Relative roles of TGF‐β1 and Wnt in the systemic regulation and aging of satellite cell responses

Muscle stem (satellite) cells are relatively resistant to cell‐autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P‐Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor‐beta (TGF‐β) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age‐dependent myogenic activity of sera TGF‐β1, and its potential cross‐talk with systemic Wnt. We found that sera TGF‐β1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF‐β1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF‐β1 were inhibitory and young sera suppressed myogenesis if TGF‐β1 was activated. Our data suggest that platelet‐derived sera TGF‐β1 levels, or endocrine TGF‐β1 levels, do not explain the age‐dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF‐β neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF‐β1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF‐β receptor kinase inhibitor, which attenuated TGF‐β signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF‐β1‐dependent block on muscle regeneration, identify physiological modalities of age‐imposed changes in TGF‐β1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.     

Regulatory factors and cell populations involved in skeletal muscle regeneration

Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF‐1 and TGF‐β1 regulate their activity. Donor‐derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra‐muscular injection. However, a range of other multipotent muscle‐ and non‐muscle‐derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases. J. Cell. Physiol. 224:7–16, 2010 © 2010 Wiley‐Liss, Inc.     

miR‐145 is differentially regulated by TGF‐β1 and ischaemia and targets Disabled‐2 expression and wnt/β‐catenin activity

The effect of wnt/β‐catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled‐2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down‐regulation of Dab2 regulates cardiac protein expression and wnt/β‐catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor‐β₁ (TGF‐β₁). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR‐145 in the 3′‐UTR of Dab2. In MSC in culture, we observed that TGF‐β₁ treatment led to rapid and sustained up‐regulation of pri–miR‐145. Through gain and loss of function studies we demonstrate that miR‐145 up‐regulation was required for the down‐regulation of Dab2 and increased β‐catenin activity in response to TGF‐β₁. To begin to define how Dab2 might regulate wnt/β‐catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham‐operated myocardium. Following AMI, Dab2 levels were rapidly up‐regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down‐regulation of myocardial pri–miR‐145 expression following AMI. Our data demonstrate a novel and critical role for miR‐145 expression as a regulator of Dab2 expression and β‐catenin activity in response to TGF‐β₁ and hypoxia.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Induction of bone formation by transforming growth factor‐β2 in the non‐human primate Papio ursinus and its modulation by skeletal muscle responding stem cells

Objectives: Four adult non‐human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor‐β₂ (hTGF‐β₂) together with muscle‐derived stem cells. Materials and methods: The hTGF‐β₂ was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein‐1, bone morphogenetic protein‐3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. Results: Heterotopic intramuscular implantation of 5 and 25 μg hTGF‐β₂ combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. Conclusions: In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF‐β₂ induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF‐β₂. Induction of bone formation by hTGF‐β₂ in the non‐human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF‐β₂; the bone induction cascade initiated by mammalian TGF‐β proteins in Papio ursinus needs to be re‐evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.     

Fibronectin synthesis by high glucose level mediated proliferation of mouse embryonic stem cells: Involvement of ANG II and TGF‐β1

The role of individual supplements necessary for the long‐term self‐renewal of embryonic stem (ES) cells is poorly characterized in feeder/serum‐free culture systems. This study sought to characterize the relationship between the effects of glucose on ES cell proliferation and fibronectin (FN) synthesis, and to assess the mechanisms responsible for these cellular effects of glucose. Treatment of the two ES cells (ES‐E14TG2a and ES‐R1) with 25 mM glucose (high glucose) increased the expression levels of FN mRNA and protein. In addition, high glucose and ANG II synergistically increased FN expression level, which coincident with data showing that high glucose increased the mRNA expression of angiotensin II (ANG II) type 1 receptor (AT₁R), angiotensinogen, and FN, but not ANG II type 2 receptor. High glucose also increased the intracellular calcium (Ca²⁺) concentration and pan‐protein kinase C (PKC) phosphorylation. Inhibition of the Ca²⁺/PKC pathway blocked high glucose‐induced FN expression. High glucose or ANG II also synergistically increased transforming growth factor‐beta1 (TGF‐β₁) expression, while pretreatment with losartan abolished the high glucose‐induced increase in TGF‐β₁ production. Moreover, TGF‐β₁‐specific small interfering RNA inhibited high glucose‐induced FN expression and c‐Jun N‐terminal kinase (JNK) activation. The JNK inhibitor SP600125 blocked high glucose‐induced FN expression and inhibited cell cycle regulatory protein expression induced by high glucose or TGF‐β₁. In this study, inhibition of AT₁R, Ca²⁺/PKC, TGF‐β₁, JNK, FN receptor blocked the high glucose‐induced DNA synthesis, increased the cell population in S phase, and the number of cells. It is concluded that high glucose increases FN synthesis through the ANG II or TGF‐β1 pathways, which in part mediates proliferation of mouse ES cells. J. Cell. Physiol. 223: 397–407, 2010. © 2010 Wiley‐Liss, Inc.     

