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Several signaling pathways are activated during hypoxia to promote angiogenesis, leading to endothelial cell patterning, interaction, and downstream signaling. Understanding the mechanistic signaling differences between normoxia and hypoxia can guide therapies to modulate angiogenesis. We present a novel mechanistic model of interacting endothelial cells, including the main pathways involved in angiogenesis. We calibrate and fit the model parameters based on well-established modeling techniques. Our results indicate that the main pathways involved in the patterning of tip and stalk endothelial cells under hypoxia differ, and the time under hypoxia affects how a reaction affects patterning. Interestingly, the interaction of receptors with Neuropilin1 is also relevant for cell patterning. Our simulations under different oxygen concentrations indicate time- and oxygen-availability-dependent responses for the two cells. Following simulations with various stimuli, our model suggests that factors such as period under hypoxia and oxygen availability must be considered for pattern control. This project provides insights into the signaling and patterning of endothelial cells under hypoxia, contributing to studies in the field.

Hypothesis: miR-433-3p is a potential therapeutic target to reduce the excess endothelial permeability seen in peripheral skeletal muscle of diabetes. Background: Peripheral arterial disease (PAD) is a complication of systemic atherosclerosis that results from occlusions in the large arteries of the leg(s). Though best known as a large vessel disease, patients with PAD also have microvascular disease including excess vascular permeability. Patients with diabetes have an increased risk to develop PAD and more severe symptoms compared to nondiabetic patients with PAD patients. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that modulate gene expression by targeting mRNA post-transcriptionally. Methods: Age-, sex-, and risk factor-controlled plasma samples were selected from a PAD biobank. Plasma samples from non-PAD controls, patients with intermittent claudication (IC), or with critical limb ischemia (CLI), with or without diabetes, were selected and miR-433-3p expression was assessed by RT-qPCR. MiR-433-3p expression was analyzed in human ECs after high glucose (HG) (25 mM) or osmolar control (25 mM Mannitol) treatment under normoxia or hypoxia-serum starvation. ECs were also treated with miR-433 inhibitor or negative control. Endothelial vascular permeability, angiogenesis, and cell viability/survival were tested. Vascular permeability by Evans Blue Dye (EBD) was measured in High Fat Diet (HFD) mice versus C57BL/6 mice treated with 100 μM hind-limb, intramuscular injections of miR-433-3p inhibitor, or scramble. Results: Plasma levels of miR-433-3p were elevated in CLI patients with diabetes. In human ECs, miR-433-3p expression was also elevated after high glucose treatment. Endothelial barrier function was impaired after HG, which was restored by miR-433 inhibition. Similarly, miR-433-3p inhibitor improved angiogenesis and cell survival after HG treatment in ECs. HFD mice demonstrated an increased vascular permeability compared to C57BL/6 mice which was attenuated by miR-433-3p inhibition. Conclusion: These data suggest miR-433-3p’s effect on vascular permeability which may contribute to the very poor clinical course seen in PAD and DM. Improved EC permeability with miR-433-3p inhibition may improve skeletal muscle perfusion and create a novel approach for patients with PAD, diabetes, or both. NIH 5R01HL150003-03 (BHA) and AHA CDA932159 (AK) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Objective:Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease. The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Id3 (inhibitor of differentiation 3) in regulating blood flow in a murine model of hindlimb ischemia (HLI).Approach and Results:HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B-cell–specific knockout of Id3 (Id3BKO) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3BKO. Consistent with these findings, ELISA (enzyme-linked immunoassay) demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when endothelial cells were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3BKOmice showed reduced density of total CD31+and αSMA+CD31+vessels.Conclusions:This study is the first to demonstrate a role for B-cell–specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in peripheral artery disease pathogenesis.

International audience; The Mars 2020 Perseverance rover is exploring a sedimentary deposit interpreted to be theremnants of a delta within Jezero crater, a 45 km diameter Late Noachian-aged crater. During itsexploration of the “lower delta” exposure of the western delta/fan in Jezero crater, Mars (Fig. 1), Perseveranceacquired image and composition data from Alagnak, a ~2 m thick, well-exposed outcrop of clastic sedimentaryrock located at Cape Nukshak (Fig. 1).This outcrop was thoroughly documented from multiple angles at a cm-scale by the Mastcam-Z camerasystem [1] and the Supercam Remote Micro-Imager (RMI) [2] on the Perseverance rover. Dip and strikemeasurements of sedimentary beds were collected from 3D reconstructions of Mastcam-Z stereo-imagescollected using the Planetary Robotics software tools PRoViP and PRo3D [3].In this study, we examine the physical sedimentology and stratigraphic context of the Alagnakoutcrop. The preliminary depositional interpretation of Alagnak is as a prograding, subaqueous fan builtthrough the deposition of many, meter-scale, gravity driven, sediment-rich flows. Flow directions rangedbetween NNE and SW, with principal directions toward SE and E.

