Biomedical subjects

Dietmar Abraham

Publications and source records attributed to Dietmar Abraham.

15 recordsLinked to original sources

PubMed2007

Target validation using RNA interference in solid tumors.

Reverse genetics is one strategy that is currently used to establish a link between a target gene and a disease phenotype. In this process, the function of a gene is inhibited and the consequence of its loss on a desired biological function, such as tumor growth and metastasis, is monitored. RNA interference (RNAi) has been found to be the most effective method to specifically inhibit gene expression. Notably, interactions between cancer cells, stromal cells, and the extracellular matrix (ECM) are crucial to angiogenesis and tumorigenesis. Tumor cells and the surrounding stroma are the principle source of growth factors and cytokines, which induce remodeling of the ECM mediated by metalloproteases (MMPs) secreted by macrophages. The production of macrophages is regulated by colony-stimulating factor (CSF)-1, which is overexpressed in several tumors. When short-interfering RNAs (siRNAs) targeting either the CSF-1 or its receptors were delivered into colon and breast cancer xenografts in mice, tumor growth was inhibited. Associated with this suppression, we observed decreased tumor vascularity, reduced expression of angiogenic factors and MMPs, and decreased macrophage recruitment to the tumors. The suppression of CSF-1 by RNA interference is therefore a powerful tool to block gene function and influence tumor-stroma interactions in solid tumor development.

Animals↗

PubMed2006

Colony-stimulating factor-1 antibody reverses chemoresistance in human MCF-7 breast cancer xenografts.

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol-linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringer's solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer-related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer.

Animals↗

PubMed2005

Reconstitution of blood supply of the denuded bronchial stump.

BACKGROUND: Our aim was to study the process of microcirculatory reconstitution in the bronchial stump after pneumonectomy. METHODS: Eighteen juvenile pigs (median weight 40.6 kg) were randomly assigned to three groups. In all animals left pneumonectomy was performed and the stapled bronchial stump (median length 3.8 cm) carefully denuded. Group I animals received coverage of the stump by intercostal flap. In group II, the stump was covered with TachoComb, an impermeable hemostatic fleece; and group III served as a control without any coverage of the stump. Animals were sacrificed at day 14 after surgery. Vascular density was evaluated in serial histologic sections at multiple levels stained with CD-31 antibody. One-way analysis of variance and the Wilcoxon test were used for data analysis. RESULTS: At autopsy, stumps of group III animals were totally covered by adjacent mediastinal structures. In group I, intercostal flaps were viable and completely healed to the bronchial stumps. There were no signs of infection or stump insufficiency in these groups. In all group II animals, empyema developed, and stumps were found necrotic at macroscopic and histologic evaluation. Statistical analysis revealed significantly lower vascular density of mature vessels in the area of the bronchial stump in group II compared with both other groups. CONCLUSIONS: Reconstitution of microcirculation of the denuded bronchial stump after pneumonectomy takes place in a centripetal way from adjacent viable tissue. Hence, the purpose of covering the bronchial stump is the improvement of blood supply rather than mechanical reinforcement.

Animals↗

PubMed2005

Differential role of TGF-beta1/bFGF and ET-1 in graft fibrosis in heart failure patients.

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)-1, transforming growth factor (TGF)-beta1, basic fibroblast growth factor (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and 16 controls, the myocardial bFGF, TGF-beta1, pro-collagen (PrCol) type 1 (PrCol1-alpha1, -alpha2) and MMP expressions were examined using real-time RT-PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF-beta1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF-beta1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF-beta1 downregulated MMP-1 and upregulated collagen 1, whereas bFGF upregulated MMP-13 in DCM tissue. Post-transplant PrCol1-alpha1, -alpha2 and ET-1 mRNA increased over time in grafts of DCM patients only, while other factors returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP-1/MMP-13, but not the excess collagen and ET-1 synthesis in cardiac grafts transplanted to DCM patients. ET-1 might be a major pathologic trigger for graft fibrosis in DCM.

Adult↗

PubMed2004

Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice.

