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1.  Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications 
Cell and Tissue Research  2012;348(1):119-130.
Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.
PMCID: PMC3316780  PMID: 22395775
Adipose-derived stromal cells; Platelet lysate; Cardiac differentiation; Human
2.  Colocalisation of intraplaque C reactive protein, complement, oxidised low density lipoprotein, and macrophages in stable and unstable angina and acute myocardial infarction 
Journal of Clinical Pathology  2006;59(2):196-201.
C reactive protein (CRP), an important serum marker of atherosclerotic vascular disease, has recently been reported to be active inside human atherosclerotic plaques.
To investigate the simultaneous presence of macrophages, CRP, membrane attack complex C5b–9 (MAC), and oxidised low density lipoprotein (oxLDL) in atherectomy specimens from patients with different coronary syndromes.
In total, 54 patients with stable angina (SA; n = 21), unstable angina (UA; n = 15), and myocardial infarction (MI; n = 18) underwent directional coronary atherectomy for coronary lesions. Cryostat sections of atherosclerotic plaques were immunohistochemically stained with monoclonal antibodies: anti‐CD68 (macrophages), anti‐5G4 (CRP), aE11 (MAC), and 12E7 (oxLDL). Immunopositive areas were evaluated in relation to fibrous and neointima tissues, atheroma, and media. Quantitative analysis was performed using image cytometry with systematic random sampling (percentage immunopositive/total tissue area).
Macrophages, CRP, MAC, and oxLDL were simultaneously present in a higher proportion of fibrous tissue and atheroma of atherectomy specimens from patients with UA and MI compared with SA (p<0.05). Quantitative analysis showed significantly higher mean percentages of macrophages in plaques from patients with MI (44%) than UA (30%; p<0.01) and SA (20%; p<0.001). Significantly higher mean percentages of CRP were also seen in MI (25%) and UA (25%) compared with SA (12%; p<0.05).
The presence of CRP, complement, and oxLDL in a high proportion of plaque tissue from patients with unstable coronary artery disease implies that these surrogate markers have important proinflammatory effects inside atherosclerotic plaques. This may increase vulnerability to plaque rupture and thrombosis, with subsequent clinical sequelae.
PMCID: PMC1860312  PMID: 16443738
acute coronary syndromes; atherosclerosis; plaque inflammation; C reactive protein
3.  The effect of monohydroxyethylrutoside on doxorubicin-induced cardiotoxicity in patients treated for metastatic cancer in a phase II study 
British Journal of Cancer  2007;97(8):1084-1089.
The purpose of this study was to investigate the cardioprotective effect of the semisynthetic flavonoid 7-monohydroxyethylrutoside (monoHER) on doxorubicin (DOX)-induced cardiotoxicity in a phase II study in patients with metastatic cancer. Eight patients with metastatic cancer were treated with DOX preceded by a 10 min i.v. infusion of 1500 mg m−2 monoHER. Five patients were examined by endomyocardial biopsy after reaching a cumulative dose of 300 mg m−2. Histopathological changes in the cardiomyocytes (Billingham score) were compared with those described in literature for patients treated with DOX only. The mean biopsy score of the patients was higher (2.7) than the mean score (1.4) of historical data of patients who received similar cumulative doses of DOX. Although there is a considerable variability in few investigated patients, it was indicative that monoHER enhanced DOX-induced cardiotoxicity. However, the antitumour activity of DOX seemed better than expected: three of the four patients with metastatic soft-tissue sarcoma had a partial remission and the fourth patient stable disease. It is likely that the relatively high dose of monoHER is responsible for the lack of cardioprotection and for the high response rate in patients with soft-tissue sarcoma possibly by depleting the glutathione defense system in both heart and tumour.
PMCID: PMC2360436  PMID: 17940501
monoHER; monohydroxyethylrutoside; doxorubicin; cardiotoxicity; clinical phase II study
4.  Accumulation of fibronectin in the heart after myocardial infarction: a putative stimulator of adhesion and proliferation of adipose-derived stem cells 
Cell and Tissue Research  2008;332(2):289-298.
