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1.
Cell ; 141(1): 178-90, 2010 Apr 02.
Article in English | MEDLINE | ID: mdl-20371353

ABSTRACT

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.


Subject(s)
Neovascularization, Physiologic/drug effects , Pregnancy Proteins/antagonists & inhibitors , Pregnancy Proteins/metabolism , Angiogenesis Inhibitors/therapeutic use , Animals , Antibodies, Monoclonal/therapeutic use , Carcinoma, Hepatocellular/blood supply , Carcinoma, Hepatocellular/prevention & control , Choroid/blood supply , Disease Models, Animal , Eye Diseases/pathology , Humans , Liver Neoplasms, Experimental/blood supply , Liver Neoplasms, Experimental/prevention & control , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Papilloma/blood supply , Papilloma/chemically induced , Papilloma/prevention & control , Placenta Growth Factor , Skin Neoplasms/blood supply , Skin Neoplasms/chemically induced , Skin Neoplasms/prevention & control
2.
Int J Mol Sci ; 24(17)2023 Aug 25.
Article in English | MEDLINE | ID: mdl-37686032

ABSTRACT

Chronic kidney disease (CKD) progression is associated with persisting oxidative stress, which impairs the NO-sGC-cGMP signaling cascade through the formation of oxidized and heme-free apo-sGC that cannot be activated by NO. Runcaciguat (BAY 1101042) is a novel, potent, and selective sGC activator that binds and activates oxidized and heme-free sGC and thereby restores NO-sGC-cGMP signaling under oxidative stress. Therefore, runcaciguat might represent a very effective treatment option for CKD/DKD. The potential kidney-protective effects of runcaciguat were investigated in ZSF1 rats as a model of CKD/DKD, characterized by hypertension, hyperglycemia, obesity, and insulin resistance. ZSF1 rats were treated daily orally for up to 12 weeks with runcaciguat (1, 3, 10 mg/kg/bid) or placebo. The study endpoints were proteinuria, kidney histopathology, plasma, urinary biomarkers of kidney damage, and gene expression profiling to gain information about relevant pathways affected by runcaciguat. Furthermore, oxidative stress was compared in the ZSF1 rat kidney with kidney samples from DKD patients. Within the duration of the 12-week treatment study, kidney function was significantly decreased in obese ZSF1 rats, indicated by a 20-fold increase in proteinuria, compared to lean ZSF1 rats. Runcaciguat dose-dependently and significantly attenuated the development of proteinuria in ZSF1 rats with reduced uPCR at the end of the study by -19%, -54%, and -70% at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo treatment. Additionally, average blood glucose levels measured as HbA1C, triglycerides, and cholesterol were increased by five times, twenty times, and four times, respectively, in obese ZSF1 compared to lean rats. In obese ZSF1 rats, runcaciguat reduced HbA1c levels by -8%, -34%, and -76%, triglycerides by -42%, -55%, and -71%, and cholesterol by -16%, -17%, and -34%, at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo. Concomitantly, runcaciguat also reduced kidney weights, morphological kidney damage, and urinary and plasma biomarkers of kidney damage. Beneficial effects were accompanied by changes in gene expression that indicate reduced fibrosis and inflammation and suggest improved endothelial stabilization. In summary, the sGC activator runcaciguat significantly prevented a decline in kidney function in a DKD rat model that mimics common comorbidities and conditions of oxidative stress of CKD patients. Thus, runcaciguat represents a promising treatment option for CKD patients, which is in line with recent phase 2 clinical study data, where runcaciguat showed promising efficacy in CKD patients (NCT04507061).


Subject(s)
Kidney , Renal Insufficiency, Chronic , Animals , Rats , Cyclic GMP , Glycated Hemoglobin , Heme , Obesity , Proteinuria , Renal Insufficiency, Chronic/drug therapy , Clinical Trials, Phase II as Topic
3.
Nature ; 454(7204): 656-60, 2008 Jul 31.
Article in English | MEDLINE | ID: mdl-18594512

ABSTRACT

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.


