Blood endothelial ALK1-BMP4 signaling axis regulates adult hair follicle stem cell activation.

Blood vessels can play dual roles in tissue growth by transporting gases and nutrients and by regulating tissue stem cell activity via signaling. Correlative evidence implicates skin endothelial cells (ECs) as signaling niches of hair follicle stem cells (HFSCs), but functional demonstration from gene depletion of signaling molecules in ECs is missing to date. Here, we show that depletion of the vasculature-factor Alk1 increases BMP4 secretion from ECs, which delays HFSC activation. Furthermore, while previous evidence suggests a lymphatic vessel role in adult HFSC activation possibly through tissue drainage, a blood vessel role has not yet been addressed. Genetic perturbation of the ALK1-BMP4 axis in all ECs or the lymphatic ECs specifically unveils inhibition of HFSC activation by blood vessels. Our work suggests a broader relevance of blood vessels, adding adult HFSCs to the EC functional repertoire as signaling niches for the adult stem cells.

Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development.

BMP signaling is crucial to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here, we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo. At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. Mechanistically, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial cell junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a 'Goldilocks' pathway in vascular biology that requires a certain signaling amplitude, regulated by SMAD6, to function properly.

Hypoxia-induced TIMAP Upregulation in Endothelial Cells and TIMAP-dependent Tumour Angiogenesis.

TIMAP, the endothelial cell-predominant protein phosphatase 1ß regulatory subunit also known as PPP1R16B, promotes in vitro endothelial cell proliferation and angiogenic sprouting. TIMAP was first identified as a target of TGF-ß1 mediated repression, but the molecular pathways regulating its expression in endothelial cells are not well-defined. This study examined the role of BMP9, hypoxia and angiogenic growth factors in the regulation of TIMAP expression and determined whether TIMAP plays a role in tumor angiogenesis and growth in vivo. BMP9, which potently activated the SMAD1/5/8 pathway in endothelial cells, significantly reduced TIMAP mRNA and protein expression. Conversely, hypoxia and the prolyl hydroxylase inhibitor Roxadustat raised TIMAP mRNA and protein levels by inhibiting the BMP9 pathway. Angiogenic growth factors, most prominent among them VEGFA and IGF-I, raised endothelial TIMAP levels partly by attenuating BMP9 pathway activation, but also through BMP pathway-independent mechanisms. Cultured breast cancer E0771 cells released mediators that raised TIMAP expression in endothelial cells, effects that were inhibited by the VEGF inhibitor Sunitinib in conjunction with the IGF-1 inhibitor Picropodophyllin. In the mouse E0771 breast cancer model in vivo, tumor growth and tumor angiogenesis were markedly attenuated in TIMAP deficient, compared to wild-type littermates. These findings indicate that TIMAP plays a critical pro-angiogenic function during tumor angiogenesis in vivo, likely through hypoxia-driven inhibition of the BMP9 pathway and through elaboration of angiogenic growth factors by tumor cells.

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors.

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

Executive summary of the 14th HHT international scientific conference.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by small, dilated clustered vessels (telangiectasias) and by larger visceral arteriovenous malformations (AVMs), which directly connect the feeding arteries with the draining veins. These lesions are fragile, prone to rupture, and lead to recurrent epistaxis and/or internal hemorrhage among other complications. Germline heterozygous loss-of-function (LOF) mutations in Bone Morphogenic Protein 9 (BMP9) and BMP10 signaling pathway genes (endoglin-ENG, activin like kinase 1 ACVRL1 aka ALK1, and SMAD4) cause different subtypes of HHT (HHT1, HHT2 and HHT-juvenile polyposis (JP)) and have a worldwide combined incidence of about 1:5000. Expert clinicians and international scientists gathered in Cascais, Portugal from September 29th to October 2nd, 2022 to present the latest scientific research in the HHT field and novel treatment strategies for people living with HHT. During the largest HHT scientific conference yet, participants included 293 in person and 46 virtually. An impressive 209 abstracts were accepted to the meeting and 59 were selected for oral presentations. The remaining 150 abstracts were presented during judged poster sessions. This review article summarizes the basic and clinical abstracts selected as oral presentations with their new observations and discoveries as well as surrounding discussion and debate. Two discussion-based workshops were also held during the conference, each focusing on mechanisms and clinical perspectives in either AVM formation and progression or current and future therapies for HHT. Our hope is that this paper will represent the current progress and the remaining unanswered questions surrounding HHT, in order to serve as an update for those within the field and an invitation to those scientists and clinicians as yet outside of the field of HHT.

