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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.

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.

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 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.