Mesenchymal Stromal Cells Facilitate Tip Cell Fusion Downstream of BMP-Mediated Venous Angiogenesis-Brief Report.

BACKGROUND: The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. METHODS: High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. RESULTS: BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. CONCLUSIONS: Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.

Characterization of Endothelial Cilia Distribution During Cerebral-Vascular Development in Zebrafish ( Danio rerio).

Objective- Endothelial cells (ECs) sense and respond to flow-induced mechanical stress, in part, via microtubule-based projections called primary cilia. However, many critical steps during vascular morphogenesis occur independent of flow. The involvement of cilia in regulating these stages of cranial vascular morphogenesis is poorly understood because cilia have not been visualized in primary head vessels. The objective of this study was to investigate involvement of cilia in regulating the early stages of cranial vascular morphogenesis. Approach and Results- Using high-resolution imaging of the Tg(kdrl:mCherry-CAAX) y171 ;(bactin::Arl13b:GFP) zebrafish line, we showed that cilia are enriched in the earliest formed cranial vessels that assemble via vasculogenesis and in angiogenic hindbrain capillaries. Cilia were more prevalent around the boundaries of putative intravascular spaces in primary and angiogenic vessels. Loss of cardiac contractility and blood flow, because of knockdown of cardiac troponin T type 2a ( tnnt2a) expression, did not affect the distribution of cilia in primary head vasculature. In later stages of development, cilia were detected in retinal vasculature, areas of high curvature, vessel bifurcation points, and during vessel anastomosis. Loss of genes crucial for cilia biogenesis ( ift172 and ift81) induced intracerebral hemorrhages in an EC-autonomous manner. Exposure to high shear stress induced premature cilia disassembly in brain ECs and was associated with intracerebral hemorrhages. Conclusions- Our study suggests a functional role for cilia in brain ECs, which is associated with the emergence and remodeling of the primary cranial vasculature. This cilia function is flow-independent, and cilia in ECs are required for cerebral-vascular stability.

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish tie1 mRNA by Long Noncoding RNA During Brain Vascular Assembly.

OBJECTIVE: Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus-tie1 antisense (tie1AS), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. APPROACH AND RESULTS: Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS. Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. CONCLUSIONS: We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS).

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signalling pathway.

Spontaneous intracranial hemorrhage is a debilitating form of stroke, often leading to death or permanent cognitive impairment. Many of the causative genes and the underlying mechanisms implicated in developmental cerebral-vascular malformations are unknown. Recent in vitro and in vivo studies in mice have shown inhibition of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway to be effective in stabilizing cranial vessels. Using a combination of pharmacological and genetic approaches to specifically inhibit the HMGCR pathway in zebrafish (Danio rerio), we demonstrate a requirement for this metabolic pathway in developmental vascular stability. Here we report that inhibition of HMGCR function perturbs cerebral-vascular stability, resulting in progressive dilation of blood vessels, followed by vessel rupture, mimicking cerebral cavernous malformation (CCM)-like lesions in humans and murine models. The hemorrhages in the brain are rescued by prior exogenous supplementation with geranylgeranyl pyrophosphate (GGPP), a 20-carbon metabolite of the HMGCR pathway, required for the membrane localization and activation of Rho GTPases. Consistent with this observation, morpholino-induced depletion of the ß-subunit of geranylgeranyltransferase I (GGTase I), an enzyme that facilitates the post-translational transfer of the GGPP moiety to the C-terminus of Rho family of GTPases, mimics the cerebral hemorrhaging induced by the pharmacological and genetic ablation of HMGCR. In embryos with cerebral hemorrhage, the endothelial-specific expression of cdc42, a Rho GTPase involved in the regulation of vascular permeability, was significantly reduced. Taken together, our data reveal a metabolic contribution to the stabilization of nascent cranial vessels, requiring protein geranylgeranylation acting downstream of the HMGCR pathway.

