Clinical Implication of Phosphodiesterase-4-Inhibition.

The pleiotropic function of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent pathways in health and disease led to the development of pharmacological phosphodiesterase inhibitors (PDE-I) to attenuate cAMP degradation. While there are many isotypes of PDE, a predominant role of PDE4 is to regulate fundamental functions, including endothelial and epithelial barrier stability, modulation of inflammatory responses and cognitive and/or mood functions. This makes the use of PDE4-I an interesting tool for various therapeutic approaches. However, due to the presence of PDE4 in many tissues, there is a significant danger for serious side effects. Based on this, the aim of this review is to provide a comprehensive overview of the approaches and effects of PDE4-I for different therapeutic applications. In summary, despite many obstacles to use of PDE4-I for different therapeutic approaches, the current data warrant future research to utilize the therapeutic potential of phosphodiesterase 4 inhibition.

[Sectio Caesarea under Gitelman Syndrome]. / Sectio caesarea unter Gitelman-Syndrom.

Gitelman syndrome is a rare inherited renal tubulopathy characterized by hypokalemia, hypomagnesemia and metabolic alcalosis. It is caused by a mutation in the SLC12A3 gene leading to a dysfunction of the thiazide-sensitive sodium chloride cotransporter and the magnesium transporters in the distal convoluted tubules. Only few reports of pregnant woman with Gitelman syndrome exist. Due to many unsolved questions about the impact on pregnancy and the maternal and fetal outcome, the obstetric and anesthesiological management remains a challenge. We discuss the case of a primary cesarean delivery in a 22-year-old woman with a new diagnosed Gitelman syndrome focusing on the anesthesiological approach.

Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation.

In sepsis and systemic inflammation, increased microvascular permeability and consecutive breakdown of microcirculatory flow significantly contribute to organ failure and death. Evidence points to a critical role of cAMP levels in endothelial cells to maintain capillary endothelial barrier properties in acute inflammation. However, approaches to verify this observation in systemic models are rare. Therefore we tested here whether systemic application of the phosphodiesterase-4-inhibitors (PD-4-Is) rolipram or roflumilast to increase endothelial cAMP was effective to attenuate capillary leakage and breakdown of microcirculatory flow in severe lipopolysaccharide (LPS)-induced systemic inflammation in rats. Measurements of cAMP in mesenteric microvessels demonstrated significant LPS-induced loss of cAMP levels which was blocked by application of rolipram. Increased endothelial cAMP by application of either PD-4-I rolipram or roflumilast led to stabilization of endothelial barrier properties as revealed by measurements of extravasated FITC-albumin in postcapillary mesenteric venules. Accordingly, microcirculatory flow in mesenteric venules was significantly increased following PD-4-I treatment and blood gas analyses indicated improved metabolism. Furthermore application of PD-4-I after manifestation of LPS-induced systemic inflammation and capillary leakage therapeutically stabilized endothelial barrier properties as revealed by significantly reduced volume resuscitation for haemodynamic stabilization. Accordingly microcirculation was significantly improved following treatment with PD-4-Is. Our results demonstrate that inflammation-derived loss of endothelial cAMP contributes to capillary leakage which was blocked by systemic PD-4-I treatment. Therefore these data suggest a highly clinically relevant and applicable approach to stabilize capillary leakage in sepsis and systemic inflammation.

Effects of crystalloids and colloids on liver and intestine microcirculation and function in cecal ligation and puncture induced septic rodents.

BACKGROUND: Septic acute liver and intestinal failure is associated with a high mortality. We therefore investigated the influence of volume resuscitation with different crystalloid or colloid solutions on liver and intestine injury and microcirculation in septic rodents. METHODS: Sepsis was induced by cecal ligation and puncture (CLP) in 77 male rats. Animals were treated with different crystalloids (NaCl 0.9% (NaCl), Ringer's acetate (RA)) or colloids (Gelafundin 4% (Gel), 6% HES 130/0.4 (HES)). After 24 h animals were re-anesthetized and intestinal (n = 6/group) and liver microcirculation (n = 6/group) were obtained using intravital microscopy, as well as macrohemodynamic parameters were measured. Blood assays and organs were harvested to determine organ function and injury. RESULTS: HES improved liver microcirculation, cardiac index and DO(2)-I, but significantly increased IL-1ß, IL-6 and TNF-α levels and resulted in a mortality rate of 33%. Gel infused animals revealed significant reduction of liver and intestine microcirculation with severe side effects on coagulation (significantly increased PTT and INR, decreased haemoglobin and platelet count). Furthermore Gel showed severe hypoglycemia, acidosis and significantly increased ALT and IL-6 with a lethality of 29%. RA exhibited no derangements in liver microcirculation when compared to sham and HES. RA showed no intestinal microcirculation disturbance compared to sham, but significantly improved the number of intestinal capillaries with flow compared to HES. All RA treated animals survided and showed no severe side effects on coagulation, liver, macrohemodynamic or metabolic state. CONCLUSIONS: Gelatine 4% revealed devastated hepatic and intestinal microcirculation and severe side effects in CLP induced septic rats, whereas the balanced crystalloid solution showed stabilization of macro- and microhemodynamics with improved survival. HES improved liver microcirculation, but exhibited significantly increased pro-inflammatory cytokine levels. Crystalloid infusion revealed best results in mortality and microcirculation, when compared with colloid infusion.

