ACVR1/ALK2-p21 signaling axis modulates proliferation of the venous endothelium in the retinal vasculature.

The proliferation of the endothelium is a highly coordinated process to ensure the emergence, expansion, and homeostasis of the vasculature. While Bone Morphogenetic Protein (BMP) signaling fine-tunes the behaviors of endothelium in health and disease, how BMP signaling influences the proliferation of endothelium and therefore, modulates angiogenesis remains largely unknown. Here, we evaluated the role of Activin A Type I Receptor (ACVR1/ALK2), a key BMP receptor in the endothelium, in modulating the proliferation of endothelial cells. We show that ACVR1/ALK2 is a key modulator for the proliferation of endothelium in the retinal vessels. Loss of endothelial ALK2 leads to a significant reduction in endothelial proliferation and results in fewer branches/endothelial cells in the retinal vessels. Interestingly, venous endothelium appears to be more susceptible to ALK2 deletion. Mechanistically, ACVR1/ALK2 inhibits the expression of CDKN1A/p21, a critical negative regulator of cell cycle progression, in a SMAD1/5-dependent manner, thereby enabling the venous endothelium to undergo active proliferation by suppressing CDKN1A/p21. Taken together, our findings show that BMP signaling mediated by ACVR1/ALK2 provides a critical yet previously underappreciated input to modulate the proliferation of venous endothelium, thereby fine-tuning the context of angiogenesis in health and disease.

Ubiquitin-conjugating enzyme V variant 1 enables cellular responses toward fibroblast growth factor signaling in endothelium.

Ubiquitination has been shown to provide an essential regulatory role in modulating the duration and amplitude of the signaling activity in angiogenesis. While successive enzymatic reactions mediated by three distinct types of enzymes commonly known as E1, E2, and E3 are required for ubiquitination, the role of E3s which govern the final step of ubiquitination has been extensively analyzed in angiogenesis. In contrast, the role of E2s, which determine the context and functional consequences of ubiquitination, remains largely unknown with respect to angiogenesis. To better elucidate the role of E2s in modulating endothelial behaviors during angiogenesis, we first systematically analyze the expression pattern of E2s in endothelial cells (ECs) using previously published scRNA-seq data and identify ubiquitin-conjugating enzyme variant 1 (UBE2V1), an unconventional E2 without innate catalytic activity, as one of the most abundantly expressed E2s in ECs. While ubiquitously expressed in diverse cell types, abrogation of UBE2V1 significantly impairs proliferation and viability of human umbilical vein endothelial cells (HUVECs) without affecting other cell types, suggesting that UBE2V1 is likely to possess nonredundant functions in ECs. Consistent with this idea, UBE2V1 appears to be critical for morphogenesis and migration of ECs during angiogenesis. Interestingly, we find that UBE2V1 is essential for fibroblast growth factor 2 (FGF2)-induced angiogenesis, but appears to have minor effects on vascular endothelial growth factor-A-induced angiogenesis in vitro as well as in vivo. Therefore, it seems that UBE2V1 could enable ECs to distinguish two related yet distinct angiogenic cues. Mechanistically, we show that UBE2V1 promotes ubiquitination of MEK kinase 1, a key mediator of FGF2 signaling, to enhance phosphorylation of extracellular signal-regulated kinase 1/2 in HUVECs. Taken together, our results illustrate the unique role of UBE2V1 as a key modulator for angiogenic behaviors in ECs.

Transcriptomic analysis identifies novel targets for individual bone morphogenetic protein type 1 receptors in endothelial cells.

Bone Morphogenetic Protein (BMP) signaling regulates diverse biological processes. Upon ligand binding, BMP receptors (BMPRs) phosphorylate SMAD1/5 and other noncanonical downstream effectors to induce transcription of downstream targets. However, the precise role of individual BMP receptors in this process remains largely unknown due to the complexity of downstream signaling and the innate promiscuity of ligand-receptor interaction. To delineate unique downstream effectors of individual BMPR1s, we analyzed the transcriptome of human umbilical endothelial cells (HUVECs) expressing three distinct constitutively active BMPR1s of which expression was detected in endothelial cells (ECs). From our analyses, we identified a number of novel downstream targets of BMPR1s in ECs. More importantly, we found that each BMPR1 possesses a distinctive set of downstream effectors, suggesting that each BMPR1 is likely to retain unique function in ECs. Taken together, our analyses suggest that each BMPR1 regulates downstream targets non-redundantly in ECs to create context-dependent outcomes of the BMP signaling.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Comparison of transcriptomic changes between zebrafish and mice upon high fat diet reveals evolutionary convergence in lipid metabolism.