Two sides of the same coin: Non-alcoholic fatty liver disease and atherosclerosis.

The prevalence of non-alcoholic fatty liver disease (NAFLD) and atherosclerosis remain high, which is primarily due to widespread adoption of a western diet and sedentary lifestyle. NAFLD, together with advanced forms of this disease such as non-alcoholic steatohepatitis (NASH) and cirrhosis, are closely associated with atherosclerotic-cardiovascular disease (ASCVD). In this review, we discussed the association between NAFLD and atherosclerosis and expounded on the common molecular biomarkers underpinning the pathogenesis of both NAFLD and atherosclerosis. Furthermore, we have summarized the mode of function and potential clinical utility of existing drugs in the context of these diseases.

Elucidating the crosstalk between endothelial-to-mesenchymal transition (EndoMT) and endothelial autophagy in the pathogenesis of atherosclerosis.

Atherosclerosis, a chronic systemic inflammatory condition, is implicated in most cardiovascular ischemic events. The pathophysiology of atherosclerosis involves various cell types and associated processes, including endothelial cell activation, monocyte recruitment, smooth muscle cell migration, involvement of macrophages and foam cells, and instability of the extracellular matrix. The process of endothelial-to-mesenchymal transition (EndoMT) has recently emerged as a pivotal process in mediating vascular inflammation associated with atherosclerosis. This transition occurs gradually, with a significant portion of endothelial cells adopting an intermediate state, characterized by a partial loss of endothelial-specific gene expression and the acquisition of "mesenchymal" traits. Consequently, this shift disrupts endothelial cell junctions, increases vascular permeability, and exacerbates inflammation, creating a self-perpetuating cycle that drives atherosclerotic progression. While endothelial cell dysfunction initiates the development of atherosclerosis, autophagy, a cellular catabolic process designed to safeguard cells by recycling intracellular molecules, is believed to exert a significant role in plaque development. Identifying the pathological mechanisms and molecular mediators of EndoMT underpinning endothelial autophagy, may be of clinical relevance. Here, we offer new insights into the underlying biology of atherosclerosis and present potential molecular mechanisms of atherosclerotic resistance and highlight potential therapeutic targets.

Investigation of Kelussia Odoratissima and Angelica Sinensis Similarities in Zebrafish-based In-vivo Bioactivity Assays and Their Chemical Composition.

BACKGROUND: Kelussia odoratissima and Angelica sinensis are two medicinal plants commonly used in Iran and China, respectively. They have been used in their indigenous traditional medicine, for various diseases including, blood refining, inflammation, cold, flu, stress, cardiovascular diseases, and nervous disorders. This study was conducted to evaluate the volatile oil composition of K. odoratissima leaves (KVL) and A. sinensis root (AVR); we also examined the biological activity of essential oils (EOs) and hydroalcoholic extracts of both plants using two different transgenic zebrafish (Danio rerio) models: angiogenesis and pancreatic beta cell (pBC) regeneration models. MATERIALS AND METHODS: Both EOs were isolated by hydrodistillation and analysed by GC and GC/MS. For viability tests, larvae were treated with different concentrations of extracts to determine an appropriate starting concentration. Hydroalcoholic extracts and EOs have been tested in a dose-dependent manner for their biological activity using tissue-specific transgenic zebrafish Tg(fli-1: EGFP) and Tg (ins: GFP-NTR) embryos and larvae. One-way ANOVA was used to compare the mean of pBC area and intersegmental vessels (ISVs) outgrowth between the treatment groups. RESULTS: Eleven compounds were in common to both oils, comprising 51.3% of KVL and 61.7% of AVR, of which 39.3% in KVL and 37.6% in AVR were phthalide structures. Results revealed that both EOs blocked ISVs formation in the Tg (fli-1: EGFP) embryos increased to 10% of the control value, while both hydroalcoholic extracts did not show any anti-angiogenesis effects in these embryos. In addition, AVR has been shown to significantly induce PBC regeneration following ablation in the Tg (ins: GFP-NTR), but its regenerative activity was lower than that of 5'-N-ethylcarboxamidoadenosine (NECA) as a positive control. Taken together, the anti-angiogenesis activity of both EOs could be attributed to the phthalide structures while for the PBC regenerative activity, other compounds including ß-Thujaplicinol, exclusively existing in AVR, might be effective. CONCLUSION: Although the genera, organs, and origin of these plants are different, their similar chemical composition and biological activities make them valuable resources for further investigation in basic medical and pharmaceutical science.