Phenylephrine versus cafedrine/theodrenaline (Akrinor) for the treatment of spinal anaesthesia-induced maternal hypotension during caesarean section: a retrospective single-centre cohort study.

OBJECTIVE: The main objective of this study was to assess the impact of phenylephrine and cafedrine/theodrenaline on the mother and newborn after spinal anaesthesia for caesarean section. SETTING: University teaching hospital. DESIGN: A single-centre retrospective data cohort study. PATIENTS: All obstetric patients who were scheduled for caesarean section in a 2-year period. INTERVENTIONS: Administration of either intravenous phenylephrine prophylactically or cafedrine/theodrenaline (Akrinor) reactively to maintain blood pressure after spinal anaesthesia. MAIN OUTCOME MEASURE: Maternal hypotension, heart rate during caesarean section and after admission to IMC, fetal arterial cord pH and base excess levels, maternal volume resuscitation and the use of rescue medication. RESULTS: 852 data sets could be included: n=440 Akrinor, n=412 in the phenylephrine cohort. During caesarean section blood pressure was slightly higher in the phenylephrine group compared with the Akrinor group, while hypotension <100 mm Hg systolic blood pressure (SBP) occurred significantly more often during arrival at the IMC after surgery when phenylephrine was used. Heart rate was lower and rescue medication was significantly more frequently given in the phenylephrine cohort. Irrespective of the medication used, women with baseline levels of <120 mm Hg SBP had a high risk to develop hypotension <100 mm Hg after spinal anaesthesia for caesarean section. While there was no statistical difference in mean umbilical arterial pH levels, the incidence of acidosis, defined as pH <7.2, was significantly higher with phenylephrine. CONCLUSION: Phenylephrine was not superior to Akrinor to treat spinal anaesthesia-induced maternal hypotension during caesarean section. TRIAL REGISTRATION NUMBER: DRKS00025795.

Standardized Colon Ascendens Stent Peritonitis in Rats - a Simple, Feasible Animal Model to Induce Septic Acute Kidney Injury.

AKI in septic patients is associated with increased mortality and poor outcome despite major efforts to refine the understanding of its pathophysiology. Here, an in vivo model is presented that combines a standardized septic focus to induce AKI and an intensive care (ICU) setup to provide an advanced hemodynamic monitoring and therapy comparable in human sepsis. Sepsis is induced by standardized colon ascendens stent peritonitis (sCASP). AKI is investigated functionally by measurement of blood and urine samples as well as histologically by evaluation of histopathological scores. Furthermore, the advanced hemodynamic monitoring and the possibility of repetitive blood gas sampling enable a differentiated analysis of severity of induced sepsis. The sCASP method is a standardized, reliable and reproducible method to induce septic AKI. The intensive care setup, continuous hemodynamic and gas exchange monitoring, low mortality rate as well as the opportunity of detailed analyses of kidney function and impairments are advantages of this setup. Therefore, the described method may serve as a new standard for experimental investigations of septic AKI.

Sphingosine-1-Phosphate Receptor-1 Agonist Sew2871 Causes Severe Cardiac Side Effects and Does Not Improve Microvascular Barrier Breakdown in Sepsis.