Hyperlipidemia is an abnormal elevation of lipid level in blood, which affects more than 100 million people in US. Zebrafish has recently emerged as a model to study pathophysiology associated with hyperlipidemia. As a poikilotherm, the innate response toward a high fat diet regimen in zebrafish is likely to be distinct from humans, and therefore, additional caution is warranted to appropriately interpret results obtained from zebrafish model. However, to date, detailed comparative analyses on similarities and dissimilarities between zebrafish and mammals, in particular, at molecular level, have not been reported yet. Here, we identified changes in hepatic specific transcriptomic profiles of zebrafish fed with a high fat diet regimen and comparatively analyzed transcriptomic changes in zebrafish and mice. While a number of previously identified risk factors for human hyperlipidemia has been upregulated in zebrafish fed with a high fat diet regimen, zebrafish hepatic transcriptome does not share high similarity with mice. Despite these differences, KEGG pathway analyses revealed that similar signaling pathways upregulated in zebrafish and mice as a response to a high fat diet. Our data show that these two species may utilize species-specific set of genes to upregulate common signaling pathways, indicating evolutionary convergence between poikilotherm and homeotherm in regulating lipid metabolism and validating the use of zebrafish as a model for human hyperlipidemia and associated diseases.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 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 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.

BMPR1A promotes ID2-ZEB1 interaction to suppress excessive endothelial to mesenchymal transition.

AIMS: Components of bone morphogenetic protein (BMP) signalling have been implicated in both pathogenesis of pulmonary arterial hypertension (PAH) and endothelial-mesenchymal transition (EndoMT). In particular, the importance of BMP type 2 receptor in these processes has been extensively analysed. However, the contribution of BMP type 1 receptors (BMPR1s) to the onset of PAH and EndoMT remains poorly understood. BMPR1A, one of BMPR1s, was recently implicated in the pathogenesis of PAH, and was found to be down-regulated in the lungs of PAH patients, neither the downstream mechanism nor its contribution to EndoMT has been described. Therefore, we aim to delineate the role of endothelial BMPR1A in modulating EndoMT and pathogenesis of PAH. METHODS AND RESULTS: We find that BMPR1A knockdown in endothelial cells (ECs) induces hallmarks of EndoMT, and deletion of endothelial Bmpr1a in adult mice (Bmpr1aiECKO) leads to development of PAH-like symptoms due to excessive EndoMT. By lineage tracing, we show that endothelial-derived smooth muscle cells are increased in endothelial Bmpr1a-deleted mice. Mechanistically, we identify ZEB1 as a primary target for BMPR1A in this setting; upon BMPR1A activation, ID2 physically interacts and sequesters ZEB1 to attenuate transcription of Tgfbr2, which in turn lowers the responses of ECs towards transforming growth factor beta (TGFß) stimulation and prevents excessive EndoMT. In Bmpr1aiECKO mice, administering endothelial targeting lipid nanoparticles containing siRNA against Tgfbr2 effectively ameliorate PAH, reiterating the importance of BMPR1A-ID2/ZEB1-TGFBR2 axis in modulating progression of EndoMT and pathogenesis of PAH. CONCLUSIONS: We demonstrate that BMPR1A is key to maintain endothelial identity and to prevent excessive EndoMT. We identify BMPR1A-induced interaction between ID2 and ZEB1 is the key regulatory step for onset of EndoMT and pathogenesis of PAH. Our findings indicate that BMPR1A-ID2/ZEB1-TGFBR2 signalling axis could serve as a potential novel therapeutic target for PAH and other EndoMT-related vascular disorders.

Matching amino acids membrane preference profile to improve activity of antimicrobial peptides.

Antimicrobial peptides (AMPs) are cationic antibiotics that can kill multidrug-resistant bacteria via membrane insertion. However, their weak activity limits their clinical use. Ironically, the cationic charge of AMPs is essential for membrane binding, but it obstructs membrane insertion. In this study, we postulate that this problem can be overcome by locating cationic amino acids at the energetically preferred membrane surface. All amino acids have an energetically preferred or less preferred membrane position profile, and this profile is strongly related to membrane insertion. However, most AMPs do not follow this profile. One exception is protegrin-1, a powerful but neglected AMP. In the present study, we found that a potent AMP, WCopW5, strongly resembles protegrin-1 and that the match between its sequence and the preferred position profile closely correlates with its antimicrobial activity. One of its derivatives, WCopW43, has antimicrobial activity comparable to that of the most effective AMPs in clinical use.

