CCR3-dependent eosinophil recruitment is regulated by sialyltransferase ST3Gal-IV.

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.

Circadian Clock and Hypoxia.

The timing of life on Earth is remarkable: between individuals of the same species, a highly similar temporal pattern is observed, with shared periods of activity and inactivity each day. At the individual level, this means that over the course of a single day, a person alternates between two states. They are either upright, active, and communicative or they lie down in a state of (un)consciousness called sleep where even the characteristic of neuronal signals in the brain shows distinctive properties. The circadian clock governs both of these time stamps-activity and (apparent) inactivity-making them come and go consistently at the same approximate time each day. This behavior thus represents the meeting of two pervasive systems: the circadian clock and metabolism. In this article, we will describe what is known about how the circadian clock anticipates daily changes in oxygen usage, how circadian clock regulation may relate to normal physiology, and to hypoxia and ischemia that can result from pathologies such as myocardial infarction and stroke.

Endothelial TDP-43 controls sprouting angiogenesis and vascular barrier integrity, and its deletion triggers neuroinflammation.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced ß-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.

Innate mechanism of mucosal barrier erosion in the pathogenesis of acquired colitis.

The colonic mucosal barrier protects against infection, inflammation, and tissue ulceration. Composed primarily of Mucin-2, proteolytic erosion of this barrier is an invariant feature of colitis; however, the molecular mechanisms are not well understood. We have applied a recurrent food poisoning model of acquired inflammatory bowel disease using Salmonella enterica Typhimurium to investigate mucosal barrier erosion. Our findings reveal an innate Toll-like receptor 4-dependent mechanism activated by previous infection that induces Neu3 neuraminidase among colonic epithelial cells concurrent with increased Cathepsin-G protease secretion by Paneth cells. These anatomically separated host responses merge with the desialylation of nascent colonic Mucin-2 by Neu3 rendering the mucosal barrier susceptible to increased proteolytic breakdown by Cathepsin-G. Depletion of Cathepsin-G or Neu3 function using pharmacological inhibitors or genetic-null alleles protected against Mucin-2 proteolysis and barrier erosion and reduced the frequency and severity of colitis, revealing approaches to preserve and potentially restore the mucosal barrier.

Therapeutic inhibition of CXCR1/2: where do we stand?

Mounting experimental evidence from in vitro and in vivo animal studies points to an essential role of the CXCL8-CXCR1/2 axis in neutrophils in the pathophysiology of inflammatory and autoimmune diseases. In addition, the pathogenetic involvement of neutrophils and the CXCL8-CXCR1/2 axis in cancer progression and metastasis is increasingly recognized. Consequently, therapeutic targeting of CXCR1/2 or CXCL8 has been intensively investigated in recent years using a wide array of in vitro and animal disease models. While a significant benefit for patients with unwanted neutrophil-mediated inflammatory conditions may be expected from a potential clinical use of inhibitors, their use in severe infections or sepsis might be problematic and should be carefully and thoroughly evaluated in animal models and clinical trials. Translating the approaches using inhibitors of the CXCL8-CXCR1/2 axis to cancer therapy is definitively a new and promising research avenue, which parallels the ongoing efforts to clearly define the involvement of neutrophils and the CXCL8-CXCR1/2 axis in neoplastic diseases. Our narrative review summarizes the current literature on the activation and inhibition of these receptors in neutrophils, key inhibitor classes for CXCR2 and the therapeutic relevance of CXCR2 inhibition focusing here on gastrointestinal diseases.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Complex regulation of alarmins S100A8/A9 and secretion via gasdermin D pores exacerbates autoinflammation in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.

A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

Sialyl Lewis X mediates interleukin-1 beta-induced trophoblast adhesion to endometrial cells during human embryo implantation.

