Jun-Dae Kim

Apelin-APJ Signaling Is a Critical Regulator of Endothelial MEF2 Activation in Cardiovascular Development

Rationale: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. Objective: This study aimed to characterize the embryonic lethal phenotype of the Apj−/− mice and to define the involved downstream signaling targets. Methods and Results: We report the first characterization of the embryonic lethality of the Apj−/− mice. More than half of the expected Apj−/− embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj−/− embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving G&agr;13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj−/− mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, G&agr;13, and MEF2 in endothelial cells, which are significantly decreased in the Apj−/− embryos and endothelial cells. Conclusions: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

Essential Role of Apelin Signaling During Lymphatic Development in Zebrafish

Objective—Apelin and its cognate receptor Aplnr/Apj are essential for diverse biological processes. However, the function of Apelin signaling in lymphatic development remains to be identified, despite the preferential expression of Apelin and Aplnr within developing blood and lymphatic endothelial cells in vertebrates. In this report, we aim to delineate the functions of Apelin signaling during lymphatic development. Approach and Results—We investigated the functions of Apelin signaling during lymphatic development using zebrafish embryos and found that attenuation of Apelin signaling substantially decreased the formation of the parachordal vessel and the number of lymphatic endothelial cells within the developing thoracic duct, indicating an essential role of Apelin signaling during the early phase of lymphatic development. Mechanistically, we found that abrogation of Apelin signaling selectively attenuates lymphatic endothelial serine–threonine kinase Akt 1/2 phosphorylation without affecting the phosphorylation status of extracellular signal–regulated kinase 1/2. Moreover, lymphatic abnormalities caused by the reduction of Apelin signaling were significantly exacerbated by the concomitant partial inhibition of serine–threonine kinase Akt/protein kinase B signaling. Apelin and vascular endothelial growth factor-C (VEGF-C) signaling provide a nonredundant activation of serine–threonine kinase Akt/protein kinase B during lymphatic development because overexpression of VEGF-C or apelin was unable to rescue the lymphatic defects caused by the lack of Apelin or VEGF-C, respectively. Conclusions—Taken together, our data present compelling evidence suggesting that Apelin signaling regulates lymphatic development by promoting serine–threonine kinase Akt/protein kinase B activity in a VEGF-C/VEGF receptor 3–independent manner during zebrafish embryogenesis.

Transmembrane protein ESDN promotes endothelial VEGF signaling and regulates angiogenesis

Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VE-cadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.

FIH-1, a Novel Interactor of Mindbomb, Functions as an Essential Anti-Angiogenic Factor during Zebrafish Vascular Development

Objective It has been shown that Mindbomb (Mib), an E3 Ubiquitin ligase, is an essential modulator of Notch signaling during development. However, its effects on vascular development remain largely unknown. Approaches and Results We identified a number of novel proteins that physically interact with Mib, including the Factor Inhibiting Hypoxia Inducible Factor 1 (FIH-1, also known as HIF1AN) from a yeast two hybrid screen, as previously reported. In cultured cells, FIH-1 colocalizes with Mib1, corroborating their potential interaction. In zebrafish embryos, FIH-1 appears to modulate VEGF-A signaling activity; depletion of fih-1 induces ectopic expression of vascular endothelial growth factor–a (vegfa) and leads to exuberant ectopic sprouts from intersegmental vessels (ISVs). Conversely, over-expression of fih-1 substantially attenuates the formation of ISVs, which can be rescued by concurrent over-expression of vegfa, indicating that FIH-1/HIF1AN may fine tune VEGF-A signaling. Conclusions Taken together, our data suggest that FIH-1 interacts with Mib E3 Ubiquitin ligase and modulates vascular development by attenuating VEGF-A signaling activity.

Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.

Alk3/Alk3b and Smad5 Mediate BMP Signaling during Lymphatic Development in Zebrafish

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.

Modulation of Endothelial BMPR2 Activity by VEGFR3 in Pulmonary Arterial Hypertension.

Background —Bone Morphogenetic Protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP type 2 receptors (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. Methods —We utilized a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between VEGFR3 and BMPR2. Additional in vitro studies were performed using human endothelial cells, including primary endothelial cells from subjects with PAH. Results —Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells (ECs) with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension (PH). Consistent with this data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells (PAECs) from human PAH subjects, and reconstitution of VEGFR3 expression in PAH PAECs restored BMP signaling responses. Conclusions —Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.Background: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. Methods: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. Results: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. Conclusions: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Diversity Is in My Veins

Endothelial cells are a highly diverse group of cells which display distinct cellular responses to exogenous stimuli. Although the aptly named vascular endothelial growth factor-A signaling pathway is hailed as the most important signaling input for endothelial cells, additional factors also participate in regulating diverse aspects of endothelial behaviors and functions. Given this heterogeneity, these additional factors seem to play a critical role in creating a custom-tailored environment to regulate behaviors and functions of distinct subgroups of endothelial cells. For instance, molecular cues that modulate morphogenesis of arterial vascular beds can be distinct from those that govern morphogenesis of venous vascular beds. Recently, we have found that bone morphogenetic protein signaling selectively promotes angiogenesis from venous vascular beds without eliciting similar responses from arterial vascular beds in zebrafish, indicating that bone morphogenetic protein signaling functions as a context-dependent regulator during vascular morphogenesis. In this review, we will provide an overview of the molecular mechanisms that underlie proangiogenic effects of bone morphogenetic protein signaling on venous vascular beds in the context of endothelial heterogeneity and suggest a more comprehensive picture of the molecular mechanisms of vascular morphogenesis during development.

Diversity Is in My Veins Role of Bone Morphogenetic Protein Signaling During Venous Morphogenesis in Zebrafish Illustrates the Heterogeneity Within Endothelial Cells

Endothelial cells are a highly diverse group of cells which display distinct cellular responses to exogenous stimuli. Although the aptly named vascular endothelial growth factor-A signaling pathway is hailed as the most important signaling input for endothelial cells, additional factors also participate in regulating diverse aspects of endothelial behaviors and functions. Given this heterogeneity, these additional factors seem to play a critical role in creating a custom-tailored environment to regulate behaviors and functions of distinct subgroups of endothelial cells. For instance, molecular cues that modulate morphogenesis of arterial vascular beds can be distinct from those that govern morphogenesis of venous vascular beds. Recently, we have found that bone morphogenetic protein signaling selectively promotes angiogenesis from venous vascular beds without eliciting similar responses from arterial vascular beds in zebrafish, indicating that bone morphogenetic protein signaling functions as a context-dependent regulator during vascular morphogenesis. In this review, we will provide an overview of the molecular mechanisms that underlie proangiogenic effects of bone morphogenetic protein signaling on venous vascular beds in the context of endothelial heterogeneity and suggest a more comprehensive picture of the molecular mechanisms of vascular morphogenesis during development.

A tale of two models: mouse and zebrafish as complementary models for lymphatic studies.

Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical importance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endothelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lymphedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in vertebrates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels.