Endothelial progenitor cells (EPC) are considered to support neovascularization and endothelial repair by being incorporated into newly formed or injured vessels and by improving vascularization in a paracrine manner by secreting proangiogenic factors. Here, we studied the role of gap junctional communication between EPC and endothelial cells in long-term co-cultures in vitro. The cultivation of endothelial cells together with mouse embryonic EPC (E 7.5) induced the spontaneous formation of angiogenic networks after 3–6 days consisting of both cell types, but not in the respective monocultures, whereas their respective cultivation on a basement matrix induced the formation of tube-like structures, as expected. The angiogenic network formation could not be mimicked by the incubation of endothelial cells with supernatants of EPC only. We therefore hypothesized that direct interaction and cell-cell communication is required to induce the angiogenic network formation in co-cultures with endothelial cells. Expression analysis demonstrated expression of the gap junctional protein connexin 43 (Cx43) in EPC. Moreover, dye injection studies as well as FACS analysis identified gap junctional communication between endothelial cells and EPC. The inhibition of gap junctions by pharmacological blockers significantly reduced the angiogenic network formation, confirming that gap junctional communication between both cell types is required for this process.

The calcium sensitizer levosimendan is used for the treatment of acute decompensated heart failure. A small portion (4–7%) of levosimendan is metabolized to the pharmacologically active metabolite OR-1896 via the inactive intermediate OR-1855. In addition, levosimendan has been shown to exert positive effects on the endothelium in vitro antagonizing vascular dysfunction and inflammation. However, the function of the levosimendan metabolites within this context is still unknown. In this study, we thus investigated the impact of the metabolites OR-1896 and OR-1855 on endothelial inflammatory processes in vitro. We observed a reduction of IL-1β-dependent endothelial adhesion molecule ICAM-1 and VCAM-1 as well as interleukin (IL) -6 expression upon levosimendan treatment but not after treatment with OR-1855 or OR-1896, as assessed by western blotting, flow cytometry, and qRT-PCR. Instead, the metabolites impaired IL-1β-induced ROS formation via inactivation of the MAPK p38, ERK1/2, and JNK. Our results suggest that the levosimendan metabolites OR-1896 and OR-1855 have certain anti-inflammatory properties, partly other than levosimendan. Importantly, they additionally show that the intermediate metabolite OR-1855 does, in fact, have pharmacological effects in the endothelium. This is interesting, as the metabolites are responsible for the long-term therapeutic effects of levosimendan, and heart failure is associated with vascular dysfunction and inflammation.

During the onset of acute inflammation, rapid trafficking of leukocytes is essential to mount appropriate immune responses towards an inflammatory insult. Monocytes are especially indispensable for counteracting the inflammatory stimulus, neutralising the noxa and reconstituting tissue homeostasis. Thus, monocyte trafficking to the inflammatory sites needs to be precisely orchestrated. In this study, we identify a regulatory network driven by miR-125a that affects monocyte adhesion and chemotaxis by the direct targeting of two adhesion molecules, i.e., junction adhesion molecule A (JAM-A), junction adhesion molecule-like (JAM-L) and the chemotaxis-mediating chemokine receptor CCR2. By investigating monocytes isolated from patients undergoing cardiac surgery, we found that acute yet sterile inflammation reduces miR-125a levels, concomitantly enhancing the expression of JAM-A, JAM-L and CCR2. In contrast, TLR-4-specific stimulation with the pathogen-associated molecular pattern (PAMP) LPS, usually present within the perivascular inflamed area, resulted in dramatically induced levels of miR-125a with concomitant repression of JAM-A, JAM-L and CCR2 as early as 3.5 h. Our study identifies miR-125a as an important regulator of monocyte trafficking and shows that the phenotype of human monocytes is strongly influenced by this miRNA, depending on the type of inflammatory stimulus.

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.

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III) TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.

Actin-dependent leukocyte trafficking and activation are critical for immune surveillance under steady state conditions and during disease states. Proper immune surveillance is of utmost importance in mammalian homeostasis and it ensures the defense against pathogen intruders, but it also guarantees tissue integrity through the continuous removal of dying cells or the elimination of tumor cells. On the cellular level, these processes depend on the precise reorganization of the actin cytoskeleton orchestrating, e.g., cell polarization, migration, and vesicular dynamics in leukocytes. The fine-tuning of the actin cytoskeleton is achieved by a multiplicity of actin-binding proteins inducing, e.g., the organization of the actin cytoskeleton or linking the cytoskeleton to membranes and their receptors. More than a decade ago, the family of leucine-rich repeat (LRR) and calponin homology (CH) domain-containing (LRCH) proteins has been identified as cytoskeletal regulators. The LRR domains are important for protein-protein interactions and the CH domains mediate actin binding. LRR and CH domains are frequently found in many proteins, but strikingly the simultaneous expression of both domains in one protein only occurs in the LRCH protein family. To date, one LRCH protein has been described in drosophila and four LRCH proteins have been identified in the murine and the human system. The function of LRCH proteins is still under investigation. Recently, LRCH proteins have emerged as novel players in leukocyte function. In this review, we summarize our current understanding of LRCH proteins with a special emphasis on their function in leukocyte biology.