BACKGROUND: Endothelial barrier dysfunction is a hallmark in the pathogenesis of sepsis. Sphingosine-1-phosphate (S1P) has been proposed to be critically involved in the maintenance of endothelial barrier function predominately by activating S1P receptor-1 (S1P1). Previous studies have shown that the specific S1P1 agonist SEW2871 improves endothelial barrier function under inflammatory conditions. However, the effectiveness of SEW2871 and potential side effects remained largely unexplored in a clinically relevant model of sepsis. Therefore, this study aimed to evaluate the effects of SEW2871 in the Colon ascendens stent peritonitis (CASP) model. METHODS: Polymicrobial sepsis was induced in Sprague-Dawley rats using CASP model that enabled the monitoring of macro-hemodynamic parameters. Twelve hours after surgery, animals received either SEW2871 or sodium chloride. Mesenteric endothelial barrier function was evaluated 24 h after sepsis induction by intravital microscopy. Organ pathology was assessed in lungs. S1P levels, blood gas analyses, and blood values were measured at different time points. In parallel the effect of SEW2871 was evaluated in human dermal microvascular endothelial cells. RESULT: In vitro SEW2871 partially stabilized TNF-α-induced endothelial barrier breakdown. However, in vivo SEW2871 caused severe cardiac side effects in septic animals leading to an increased lethality. Sepsis-induced endothelial barrier dysfunction was not attenuated by SEW2871 as revealed by increased FITC-albumin extra-vasation, requirement of intravasal fluid replacement, and pulmonary edema. Interestingly, Sham-operated animals did not present any side effects after SEW2871 treatment. CONCLUSION: Our study demonstrates that the application of SEW2871 causes severe cardiac side effects and cannot attenuate the inflammation-induced endothelial barrier breakdown in a clinically relevant sepsis model, suggesting that the time point of administration and the pro-inflammatory milieu play a pivotal role in the therapeutic benefit of SEW2871.

Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo.

BACKGROUND: Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. METHODS AND RESULTS: In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. CONCLUSION AND SIGNIFICANCE: Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.

Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis.

AIMS: Microvascular endothelial barrier breakdown in sepsis precedes organ failure and death in patients. We tested the hypothesis that the formation of endothelium-derived soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) is involved in inflammation-induced endothelial barrier disruption. METHODS AND RESULTS: Incubation of human dermal microvascular endothelial cells (HDMEC) with tumour necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) led to endothelial barrier disruption which correlated with significantly increased sVE-cadherin at a size of ∼90 kDa in cell culture supernatants. Inhibition of the VE-cadherin-cleaving disintegrin and metalloproteinase ADAM10 using GI254023X attenuated inflammation-induced formation of sVE-cadherin and endothelial barrier disruption, suggesting ADAM10-mediated shedding as a mechanism underlying sVE-cadherin release. Formation of VE-cadherin fragments at 90 and 110 kDa was observed when recombinant VE-cadherin (rVE-cadherin) was digested with recombinant ADAM10. Mass spectrometry of the VE-cadherin fragments showed that they originated from cleavage of the extracelluar domain and thereby several cleavage sites of ADAM10 were identified. Atomic force microscopy measurements demonstrated that cell culture supernatants containing sVE-cadherin and application of rVE-cadherin blocked VE-cadherin binding. Accordingly rVE-cadherin dose-dependently led to loss of endothelial barrier functions in HDMEC monolayers. Finally, in patients suffering from severe sepsis or septic shock with clinical signs of a microvascular leackage, serum levels of sVE-cadherin were significantly increased. CONCLUSION: Taken together, formation of sVE-cadherin is associated and contributes to inflammation-induced breakdown of endothelial barrier functions by inhibition of VE-cadherin binding. The underlying mechanism of VE-cadherin cleavage involves ADAM10 and appears to be of clinical relevance since sVE-cadherin was augmented in patients with severe sepsis.

Balanced Hydroxyethylstarch (HES 130/0.4) Impairs Kidney Function In-Vivo without Inflammation.

Volume therapy is a standard procedure in daily perioperative care, and there is an ongoing discussion about the benefits of colloid resuscitation with hydroxyethylstarch (HES). In sepsis HES should be avoided due to a higher risk for acute kidney injury (AKI). Results of the usage of HES in patients without sepsis are controversial. Therefore we conducted an animal study to evaluate the impact of 6% HES 130/0.4 on kidney integrity with sepsis or under healthy conditions Sepsis was induced by standardized Colon Ascendens Stent Peritonitis (sCASP). sCASP-group as well as control group (C) remained untreated for 24 h. After 18 h sCASP+HES group (sCASP+VOL) and control+HES (C+VOL) received 50 ml/KG balanced 6% HES (VOL) 130/0.4 over 6 h. After 24 h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea), cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL) as well as histopathology were analysed. In vitro human proximal tubule cells (PTC) were cultured +/- lipopolysaccharid (LPS) and with 0.1-4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL) deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion.

