechanically injured. In conclusion, anxA5 down-regulates surface-expressed TF by activating the novel portal of cell entry. This mechanism may be part of a more general autocrine function of anxA5 to regulate the plasma membrane receptor repertoir under stress conditions associated with the surface expression of PtdSer.

Animals↗
PubMed2004

Cell surface-expressed phosphatidylserine and annexin A5 open a novel portal of cell entry.

Expression of phosphatidylserine (PtdSer) at the cell surface is part of the membrane dynamics of apoptosis. Expressed phosphatidylserine functions as an "eat me" flag toward phagocytes. Here, we report that the expressed phosphatidylserine forms part of a hitherto undescribed pinocytic pathway. Annexin A5, a phosphatidylserine-binding protein, binds to and polymerizes through protein-protein interactions on membrane patches expressing phosphatidylserine. The two-dimensional protein network of annexin A5 at the surface prevents apoptotic body formation without interfering with the progression of apoptosis as demonstrated by activation of caspase-3, PtdSer exposure, and DNA fragmentation. The annexin A5 protein network bends the membrane patch nanomechanically into the cell and elicits budding, endocytic vesicle formation, and cytoskeleton-dependent trafficking of the endocytic vesicle. Annexin A1, which binds to PtdSer without forming a two-dimensional protein network, does not induce the formation of endocytic vesicles. This novel pinocytic pathway differs from macropinocytosis, which is preceded by membrane ruffling and actin polymerization. We clearly showed that actin polymerization is not involved in budding and endocytic vesicle formation but is required for intracellular trafficking. The phosphatidylserine-annexin A5-mediated pinocytic pathway is not restricted to cells in apoptosis. We demonstrated that living tumor cells can take up substances through this novel portal of cell entry. This opens new avenues for targeted drug delivery and cell entry.

Annexin A5↗
PubMed2004

The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5.

We have demonstrated that imaging of programmed cell death (PCD) in patients is possible using 99mTc-Annexin A5. Because of the short half-life of the technetium label it is important to limit the time span between the preparation of 99mTc-Annexin A5 and its administration into the patient. Therefore methods of quality control that determine the biological active fraction in the 99mTc-Annexin A5 should be not only accurate and precise but also rapid. We report the development and validation of a rapid, simple assay measuring the biological active fraction of 99mTc-Annexin A5. The assay is based on a solid phase of paramagnetic beads which are coated with phospholipids. Annexin A5 binds to these beads with high affinity if phosphatidyl serine is present within the phospholipid coat. Furthermore the binding depends on Ca2+ ions and functional Ca2+/phospholipid binding sites of Annexin A5. The bead assay is specific, stability-indicating, repeatable, and reproducible. It allows one to determine within 25 min the biological active fraction of a 99mTc-Annexin A5 preparation. We dubbed this assay the ApoCorrect assay.

Annexin A5↗
PubMed2003

Targeting of apoptotic macrophages and experimental atheroma with radiolabeled annexin V: a technique with potential for noninvasive imaging of vulnerable plaque.

BACKGROUND: Apoptosis is common in advanced human atheroma and contributes to plaque instability. Because annexin V has a high affinity for exposed phosphatidylserine on apoptotic cells, radiolabeled annexin V may be used for noninvasive detection of apoptosis in atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the infradiaphragmatic aorta followed by 12 weeks of cholesterol diet; 5 controls were studied without manipulation. Animals were injected with human recombinant annexin V labeled with technetium-99m before imaging. Aortas were explanted for ex vivo imaging, macroautoradiography, and histological characterization of plaque. Radiolabeled annexin V cleared rapidly from the circulation (T1/2, alpha 9 and beta 46 minutes). There was intense uptake of radiolabel within lesions by 2 hours; no uptake was seen in controls. The results were confirmed in the ex vivo imaging of the explanted aorta. Quantitative annexin uptake was 9.3-fold higher in lesion versus nonlesion areas; the lesion-to-blood ratio was 3.0+/-0.37. Annexin uptake paralleled lesion severity and macrophage burden; no correlation was observed with smooth muscle cells. DNA fragmentation staining of apoptotic nuclei was increased in advanced lesions with evolving necrotic cores, predominantly in macrophages; the uptake of radiolabel correlated with the apoptotic index. CONCLUSIONS: Because annexin V clears rapidly from blood and targets apoptotic macrophage population, it should constitute an attractive imaging agent for the noninvasive detection of unstable atherosclerotic plaques.