International audience; Orbital and rover observations of relict geomorphic features and stratigraphic architectures indicate Mars once had a warmer, wetter climate. Constraining the character, relative timing and persistence of ancient aqueous activity on Mars is possible through detailed interrogation of the stratal geometry of aqueously deposited sedimentary bodies. Such analyses inform interpretations of Martian climate evolution, potential habitability, and search strategies for rocks that might contain potential biosignatures. NASA’s Mars 2020 Perseverance rover mission is seeking signs of ancient life in Jezero crater and is collecting a cache of Martian rock and soil samples for planned return to Earth by a future mission.A prominent sedimentary fan deposit at the western margin of Jezero crater has been interpreted to be a riverdelta that built into an ancient lake basin during the Late Noachian-Early Hesperian epochs on Mars (~3.6-3.8Ga) [1, 2]. The Perseverance rover landed on 18 February 2021 ~2.2 km from the western fan. In March-April 2022, the rover conducted a rapid traverse along the eastern and southeastern side of Jezero fan onlyobtaining a few remote sensing observations along the way. In April 2022, the rover arrived at the base of theancient delta in the Three Forks region of the crater floor adjacent to the delta front (Fig. 1). During the ‘rapidtraverse’ and the exploration of two sections at the delta front - Cape Nukshak and Hawksbill Gap –Perseverance obtained striking images from the Mastcam-Z and SuperCam’s Remote Micro-Imagerinstruments of the stratigraphy exposed stratigraphically higher up in the fan’s erosional front [3]. Images providenew views of the stratigraphy exposed in the erosional front of the western Jezero delta; in particular, showingsections of the delta previously not visible from long distance observations and at much higher resolution.Here, we report its stratigraphy and sedimentology, which provide new constraints on the nature of the fandeposits, and therefore paleoenvironmental implications.

International audience; The NASA Mars2020 rover Perseverance has been traversing series that represent the transition from crater floor lithologies to deposits of the Jezero western delta since Sol 422 of rover operations [1]. During that time, the mission has explored the exposed stratigraphic succession at the delta front, named the Shenandoah formation [2]. Here we analyse Mastcam-Z mosaics and 3D data products derived from Planetary Robotics processing and viewing tools (PRoViP and PRo3D [3]) to map the 3D geometry of key stratigraphic boundaries and document the 3D stratigraphic architecture at the sub-km- to m-scale within the Shenandoah formation.

Inflammatory cytokine mediated responses are important in the development of many diseases that are associated with angiogenesis. Targeting angiogenesis as a prominent strategy has shown limited effects in many contexts such as peripheral arterial disease (PAD) and cancer. One potential reason for the unsuccessful outcome is the mutual dependent role between inflammation and angiogenesis. Inflammation-based therapies primarily target inflammatory cytokines such as interleukin-6 (IL-6) in T cells, macrophages, cancer cells, muscle cells, and there is a limited understanding of how these cytokines act on endothelial cells. Thus, we focus on one of the major inflammatory cytokines, IL-6, mediated intracellular signaling in endothelial cells by developing a detailed computational model. Our model quantitatively characterized the effects of IL-6 classic and trans-signaling in activating the signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), and mitogen-activated protein kinase (MAPK) signaling to phosphorylate STAT3, extracellular regulated kinase (ERK) and Akt, respectively. We applied the trained and validated experiment-based computational model to characterize the dynamics of phosphorylated STAT3 (pSTAT3), Akt (pAkt), and extracellular regulated kinase (pERK) in response to IL-6 classic and/or trans-signaling. The model predicts that IL-6 classic and trans-signaling induced responses are IL-6 and soluble IL-6 receptor (sIL-6R) dose-dependent. Also, IL-6 trans-signaling induces stronger downstream signaling and plays a dominant role in the overall effects from IL-6. In addition, both IL-6 and sIL-6R levels regulate signaling strength. Moreover, our model identifies the influential species and kinetic parameters that specifically modulate the pSTAT3, pAkt, and pERK responses, which represent potential targets for inflammatory cytokine mediated signaling and angiogenesis-based therapies. Overall, the model predicts the effects of IL-6 classic and/or trans-signaling stimulation quantitatively and provides a framework for analyzing and integrating experimental data. More broadly, this model can be utilized to identify targets that influence inflammatory cytokine mediated signaling in endothelial cells and to study the effects of angiogenesis- and inflammation-based therapies.

The light-toned sedimentary layers that outcrop widely throughout Mars’ Southern Highlands have long been an enigma in uncovering the climatic history of Mars. Although these units seem to contain unique records of fluctuating surface conditions, the role of water in their formation is debated. A distinctive property of many such deposits is their elevated thermal inertia relative to that of surrounding materials. This temperature-controlling trait is often interpreted to indicate induration resulting from aqueous processes. However, prevalent erosional landforms suggest that the deposits host much weaker materials than neighboring units. We address this apparent contradiction by disentangling the relationships between thermal inertia, mineralogy, and erosion susceptibility and by quantifying the cohesion of layered deposits in the Arabia Terra region. We demonstrate that variations in dust cover associated with relative erodibility and eolian abrasion can be inconspicuous controls on apparent thermal inertia. We ultimately find that these deposits are not as cohesive as would be expected from a high water-to-rock ratio setting either during or after deposition. If water-rich surface conditions existed in the region after the Noachian, these deposits suggest they may have only been intermittent and fleeting.

