Time for primary review: 37 days Aims Peroxisome proliferator-activated receptor-a (PPARa) is a n... more Time for primary review: 37 days Aims Peroxisome proliferator-activated receptor-a (PPARa) is a nuclear receptor regulating cardiac metabolism that also has anti-inflammatory properties. Since the activation of inflammatory signalling pathways is considered to be important in cardiac hypertrophy and fibrosis, it is anticipated that PPARa modulates cardiac remodelling. Accordingly, in this study the hypothesis was tested that the absence of PPARa aggravates the cardiac hypertrophic response to pressure overload. Methods and results Male PPARa2/2 and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days. TAC resulted in a more pronounced increase in ventricular weight and left ventricular (LV) wall thickness in PPARa2/2 than in wild-type mice. Compared with sham-operated mice, TAC did not affect cardiac function in wild-type mice, but significantly depressed LV ejection fraction and LV contractility in PPARa2/2 mice. Moreover, after TAC mRNA levels of hypertrophic (atrial natriuretic factor, a-skeletal actin), fibrotic (collagen 1, matrix metalloproteinase-2), and inflammatory (interleukin-6, tumour necrosis factor-a, cyclo-oxygenase-2) marker genes were higher in PPARa2/2 than in wild-type mice. The mRNA levels of genes involved in fatty acid metabolism (long-chain acyl-CoA synthetase, hydroxyacyl-CoA dehydrogenase) were decreased in PPARa2/2 mice, but were not further compromised by TAC. Conclusion The present findings show that the absence of PPARa results in a more pronounced hypertrophic growth response and cardiac dysfunction that are associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. These findings indicate that PPARa exerts salutary effects during cardiac hypertrophy.
Objective Until today, the true pathophysiology of hemorrhoidal disease (HD) has not yet been unr... more Objective Until today, the true pathophysiology of hemorrhoidal disease (HD) has not yet been unraveled. More and more evidence guides us towards the hypothesis that reduced connective tissue stability is associated with a higher incidence of hemorrhoids. The present study aimed to compare the quantity and quality of collagen, and vessel morphometrics, in patients with symptomatic HD compared with normal controls. Methods Twenty-two samples of grade III and grade IV HD tissue from patients undergoing a hemorrhoidectomy between January 2004 and June 2015 were included in the study group. Samples of 15 individuals without symptomatic HD who donated their body to science and died a natural death served as controls. The quantity and quality of anal collagen, and anal vessel morphometrics were objectified. The quality of collagen was subdivided in young (immature) and old (mature) collagen. Results Patients with HD had an increased percentage of total anal collagen (62.1 ± 13.8 versus 18...
Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease pr... more Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure.
Basement membrane deposition in benign and malignant naevo-melanocytic lesions: an immunohistoche... more Basement membrane deposition in benign and malignant naevo-melanocytic lesions: an immunohistochemical study with antibodies to type IV collagen and laminin Patterns of bascmcnt membrane deposition wcrc investigated in bcnign and malignant nacvo-melanocytic lcsions using antibodies t o typc IV collagen and laminin. Parafin sections rcquircd pretreatment with 6 M guanidine-HCI in addition t o pepsin prctreatment. Basement mcmbrane deposition was found around clusters as well as individual nacvo-melanocytic cells in contact with dermal stroma. However, bctwccn kcratinocytcs and intrd-cpidcrmally located nacvo-mclanocytic cells, bascmcnt membrane immunostaining could not bc dctcctcd. Tumour cellstromal interaction is apparently a prerequisitc for basement niembranc dcposition in naevo-mclanocytic lesions. Bascmcnt membrane discontinuitics, in the absence of inflammatory infiltratc. appeared, in doubtful cascs, to be evidencc i n favour of malignant melanoma. Thc gcncral pattern of basemcnt rncmbrane deposition in benign and malignant lesions was found to be similar and thcrclorc of no help in diffcrcntial diagnosis. ldcntification of hyaline bodics, which show ininiunorcactivity with antibodies to bascrnent membrane components, may be hclpful in distinguishing bctwccn Spitz naevi and malignant melanomas. Dctcction of vascular invasion, a prognostic indicator in malignant melanoma, is facilitated by bascmcnt mcmbranc immunostaining.
Calcium channel antagonists (CCAs) have been proposed to prevent cardiac events after myocardial ... more Calcium channel antagonists (CCAs) have been proposed to prevent cardiac events after myocardial infarction (MI). However, unwanted effects, such as negative inotropy, limit their use in many cases. The aim of this study was to compare the effects of long-term treatment with the CCAs, mibefradil, verapamil, and amlodipine, administered before and after chronic MI on myocardial remodeling and cardiac function. MI was induced by permanent ligation of the left coronary artery in male Wistar rats. Infarcted animals were treated with placebo, mibefradil (10 mg/kg/d po), verapamil (8 mg/kg bid po), or amlodipine (4 mg/kg/d po). Treatment was started 7 days before or 3 h after MI induction. Six weeks after MI, mean arterial blood pressure (MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), and cardiac contractility (dP/dt max) were measured. Morphometric parameters such as infarct size (IS), left ventricular dilation (LVD), septal thickness (ST), and cardiac fibrosis were determined in picrosirius red-stained hearts. Six weeks after MI, MAP and dP/dt max were decreased, whereas LVEDP and HR were increased in placebo-treated controls. The hearts featured an IS of 45%, left ventricular dilation, cardiac fibrosis, and septal thinning. MAP of all CCA-treated animals was increased, whereas LVEDP was decreased and dP/dt max increased 7-day pre-and 3-h post-MI started in mibefradil-and amlodipine-treated animals, but not in verapamil-treated animals. In contrast to amlodipine treatment, before and after MI started mibefradil and verapamil treatment decreased HR. Pretreatment with all CCA reduced IS and increased ST, whereas only mibefradil and amlodipine pretreatment prevented LVD and cardiac fibrosis. After MI started treatment with mibefradil and amlodipine reduced IS and cardiac fibrosis, and increased ST. Long-term treatment with the CCAs mibefradil, verapamil, and amlodipine reduced myocardial remodeling and improved cardiac function in MI-induced heart failure in rats.