The Role of BMP Signaling in Endothelial Heterogeneity.

Bone morphogenetic proteins (BMPs), which compose the largest group of the transforming growth factor-ß (TGF-ß) superfamily, have been implied to play a crucial role in diverse physiological processes. The most intriguing feature of BMP signaling is that it elicits heterogeneous responses from cells with equivalent identity, thus permitting highly context-dependent signaling outcomes. In endothelial cells (ECs), which are increasingly perceived as a highly heterogeneous population of cells with respect to their morphology, function, as well as molecular characteristics, BMP signaling has shown to elicit diverse and often opposite effects, illustrating the innate complexity of signaling responses. In this review, we provide a concise yet comprehensive overview of how outcomes of BMP signaling are modulated in a context-dependent manner with an emphasis on the underlying molecular mechanisms and summarize how these regulations of the BMP signaling promote endothelial heterogeneity.

Bone Morphogenetic Protein Signaling Restricts Proximodistal Extension of the Ventral Fin Fold.

Unpaired fins, which are the most ancient form of locomotory appendages in chordates, had emerged at least 500 million years ago. While it has been suggested that unpaired fins and paired fins share structural similarities, cellular and molecular mechanisms that regulate the outgrowth of the former have not been fully elucidated yet. Using the ventral fin fold in zebrafish as a model, here, we investigate how the outgrowth of the unpaired fin is modulated. We show that Bone Morphogenetic Protein (BMP) signaling restricts extension of the ventral fin fold along the proximodistal axis by modulating diverse aspects of cellular behaviors. We find that lack of BMP signaling, either caused by genetic or chemical manipulation, prolongs the proliferative capacity of epithelial cells and substantially increases the number of cells within the ventral fin fold. In addition, inhibition of BMP signaling attenuates the innate propensity of cell division along the anteroposterior axis and shifts the orientation of cell division toward the proximodistal axis. Moreover, abrogating BMP signaling appears to induce excessive distal migration of cells within the ventral fin fold, and therefore precipitates extension along the proximodistal axis. Taken together, our data suggest that BMP signaling restricts the outgrowth of the ventral fin fold during zebrafish development.

Analyses of Avascular Mutants Reveal Unique Transcriptomic Signature of Non-conventional Endothelial Cells.

Endothelial cells appear to emerge from diverse progenitors. However, to which extent their developmental origin contributes to define their cellular and molecular characteristics remains largely unknown. Here, we report that a subset of endothelial cells that emerge from the tailbud possess unique molecular characteristics that set them apart from stereotypical lateral plate mesoderm (LPM)-derived endothelial cells. Lineage tracing shows that these tailbud-derived endothelial cells arise at mid-somitogenesis stages, and surprisingly do not require Npas4l or Etsrp function, indicating that they have distinct spatiotemporal origins and are regulated by distinct molecular mechanisms. Microarray and single cell RNA-seq analyses reveal that somitogenesis- and neurogenesis-associated transcripts are over-represented in these tailbud-derived endothelial cells, suggesting that they possess a unique transcriptomic signature. Taken together, our results further reveal the diversity of endothelial cells with respect to their developmental origin and molecular properties, and provide compelling evidence that the molecular characteristics of endothelial cells may reflect their distinct developmental history.

Cell Electrical Impedance as a Novel Approach for Studies on Senescence Not Based on Biomarkers.

Senescence of cardiac myocytes is frequently associated with heart diseases. To analyze senescence in cardiac myocytes, a number of biomarkers have been isolated. However, due to the complex nature of senescence, multiple markers are required for a single assay to accurately depict complex physiological changes associated with senescence. In single cells, changes in both cytoplasm and cell membrane during senescence can affect the changes in electrical impedance. Based on this phenomenon, we developed MEDoS, a novel microelectrochemical impedance spectroscopy for diagnosis of senescence, which allows us to precisely measure quantitative changes in electrical properties of aging cells. Using cardiac myocytes isolated from 3-, 6-, and 18-month-old isogenic zebrafish, we examined the efficacy of MEDoS and showed that MEDoS can identify discernible changes in electrical impedance. Taken together, our data demonstrated that electrical impedance in cells at different ages is distinct with quantitative values; these results were comparable with previously reported ones. Therefore, we propose that MEDoS be used as a new biomarker-independent methodology to obtain quantitative data on the biological senescence status of individual cells.