Gudrun Valdimarsdottir

Balancing the activation state of the endothelium via two distinct TGF‐β type I receptors

The generation of mice lacking specific components of the transforming growth factor‐β (TGF‐β) signal tranduction pathway shows that TGF‐β is a key player in the development and physiology of the cardiovascular system. Both pro‐ and anti‐angiogenic properties have been ascribed to TGF‐β, for which the molecular mechanisms are unclear. Here we report that TGF‐β can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF‐β induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF‐β/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF‐β/ALK1 pathway induces endothelial cell migration and proliferation. We identified genes that are induced specifically by TGF‐β‐mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF‐β/ALK1‐induced (and Smad‐dependent) migration, while induction of plasminogen activator inhibitor‐1 by activated ALK5 may contribute to the TGF‐β‐induced maturation of blood vessels. Our results suggest that TGF‐β regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signalling.

Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFβ/ALK5 signaling

Transforming growth factor-beta (TGFbeta) regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways. Activin receptor-like kinase-1 (ALK1) induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation and migration, while ALK5 promotes Smad2/3 activation and inhibits both processes. Here, we report that ALK5 is important for TGFbeta/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGFbeta/ALK1-induced responses. More specifically, we show that ALK5 mediates a TGFbeta-dependent recruitment of ALK1 into a TGFbeta receptor complex and that the ALK5 kinase activity is required for optimal ALK1 activation. TGFbeta type II receptor is also required for ALK1 activation by TGFbeta. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5 but also directly antagonizes ALK5/Smad signaling.

Endoglin promotes endothelial cell proliferation and TGF-β/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor‐β (TGF‐β) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF‐β signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor‐like kinase 1 (ALK1), an endothelial specific TGF‐β type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF‐β/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF‐β/ALK1 signalling, which indirectly inhibits TGF‐β/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF‐β/ALK1 signalling is reduced and TGF‐β/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF‐β‐induced growth arrest by indirectly reducing TGF‐β/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.

Stimulation of id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells.

Background—Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the proliferation, differentiation, and migration of a large variety of cell types. Like other members of the transforming growth factor-&bgr; family, BMPs elicit their cellular effects through activating specific combinations of type I and type II serine/threonine kinase receptors and their downstream effector proteins, which are termed Smads. In the present study, we investigated BMP receptor/Smad expression and signaling in endothelial cells (ECs) and examined the effects of BMP on EC behavior. Methods and Results—Immunohistochemical analysis of tissue sections of human colon and mouse heart and aorta showed that BMP receptors are expressed in ECs in vivo. Bovine aortic ECs and mouse embryonic ECs were found to express BMP receptors and their Smads. BMP receptor activation induced the phosphorylation of specific Smad proteins and promoted EC migration and tube formation. Id1 was identified as a BMP/Smad target in ECs. Ectopic expression of Id1 mimicked BMP-induced effects. Importantly, specific interference with Id1 expression blocked BMP-induced EC migration. Conclusions—The BMP/Smad pathway can potently activate the endothelium. Id1 expression is strongly induced by BMP in ECs. Ectopic expression of Id1 induces EC migration and tube formation. Moreover, Id1 played a critical role in mediating BMP-induced EC migration.

Controlling the angiogenic switch: a balance between two distinct TGF-b receptor signaling pathways.

Biochemical studies in endothelial cells (ECs) and genetic studies in mice and humans have yielded major insights into the role of transforming growth factor beta (TGF-beta) and its downstream Smad effectors in embryonic vascular morphogenesis and in the establishment and maintenance of vessel wall integrity. These studies showed that TGF-beta signaling is of critical importance for normal vascular development and physiology. They also indicated the involvement of two distinct TGF-beta signaling cascades within ECs, namely the activin receptor-like kinase 5 (ALK5)-Smad2/3 pathway and the ALK1-Smad1/5 pathway. Aberrant TGF-beta signaling forms the basis for several vascular disorders such as hereditary hemorrhagic telengiectasia and primary pulmonary hypertension as well as neovascularization during tumorigenesis. This review describes the role of TGF-beta in angiogenesis and some of the controversial issues concerning TGF-beta signaling through ALK1 and ALK5 in ECs.

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP‐Jκ/CBF‐1 and GC‐rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1‐induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.

Brief reviewsControlling the Angiogenic Switch: A Balance between Two Distinct TGF-b Receptor Signaling Pathways

Biochemical studies in endothelial cells (ECs) and genetic studies in mice and humans have yielded major insights into the role of transforming growth factor beta (TGF-beta) and its downstream Smad effectors in embryonic vascular morphogenesis and in the establishment and maintenance of vessel wall integrity. These studies showed that TGF-beta signaling is of critical importance for normal vascular development and physiology. They also indicated the involvement of two distinct TGF-beta signaling cascades within ECs, namely the activin receptor-like kinase 5 (ALK5)-Smad2/3 pathway and the ALK1-Smad1/5 pathway. Aberrant TGF-beta signaling forms the basis for several vascular disorders such as hereditary hemorrhagic telengiectasia and primary pulmonary hypertension as well as neovascularization during tumorigenesis. This review describes the role of TGF-beta in angiogenesis and some of the controversial issues concerning TGF-beta signaling through ALK1 and ALK5 in ECs.

Controlling cell fate by bone morphogenetic protein receptors

Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the fate of different cell types, including mesenchymal and endothelial cells. BMPs inhibit myogenic differentiation, but promote the differentiation of mesenchymal cells into osteoblasts. Furthermore, endothelial migration and tube formation are stimulated by BMPs. Like other members of the transforming growth factor-beta (TGF-beta) superfamily, BMPs elicit their cellular effects via specific types I and II serine/threonine receptors. The activated BMP type I receptor phosphorylates specific receptor-regulated (R)-Smad proteins, which assemble into heteromeric complexes with common partner (Co)-Smad4. Heteromeric Smad complexes efficiently translocate into the nucleus, where they regulate the transcription of target genes. Inhibitors of differentiation (Id) are genes that are specifically induced by BMPs in tissues of different origin. Promoter analysis of Id1 indicates three distinct sequence elements that are sufficient and essential for efficient BMP-induced activation. Furthermore, recent studies reveal an important effector function for Id1 in various BMP-induced biological responses.

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.

Transient disruption of autocrine TGF-beta signaling leads to enhanced survival and proliferation potential in single primitive human hemopoietic progenitor cells.

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-β negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-β type II receptor (TβRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-β signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TβRIIDN and green fluorescence protein in the same CD34+CD38−Lin− cells were constructed. Overexpression of TβRIIDN specifically disrupted TGF-β-mediated signaling. Autocrine TGF-β signaling in CD34+CD38−Lin− cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-β signaling in CD34+CD38−Lin− cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34+CD38−Lin− cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-β-negative regulatory loop of primitive hemopoietic progenitor cells.