Biology subjects

Stratman, A. N.

Publications and source records attributed to Stratman, A. N..

18 recordsLinked to original sources

bioRxiv2025

Neutrophil TLR2 signaling promotes lipid accumulation and vascular plaque growth

Neutrophils are short-lived cells that are produced by the billions every day to circulate throughout the body and surveil all tissues. They are a key component of the innate immune system that play essential roles in antimicrobial immunity, but can also instigate sterile inflammatory diseases like gout and cancer. Immunometabolic paradigms that were developed by studying T cells and macrophages establish that cellular metabolic programming dictates immune function. Neutrophils have long been known as glucose-reliant and highly glycolytic. But surprising neutrophil heterogeneity has recently been described, and roles for lipids have been reported in both granulopoiesis and mature neutrophil effector function. Therefore, we set out to uncover how neutrophils acquire lipids from their environment and how this influences their functionality in the context of lipotoxicity. We found that neutrophils take up both free fatty acids and complex lipoproteins, but that their uptake is regulated through different signaling pathways. Neutrophil lipoprotein uptake is inducible by certain TLR2 signals, and this causes neutrophils to depolymerize their actin fibers and stop moving. Using a mouse model of atherosclerosis, we show that neutrophils in the plaque are lipid-laden and that neutrophil-deficient mice are protected from atherosclerotic plaque growth. Lipoprotein uptake causes neutrophils to recruit macrophages, conditional ablation of TLR2 on neutrophils prevents their lipid uptake and storage, and these mice are also protected against atherosclerosis. Our work highlights an important understudied role for lipids in neutrophil biology, and the importance of studying different lipid classes and different signaling pathways in neutrophils as compared to other myeloid populations.

immunology↗

bioRxiv2025

Ablation of VEGFA following a lumbar intervertebral disc injury attenuates intradiscal neurovascular features and prevents chronic low back pain symptoms

Despite its enormous burden on patients and society, chronic low back pain (LBP) has no effective therapeutic options. Innervation of the degenerating intervertebral disc (IVD) is suspected to cause discogenic LBP, but the mechanisms that orchestrate the IVDs neo-innervation and subsequent symptoms of LBP remain unknown. We hypothesize that Vascular Endothelial Growth Factor-A (VEGFA) critically mediates the neurite invasion in the IVD and contributes to chronic LBP. Initiating IVD degeneration through a mechanical injury, we evaluated the progression of neurovascular features into the IVD, as well as ensuring LBP symptoms and locomotive impairments at acute (3-weeks) and chronic (12-weeks) timepoints following the IVD injury. To determine the role of VEGFA, we utilized a mouse model with ubiquitously inducible recombination of the floxed Vegfa allele (UBC-CreERT2; Vegfafl/fl). The ablation of VEGFA after an IVD injury attenuated de novo neurite and vessel infiltration and impeded the expression of TRPA1, a nociceptive ion channel, in the dorsal root ganglion. The VEGFA-null animals, despite IVD degeneration, exhibited alleviated mechanical allodynia and improved locomotive performance. To determine the effects of IVD-derived VEGFA on endothelial cells and neurons, we co-cultured HMEC-1 endothelial cells and SH-SY5Y neurons with VEGFA-silenced human primary IVD cells. The endothelial cells co-cultured with VEGFA-silenced IVD cells exhibited reduced vessel growth and shifted their transcriptome and secretome from angiogenic to lymphangiogenic. The neurons co-cultured VEGFA-silenced IVD cells showed slowed growth and attenuated transcriptional programs for growth and elongation. These results show that VEGFA directs the growth of intradiscal vessels and neurites that cause low back pain and impaired function, and the inhibition of IVD-derived VEGFA during degeneration may be sufficient to prevent chronic pain behavior and motor impairment associated with discogenic low back pain. One Sentence SummaryVEGFA is a key mediator of neurovascular infiltration in the degenerating intervertebral disc and an essential driver of chronic low back pain, whose ablation prevents pain-related behaviors.

