Stimulation of the EP3 receptor causes lung oedema by activation of TRPC6 in pulmonary endothelial cells.

BACKGROUND: Prostaglandin E2 (PGE2) increases pulmonary vascular permeability by activation of the PGE2 receptor 3 (EP3), which may explain adverse pulmonary effects of the EP1/EP3 receptor agonist sulprostone in patients. In addition, PGE2 contributes to pulmonary oedema in response to platelet-activating factor (PAF). PAF increases endothelial permeability by recruiting the cation channel transient receptor potential canonical 6 (TRPC6) to endothelial caveolae via acid sphingomyelinase (ASMase). Yet, the roles of PGE2 and EP3 in this pathway are unknown. We hypothesised that EP3 receptor activation may increase pulmonary vascular permeability by activation of TRPC6, and thus, synergise with ASMase-mediated TRPC6 recruitment in PAF-induced lung oedema. METHODS: In isolated lungs, we measured increases in endothelial calcium (ΔCa2+) or lung weight (Δweight), and endothelial caveolar TRPC6 abundance as well as phosphorylation. RESULTS: PAF-induced ΔCa2+ and Δweight were attenuated in EP3-deficient mice. Sulprostone replicated PAF-induced ΔCa2+ and Δweight which were blocked by pharmacological/genetic inhibition of TRPC6, ASMase or Src-family kinases (SrcFK). PAF, but not sulprostone, increased TRPC6 abundance in endothelial caveolae. Immunoprecipitation revealed PAF- and sulprostone-induced tyrosine-phosphorylation of TRPC6 that was prevented by inhibition of phospholipase C (PLC) or SrcFK. PLC inhibition also blocked sulprostone-induced ΔCa2+ and Δweight, as did inhibition of SrcFK or inhibitory G-protein (Gi) signalling. CONCLUSIONS: EP3 activation triggers pulmonary oedema via Gi-dependent activation of PLC and subsequent SrcFK-dependent tyrosine phosphorylation of TRPC6. In PAF-induced lung oedema, this TRPC6 activation coincides with ASMase-dependent caveolar recruitment of TRPC6, resulting in rapid endothelial Ca2+ influx and barrier failure.

Thoracic epidural anesthesia decreases endotoxin-induced endothelial injury.

BACKGROUND: The sympathetic nervous system is considered to modulate the endotoxin-induced activation of immune cells. Here we investigate whether thoracic epidural anesthesia with its regional symapathetic blocking effect alters endotoxin-induced leukocyte-endothelium activation and interaction with subsequent endothelial injury. METHODS: Sprague Dawley rats were anesthetized, cannulated and hemodynamically monitored. E. coli lipopolysaccharide (Serotype 0127:B8, 1.5 mg x kg(-1) x h(-1)) or isotonic saline (controls) was infused for 300 minutes. An epidural catheter was inserted for continuous application of lidocaine or normal saline in endotoxemic animals and saline in controls. After 300 minutes we measured catecholamine and cytokine plasma concentrations, adhesion molecule expression, leukocyte adhesion, and intestinal tissue edema. RESULTS: In endotoxemic animals with epidural saline, LPS significantly increased the interleukin-1ß plasma concentration (48%), the expression of endothelial adhesion molecules E-selectin (34%) and ICAM-1 (42%), and the number of adherent leukocytes (40%) with an increase in intestinal myeloperoxidase activity (26%) and tissue edema (75%) when compared to healthy controls. In endotoxemic animals with epidural infusion of lidocaine the values were similar to those in control animals, while epinephrine plasma concentration was 32% lower compared to endotoxemic animals with epidural saline. CONCLUSIONS: Thoracic epidural anesthesia attenuated the endotoxin-induced increase of IL-1ß concentration, adhesion molecule expression and leukocyte-adhesion with subsequent endothelial injury. A potential mechanism is the reduction in the plasma concentration of epinephrine.

Lung endothelial Ca2+ and permeability response to platelet-activating factor is mediated by acid sphingomyelinase and transient receptor potential classical 6.

