The influence of M-CSF on fracture healing in a mouse model.

Macrophage colony-stimulating factor 1 (M-CSF) is known to play a critical role during fracture repair e.g. by recruiting stem cells to the fracture site and impacting hard callus formation by stimulating osteoclastogenesis. The aim of this experiment was to study the impact of systemic M-CSF application and its effect on bony healing in a mouse model of femoral osteotomy. Doing so, we studied 61 wild type (wt) mice (18-week-old female C57BL/6) which were divided into three groups: (1) femoral osteotomy, (2) femoral osteotomy + stabilization with external fixator and (3) femoral osteotomy + stabilization with external fixator + systemic M-CSF application. Further, 12 op/op mice underwent femoral osteotomy and served as proof of concept. After being sacrificed at 28 days bony bridging was evaluated ex vivo with µCT, histological and biomechanical testing. Systemic M-CSF application impacted osteoclasts numbers, which were almost as low as found in op/op mice. Regarding callus size, the application of M-CSF in wt mice resulted in significantly larger calluses compared to wt mice without systemic M-CSF treatment. We further observed an anabolic effect of M-CSF application resulting in increased trabecular thickness compared to wt animals without additional M-CSF application. Systemic M-CSF application did not alter biomechanical properties in WT mice. The impact of M-CSF application in a mouse model of femoral osteotomy was oppositional to what we were expecting. While M-CSF application had a distinct anabolic effect on callus size as well as trabecular thickness, this on bottom line did not improve biomechanical properties. We hypothesize that in addition to the well-recognized negative effects of M-CSF on osteoclast numbers this seems to further downstream cause a lack of feedback on osteoblasts. Ultimately, continuous M-CSF application in the absence of co-stimulatory signals (e.g. RANKL) might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts.

Frizzled2 signaling regulates growth of high-risk neuroblastomas by interfering with ß-catenin-dependent and ß-catenin-independent signaling pathways.

Frizzled2 (FZD2) is a receptor for Wnts and may activate both canonical and non-canonical Wnt signaling pathways in cancer. However, no studies have reported an association between FZD2 signaling and high-risk NB so far. Here we report that FZD2 signaling pathways are critical to NB growth in MYCN-single copy SK-N-AS and MYCN-amplified SK-N-DZ high-risk NB cells. We demonstrate that stimulation of FZD2 by Wnt3a and Wnt5a regulates ß-catenin-dependent and -independent Wnt signaling factors. FZD2 blockade suppressed ß-catenin-dependent signaling activity and increased phosphorylation of PKC, AKT and ERK in vitro, consistent with upregulation of ß-catenin-independent signaling activity. Finally, FZD2 small interfering RNA knockdown suppressed tumor growth in murine NB xenograft models associated with suppressed ß-catenin-dependent signaling and a less vascularized phenotype in both NB xenografts. Together, our study suggests a role for FZD2 in high-risk NB cell growth and provides a potential candidate for therapeutic inhibition in FZD2-expressing NB patients.

Effects of azithromycin and tanomastat on experimental bronchiolitis obliterans.

OBJECTIVE: Azithromycin has become a standard of care in therapy of bronchiolitis obliterans following lung transplantation. Matrix metalloprotease-9 broncho-alveolar lavage levels increase in airway neutrophilia and bronchiolitis obliterans. Interleukin-17 may play a role in lung allograft rejection, and interleukin-12 is downregulated in bronchiolitis obliterans. Whether these mechanisms can be targeted by azithromycin remains unclear. METHODS: Bronchiolitis obliterans was induced by transplantation of Fischer F344 rat left lungs to Wistar Kyoto rats. Allografts with azithromycin therapy from day 1 to 28 or 56 and mono- or combination therapy with the broad-spectrum matrix metalloprotease inhibitor tanomastat from day 1 to 56 were compared to control allografts and isografts. Graft histology was assessed, and tissue cytokine expression studied using Western blotting and immunofluorescence. RESULTS: The chronic airway rejection score in the azithromycin group did not change between 4 and 8 weeks after transplantation, whereas it significantly worsened in control allografts (P = .041). Azithromycin+tanomastat prevented complete allograft fibrosis, which occurred in 40% of control allografts. Azithromycin reduced interleukin-17 expression (P = .049) and the number of IL-17(+)/CD8(+) lymphocytes at 4 weeks, and active matrix metalloprotease-9 at 8 weeks (P = .017), and increased interleukin-12 expression (P = .025) at 8 weeks following transplantation versus control allografts. CONCLUSIONS: The expression of interleukin-17 and matrix metalloprotease-9 in bronchiolitis obliterans may be attenuated by azithromycin, and the decrease in interleukin-12 expression was prevented by azithromycin. Combination of azithromycin with a matrix metalloprotease inhibitor is worth studying further because it prevented complete allograft fibrosis in this study.