Subject(s)
Neoplasms/blood supply , Neovascularization, Pathologic/metabolism , Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-3/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Dipeptides/pharmacology , Down-Regulation , Endothelial Cells/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Ligands , Mice , Mice, Inbred BALB C , Mice, Transgenic , Neoplasms/drug therapy , Neovascularization, Pathologic/genetics , Receptors, Notch/metabolism , Signal Transduction
4.
EMBO J ; 27(19): 2603-15, 2008 Oct 08.
Article in English | MEDLINE | ID: mdl-18772882

ABSTRACT

Loss of expression of the cell-cell adhesion molecule E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) in development and in the progression from epithelial tumours to invasive and metastatic cancers. Here, we demonstrate that the loss of E-cadherin function upregulates expression of the neuronal cell adhesion molecule (NCAM). Subsequently, a subset of NCAM translocates from fibroblast growth factor receptor (FGFR) complexes outside lipid rafts into lipid rafts where it stimulates the non-receptor tyrosine kinase p59(Fyn) leading to the phosphorylation and activation of focal adhesion kinase and the assembly of integrin-mediated focal adhesions. Ablation of NCAM expression during EMT inhibits focal adhesion assembly, cell spreading and EMT. Conversely, forced expression of NCAM induces epithelial cell delamination and migration, and high NCAM expression correlates with tumour invasion. These results establish a mechanistic link between the loss of E-cadherin expression, NCAM function, focal adhesion assembly and cell migration and invasion.


Subject(s)
Cadherins/metabolism , Focal Adhesions/metabolism , Neural Cell Adhesion Molecules/metabolism , Animals , Cadherins/genetics , Cell Line , Cell Movement/physiology , Epithelium/physiology , Gene Expression Regulation , Humans , Membrane Microdomains/metabolism , Mesoderm/physiology , Mice , Mice, Knockout , Neoplasm Invasiveness , Neoplasms/metabolism , Neoplasms/pathology , Neural Cell Adhesion Molecules/genetics , Neurons/cytology , Neurons/metabolism , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Receptors, Fibroblast Growth Factor/metabolism
5.
Br J Pharmacol ; 179(11): 2476-2489, 2022 06.
Article in English | MEDLINE | ID: mdl-34096053

ABSTRACT

BACKGROUND AND PURPOSE: Generation of cGMP via NO-sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem-free form, apo-sGC. Apo-sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo-sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function. EXPERIMENTAL APPROACH: Two novel sGC activators, runcaciguat and BAY-543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre-constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L-NAME-induced NO deficiency, and in the presence of oxidative stress induced by ODQ. KEY RESULTS: Mouse glomeruli showed NO-induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO-induced cGMP generation. In the presence of ODQ, NO-induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L-NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ-perfused kidneys. CONCLUSION AND IMPLICATION: sGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease-relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.


Subject(s)
Renal Insufficiency, Chronic , Vasodilation , Animals , Cyclic GMP , Female , Guanylate Cyclase , Humans , Kidney , Male , Mice , NG-Nitroarginine Methyl Ester , Nitric Oxide , Renal Insufficiency, Chronic/drug therapy , Soluble Guanylyl Cyclase
6.
Naunyn Schmiedebergs Arch Pharmacol ; 394(12): 2363-2379, 2021 12.
Article in English | MEDLINE | ID: mdl-34550407

ABSTRACT

Chronic kidney diseaQueryse (CKD) is associated with oxidative stress which can interrupt the nitric oxide (NO)/soluble guanylyl cyclase (sGC) signaling and decrease cyclic guanosine monophosphate (cGMP) production. Low cGMP concentrations can cause kidney damage and progression of CKD. The novel sGC activator runcaciguat targets the oxidized and heme-free form of sGC, restoring cGMP production under oxidative stress. The purpose of this study is to investigate if runcaciguat could provide an effective treatment for CKD. Runcaciguat was used for the treatment not only in rat CKD models with different etiologies and comorbidities, namely of hypertensive rats, the renin transgenic (RenTG) rat, and angiotensin-supplemented (ANG-SD) rat, but also in rats with diabetic and metabolic CKD, the Zucker diabetic fatty (ZDF) rat. The treatment duration was 2 to 42 weeks and runcaciguat was applied orally in doses from 1 to 10 mg/kg/bid. In these different rat CKD models, runcaciguat significantly reduced proteinuria (urinary protein to creatinine ratio; uPCR). These effects were also significant at doses which did not or only moderately decrease systemic blood pressure. Moreover, runcaciguat significantly decreased kidney injury biomarkers and attenuated morphological kidney damages. In RenTG rats, runcaciguat improved survival rates and markers of heart injury. These data demonstrate that the sGC activator runcaciguat exhibits cardio-renal protection at doses which did not reduce blood pressure and was effective in hypertensive as well as diabetic and metabolic CKD models. These data, therefore, suggest that runcaciguat, with its specific mode of action, represents an efficient treatment approach for CKD and associated CV diseases.


