Regulation of endothelial and epithelial barrier functions by peptide hormones of the adrenomedullin family.
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ABSTRACT:
SUBMITTER: Garcia-Ponce A
PROVIDER: S-EPMC5214765 | biostudies-other | 2016
REPOSITORIES: biostudies-other
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Project description:BACKGROUND: Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. RESULTS: EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. CONCLUSION: This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions.
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Project description:Natriuretic peptides (NPs) are a family of cardiac- and vascular-derived hormones that are well known for regulating blood pressure, but their expression in the brain poses an intriguing yet unanswered question concerning their roles in the nervous system. Here, we report several unique activities of these hormones in regulating axonal development of dorsal root ganglion (DRG) neurons in the spinal cord. First, the C-type NP (CNP) is expressed in a restricted area of the dorsal spinal cord and provides a cue that is necessary for bifurcation of central sensory afferents. Second, in the culture of embryonic DRG neurons, CNP stimulates branch formation, induces axon outgrowth, and attracts growth cones. Furthermore, these activities are mediated by cyclic guanosine-3',5'-monophosphate (cGMP) signaling and can be elicited by other members of NP hormones. Thus, NPs represent a new class of extracellular factors that regulate key axonal processes during development. Because their receptors are present in many regions of the embryonic and adult brain, we propose that these hormones have wide influence on the development and function of the nervous system.
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Project description:IntroductionThe epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood.MethodsIn a close-contact co-culture model of human airway epithelial and endothelial cells, cellular crosstalk was analyzed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy, and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system, the temporal release of mediators was analyzed in the co-culture model.ResultsWithin 4 h of challenge, double-stranded RNA induced the release of TNF-α by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8 h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-α.ConclusionBy facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signaling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation.
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Project description:Increased vascular permeability is a hallmark of several cardiovascular anomalies, including ischaemia/reperfusion injury and inflammation. During both ischaemia/reperfusion and inflammation, massive amounts of various nucleotides, particularly adenosine 5'-triphosphate (ATP) and adenosine, are released that can induce a plethora of signalling pathways via activation of several purinergic receptors and may affect endothelial barrier properties. The nature of the effects on endothelial barrier function may depend on the prevalence and type of purinergic receptors activated in a particular tissue. In this review, we discuss the influence of the activation of various purinergic receptors and downstream signalling pathways on vascular permeability during pathological conditions.
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Project description:Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1alpha on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1alpha enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage.
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Project description:Mucosal tissues represent surfaces that are exposed to the outside world and provide a conduit for internal and external communication. Tissues such as the intestine and the lung are lined by layer(s) of epithelial cells that, when organized in three dimensions, provide a critical barrier to the flux of luminal contents. This selective barrier is provided through the regulated expression of junctional proteins and mucins. Tissue oxygen metabolism is central to the maintenance of homeostasis in the mucosa. In some organs (e.g., the colon), low baseline Po2 determines tissue metabolism and results in basal expression of the transcription factor, hypoxia-inducible factor (HIF), which is enhanced after ischemia/inflammation. Recent studies have indicated that HIF contributes fundamentally to the expression of barrier-related genes and in the regulation of barrier-adaptive responses within the mucosa. Here, we briefly review recent literature on the topic of hypoxia and HIF regulation of barrier in mucosal health and during disease.
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Project description:Epithelial cell function is modulated by mechanical forces imparted by the extracellular environment. The transmission of forces onto the cytoskeleton by modalities such as mechanical stress and matrix stiffness is necessary to address by the development of new experimental models that permit finely tuned cell mechanical challenges. Herein, we developed an epithelial tissue culture model, named the 3D Oral Epi-mucosa platform, to investigate the role mechanical cues in the epithelial barrier. In this platform, low-level mechanical stress (0.1 kPa) is applied to oral keratinocytes, which lie on 3D fibrous collagen (Col) gels whose stiffness is modulated by different concentrations or the addition of other factors such as fibronectin (FN). Our results show that cells lying on intermediate Col (3 mg/mL; stiffness = 30 Pa) demonstrated lower epithelial leakiness compared with soft Col (1.5 mg/mL; stiffness = 10 Pa) and stiff Col (6 mg/mL; stiffness = 120 Pa) gels, indicating that stiffness modulates barrier function. In addition, the presence of FN reversed the barrier integrity by inhibiting the interepithelial interaction via E-cadherin and Zonula occludens-1. Overall, the 3D Oral Epi-mucosa platform, as a new in vitro system, will be utilized to identify new mechanisms and develop future targets involved in mucosal diseases.
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Project description:Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The ? subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-? and whether this subunit is involved in the regulation of their barrier function.The presence of ?, ?, and ? subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-? in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-?.HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-?. Both TIP peptide, which specifically binds to ENaC-?, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-? depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-? and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-? and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-? in both barrier function and liquid clearance.Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-? in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-? should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.
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Project description:Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bbeta15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bbeta15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bbeta15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bbeta15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bbeta15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bbeta15-42 is confirmed in Fyn(-/-) mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bbeta15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.
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