Integrin expression during reverse remodeling in the myocardium of heart failure patients.

BACKGROUND: The main anchoring proteins of myocardial cells with each other and with the extracellular matrix are integrins present in the membranes of myocardial cells. These integrins are important for maintaining the architecture of the myocardial tissue and the mechanotransduction in the heart. Heart failure leads to various alterations in the myocardium, such as changes in morphology, and in expression of mRNAs, miRNAs, and proteins. Left ventricular assist device (LVAD) support in heart failure patients has been described to induce reverse remodeling of the myocardium and thus to (some degree of) reversal of the aforementioned alterations. In this study, we evaluated whether changes in expression of integrins α-1, -3, -5, -6, -7, -10, -11 and ß-1, -3, -5 and -6 play a role during reverse remodeling. METHODS: Three-step immunoperoxidase staining procedures were applied on frozen heart tissue sections to locate the various integrins tested. Integrin mRNA expression was established by standard Q-PCR procedures. RESULTS: It was shown that mRNA expression of several integrins changes significantly during LVAD support, however without subsequent changes in immunohistochemical detectable quantities. Various integrins showed different locations within the myocardium. CONCLUSION: LVAD-induced reversed remodeling did not result in significant integrin protein expression, although changes in integrin mRNA expression suggested an adaptation to unloading.

Osteopontin: a potential biomarker for heart failure and reverse remodeling after left ventricular assist device support.

BACKGROUND: Left ventricular assist device (LVAD) support in end-stage heart failure (HF) leads to recovery of the patient's condition, size reduction of cardiomyocytes, and also volume reduction and change in the composition of the extracellular matrix (ECM). Myocardial expression of ECM osteopontin (OPN) protein increases with the severity of HF. We analyzed whether OPN messenger RNA expression in heart tissue and/or OPN protein in plasma are associated with reverse remodeling during LVAD support. METHODS: Plasma and heart tissue specimens of 22 end-stage HF patients before and after LVAD implantation and subsequent heart transplantation (HTx) were used to determine the concentrations of OPN protein (EIA) and OPN messenger RNA (mRNA) by quantitative polymerase chain reaction. Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to locate OPN protein and mRNA. RESULTS: The high OPN protein levels in plasma of HF patients did not differ significantly before and after LVAD support in ischemic heart disease (IHD) (pre-LVAD 167 ± 32 ng/ml; post-LVAD 165 ± 28 ng/ml) and in dilated cardiomyopathy (DCM) (pre-LVAD 99 ± 12 ng/ml; post-LVAD (142 ± 6 ng/ml). The OPN plasma levels after HTx decreased to control levels (IHD, 48 ± 6; DCM, 40 ± 5; control, 31 ± 3 ng/ml). In contrast, expression of OPN mRNA in heart biopsy specimens decreased significantly after LVAD support (the relative quantity decreased > 90% in IHD and 50% in DCM). ISH and IHC revealed that OPN was present in cardiomyocytes and in the ECM. CONCLUSIONS: Levels of OPN mRNA in the myocardium of HF patients showed a significant decrease after LVAD support but OPN protein expression did not. LVAD support only induced a decrease of OPN plasma levels in individual patients, whereas OPN plasma levels reduced significantly in all patients after HTx.

Expression of BNIP3 in invasive breast cancer: correlations with the hypoxic response and clinicopathological features.

BACKGROUND: Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a pro-apoptotic member of the Bcl-2 family induced under hypoxia. Low or absent expression has recently been described in human tumors, including gastrointestinal tumors, resulting in poor prognosis. Little is known about BNIP3 expression in invasive breast cancer. The aim of the present study was to investigate the expression of BNIP3 in invasive breast cancer at the mRNA and protein level in correlation with the hypoxic response and clinicopathological features. METHODS: In 40 cases of invasive breast cancer, BNIP3 mRNA in situ hybridization was performed on frozen sections with a digoxigenin labeled anti-BNIP3 probe. Paraffin embedded sections of the same specimens were used to determine protein expression of BNIP3, Hypoxia Inducible Factor 1 alpha (HIF-1alpha) and its downstream targets Glucose Transporter 1 (Glut-1) and Carbonic Anhydrase (CAIX) by immunohistochemistry. RESULTS: BNIP3 mRNA was expressed in 16/40 (40%) of the cases and correlated with BNIP3 protein expression (p = 0.0218). Neither BNIP3 protein nor mRNA expression correlated with expression of HIF-1alpha expression or its downstream targets. Tumors which showed loss of expression of BNIP3 had significantly more often lymph node metastases (82% vs 39%, p = 0.010) and showed a higher mitotic activity index (p = 0.027). BNIP3 protein expression was often nuclear in normal breast, but cytoplasmic in tumor cells. CONCLUSION: BNIP3 expression is lost in a significant portion of invasive breast cancers, which is correlated with poor prognostic features such as positive lymph node status and high proliferation, but not with the hypoxic response.

Histopathological comparison between endofibrosis of the high-performance cyclist and atherosclerosis in the external iliac artery.

INTRODUCTION: High performance athletes, predominantly professional cyclists, can develop symptomatic arterial flow restriction in one or both legs during exercise. The ischemic symptoms are caused by endofibrosis and/or kinking of the external iliac artery. Because these athletes are young and have no classic risk factors for atherosclerosis, endofibrosis and atherosclerosis are considered different disease entities. We compared histology of endofibrotic lesions from young sportsmen with atherosclerotic lesions of the external iliac artery in elderly individuals. METHODS AND RESULTS: Nineteen external iliac endarterectomy specimens from 18 cyclists (age 29 +/- 8 years) were compared with 42 external iliac segments from 22 elderly individuals (82 +/- 10 years). Ten arteries from elderly individuals revealed an intimal area that was >or=25% of the area encompassed by the internal elastic lamina and were considered atherosclerotic lesions. Stenosis was higher in patients (65% [interquartile range 50-75]) than in controls (11% [7-24]) (P < .0001). The endofibrotic lesions revealed loose connective tissue with moderate to high cellularity. Both in endofibrosis and atherosclerosis, most cells in the lesion were smooth muscle actin positive. In the endofibrosis specimens, loose fibers of collagen were observed, whereas in the atherosclerotic lesions collagen was mostly densely packed. Calcification of the lesion was not observed in endofibrotic lesions, whereas calcium deposition was observed in 80% of atherosclerotic lesions. Lymphocytes were present in 21% of endofibrotic lesions and in 80% of atherosclerotic cases. Macrophages were observed in 16% of endofibrotic lesions and in all atherosclerotic plaques. Luminal thrombosis was observed in one case of endofibrosis. CONCLUSION: In the external iliac artery, atherosclerotic lesions and endofibrotic lesions of high performance cyclists have distinct morphologic characteristics. Endofibrosis in the external iliac artery may serve as soil for luminal thrombosis.

Type IV collagen degradation in the myocardial basement membrane after unloading of the failing heart by a left ventricular assist device.

After left ventricular assist device (LVAD) support in patients with end-stage cardiomyopathy, cardiomyocytes decrease in size. We hypothesized that during this process, known as reverse remodeling, the basement membrane (BM), which is closely connected to, and forms the interface between the cardiomyocytes and the extracellular matrix, will be severely affected. Therefore, the changes in the myocardial BM in patients with end-stage heart failure before and after LVAD support were studied. The role of MMP-2 in this process was also investigated. Transmission electron microscopy showed that the BM thickness decreased post-LVAD compared to pre-LVAD. Immunohistochemistry indicated a reduced immunoreactivity for type IV collagen in the BM after LVAD support. Quantitative PCR showed a similar mRNA expression for type IV collagen pre- and post-LVAD. MMP-2 mRNA almost doubled post-LVAD (P<0.01). In addition, active MMP-2 protein as identified by gelatin zymography and confirmed by Western blot analysis was detected after LVAD support and in controls, but not before LVAD support. Active MMP was localized in the BM of the cardiomyocyte, as detected by type IV collagen in situ zymography. Furthermore, in situ hybridization/immunohistochemical double staining showed that MMP-2 mRNA was expressed in cardiomyocytes, macrophages, T-cells and endothelial cells. Taken together, these findings show reduced type IV collagen content in the BM of cardiomyocytes after LVAD support. This reduction is at least in part the result of increased MMP-2 activity and not due to reduced synthesis of type IV collagen.

The chemokine and chemokine receptor profile of infiltrating cells in the wall of arteries with cardiac allograft vasculopathy is indicative of a memory T-helper 1 response.