Longitudinal Assessment of Curaçao Criteria in Children with Hereditary Hemorrhagic Telangiectasia.

OBJECTIVE: To assess the utility of the Curaçao criteria by age over time in children with hereditary hemorrhagic telangiectasia (HHT). STUDY DESIGN: This was a single-center, retrospective analysis of patients attending the HHT clinic at the Hospital for Sick Children (Toronto, Canada) between 2000 and 2019. The evaluation of the Curaçao criteria was completed during initial and follow-up visits. Screening for pulmonary and brain arteriovenous malformations was completed at 5 yearly intervals. RESULTS: A total of 116 patients with genetic confirmation of HHT were included in the analysis. At initial screening at a median (IQR) age of 8.4 (2.8, 12.9) years, 41% met criteria for a definite clinical diagnosis (≥3 criteria). In children <6 years at presentation, only 23% fulfilled at least 3 criteria initially. In longitudinal follow-up, 63% reached a definite clinical diagnosis, with a median (IQR) follow-up duration of 5.2 (3.2, 7.9) years (P = .005). Specifically, more patients met the epistaxis and telangiectasia criteria at last visit compared with initial (79% vs 60%; P = .006; 47% vs 30%; P = .02) but not for the arteriovenous malformation criterion (59% vs 57%; P = .65). CONCLUSIONS: In the pediatric population, most patients do not meet definite clinical criteria of HHT at initial presentation. Although the number of diagnostic criteria met increased over time, mainly due to new onset of epistaxis and telangiectasia, accuracy remained low during follow-up visits. Relying solely on clinical criteria may lead to underdiagnosis of HHT in children.

LDL Transcytosis by the Arterial Endothelium-Atherosclerosis by a Thousand Cuts?

PURPOSE OF REVIEW: The accumulation of LDL in the arterial intima is an initiating event in atherosclerosis. After decades of controversy, it is now clear that transcytosis of LDL across an intact endothelial monolayer contributes to its intimal deposition. We review recent observations in this field and address the question of whether LDL transcytosis can be manipulated therapeutically. RECENT FINDINGS: The development of a live-cell imaging method for studying transcytosis using total internal reflection fluorescence (TIRF) microscopy has catalyzed recent discoveries. LDL transcytosis is mediated by SR-BI and ALK1. Estrogen down-regulates SR-BI and inhibits LDL transcytosis, while the nuclear structural protein HMGB1 promotes LDL transcytosis. LDL transcytosis by ALK1 is independent of the receptor's kinase activity and is antagonized by BMP9, ALK1's canonical ligand. Inflammation stimulates LDL transcytosis. Identifying the function and mechanisms of LDL transcytosis may ultimately permit its therapeutic manipulation.

BMP9 and BMP10: Two close vascular quiescence partners that stand out.

Bone morphogenetic proteins (BMPs) are dimeric transforming growth factor ß (TGFß) family cytokines that were first described in bone and cartilage formation but have since been shown to be involved in many pleiotropic functions. In human, there are 15 BMP ligands, which initiate their cellular signaling by forming a complex with two copies of type I receptors and two copies of type II receptors, both of which are transmembrane receptors with an intracellular serine/threonine kinase domain. Within this receptor family, ALK1 (activin receptor-like kinase 1), which is a type I receptor mainly expressed on endothelial cells, and BMPRII (BMP Receptor type II), a type II receptor also highly expressed on endothelial cells, have been directly linked to two rare vascular diseases: hereditary hemorrhagic telangiectasia (HHT), and pulmonary arterial hypertension (PAH), respectively. BMP9 (gene name GDF2) and BMP10, two close members of the BMP family, are the only known ligands for the ALK1 receptor. This specificity gives them a unique role in physiological and pathological angiogenesis and tissue homeostasis. The aim of this current review is to present an overview of what is known about BMP9 and BMP10 on vascular regulation with a particular emphasis on recent results and the many questions that remain unanswered regarding the roles and specificities between BMP9 and BMP10.