Colony-stimulating factor (CSF)-1 is the primary regulator of tissue macrophage production. CSF-1 expression is correlated with poor prognosis in breast cancer and is believed to enhance mammary tumor progression and metastasis through the recruitment and regulation of tumor-associated macrophages. Macrophages produce matrix metalloproteases (MMPs) and vascular endothelial growth factor, which are crucial for tumor invasion and angiogenesis. Given the important role of CSF-1, we hypothesized that blockade of CSF-1 or the CSF-1 receptor (the product of the c-fms proto-oncogene) would suppress macrophage infiltration and mammary tumor growth. Human MCF-7 mammary carcinoma cell xenografts in mice were treated with either mouse CSF-1 antisense oligonucleotide for 2 weeks or five intratumoral injections of either CSF-1 small interfering RNAs or c-fms small interfering RNAs. These treatments suppressed mammary tumor growth by 50%, 45%, and 40%, respectively, and selectively down-regulated target protein expression in tumor lysates. Host macrophage infiltration; host MMP-12, MMP-2, and vascular endothelial growth factor A expression; and endothelial cell proliferation within tumors of treated mice were decreased compared with tumors in control mice. In addition, mouse survival significantly increased after CSF-1 blockade. These studies demonstrate that CSF-1 and CSF-1 receptor are potential therapeutic targets for the treatment of mammary cancer.

Animals↗

PubMed2004

Prediction of lung-transplant rejection by hepatocyte growth factor.

BACKGROUND: Graft rejection is a major complication of lung transplantation. No serological marker of rejection is in common use. Hepatocyte growth factor (HGF) is highly expressed in the lung and produced after acute lung injury; serum concentrations increase in inflammatory lung diseases. We investigated whether HGF could be an accurate marker for prediction of lung-graft rejection. METHODS: Serum concentrations of HGF were measured by ELISA in 109 patients who had undergone lung transplantation (65 for chronic obstructive pulmonary disease; 23 for cystic fibrosis; 21 for idiopathic lung fibrosis), comparing those who had no subsequent events and those with episodes of infection or rejection, as well as in 12 healthy controls. FINDINGS: The mean baseline serum HGF concentration was 645 ng/L (SD 259) in controls and 1358 ng/L (603) in the patients before transplantation. After transplantation the mean concentration in patients with no events was 1147 ng/L (510) compared with 1559 ng/L (323) in patients with infection (p=0.001 vs controls; change from pretransplant value not significant). Patients with rejection had significantly higher concentrations than all other groups (3972 ng/L [1463], p<0.0001). Logistic regression identified HGF as a predictor for lung graft rejection (p=0.012). After steroid treatment, HGF concentrations returned almost to the preoperative values within 3 days. INTERPRETATION: HGF might be a marker for graft rejection in lung transplantation. A potential link between viral infection, mainly cytomegalovirus, and HGF, however, remains to be investigated.

Acute Disease↗

PubMed2004

Upregulated hypoxia-inducible factor-1 DNA binding activity to the vascular endothelial growth factor-A promoter mediates increased vascular permeability in donor lung grafts.

BACKGROUND: Transplantation-induced hypoxia results in enhanced vascular permeability and tissue vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) overexpression in donor lung grafts. Promoter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF; and ET-1 potently stimulates VEGF-A production. We hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in lung grafts. METHODS: We studied the mRNA and protein expression of HIF-1 and its protein-binding capacity to the HBS of the VEGF gene in biopsies of preserved donor and control lungs, using real-time reverse transcription-polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay. Wet-to-dry lung weight ratio was measured in donor and control lungs. RESULTS: While HIF-1 alpha mRNA expression was unchanged, HIF-1 beta was downregulated (p < 0.05) in donor versus control lungs. Protein expression of both, HIF-1 alpha and -beta was significantly upregulated in donor lung grafts. HIF-1 binding to the HBS of the VEGF promoter as well as tissue fluid content were increased in donor lung biopsies versus controls (p < 0.05). CONCLUSIONS: These data indicate that upregulated HIF-1 DNA binding activity to the HBS of VEGF-A most likely contributes to elevated VEGF levels in preserved lung grafts. Unchanged HIF-1 alpha mRNA expression did not affect HIF-1 alpha protein levels. Endothelin-1 increases HIF-1 alpha accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1 alpha protein stability most likely leads to transcriptional activation of VEGF during lung graft preservation. Targeting HIF might be of benefit to counteract edema formation in preserved lung grafts.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed2003

Impaired VE-cadherin/beta-catenin expression mediates endothelial cell degeneration in dilated cardiomyopathy.