Subject(s)
Cyclopropanes , Diabetes Mellitus, Experimental , Hypertension , Renal Insufficiency, Chronic , Animals , Male , Rats , Blood Pressure/drug effects , Cyclic GMP/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Activators/administration & dosage , Enzyme Activators/pharmacology , Hypertension/complications , Hypertension/drug therapy , Rats, Sprague-Dawley , Rats, Transgenic , Rats, Zucker , Renal Insufficiency, Chronic/etiology , Renal Insufficiency, Chronic/prevention & control , Soluble Guanylyl Cyclase/drug effects , Soluble Guanylyl Cyclase/metabolism , Time Factors , Cyclopropanes/pharmacology , Cyclopropanes/therapeutic use
7.
J Med Chem ; 64(9): 5323-5344, 2021 05 13.
Article in English | MEDLINE | ID: mdl-33872507

ABSTRACT

Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45, BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.


Subject(s)
Drug Design , Enzyme Activators/chemistry , Soluble Guanylyl Cyclase/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Cytochrome P-450 CYP3A/chemistry , Cytochrome P-450 CYP3A/metabolism , Dogs , Enzyme Activators/metabolism , Enzyme Activators/pharmacology , Enzyme Activators/therapeutic use , Half-Life , Heart Rate/drug effects , Hemodynamics/drug effects , Hypertension/drug therapy , Hypertension/pathology , Molecular Dynamics Simulation , Rats , Rats, Inbred SHR , Solubility , Soluble Guanylyl Cyclase/metabolism , Structure-Activity Relationship
8.
Mol Cancer Ther ; 8(1): 55-63, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19139113

ABSTRACT

Halting tumor growth by interfering with tumor-induced angiogenesis is an attractive therapeutic approach. Such treatments include humanized antibodies blocking the activity of vascular endothelial growth factor (VEGF)-A (bevacizumab), soluble VEGF receptor (VEGFR) constructs (VEGF-Trap), or small-molecule inhibitors of VEGFR signaling, including PTK787/ZK222584 (PTK/ZK), sorafenib, and sunitinib. PTK/ZK has been shown previously to specifically block VEGF-induced phosphorylation of VEGFR-1, -2 and -3 and thereby to inhibit endothelial cell proliferation, differentiation, and tumor angiogenesis. We have investigated the effect of PTK/ZK on tumor angiogenesis and tumor lymphangiogenesis using the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis. In Rip1Tag2 mice, tumor angiogenesis is predominantly mediated by VEGF-A, and as expected, PTK/ZK efficiently impaired tumor blood vessel angiogenesis and tumor growth. Double-transgenic Rip1Tag2;Rip1VEGF-C and Rip1Tag2;Rip1VEGF-D mice not only exhibit VEGF-A-dependent blood vessel angiogenesis but also tumor lymphangiogenesis induced by the transgenic expression of VEGF-C or -D. In these mouse models, PTK/ZK also repressed tumor blood vessel angiogenesis and tumor growth yet failed to affect tumor lymphangiogenesis and lymphogenic metastasis. Adenoviral delivery of soluble VEGFR-3 also did not prevent tumor lymphangiogenesis in these mice. In contrast, spontaneous tumor lymphangiogenesis, as observed by the stochastic expression of VEGF-C and -D in tumors of neural cell adhesion molecule-deficient Rip1Tag2 mice, was repressed by PTK/ZK and soluble VEGFR-3. The results indicate that the time of onset and the levels of VEGF-C/D expression may be critical variables in efficiently repressing tumor lymphangiogenesis and that pathways other than VEGFR signaling may be involved in tumor lymphangiogenesis.


Subject(s)
Lymphangiogenesis/drug effects , Neoplasms/drug therapy , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Phthalazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyridines/therapeutic use , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Cell Proliferation/drug effects , Disease Progression , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/blood supply , Neoplasms/enzymology , Receptors, Vascular Endothelial Growth Factor/metabolism
9.
Nucleic Acids Res ; 30(14): 3245-52, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12136106

ABSTRACT

SOX9 transcription factor is involved in chondrocyte differentiation and male sex determination. Heterozygous defects in the human SOX9 gene cause campomelic dysplasia. The mechanisms behind SOX9 function are not understood despite the description of different target genes. This study therefore sets out to identify SOX9-associated proteins to unravel how SOX9 interacts with the cellular transcription machinery. We report the ability of SOX9 to interact with TRAP230, a component of the thyroid hormone receptor-associated protein (TRAP) complex. Both in vitro and in vivo assays have confirmed that the detected interaction is specific and occurs endogenously in cells. Using co-transfection experiments, we have also shown that the TRAP230 interacting domain can act in a dominant-negative manner regarding SOX9 activity. Our results add SOX9 to the list of activators that communicate with the general transcription machinery through the TRAP complex and suggest a basis for the collaboration of SOX9 with different coactivators that could contact the same coactivator/integrator complex.