The TGFß type I receptor TGFßRI functions as an inhibitor of BMP signaling in cartilage.

The type I TGFß receptor TGFßRI (encoded by Tgfbr1) was ablated in cartilage. The resulting Tgfbr1Col2 mice exhibited lethal chondrodysplasia. Similar defects were not seen in mice lacking the type II TGFß receptor or SMADs 2 and 3, the intracellular mediators of canonical TGFß signaling. However, we detected elevated BMP activity in Tgfbr1Col2 mice. As previous studies showed that TGFßRI can physically interact with ACVRL1, a type I BMP receptor, we generated cartilage-specific Acvrl1 (Acvrl1Col2 ) and Acvrl1/Tgfbr1 (Acvrl1/Tgfbr1Col2) knockouts. Loss of ACVRL1 alone had no effect, but Acvrl1/Tgfbr1Col2 mice exhibited a striking reversal of the chondrodysplasia seen in Tgfbr1Col2 mice. Loss of TGFßRI led to a redistribution of the type II receptor ACTRIIB into ACVRL1/ACTRIIB complexes, which have high affinity for BMP9. Although BMP9 is not produced in cartilage, we detected BMP9 in the growth plate, most likely derived from the circulation. These findings demonstrate that the major function of TGFßRI in cartilage is not to transduce TGFß signaling, but rather to antagonize BMP signaling mediated by ACVRL1.

Role of soluble endoglin in BMP9 signaling.

Endoglin (ENG) is a coreceptor of the transforming growth factor-ß (TGFß) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.

Low-Dose-Rate Radiation-Induced Secretion of TGF-ß3 Together with an Activator in Small Extracellular Vesicles Modifies Low-Dose Hyper-Radiosensitivity through ALK1 Binding.

Hyper-radiosensitivity (HRS) is the increased sensitivity to low doses of ionizing radiation observed in most cell lines. We previously demonstrated that HRS is permanently abolished in cells irradiated at a low dose rate (LDR), in a mechanism dependent on transforming growth factor ß3 (TGF-ß3). In this study, we aimed to elucidate the activation and receptor binding of TGF-ß3 in this mechanism. T-47D cells were pretreated with inhibitors of potential receptors and activators of TGF-ß3, along with addition of small extracellular vesicles (sEVs) from LDR primed cells, before their radiosensitivity was assessed by the clonogenic assay. The protein content of sEVs from LDR primed cells was analyzed with mass spectrometry. Our results show that sEVs contain TGF-ß3 regardless of priming status, but only sEVs from LDR primed cells remove HRS in reporter cells. Inhibition of the matrix metalloproteinase (MMP) family prevents removal of HRS, suggesting an MMP-dependent activation of TGF-ß3 in the LDR primed cells. We demonstrate a functional interaction between TGF-ß3 and activin receptor like kinase 1 (ALK1) by showing that TGF-ß3 removes HRS through ALK1 binding, independent of ALK5 and TGF-ßRII. These results are an important contribution to a more comprehensive understanding of the mechanism behind TGF-ß3 mediated removal of HRS.

BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway.

Activin A, a member of transforming growth factor-ß superfamily, is involved in the regulation of cellular differentiation and promotes tissue healing. Previously, we reported that expression of activin A was upregulated around the damaged periodontal tissue including periodontal ligament (PDL) tissue and alveolar bone, and activin A promoted PDL-related gene expression of human PDL cells (HPDLCs). However, little is known about the biological function of activin A in alveolar bone. Thus, this study analyzed activin A-induced biological functions in preosteoblasts (Saos2 cells). Activin A promoted osteoblastic differentiation of Saos2 cells. Activin receptor-like kinase (ALK) 1, an activin type I receptor, was more strongly expressed in Saos2 cells than in HPDLCs, and knockdown of ALK1 inhibited activin A-induced osteoblastic differentiation of Saos2 cells. Expression of ALK1 was upregulated in alveolar bone around damaged periodontal tissue when compared with a nondamaged site. Furthermore, activin A promoted phosphorylation of Smad1/5/9 during osteoblastic differentiation of Saos2 cells and knockdown of ALK1 inhibited activin A-induced phosphorylation of Smad1/5/9 in Saos2 cells. Collectively, these findings suggest that activin A promotes osteoblastic differentiation of preosteoblasts through the ALK1-Smad1/5/9 pathway and could be used as a therapeutic product for the healing of alveolar bone as well as PDL tissue.