Background: The recombinant IL-1 receptor antagonist anakinra—currently approved for the treatment of autoinflammatory diseases—blocks IL-1β-mediated inflammatory signaling. As inflammation is a major driver of cancer, we hypothesized that anakinra might be able to mitigate glioblastoma (GBM) aggressiveness. Methods: Primary GBM or T98G cells were incubated alone or with peripheral blood mononuclear cells (PBMCs) and were subsequently treated with IL-1β and/or anakinra. T cells were obtained by magnetic bead isolation. Protein and mRNA expression were quantified by SDS-PAGE, qRT-PCR, and ELISA, respectively. Cell proliferation and apoptosis were analyzed via flow cytometry. Chemotaxis was studied via time-lapse microscopy. Results: Upon IL-1β stimulation, anakinra attenuated proinflammatory gene expression in both GBM cells and PBMCs, and mitigated tumor migration and proliferation. In a more lifelike model replacing IL-1β stimulation by GBM–PBMC co-culture, sole presence of PBMCs proved sufficient to induce a proinflammatory phenotype in GBM cells with enhanced proliferation and migration rates and attenuated apoptosis. Anakinra antagonized these pro-tumorigenic effects and, moreover, reduced inflammatory signaling in T cells without compromising anti-tumor effector molecules. Conclusion: By dampening the inflammatory crosstalk between GBM and immune cells, anakinra mitigated GBM aggressiveness. Hence, counteracting IL-1β-mediated inflammation might be a promising strategy to pursue.

During acute inflammation, the recruitment of leukocytes from the blood stream into the inflamed tissue is a well-described mechanism encompassing the interaction of endothelial cells with leukocytes allowing leukocytes to reach the site of tissue injury or infection where they can fulfill their function such as phagocytosis. This process requires a fine-tuned regulation of a plethora of signaling cascades, which are still incompletely understood. Here, connexin 43 (Cx43) and pannexin 1 (Panx1) are known to be pivotal for the correct communication of endothelial cells with leukocytes. Pharmacological as well as genetic approaches provide evidence that endothelial Cx43-hemichannels and Panx1-channels release signaling molecules including ATP and thereby regulate vessel function and permeability as well as the recruitment of leukocytes during acute inflammation. Furthermore, Cx43 hemichannels and Panx1-channels in leukocytes release signaling molecules and can mediate the activation and function of leukocytes in an autocrine manner. The focus of the present review is to summarize the current knowledge of the role of Cx43 and Panx1 in endothelial cells and leukocytes in the vasculature during acute inflammation and to discuss relevant molecular mechanisms regulating Cx43 and Panx1 function.

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.

BACKGROUND:Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence.METHODS:Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay.FINDINGS:We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1β as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1β-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88.INTERPRETATION:Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. FUND: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research.

Myoendothelial gap junctions are involved in the regulation of vascular tone. The major connexins described in the vascular system are Cx37, Cx40, Cx43, and Cx45 with all but Cx45 found in myoendothelial connections. Although many reports on post-translational modifications of these connexins are available, only few groups have investigated their role in controlling myoendothelial communication and signal propagation. In particular, myoendothelial gap junctions serve as essential feedback pathways between vascular smooth muscle cells and endothelial cells in the regulation of vessel responses. In conclusion, myoendothelial gap junctions coordinate and shift the overall response of vessels toward relaxation and consequently limit the constriction of vessels.

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.

Vascular endothelial cells, as well as smooth muscle cells, show heterogeneity with regard to their receptor expression and reactivity. For the vascular wall to act as a functional unit, the various cells' responses require integration. Such an integration is not only required for a homogeneous response of the vascular wall, but also for the vasomotor behaviour of consecutive segments of the microvascular arteriolar tree. As flow resistances of individual sections are connected in series, sections require synchronization and coordination to allow effective changes of conductivity and blood flow. A prerequisite for the local coordination of individual vascular cells and different sections of an arteriolar tree is intercellular communication. Connexins are involved in a dual manner in this coordination. (i) By forming gap junctions between cells, they allow an intercellular exchange of signalling molecules and electrical currents. In particular, the spread of electrical currents allows for coordination of cell responses over longer distances. (ii) Connexins are able to interact with other proteins to form signalling complexes. In this way, they can modulate and integrate individual cells' responses also in a channel-independent manner. This review outlines mechanisms allowing the vascular connexins to exert their coordinating function and to regulate the vasomotor reactions of blood vessels both locally, and in vascular networks. Wherever possible, we focus on the vasomotor behaviour of small vessels and arterioles which are the main vessels determining vascular resistance, blood pressure and local blood flow.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1 alpha (HIF-1 alpha), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and nonspecific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanopartidelentivirus (MNP-LV) complexes allowing site -directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1 alpha dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1 a from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1 a DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-la activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

Objective-Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. Approach and Results-Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4] oxadiazolo[ 4,3-a] quinoxalin-1-one). Conclusions-Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium. Visual Overview-An online visual overview is available for this article.

Objective— Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. Approach and Results— Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction–dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Conclusions— Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.