International audience; A key exploration target for the Perseverance rover mission is a sedimentary fan deposit at the western margin of Jezero crater, which has been interpretated to be a river delta that built into a lake basin during the Late Noachian-Early Hesperian epochs on Mars (~3.6-3.8 Ga). Images from the Mastcam-Z and SuperCam Remote Micro-Imager instruments provide striking views of the stratigraphy exposed in the fan’s erosional front. Here, we report its stratigraphy and sedimentology, which place constraints on the nature of the fan deposits and their paleoenvironmental implications.Multiple Mastcam-Z mosaics show spectacular views of the stratigraphy of a prominent embayment in the delta scarp that has been informally named Hawksbill Gap, the lower section of which the rover is investigating and sampling. The prominent cliffs of the eastern and western margin of Hawksbill Gap show distinctive stratal geometries with complex stratigraphic relations. The basal succession comprises poorly exposed thinly bedded, planar laminated sandstones that are interpreted as the deposits of low-density turbidity currents. A locally prominent, resistant unit named Rocky Top comprises planar stratified pebbly sandstones also likely to be high-density turbidite deposits. The stratigraphic mid-sections of the scarps are characterized by packages of decameter-scale inclined tabular strata. These tabular beds are locally conglomeratic but predominantly comprise finer-than-conglomerate lithologies, likely pebbly sandstones. Interstratified within these are poorly sorted matrix-supported conglomerates interpreted to be debris flow deposits. The inclined strata are overlain across a sharp truncation surface by generally planar parallel thin-bedded horizontal strata that we interpret as topset beds. Conglomerate beds containing bouldersare located within the overlying topset strata. The stratal patterns are broadly consistent with deposition in a Gilbert-type delta setting with basal strata representing deposition from sediment gravity flows, inclined strata representing foreset beds, and overlying topset beds deposition from fluvial processes in a delta top environment. The boulder conglomerates indicate sediment-transport on the delta top by episodic high-discharge floods.

The interleukin-21 receptor (IL-21R) can be upregulated in endothelial cells (EC) from ischemic muscles in mice following hind-limb ischemia (HLI), an experimental peripheral arterial disease (PAD) model, blocking this ligand–receptor pathway-impaired STAT3 activation, angiogenesis, and perfusion recovery. We sought to identify mRNA and microRNA transcripts that were differentially regulated following HLI, based on the ischemic muscle having intact, or reduced, IL-21/IL21R signaling. In this comparison, 200 mRNAs were differentially expressed but only six microRNA (miR)/miR clusters (and among these only miR-30b) were upregulated in EC isolated from ischemic muscle. Next, myoglobin-overexpressing transgenic (MgTG) C57BL/6 mice examined following HLI and IL-21 overexpression displayed greater angiogenesis, better perfusion recovery, and less tissue necrosis, with increased miR-30b expression. In EC cultured under hypoxia serum starvation, knock-down of miR-30b reduced, while overexpression of miR-30b increased IL-21-mediated EC survival and angiogenesis. In Il21r−/− mice following HLI, miR-30b overexpression vs. control improved perfusion recovery, with a reduction of suppressor of cytokine signaling 3, a miR-30b target and negative regulator of STAT3. Together, miR-30b appears both necessary and sufficient for IL21/IL-21R-mediated angiogenesis and may present a new therapeutic option to treat PAD if the IL21R is not available for activation.

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to ’see’ (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells - specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster and weighted to intracellular expression, due to faster internalization and lower recycling, resulting in increased overall degradation. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.

International audience; NASA’s Mars 2020 Perseverance rover mission is seeking signs of ancient life in Jezero crater and is collecting a cache of Martian rock and soil samples for planned return to Earth by a future mission. A key exploration target for the mission is a prominent sedimentary fan deposit at the western margin of Jezero crater that has been interpretated to be a river delta that built into an ancient lake basin during the Late Noachian-Early Hesperian epochs on Mars (~3.6-3.8 Ga) [1, 2]. Long distance observations of a remnant butte (informally named Kodiak) related to the western fan demonstrated that it comprised two distinct Gilbert-type delta units [2, 3].In her approach to the western fan, Perseverance drove alongside the east-facing scarp of the western fan and arrived at a key location called Three Forks - a setting off point for delta exploration - in April 2022. Images from the Mastcam-Z and SuperCam Remote Micro-Imager instrumentsprovide new views of the stratigraphy exposed in the erosional front of the western Jezero delta; in particular, showing sections of the delta previously not visible from long distance observations and at much higher resolution. These observations provide the first direct evidence of delta geometries in the main western fan deposit. Here, we report its stratigraphy and sedimentology, which providenew constraints on the nature of the fan deposits, and therefore paleoenvironmental implications.