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Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease.

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function–associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the β2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of β2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3–dependent conformational activation of LFA-1.

In recent years, the interest in extracellular vesicles (EV) functions has been growing. However, methods for isolating these small vesicles from tissue are still not trivial. Few protocols are available that allows EV isolation from whole tissue samples, including the heart. Those protocols are based on organ perfusion using the Langendorff method. In this work, aiming to analysing in vivo biology of small EVs, we implemented a simple method to obtain enrichment of these vesicles from murine heart tissue. We tested a titration of Liberase for tissue digestion, which was subjected to differential ultracentrifugation combined with iodixanol cushion and presented the step-by-step procedure of this protocol. Validation was done with Nanoparticle Tracking Analysis, transmission Electron Microscope and Western Blot analysis of EV markers and organelles contaminants. Furthermore, we tested the suitability of the protocol for isolating EVs from heart tissue obtained from a pre-clinical translational non-human primate animal model. Therefore, this protocol may be suitable for isolating vesicle from human heart tissue. Additionally, this method can potentially be applied beyond heart tissue.

Cardiovascular diseases are the leading cause of mortality worldwide. Understanding the mechanisms at the basis of these diseases is necessary in order to generate therapeutic approaches. Recently, cardiac tissue engineering and induced pluripotent stem cell (iPSC) reprogramming has led to a skyrocketing number of publications describing cardiovascular regeneration as a promising option for cardiovascular disease treatment. Generation of artificial tissue and organoids derived from induced pluripotent stem cells is in the pipeline for regenerative medicine. The present review summarizes the multiple approaches of heart regeneration with a special focus on iPSC application. In particular, we describe the strength of iPSCs as a tool to study the molecular mechanisms driving cardiovascular pathologies, as well as their potential in drug discovery. Moreover, we will describe some insights into novel discoveries of how stem-cell-secreted biomolecules, such as exosomes, could affect cardiac regeneration, and how the fine tuning of the immune system could be a revolutionary tool in the modulation of heart regeneration.

During inflammation, leukocyte recruitment has to be tightly controlled to prevent overwhelming leukocyte infiltration, activation, and, consequently, organ damage. A central regulator of leukocyte recruitment is Rac1. In this study, we analyzed the effects of the RacGAP ArhGAP15 on leukocyte recruitment. Using ArhGAP15-deficient mice, reduced neutrophil adhesion and transmigration in the TNF-α–inflamed cremaster muscle and a prolongation of chemokine-dependent leukocyte adhesion could be observed. In a murine model of sterile kidney injury, reduced neutrophil infiltration, and serum creatinine levels were apparent. Further in vitro and in vivo analyses revealed a defective intravascular crawling capacity, resulting from increased affinity of the β2-integrin Mac-1 after prolonged chemokine stimulation of neutrophils. LFA-1 activity regulation was not affected. Summarizing, ArhGAP15 specifically regulates Mac-1, but not LFA-1, and affects leukocyte recruitment by controlling postadhesion strengthening and intravascular crawling in a Mac-1–dependent manner. In conclusion, ArhGAP15 is involved in the time-dependent regulation of leukocyte postadhesion in sterile inflammation.

