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Prostaglandin D2 (PGD2) released from immune cells or other cell types activates its receptors, D prostanoid receptor (DP)1 and 2 (DP1 and DP2), to promote inflammatory responses in allergic and lung diseases. Prostaglandin-mediated inflammation may also contribute to vascular diseases such as abdominal aortic aneurysm (AAA). However, the role of DP receptors in the pathogenesis of AAA has not been systematically investigated. In the present study, DP1-deficient mice and pharmacological inhibitors of either DP1 or DP2 were tested in two distinct mouse models of AAA formation: angiotensin II (AngII) infusion and calcium chloride (CaCl2) application. DP1-deficient mice [both heterozygous (DP1+/−) and homozygous (DP1−/−)] were protected against CaCl2-induced AAA formation, in conjunction with decreased matrix metallopeptidase (MMP) activity and adventitial inflammatory cell infiltration. In the AngII infusion model, DP1+/− mice, but not DP1−/− mice, exhibited reduced AAA formation. Interestingly, compensatory up-regulation of the DP2 receptor was detected in DP1−/− mice in response to AngII infusion, suggesting a potential role for DP2 receptors in AAA. Treatment with selective antagonists of DP1 (laropiprant) or DP2 (fevipiprant) protected against AAA formation, in conjunction with reduced elastin degradation and aortic inflammatory responses. In conclusion, PGD2 signaling contributes to AAA formation in mice, suggesting that antagonists of DP receptors, which have been extensively tested in allergic and lung diseases, may be promising candidates to ameliorate AAA.

OBJECTIVE: Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease (PAD). The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Inhibitor of differentiation 3 (Id3) in regulating blood flow in a murine model of hind limb 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 (Id3(BKO)) 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 Id3(BKO). Consistent with these findings, ELISA demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when ECs were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3(BKO) mice 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 PAD pathogenesis.

OBJECTIVE: Angiogenic factor AGGF1 promotes angiogenesis as potently as VEGFA, and regulates endothelial cell (EC) proliferation, migration, specification of multi-potent hemangioblasts and venous ECs, hematopoiesis, and vascular development, and causes vascular disease Klippel-Trenaunay syndrome (KTS) when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. APPROACH AND RESULTS: Using functional blocking studies with neutralizing antibodies, we identified α5β1 as the receptor for AGGF1 on ECs. AGGF1 interacts with α5β1 and activates FAK, Src, and AKT. Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the Angiogenic Domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The Angiogenic Domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the Angiogenic Domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease (PAD). A 40-mer or 15-mer peptide containing the Angiogenic Domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from –RDD- to –RGD- (a classical RGD integrin binding motif) failed to block AGGF1 function. CONCLUSIONS: We have identified integrin α5β1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of α5β1-FAK-Src-AKT for angiogenesis. Our results identify a FQRDDAPAS Angiogenic Domain of AGGF1 crucial for its interaction with α5β1 and signaling.

Summary The angiopoietin-Tie signaling pathway is an important vascular signaling pathway involved in angiogenesis, vascular stability, and quiescence. Dysregulation in the pathway is linked to the impairments in vascular function associated with many diseases, including cancer, ocular diseases, systemic inflammation, and cardiovascular diseases. The present study uses a computational signaling pathway model validated against experimental data to quantitatively study various mechanistic aspects of the angiopoietin-Tie signaling pathway, including receptor activation, trafficking, turnover, and molecular mechanisms of its regulation. The model provides mechanistic insights into the controversial role of Ang2 and its regulators vascular endothelial protein tyrosine phosphatase (VE-PTP) and Tie1 and predicts synergistic effects of inhibition of VE-PTP, Tie1, and Tie2 cleavage on enhancing the vascular protective actions of Tie2., Graphical Abstract, Highlights • Mechanistically detailed computational model of the angiopoietin-Tie pathway • Receptor internalization dynamics modulate effectiveness of VE-PTP • Inhibition of VE-PTP, Tie1, and Tie2 shedding synergistically enhances Tie2 activation • Tie1 silencing modulates the effectiveness of inhibiting VE-PTP and Tie2 shedding, Biological Sciences; Cell Biology; Bioinformatics

Magnetic cyclotron waves were discovered by the Cassini-Huygens spacecraft in Saturn's atmospheric torus’ magnetic layer. They are left-handed and propagate at a minor angle to the ambient magnetic field in most areas because their frequency is close to the frequency of the aqua ions. The ion cyclotron instability caused by Saturn's neutral cloud ions helps explain their formation. They can be classified as n = 2 mode fluctuations because of the ion-ring distribution. We planned the characteristics of these waves in advance of starting this project. Our dispersion growth rates are evaluated using kinetic method analysis as well. The results were calculated and explained for the exemplary values of the magnetosphere parameters suitable for Saturn. Another potential free energy source for ion cyclotrons is temperature anisotropy. Instead of the standard Maxwell distribution, a ring distribution is employed in this study. The focus of this research is EMIC waves’ oblique propagation in the magnetic field, which changes their temperature anisotropy, ion energy density, and propagation angle. The interaction of relativistic particles with ion cyclotron waves is also included in this extension. EMIC wave size decreases with the increasing density of particles, as shown by a numerical study. A comparison of planetary studies based on data from space plasma environments and magnetospheric systems produced these results. HIGHLIGHTS Temperature anisotropy - free energy source for Ion Cyclotron waves EMIC wave size decreases with the increasing density of particles Saturn's neutral cloud ions helps the formation of ion cyclotron instability GRAPHICAL ABSTRACT