Stem cell therapy is a promising treatment after myocardial infarction (MI). A major problem in stem cell therapy, however, is that only a small proportion of stem cells applied to the heart can survive and differentiate into cardiomyocytes. We hypothesized that fibronectin in the heart after MI might positively affect stem cell adhesion and proliferation at the site of injury. Therefore, we investigated the kinetics of attachment and proliferation of adipose-tissue-derived stem cells (ASC) on fibronectin and analysed the time frame and localization of fibronectin accumulation in the human heart after MI. ASCs were seeded onto fibronectin-coated and uncoated culture wells. The numbers of adhering ASC were quantified after various incubation periods (5–30 min) by using DNA quantification assays. The proliferation of ASC was quantified after culturing ASC for various periods (0–9 days) by using DNA assays. Fibronectin accumulation after MI was quantified by immunohistochemical staining of heart sections from 35 patients, after different infarction periods (0–14 days old). We found that ASC attachment and proliferation on fibronectin-coated culture wells was significantly higher than on uncoated wells. Fibronectin deposition was significantly increased from 12 h to 14 days post-infarction, both in the infarction area and in the border-zone, compared with the uninfarcted heart. Our results suggest that a positive effect of fibronectin on stem cells in the heart can only be achieved when stem cell therapy is applied at least 12 h after MI, when the accumulation of fibronectin occurs in the infarcted heart.
PMCID: PMC2295254  PMID: 18305959
Stem cell; Heart; Adhesion; Infarction; Adipose tissue; Human
5.  IgM colocalises with complement and C reactive protein in infarcted human myocardium 
Journal of Clinical Pathology  2005;58(4):382-388.
Aims: Reperfusion of ischaemic myocardium after acute myocardial infarction (AMI) can induce ischaemia/reperfusion (I/R) injury, as a result of local activation of the complement system. C reactive protein (CRP) is involved in this activation. This study analysed the potential role of IgM in complement activation in the infarcted human myocardium.
Methods: Immunochemical analysis was carried out on heart specimens from 59 patients who died from AMI. Serial slides of frozen tissue from the infarction site were stained for IgM, complement factors C3d and C5b–9 (membrane attack complex), and CRP.
Results: IgM deposits were found on the plasma membrane, cross striations, and in the cytoplasm of jeopardised cardiomyocytes in infarcts of one to five days duration. IgM depositions were remarkably similar to those of CRP and both complement factors. The relative staining intensities of IgM and CRP varied greatly among patients.
Conclusions: Similar to CRP, IgM targets complement locally to jeopardised cardiomyocytes in the human heart after AMI. Localisation patterns and relative staining intensities suggest that IgM and CRP recognise similar epitopes in the ischaemic heart, but that the relative contribution of each protein to complement activation in the ischaemic myocardium differs among patients.
PMCID: PMC1770638  PMID: 15790702
IgM; immunology; inflammation; myocardial infarction
6.  Anti-inflammatory agents and monoHER protect against DOX-induced cardiotoxicity and accumulation of CML in mice 
British Journal of Cancer  2007;96(6):937-943.
Cardiac damage is the major limiting factor for the clinical use of doxorubicin (DOX). Preclinical studies indicate that inflammatory effects may be involved in DOX-induced cardiotoxicity. Nɛ-(carboxymethyl) lysine (CML) is suggested to be generated subsequent to oxidative stress, including inflammation. Therefore, the aim of this study was to investigate whether CML increased in the heart after DOX and whether anti-inflammatory agents reduced this effect in addition to their possible protection on DOX-induced cardiotoxicity. These effects were compared with those of the potential cardioprotector 7-monohydroxyethylrutoside (monoHER).
BALB/c mice were treated with saline, DOX alone or DOX preceded by ketoprofen (KP), dexamethasone (DEX) or monoHER. Cardiac damage was evaluated according to Billingham. Nɛ-(carboxymethyl) lysine was quantified immunohistochemically.
Compared to saline, a 21.6-fold increase of damaged cardiomyocytes was observed in mice treated with DOX (P<0.001). Addition of KP, DEX or monoHER before DOX significantly reduced the mean ratio of abnormal cardiomyocytes in comparison to mice treated with DOX alone (P⩽0.02). In addition, DOX induced a significant increase in the number of CML-stained intramyocardial vessels per mm2 (P=0.001) and also in the intensity of CML staining (P=0.001) compared with the saline-treated group. Nɛ-(carboxymethyl) lysine positivity was significantly reduced (P⩽0.01) by DOX-DEX, DOX-KP and DOX-monoHER. These results confirm that inflammation plays a role in DOX-induced cardiotoxicity, which is strengthened by the observed DOX-induced accumulation of CML, which can be reduced by anti-inflammatory agents and monoHER.