Immunucytachemical detection of basement membrane antigens in the histopathological evaluation of... more Immunucytachemical detection of basement membrane antigens in the histopathological evaluation of laryngeal dysplasia and neoplasia Immunocytochemical detection of intrinsic components of the basement membrane (type IV collagen and laminin) was performed in biopsies of carcinoma in situ, dysplasia and hyperplasia of the laryngeal mucosa. We found a distinct and continuous basement membrane, containing both laminin and type IV collagen, at the border between epithelial cells and mesenchymal stroma in normal as well as in hyperplastic and dysplastic mucosa. In contrast, irregular discontinuities were found in some cases of carcinoma in situ and in invasive carcinoma. In addition, immunoreactivity for intracytoplasmic basement membrane components was noticed occasionally in neoplastic epithelial cells. In areas of inflammation, infiltration of leucocytes into the epithelium was occasionally accompanied by sharply defined small interruptions of the basement membrane. Our results indicate that immunohistochemical detection of basement membrane components can be of value for the demonstration of microinvasive growth in laryngeal cancer.
We explored effects of a1-adrenoreceptor blockade with prazosin on the increased vascular smooth ... more We explored effects of a1-adrenoreceptor blockade with prazosin on the increased vascular smooth muscle cell (SMC) DNA synthesis induced by angiotensin II (Ang II) in rats. Ang II was infused with or without prazosin or its solvent. Observations were compared with those in rats receiving saline or solvent. In group A, Ang II was infused for 2 weeks by subcutaneously implanted osmotic minipumps at a rate of 35 ng/100 g per minute. Group B received Ang II together with the a1-adrenoreceptor antagonist prazosin (0.35 gg/100 g per minute). Group C received Ang II and 50% dimethyl sulfoxide (DMSO), the solvent of prazosin; group D received 50%S DMSO; and group E received 0.9%Yo NaCI (Ang II vehicle). All animals were infused with 5-bromo-2'-deoxyuridine for 2 weeks via separate minipumps to measure DNA synthesis. Ang II significantly increased the fraction of DNA synthesizing SMCs in the media of the thoracic aorta from 0.4+0.1 % (mean± SD) in group E (n=6) to 10.8±7.0% in group A (n=8). Addition of prazosin to Ang II reduced the labeling fraction of SMCs to 3.0+2.2% (group B, n=9). The remaining SMC DNA synthesis in the prazosin-treated group was probably due to the effects of the solvent of prazosin, i.e., 50% DMSO, since infusion of 50%o DMSO alone increased the labeling fraction to 4.1±+2.0%o (group D, n =6). Comparable results were obtained in the carotid artery. Both systolic blood pressures and medial cross-sectional areas increased during Ang II infusion, but these parameters were not altered by prazosin or DMSO. We conclude that cal-adrenoreceptors are, either indirectly or by parallel stimulation, involved in the Ang lI-induced increase of medial SMC DNA synthesis in the rat. Stimulation of al-adrenoreceptors rather than an elevated blood pressure stimulated vascular SMC DNA synthesis. (Circulation Research 1992;70:1122-1127) KEY WORDs * smooth muscle cell * DNA synthesis * angiotensin H * eta-adrenoreceptor T here is considerable interest in control mechanisms of accelerated smooth muscle cell (SMC) DNA synthesis as a critical event in the pathogenesis of hypertension and atherosclerosis.1-3 Although most attention has been devoted to polypeptide growth factors as initiators of this response, recent studies indicate that the variety of substances that may be involved in the regulation of vascular SMC DNA synthesis includes endogenous regulators of arterial blood pressure, such as angiotensin II (Ang II).4-6 Evidence that Ang II increases vascular SMC DNA synthesis in vivo includes effects of inhibitors as well as agonists. The thoracic aorta of the two-kidney, one-clip Goldblatt hypertensive rat, an Ang II-dependent model From the Departments of Pathology (E.
Journal of Molecular and Cellular Cardiology, Sep 1, 1997
Fibronectin is a known chemoattractant for several cell types which play a role in the wound heal... more Fibronectin is a known chemoattractant for several cell types which play a role in the wound healing process, like fibroblasts, endothelial cells and macrophages. In addition, fibronectin generates a scaffold to which other extracellular matrix components can attach. The possible involvement of fibronectin in the wound healing process after myocardial infarction (MI) was investigated by studying the expression of fibronectin isoforms after induction of a MI in the rat. Deposition of plasma (pFN) and cellular fibronectin (cFN) protein was determined immunohistochemically, using monoclonal antibodies specific for cFN and polyclonal anti-human total FN (tFN antibodies). Expression of the mRNAs of total cFN and the embryonic isoforms EIIIA and EIIIB was investigated, using in situ hybridization (ISH). The ratio between EIIIA containing fibronectin (EIIIA+-FN) mRNA and total cFN mRNA was determined using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). cFN protein was present from day 4 until day 35 after infarction and was located around the area of infarction, in the epi- and endomyocardium and in the wall of larger vessels. pFN was found in the infarcted cardiomyocytes 1 day after the induction of the MI. From day 4 on pFN protein deposition was found in the border zone of the infarction and in the wall of larger vessels. pFN immunoreactivity remained present at high levels around the area of infarction and in the vessel wall throughout the entire period of investigation (90 days). From day 35 after the infarction pFN protein was detected in cardiomyocytes of the right ventricle and septum. cFN mRNA, determined by in situ hybridization, was present in the border zone of the infarcted area as early as 1 day after MI, and its expression peaked at 4 days after MI. Four days after MI the mRNA's coding for both the embryonic isoforms EIIIA and EIIIB could also be detected in the same area. Because expression of the EIIIA isoform was more abundant than the EIIIB isoform we only determined the percentage of the EIIIIA containing isoform from total FN. EIIIA+ mRNA was elevated 1 day after MI. We conclude that various fibronectin isoforms including the embryonic isoforms accumulate in the heart after MI. This suggests that these isoforms may play a role in the wound healing process in the left ventricle of the infarcted heart.