Differential responses to oxidative stress and calcium influx on expression of the transforming growth factor‐β family in myoblasts and myotubes

Changes in gene expression of TGF‐β family members and their receptors in response to treatment with H₂O₂ and a calcium ionophore, A23187, were examined in C2C12 myoblasts and myotubes. The expression of Myf5, an initial regulator of myogenesis, was increased by A23187, and H₂O₂ inhibited the up‐regulation of Myf5. Treatment with H₂O₂ decreased the expression of MHC IIb, a protein component of the myofibrils, irrespective of the presence of A23187, suggesting an inhibitory role of oxidative stress for myogenesis. Expression of ligands and receptors for the TGF‐β family was modulated in response to H₂O₂ and A23187. Treatment with H₂O₂ decreased expression of TGF‐β3, BMP‐4, ALK4, ALK5, and ActRIIB, and increased expression of inhibin α and inhibin βA in either the myoblast stage or the myotube stage, or both. A23187 potentiated down‐regulation of BMP‐4 and ALK4 expression, and up‐regulation of TGF‐β1, TGF‐β2, inhibin α, inhibin βA, ALK2, and ALK3 expression. These results indicate that oxidative stress and Ca²⁺ influx affect expression of the TGF‐β family in C2C12 myoblasts and myotubes. Copyright © 2009 John Wiley & Sons, Ltd.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

High glucose regulates cyclin D1/E of human mesenchymal stem cells through TGF‐β1 expression via Ca2+/PKC/MAPKs and PI3K/Akt/mTOR signal pathways

The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill‐defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D ‐glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [³H]‐thymidine incorporation and cell‐cycle regulatory protein expression levels compared with 5 mM D ‐glucose or 25 mM L ‐glucose. In addition, high glucose increased transforming growth factor‐β1 (TGF‐β₁) mRNA and protein expression levels. High glucose‐induced cell‐cycle regulatory protein expression levels and [³H]‐thymidine incorporation, which were inhibited by TGF‐β₁ siRNA transfection and TGF‐β₁ neutralizing antibody treatment. High glucose‐induced phosphorylation of protein kinase C (PKC), p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time‐dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10^?6 M; bisindolylmaleimide I, 10^?6 M), LY 294002 (PI3 kinase inhibitor, 10^?6 M), Akt inhibitor (10^?5 M), PD 98059 (p44/42 MAPKs inhibitor, 10^?5 M), SB 203580 (p38 MAPK inhibitor, 10^?6 M), and rapamycin (mTOR inhibitor, 10^?8 M) blocked the high glucose‐induced cellular proliferation and TGF‐β₁ protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF‐β₁ expression via Ca²⁺/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways. J. Cell. Physiol. 224:59–70, 2010 © 2010 Wiley‐Liss, Inc.     

Enhanced alleviation of aGVHD by TGF‐β1‐modified mesenchymal stem cells in mice through shifting MΦ into M2 phenotype and promoting the differentiation of Treg cells

Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is the only curative method in treating haematologic malignant diseases. Graft‐versus‐host disease (GVHD) is a common complication post–allo‐HSCT, which can be life‐threatening. Mesenchymal stem cells (MSCs) as an adult stem cell with immunoregulatory function have demonstrated efficacy in steroid resistant acute GVHD (aGVHD). However, the outcome of aGVHD treated with MSCs in clinical trials varied and its underlying mechanism is still unclear. TGF‐β1 is a potent cytokine, which plays a key role in immunoregulation. In the present study, we firstly transduced the lentivirus vector containing TGF‐β1 gene with mouse bone marrow‐derived MSCs. Then, we investigated the immunosuppressive effect of TGF‐β1 gene‐modified MSCs on lymphocytes in vitro and its preventive and therapeutical effects on murine aGVHD model in vivo. Murine MSC was successfully isolated and identified. TGF‐β1 was efficiently transduced into mouse MSCs, and high level TGF‐β1 was detected. MSC‐TGF‐β1 shared the same morphology and immunotypic features of normal MSC. In vitro, MSC‐TGF‐β1 showed enhanced immunosuppressive function on lymphocyte proliferation. In vivo, MSC‐TGF‐β1 showed enhanced amelioration on the severity of aGVHD both in prophylactic and therapeutic murine models. Finally, the macrophages (MØs) derived from MSC‐TGF‐β1–treated mice showed a remarkably increasing of anti‐inflammatory M2‐like phenotype. Furthermore, the differentiation of CD4+ CD25+ Foxp3+ Treg cells was significantly increased in MSC‐TGF‐β1–treated group. Taken together, we proved that MSC‐TGF‐β1 showed enhanced alleviation of aGVHD severity in mice by skewing macrophages into a M2 like phenotype or increasing the proportion of Treg cells, which opens a new frontier in the treatment of aGVHD.     