Endothelial cilia dysfunction in pathogenesis of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is associated with defective capillary network, leading to dilated superficial vessels and arteriovenous malformations (AVMs) in which arteries connect directly to the veins. Loss or haploinsufficiency of components of TGF-ß signaling, ALK1, ENG, SMAD4, and BMP9, have been implicated in the pathogenesis AVMs. Emerging evidence suggests that the inability of endothelial cells to detect, transduce and respond to blood flow, during early development, is an underpinning of AVM pathogenesis. Therefore, components of endothelial flow detection may be instrumental in potentiating TGF-ß signaling in perfused blood vessels. Here, we argue that endothelial cilium, a microtubule-based and flow-sensitive organelle, serves as a signaling hub by coupling early flow detection with potentiation of the canonical TGF-ß signaling in nascent endothelial cells. Emerging evidence from animal models suggest a role for primary cilia in mediating vascular development. We reason, on recent observations, that endothelial cilia are crucial for vascular development and that embryonic loss of endothelial cilia will curtail TGF-ß signaling, leading to associated defects in arteriovenous development and impaired vascular stability. Loss or dysfunction of endothelial primary cilia may be implicated in the genesis of AVMs due, in part, to inhibition of ALK1/SMAD4 signaling. We speculate that AVMs constitute part of the increasing spectrum of ciliopathy-associated vascular defects.

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

RhoA activation-mediated vascular permeability in capillary malformation-arteriovenous malformation syndrome: a hypothesis.

Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is a class of capillary anomalies that are associated with arteriovenous malformations and arteriovenous fistulas, which carry a risk of hemorrhages. There are no broadly effective pharmacological therapies currently available. Most CM-AVMs are associated with a loss of RASA1, resulting in constitutive activation of RAS signaling. However, protein interaction analysis revealed that RASA1 forms a complex with Rho GTPase-activating protein (RhoGAP), a negative regulator of RhoA signaling. Herein, we propose that loss of RASA1 function results in constitutive activation of RhoA signaling in endothelial cells, resulting in enhanced vascular permeability. Therefore, strategies aimed at curtailing RhoA activity should be tested as an adjunctive therapeutic approach in cell culture studies and animal models of RASA1 deficiency.

Establishing a new animal model for muscle regeneration studies.

Skeletal muscle injuries are one of the most common problems in the worldwide which impose a substantial financial burden to the health care system. Accordingly, it widely accepted that muscle regeneration is a promising approach that can be used to treat muscle injury patients. However, the underlying mechanisms of muscle regeneration have yet to be elucidated. The muscle structure and muscle-related gene expression are highly conserved between human and zebrafish. Therefore, the zebrafish can be considered as an ideal animal model in muscle regeneration studies. In this study, Tol2 transposase was applied to produce Tg(mylpfa: cfp-nfsB) zebrafish model that express a fusion protein composed of cyan fluorescent protein (CFP) and nitrorudactase (NTR) under control of mylpfa promoter. The results showed that MTZ (Metronidazole) treatment of Tg(mylpfa:cfp-nfsB) zebrafish larvae can lead to muscle injury by selective ablation of muscle cells. And also, results confirmed the muscle regeneration ability of the transgenic larvae after withdrawal of Mtz for three days. Overall, The results of this study suggest that the Tg(mylpfa:cfp-nfsB) zebrafish model can be used in muscle regeneration study in order to elucidate the mechanisms of this process.