The cardiac extracellular matrix consists of a three-dimensional structural network of interstiti... more The cardiac extracellular matrix consists of a three-dimensional structural network of interstitial collagens to which other matrix components are attached. The main physiological functions of this network are to retain tissue integrity and cardiac pump function. Collagen deposition is controlled and can be modulated by hormonal factors, growth factors, cytokines, regulatory proteins and/or hemodynamic factors. Increased collagen deposition is a prerequisite to prevent dilatation of the infarcted area. Excessive accumulation of collagen leads to ventricular diastolic and systolic dysfunction and ultimately contributes to heart failure. An appropriate balance of extracellular matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. A disbalance in the extracellular matrix turnover either by decreased matrix synthesis and/or increased degradation leads to less than normal extracellular matrix in the myocardium which in its turn may lead to cardiac dilatation or even rupture. Extracellular matrix degrading enzymes expressed after myocardial infarction belong to the families of serine and matrix metalloproteinases (MMPs) and are secreted as latent proenzymes that have to be activated. It is crucial to keep the activity of these enzymes under tight control by either influencing the synthesis, activation or inhibition by tissue inhibitors of MMPs (TIMPs) or alpha2-macroglobulin. First studies using MMP inhibitors in experimental models of myocardial infarction seem to give attenuation of ventricular geometry but not always improvement of cardiac function. A central role in the activation of MMPs plays the plasminogen-plasmin system. Invasion of inflammatory cells and hitherto the rest of the wound healing cascade is inhibited in plasminogen or uPA deficient mice, most likely by the inhibition of MMP activity. Regulating the balance of extracellular matrix remodeling either by extracellular matrix synthesis or degradation might be one of the possible prevention mechanisms for heart failure. But also regeneration of the vascular and cardiomyocyte network might be potential new treatments for people with heart failure.
The plasminogen system plays an important role in the proteolytic degradation of extracellular ma... more The plasminogen system plays an important role in the proteolytic degradation of extracellular matrices during wound healing. In the present study we investigated the impact of the plasminogen system on cardiac wound healing and function after myocardial infarction. Myocardial infarction was induced in plasminogen-deficient mice (Plg؊/؊) and in wild-type controls (Plg؉/؉). Structural analysis 1, 2, and 5 weeks after infarction revealed that infarct healing was virtually abolished in Plg؊/؊ mice, indicating that the plasminogen system is required for the repair process of the heart after infarction. In the absence of plasminogen, inflammatory cells did not migrate into the infarcted myocardium. Necrotic cardiomyocytes were not removed and the formation of granulation tissue and fibrous tissue did not occur. In these nonhealing infarcted hearts, LV dilatation was not altered. In addition, gelatinolytic activity of MMP-2 and MMP-9 was depressed in the Plg؊/؊ infarcted hearts, suggesting that the plasmin effect on infarct healing may be mediated by MMPs. Surprisingly, cardiac function was only attenuated to a rather small extent in the Plg؊/؊ infarcted mice when compared to the wild-types. This study provides direct prove that plasmin-mediated proteolysis plays a central role in cardiac wound healing after myocardial infarction in mice.
In this paper we describe the development of basement membrane (BM) reactive monoclonal antibodie... more In this paper we describe the development of basement membrane (BM) reactive monoclonal antibodies (MA), by immunization of mice with intact denuded BM. The MA raised against denuded amniotic BM (clones 1052, 1053 and 1065) showed heterogeneous staining patterns. MA 1052 and 1053 reacted with epithelial BM of the epidermis and epidermal adnexa and furthermore with the epithelial alveolar BM in the lung and the superficial part of the epithelial BM in the gastrointestinal tract. MA 1065 showed immunoreactivity with the epithelial BM of epidermis and epidermal adnexa and the epithelial BM of trachea and oesophagus, and furthermore pericellular staining of the basal keratinocytes and basal corneal epithelial cells. MA 1087, raised against human glomerular BM, showed immunoreactivity with all BM, except the central epithelial BM in the cornea. The precise localization of the target epitopes in the BM was investigated on chemically cleaved human skin. Reactivity for the MA occurred predominantly in the BM lamina adherent to the dermis, suggesting that the target epitopes reside in the lamina densa and/or lamina fibroreticularis. We furthermore examined the nature of the epitopes by preincubation of tissue sections with various enzymes prior to immunohistochemistry. The reactivity of the target epitopes was not affected by bacterial coltagenase, but after various protease treatments the reactivity disappeared, suggesting that the epitopes are not localized on the triple helical part of collagenous proteins.
Basement membrane (BM) deposition at the inter-face of tumor cells and stroma was studied in 27 b... more Basement membrane (BM) deposition at the inter-face of tumor cells and stroma was studied in 27 bronchogenic squamous cell carcinomas. Specimens from peripheral and central parts of each tumor were collected. These were frozen, formalin fixed and paraffin embedded or fixed in Karnovsky's fixative, and processed for electron microscopy. With the use of antibodies to type IV collagen and laminin, the BM was visualized by light microscopy with an indirect immunoperoxidase technique. Light microscopic findings were compared to ultrastructural observations. The peripheral parts of the tumors showed continuous BM in a recognizable preexisting alveolar pattern without evidence of invasive growth into the alveolar septa. In contrast, central parts showed highly variable BM deposition ranging from continuous to almost completely absent. Alveolar patterns were not observed in the tumor centers. The stromal compartment of the tumor centers contained many spindle cells with irregular pericellular BM-like material that could be identified ultrastructurally as myofibroblasts. Electron microscopy and immunohistochemistry yielded virtually identical results. It is concluded that invasive growth in bronchogenic squamous cell carcinomas occurs in central parts of the tumor when the tumor periphery shows expansive growth without invasion of alveolar septa. The situation is different in invasive squamous cell carcinomas originating from other organs because of anatomical differences between the lung and solid organs.
Background-Adipose tissue (AT) dysfunction in obesity contributes to chronic, low-grade inflammat... more Background-Adipose tissue (AT) dysfunction in obesity contributes to chronic, low-grade inflammation that predisposes to type 2 diabetes mellitus and cardiovascular disease. Recent in vitro studies suggest that AT hypoxia may induce inflammation. We hypothesized that adipose tissue blood flow (ATBF) regulates AT oxygen partial pressure (AT PO 2), thereby affecting AT inflammation and insulin sensitivity. Methods and Results-We developed an optochemical measurement system for continuous monitoring of AT PO 2 using microdialysis. The effect of alterations in ATBF on AT PO 2 was investigated in lean and obese subjects with both pharmacological and physiological approaches to manipulate ATBF. Local administration of angiotensin II (vasoconstrictor) in abdominal subcutaneous AT decreased ATBF and AT PO 2 , whereas infusion of isoprenaline (vasodilator) evoked opposite effects. Ingestion of a glucose drink increased ATBF and AT PO 2 in lean subjects, but these responses were blunted in obese individuals. However, AT PO 2 was higher (hyperoxia) in obese subjects despite lower ATBF, which appears to be explained by lower AT oxygen consumption. This was accompanied by insulin resistance, lower AT capillarization, lower AT expression of genes encoding proteins involved in mitochondrial biogenesis and function, and higher AT gene expression of macrophage infiltration and inflammatory markers. Conclusions-Our findings establish ATBF as an important regulator of AT PO 2. Nevertheless, obese individuals exhibit AT hyperoxia despite lower ATBF, which seems to be explained by lower AT oxygen consumption. This is accompanied by insulin resistance, impaired AT capillarization, and higher AT gene expression of inflammatory cell markers.