PMCID: PMC2360105  PMID: 17325706
doxorubicin; cardiotoxicity; inflammation; Nɛ-(carboxymethyl)lysine; monoHER; anti-inflammatory agents
7.  Amyloid in the cardiovascular system: a review 
Journal of Clinical Pathology  2005;58(2):125-133.
The cardiovascular system is a common target of amyloidosis. This review presents the current clinical and diagnostic approach to amyloidosis, with the emphasis on cardiovascular involvement. It summarises recent nomenclature, classification, and pathogenesis of amyloidosis. In addition, non-invasive possibilities are discussed, together with endomyocardial biopsies in the diagnosis of cardiac amyloidosis. Finally, recent advances in treatment and prognostic implications are presented.
PMCID: PMC1770576  PMID: 15677530
8.  Myocardial air collections as a result of infection with a gas producing strain of Escherichia coli  
Journal of Clinical Pathology  2004;57(6):660-661.
Certain strains of Escherichia coli have been shown to cause gas accumulation in—for example, emphysematous pyelonephritis. This paper describes a patient with intramyocardial air collections resulting from an intramyocardial infection with gas forming E coli.
PMCID: PMC1770323  PMID: 15166278
myocardial abscess; Escherichia coli
9.  Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction 
Journal of Clinical Pathology  2003;56(3):194-199.
Background/Aims: Recent studies indicate the presence of reactive oxygen species (ROS) producing homologues of the enzymatic subunit (Nox2) of phagocytic NADPH oxidase in non-phagocytic cells. Interestingly, in these cells, ROS produced by the Nox2 homologue(s) was shown to play a role in various regulatory processes, including cell death, proliferation, and aging. The purpose of this study was to investigate whether human cardiomyocytes express Nox2.
Methods: The expression of Nox2 was studied in human cardiomyocytes using western blot and immunohistochemical analysis. To analyse the putative expression of Nox2 in human heart disease, cardiac samples from patients who had died subsequent to acute myocardial infarction (AMI) were studied.
Results: Both in western blot and immunohistochemical studies, Nox2 expression was found in normal human cardiomyocytes. In patients with AMI, a significant increase in Nox2 expression was found both in viable and in jeopardised cardiomyocytes in the infarcted area. In addition, in the “remote from infarction” area, Nox2 expression was present in cardiomyocytes, but was not increased.
Conclusions: Nox2 or its homologue(s) is expressed in normal and jeopardised human cardiomyocytes. This expression is increased in patients with AMI, suggesting a role for this ROS producing Nox2 homologue(s) in the human heart after AMI.
PMCID: PMC1769897  PMID: 12610097
acute myocardial infarction; Nox2; cardiomyocytes; immunohistochemistry; protein expression
10.  Apoptosis in myocardial ischaemia and infarction 
Journal of Clinical Pathology  2002;55(11):801-811.
Recent studies indicate that, in addition to necrosis, apoptosis also plays a role in the process of tissue damage after myocardial infarction, which has pathological and therapeutic implications. This review article will discuss studies in which the role and mechanisms of apoptosis in myocardial infarction were analysed in vivo and in vitro in humans and in animals.
PMCID: PMC1769793  PMID: 12401816
apoptosis; myocardial ischaemia; myocardial infarction; review
11.  NADPH oxidase(s): new source(s) of reactive oxygen species in the vascular system? 
Journal of Clinical Pathology  2002;55(8):561-568.
Reactive oxygen species play an important role in a variety of (patho)physiological vascular processes. Recent publications have produced evidence of a role for putative non-phagocyte NADP oxidase(s) in the vascular production of reactive oxygen species. In the present review, we discuss the detection of the different components of NADP oxidase(s) in the vascular system, together with the putative role of reactive oxygen species produced by vascular NADPH oxidase(s), in both in vitro and in vivo studies.
PMCID: PMC1769734  PMID: 12147646
NADPH oxidase; phagocytes; reactive oxygen species; atherosclerosis
12.  Localisation of C reactive protein in infarcted tissue sites of multiple organs during sepsis 
Journal of Clinical Pathology  2002;55(2):152-153.