Corrigendum: Cystathionine ß-Synthase Is Necessary for Axis Development in vivo.

[This corrects the article DOI: 10.3389/fcell.2018.00014.].

Cystathionine ß-Synthase Is Necessary for Axis Development in Vivo.

The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H2S) from cysteine. Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H2S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H2S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H2S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H2S levels, pathways that are controlled by Cbsb, and thus is essential for development.

Signaling Molecules Governing Pluripotency and Early Lineage Commitments in Human Pluripotent Stem Cells.

Signaling in pluripotent stem cells is a complex and dynamic process involving multiple mediators, finely tuned to balancing pluripotency and differentiation states. Characterizing and modifying the necessary signaling pathways to attain desired cell types is required for stem-cell applications in various fields of regenerative medicine. These signals may help enhance the differentiation potential of pluripotent cells towards each of the embryonic lineages and enable us to achieve pure in vitro cultures of various cell types. This review provides a timely synthesis of recent advances into how maintenance of pluripotency in hPSCs is regulated by extrinsic cues, such as the fibroblast growth factor (FGF) and ACTIVIN signaling pathways, their interplay with other signaling pathways, namely, wingless- type MMTV integration site family (WNT) and mammalian target of rapamycin (mTOR), and the pathways governing the determination of multiple lineages.

Disruption of pdgfra alters endocardial and myocardial fusion during zebrafish cardiac assembly.

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models is embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development.

Etiology of intracerebral hemorrhage (ICH): novel insights from Zebrafish embryos.

Intracerebral hemorrhage (ICH) is the most severe subtype of stroke. Treatment options are scarce and given the high morbidity and mortality, relatively ineffective. Since patients with ICH may have an unknown heritable component, the need to identify potential risk factors necessitates the use of animal models to elucidate the genetic underpinnings of neurovascular development and, thereby, identify candidate regulatory pathways that are likely to be disrupted in patients with ICH. Zebrafish (Danio rerio) exhibits the anatomical and physiological complexity of a closed circulatory system observed in all vertebrates (with arteries, veins and capillaries). Moreover, studies over the last decade, aided by the application of chemical mutagenesis screens, morpholino mediated knockdown approaches and tissue-specific transgenic markers, have paved the way for the identification of several genes and signaling pathways that regulate developmental neurovascular stabilization. We hypothesize that mutations in these genes or pharmacological perturbations of these gene-products may account, at least in part, for the etiology of some forms of spontaneous ICH in humans.

Could pharmacological curtailment of the RhoA/Rho-kinase pathway reverse the endothelial barrier dysfunction associated with Ebola virus infection?

Activation of the RhoA/Rho-kinase (ROCK) pathway induces endothelial barrier dysfunction and increased vascular permeability, which is a hallmark of various life-threatening vascular pathologies. Therapeutic approaches aimed at inhibiting the RhoA/ROCK pathway have proven effective in the attenuation of vascular leakage observed in animal models of endotoxin-induced lung injury/sepsis, edema, autoimmune disorders, and stroke. These findings suggest that treatments targeting the ROCK pathway might be of benefit in the management of the Ebola virus disease (EVD), which is characterized by severe vascular leak, likely involving pro-inflammatory cytokines, such as tumor necrosis factor-alpha, released from virus-infected macrophages. In this paper, we review evidence from in vivo and in vitro models of vascular leakage, suggesting that the RhoA/ROCK pathway is an important therapeutic target for the reversal of the vascular permeability defects associated with EVD. Future studies should explore the efficacy of pharmacological inhibition of RhoA/ROCK pathway on reversing the endothelial barrier dysfunction in animal models of EVD and other hemorrhagic fever virus infections as part of an adjunctive therapy. Such experimental studies should focus, in particular, on the small molecule fasudil (HA-1077), a derivative of isoquinoline, which is a safe and clinically approved inhibitor of ROCK, making it an excellent candidate in this context.