Cell surface carbohydrate antigens sialyl Lewis X (sLeX) and Lewis Y (LeY) are paramount glycoconjugates and are abundantly expressed in the receptive endometrium. Furthermore, among the important biological functions of both antigens is their role in leukocytes adhesion and extravasation. Interleukin-1 beta (IL-1ß) is involved in the process of human embryo implantation and placenta development. Here, we used an in vitro model to investigate whether sLeX and LeY are playing a role in the embryo implantation process mediated by IL-1ß. Our results are showing that the expression of cell surface sLeX was enhanced in endometrial RL95-2 cells after exposure to IL-1ß. RT-qPCR detection indicated that the transcript level of glycosyltransferase gene fucosyltransferase 3 (FUT3) was significantly elevated and that of FUT4/7 and ST3 beta-galactoside alpha-2,3-sialyltransferase 3/4 (ST3GAL3/4) were decreased by treatment with IL-1ß. Modulatory role of glycosyltransferase FUT3 on sLeX biosynthesis was determined by FUT3 siRNA transfection in RL95-2 cells. Results showed that the expression level of sLeX was suppressed, but no change was observed in regard to LeY. Moreover, IL-1ß promoted the HTR-8/SVneo trophoblast spheroids attachment to the RL95-2 endometrial monolayer, which was partially blocked by anti-sLeX antibody and FUT3 knockdown. Gene expression analysis of the RNA-seq transcriptome data from human secretory endometrium demonstrated a significantly higher level of FUT3 in the mid-secretory phase compared to the early secretory phase, which was correlated with the expression of IL1B. In summary, the inflammatory microenvironment at the fetomaternal interface can regulate the glycosylation pattern of endometrial cells at the time of implantation. SLeX can be significantly induced by IL-1ß via increasing FUT3 expression, which facilitates the trophoblast adhesion during embryo implantation.

Intravital calcium imaging in myeloid leukocytes identifies calcium frequency spectra as indicators of functional states.

The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca2+ signals with Ca2+ reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca2+ transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca2+ signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca2+ channel Orai1 and Gαi-coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca2+ signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca2+ oscillations. Together, these findings establish Ca2+ frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo.

Targeting Aberrantly Elevated Sialyl Lewis A as a Potential Therapy for Impaired Endometrial Selection Ability in Unexplained Recurrent Miscarriage.

Background: Carbohydrate Lewis antigens including sialyl Lewis A (sLeA), sialyl Lewis X (sLeX), Lewis X (LeX), and Lewis Y (LeY) are the commonest cell surface glycoconjugates that play pivotal roles in multiple biological processes, including cell adhesion and cell communication events during embryogenesis. SLeX, LeY, and associated glycosyltransferases ST3GAL3 and FUT4 have been reported to be involved in human embryo implantation. While the expression pattern of Lewis antigens in the decidua of unexplained recurrent miscarriage (uRM) patients remains unclear. Methods: Paraffin-embedded placental tissue slides collected from patients experiencing early miscarriages (6-12 weeks) were analyzed using immunohistochemical (IHC) and immunofluorescent (IF) staining. An in vitro assay was developed using endometrial cell line RL95-2 and trophoblast cell line HTR-8/SVneo. Modulatory effect of potential glycosyltransferase on Lewis antigens expression was investigated by target-specific small interfering RNA (siRNA) knockdown in RL95-2 cells. HTR-8/SVneo cells spheroids adhesion assay was applied to investigate the intrinsic role of Lewis antigens in the abnormal implantation process of uRM. The expression of Lewis antigens in RL95-2 cells in response to the treatment with pro-implantation cytokine IL-1ß was further measured by flow cytometry and immunocytochemical (ICC) staining. Results: IHC staining revealed that Lewis antigens are mainly expressed in the luminal and glandular epithelium, IF staining further indicated the cellular localization at the apical membrane of the epithelial cells. FUTs, ST3GALs, and NEU1 located in both stromal and epithelial cells. We have found that the expression of sLeA, LeX, FUT3/4, and ST3GAL3/4 are significantly upregulated in the RM group, while FUT1 is downregulated. SLeX, LeY, ST3GAL6, and NEU1 showed no significant differences between groups. FUT3 knockdown in RL95-2 cells significantly decreased the expression of sLeA and the spheroids adhesion to endometrial monolayer. Anti-sLeA antibody can remarkably suppress both the basal and IL-1ß induced adhesion of HTR-8/SVneo spheroids to RL95-2 cells monolayer. While further flow cytometry and ICC detection indicated that the treatment of RL95-2 cells with IL-1ß significantly increases the surface expression of LeX, but not sLeA. Conclusions: SLeA, LeX, and pertinent glycosyltransferase genes FUT1/3/4 and ST3GAL3/4 are notably dysregulated in the decidua of uRM patients. FUT3 accounts for the synthesis of sLeA in RL95-2 cells and affects the endometrial receptivity. Targeting aberrantly elevated sLeA may be a potential therapy for the inappropriate implantation in uRM.