This report hypothesises an active role for the acute phase protein, C reactive protein (CRP), in local inflammatory reactions. This was studied in infarction sites from liver and kidney in a patient who died as a result of multiple complications after cholecystectomy. In this patient, a general acute phase protein reaction was induced, with an increase in plasma CRP. In infarction sites of kidney and liver, colocalisation of CRP and activated complement were found, whereas non-infarct sites were negative for CRP and complement. These results suggest that CRP directly participates in local inflammatory processes, possibly via complement activation, after binding of a suitable ligand.
PMCID: PMC1769593  PMID: 11865015
C reactive protein; infarcted tissue; sepsis; complement
14.  Role of plaque inflammation in acute and recurrent coronary syndromes 
Netherlands Heart Journal  2004;12(3):106-109.
Inflammation plays an important role in the initiation, development, progression and complications of atherosclerotic vascular disease. Our present knowledge of the elementary role of inflammation for the onset of plaque rupture in atherosclerotic coronary lesions primarily stems from autopsy studies. However, the introduction of directional coronary atherectomy catheters has provided a unique opportunity to directly investigate the role of inflammation in coronary syndromes. In this report we describe the role of coronary plaque inflammation, as determined by immunohistochemistry, on the presentation of coronary syndromes and on the clinical outcome following percutaneous interventions.
PMCID: PMC2497049
coronary syndromes; C-reactive protein; inflammation; plaque; restenosis
15.  Apolipoprotein H, a new mediator in the inflammatory changes ensuing in jeopardised human myocardium 
Journal of Clinical Pathology  2000;53(11):863-867.
Aim—To investigate the presence of membrane "flip flop" in ischaemic human myocardium, we assessed depositions of apolipoprotein H (apoH; ß2-glycoprotein 1) in ischaemic myocardium. Serum protein apoH can bind to negatively charged phospholipids and can also inhibit blood coagulation in vitro. We hypothesised that, because of its affinity for phosphatidyl serine, apoH might bind to "flip flopped" cells and would therefore be useful as a marker for membrane flip flop in vivo.
Methods—Myocardial tissue specimens were obtained from patients who had died within 14 days of acute myocardial infarction.
Results—Immunohistochemical analysis of these specimens revealed that apoH was selectively deposited in infarcted areas of human myocardium of at least one day's duration. Depositions of apoH were not found in non-ischaemic myocardial tissue samples obtained from patients who died from other (extracardial) causes. In vitro experiments with the human leukaemia T cell line Jurkat, subjected to apoptosis by etoposide, showed that apoH was bound to the membrane of apoptotic cells. However, these experiments also indicated that flip flop itself is not sufficient for apoH binding. In addition, Jurkat cells that bound apoH were positive for activated complement complexes, as was also found in the human heart.
Conclusions—These results suggest that apoH is involved in the inflammatory processes that occur in ischaemic myocardium.
Key Words: myocardium • apolipoprotein H • inflammation • complement
PMCID: PMC1731112  PMID: 11127271
16.  Microorganisms in the aetiology of atherosclerosis 
Journal of Clinical Pathology  2000;53(9):647-654.
Recent publications have suggested that infective pathogens might play an important role in the pathogenesis of atherosclerosis. This review focuses on these microorganisms in the process of atherosclerosis. The results of in vitro studies, animal studies, tissue studies, and serological studies will be summarised, followed by an overall conclusion concerning the strength of the association of the microorganism with the pathogenesis of atherosclerosis. The role of the bacteria Chlamydia pneumoniae and Helicobacter pylori, and the viruses human immunodeficiency virus, coxsackie B virus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and measles virus will be discussed.
Key Words: atherosclerosis • Chlamydia pneumoniae • Helicobacter pylori
PMCID: PMC1731245  PMID: 11041053
17.  C-reactive protein as a pro-inflammatory mediator in cardiovascular disease by its ability to activate complement: additional proof and hypothetical mechanisms 
Netherlands Heart Journal  2002;10(4):189-197.
This study was financially supported by the Netherlands Heart Foundation, grant numbers 93-119 and 97-088. Dr. Niessen is a recipient of the Dr. E. Dekker programme of the Netherlands Heart Foundation (D99025).
PMCID: PMC2499743
myocardial infarction; myocardial ischaemia; inflammation; inflammatory mediators; C-reactive protein

Results 1-18 (18)