Modulating the tumor microenvironment with RNA interference as a cancer treatment strategy.

The tumor microenvironment is composed of accessory cells and immune cells in addition to extracellular matrix (ECM) components. The stromal compartment interacts with cancer cells in a complex crosstalk to support tumor development. Growth factors and cytokines produced by stromal cells support the growth of tumor cells and promote interaction with the vasculature to enhance tumor progression and invasion. The activation of autocrine and paracrine oncogenic signaling pathways by growth factors, cytokines, and proteases derived from both tumor cells and the stromal compartment is thought to play a major role in assisting tumor cells during metastasis. Consequently, targeting tumor-stroma interactions by RNA interference (RNAi)-based approaches is a promising strategy in the search for novel treatment modalities in human cancer. Recent advances in packaging technology including the use of polymers, peptides, liposomes, and nanoparticles to deliver small interfering RNAs (siRNAs) into target cells may overcome limitations associated with potential RNAi-based therapeutics. Newly developed nonviral gene delivery approaches have shown improved anticancer efficacy suggesting that RNAi-based therapeutics provide novel opportunities to elicit significant gene silencing and induce regression of tumor growth. This chapter summarizes our current understanding of the tumor microenvironment and highlights some potential targets for therapeutic intervention with RNAi-based cancer therapeutics.

Single-dose GSTP1 prevents infarction-induced heart failure.

BACKGROUND: Heart failure (HF) is a common and often fatal complication of myocardial infarction (MI). Glutathione S-transferase P1-1 (GSTP1) has antiapoptotic and antiinflammatory effects and is a specific serum marker in HF patients. However, its role in HF treatment is unknown. METHODS AND RESULTS: GSTP1 effect was examined in a rat MI-induced HF model. Magnetic resonance imaging was used to examine cardiac function. GSTP1 and tumor necrosis factor α receptor-associated factor 2 (TRAF2) mRNA and protein expression were elevated in failing myocardium, although GSTP-1 binding activity to TRAF2 was not changed versus control. HF was associated with higher active JNK1 and p38 protein expression but reduced GSTP-1 binding activity to JNK1 and p38. Recombinant GSTP1 inhibited JNK1 and p38 and enhanced its own binding activity to TRAF2 and JNK1 in vitro. In the HF model, single-dose GSTP1 treatment reduced infarct area, apoptosis, and the expression of JNK1, p38, nuclear factor κB, and proinflammatory cytokines and improved thinning ratio, cardiac index and output, stroke volume, ejection fraction, regional wall motion, and survival compared with control. CONCLUSIONS: GSTP1 application early after MI results in long-term beneficial structural and functional effects that prevent progression to HF. GSTP1 could be a novel adjunct myocardial salvage approach in patients after MI.

Egr-1 upregulates Siva-1 expression and induces cardiac fibroblast apoptosis.

The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity.

BACKGROUND: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential. METHODS: We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197. RESULTS: In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo. CONCLUSIONS: These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

A Rac1/Cdc42 GTPase-specific small molecule inhibitor suppresses growth of primary human prostate cancer xenografts and prolongs survival in mice.

