Marion Scharpfenecker

BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis.

Genetic studies in mice and humans have shown that the transforming growth factor-β (TGF-β) type-I receptor activin receptor-like kinase 1 (ALK1) and its co-receptor endoglin play an important role in vascular development and angiogenesis. Here, we demonstrate that ALK1 is a signalling receptor for bone morphogenetic protein-9 (BMP-9) in endothelial cells (ECs). BMP-9 bound with high affinity to ALK1 and endoglin, and weakly to the type-I receptor ALK2 and to the BMP type-II receptor (BMPR-II) and activin type-II receptor (ActR-II) in transfected COS cells. Binding of BMP-9 to ALK2 was greatly facilitated when BMPR-II or ActR-II were co-expressed. Whereas BMP-9 predominantly bound to ALK1 and BMPR-II in ECs, it bound to ALK2 and BMPR-II in myoblasts. In addition, we observed binding of BMP-9 to ALK1 and endoglin in glioblastoma cells. BMP-9 activated Smad1 and/or Smad5, and induced ID1 protein and endoglin mRNA expression in ECs. Furthermore, BMP-9 was found to inhibit basic fibroblast growth factor (bFGF)-stimulated proliferation and migration of bovine aortic ECs (BAECs) and to block vascular endothelial growth factor (VEGF)-induced angiogenesis. Taken together, these results suggest that BMP-9 is a physiological ALK1 ligand that plays an important role in the regulation of angiogenesis.

VEGF and inhibitors of TGFβ type-I receptor kinase synergistically promote blood-vessel formation by inducing α5-integrin expression

Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGFβ) are potent regulators of angiogenesis. How VEGF and TGFβ signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGFβ type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Sub-optimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGFβ type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins α5 and β3. Specific downregulation of α5-integrin expression or functional blocking of α5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an α5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of α5-integrin expression is a key determinant by which inhibitors of TGFβ type-I receptor kinase and VEGF synergistically promote angiogenesis.

Ionizing Radiation Shifts the PAI-1/ID-1 Balance and Activates Notch Signaling in Endothelial Cells

PURPOSE Transforming growth factor-beta (TGF-beta) and Notch signaling pathways are important regulators of vascular homeostasis and vessel remodeling; mutations in these pathways can lead to vascular disorders. Similar vascular phenotypes develop in the normal tissues of cancer patients as a long-term effect of radiotherapy. Irradiation most severely affects the capillaries, which become leaky and dilated and might eventually rupture. To investigate the mechanism of such capillary damage, we studied the effect of TGF-beta and Notch signaling in microvascular endothelial cells. METHODS AND MATERIALS Human microvascular endothelial cells were irradiated with 5 or 10 Gy and activation of TGF-beta and Notch signaling pathways was assessed by biochemical methods and a cell migration assay. RESULTS Ionizing radiation induced Smad2 phosphorylation and nuclear translocation and increased mRNA and protein expression of the activin-like kinase 5 (ALK5) target gene plasminogen activator inhibitor-1 (PAI-1). At the same time, we observed diminished Smad1/5/8 activation and downregulation of the ALK1 downstream target, inhibitor of DNA binding-1 (ID-1). We also measured an upregulation of the Notch ligand Jagged-1 and the target gene Hey1. Decreased inhibitor of DNA binding-1 levels coincided with a reduced ability of the cells to migrate. CONCLUSION Ionizing radiation shifts the balance from ALK1 to ALK5 signaling and activates the Notch pathway in endothelial cells. This combination of anti-angiogenic signals contributes to reduced cell migration after irradiation.

Endoglin haploinsufficiency reduces radiation-induced fibrosis and telangiectasia formation in mouse kidneys

BACKGROUND AND PURPOSE Endoglin is a transforming growth factor beta (TGF-beta) co-receptor mainly expressed in dividing endothelial cells. It regulates cell proliferation and survival and is upregulated at sites of vessel repair. Mutations in endoglin have been linked to the vascular disease hereditary hemorrhagic telangiectasia (HHT). HHT patients display dilated capillaries (telangiectasia) that are prone to rupture. Cancer patients receiving radiotherapy develop similar vascular damage in normal tissues lying in the irradiation field. If located in the mucosa, irradiation-induced telangiectasia can lead to severe bleeding. Therefore, this study was aimed at investigating the role of endoglin in radiation-induced telangiectasia formation. MATERIALS AND METHODS Kidneys of endoglin heterozygous (Eng(+/-)) or wild type mice were irradiated with 16 Gy. Mice were sacrificed after 20 weeks and changes in gene expression and protein levels were analysed. RESULTS Expression of TGF-beta target genes involved in radiation-induced fibrosis and fibrosis development in the kidney decreased in Eng(+/-) compared to wild type mice. Unexpectedly, Eng(+/-) mice also displayed reduced telangiectasia formation in the irradiated kidney. CONCLUSIONS Endoglin plays an important role in the development of irradiation-induced normal tissue damage. Future studies will show whether interfering with endoglin functions protects tissues from late radiation toxicity.