Tetraspanin 1 as a mediator of fibrosis inhibits EMT process and Smad2/3 and beta‐catenin pathway in human pulmonary fibrosis

Tetraspanin 1(TSPAN1) as a clinically relevant gene target in cancer has been studied, but there is no direct in vivo or vitro evidence for pulmonary fibrosis (PF). Using reanalysing Gene Expression Omnibus data, here, we show for the first time that TSPAN1 was markedly down‐regulated in lung tissue of patient with idiopathic PF (IPF) and verified the reduced protein expression of TSPAN1 in lung tissue samples of patient with IPF and bleomycin‐induced PF mice. The expression of TSPAN1 was decreased and associated with transforming growth factor‐β1 (TGF‐β₁)‐induced molecular characteristics of epithelial‐to‐mesenchymal transition (EMT) in alveolar epithelial cells (AECs). Silencing TSPAN1 promoted cell migration, and the expression of alpha‐smooth muscle actin, vimentin and E‐cadherin in AECs with TGF‐β₁ treatment, while exogenous TSPAN1 has the converse effects. Moreover, silencing TSPAN1 promotes the phosphorylation of Smad2/3 and stabilizes beta‐catenin protein, however, overexpressed TSPAN1 impeded TGF‐β₁‐induced activation of Smad2/3 and beta‐catenin pathway in AECs. Together, our study implicates TSPAN1 as a key regulator in the process of EMT in AECs of IPF.     

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

Soluble factors from ASCs effectively direct control of chondrogenic fate

Background and objectives: Adipose tissue‐derived stem cells (ASCs) have great potential for regenerative medicine. For molecular understanding of specific functional molecules present in ASCs, we analysed 756 proteins including specific chondrogenic functional factors, using high‐throughput nano reverse‐phase liquid chromatography–electrospray ionization–tandem mass spectrometry. Materials, methods and results: Of these proteins, 33 were identified as chondrogenic factors or proteins including type 2 collagen, biglycan, insulin‐like growth factor‐binding protein and transforming growth factor‐beta 1 (TGF‐β1). ASCs are a possible cell source for cartilage regeneration as they are able to secrete a number of functional cytokines including chondrogenesis‐inducing molecules such as TGF‐β1 and bone morphogenetic protein 4 (BMP4). The chondrogenic phenotype of cultured ASCs was effectively induced by ASC‐culture media (CM) containing BMP4 and TGF‐β1, and maintained after pre‐treatment for 14 days in vitro and subcutaneous implantation in vivo. Chondrogenic differentiation efficiency of cultured ASCs and cultured mouse skin‐derived progenitor cells (SPCs) depended absolutely on ASC CM‐fold concentration. Cell density was also a very important factor for chondrogenic behaviour development during differentiation of ASCs and SPCs. Conclusion: ASC CM‐derived TGF‐β1‐induced chondrogenic differentiation of ASCs resulted in significant reduction in chondrogenic activity after inhibition of the p38 pathway, revealing involvement of this MAPK pathway in TGF‐β1 signalling. On the other hand, TGF‐β1 signalling also led to SMAD activation that could directly increase chondrogenic activity of ASCs.     

Transforming growth factor‐beta 1 (TGF‐β1) induces angiogenesis through vascular endothelial growth factor (VEGF)‐mediated apoptosis

VEGF and TGF‐β1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF‐β1 induces apoptosis. We have previously shown that VEGF/VEGF receptor‐2 (VEGFR2) signaling mediates TGF‐β1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF‐mediated apoptosis is required for TGF‐β1 induction of angiogenesis. In vitro the apoptotic effect of TGF‐β1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF‐β1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord‐like structures by TGF‐β1 with an effect comparable to that of z‐VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF‐β1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF‐β1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF‐β1 induction of both apoptosis and angiogenesis, an effect similar to that of z‐VAD. Thus, TGF‐β1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF‐mediated apoptosis as a potential target to control angiogenesis. J. Cell. Physiol. 219: 449–458, 2009. © 2009 Wiley‐Liss, Inc.