Animals↗
PubMed2003

Hemodynamic and renal effects of low-dose brain natriuretic peptide infusion in humans: a randomized, placebo-controlled crossover study.

Brain natriuretic peptide (BNP) is a cardiac hormone with natriuretic activity. The aim of this study was to investigate the cardiovascular effects of pathophysiological levels of BNP on central hemodynamics, cardiac function, renal hemodynamics and function, and microvascular hemodynamics in healthy subjects. In this double-blind, placebo-controlled crossover study, we intravenously infused BNP (4 pmol. kg-1. min-1) or placebo for 1 h on two separate days in 12 healthy subjects (mean age, 60 +/- 5 yr). Nailfold and conjunctival capillary density, finger-skin (thermoregulatory) microvascular blood flow, and cardiac output were studied before and after infusion using intravital videomicroscopy, laser-Doppler fluxmetry, and echocardiography, respectively. Furthermore, during infusion, we measured the effective renal plasma flow and glomerular filtration rate using p-aminohippurate and inulin clearances. Blood pressure and heart rate were monitored for all measurements. Compared with placebo, BNP significantly decreased stroke volume with a tendency to decrease cardiac output. With subjects in the sitting position, mean arterial pressure decreased and heart rate increased after BNP infusion, whereas with subjects in the supine position, these variables remained unchanged. BNP increased natriuresis, diuresis, glomerular filtration rate, filtration fraction, and filtered load of Na+ compared with placebo, whereas effective renal plasma flow did not change. BNP did not affect the microvascular capillary density of conjunctiva and skin, microvascular blood flow, total skin oxygen capacity, and postocclusive recruitment. These results suggest that BNP has predominantly central and renal hemodynamic effects; however, it does not influence peripheral microcirculation in skin and conjunctiva.

Blood Pressure↗
PubMed2003

Microvascular abnormalities in chronic heart failure: a cross-sectional analysis.

OBJECTIVES: Similar to what has been found in hypertension, elevated peripheral resistance in chronic heart failure (CHF) might be related to microvascular constriction and rarefaction. Our objective was to evaluate both structural and functional microvascular changes in patients with CHF in relation to left ventricular function and neurohumoral activation. METHODS: In 25 patients with mild and severe CHF (New York Heart Association class I-II [n = 11] and class III-IV [n = 14]) and 10 age-matched healthy subjects, we studied microvascular density, diameters, and morphology of the bulbar conjunctiva and skin nailfold using intravital microscopy. RESULTS: Total conjunctival microvascular density was higher in patients with mild heart failure compared with healthy controls, whereas it was lower in severe heart failure (medians, 6.75, 4.31, and 3.56 mm/mm2, respectively, p < 0.01). In patients with heart failure, venular density was correlated with left ventricular ejection fraction. Nailfold capillary recruitment during postocclusive reactive hyperemia, a measure of functional reserve capacity, was impaired in patients with severe CHF (p < 0.05). Furthermore, in severe CHF, more abnormal capillaries and enlarged diameters were found in the nailfold (p < 0.05). CONCLUSIONS: In heart failure, several microvascular abnormalities occur that differ, depending on the severity of this condition.

Aged↗
PubMed2003

Apoptosis as a therapeutic target in acutely ischemic myocardium.