Deregulated Rho GTPases Rac1 and Cdc42 have been discovered in various tumors, including prostate and Rac protein expression significantly increases in prostate cancer. The Rac and Cdc42 pathways promote the uncontrolled proliferation, invasion and metastatic properties of human cancer cells. We synthesized the novel compound AZA1 based on structural information of the known Rac1 inhibitor NSC23766. In the current study we investigated the effects of inhibition of these pathways by AZA1 on prostate tumorigenicity by performing preclinical studies using a xenograft mouse model of prostate cancer. In androgen-independent prostate cancer cells, AZA1 inhibited both Rac1 and Cdc42 but not RhoA GTPase activity in a dose-dependent manner and blocked cellular migration and proliferation. Cyclin D1 expression significantly decreased following Rac1/Cdc42 inhibition in prostate cancer cells. AZA1 treatment also down-regulated PAK and AKT activity in prostate cancer cells, associated with induction of the pro-apoptotic function of BAD by suppression of serine-112 phosphorylation. Daily systemic administration of AZA1 for 2 weeks reduced growth of human 22Rv1 prostate tumor xenografts in mice and improved the survival of tumor-bearing animals significantly. These data suggest a role of AZA1 in blocking Rac1/Cdc42-dependent cell cycle progression, cancer cell migration and increase of cancer cell apoptosis involving down-regulation of the AKT and PAK signaling pathway in prostate cancer cells. We therefore propose that a small-molecule inhibitor therapy targeting Rac1/Cdc42 Rho GTPase signaling pathways may be used as a novel treatment for patients with advanced prostate cancer.

Elevated CSF1 serum concentration predicts poor overall survival in women with early breast cancer.

Colony-stimulating factor 1 (CSF1) is a key regulator of mammary gland development, and a modulator of tissue macrophages. Expression of the CSF1 receptor gene C-FMS (CSF1R) is strongly associated with poor outcome in breast cancer and results in tumor cell invasiveness and pro-metastatic behavior in vitro. However, CSF1's role as a predictive factor in breast cancer remains unclear. We have prospectively measured circulating CSF1 using ELISA in 572 women with early breast cancer and in 688 women with benign breast lesions, and correlated these concentrations with overall survival (OS), nodal status, and other clinical and histological parameters. Serum CSF1 concentrations were significantly elevated in patients with early breast cancer when compared with those with benign tumors (P<0.0001). Within breast cancer patients, CSF1 was higher in women with axillary lymph nodes (P=0.03). Serum CSF1 correlated with tumor size (P=0.002), age (P<0.001), and Ki67 expression (P=0.006). Log CSF1 serum concentrations were predictive of poor survival in both univariate (hazard ratio (HR): 3.77, 95% CI: 1.65-8.65, P=0.002) and multivariate analyses (HR: 3.1, 95% CI: 1.03-9.33, P=0.04). Post- but not premenopausal women with CSF1 serum concentrations >873  pg/ml experienced a significantly poorer outcome (P=0.004 log-rank test). Serum CSF1 concentrations are elevated in women with malignant breast tumors. In early breast cancer, elevated serum CSF1 is associated with nodal involvement, and in postmenopausal women also with poor OS.

Inhibition of stromal PlGF suppresses the growth of prostate cancer xenografts.

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.

DIGE proteome analysis reveals suitability of ischemic cardiac in vitro model for studying cellular response to acute ischemia and regeneration.

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.

The influence of low molecular weight heparin on the expression of osteogenic growth factors in human fracture healing.

PURPOSE: Anticoagulant therapy with low molecular weight heparins (LMWH) and mechanical compression is considered the gold standard for the prevention of thrombosis. However, evidence exists that LMWHs impair bone metabolism. The aim of this study was therefore to analyse alterations in the expression of M-CSF, VEGF and TGF-ß1 after treatment with enoxaparin in patients with long bone fracture to investigate the effect of LMWH on human fracture healing. METHODS: A total of 81 patients with long bone fractures were included in the study and divided into two groups. One group comprised patients who received enoxaparin and the other group, patients who did not receive enoxaparin postoperatively. Growth factor levels were analysed in patients' serum and different groups were retrospectively compared. RESULTS: M-CSF serum concentrations were found to be significantly higher only at 48 weeks after surgery in enoxaparin. Mean overall VEGF serum concentration was higher in patients with enoxaparin. TGF-ß1 serum concentrations were higher at 48 weeks after surgery in patients with enoxaparin. CONCLUSION: This is the first comparative systemic measurement of M-CSF, VEGF and TGF-ß1 serum levels in patients with and without enoxaparin after long bone fracture. Significant differences of the expression of the growth factors after enoxaparin therapy were only observed at week 48 after surgery for M-CSF and TGF-ß1.

Association of CD14+ monocyte-derived progenitor cells with cardiac allograft vasculopathy.