Visual Abstract, Highlights • A first-of-a-kind systems biology computational model is presented that describes multiscale regulation of perfusion recovery in experimental peripheral arterial disease. • Multilevel model calibration and validation enable high-resolution model simulations for experimental peripheral arterial disease (mouse HLI). • An integrative model-based mechanistic characterization of the intracellular, cellular, and tissue-level features critical for the dynamic reconstitution of perfusion following different patterns of occlusion-induced ischemia in HLI is described. • Using a model-based virtual HLI mouse population, pharmacologic inhibition of cell necrosis is predicted as a strategy with high therapeutic potential to improve perfusion recovery; in real HLI mice, the positive impact of this new strategy is then experimentally studied and confirmed., Summary In peripheral arterial disease (PAD), the degree of endogenous capacity to modulate revascularization of limb muscle is central to the management of leg ischemia. To characterize the multiscale and multicellular nature of revascularization in PAD, we have developed the first computational systems biology model that mechanistically incorporates intracellular, cellular, and tissue-level features critical for the dynamic reconstitution of perfusion after occlusion-induced ischemia. The computational model was specifically formulated for a preclinical animal model of PAD (mouse hindlimb ischemia [HLI]), and it has gone through multilevel model calibration and validation against a comprehensive set of experimental data so that it accurately captures the complex cellular signaling, cell–cell communication, and function during post-HLI perfusion recovery. As an example, our model simulations generated a highly detailed description of the time-dependent spectrum-like macrophage phenotypes in HLI, and through model sensitivity analysis we identified key cellular processes with potential therapeutic significance in the pathophysiology of PAD. Furthermore, we computationally evaluated the in vivo effects of different targeted interventions on post-HLI tissue perfusion recovery in a model-based, data-driven, virtual mouse population and experimentally confirmed the therapeutic effect of a novel model-predicted intervention in real HLI mice. This novel multiscale model opens up a new avenue to use integrative systems biology modeling to facilitate translational research in PAD.

The prevalence of peripheral arterial disease (PAD) in the United States exceeds 10 million people, and PAD is a significant cause of morbidity and mortality across the globe. PAD is typically caused by atherosclerotic obstructions in the large arteries to the leg(s). The most common clinical consequences of PAD include pain on walking (claudication), impaired functional capacity, pain at rest, and loss of tissue integrity in the distal limbs that may lead to lower extremity amputation. Patients with PAD also have higher than expected rates of myocardial infarction, stroke, and cardiovascular death. Despite advances in surgical and endovascular procedures, revascularization procedures may be suboptimal in relieving symptoms, and some patients with PAD cannot be treated because of comorbid conditions. In some cases, relieving obstructive disease in the large conduit arteries does not assure complete limb salvage because of severe microvascular disease. Despite several decades of investigational efforts, medical therapies to improve perfusion to the distal limb are of limited benefit. Whereas recent studies of anticoagulant (eg, rivaroxaban) and intensive lipid lowering (such as PCSK9 [proprotein convertase subtilisin/kexin type 9] inhibitors) have reduced major cardiovascular and limb events in PAD populations, chronic ischemia of the limb remains largely resistant to medical therapy. Experimental approaches to improve limb outcomes have included the administration of angiogenic cytokines (either as recombinant protein or as gene therapy) as well as cell therapy. Although early angiogenesis and cell therapy studies were promising, these studies lacked sufficient control groups and larger randomized clinical trials have yet to achieve significant benefit. This review will focus on what has been learned to advance medical revascularization for PAD and how that information might lead to novel approaches for therapeutic angiogenesis and arteriogenesis for PAD.

INTRODUCTION: Vascular endothelial growth factor (VEGF)-A is a sought therapeutic target for the treatment of PAD because of its potent role in angiogenesis. However, no therapeutic benefit was achieved in VEGF-A clinical trials, suggesting that our understanding of VEGF-A biology and ischemic angiogenic processes needs development. Alternate splicing in VEGF-A produces pro- and anti-angiogenic VEGF-A isoforms; the only difference being a 6-amino acid switch in the C-terminus of the final 8(th) exon of the gene. This finding has changed our understanding of VEGF-A biology and may explain the lack of benefit in VEGF-A clinical trials. It presents new therapeutic opportunities for peripheral arterial disease (PAD) treatment. AREAS COVERED: This review examines a newly recognized “anti-angiogenic” VEGF-A isoform and its role in ischemic angiogenesis regulation. A literature search was conducted to include: 1) reports on the predicted mechanism by which the anti-angiogenic VEGF-A isoform would inhibit angiogenesis, 2) findings of the unexpected mechanism of action, and 3) how this mechanism revealed novel signaling pathways that may enhance future therapeutics in PAD. EXPERT OPINION: Inhibiting a specific anti-angiogenic VEGF-A isoform in ischemic muscle promotes perfusion recovery in preclinical PAD. Additional efforts focused on the production of these isoforms, and the pathways altered by the presence and removal of different VEGF receptor-ligand interactions, and how this new data may allow bench to bedside progress offers new approaches to PAD.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.