The TGF-β co-receptor endoglin regulates macrophage infiltration and cytokine production in the irradiated mouse kidney

BACKGROUND AND PURPOSE We previously showed that mice with reduced levels of the transforming growth factor-beta (TGF-β) co-receptor endoglin (Eng(+/-) mice) develop less fibrosis and vascular damage after kidney irradiation than their wild type (Eng(+/+) mice) littermates; however, the underlying mechanism was unclear. Results from current studies suggest that this occurs via modulation of the radiation-induced inflammatory response. MATERIALS AND METHODS Kidneys of Eng(+/+) and Eng(+/-) mice were irradiated with 16Gy. Mice were sacrificed at 20weeks after irradiation and gene expression and protein levels were analyzed. RESULTS Kidney irradiation triggered the infiltration of macrophages in both Eng(+/+) and Eng(+/-) mice, however, levels of macrophage-produced cytokines interleukin 1 beta (Il1b) and interleukin 6 (Il6) were reduced in irradiated Eng(+/-) compared to Eng(+/+) mice. Double immuno-stainings confirmed that IL-6 was produced by macrophages, whereas IL-1β was mainly detected in other cell types. Accordingly, inflammatory cell precursors derived from the bone marrow of Eng(+/-) mice showed impaired ability to express Il1b and Il6 compared to wild type mice. CONCLUSIONS Endoglin promotes kidney inflammation after irradiation by regulating macrophage infiltration and interleukin production, thereby promoting pathogenic changes after radiation exposure.

ALK1 heterozygosity delays development of late normal tissue damage in the irradiated mouse kidney

BACKGROUND AND PURPOSE Activin receptor-like kinase 1 (ALK1) is a transforming growth factor β (TGF-β) receptor, which is mainly expressed in endothelial cells regulating proliferation and migration in vitro and angiogenesis in vivo. Endothelial cells also express the co-receptor endoglin, which modulates ALK1 effects on endothelial cells. Our previous studies showed that mice with reduced endoglin levels develop less irradiation-induced vascular damage and fibrosis, caused by an impaired inflammatory response. This study was aimed at investigating the role of ALK1 in late radiation toxicity. MATERIAL AND METHODS Kidneys of ALK(+/+) and ALK1(+/-) mice were irradiated with 14 Gy. Mice were sacrificed at 10, 20, and 30 weeks after irradiation and gene expression and protein levels were analyzed. RESULTS Compared to wild type littermates, ALK1(+/-) mice developed less inflammation and fibrosis at 20 weeks after irradiation, but displayed an increase in pro-inflammatory and pro-fibrotic gene expression at 30 weeks. In addition, ALK1(+/-) mice showed superior vascular integrity at 10 and 20 weeks after irradiation which deteriorated at 30 weeks coinciding with changes in the VEGF pathway. CONCLUSIONS ALK1(+/-) mice develop a delayed normal tissue response by modulating the inflammatory response and growth factor expression after irradiation.

Endoglin haploinsufficiency attenuates radiation-induced deterioration of kidney function in mice

BACKGROUND AND PURPOSE Endoglin is a transforming growth receptor beta (TGF-β) co-receptor, which plays a crucial role in the development of late normal tissue damage. Mice with halved endoglin levels (Eng(+/-) mice) develop less inflammation, vascular damage and fibrosis after kidney irradiation compared to their wild type littermates (Eng(+/+) mice). This study was aimed at investigating whether reduced tissue damage in Eng(+/-) mice also results in superior kidney function. MATERIAL AND METHODS Kidneys of Eng(+/+) and Eng(+/-) mice were irradiated with a single dose of 14 Gy. Functional kidney parameters and kidney histology were analysed at 20, 30 and 40 weeks after irradiation. RESULTS Eng(+/-) mice displayed improved kidney parameters (haematocrit, BUN) compared to Eng(+/+) mice at 40 weeks after irradiation. Irradiation of Eng(+/+) kidneys damaged the vascular network and led to an increase in PDGFR-β positive cells, indicative of fibrosis-promoting myofibroblasts. Compared to Eng(+/+) kidneys, vascular perfusion and number of PDGFR-β positive cells were reduced in Eng(+/-) control mice; however, this did not further deteriorate after irradiation. CONCLUSIONS Taken together, we show that not only kidney morphology, but also kidney function is improved after irradiation in Eng(+/-) compared to Eng(+/+) mice.