OBJECTIVE: The pathogenesis of cardiac allograft vasculopathy after heart transplant remains controversial. Histologically, cardiac allograft vasculopathy is characterized by intimal hyperplasia of the coronary arteries induced by infiltrating cells. The origin of these infiltrating cells in cardiac allograft vasculopathy is unclear. Endothelial progenitor cells are reportedly involved in cardiac allograft vasculopathy; however, the role of CD14(+) monocyte-derived progenitor cells in cardiac allograft vasculopathy pathogenesis remains unknown. METHODS: Monocyte-derived progenitor cells were isolated from blood mononuclear cell fractions obtained from 25 patients with cardiac allograft vasculopathy and 25 patients without cardiac allograft vasculopathy. RESULTS: Both patients with cardiac allograft vasculopathy and those without cardiac allograft vasculopathy had CD45(+), CD34(+), CD14(+), CD141(-), CD31(-) monocyte-derived progenitor cells that differentiated into mesenchymal lineages. Monocyte-derived progenitor cells formed significantly higher numbers of colonies in patients with cardiac allograft vasculopathy than in those without cardiac allograft vasculopathy; this correlated with posttransplant follow-up time. Importantly, monocyte-derived progenitor cells from patients with cardiac allograft vasculopathy expressed significantly more α smooth muscle actin and proliferated at a higher rate than did monocyte-derived progenitor cells of patients without cardiac allograft vasculopathy. In vitro experiments suggested a paracrine control mechanism in proliferation of monocyte-derived progenitor cells in cardiac allograft vasculopathy. CONCLUSIONS: These results indicate that monocyte-derived progenitor cells are associated with cardiac allograft vasculopathy, have the ability to transdifferentiate into smooth muscle cells, and thus may contribute to intimal hyperplasia of coronary arteries in cardiac allograft vasculopathy. Targeting monocyte-derived progenitor cell recruitment could be beneficial in cardiac allograft vasculopathy treatment.

Endothelin-1 governs proliferation and migration of bronchoalveolar lavage-derived lung mesenchymal stem cells in bronchiolitis obliterans syndrome.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) has an incidence of 57% at 5 years after lung transplantation, accounts for 30% of all deaths 3 years posttransplant and because treatment options are extremely limited, it constitutes a significant health care problem. Adult mesenchymal stem cells (MSCs) play a role in lung turnover; however, their role in BOS remains unknown. METHODS: MSCs were isolated from bronchoalveolar lavage (BAL) in 101 lung allograft recipients. BAL was screened by protein array and MSCs were analyzed by real-time polymerase chain reaction, proliferation, migration, and enzyme linked immunosorbent assays. RESULTS: Multipotent MSCs were isolated from BAL of lung recipients independent of BOS presence. However, MSCs from BOS patients proliferated at higher rates (P<0.001) and were associated with higher α-smooth muscle actin (P = 0.03) but lower surfactant protein B (P = 0.02) compared with those from no-BOS patients. Histological analysis revealed that MSCs are abundant in lung tissue of BOS patients. MSCs from BOS patients produced higher endothelin-1 (ET-1) amounts (P<0.001) compared with those from no-BOS; and ET-1 stimulated whereas ET-1 blockade suppressed MSC proliferation, migration, and differentiation. CONCLUSIONS: These results indicate that MSCs are associated with BOS and are governed by ET-1. Targeting MSCs by ET-1 blockade might be useful in BOS treatment.

Improvement of cardiac function in the failing rat heart after transfer of skeletal myoblasts engineered to overexpress placental growth factor.

BACKGROUND: Transplant of skeletal myoblasts is an attractive alternative to repair irreversibly damaged myocardium in ischemic heart failure. We investigated whether transplant of myoblasts overexpressing placental growth factor would stimulate angiogenesis and enhance myoblast survival in a rat heart failure model. METHODS: Three weeks after myocardial infarction, Sprague-Dawley rats in heart failure received intramyocardial injections of Ringer solution (control) or autologous myoblasts, unmodified or transfected with placental growth factor expression plasmid. Sham-operated animals served as noninfarct controls. Cardiac function was assessed by echocardiography to 86 days after engraftment. Immunocytochemistry and fluorescence imaging were used to investigate vessel formation, grafted myoblast survival, infarct wall thickness, and infarct size. Quantitative real-time reverse transcriptase polymerase chain reaction and Western blotting measured tissue messenger RNA and protein expressions. RESULTS: Left ventricular function significantly improved with time, and fractional shortening on day 86 was significantly enhanced in transfected myoblast group relative to control (P < .01) and unmodified myoblast (P < .05) groups. Vascular density (P < .01) and myoblast survival (P < .05) were enhanced in rats treated with transfected myoblasts relative to other groups (P < .05). Mean fraction of fibrotic scar tissue was decreased in unmodified and transfected myoblast groups relative to controls on day 86 (P < .05), and left ventricular wall thickness was significantly increased in transfected myoblast group relative to other groups (P < .05). CONCLUSIONS: Intramyocardial injections of autologous myoblasts overexpressing placental growth factor improved cardiac function, attenuated adverse cardiac remodeling, induced angiogenesis, and probably enhanced survival of grafted myoblasts.