Differential regulation of TGFß type-I receptor expressions in TGFß1-induced myofibroblast differentiation.

Fibroblast-to-myofibroblast (FibroMF) differentiation is crucial for embryogenesis and organ fibrosis. Although transforming growth factor-ß (TGFß) is the primary mediator of FibroMF differentiation, the type-I receptor (TGFßRI) responsible for this has not yet been confirmed. In the current study, we investigated the ALK1 and ALK5 expressions in TGFß1-stimulated NIH 3T3 fibroblasts to compare with the data from the Gene Expression Omnibus (GEO) repository. In our results, whereas TGFß1 treatment promoted FibroMF differentiation accompanied by increased ALK5 expression and reduced ALK1 expression, TGFß1-induced FibroMF differentiation and increased α-smooth muscle actin (αSMA) and ALK5 expression were inhibited by co-treatment with ALK5 inhibitor SB431542. GEO database analysis indicated increased ALK5 expression and reduced ALK1 expression in fibrotic compared to normal mouse or human tissues correlating with organ fibrosis progression. Finally, the inhibitors of Akt, mTOR, and ß-catenin suppressed TGFß1-induced ALK5 expression, indicating that the Akt pathway promotes FibroMF differentiation via ALK5 expression and fibrosis.

The TGFß pathway is a key player for the endothelial-to-hematopoietic transition in the embryonic aorta.

The embryonic aorta produces hematopoietic stem and progenitor cells from a hemogenic endothelium localized in the aortic floor through an endothelial to hematopoietic transition. It has been long proposed that the Bone Morphogenetic Protein (BMP)/Transforming Growth Factor ß (TGFß) signaling pathway was implicated in aortic hematopoiesis but the very nature of the signal was unknown. Here, using thorough expression analysis of the BMP/TGFß signaling pathway members in the endothelial and hematopoietic compartments of the aorta at pre-hematopoietic and hematopoietic stages, we show that the TGFß pathway is preferentially balanced with a prominent role of Alk1/TgfßR2/Smad1 and 5 on both chicken and mouse species. Functional analysis using embryonic stem cells mutated for Acvrl1 revealed an enhanced propensity to produce hematopoietic cells. Collectively, we reveal that TGFß through the Alk1/TgfßR2 receptor axis is acting on endothelial cells to produce hematopoiesis.

Activin receptor-like kinase 1 is associated with immune cell infiltration and regulates CLEC14A transcription in cancer.

Cancer cells sustain their metabolic needs through nutrients and oxygen supplied by the bloodstream. The requirement for tumor angiogenesis has been therapeutically exploited in the clinical setting mainly by means of inhibition of the vascular endothelial growth factor family of ligands and receptors. Despite promising results in preclinical models, the benefits for patients proved to be limited. Inadequate efficacy similarly halted the development of agents impinging on the activity of the activin receptor-like kinase (ALK)1, a member of the transforming growth factor-ß superfamily. Notwithstanding its characterization as an endothelial cell marker, the full spectrum of biological processes associated with ALK1 is essentially unexplored. Here, we present data revealing the genetic network associated with ACVRL1 (the gene encoding for ALK1) expression in human cancer tissues. Computational analysis unveiled a hitherto unknown role for ACVRL1 in relation to genes modulating the functionality of the immune cell compartment. Moreover, we generated a signature of 8 genes co-expressed with ACVRL1 across different tumor types and characterized the c-type lectin domain containing protein (CLEC)14A as a potential downstream target of ACVRL1. Considering the lack of reagents for ALK1 detection that has hampered the field to date, our work provides the opportunity to validate the 8-gene signature and CLEC14A as biomarkers for ALK1 activity. Ultimately, this may help revisit the clinical development of already existing ALK1-blocking compounds as precision medicines for cancer.

Alk1 controls arterial endothelial cell migration in lumenized vessels.

Heterozygous loss of the arterial-specific TGFß type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs.

ADMP controls the size of Spemann's organizer through a network of self-regulating expansion-restriction signals.