BACKGROUND: The cross-talk between vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang), and VE-cadherin coregulates endothelial cell (EC) survival. Cardiac expression of VEGF-A but not its receptor KDR is blunted in dilated cardiomyopathy (DCM). Whether VE-cadherin/Ang function is affected in DCM is unknown. METHODS AND RESULTS: The myocardial expression of VE-cadherin/beta-catenin, Ang-1, Ang-2, and their receptor Tie-2 was examined in DCM, ischemic cardiomyopathy (ICM), and in control subjects through the use of real-time RT-PCR, Western blotting, and immunocytochemistry. EC degeneration was quantified by TEM. RNA interference against VE-cadherin and VEGF deprivation and stimulation were applied to cultured DCM myocardium and human microvascular ECs to examine the interplay between VEGF, VE-cadherin/beta-catenin, and Ang-2. Analysis of tissue sections with similar rates of EC degeneration in both patient groups showed that VE-cadherin/beta-catenin expression was downregulated in DCM only (P<0.05). Although Ang-1 was not changed, Ang-2 expression was downregulated and Tie-2 protein expression was upregulated both in DCM and ICM (P<0.05). The ratio of degenerated to normal ECs was significantly higher in DCM versus ICM (P<0.05). Targeted VE-cadherin gene silencing in cultured human ECs resulted in similar degenerative effects observed in myocardial ECs of DCM patients. In vitro experiments indicated that VE-cadherin/beta-catenin expression is independent of VEGF. CONCLUSIONS: These results indicate for the first time that the EC survival is impaired in myocardium of patients with DCM involving VE-cadherin/beta-catenin, probably independent of VEGF. Targeting VE-cadherin might be of benefit to counteract the selective EC pathology in DCM.

Adult↗

PubMed2003

Low dose carboplatin combined with angiostatic agents prevents metastasis in human testicular germ cell tumor xenografts.

PURPOSE: Low dose chemotherapy combined with angiogenesis inhibitors has been shown to be more effective for experimental tumor treatment than chemotherapy alone. To our knowledge whether germ cell tumors could benefit from this treatment strategy remains to be evaluated. We examined the efficacy of angiostatic thrombospondin-1 (TSP-1), endostatin and combined angiostatic/low dose carboplatin in mice xenografted with human nonseminomatous germ cell tumor. MATERIALS AND METHODS: We monitored tumor progression and angiogenesis in the established model of human nonseminomatous germ cell tumor xenograft in 120 SCID mice using intravital video microscopy, immunocytochemistry and real-time polymerase chain reaction. Mice received TSP-1 (20 mg/kg daily) or endostatin (10 mg/kg daily) subcutaneously (via osmotic mini pumps) for 2 weeks starting 15 days after cancer cell grafting, carboplatin cycled twice (30 mg/kg intraperitoneally days 14 and 21 after cancer cell grafting), or a combination of carboplatin with TSP-1 or endostatin. Untreated, sham and tumor bearing mice treated with Ringer's solution served as controls. RESULTS: Primary tumor development was not affected in mice treated with TSP-1, endostatin or carboplatin alone. All animals had metastases at 6 months, while metastasis did not develop following the combination of carboplatin with TSP-1 or endostatin. This combined therapy suppressed tumor angiogenesis, enhanced apoptosis in tumor cells and decreased vascular endothelial growth factor-A tissue mRNA expression vs controls (p <0.05). CONCLUSIONS: These data indicate that angiostatic agents added to low dose carboplatin have the ability to suppress the progression of human germ cell tumor xenografts toward a metastatic phenotype. Therefore, this treatment strategy might be beneficial to prevent metastasis in germ cell tumors.

Angiogenesis Inhibitors↗

PubMed2002

Colony-stimulating factor-1 antisense treatment suppresses growth of human tumor xenografts in mice.