RATIONALE: Platelet-activating factor (PAF) increases lung vascular permeability within minutes by activation of acid sphingomyelinase (ASM) and a subsequent nitric oxide (NO)-inhibitable and Ca(2+)-dependent loss in barrier function. OBJECTIVES: To elucidate the molecular mechanisms underlying this response. METHODS: In isolated perfused rat and mouse lungs, endothelial Ca(2+) concentration ([Ca(2+)](i)) was quantified by real-time fluorescence imaging, and caveolae of endothelial cells were isolated and probed for Ca(2+) entry channels. Regulation of transient receptor potential classical (TRPC) 6-mediated currents in lung endothelial cells was assessed by patch clamp technique. MEASUREMENTS AND MAIN RESULTS: PAF increased lung weight gain and endothelial [Ca(2+)](i). This response was abrogated by inhibitors of ASM or in ASM-deficient mice, and replicated by lung perfusion with exogenous ASM or C2-ceramide. PAF increased the caveolar abundance of TRPC6 channels, which was similarly blocked by ASM inhibition. PAF-induced increases in lung endothelial [Ca(2+)](i), vascular filtration coefficient, and edema formation were attenuated by the TRPC inhibitor SKF96365 and in TRPC6-deficient mice, whereas direct activation of TRPC6 replicated the [Ca(2+)](i) and edema response to PAF. The exogenous NO donor PapaNONOate or the cyclic guanosine 3',5'-monophosphate analog 8Br-cGMP blocked the endothelial [Ca(2+)](i) and permeability response to PAF, in that they directly blocked TRPC6 channels without interfering with their PAF-induced recruitment to caveolae. CONCLUSIONS: The present findings outline a new signaling cascade in the induction of PAF-induced lung edema, in that stimulation of ASM causes recruitment of TRPC6 channels to caveolae, thus allowing for Ca(2+) influx and subsequent increases in endothelial permeability that are amplified in the absence of endothelial NO synthesis.

Vascular barrier regulation by PAF, ceramide, caveolae, and NO - an intricate signaling network with discrepant effects in the pulmonary and systemic vasculature.

Increased endothelial permeability and vascular barrier failure are hallmarks of inflammatory responses in both the pulmonary and the systemic circulation. Platelet-activating factor (PAF) has been implicated as an important lipid mediator in the formation of pulmonary and extrapulmonary edema. Ostensibly, the PAF-induced signaling pathways in endothelial cells utilize similar structures and molecules including acid sphingomyelinase, ceramide, caveolae, endothelial nitric oxide synthase, and nitric oxide, in pulmonary and systemic microvessels. Yet, the constituents of these signaling pathways act and respond in distinctly different and frequently opposing ways in the lung versus organs of the systemic circulation. By confronting seemingly discrepant findings from the literature, we reconstruct the differential signaling pathways by which PAF regulates edema formation in the systemic and the pulmonary vascular bed, and trace this dichotomy from the level of myosin light chain kinase via the regulation of endothelial nitric oxide synthase and sphingomyelinase signaling to the level of caveolar trafficking. Here, we propose that PAF regulates vascular barrier function in individual organs by opposing signaling pathways that culminate in increased respectively decreased nitric oxide synthesis in the systemic and the pulmonary endothelium. The present review may provide a physiological explanation for the overall disappointing results of previous pharmacological strategies in conditions of generalized barrier failure such as sepsis, and instead advertises the development of organ-specific interventions by targeting the individual composition or trafficking of endothelial caveolae.

Chronic kidney disease induces a systemic microangiopathy, tissue hypoxia and dysfunctional angiogenesis.

Chronic kidney disease (CKD) is associated with excessive mortality from cardiovascular disease (CVD). Endothelial dysfunction, an early manifestation of CVD, is consistently observed in CKD patients and might be linked to structural defects of the microcirculation including microvascular rarefaction. However, patterns of microvascular rarefaction in CKD and their relation to functional deficits in perfusion and oxygen delivery are currently unknown. In this in-vivo microscopy study of the cremaster muscle microcirculation in BALB/c mice with moderate to severe uremia, we show in two experimental models (adenine feeding or subtotal nephrectomy), that serum urea levels associate incrementally with a distinct microangiopathy. Structural changes were characterized by a heterogeneous pattern of focal microvascular rarefaction with loss of coherent microvascular networks resulting in large avascular areas. Corresponding microvascular dysfunction was evident by significantly diminished blood flow velocity, vascular tone, and oxygen uptake. Microvascular rarefaction in the cremaster muscle paralleled rarefaction in the myocardium, which was accompanied by a decrease in transcription levels not only of the transcriptional regulator HIF-1α, but also of its target genes Angpt-2, TIE-1 and TIE-2, Flkt-1 and MMP-9, indicating an impaired hypoxia-driven angiogenesis. Thus, experimental uremia in mice associates with systemic microvascular disease with rarefaction, tissue hypoxia and dysfunctional angiogenesis.