neuroscience↗

bioRxiv2024

The E3 Ubiquitin Ligase Trip12 attenuates Wnt9a/Fzd9b signaling during hematopoietic stem cell development

Wnt signaling is essential for both the development and homeostasis of diverse cellular lineages, including hematopoietic stem cells. Organism-wide, Wnt signals are tightly regulated, as overactivation of the pathway can lead to tumorigenesis. Although numerous Wnt ligands and Frizzled (Fzd) receptors exist, how particular Wnt/Fzd pairings are established and how their signals are regulated is poorly understood. We have previously identified the requirements of the cognate pairing of Wnt9a and Fzd9b for early hematopoietic stem cell proliferation. However, the specific signals governing activation, but equally important, the molecular mechanisms required to turn the signal off, are unknown. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) is specifically required to ubiquitinate the third intracellular loop of Fzd9b at K437, targeting it for lysosomal degradation. In contrast to other ubiquitin ligases described to regulate the cell surface availability of multiple Fzds broadly, our data indicate that Trip12 is selective for Fzd9b. We further demonstrate that this occurs through ubiquitination at K437 of Fzd9b in the third intracellular loop, ultimately leading to a decrease in Fzd9b receptor availability and in Wnt9a/Fzd9b signaling that impacts hematopoietic stem cell proliferation in zebrafish. Our results point to specific mechanisms driving the availability of different Fzd receptors. Determining how particular Fzd abundance is regulated at the membrane will be critical to developing specific therapies for human intervention. One sentence summaryTrip12 ubiquitinates Fzd9b

cell biology↗

bioRxiv2024

Role of U11/U12 minor spliceosome gene ZCRB1 in Ciliogenesis and WNT Signaling

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally, zcrb1 CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.

cell biology↗

bioRxiv2024

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

developmental biology↗

bioRxiv2024

Oncogenic KRAS Mutations Confer a Unique Mechanotransduction Response to Peristalsis in Colorectal Cancer Cells

Colorectal cancer (CRC) tumors start as precancerous polyps on the inner lining of the colon or rectum, where they are exposed to the mechanics of colonic peristalsis. Our previous work leveraged a custom-built peristalsis bioreactor to demonstrate that colonic peristalsis led to cancer stem cell enrichment in colorectal cancer cells. However, this malignant mechanotransductive response was confined to select CRC lines that harbored an oncogenic mutation in the KRAS gene. In this work, therefore, we explored the involvement of activating KRAS mutations on peristalsis-associated mechanotransduction in CRC. Peristalsis enriched the cancer stem cell marker LGR5 in KRAS mutant (G13D, etc.) lines, in a Wnt-independent manner. Conversely, LGR5 enrichment in wild type KRAS lines exposed to peristalsis were minimal. LGR5 enrichment downstream of peristalsis translated to increased tumorigenicity in vivo in KRAS mutant vs. wild type lines. Differences in mechanotransduction response was additionally apparent via unbiased gene set enrichment analysis, where many unique pathways were enriched in wild type vs. mutant lines, in response to peristalsis. Interestingly, peristalsis also triggered {beta}-catenin nuclear localization independent of Wnt, particularly in KRAS mutant lines. The central involvement of KRAS in the mechanotransductive responses was validated via gain and loss of function strategies. {beta}-catenin activation and LGR5 enrichment downstream of peristalsis converged to the activation of the MEK/ERK kinase cascade, that remains active in cells that harbor oncogenic KRAS mutations. Taken together, our results demonstrated that oncogenic KRAS mutations conferred a unique peristalsis-associated mechanotransduction response to colorectal cancer cells, resulting in cancer stem cell enrichment and increased tumorigenicity. These mechanosensory connections can be leveraged in improving the sensitivity of emerging therapies that target oncogenic KRAS.

