Abstract FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Live-imaging of temporally controlled sFLT1 release from the endoplasmic reticulum showed clathrin-dependent sFLT1 trafficking at the Golgi into secretory vesicles that then trafficked to the plasma membrane. Depletion of STX6 altered vessel sprouting in 3D, suggesting that endothelial cell sFLT1 secretion influences proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.
ABSTRACT Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here we show that the nuclear-localized LINC complex protein SUN1 regulates vascular sprouting and barrier function via effects on endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted barrier function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and barrier function. SUMMARY The nuclear membrane protein SUN1 promotes blood vessel formation and barrier function by stabilizing endothelial cell-cell junctions. Communication between SUN1 and endothelial cell junctions relies upon proper microtubule dynamics and Rho signaling far from the nucleus, revealing long-range cellular communication from the nucleus to the cell periphery that is important for vascular development and function.
We developed an agent-based model of endothelial sprout initiations based on time-lapse confocal imaging in vitro that outperforms Monte Carlo simulations, suggesting that sprout location and frequency are not purely stochastic behaviors.
Abstract Background: Functional and molecular changes often precede gross anatomical changes in cancer, so early assessment of a tumor’s functional and molecular response to therapy can help reduce a patient’s exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. Clear-cell renal cell carcinoma (ccRCC) is an aggressive and hyper-vascular form of renal cancer that is often treated with anti-angiogenic and Notch Inhibition therapies, which target the vasculature feeding the disease. The purpose of this work is to show that ultrasound microvascular imaging can provide indications of response to antiangiogenic and Notch Inhibition therapies prior to measurable changes in tumor size. Methods: Mice bearing 786-O ccRCC xenograft tumors were treated with SU (Sunitnib malate, Selleckchem, TX), an antiangiogenic drug, and a combination of SU and the Notch inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY) therapies (n=8). A 3D ultrasound system (SonoVol Inc., Research Triangle Park, NC), in addition to microbubble ultrasound contrast agents, was used to obtain a measurement of microvascular density over time and assess the response of the tumors to the therapies. CD31 immunohistochemistry was performed to serve as a gold standard for comparison against imaging results. Statistical tests included: Spearman correlation to compare imaging and histology; Kruskal-Wallis analysis with Tukey multiple comparison post-test to determine if the vessel density or tumor volume were significantly different between the treatment groups; and receiver operating characteristic (ROC) curve analysis to determine sensitivity/specificity for separating treated/untreated groups. Results: Data indicated that ultrasound-derived microvascular density can detect response to antiangiogenic and Notch inhibition therapies a week prior to changes in tumor volume. Furthermore, the imaging measurements of vasculature are strongly correlated with physiological characteristics of the tumors as measured by histology (p=0.75). Moreover, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups 1 week after the start of treatment with a high sensitivity and specificity of 94% and 86%, respectively. Conclusion: This work shows vascular density measurements that are strongly correlated with histology can be obtained using ultrasound, and that imaging-derived vessel density metrics may be a better tool for assessing the response of ccRCC to antiangiogenic and Notch inhibition therapies than anatomical size measurements. Note: This abstract was not presented at the meeting. Citation Format: Juan D. Rojas, Virginie Papadopoulou, Tomasz Czernuszewicz, Rajalekha Rajamahendiran, Anna Chytil, Yun-Chen Chiang, Diana Chong, Victoria L. Bautch, Wendy K. Rathmell, Stephen Aylward, Ryan Gessner, Paul Dayton. Early treatment response detected in a murine clear cell renal cell carcinoma model in response to combination therapy with antiangiogenic and notch inhibition therapy using a non-invasive imaging tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1958.
Blood vessel formation is essential for vertebrate development and is primarily achieved by angiogenesis - endothelial cell sprouting from pre-existing vessels. Vessel networks expand when sprouts form new connections, a process whose regulation is poorly understood. Here, we show that vessel anastomosis is spatially regulated by Flt1 (VEGFR1), a VEGFA receptor that acts as a decoy receptor. In vivo, expanding vessel networks favor interactions with Flt1 mutant mouse endothelial cells. Live imaging in human endothelial cells in vitro revealed that stable connections are preceded by transient contacts from extending sprouts, suggesting sampling of potential target sites, and lowered Flt1 levels reduced transient contacts and increased VEGFA signaling. Endothelial cells at target sites with reduced Flt1 and/or elevated protrusive activity were more likely to form stable connections with incoming sprouts. Target cells with reduced membrane-localized Flt1 (mFlt1), but not soluble Flt1, recapitulated the bias towards stable connections, suggesting that relative mFlt1 expression spatially influences the selection of stable connections. Thus, sprout anastomosis parameters are regulated by VEGFA signaling, and stable connections are spatially regulated by endothelial cell-intrinsic modulation of mFlt1, suggesting new ways to manipulate vessel network formation.
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