Identification of distinct vascular mural cell populations during zebrafish embryonic development.

BACKGROUND: Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells. However, the overlapping and distinct expression patterns of these markers in tandem have not been fully described. RESULTS: Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular cell populations on the cranial, axial, and intersegmental vessels between 1 and 5 days postfertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord-a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. CONCLUSION: Together, our findings highlight the variability and versatility of tracking both pdgfrb and tagln expression in mural cells of the developing zebrafish embryo and reveal unexpected embryonic cell populations that express pdgfrb and tagln.

CXCR3-CXCL11 signaling restricts angiogenesis and promotes pericyte recruitment.

Endothelial cell (EC)-pericyte interactions are known to remodel in response to hemodynamic forces, yet there is a lack of mechanistic understanding of the signaling pathways that underlie these events. Here, we have identified a novel signaling network regulated by blood flow in ECs-the chemokine receptor, CXCR3, and one of its ligands, CXCL11-that delimits EC angiogenic potential and suppresses pericyte recruitment during development through regulation of pdgfb expression in ECs. In vitro modeling of EC-pericyte interactions demonstrates that suppression of EC-specific CXCR3 signaling leads to loss of pericyte association with EC tubes. In vivo, phenotypic defects are particularly noted in the cranial vasculature, where we see a loss of pericyte association with and expansion of the vasculature in zebrafish treated with the Cxcr3 inhibitor AMG487. We also demonstrate using flow modeling platforms that CXCR3-deficient ECs are more elongated, move more slowly, and have impaired EC-EC junctions compared to their control counterparts. Together these data suggest that CXCR3 signaling in ECs drives vascular stabilization events during development.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

Objective: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (K ATP ) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with distinct hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Approach and Results: Whole-cell voltage-clamp of isolated aortic and mesenteric VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no difference in voltage-gated K + (K v ) or Ca 2+ currents. K v and Ca 2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. Pinacidil-sensitive K ATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, and were considerably larger in CS hiPSC-VSMCs. Consistent with lack of any compensatory modulation of other currents, this resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Increased compliance and dilation in isolated CS mouse aortae, was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular K ATP GoF. Conclusions: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. The results further indicate that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by K ATP overactivity within VSMCs.

Identification of overlapping and distinct mural cell populations during early embryonic development.

Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells (vSMCs). However, the expression patterns of these markers used in tandem have not been fully described. Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular populations in the cranial, axial, and intersegmental vessels between 1 and 5 days post-fertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord- a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique sclerotome-derived mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. Together, our findings highlight the variability and versatility of tracking pdgfrb and tagln expression in mural cells of the developing zebrafish embryo.

High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products.

Targeted mutagenesis in zebrafish, fruit flies, and C. elegans has been significantly improved over the years through CRISPR technology. CRISPR enables researchers to efficiently examine cellular pathways by inducing small, targeted mutations in vivo. Though these mutations are commonly random insertions or deletions (indels), they often result in functionally disrupted alleles of a target gene if the CRISPR components are appropriately designed. However, current protocols used to identify the presence of CRISPR-generated indels are often labor intensive, time-consuming, or expensive. Here, we describe a straightforward, high-throughput method for identifying the presence of mutations by using a fragment analyzer platform which allows for DNA fragment sizing through high-resolution capillary gel-electrophoresis. Following this protocol, small indels-down to 2 base pairs-can be quickly and reliably identified, thus allowing for large-scale genotyping of newly-generated or stable mutant lines.

The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development.

ATP-dependent chromatin-remodeling complexes epigenetically modulate transcription of target genes to impact a variety of developmental processes. Our lab previously demonstrated that CHD4-a central ATPase and catalytic enzyme of the NuRD chromatin-remodeling complex-plays an important role in murine embryonic endothelial cells by transcriptionally regulating vascular integrity at midgestation. Since NuRD complexes can incorporate the ATPase CHD3 as an alternative to CHD4, we questioned whether the CHD3 enzyme likewise modulates vascular development or integrity. We generated a floxed allele of Chd3 but saw no evidence of lethality or vascular anomalies when we deleted it in embryonic endothelial cells in vivo (Chd3ECKO). Furthermore, double-deletion of Chd3 and Chd4 in embryonic endothelial cells (Chd3/4ECKO) did not dramatically alter the timing and severity of embryonic phenotypes seen in Chd4ECKO mutants, indicating that CHD3 does not play a cooperative role with CHD4 in early vascular development. However, excision of Chd3 at the epiblast stage of development with a Sox2-Cre line allowed us to generate global heterozygous Chd3 mice (Chd3Δ/+), which were subsequently intercrossed and revealed partial lethality of Chd3Δ/Δ mutants prior to weaning. Tissues from surviving Chd3Δ/Δ mutants helped us confirm that CHD3 was efficiently deleted in these animals and that CHD3 is highly expressed in the gonads and brains of adult wildtype mice. Therefore, Chd3-flox mice will be beneficial for future studies about roles for this chromatin-remodeling enzyme in viable embryonic development and in gonadal and brain physiology.

Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis.

Receptor-interacting protein kinase 3 (RIPK3) was recently implicated in promoting atherosclerosis progression through a proposed role in macrophage necroptosis. However, RIPK3 has been connected to numerous other cellular pathways, which raises questions about its actual role in atherosclerosis. Furthermore, RIPK3 is expressed in a multitude of cell types, suggesting that it may be physiologically relevant to more than just macrophages in atherosclerosis. In this study, Ripk3 was deleted in macrophages, endothelial cells, vascular smooth muscle cells or globally on the Apoe-/- background using Cre-lox technology. To induce atherosclerosis progression, male and female mice were fed a Western diet for three months before tissue collection and analysis. Surprisingly, necroptosis markers were nearly undetectable in atherosclerotic aortas. Furthermore, en face lesion area was increased in macrophage- and endothelial-specific deletions of Ripk3 in the descending and abdominal regions of the aorta. Analysis of bone-marrow-derived macrophages and cultured endothelial cells revealed that Ripk3 deletion promotes expression of monocyte chemoattractant protein 1 (MCP-1) and E-selectin in these cell types, respectively. Western blot analysis showed upregulation of MCP-1 in aortas with Ripk3-deficient macrophages. Altogether, these data suggest that RIPK3 in macrophages and endothelial cells protects against atherosclerosis through a mechanism that likely does not involve necroptosis. This protection may be due to RIPK3-mediated suppression of pro-inflammatory MCP-1 expression in macrophages and E-selectin expression in endothelial cells. These findings suggest a novel and unexpected cell-type specific and athero-protective function for RIPK3.This article has an associated First Person interview with the first author of the paper.

The NuRD chromatin-remodeling complex enzyme CHD4 prevents hypoxia-induced endothelial Ripk3 transcription and murine embryonic vascular rupture.

Physiological hypoxia can trigger transcriptional events that influence many developmental processes during mammalian embryogenesis. One way that hypoxia affects transcription is by engaging chromatin-remodeling complexes. We now report that chromodomain helicase DNA binding protein 4 (CHD4), an enzyme belonging to the nucleosome remodeling and deacetylase (NuRD) chromatin-remodeling complex, is required for transcriptional repression of the receptor-interacting protein kinase 3 (Ripk3)-a critical executor of the necroptosis cell death program-in hypoxic murine embryonic endothelial cells. Genetic deletion of Chd4 in murine embryonic endothelial cells in vivo results in upregulation of Ripk3 transcripts and protein prior to vascular rupture and lethality at midgestation, and concomitant deletion of Ripk3 partially rescues these phenotypes. In addition, CHD4 binds to and prevents acetylation of the Ripk3 promoter in cultured endothelial cells grown under hypoxic conditions to prevent excessive Ripk3 transcription. These data demonstrate that excessive RIPK3 is detrimental to embryonic vascular integrity and indicate that CHD4 suppresses Ripk3 transcription when the embryonic environment is particularly hypoxic prior to the establishment of fetal-placental circulation at midgestation. Altogether, this research provides new insights into regulators of Ripk3 transcription and encourages future studies into the mechanism by which excessive RIPK3 damages embryonic blood vessels.

OKN-007 decreases VEGFR-2 levels in a preclinical GL261 mouse glioma model.

Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker vascular endothelial growth factor receptor 2 (VEGFR-2) may provide an accurate evaluation for angiogenesis during a therapeutic response. With the use of molecular magnetic resonance imaging (mMRI), an in vitro cell assay indicated significantly decreased T1 relaxation values when tumor endothelial cells (TEC), which positively expressed VEGFR-2 (Western blot), were in the presence of the VEGFR-2 probe compared to TEC alone (P < 0.001). For in vivo mMRI evaluations, we assessed VEGFR-2 levels in untreated and OKN-007-treated GL261 mouse gliomas. Regarding treatment response, OKN-007 was also able to significantly decrease tumor volumes (P < 0.01) and increase survival (P < 0.001) in treated animals. Regarding in vivo detection of VEGFR-2, OKN-007 was found to significantly decrease the amount of VEGFR-2 probe (P < 0.05) compared to an untreated control group. Fluorescence imaging for the VEGFR-2 probe indicated that there was colocalization with the endothelial marker CD31 in an untreated tumor bearing mouse and decreased levels for an OKN-007-treated animal. Immuno-fluorescence imaging for VEGFR-2 indicated that OKN-007 treatment significantly decreased VEGFR-2 levels (P < 0.0001) when compared to untreated tumors. Immuno-electron microscopy was used with gold-labeled anti-biotin to detect the anti-VEGFR-2 probe within the plasma membrane of GL261 tumor endothelial cells. This is the first attempt at detecting in vivo levels of VEGFR-2 in a mouse GL261 glioma model and assessing the anti-angiogenic capability of an anticancer nitrone. The results indicate that OKN-007 treatment substantially decreased VEGFR-2 levels in a GL261 glioma model, and can be considered as an anti-angiogenic therapy in human gliomas.