bioengineering↗

bioRxiv2024

Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

cell biology↗

bioRxiv2023

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries, playing a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular diseases and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating differentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on the zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that the arterial expression of CoW endothelial cells (ECs) occurs after their migration from the cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors upon recruitment to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity wall shear stress. Furthermore, pulsatile blood flow is required for differentiation of human brain pdgfrb+ mural cells into VSMCs as well as VSMC differentiation on zebrafish CoW arteries. Consistently, the flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight the role of blood flow activation of endothelial klf2a as a mechanism regulating the initial VSMC differentiation on vertebrate brain arteries.

developmental biology↗

bioRxiv2023

FAM210B is dispensable for erythroid differentiation in adult mice

Iron is required for redox homeostasis but poses toxicity risks due to its redox activity. Erythropoiesis hence requires tight regulation of iron utilization for hemoglobin synthesis. The requirement for iron in erythropoiesis has necessitated the evolution of mechanisms to handle the iron required for hemoglobinization. FAM210B was identified as a regulator of mitochondrial iron import and heme synthesis in erythroid cell culture and zebrafish models. Here, we demonstrate that while FAM210B is required for erythroid differentiation and heme synthesis under standard cell culture conditions, holotransferrin supplementation was sufficient to chemically complement the iron-deficient phenotype. To investigate the role of FAM210B in erythropoiesis, we used knockout mice. While Fam210b-/- mice were viable and did not exhibit overt erythropoietic defects in the bone marrow, the male mice exhibited an increase in serum transferrin suggesting sex-specific alterations in systemic iron sensing. Upon phlebotomy-induced stress erythropoiesis, Fam210b-/- mice exhibited differences in serum transferrin levels, and more starkly, had markedly smaller spleens indicating defects in stress response. Fam210b-/- males had defects in neutrophil and monocyte numbers, as well as decreased erythroid progenitor numbers during erythropoietic stress. Together, our findings show that Fam210b plays a key role in splenic response to erythropoietic stress Our findings reveal a critical role for FAM210B in mediating splenic stress erythropoiesis and suggest it may act as a sex-specific regulator potentially linked to androgen signaling.

developmental biology↗

bioRxiv2023

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.

developmental biology↗

bioRxiv2023

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

ObjectiveCantu 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 (KATP) 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 ResultsWhole-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+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. Pinacidil-sensitive KATP 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 KATP GoF. ConclusionsThe 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 KATP overactivity within VSMCs.

physiology↗

bioRxiv2023

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. Summary StatementDetailed analysis of pdgfrb/tagln vascular expression patterns in embryonic zebrafish reveals novel regions of tagln-positive populations such as the hypochord and a mural cell progenitor population adjacent to the midline.

developmental biology↗

bioRxiv2022

Regulation of iron transport is required for terminal erythroid differentiation even under iron-replete conditions that are sufficient for hemoglobinization

Iron metabolism drives key erythropoietic processes, including hemoglobinization, survival, and proliferation. Here, we developed in vivo methods to interrogate how iron regulates erythropoiesis and report that mitochondrial iron transport via MFRN1 is essential for erythroid cell cycle progression. mfrn1 embryos had severely decreased erythroid cell number caused by cell cycle arrest at G2/M. They had enlarged nuclei, suggesting a mitotic defect. Iron supplementation rescued the cell cycle defect, implicating mitochondrial iron deficiency as its cause. In contrast, fpn1 mutants, anemic from systemic iron deficiency, had less severe decreases in erythroid mitochondrial iron than mfrn1 mutants and no proliferative defects. scRNAseq and FACS analyses for cd41 (thrombocytic) and gata1 reporters indicated that developmental defects in mfrn1 mutants were largely erythroid restricted. mfrn1 mutant gata1+ erythroid progenitors were severely decreased at 3 dpf, and a further decrease in globin- expressing terminally differentiating erythroid cells. While wild-type erythroid cells mostly lost expression of the gata1 progenitor marker by 3 dpf, mfrn1 mutant erythroid cells retained gata1 expression. These data are consistent with a model where mitochondrial iron transport facilitates development of gata1+ erythroid progenitors and is required for the completion of erythropoiesis by facilitating mitosis in the terminal cell cycles. Key pointsO_LIMitochondrial iron import through MFRN1 is required for progress through the G2/M checkpoint in the terminal cell cycles leading to terminal erythroid differentiation. C_LIO_LIMitochondrial iron supply through MFRN1 is required for maintenance/proliferation of erythroid progenitors and terminal maturation into globin expressing erythroid cells. C_LI