Endothelin-1 is a useful biomarker for early detection of bronchiolitis obliterans in lung transplant recipients.

OBJECTIVES: Bronchiolitis obliterans (BO) is a severe complication limiting long-term survival after lung transplantation. To date, no cure exists for BO, and the mechanisms leading to BO are not well understood. Endothelin-1 (ET-1) is a potent mitogenic and profibrotic peptide produced by pulmonary vascular endothelial cells that play a role in the pathophysiology of lung allograft dysfunction. Whether ET-1 could predict BO syndrome (BOS) development is unknown. METHODS: Transbronchial biopsy specimens and serum and bronchoalveolar lavage were obtained from 30 lung transplantation patients with and 30 without BOS at 3 points. The serum and bronchoalveolar lavage ET-1 concentrations were measured by enzyme-linked immunosorbent assay, and the ET-1 mRNA expression in the transbronchial biopsy specimens was examined using real-time polymerase chain reaction. RESULTS: The pretransplant ET-1 serum concentrations were greater in the patients with BOS (P = .02); and ET-1 mRNA was significantly upregulated in the lung grafts of those with versus those without BOS at 3 and 12 months after transplant (P = .01). At 3 and 12 months after transplantation, the ET-1 concentrations were significantly elevated in the serum (P < .01 and P < .0001, respectively) and bronchoalveolar lavage (P < .01 and P = .02, respectively) of patients with compared with those without BOS. On logistic regression analysis, the pretransplant and 3-month post-transplant serum ET-1 level predicted for BOS (odds ratio, 1.01; 95% confidence interval, 1.004-1.025; P < .007; odds ratio, 2.9; 95% confidence interval, 1.01-8.52; P < .001). The serum ET-1 level at 12 months was diagnostic for BOS (odds ratio, 3.9; 95% confidence interval, 1.42-10.80; P = .008). CONCLUSIONS: Elevated serum ET-1 concentrations were predictive of BOS, and the assessment of circulating ET-1 might be beneficial in diagnosing and monitoring BO.

Donor myocardial apollon mRNA is associated with cardiac allograft rejection.

BACKGROUND: Endomyocardial biopsy (EMB) is the "gold standard" in cardiac allograft acute cellular rejection (ACR) diagnosis, but it is invasive. ACR prediction at organ harvest could reduce EMB frequency in low-risk recipients. ACR is reportedly associated with apoptosis and Apollon inhibits apoptosis and promotes cell survival. It is unknown whether donor myocardial tissue Apollon mRNA could predict ACR. METHODS: Left ventricular biopsies (LVBs) were obtained from 336 donors before and after aortic cross-clamping (ACC) and at 10, 30 and 60 minutes of reperfusion in recipients. Tissue Apollon messenger RNA (mRNA) expression was examined by real-time reverse transcript-polymerase chain reaction (RT-PCR) and correlated with ACR episodes. RESULTS: Apollon LVB expression was significantly decreased before and after ACC in donors and at 10 minutes of reperfusion (p < or = 0.0233) in recipients who developed Grade 2R and 3R ACR as compared with those with Grade 1R or no ACR. In addition, Apollon myocardial expression was significantly decreased at 10 minutes of reperfusion in patients who developed Grade 3R ACR compared with all other ACR grades (p < or = 0.0425). In receiver-operating characteristic curve analysis, at a cut-off level of 75.66 arbitrary units, Apollon mRNA before ACC in donors identified combined Grade 2R and 3R ACR with a sensitivity of 94% and a specificity of 96% (p < 0.0001; area under the curve [AUC] = 0.965; odds ratio [OR] = 0.958; 95% confidence interval [CI] 0.942 to 0.974). CONCLUSIONS: Apollon is a sensitive and specific predictor of ACR in donated cardiac allografts and may have important implications in the choice of surveillance.