Humanized ß2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

The use of cell-based reporter systems has provided valuable insights into the molecular mechanisms of integrin activation. However, current models have significant drawbacks because their artificially expressed integrins cannot be regulated by either physiological stimuli or endogenous signaling pathways. Here, we report the generation of a Hoxb8 cell line expressing human ß2 integrin that functionally replaced the deleted mouse ortholog. Hoxb8 cells are murine hematopoietic progenitor cells that can be efficiently differentiated into neutrophils and macrophages resembling their primary counterparts. Importantly, these cells can be stimulated by physiological stimuli triggering classical integrin inside-out signaling pathways, ultimately leading to ß2 integrin conformational changes that can be recorded by the conformation-specific antibodies KIM127 and mAb24. Moreover, these cells can be efficiently manipulated via the CRISPR/Cas9 technique or retroviral vector systems. Deletion of the key integrin regulators talin1 and kindlin3 or expression of ß2 integrins with mutations in their binding sites abolished both integrin extension and full activation regardless of whether only one or both activators no longer bind to the integrin. Moreover, humanized ß2 integrin Hoxb8 cells represent a valuable new model for rapidly testing the role of putative integrin regulators in controlling ß2 integrin activity in a physiological context.

Cell-Crossing Functional Network Driven by microRNA-125a Regulates Endothelial Permeability and Monocyte Trafficking in Acute Inflammation.

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications.

Soluble uric acid inhibits ß2 integrin-mediated neutrophil recruitment in innate immunity.

Neutrophils are key players during host defense and sterile inflammation. Neutrophil dysfunction is a characteristic feature of the acquired immunodeficiency during kidney disease. We speculated that the impaired renal clearance of the intrinsic purine metabolite soluble uric acid (sUA) may account for neutrophil dysfunction. Indeed, hyperuricemia (HU, serum UA of 9-12 mg/dL) related or unrelated to kidney dysfunction significantly diminished neutrophil adhesion and extravasation in mice with crystal- and coronavirus-related sterile inflammation using intravital microscopy and an air pouch model. This impaired neutrophil recruitment was partially reversible by depleting UA with rasburicase. We validated these findings in vitro using either neutrophils or serum from patients with kidney dysfunction-related HU with or without UA depletion, which partially normalized the defective migration of neutrophils. Mechanistically, sUA impaired ß2 integrin activity and internalization/recycling by regulating intracellular pH and cytoskeletal dynamics, physiological processes that are known to alter the migratory and phagocytic capability of neutrophils. This effect was fully reversible by blocking intracellular uptake of sUA via urate transporters. In contrast, sUA had no effect on neutrophil extracellular trap formation in neutrophils from healthy subjects or patients with kidney dysfunction. Our results identify an unexpected immunoregulatory role of the intrinsic purine metabolite sUA, which contrasts the well-known immunostimulatory effects of crystalline UA. Specifically targeting UA may help to overcome certain forms of immunodeficiency, for example in kidney dysfunction, but may enhance sterile forms of inflammation.

Gut Microbiota and Short Chain Fatty Acids: Implications in Glucose Homeostasis.

Gut microbiota encompasses a wide variety of commensal microorganisms consisting of trillions of bacteria, fungi, and viruses. This microbial population coexists in symbiosis with the host, and related metabolites have profound effects on human health. In this respect, gut microbiota plays a pivotal role in the regulation of metabolic, endocrine, and immune functions. Bacterial metabolites include the short chain fatty acids (SCFAs) acetate (C2), propionate (C3), and butyrate (C4), which are the most abundant SCFAs in the human body and the most abundant anions in the colon. SCFAs are made from fermentation of dietary fiber and resistant starch in the gut. They modulate several metabolic pathways and are involved in obesity, insulin resistance, and type 2 diabetes. Thus, diet might influence gut microbiota composition and activity, SCFAs production, and metabolic effects. In this narrative review, we discuss the relevant research focusing on the relationship between gut microbiota, SCFAs, and glucose metabolism.

Endothelial Retargeting of AAV9 In Vivo.

Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2CNN AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2CNN AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE-/- mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2CNN AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2CNN AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2CNN AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2CNN AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2CNN AAV9-gRNAeNOS . Therefore, G2CNN AAV9 may enable gene transfer in vascular and atherosclerosis models.