biochemistry↗

bioRxiv2022

Mechanotransductive feedback control of endothelial cell motility and vascular morphogenesis

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

cell biology↗

bioRxiv2021

Adaptation of peristaltic pumps for laminar flow experiments

Endothelial cells (ECs) are the primary cellular constituent of blood vessels that are in direct contact with hemodynamic forces over the course of a lifetime. Throughout the body, vessels experience different types of blood flow patterns and rates that alter vascular architecture and cellular behavior. Because of the complexities of studying blood flow in an intact organism, particularly during development, modeling of blood flow in vitro has become a powerful technique for studying hemodynamic dependent signaling mechanisms in ECs. While commercial flow systems that recirculate fluids exist, many commercially available pumps are peristaltic and best model pulsatile flow conditions. However, there are many important in vivo situations in which ECs experience laminar flow conditions, such as along long, straight stretches of the vasculature. To understand EC function under these situations, it is important to be able to consistently model laminar flow conditions in vitro. Here, we outline a method to reliably adapt commercially available peristaltic pumps to reproducibly study laminar flow conditions. Our proof of concept study focuses on 2-dimensional (2D) models but could be further adapted to 3-dimensional (3D) environments to better model in vivo scenarios such as organ development. Our studies make significant inroads into solving technical challenges associated with flow modeling, and allow us to conduct functional studies towards understanding the mechanistic role of flow forces on vascular architecture, cellular behavior, and remodeling during a variety of physiological contexts.

cell biology↗

bioRxiv2021

In vivo dissection of Rhoa function in vascular development using zebrafish

RationaleThe small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. ObjectiveIn this study we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. Methods and ResultsWe use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant-negative, or constitutively active forms of rhoaa in ECs, and a pharmacologic inhibitor of ROCK1/2 to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis. ConclusionsOur results indicate that either too much or too little RHOA activity leads to vascular dysfunction in vivo.

developmental biology↗

bioRxiv2019

A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

The preferential accumulation of vascular smooth muscle cells on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. During zebrafish embryogenesis the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vascular smooth muscle cells (vSMCs). We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries in vivo. Additionally, we demonstrate that expression of the well-characterized blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signaling axis leads to the differential acquisition of smooth muscle cells at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

cell biology↗

bioRxiv2018

Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth (1-6). However, the ability of tumors to overproduce pro-angiogenic ligands and overcome targeted inhibitory therapies has hampered this approach (7, 8). A novel way to circumvent this problem might be to target the resynthesis of critical substrates consumed during intracellular transduction of pro-angiogenic signals in endothelial cells, thus harnessing the tumors own production of excess stimulatory ligands to deplete adjacent host endothelial cells of the capacity to respond to these signals (9-12). Here we show using zebrafish and human endothelial cells in vitro that endothelial cells deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased VEGF stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) a key substrate for VEGF signal transduction, resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling (9-22). Using murine tumor allograft models (23) we show that either systemic or endothelial cell specific suppression of phosphoinositide recycling results in reduced tumor growth and reduced tumor angiogenesis. Our results suggest that inhibition of phosphoinositide recycling may provide a useful anti-angiogenic approach, and highlights the general potential of targeting the resynthesis of rate limiting signaling substrates as a valuable therapeutic strategy.\n\nSUMMARY STATEMENTTargeting phosphoinositide recycling during tumor angiogenesis provides a potentially uniquely effective anti-cancer therapy.

cell biology↗