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication... more This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
C arotid endarterectomy (CEA) has been shown to prevent stroke in patients with high-grade caroti... more C arotid endarterectomy (CEA) has been shown to prevent stroke in patients with high-grade carotid artery stenosis. 1 It has, however, become increasingly clear that the degree of luminal stenosis alone is not the most optimal clinical decision parameter. Patients with almost occluded carotid arteries may remain completely asymptomatic throughout their lifetime, whereas strokes may occur in the absence of severe carotid stenosis because of outward arterial remodeling of a vulnerable plaque. 2 Hence, novel targets for noninvasive imaging of Background-18 F-fluorocholine (18 F-FCH) uptake is associated with cell proliferation and activity in tumor patients. We hypothesized that 18 F-FCH could similarly be a valuable imaging tool to identify vulnerable plaques and associated intraplaque inflammation and atheroma cell proliferation. Methods and Results-Ten consecutive stroke patients (90% men, median age 66.5 years, range, 59.4-69.7) with ipsilateral >70% carotid artery stenosis and who underwent carotid endarterectomy were included in the study. Before carotid endarterectomy, all patients underwent positron emission tomography to assess maximum 18 F-FCH uptake in ipsilateral symptomatic carotid plaques and contralateral asymptomatic carotid arteries, which was corrected for background activity, resulting in a maximum target-to-background ratio (TBRmax). Macrophage content was assessed in all carotid endarterectomy specimens as a percentage of CD68 +-staining per whole plaque area (plaqueCD68 +) and as a maximum CD68 + percentage (maxCD68 +) in the most inflamed section/plaque. Dynamic positron emission tomography imaging demonstrated that an interval of 10 minutes between 18 F-FCH injection and positron emission tomography acquisition is appropriate for carotid plaque imaging. TBRmax in ipsilateral symptomatic carotid plaques correlated significantly with plaqueCD68 + (Spearman's ρ=0.648, P=0.043) and maxCD68 + (ρ=0.721, P=0.019) in the 10 corresponding carotid endarterectomy specimens. TBRmax was significantly higher (P=0.047) in ipsilateral symptomatic carotid plaques (median: 2.0; interquartile range [Q1-Q3], 1.5-2.5) compared with the contralateral asymptomatic carotid arteries (median: 1.4; Q1-Q3, 1.3-1.6). TBRmax was not significantly correlated to carotid artery stenosis (ρ=0.506, P=0.135). Conclusions-In vivo uptake of 18 F-FCH in human carotid atherosclerotic plaques correlated strongly with degree of macrophage infiltration and recent symptoms, thus 18 F-FCH positron emission tomography is a promising tool for the evaluation of vulnerable plaques. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01899014.
American Journal of Respiratory Cell and Molecular Biology, Jun 1, 2010
Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cel... more Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cells, destruction of lung parenchyma, and airway wall remodeling. Hyaluronan (HA) is a component of the extracellular matrix, and low-molecular-weight (LMW) HA fragments have proinflammatory capacities. We evaluated the presence of HA in alveolar and airway walls of C57BL/6 mice that were exposed to air or cigarette smoke (CS) for 4 weeks (subacute) or 24 weeks (chronic). We measured deposition of the extracellular matrix proteins collagen and fibronectin in airway walls and determined the molecular weight of HA purified from lung tissue. In addition, we studied the expression of HA-modulating genes by RT-PCR. HA staining in alveolar walls was significantly enhanced upon chronic CS exposure, whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure. This differed from the deposition of collagen and fibronectin, which are only elevated at the chronic time point. In lungs of CS-exposed mice, the molecular weight of HA clearly shifted toward more LMW HA fragments. CS exposure significantly increased the mRNA expression of the HA synthase gene Has3 in total lung tissue, whereas the expression of Has1 was decreased. These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW) HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes. This may contribute to airway wall remodeling and pulmonary inflammation in COPD.
Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molec... more Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture. Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed. The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts. These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans.
Time for primary review: 31 days Aims Our objective was to study the effect of the genetic backgr... more Time for primary review: 31 days Aims Our objective was to study the effect of the genetic background on the wound healing process after myocardial infarction (MI) in mice. Methods and results MI was induced in five different mouse strains (BalbC, C57Bl6, FVB, 129S6, and Swiss). At 3, 14, and 28 days after MI, cardiac dimensions were monitored by echocardiography and histology, whereas cardiac function was determined by direct intraventricular pressure measurements (dP/ dt). Furthermore, matrix metalloproteinases were measured by zymography, and mRNA expression by quantitative PCR. Infarct rupture, which typically occurred at 3-6 days post-MI, was most frequent in 129S6 mice (62%), followed by C57Bl6 (36%), FVB (29%), Swiss (23%), and BalbC (5%). The high incidence of infarct rupture in 129S6 mice was associated with high systolic blood pressure and increased influx of inflammatory cells. Cardiac dilatation was most marked in Swiss mice and least prominent in 129S6 mice. The degree of dilatation was associated with a reduced ejection fraction and decreased dP/dt values at 14 and 28 days post-MI. At day 14 and 28 post-MI, secondary thinning of the infarct area was marked in BalbC, FVB, and Swiss, but absent in C57Bl6 and 129S6 mice. In the latter two groups, this was paralleled by the highest number of myofibroblasts at day 14 post-MI. Conclusion The outcome of infarct healing in mice strongly depends on genetic background. On the basis of our results, we suggest that for studies on infarct rupture, the 129S6 mouse is the background of choice, whereas BalbC and Swiss mice are the preferred models to study infarct thinning post-MI.