BACKGROUND: Despite improvement in short-term patient survival after heart transplantation (HTx), long-term survival rates have not improved much, mainly because of cardiac allograft vasculopathy (CAV). Cytokines and chemokines are considered to play an important role in CAV development. METHODS AND RESULTS: We focused on coronary arteries of HTx patients and made an inventory of the infiltrating cells and the expression of cytokines as well as chemokines and chemokine receptors (C+CR) in the different layers of the vessel wall with CAV. Tissue slides were stained for a variety of cell markers (CD3, CD4, CD8, CD20, CD68, CD79a), chemokines (monokine induced by interferon [MIG], interferon-inducible protein 10 [IP-10], interferon-inducible T cell-alpha chemoattractant [ITAC], RANTES [regulated on activation normal T cell expressed and secreted], and fractalkine), and chemokine receptors (CXCR3, CCR5, and CX3CR1). In reference coronary arteries (not transplanted), almost no infiltrating cells were found, and in transplanted hearts with CAV (HTx+CAV), a large number of T cells were observed (CD4:CD8=2:1), mainly localized in the neointima and adventitia. Most of these T cells appeared to be activated (human leukocyte antigen DR positive). Coronary arteries from transplanted hearts without CAV (HTx-CAV), HTx+CAV, and references were also analyzed for cytokine and C+CR mRNA expression with the use of quantitative polymerase chain reaction. Interferon-gamma was highly expressed in HTx+CAV compared with HTx-CAV. Interleukin-4 and interleukin-10 were expressed at the same level in both HTx groups and references. In HTx+CAV, all C+CR, but especially the T-helper 1 (TH1) C+CR, were more abundant than in the HTx-CAV and references. However, TH2 CCR4 expression did not differ significantly between both HTx groups. CONCLUSIONS: In coronary arteries with CAV, most T cells are CD4+ and express human leukocyte antigen DR. These activated TH cells are mainly memory TH1 cells on the basis of their C+CR profile and cytokine expression.

Brain natriuretic peptide is produced both by cardiomyocytes and cells infiltrating the heart in patients with severe heart failure supported by a left ventricular assist device.

BACKGROUND: Brain natriuretic peptide (BNP) is a cardiac neurohormone synthesized in cardiac ventricles as a result of increased wall stress. Left ventricular assist device (LVAD) support in patients with end-stage heart failure results in reduced wall stress and therefore may change BNP levels in the heart. METHODS: BNP plasma levels were measured in 17 patients with end-stage HF before LVAD implantation and at 1 week, 1 month, and 3 months after LVAD support. BNP-messenger RNA (mRNA) expression in cardiac biopsy specimens of 27 patients before and after LVAD support was determined by quantitative polymerase chain reaction. Immunohistochemistry (IHC) and IHC-double staining was used in biopsy specimens from 32 patients before and after LVAD support to localize the BNP protein expression in the heart. RESULTS: BNP plasma levels significantly decreased from 1,872 +/- 1,098 pg/ml before implantation to 117 +/- 91 pg/ml at 3 months after LVAD implantation. This decrease in plasma levels was accompanied by a significant decrease in mRNA expression (relative quantity) in the heart. IHC and IHC-double staining showed BNP immunoreactivity in the cardiomyocytes, endothelial cells, infiltrating T cells, and macrophages. CONCLUSIONS: The significant decrease in serum BNP concentration after LVAD support coincides with a decrease in BNP mRNA and protein expression in the heart. BNP is produced in the left ventricle not only by cardiomyocytes but also by endothelial cells, T cells, and macrophages. Unloading of the left ventricle by a LVAD results in decreased BNP expression in the heart and plasma and may play an important role in the reverse remodeling process of the heart.

Similar left and right ventricular sarcomere structure after support with a left ventricular assist device suggests the utility of right ventricular biopsies to monitor left ventricular reverse remodeling.