The ALK inhibitor crizotinib showed promising therapeutic efficacy for relapsed/refractory Anaplastic Large Cell Lymphoma (R/R ALCL). However, a fraction of ALK+ R/R ALCL patients do not achieve complete remission due to crizotinib resistance that develops within the first 3 months of therapy. In patients that achieve complete remission, crizotinib discontinuation causes rapid disease relapses due to the expansion of persister lymphoma cells never completely eradicated by the ALK inhibitor. There is, in fact, growing evidence that ALK+ ALCL can persist for years in patients being undetectable. ALCL grows around blood and lymphatic vessels in the lymph node. We hypothesize that this perivascular niche provides pro-survival signals contributing to ALK+ ALCL persistence and TKI resistance. By RNA-seq analysis on ALK+ ALCL cells and scRNA-seq analysis in one ALK+ ALCL primary sample, we found that ALK+ cells expressed the C-C chemokine receptor type 7 (CCR7), while endothelial cells and fibroblasts expressed the unique CCR7 ligands, the chemokine (C-C motif) ligand 19 (CCL19) and 21 (CCL21). Therefore, we explored whether the CCL19/21-CCR7 chemokine-receptor signaling axis could be involved in the persistence of ALK+ ALCL cells during ALK inhibitor treatment. We show that treatment with crizotinib caused upregulation of CCR7 in ALK+ ALCL cells via STAT3, as demonstrated by ChIP-seq data. Besides, stimulation of ALK+ ALCL cells with both CCL19/21 potently activated the MAPK signaling and sustained MAPK activation during ALK inhibition by crizotinib. Mechanistically, we demonstrate that this MAPK activation was mediated by PI3Kγ-dependent CCR7 signaling. This effect was more marked in human ALK+ ALCL cells that express high levels of PI3Kγ, while it was strongly reduced in murine lymphoma PI3KγKO cells, generated from NPM-ALK transgenic mice crossed with PI3KγKO mice. Treatment with the PI3Kγ/δ dual inhibitor duvelisib abrogated the MAPK phosphorylation induced by CCL19/21. When we knocked-out the CCR7 gene via CRISPR/Cas9, human ALK+ ALCL showed markedly reduced activation of the MAPK pathway upon stimulation with CCL19/21. Next, we developed a microphysiological model of ALCL cells in the perivascular niche with a 3D vasculature using a microfluidic chip. In this model, ALCL cells circulate inside the chip in continuous contact with a perfusable vasculature. We, then, demonstrated that the presence of endothelial cells conferred resistance to crizotinib and sustained cell viability of CCR7WT cells, whereas the protective effect was lost in CCR7KO cells. In in vivo experiments, CCR7 was required for lymphoma cell survival and diffusion to the brain during crizotinib treatment. Overall, our results suggest that the perivascular niche could promote the survival of ALK+ ALCL persister cells and protect them from the effect of ALK TKIs via the CCL19/21-CCR7 axis. The disruption of this survival axis could contribute to eradicating minimal residual disease in combination with ALK TKI. Citation Format: Cristina Mastini, Marco Campisi, Carlotta Costa, Chiara Ambrogio, Giulia Germena, Silvia Peola, Cinzia Martinengo, Enrico Patrucco, Ines Mota, Maddalena Arrigoni, Martina Olivero, Raffaele Calogero, Valeria Chiono, Roger D. Kamm, Emilio Hirsch, Jon C. Aster, Claudia Voena, Roberto Chiarle. The perivascular niche protects ALK+ lymphoma cells from ALK inhibition through the CCL19/21-CCR7 axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT018.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. Src family kinases (SFKs) activation is required for integrin and chemokine signaling as well as immune cell function. The receptor-like protein tyrosine phosphatase CD45 positively regulates chemoattractant signaling acting on SFK activity. To further investigate the role of CD45 in neutrophil recruitment and function, we analyzed transgenic mice carrying a single point mutation (CD45E613R), which constitutively activates CD45. By using intravital microscopy experiments, we demonstrated that different steps of the leukocyte recruitment cascade were affected in CD45E613R mutant mice. The rolling velocity of CD45E613R mutant neutrophils was decreased compared with wild-type neutrophils that subsequently resulted in an increased number of adherent cells. The analysis of β2 integrins LFA-1 and macrophage-1 Ag (Mac-1) showed that in CD45E613R mutant neutrophils LFA-1 adhesiveness was impaired, and avidity was enhanced, whereas Mac-1 adhesiveness was increased. Because of the increased Mac-1 adhesiveness, neutrophil crawling was impaired in CD45E613R mutant compared with wild-type neutrophils. In an Escherichia coli lung infection model, CD45E613R mice displayed a decreased neutrophil recruitment into the alveolar compartment, which resulted in an increased number of CFUs in the lung. Our data demonstrate that the CD45E613R mutation modulates integrin activation and leukocyte recruitment during inflammation.

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.

A subset of PSGL-1 is constitutively associated with L-selectin and signals through Src family kinases to activate LFA-1, which regulates neutrophil slow rolling and recruitment., Neutrophils are recruited from the blood to sites of inflammation, where they contribute to immune defense but may also cause tissue damage. During inflammation, neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function–associated antigen 1 (LFA-1), which can be induced by selectin engagement. Here, we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1, the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1–L-selectin complex signals through Src family kinases, ITAM domain–containing adaptor proteins, and other kinases to ultimately result in LFA-1 activation. The PSGL-1–L-selectin complex–induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow.

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

Mobilization of neutrophils from the bone marrow determines neutrophil blood counts and thus is medically important. Balanced neutrophil mobilization from the bone marrow depends on the retention-promoting chemokine CXCL12 and its receptor CXCR4 and the egression-promoting chemokine CXCL2 and its receptor CXCR2. Both pathways activate the small guanosine triphosphatase Rac, leaving the role of this signaling event in neutrophil retention and egression ambiguous. On the assumption that active Rac determines persistent directional cell migration, we generated a mathematical model to link chemokine-mediated Rac modulation to neutrophil egression time. Our computer simulation indicated that, in the bone marrow, where the retention signal predominated, egression time strictly depended on the time it took Rac to return to its basal activity (namely, adaptation). This prediction was validated in mice lacking the Rac inhibitor ArhGAP15. Neutrophils in these mice showed prolonged Rac adaptation and cell-autonomous retention in the bone marrow. Our model thus demonstrates that mobilization in the presence of two spatially defined opposing chemotactic cues strictly depends on inhibitors shaping the time course of signal adaptation. Furthermore, our findings might help to find new modes of intervention to treat conditions characterized by excessively low or high circulating neutrophils.