Time for primary review: 37 days Aims Peroxisome proliferator-activated receptor-a (PPARa) is a n... more Time for primary review: 37 days Aims Peroxisome proliferator-activated receptor-a (PPARa) is a nuclear receptor regulating cardiac metabolism that also has anti-inflammatory properties. Since the activation of inflammatory signalling pathways is considered to be important in cardiac hypertrophy and fibrosis, it is anticipated that PPARa modulates cardiac remodelling. Accordingly, in this study the hypothesis was tested that the absence of PPARa aggravates the cardiac hypertrophic response to pressure overload. Methods and results Male PPARa2/2 and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days. TAC resulted in a more pronounced increase in ventricular weight and left ventricular (LV) wall thickness in PPARa2/2 than in wild-type mice. Compared with sham-operated mice, TAC did not affect cardiac function in wild-type mice, but significantly depressed LV ejection fraction and LV contractility in PPARa2/2 mice. Moreover, after TAC mRNA levels of hypertrophic (atrial natriuretic factor, a-skeletal actin), fibrotic (collagen 1, matrix metalloproteinase-2), and inflammatory (interleukin-6, tumour necrosis factor-a, cyclo-oxygenase-2) marker genes were higher in PPARa2/2 than in wild-type mice. The mRNA levels of genes involved in fatty acid metabolism (long-chain acyl-CoA synthetase, hydroxyacyl-CoA dehydrogenase) were decreased in PPARa2/2 mice, but were not further compromised by TAC. Conclusion The present findings show that the absence of PPARa results in a more pronounced hypertrophic growth response and cardiac dysfunction that are associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. These findings indicate that PPARa exerts salutary effects during cardiac hypertrophy.
Objective Until today, the true pathophysiology of hemorrhoidal disease (HD) has not yet been unr... more Objective Until today, the true pathophysiology of hemorrhoidal disease (HD) has not yet been unraveled. More and more evidence guides us towards the hypothesis that reduced connective tissue stability is associated with a higher incidence of hemorrhoids. The present study aimed to compare the quantity and quality of collagen, and vessel morphometrics, in patients with symptomatic HD compared with normal controls. Methods Twenty-two samples of grade III and grade IV HD tissue from patients undergoing a hemorrhoidectomy between January 2004 and June 2015 were included in the study group. Samples of 15 individuals without symptomatic HD who donated their body to science and died a natural death served as controls. The quantity and quality of anal collagen, and anal vessel morphometrics were objectified. The quality of collagen was subdivided in young (immature) and old (mature) collagen. Results Patients with HD had an increased percentage of total anal collagen (62.1 ± 13.8 versus 18...
Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease pr... more Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure.
Basement membrane deposition in benign and malignant naevo-melanocytic lesions: an immunohistoche... more Basement membrane deposition in benign and malignant naevo-melanocytic lesions: an immunohistochemical study with antibodies to type IV collagen and laminin Patterns of bascmcnt membrane deposition wcrc investigated in bcnign and malignant nacvo-melanocytic lcsions using antibodies t o typc IV collagen and laminin. Parafin sections rcquircd pretreatment with 6 M guanidine-HCI in addition t o pepsin prctreatment. Basement mcmbrane deposition was found around clusters as well as individual nacvo-melanocytic cells in contact with dermal stroma. However, bctwccn kcratinocytcs and intrd-cpidcrmally located nacvo-mclanocytic cells, bascmcnt membrane immunostaining could not bc dctcctcd. Tumour cellstromal interaction is apparently a prerequisitc for basement niembranc dcposition in naevo-mclanocytic lesions. Bascmcnt membrane discontinuitics, in the absence of inflammatory infiltratc. appeared, in doubtful cascs, to be evidencc i n favour of malignant melanoma. Thc gcncral pattern of basemcnt rncmbrane deposition in benign and malignant lesions was found to be similar and thcrclorc of no help in diffcrcntial diagnosis. ldcntification of hyaline bodics, which show ininiunorcactivity with antibodies to bascrnent membrane components, may be hclpful in distinguishing bctwccn Spitz naevi and malignant melanomas. Dctcction of vascular invasion, a prognostic indicator in malignant melanoma, is facilitated by bascmcnt mcmbranc immunostaining.
Calcium channel antagonists (CCAs) have been proposed to prevent cardiac events after myocardial ... more Calcium channel antagonists (CCAs) have been proposed to prevent cardiac events after myocardial infarction (MI). However, unwanted effects, such as negative inotropy, limit their use in many cases. The aim of this study was to compare the effects of long-term treatment with the CCAs, mibefradil, verapamil, and amlodipine, administered before and after chronic MI on myocardial remodeling and cardiac function. MI was induced by permanent ligation of the left coronary artery in male Wistar rats. Infarcted animals were treated with placebo, mibefradil (10 mg/kg/d po), verapamil (8 mg/kg bid po), or amlodipine (4 mg/kg/d po). Treatment was started 7 days before or 3 h after MI induction. Six weeks after MI, mean arterial blood pressure (MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), and cardiac contractility (dP/dt max) were measured. Morphometric parameters such as infarct size (IS), left ventricular dilation (LVD), septal thickness (ST), and cardiac fibrosis were determined in picrosirius red-stained hearts. Six weeks after MI, MAP and dP/dt max were decreased, whereas LVEDP and HR were increased in placebo-treated controls. The hearts featured an IS of 45%, left ventricular dilation, cardiac fibrosis, and septal thinning. MAP of all CCA-treated animals was increased, whereas LVEDP was decreased and dP/dt max increased 7-day pre-and 3-h post-MI started in mibefradil-and amlodipine-treated animals, but not in verapamil-treated animals. In contrast to amlodipine treatment, before and after MI started mibefradil and verapamil treatment decreased HR. Pretreatment with all CCA reduced IS and increased ST, whereas only mibefradil and amlodipine pretreatment prevented LVD and cardiac fibrosis. After MI started treatment with mibefradil and amlodipine reduced IS and cardiac fibrosis, and increased ST. Long-term treatment with the CCAs mibefradil, verapamil, and amlodipine reduced myocardial remodeling and improved cardiac function in MI-induced heart failure in rats.