Phosphodiesterase 4 inhibition dose dependently stabilizes microvascular barrier functions and microcirculation in a rodent model of polymicrobial sepsis.

BACKGROUND: Breakdown of microvascular endothelial barrier functions contributes to disturbed microcirculation, organ failure, and death in sepsis. Increased endothelial cAMP levels by systemic application of phosphodiesterase 4 inhibitors (PD-4-I) have previously been demonstrated to protect microvascular barrier properties in a model of systemic inflammation (systemic inflammatory response syndrome) suggesting a novel therapeutic option to overcome this problem. However, in a clinically relevant model of polymicrobial sepsis long-term effects, immunomodulatory effects and effectivity of PD-4-I to stabilize microvascular barrier functions and microcirculation remained unexplored. METHODS: We induced polymicrobial sepsis using the colon ascendens stent peritonitis (CASP) model in which we performed macrohemodynamic and microhemodynamic monitoring with and without systemic intravenous application of different doses of PD-4-I rolipram in Sprague-Dawley rats over 26 h. RESULTS: All animals with CASP showed clinical and laboratory signs of sepsis and peritonitis. Whereas macrohemodynamic adverse effects were not evident, application of PD-4-I led to stabilization of endothelial barrier properties as revealed by reduced extravasation of fluorescein isothiocyanate-albumin. However, only low-dose application of 1 mg/kg body weight per hour of PD-4-I improved microcirculatory flow in the CASP model, whereas high-dose therapy of 3 mg/kg BW per hour PDI-4-I had adverse effects. Accordingly, sepsis-induced acute kidney injury and lung edema were prevented by PD-4-I treatment. Furthermore, PD-4-I showed immunomodulatory effects as revealed by decreased interleukin 1α (IL-1α), IL-1ß, IL-12, and tumor necrosis factor α levels following PD-4-I treatment, which appeared not to correlate with barrier-stabilizing effects of rolipram. CONCLUSIONS: These data provide further evidence that systemic application of PD-4-I could be suitable for therapeutic microvascular barrier stabilization and improvement of microcirculatory flow in sepsis.

Rosiglitazone affects nitric oxide synthases and improves renal outcome in a rat model of severe ischemia/reperfusion injury.

Background. Nitric oxide (NO)-signal transduction plays an important role in renal ischemia/reperfusion (I/R) injury. NO produced by endothelial NO-synthase (eNOS) has protective functions whereas NO from inducible NO-synthase (iNOS) induces impairment. Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg) was administered i.p. to SD-rats (f) subjected to bilateral renal ischemia (60 min). Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NO(x)-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3) was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion) and reduces histomorphological injury. Additionally, RGZ reduces NO(x) plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury.

The impact of crystalloid and colloid infusion on the kidney in rodent sepsis.

PURPOSE: Volume replacement remains one of the pillars of sepsis therapy. The effect of different volume solutions on kidney function in sepsis still remains unclear. We therefore determined the impact of crystalloid and colloid solutions on kidney function in a rodent model of abdominal sepsis induced by cecal ligation and puncture (CLP). METHODS: Anesthetized rats underwent the CLP procedure, whereas control animals were sham operated. Septic animals were treated with crystalloid and colloid solutions. Hemodynamic variables and blood gases were measured. After 24 h animals were re-anesthetized, the kidneys were harvested, and creatinine (crea), urea, cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) were investigated. RESULTS: Septic animals exhibited a mortality rate of 19% after 24 h. Gelatin-treated animals showed significantly increased levels of crea and urea. Colloids [gelatin 4% (Gel) or hydroxyethyl starch 6% 130/0.4 (HES)] as volume replacement resulted in elevated levels of NGAL. The histopathological observations revealed that Gel- and HES-treated animals showed vesicles within epithelial cells of the tubulus system and an overall increased injury. In contrast, total injury scores in groups treated with crystalloids [0.9% NaCl (NaCl) and Sterofundin ISO (SteroIso)] were not significantly different compared to sham-treated animals. CONCLUSION: None of the examined volume solution was inert to the kidney. In a CLP rodent sepsis model, animals infused with balanced crystalloid SteroIso exhibited the least effects on kidney function. Both hydroxyethyl starch 6% 130/0.4 and gelatin 4% derogated the kidney, whereas gelatin was more harmful when compared with hydroxyethyl starch.