Matrix metalloproteinases (MMPs) foster cellular invasion by disrupting extracellular matrix barriers and thereby facilitate tumor development. MMPs are synthesized by both cancer cells and adjacent stromal cells, primarily macrophages. The production of macrophages is regulated by colony-stimulating factor-1 (CSF-1). Tissue CSF-1 expression increased significantly in embryonic and colon cancer xenografts. We, therefore, hypothesized that blocking CSF-1 may suppress tumor growth by decelerating macrophage-mediated extracellular matrix breakdown. Cells expressing CSF-1 and mice xenografted with CSF-1 receptor (c-fms)- and CSF-1-negative malignant human embryonic or colon cancer cells were treated with mouse CSF-1 antisense oligonucleotides. Two weeks of CSF-1 antisense treatment selectively down-regulated CSF-1 mRNA and protein tissue expression in tumor lysates. CSF-1 blockade suppressed the growth of embryonic tumors to dormant levels and the growth of the colon carcinoma by 50%. In addition, tumor vascularity and the expression of MMP-2 and angiogenic factors were reduced. Six-month survival was observed in colon carcinoma mice only after CSF-1 blockade, whereas controls were all dead at day 65. These results suggest that human embryonic and colon cancer cells up-regulate host CSF-1 and MMP-2 expression. Because the cancer cells used were CSF-1 negative, CSF-1 antisense targeted tumor stromal cell CSF-1 production. CSF-1 blockade could be a novel strategy in treatment of solid tumors.

Animals↗

PubMed2002

VEGF-A and -C but not -B mediate increased vascular permeability in preserved lung grafts.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor, expressed in the lung. Overexpression of VEGF is associated with increased vascular permeability in the early stage of acute lung injury in mice. The role of various forms of VEGF in transplantation-induced lung injury is not well understood. METHODS: VEGF mRNA and protein expression was measured in biopsies of preserved donor lung grafts as well as in control lung biopsies, using real-time reverse transcriptase-polymerase chain reaction and Western blot analysis. VEGF tissue expression was also evaluated by immunocytochemistry. Serum VEGF was measured in recipients after transplantation and in controls using ELISA. RESULTS: Although VEGF-A and VEGF-C protein expression was up-regulated, their mRNA levels were decreased in donor versus control lung biopsies (P<0.05). VEGF-B mRNA was decreased, but its protein level was unchanged in donors. Flt-1 was unchanged, KDR gene expression was down-regulated in donors (P<0.05), and both receptors' protein expression was under the detection level in donor and control lungs. VEGF-A was detected in pulmonary vessels and bronchi, whereas VEGF-C was only detectable in vessels of both donor and control lungs. After transplantation, serum VEGF increased (P<0.05) and returned to control baseline levels 12 weeks after surgery. Wet-to-dry lung weight was increased in donor versus control lungs. CONCLUSIONS: These results indicate that unventilated hypoxia increases vascular permeability in lung grafts and that this process is mainly regulated at VEGF-A and VEGF-C translational but not transcriptional level. Selective VEGF antagonism during graft preservation might be of benefit to counteract edema formation.

Capillary Permeability↗

PubMed2002

Selective upregulation of vascular endothelial growth factor receptors neuropilin-1 and -2 in human neuroblastoma.

BACKGROUND: Recent studies show that vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR, and a series of other angiogenic molecules, are upregulated in advanced but not low stage human neuroblastoma. Neuropilin-1 and 2 (NRP) are novel specific receptors of VEGF(165), whose role is unknown in human neuroblastoma. METHODS: Tissue biopsies of 37 children with Stage I-IV neuroblastoma were obtained, as well as biopsies of 7 normal adrenals as controls. The mRNA expression of VEGF(165) and its receptors Flt-1, KDR, NRP1, and NRP2 was evaluated by real-time reverse transcription polymerase chain reaction. NRP protein expression was detected by immunocytochemistry and Western blotting. RESULTS: VEGF(165) mRNA was upregulated in Stage III and IV and Flt-1 and KDR gene expression was increased in Stage III, while NRP1 and 2 mRNA and protein levels were higher in Stages I-IV vs. controls (P < 0.05). NRP was expressed in vascular endothelial but not tumor cells. CONCLUSIONS: These results show for the first time that human neuroblastoma expresses NRP, and that NRP co-regulates VEGF angiogenic effect in human neuroblastoma. NRP might be a sensitive angiogenic measure of VEGF systems in neuroblastoma, particularly in its early stages.