Immunucytachemical detection of basement membrane antigens in the histopathological evaluation of... more Immunucytachemical detection of basement membrane antigens in the histopathological evaluation of laryngeal dysplasia and neoplasia Immunocytochemical detection of intrinsic components of the basement membrane (type IV collagen and laminin) was performed in biopsies of carcinoma in situ, dysplasia and hyperplasia of the laryngeal mucosa. We found a distinct and continuous basement membrane, containing both laminin and type IV collagen, at the border between epithelial cells and mesenchymal stroma in normal as well as in hyperplastic and dysplastic mucosa. In contrast, irregular discontinuities were found in some cases of carcinoma in situ and in invasive carcinoma. In addition, immunoreactivity for intracytoplasmic basement membrane components was noticed occasionally in neoplastic epithelial cells. In areas of inflammation, infiltration of leucocytes into the epithelium was occasionally accompanied by sharply defined small interruptions of the basement membrane. Our results indicate that immunohistochemical detection of basement membrane components can be of value for the demonstration of microinvasive growth in laryngeal cancer.
We explored effects of a1-adrenoreceptor blockade with prazosin on the increased vascular smooth ... more We explored effects of a1-adrenoreceptor blockade with prazosin on the increased vascular smooth muscle cell (SMC) DNA synthesis induced by angiotensin II (Ang II) in rats. Ang II was infused with or without prazosin or its solvent. Observations were compared with those in rats receiving saline or solvent. In group A, Ang II was infused for 2 weeks by subcutaneously implanted osmotic minipumps at a rate of 35 ng/100 g per minute. Group B received Ang II together with the a1-adrenoreceptor antagonist prazosin (0.35 gg/100 g per minute). Group C received Ang II and 50% dimethyl sulfoxide (DMSO), the solvent of prazosin; group D received 50%S DMSO; and group E received 0.9%Yo NaCI (Ang II vehicle). All animals were infused with 5-bromo-2'-deoxyuridine for 2 weeks via separate minipumps to measure DNA synthesis. Ang II significantly increased the fraction of DNA synthesizing SMCs in the media of the thoracic aorta from 0.4+0.1 % (mean± SD) in group E (n=6) to 10.8±7.0% in group A (n=8). Addition of prazosin to Ang II reduced the labeling fraction of SMCs to 3.0+2.2% (group B, n=9). The remaining SMC DNA synthesis in the prazosin-treated group was probably due to the effects of the solvent of prazosin, i.e., 50% DMSO, since infusion of 50%o DMSO alone increased the labeling fraction to 4.1±+2.0%o (group D, n =6). Comparable results were obtained in the carotid artery. Both systolic blood pressures and medial cross-sectional areas increased during Ang II infusion, but these parameters were not altered by prazosin or DMSO. We conclude that cal-adrenoreceptors are, either indirectly or by parallel stimulation, involved in the Ang lI-induced increase of medial SMC DNA synthesis in the rat. Stimulation of al-adrenoreceptors rather than an elevated blood pressure stimulated vascular SMC DNA synthesis. (Circulation Research 1992;70:1122-1127) KEY WORDs * smooth muscle cell * DNA synthesis * angiotensin H * eta-adrenoreceptor T here is considerable interest in control mechanisms of accelerated smooth muscle cell (SMC) DNA synthesis as a critical event in the pathogenesis of hypertension and atherosclerosis.1-3 Although most attention has been devoted to polypeptide growth factors as initiators of this response, recent studies indicate that the variety of substances that may be involved in the regulation of vascular SMC DNA synthesis includes endogenous regulators of arterial blood pressure, such as angiotensin II (Ang II).4-6 Evidence that Ang II increases vascular SMC DNA synthesis in vivo includes effects of inhibitors as well as agonists. The thoracic aorta of the two-kidney, one-clip Goldblatt hypertensive rat, an Ang II-dependent model From the Departments of Pathology (E.
Journal of Molecular and Cellular Cardiology, Sep 1, 1997
Fibronectin is a known chemoattractant for several cell types which play a role in the wound heal... more Fibronectin is a known chemoattractant for several cell types which play a role in the wound healing process, like fibroblasts, endothelial cells and macrophages. In addition, fibronectin generates a scaffold to which other extracellular matrix components can attach. The possible involvement of fibronectin in the wound healing process after myocardial infarction (MI) was investigated by studying the expression of fibronectin isoforms after induction of a MI in the rat. Deposition of plasma (pFN) and cellular fibronectin (cFN) protein was determined immunohistochemically, using monoclonal antibodies specific for cFN and polyclonal anti-human total FN (tFN antibodies). Expression of the mRNAs of total cFN and the embryonic isoforms EIIIA and EIIIB was investigated, using in situ hybridization (ISH). The ratio between EIIIA containing fibronectin (EIIIA+-FN) mRNA and total cFN mRNA was determined using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). cFN protein was present from day 4 until day 35 after infarction and was located around the area of infarction, in the epi- and endomyocardium and in the wall of larger vessels. pFN was found in the infarcted cardiomyocytes 1 day after the induction of the MI. From day 4 on pFN protein deposition was found in the border zone of the infarction and in the wall of larger vessels. pFN immunoreactivity remained present at high levels around the area of infarction and in the vessel wall throughout the entire period of investigation (90 days). From day 35 after the infarction pFN protein was detected in cardiomyocytes of the right ventricle and septum. cFN mRNA, determined by in situ hybridization, was present in the border zone of the infarcted area as early as 1 day after MI, and its expression peaked at 4 days after MI. Four days after MI the mRNA's coding for both the embryonic isoforms EIIIA and EIIIB could also be detected in the same area. Because expression of the EIIIA isoform was more abundant than the EIIIB isoform we only determined the percentage of the EIIIIA containing isoform from total FN. EIIIA+ mRNA was elevated 1 day after MI. We conclude that various fibronectin isoforms including the embryonic isoforms accumulate in the heart after MI. This suggests that these isoforms may play a role in the wound healing process in the left ventricle of the infarcted heart.