BACKGROUND: To evaluate whether the morphology of the contractile filaments in cardiomyocytes of patients with end-stage heart failure, treated with a left ventricular assist device (LVAD), is identical in the left- and right ventricle (LV, RV) and in the interventricular septum (IVS) and can be monitored by biopsies taken with a bioptome. The application of an LVAD as a bridge to recovery of cardiac function requires monitoring of myocyte recovery. The use of RV biopsies for this purpose might be feasible, if morphologic findings in the RV coincide with those in the LV. METHODS AND RESULTS: At the time of heart transplantation, myocardial biopsies of LV, RV and IVS from 13 patients after LVAD support were compared using immunohistochemistry with monoclonal antibodies against contractile proteins. Additionally, in five of these patients, small biopsies obtained with a diagnostic bioptome were compared with large transmural biopsies of the same region. Hemodynamic monitoring was performed when the patients were fully recovered from the implantation, to rule out persistent RV failure. The staining pattern of actin, myosin, tropomyosin, troponin T and C was identical in the biopsies of LV, RV and IVS. Small biopsies taken with a bioptome appeared to be representative for the larger biopsies. Hemodynamic monitoring showed absence of RV failure in our study group. CONCLUSION: In the absence of RV failure, morphology of the contractile myofilaments after LVAD support for 215+/-143 days is identical in LV, RV and IVS. This may allow monitoring of the possible occurrence of LV reverse remodeling by RV biopsies.

Cardiomyocyte death in patients with end-stage heart failure before and after support with a left ventricular assist device: low incidence of apoptosis despite ubiquitous mediators.

BACKGROUND: Left ventricular assist device (LVAD) implantation in patients with end-stage heart failure results in impressive hemodynamic improvement. The effects on myocardial apoptosis and its mediators are unknown. METHODS: Myocardial biopsies from 17 patients at the time of LVAD implantation and after explantation, at the time of heart transplantation (HTx), were examined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) reaction and with antibodies against Fas ligand (FasL), Fas, tumor necrosis factor (TNF)-alpha receptor 1 (TNF-R1), TNF-alpha receptor 2 (TNF-R2), TNF-alpha, TNF-alpha-converting enzyme (TACE), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) (PAR), caspase-3 and FLICE inhibitory protein (FLIP). RESULTS: Apoptosis incidence was low: 0.8% (range 0% to 3%) positive cardiomyocytes nuclei before support, and 0.1% (range 0% to 0.6%) after support (p < 0.01). This was accompanied by low expression of caspase-3 and high expression of the DNA repair enzyme, PARP. Its product, PAR, increased after support. Mediators and receptors inducing apoptosis as well as FLIP were widely present before and after support. CONCLUSIONS: Despite the abundant presence of mediators and receptors inducing apoptosis, the incidence of apoptosis itself was low before and after mechanical support. The abundant expression of FLIP may suggest an important role for this protein in the inhibition of cardiomyocyte death.

Left ventricular assist device in end-stage heart failure: persistence of structural myocyte damage after unloading. An immunohistochemical analysis of the contractile myofilaments.

OBJECTIVES: We sought to evaluate the contractile proteins in cardiomyocytes of patients with end-stage heart failure (HF) before and after mechanical support with a left ventricular assist device (LVAD). BACKGROUND: Improvement of myocyte dysfunction has been suggested after LVAD support. METHODS: Fourteen patients' myocardial biopsies taken at the time of LVAD implantation and after explantation, at the time of heart transplantation, were processed for routine hematoxylin-eosin staining and immunohistochemistry using monoclonal antibodies against actin, myosin, tropomyosin, troponin C and T and titin. A grading scale from 1 (abnormal staining of all myocytes, no cross-striation) to 5 (normal fiber anatomy and striation) was used. The cross-sectional area of cardiomyocytes was also measured. RESULTS: The cardiomyocytes' cross-sectional area decreased after support, from 519 +/- 94 microm(2) to 319 +/- 53 microm(2) (p < 0.001). Actin, tropomyosin, troponin C, troponin T and titin at the time of LVAD implantation showed widespread distortion of architecture; their grades were 1.4 +/- 0.6, 2.3 +/- 1.0, 2.1 +/- 0.9, 2.1 +/- 1.2 and 2.0 +/- 0.6, respectively. In contrast, myosin morphology was preserved (4.6 +/- 0.7). After LVAD support, actin, tropomyosin, troponin C, troponin T and titin showed improvement (grades 2.7 +/- 1.3 [p = 0.004], 3.2 +/- 1.2 [p = 0.021], 3.3 +/- 0.9 [p = 0.004], 3.0 +/- 1.1 [p = 0.048] and 3.1 +/- 0.9 [p = 0.001], respectively), but no normalization. The myosin pattern deteriorated slightly (3.6 +/- 1.6 [p = 0.058]). CONCLUSIONS: After LVAD support, during a period of 213 +/- 135 days in patients with end-stage HF, despite a decrease in the size of the cardiomyocytes, severe structural myocyte damage persisted. This does not support complete recovery of myocyte histologic features.