BACKGROUND: The bone morphogenetic protein (BMP) signaling gradient is central for dorsoventral patterning in amphibian embryos. This gradient is established through the interaction of several BMPs and BMP antagonists and modulators, some secreted by Spemann's organizer, a cluster of cells coordinating embryonic development. Anti-dorsalizing morphogenetic protein (ADMP), a BMP-like transforming growth factor beta ligand, negatively affects the formation of the organizer, although it is robustly expressed within the organizer itself. Previously, we proposed that this apparent discrepancy may be important for the ability of ADMP to scale the BMP gradient with embryo size, but how this is achieved is unclear. RESULTS: Here we report that ADMP acts in the establishment of the organizer via temporally and mechanistically distinct signals. At the onset of gastrulation, ADMP is required to establish normal organizer-specific gene expression domains, thus displaying a dorsal, organizer-promoting function. The organizer-restricting, BMP-like function of ADMP becomes apparent slightly later, from mid-gastrula. The organizer-promoting signal of ADMP is mediated by the activin A type I receptor, ACVR1 (also known as activin receptor-like kinase-2, ALK2). ALK2 is expressed in the organizer and is required for organizer establishment. The anti-organizer function of ADMP is mediated by ACVRL1 (ALK1), a putative ADMP receptor expressed in the lateral regions flanking the organizer that blocks expansion of the organizer. Truncated ALK1 prevents the organizer-restricting effects of ADMP overexpression, suggesting a ligand-receptor interaction. We also present a mathematical model of the regulatory network controlling the size of the organizer. CONCLUSIONS: We show that the opposed, organizer-promoting and organizer-restricting roles of ADMP are mediated by different receptors. A self-regulating network is proposed in which ADMP functions early through ALK2 to expand its own expression domain, the organizer, and later functions through ALK1 to restrict this domain. These effects are dependent on ADMP concentration, timing, and the spatial localization of the two receptors. This self-regulating temporal switch may control the size of the organizer and the genes expressed within in response to genetic and external stimuli during gastrulation.

Bioreactor-Controlled Physoxia Regulates TGF-ß Signaling to Alter Extracellular Matrix Synthesis by Human Chondrocytes.

Culturing articular chondrocytes under physiological oxygen tension exerts positive effects on their extracellular matrix synthesis. The underlying molecular mechanisms which enhance the chondrocytic phenotype are, however, still insufficiently elucidated. The TGF-ß superfamily of growth factors, and the prototypic TGF-ß isoforms in particular, are crucial in maintaining matrix homeostasis of these cells. We employed a feedback-controlled table-top bioreactor to investigate the role of TGF-ß in microtissues of human chondrocytes over a wider range of physiological oxygen tensions (i.e., physoxia). We compared 1%, 2.5%, and 5% of partial oxygen pressure (pO2) to the 'normoxic' 20%. We confirmed physoxic conditions through the induction of marker genes (PHD3, VEGF) and oxygen tension-dependent chondrocytic markers (SOX9, COL2A1). We identified 2.5% pO2 as an oxygen tension optimally improving chondrocytic marker expression (ACAN, COL2A1), while suppressing de-differentiation markers (COL1A1, COL3A1). Expression of TGF-ß isoform 2 (TGFB2) was, relatively, most responsive to 2.5% pO2, while all three isoforms were induced by physoxia. We found TGF-ß receptors ALK1 and ALK5 to be regulated by oxygen tension on the mRNA and protein level. In addition, expression of type III co-receptors betaglycan and endoglin appeared to be regulated by oxygen tension as well. R-Smad signaling confirmed that physoxia divergently regulated phosphorylation of Smad1/5/8 and Smad2/3. Pharmacological inhibition of canonical ALK5-mediated signaling abrogated physoxia-induced COL2A1 and PAI-1 expression. Physoxia altered expression of hypertrophy markers and that of matrix metalloproteases and their activity, as well as expression ratios of specific proteins (Sp)/Krüppel-like transcription factor family members SP1 and SP3, proving a molecular concept of ECM marker regulation. Keeping oxygen levels tightly balanced within a physiological range is important for optimal chondrocytic marker expression. Our study provides novel insights into transcriptional regulations in chondrocytes under physoxic in vitro conditions and may contribute to improving future cell-based articular cartilage repair strategies.

Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.