The cardiac extracellular matrix consists of a three-dimensional structural network of interstiti... more The cardiac extracellular matrix consists of a three-dimensional structural network of interstitial collagens to which other matrix components are attached. The main physiological functions of this network are to retain tissue integrity and cardiac pump function. Collagen deposition is controlled and can be modulated by hormonal factors, growth factors, cytokines, regulatory proteins and/or hemodynamic factors. Increased collagen deposition is a prerequisite to prevent dilatation of the infarcted area. Excessive accumulation of collagen leads to ventricular diastolic and systolic dysfunction and ultimately contributes to heart failure. An appropriate balance of extracellular matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. A disbalance in the extracellular matrix turnover either by decreased matrix synthesis and/or increased degradation leads to less than normal extracellular matrix in the myocardium which in its turn may lead to cardiac dilatation or even rupture. Extracellular matrix degrading enzymes expressed after myocardial infarction belong to the families of serine and matrix metalloproteinases (MMPs) and are secreted as latent proenzymes that have to be activated. It is crucial to keep the activity of these enzymes under tight control by either influencing the synthesis, activation or inhibition by tissue inhibitors of MMPs (TIMPs) or alpha2-macroglobulin. First studies using MMP inhibitors in experimental models of myocardial infarction seem to give attenuation of ventricular geometry but not always improvement of cardiac function. A central role in the activation of MMPs plays the plasminogen-plasmin system. Invasion of inflammatory cells and hitherto the rest of the wound healing cascade is inhibited in plasminogen or uPA deficient mice, most likely by the inhibition of MMP activity. Regulating the balance of extracellular matrix remodeling either by extracellular matrix synthesis or degradation might be one of the possible prevention mechanisms for heart failure. But also regeneration of the vascular and cardiomyocyte network might be potential new treatments for people with heart failure.
The plasminogen system plays an important role in the proteolytic degradation of extracellular ma... more The plasminogen system plays an important role in the proteolytic degradation of extracellular matrices during wound healing. In the present study we investigated the impact of the plasminogen system on cardiac wound healing and function after myocardial infarction. Myocardial infarction was induced in plasminogen-deficient mice (Plg؊/؊) and in wild-type controls (Plg؉/؉). Structural analysis 1, 2, and 5 weeks after infarction revealed that infarct healing was virtually abolished in Plg؊/؊ mice, indicating that the plasminogen system is required for the repair process of the heart after infarction. In the absence of plasminogen, inflammatory cells did not migrate into the infarcted myocardium. Necrotic cardiomyocytes were not removed and the formation of granulation tissue and fibrous tissue did not occur. In these nonhealing infarcted hearts, LV dilatation was not altered. In addition, gelatinolytic activity of MMP-2 and MMP-9 was depressed in the Plg؊/؊ infarcted hearts, suggesting that the plasmin effect on infarct healing may be mediated by MMPs. Surprisingly, cardiac function was only attenuated to a rather small extent in the Plg؊/؊ infarcted mice when compared to the wild-types. This study provides direct prove that plasmin-mediated proteolysis plays a central role in cardiac wound healing after myocardial infarction in mice.
In this paper we describe the development of basement membrane (BM) reactive monoclonal antibodie... more In this paper we describe the development of basement membrane (BM) reactive monoclonal antibodies (MA), by immunization of mice with intact denuded BM. The MA raised against denuded amniotic BM (clones 1052, 1053 and 1065) showed heterogeneous staining patterns. MA 1052 and 1053 reacted with epithelial BM of the epidermis and epidermal adnexa and furthermore with the epithelial alveolar BM in the lung and the superficial part of the epithelial BM in the gastrointestinal tract. MA 1065 showed immunoreactivity with the epithelial BM of epidermis and epidermal adnexa and the epithelial BM of trachea and oesophagus, and furthermore pericellular staining of the basal keratinocytes and basal corneal epithelial cells. MA 1087, raised against human glomerular BM, showed immunoreactivity with all BM, except the central epithelial BM in the cornea. The precise localization of the target epitopes in the BM was investigated on chemically cleaved human skin. Reactivity for the MA occurred predominantly in the BM lamina adherent to the dermis, suggesting that the target epitopes reside in the lamina densa and/or lamina fibroreticularis. We furthermore examined the nature of the epitopes by preincubation of tissue sections with various enzymes prior to immunohistochemistry. The reactivity of the target epitopes was not affected by bacterial coltagenase, but after various protease treatments the reactivity disappeared, suggesting that the epitopes are not localized on the triple helical part of collagenous proteins.
Basement membrane (BM) deposition at the inter-face of tumor cells and stroma was studied in 27 b... more Basement membrane (BM) deposition at the inter-face of tumor cells and stroma was studied in 27 bronchogenic squamous cell carcinomas. Specimens from peripheral and central parts of each tumor were collected. These were frozen, formalin fixed and paraffin embedded or fixed in Karnovsky's fixative, and processed for electron microscopy. With the use of antibodies to type IV collagen and laminin, the BM was visualized by light microscopy with an indirect immunoperoxidase technique. Light microscopic findings were compared to ultrastructural observations. The peripheral parts of the tumors showed continuous BM in a recognizable preexisting alveolar pattern without evidence of invasive growth into the alveolar septa. In contrast, central parts showed highly variable BM deposition ranging from continuous to almost completely absent. Alveolar patterns were not observed in the tumor centers. The stromal compartment of the tumor centers contained many spindle cells with irregular pericellular BM-like material that could be identified ultrastructurally as myofibroblasts. Electron microscopy and immunohistochemistry yielded virtually identical results. It is concluded that invasive growth in bronchogenic squamous cell carcinomas occurs in central parts of the tumor when the tumor periphery shows expansive growth without invasion of alveolar septa. The situation is different in invasive squamous cell carcinomas originating from other organs because of anatomical differences between the lung and solid organs.
Background-Adipose tissue (AT) dysfunction in obesity contributes to chronic, low-grade inflammat... more Background-Adipose tissue (AT) dysfunction in obesity contributes to chronic, low-grade inflammation that predisposes to type 2 diabetes mellitus and cardiovascular disease. Recent in vitro studies suggest that AT hypoxia may induce inflammation. We hypothesized that adipose tissue blood flow (ATBF) regulates AT oxygen partial pressure (AT PO 2), thereby affecting AT inflammation and insulin sensitivity. Methods and Results-We developed an optochemical measurement system for continuous monitoring of AT PO 2 using microdialysis. The effect of alterations in ATBF on AT PO 2 was investigated in lean and obese subjects with both pharmacological and physiological approaches to manipulate ATBF. Local administration of angiotensin II (vasoconstrictor) in abdominal subcutaneous AT decreased ATBF and AT PO 2 , whereas infusion of isoprenaline (vasodilator) evoked opposite effects. Ingestion of a glucose drink increased ATBF and AT PO 2 in lean subjects, but these responses were blunted in obese individuals. However, AT PO 2 was higher (hyperoxia) in obese subjects despite lower ATBF, which appears to be explained by lower AT oxygen consumption. This was accompanied by insulin resistance, lower AT capillarization, lower AT expression of genes encoding proteins involved in mitochondrial biogenesis and function, and higher AT gene expression of macrophage infiltration and inflammatory markers. Conclusions-Our findings establish ATBF as an important regulator of AT PO 2. Nevertheless, obese individuals exhibit AT hyperoxia despite lower ATBF, which seems to be explained by lower AT oxygen consumption. This is accompanied by insulin resistance, impaired AT capillarization, and higher AT gene expression of inflammatory cell markers.