Subject(s)
Carrier Proteins/metabolism , High Mobility Group Proteins/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Binding, Competitive , COS Cells , Carrier Proteins/chemistry , Carrier Proteins/genetics , Chondrocytes/cytology , Chondrocytes/metabolism , Gene Expression , High Mobility Group Proteins/genetics , Humans , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Receptors, Thyroid Hormone/metabolism , SOX9 Transcription Factor , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Transcriptional Activation/genetics , Tumor Cells, Cultured , Two-Hybrid System Techniques
10.
PLoS One ; 5(11): e14109, 2010 Nov 24.
Article in English | MEDLINE | ID: mdl-21124841

ABSTRACT

BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing ß-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.


Subject(s)
Insulin-Secreting Cells/metabolism , Neuroendocrine Tumors/metabolism , Pancreatic Neoplasms/metabolism , Vascular Endothelial Growth Factor B/metabolism , Animals , Disease Models, Animal , Female , Humans , Immunoblotting , Immunohistochemistry , Insulin-Secreting Cells/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/pathology , Pancreas/blood supply , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Rats , Reverse Transcriptase Polymerase Chain Reaction , Tumor Burden , Vascular Endothelial Growth Factor B/genetics
11.
Blood ; 109(4): 1503-6, 2007 Feb 15.
Article in English | MEDLINE | ID: mdl-17032923

ABSTRACT

To generate transgenic mice that express Cre-recombinase exclusively in the megakaryocytic lineage, we modified a mouse bacterial artificial chromosome (BAC) clone by homologous recombination and replaced the first exon of the platelet factor 4 (Pf4), also called CXCL4, with a codon-improved Cre cDNA. Several strains expressing the transgene were obtained and one strain, Q3, was studied in detail. Crossing Q3 mice with the ROSA26-lacZ reporter strain showed that Cre-recombinase activity was confined to megakaryocytes. These results were further verified by crossing the Q3 mice with a strain containing loxP-flanked integrin beta1. Excision of this conditional allele in megakaryocytes was complete at the DNA level, and platelets were virtually devoid of the integrin beta1 protein. The Pf4-Cre transgenic strain will be a valuable tool to study megakaryopoiesis, platelet formation, and platelet function.


Subject(s)
Blood Platelets/physiology , Cell Lineage , Megakaryocytes/physiology , Platelet Factor 4/genetics , Transgenes , Animals , Chromosomes, Artificial, Bacterial , Integrases/analysis , Integrases/genetics , Mice , Mice, Transgenic , Recombination, Genetic
12.
Cancer Res ; 67(22): 10840-8, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-18006829

ABSTRACT

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.


Subject(s)
Insulin-Secreting Cells/metabolism , Neovascularization, Pathologic , Pancreatic Neoplasms/pathology , Pregnancy Proteins/physiology , Vascular Endothelial Growth Factor A/metabolism , Animals , Collagen/chemistry , Dimerization , Enzyme-Linked Immunosorbent Assay , Humans , Islets of Langerhans/metabolism , Methylmethacrylate/chemistry , Mice , Mice, Transgenic , Neoplasm Invasiveness , Pancreatic Neoplasms/metabolism , Placenta Growth Factor , Pregnancy Proteins/metabolism
13.
Blood ; 110(8): 3049-55, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17638848

ABSTRACT

The tumor suppressor Smad4 mediates signaling by the transforming growth factor beta (TGF-beta) superfamily of ligands. Previous studies showed that several TGF-beta family members exert important functions in hematopoiesis. Here, we studied the role of Smad4 in adult murine hematopoiesis using the inducible Mx-Cre/loxP system. Mice with homozygous Smad4 deletion (Smad4(Delta/Delta)) developed severe anemia 6 to 8 weeks after induction (mean hemoglobin level 70 g/L). The anemia was not transplantable, as wild-type mice reconstituted with Smad4(Delta/Delta) bone marrow cells had normal peripheral blood counts. These mice did not develop an inflammatory disease typical for mice deficient in TGF-beta receptors I and II, suggesting that the suppression of inflammation by TGF-beta is Smad4 independent. The same results were obtained when Smad4 alleles were deleted selectively in hematopoietic cells using the VavCre transgenic mice. In contrast, lethally irradiated Smad4(Delta/Delta) mice that received wild-type bone marrow cells developed anemia similar to Smad4(Delta/Delta) mice that did not receive a transplant. Liver iron stores were decreased and blood was present in stool, indicating that the anemia was due to blood loss. Multiple polyps in stomach and colon represent a likely source of the bleeding. We conclude that Smad4 is not required for adult erythropoiesis and that anemia is solely the consequence of blood loss.