A Randomized Phase II Study Adding Axitinib to Pemetrexed-Cisplatin in Patients with Malignant Pleural Mesothelioma: A Single-Center Trial Combining Clinical and Translational Outcomes

Introduction: Mesothelioma often presents with a high vessel count and increased vascular growth factors levels. Interference with angiogenesis may therefore improve outcome. This study reports on clinical and translational parameters in patients treated with the small molecule tyrosine kinase inhibitor axitinib and chemotherapy. Methods: Chemonaive patients with mesothelioma were eligible. Patients received pemetrexed (500 mg/m2 every 3 weeks) and cisplatin (75 mg/m2 every 3 weeks) and were randomized to receive axitinib daily (two 5‐mg tablets on days 2–19) or observation. Before treatment and after three cycles of chemotherapy, a thoracoscopy was performed to evaluate vascular changes. Results: Twenty‐five patients were randomized after a successful lead‐in with six patients who received axitinib. Median follow‐up was 45 months. In all but one patient, it was feasible to perform a second thoracoscopy. However, there was more grade 3 or 4 neutropenia leading to pneumonia in the axitinib group. The rates of partial response and stable disease in the axitinib arm were 36% and 43% compared with 18% and 73% in the chemotherapy‐only arm. Median progression‐free survival and overall survival (5.8 and 18.9 months versus 8.3 and 18.5 months) were not different between the two groups. Axitinib reduced vessel number and vessel immaturation. Yet, the mRNA levels of a number of vascular growth factors, their receptors, serum VEGF levels, and activation of tissue vascular endothelial growth factor receptor 2 were increased. Gene expression of platelet‐derived growth factor receptor beta, fms‐related tyrosine kinase 1, and fms‐related tyrosine kinase 4 even correlated with outcome. Conclusions: Axitinib was well tolerated in combination with cisplatin and pemetrexed. Despite the lack of a clinical benefit, axitinib reduced angiogenesis. Whether changes in differentially expressed growth factors in tissue and serum may serve as a biomarker needs further investigation.

Bone marrow-derived macrophages incorporate into the endothelium and influence vascular and renal function after irradiation

Abstract Purpose: We recently demonstrated that endoglin, an ancillary transforming growth factor beta (TGF-β) receptor, modulates vascular damage and fibrosis formation and influences renal function after kidney irradiation. We also suggested that this was partially accomplished by endoglin-mediated regulation of cytokine production in macrophages. Endoglin is expressed on both endothelial cells and on activated macrophages. Therefore, in the current study, we addressed the respective contribution of altered endoglin levels in the different cellular compartments to the development of kidney toxicity after irradiation. Materials and methods: Female endoglin wild-type (Eng+/+ or WT) or heterozygous (Eng+/− or HET) mice were subjected to total body irradiation (2 × 6 Gy with a 6-hour interval) followed by kidney irradiation (1 × 3 Gy). Recipient mice were then transplanted with 4 × 10E6 green fluorescent protein heterozygous (GFP+/−) bone marrow cells from either Eng+/+ or Eng+/− male donor mice. Chimerism was determined 6 weeks thereafter. Blood samples were taken every 10 weeks after irradiation and at sacrifice at 35 weeks. One kidney was used to isolate macrophages; the other kidney was used for histology and to determine cytokine and chemokine concentrations. Results: In all treatment groups, the majority of infiltrating macrophages were bone marrow-derived and this was not altered by endoglin. Bone marrow cells accumulated in damaged tissue areas in the interstitium, but also incorporated into the vasculature. Reducing endoglin levels in macrophages, but not in the endothelium, led to improved renal function (hematocrit, blood urea nitrogen) after irradiation. This was probably promoted by lowered production of pro-inflammatory cytokines and chemokines in macrophages. Other measurements of tissue toxicity (pericyte coverage, fibrosis, damage score) were not altered by differential endoglin expression. Conclusions: Endoglin regulates pro-inflammatory macrophage properties thereby influencing vascular and renal function after kidney irradiation.

Thalidomide ameliorates inflammation and vascular injury but aggravates tubular damage in the irradiated mouse kidney.

PURPOSE The late side effects of kidney irradiation include vascular damage and fibrosis, which are promoted by an irradiation-induced inflammatory response. We therefore treated kidney-irradiated mice with the anti-inflammatory and angiogenesis-modulating drug thalidomide in an attempt to prevent the development of late normal tissue damage and radiation nephropathy in the mouse kidney. METHODS AND MATERIALS Kidneys of C57Bl/6 mice were irradiated with a single dose of 14 Gy. Starting from week 16 after irradiation, the mice were fed with thalidomide-containing chow (100 mg/kg body weight/day). Gene expression and kidney histology were analyzed at 40 weeks and blood samples at 10, 20, 30, and 40 weeks after irradiation. RESULTS Thalidomide improved the vascular structure and vessel perfusion after irradiation, associated with a normalization of pericyte coverage. The drug also reduced infiltration of inflammatory cells but could not suppress the development of fibrosis. Irradiation-induced changes in hematocrit and blood urea nitrogen levels were not rescued by thalidomide. Moreover, thalidomide worsened tubular damage after irradiation and also negatively affected basal tubular function. CONCLUSIONS Thalidomide improved the inflammatory and vascular side effects of kidney irradiation but could not reverse tubular toxicity, which probably prevented preservation of kidney function.