Rac and PI3Ks are intracellular signal transducers able to regulate multiple signaling pathways fundamental for cell behavior. PI3Ks are lipid kinases that produce phosphorylated lipids which, in turn, transduce extracellular cues within the cell, while Rac is a small G protein that impacts on actin organization. Compelling evidence indicates that in multiple circumstances the 2 signaling pathways appear intermingled. For instance, phosphorylated lipids produced by PI3Ks recruit and activate GEF and GAP proteins, key modulators of Rac function. Conversely, PI3Ks interact with activated Rac, leading to Rac signaling amplification. This review summarizes the molecular mechanisms underlying the cross-talk between Rac and PI3K signaling in 2 different processes, cell migration and ROS production.

Cell migration is a key event in physiological processes such as embryonic development, tissue repair, angiogenesis and immune responses. Alteration of the migration program is an important component in multiple pathologies, including chronic inflammation, autoimmunity and tumor metastasis. Understanding of the precise mechanisms at the basis of cellular migration may lead to the identification of novel therapeutic approach for these diseases. Recent evidences show that the interplay between the lipid kinases phosphatidylinositol 3-kinase (PI3Ks) and small GTPases play a critical role in driving cell migration. In this review we will describe the role of these molecules and the interaction between their signal cascades in leukocyte polarization and amoeboid migration.

Epidemiological studies have established a positive correlation between cancer and metabolic disorders, suggesting that aberrant cell metabolism is a common feature of nearly all tumors. To meet their demand of building block molecules, cancer cells switch to a heavily glucose-dependent metabolism. As insulin triggers glucose uptake, most tumors are or become insulin-dependent. However, the effects of insulin and of other similar growth factors are not only limited to metabolic control but also favor tumor growth by stimulating proliferation and survival. A key signaling event mediating these metabolic and proliferative responses is the activation of the phosphatidylinositol-3 kinases (PI3K) pathway. In this review, we will thus discuss the current concepts of tumor metabolism and the opportunity of PI3K-targeted therapies to exploit the "sweet tooth" of cancer cells.

In this report, we confirm that the RhoA-binding protein citron kinase (CIT-K) is required for midbody abscission in late cytokinesis, while it has little or no role in early cytokinesis. Moreover, we show that CIT-K, despite being commonly considered a RhoA effector, promotes midbody stability through RhoA and anillin during late cytokinesis., The small GTPase RhoA plays a crucial role in the different stages of cytokinesis, including contractile ring formation, cleavage furrow ingression, and midbody abscission. Citron kinase (CIT-K), a protein required for cytokinesis and conserved from insects to mammals, is currently considered a cytokinesis-specific effector of active RhoA. In agreement with previous observations, we show here that, as in Drosophila cells, CIT-K is specifically required for abscission in mammalian cells. However, in contrast with the current view, we provide evidence that CIT-K is an upstream regulator rather than a downstream effector of RhoA during late cytokinesis. In addition, we show that CIT-K is capable of physically and functionally interacting with the actin-binding protein anillin. Active RhoA and anillin are displaced from the midbody in CIT-K-depleted cells, while only anillin, but not CIT-K, is affected if RhoA is inactivated in late cytokinesis. The overexpression of CIT-K and of anillin leads to abscission delay. However, the delay produced by CIT-K overexpression can be reversed by RhoA inactivation, while the delay produced by anillin overexpression is RhoA-independent. Altogether, these results indicate that CIT-K is a crucial abscission regulator that may promote midbody stability through active RhoA and anillin.

In phagocytes, GTPases of the Rac family control crucial antimicrobial functions. The RacGAP ArhGAP15 negatively modulates Rac activity in leukocytes, but its in vivo role in innate immunity remains largely unknown. Here we show that neutrophils and macrophages derived from mice lacking ArhGAP15 presented higher Rac activity but distinct phenotypes. In macrophages, the loss of ArhGAP15 induced increased cellular elongation and membrane protrusions but did not modify chemotactic responses. Conversely, the lack of ArhGAP15 in neutrophils affected critical Rac-dependent antimicrobial functions, specifically causing enhanced chemotactic responses, straighter directional migration, amplified reactive oxygen species production, increased phagocytosis, and improved bacterial killing. In vivo, in a model of severe abdominal sepsis, these effects contributed to increase neutrophil recruitment to the site of infection, thereby limiting bacterial growth, controlling infection spread, reducing systemic inflammation, and ultimately improving survival in ArhGAP15-null mice. Altogether, these results demonstrate the relevance of ArhGAP15 in the selective regulation of multiple neutrophil functions, suggesting that ArhGAP15 targeting might be beneficial in specific pathologic settings like severe sepsis.

Phagocytes, like neutrophils and macrophages, are specialized cells evolved to clear infectious pathogens. This function resides at the core of innate immunity and requires a series of concerted events that lead first to migration to the infected tissue and then to the killing of the invading pathogens. Molecular mechanisms underlying these processes are starting to emerge and point to the interplay between two families of crucial proteins: the PI3K lipid kinases and the Rac GTPases. This review focuses on how these two protein families contribute to migration, phagocytosis, and reactive oxygen species production, as well as their epistatic and feedback relations that finely tune these crucial aspects of the immune response.