Subject(s)
Anemia/etiology , Erythropoiesis/physiology , Gastrointestinal Hemorrhage/etiology , Intestinal Polyposis/etiology , Smad4 Protein/deficiency , Animals , Bone Marrow Transplantation , Flow Cytometry , Intestinal Polyposis/pathology , Iron Deficiencies , Liver/chemistry , Liver/metabolism , Mice , Mice, Transgenic , Polyps/pathology , Reverse Transcriptase Polymerase Chain Reaction , Stomach Diseases/pathology , Transforming Growth Factor beta/metabolism
14.
Am J Pathol ; 170(4): 1348-61, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17392173

ABSTRACT

In many human carcinomas, expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) correlates with up-regulated lymphangiogenesis and regional lymph node metastasis. Here, we have used the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis to investigate the functional role of VEGF-D in the induction of lymphangiogenesis and tumor progression. Expression of VEGF-D in beta cells of single-transgenic Rip1VEGF-D mice resulted in the formation of peri-insular lymphatic lacunae, often containing leukocyte accumulations and blood hemorrhages. When these mice were crossed to Rip1Tag2 mice, VEGF-D-expressing tumors also exhibited peritumoral lymphangiogenesis with lymphocyte accumulations and hemorrhages, and they frequently developed lymph node and lung metastases. Notably, tumor outgrowth and blood microvessel density were significantly reduced in VEGF-D-expressing tumors. Our results demonstrate that VEGF-D induces lymphangiogenesis, promotes metastasis to lymph nodes and lungs, and yet represses hemangiogenesis and tumor outgrowth. Because a comparable transgenic expression of vascular endothelial growth factor-C (VEGF-C) in Rip1Tag2 has been shown previously to provoke lymphangiogenesis and lymph node metastasis in the absence of any distant metastasis, leukocyte infiltration, or angiogenesis-suppressing effects, these results reveal further functional differences between VEGF-D and VEGF-C.


Subject(s)
Lymphangiogenesis/physiology , Neoplasm Metastasis/physiopathology , Vascular Endothelial Growth Factor D/physiology , Animals , Cells, Cultured , Female , Genotype , Humans , Immunoblotting , Leukocytes/metabolism , Leukocytes/pathology , Lung Neoplasms/genetics , Lung Neoplasms/physiopathology , Lung Neoplasms/secondary , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphangiogenesis/genetics , Lymphatic Vessels/metabolism , Lymphatic Vessels/pathology , Lymphatic Vessels/ultrastructure , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Neoplasm Metastasis/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/physiopathology , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/ultrastructure , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/physiology , Vascular Endothelial Growth Factor D/genetics , Vascular Endothelial Growth Factor D/metabolism
15.
Blood ; 103(12): 4511-3, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-14988151

ABSTRACT

To derive an efficient system for gene silencing in human hematopoietic stem cells (HSCs) we modified a lentiviral vector for small interfering RNA (siRNA) delivery. For this purpose, an H1 promoter-driven siRNA expression cassette was introduced into a lentiviral vector, and the p53 mRNA was chosen as a target for siRNA-mediated gene silencing. Using the recombinant lentivirus we infected human cord blood-derived CD34+ cells and obtained a transfection efficiency of up to 50%, as determined by expression of enhanced green fluorescent protein (EGFP). In EGFP-positive long-term culture-initiating cell (LTC-IC)- and colony-forming unit cell (CFU-C)-derived cells, we observed a reduction of p53 mRNA of up to 95%. Importantly, this reduction remained stable during several weeks of cell culture. Furthermore, p53 gene silencing resulted in decreased p21 mRNA levels and reduced the sensitivity of CD34+ cells toward the cytotoxic drug etoposide. Thus, lentiviral delivery of siRNA can allow for efficient and stable gene silencing in human HSCs and will be very valuable for further gene function studies.


Subject(s)
Gene Silencing , Hematopoietic Stem Cells/physiology , Lentivirus/genetics , RNA, Small Interfering/pharmacokinetics , Antigens, CD/blood , Antigens, CD34/blood , Base Sequence , DNA Primers , Genes, Reporter , Genes, p53 , Green Fluorescent Proteins , Hematopoietic Stem Cells/cytology , Humans , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Promoter Regions, Genetic , RNA, Small Interfering/genetics
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