Blotting, Western↗

PubMed2002

Co-expression of endothelin-1 and vascular endothelial growth factor mediates increased vascular permeability in lung grafts before reperfusion.

Endothelin-1 (ET-1) protein levels increase in lung transplant recipients, although the source of ET-1 remains uncertain. Evidence is accumulating that ET-1 coregulates the expression of vascular endothelial growth/permeability factor (VEGF-A). By using real-time polymerase chain reaction and Western blotting, we describe upregulated ET-1 mRNA and VEGF-A protein levels as well as increased fluid content in donor lung-tissue biopsies before reperfusion. Hypoxia and ET-1 co-upregulate VEGF-A overexpression, therefore temporary VEGF-A antagonism during graft preservation might be of benefit in lung transplantation.

Adult↗

PubMed2002

Efficient carboplatin single therapy in a mouse model of human testicular nonseminomatous germ cell tumor.

PURPOSE: Cisplatin based combination therapy has shown excellent clinical results in patients with testicular nonseminomatous germ cell tumor but chemotherapy induced morbidity and reduced patient compliance are limiting factors in this regimen. To decrease cisplatin based combination therapy induced morbidity we examined carboplatin versus etoposide single therapy in an animal model. MATERIALS AND METHODS: A total of 180 SCID mice bearing testicular nonseminomatous germ cell tumor xenografts received 120 mg./kg. carboplatin as a single cycle, 60 or 30 mg./kg. carboplatin cycled twice, 80, 50 or 30 mg./kg. etoposide cycled twice, or Ringer solution as the control. An additional 20 sham treated and 20 untreated mice also served as controls. Histological and immunocytochemical testing, in vivo microscopy, vascular corrosion casting, serum tumor markers, complete blood count and real-time polymerase chain reaction were used to monitor therapy efficacy. RESULTS: Carboplatin at 60 mg./kg. cycled twice eradicated the tumor and significantly reduced vascular density and vascular endothelial growth factor-A messenger RNA (p <0.05). Elevated tumor markers returned to baseline after carboplatin administration. Therapy was well tolerated, resulting thrombocytopenia disappeared 6 weeks after therapy and the animals were tumor-free 6 months after treatment. Although 120 mg./kg. carboplatin eradicated the tumor, it resulted in extensive mortality and morbidity. Single treatment 30, 50 and 80 mg./kg. etoposide failed. CONCLUSIONS: Carboplatin single therapy was highly effective in our nonseminomatous germ cell tumor model and it may be examined in future clinical trials in patients with high risk stage I nonseminomatous germ cell cancer for reducing cisplatin based combination therapy induced morbidity. Vascular density and vascular endothelial growth factor messenger RNA were elevated in our animal model and deserve further study in nonseminomatous germ cell tumor cases as potential risk factors.

Animals↗

PubMed2001

Dephosphorylation of Ser-259 regulates Raf-1 membrane association.

Mitogenic stimulation of Raf-1 is a complex yet incompletely understood process involving membrane relocalization and phosphorylation of activating residues. We recently reported that Raf-1-associated protein phosphatase 2A contributes to kinase activation, an effect mediated via Ser-259 of Raf-1. Here, we show that mitogens stimulate Ser-259 dephosphorylation and Raf-1/protein phosphatase 2A association concomitantly with membrane accumulation and activation of Raf-1. Blocking Ser-259 dephosphorylation inhibits the two latter events, but it does not prevent activation of a S259A Raf-1 mutant, which is preferentially localized at the membrane independently of mitogenic stimulation. Inhibition of Ser-259 dephosphorylation has no effect on the activation of membrane-tethered Raf-1 (Raf-1CAAX). These data show that Ser-259 dephosphorylation contributes to Raf-1 activation by supporting its membrane accumulation rather than by increasing the specific activity of the kinase and provide a mechanistic basis for the support of kinase activation by Raf-1-associated protein phosphatase 2A.

Animals↗