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.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non–small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell transcriptome profiling of hearts with inducible cardiomyocyte-specific Wnt activation (β-cat^Δex3) as well as with compensatory and failing hypertrophic remodeling. We show that functional enrichment analysis points to an involvement of extracellular vesicles (EVs) related processes in hearts of β-cat^Δex3 mice. A proteomic analysis of in vivo cardiac derived EVs from β-cat^Δex3 hearts has identified differentially enriched proteins involving 20 S proteasome constitutes, protein quality control (PQC), chaperones and associated cardiac proteins including α-Crystallin B (CRYAB) and sarcomeric components. The hypertrophic model confirms that cardiomyocytes reacted with an acute early transcriptional upregulation of exosome biogenesis processes and chaperones transcripts including CRYAB, which is ameliorated in advanced remodeling. Finally, human induced pluripotent stem cells (iPSC)-derived cardiomyocytes subjected to pharmacological Wnt activation recapitulated the increased expression of exosomal markers, CRYAB accumulation and increased PQC signaling. These findings reveal that secretion of EVs with a proteostasis signature contributes to early patho-physiological adaptation of cardiomyocytes, which may serve as a read-out of disease progression and can be used for monitoring cellular remodeling in vivo with a possible diagnostic and prognostic role in the future.

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.

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.

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.