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication... more This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
C arotid endarterectomy (CEA) has been shown to prevent stroke in patients with high-grade caroti... more C arotid endarterectomy (CEA) has been shown to prevent stroke in patients with high-grade carotid artery stenosis. 1 It has, however, become increasingly clear that the degree of luminal stenosis alone is not the most optimal clinical decision parameter. Patients with almost occluded carotid arteries may remain completely asymptomatic throughout their lifetime, whereas strokes may occur in the absence of severe carotid stenosis because of outward arterial remodeling of a vulnerable plaque. 2 Hence, novel targets for noninvasive imaging of Background-18 F-fluorocholine (18 F-FCH) uptake is associated with cell proliferation and activity in tumor patients. We hypothesized that 18 F-FCH could similarly be a valuable imaging tool to identify vulnerable plaques and associated intraplaque inflammation and atheroma cell proliferation. Methods and Results-Ten consecutive stroke patients (90% men, median age 66.5 years, range, 59.4-69.7) with ipsilateral >70% carotid artery stenosis and who underwent carotid endarterectomy were included in the study. Before carotid endarterectomy, all patients underwent positron emission tomography to assess maximum 18 F-FCH uptake in ipsilateral symptomatic carotid plaques and contralateral asymptomatic carotid arteries, which was corrected for background activity, resulting in a maximum target-to-background ratio (TBRmax). Macrophage content was assessed in all carotid endarterectomy specimens as a percentage of CD68 +-staining per whole plaque area (plaqueCD68 +) and as a maximum CD68 + percentage (maxCD68 +) in the most inflamed section/plaque. Dynamic positron emission tomography imaging demonstrated that an interval of 10 minutes between 18 F-FCH injection and positron emission tomography acquisition is appropriate for carotid plaque imaging. TBRmax in ipsilateral symptomatic carotid plaques correlated significantly with plaqueCD68 + (Spearman's ρ=0.648, P=0.043) and maxCD68 + (ρ=0.721, P=0.019) in the 10 corresponding carotid endarterectomy specimens. TBRmax was significantly higher (P=0.047) in ipsilateral symptomatic carotid plaques (median: 2.0; interquartile range [Q1-Q3], 1.5-2.5) compared with the contralateral asymptomatic carotid arteries (median: 1.4; Q1-Q3, 1.3-1.6). TBRmax was not significantly correlated to carotid artery stenosis (ρ=0.506, P=0.135). Conclusions-In vivo uptake of 18 F-FCH in human carotid atherosclerotic plaques correlated strongly with degree of macrophage infiltration and recent symptoms, thus 18 F-FCH positron emission tomography is a promising tool for the evaluation of vulnerable plaques. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01899014.
American Journal of Respiratory Cell and Molecular Biology, Jun 1, 2010
Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cel... more Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cells, destruction of lung parenchyma, and airway wall remodeling. Hyaluronan (HA) is a component of the extracellular matrix, and low-molecular-weight (LMW) HA fragments have proinflammatory capacities. We evaluated the presence of HA in alveolar and airway walls of C57BL/6 mice that were exposed to air or cigarette smoke (CS) for 4 weeks (subacute) or 24 weeks (chronic). We measured deposition of the extracellular matrix proteins collagen and fibronectin in airway walls and determined the molecular weight of HA purified from lung tissue. In addition, we studied the expression of HA-modulating genes by RT-PCR. HA staining in alveolar walls was significantly enhanced upon chronic CS exposure, whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure. This differed from the deposition of collagen and fibronectin, which are only elevated at the chronic time point. In lungs of CS-exposed mice, the molecular weight of HA clearly shifted toward more LMW HA fragments. CS exposure significantly increased the mRNA expression of the HA synthase gene Has3 in total lung tissue, whereas the expression of Has1 was decreased. These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW) HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes. This may contribute to airway wall remodeling and pulmonary inflammation in COPD.
Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molec... more Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture. Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed. The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts. These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans.
Time for primary review: 31 days Aims Our objective was to study the effect of the genetic backgr... more Time for primary review: 31 days Aims Our objective was to study the effect of the genetic background on the wound healing process after myocardial infarction (MI) in mice. Methods and results MI was induced in five different mouse strains (BalbC, C57Bl6, FVB, 129S6, and Swiss). At 3, 14, and 28 days after MI, cardiac dimensions were monitored by echocardiography and histology, whereas cardiac function was determined by direct intraventricular pressure measurements (dP/ dt). Furthermore, matrix metalloproteinases were measured by zymography, and mRNA expression by quantitative PCR. Infarct rupture, which typically occurred at 3-6 days post-MI, was most frequent in 129S6 mice (62%), followed by C57Bl6 (36%), FVB (29%), Swiss (23%), and BalbC (5%). The high incidence of infarct rupture in 129S6 mice was associated with high systolic blood pressure and increased influx of inflammatory cells. Cardiac dilatation was most marked in Swiss mice and least prominent in 129S6 mice. The degree of dilatation was associated with a reduced ejection fraction and decreased dP/dt values at 14 and 28 days post-MI. At day 14 and 28 post-MI, secondary thinning of the infarct area was marked in BalbC, FVB, and Swiss, but absent in C57Bl6 and 129S6 mice. In the latter two groups, this was paralleled by the highest number of myofibroblasts at day 14 post-MI. Conclusion The outcome of infarct healing in mice strongly depends on genetic background. On the basis of our results, we suggest that for studies on infarct rupture, the 129S6 mouse is the background of choice, whereas BalbC and Swiss mice are the preferred models to study infarct thinning post-MI.
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Papers by Jack Cleutjens