Persistent plasminogen activator inhibitor 1 gene expression in cardiac transplant recipients with idiopathic dilated cardiomyopathy.

OBJECTIVES: Plasminogen activator inhibitor 1 is the primary regulator of urokinase plasminogen activator and tissue plasminogen activator. Plasminogen activator inhibitor 1 is essential in the control of the thrombotic/fibrinolytic balance and is a marker of endothelial cell injury. Idiopathic dilated cardiomyopathy is reportedly associated with endothelial cell dysfunction. Whether endothelial cell damage plays a role in patients with dilated cardiomyopathy after cardiac transplantation remains unknown. METHODS: In this study explanted hearts of cardiac transplant recipients with ischemic cardiomyopathy and dilated cardiomyopathy, as well as control myocardial tissue, were investigated for expression of urokinase plasminogen activator, tissue plasminogen activator, urokinase plasminogen activator receptor, and plasminogen activator inhibitor 1 and 2. Furthermore, plasminogen activator inhibitor 1 expression was examined in endomyocardial biopsy specimens and sera of patients with ischemic cardiomyopathy and those with dilated cardiomyopathy during the first posttransplantation year. The effect of the patient's serum on endothelial cells was assessed in vitro to examine the role of circulating endothelial cell damage-related factors. RESULTS: Plasminogen activator inhibitor 1 expression was upregulated in ischemic cardiomyopathy and dilated cardiomyopathy myocardial tissue versus that seen in control tissue. After transplantation, plasminogen activator inhibitor 1 expression returned to control levels in patients with ischemic cardiomyopathy. In patients with dilated cardiomyopathy, plasminogen activator inhibitor 1 expression increased at 24 weeks after transplantation in both biopsy specimens and sera versus that seen in control tissue. Sera of patients with dilated cardiomyopathy, but not that of patients with ischemic cardiomyopathy, inhibited vascular endothelial growth factor A-induced proliferation of endothelial cells, although downstream target gene activation of early growth response factor 1 and NGFI-A binding protein 2 was not affected. CONCLUSIONS: These data suggest for the first time that the endothelial cell damage-related process recurs in patients with dilated cardiomyopathy after transplantation, which, independently of vascular endothelial growth factor, is associated with increased plasminogen activator inhibitor 1 expression, and that this pathology might play a role in allograft remodeling in patients with dilated cardiomyopathy.

Apollon/RNF41 myocardial messenger RNA diagnoses cardiac allograft apoptosis in rejection.

BACKGROUND: Endomyocardial biopsy (EMB) remains the gold standard for acute cellular rejection (ACR) diagnosis in cardiac transplantation yet is subject to interobserver variability. A method that could avoid discordant EMB analysis would be desirable. The apoptosis rate in EMB correlates with ACR severity. Apollon inhibits apoptosis, and RNF41 catalyzes its degradation. Whether tissue Apollon/RNF41 could diagnose ACR is not known. This study addressed this issue. METHODS: Apollon/RNF41 messenger RNA (mRNA) was measured by real time reverse-transcriptase polymerase chain reaction and apoptosis was quantified with TUNEL assays in EMBs of 268 transplant recipients. EMBs were obtained at 1, 2, 3, 4, 7, 12, 24, and 52 posttransplant weeks. RESULTS: At all time points posttransplant, Apollon mRNA decreased significantly in EMBs with ACR grades 2R/3R combined (P

Stromal cell-derived CSF-1 blockade prolongs xenograft survival of CSF-1-negative neuroblastoma.

The molecular mechanisms of tumor-host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony-stimulating factor-1 (CSF-1) production to recruit tumor-associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF-1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF-1-expressing SK-N-AS and CSF-1-negative SK-N-DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF-1. Significant suppression of both SK-N-AS and SK-N-DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)-12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF-1 blockade. Furthermore, Tie-2-positive and -negative TAMs recruited by host CSF-1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host-CSF-1 blockade prolonged survival only in CSF-1-negative SK-N-DZ NB. These studies demonstrated that increased CSF-1 production by host cells enhances TAM recruitment and NB growth and that the CSF-1 phenotype of NB tumor cells adversely affects survival.