In addition to necrosis, apoptosis makes a significant contribution to cell death following acute myocardial ischemia; apoptosis is particularly enhanced during reperfusion. Several studies are beginning to indicate that the cell death by apoptosis may be preventable. A boost in the research in this direction has been provided by noninvasive means of detection of apoptosis, such as that achieved by technetium-99m labeled Annexin-A5 scintigraphy. Various levels within the apoptotic cascade are being investigated as therapeutic targets. Caspase inhibitors, mitochondrial stabilizers, and Na+-H+ exchanger inhibitors reduce cell death by apoptosis and infarct size. Although most of the evidence at present is limited to the laboratory animals, this approach seems to hold a promise for the future.

Acute Disease↗
PubMed2003

Bringing cell death alive.

The role of apoptosis in ischemia and reperfusion of the heart has been widely debated. This controversy has been continued because of the lack of an apoptosis detection method that allowed obtaining detailed kinetic and quantitative information on apoptosis. Here we focus on recent findings that look into the detection of apoptosis following ischemia and reperfusion in the heart in animal models and in patients using Annexin-A5 based image technology. Following cardiac cell damage, one major characteristic finding is that apoptotic cells express phosphatidylserines (PS) on the outer leaflet of their cell membrane, serving as a "remove me" signal for the immune system. Annexin-A5, a native plasma protein with a high affinity for PS, can be used to measure this mode of cell death. Several Annexin-A5 based imaging systems have been developed to measure apoptosis from cell culture up to patients. In this review, implications, limitations, and clinical relevance of cell death imaging will be discussed.

Animals↗
PubMed2003

In vivo detection of cell death in the area at risk in acute myocardial infarction.

UNLABELLED: Annexin A5 is a phospholipid binding protein with high affinity for phosphatidyl-serine, which is externalized by cells undergoing programmed cell death. An increased programmed cell death rate has been reported in the heart after myocardial infarction (MI). The aim of this study was to correctly localize annexin A5 uptake in vivo and to determine the area at risk in humans with acute MI. METHODS: Nine patients were studied. Before reperfusion was achieved, (99m)Tc-sestamibi was injected intravenously. Myocardial (99m)Tc-sestamibi perfusion scintigraphy was performed after reperfusion. Thereafter, (99m)Tc-labeled annexin A5 was administered intravenously, followed by scintigraphic imaging of the heart. Myocardial (99m)Tc-sestamibi scintigraphy was repeated 1-3 wk after the MI onset. (99m)Tc-Annexin uptake was also studied in the subacute phase of the MI in 2 patients. RESULTS: All patients clearly showed perfusion defects on (99m)Tc-sestamibi scintigraphy in concordance with the infarct location. Furthermore, all patients showed accumulation of (99m)Tc-annexin A5 at the infarct site, indicating that cardiomyocytes with externalized phosphatidyl-serine are present in the infarct area. (99m)Tc-sestamibi defects determined 1-3 wk after the MI onset were significantly smaller than the defects in the acute phase. (99m)Tc-annexin uptake was absent in the 2 patients studied in the subacute phase. CONCLUSION: In acute MI, an increase of programmed cell death can be correctly localized in vivo in the area at risk. Furthermore, the decrease in (99m)Tc-sestamibi defect size in the subacute phase of the MI further suggests that in parts of the area at risk, reversible myocardial damage rather than necrosis is present in cardiomyocytes.

Adult↗
PubMed2002

Visualization of cell death in vivo with the annexin A5 imaging protocol.

Annexin A5 binds to phosphatidylserine (PS), which is one of the "eat me" signals at the surface of the apoptotic cell. This property has been the driving force for the research of annexin A5 as a probe to measure apoptosis in vitro and in vivo. A non-invasive imaging protocol using annexin A5 has been developed and applied successfully to measure programmed cell death programmed cell death (PCD) in patients. This review highlights the aspects of this development and discusses clinical relevance, limitations and future perspectives of this approach of visualizing cell death.

Animals↗