Johannes Wedel

N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

Since stimulation of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) mitigates acute kidney injury (AKI) and endogenous N-acyl dopamine derivatives are able to activate TRPV1, we tested if synthetic N-octanoyl-dopamine (NOD) activates TRPV1 and if it improves AKI. These properties of NOD and its intrinsic anti-inflammatory character were compared with those of dopamine (DA). TRPV1 activation and anti-inflammatory properties of NOD and DA were tested using primary cell cultures in vitro. The influence of NOD and DA on AKI was tested in a prospective, randomized, controlled animal study with 42 inbred male Lewis rats (LEW, RT1), treated intravenously with equimolar concentrations of DA or NOD one hour before the onset of warm ischemia and immediately before clamp release. NOD, but not DA, activates TRPV1 channels in isolated dorsal root ganglion neurons (DRG) that innervate several tissues including kidney. In TNFα stimulated proximal tubular epithelial cells, inhibition of NFκB and subsequent inhibition of VCAM1 expression by NOD was significantly stronger than by DA. NOD improved renal function compared to DA and saline controls. Histology revealed protective effects of NOD on tubular epithelium at day 5 and a reduced number of monocytes in renal tissue of DA and NOD treated rats. Our data demonstrate that NOD but not DA activates TRPV1 and that NOD has superior anti-inflammatory properties in vitro. Although NOD mitigates deterioration in renal function after AKI, further studies are required to assess to what extend this is causally related to TRPV1 activation and/or desensitization.

Different design of enzyme-triggered CO-releasing molecules (ET-CORMs) reveals quantitative differences in biological activities in terms of toxicity and inflammation

Acyloxydiene–Fe(CO)3 complexes can act as enzyme-triggered CO-releasing molecules (ET-CORMs). Their biological activity strongly depends on the mother compound from which they are derived, i.e. cyclohexenone or cyclohexanedione, and on the position of the ester functionality they harbour. The present study addresses if the latter characteristic affects CO release, if cytotoxicity of ET-CORMs is mediated through iron release or inhibition of cell respiration and to what extent cyclohexenone and cyclohexanedione derived ET-CORMs differ in their ability to counteract TNF-α mediated inflammation. Irrespective of the formulation (DMSO or cyclodextrin), toxicity in HUVEC was significantly higher for ET-CORMs bearing the ester functionality at the outer (rac-4), as compared to the inner (rac-1) position of the cyclohexenone moiety. This was paralleled by an increased CO release from the former ET-CORM. Toxicity was not mediated via iron as EC50 values for rac-4 were significantly lower than for FeCl2 or FeCl3 and were not influenced by iron chelation. ATP depletion preceded toxicity suggesting impaired cell respiration as putative cause for cell death. In long-term HUVEC cultures inhibition of VCAM-1 expression by rac-1 waned in time, while for the cyclohexanedione derived rac-8 inhibition seems to increase. NFκB was inhibited by both rac-1 and rac-8 independent of IκBα degradation. Both ET-CORMs activated Nrf-2 and consequently induced the expression of HO-1. This study further provides a rational framework for designing acyloxydiene–Fe(CO)3 complexes as ET-CORMs with differential CO release and biological activities. We also provide a better understanding of how these complexes affect cell-biology in mechanistic terms.

Anserine inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated with histidine dipeptide concentration

BACKGROUND We reported an association of a particular allele of the carnosinase (CNDP1 Mannheim) gene with reduced serum carnosinase (CN1) activity and absence of nephropathy in diabetic patients. Carnosine protects against the adverse effects of high glucose levels but serum carnosine concentration was generally low. METHODS We measured the concentration of two further histidine dipeptides, anserine and homocarnosine, via HPLC. CN1 activity was measured fluorometically and for concentration we developed a capture ELISA. RESULTS We found an association between the CNDP1 Mannheim allele and reduced serum CN1 activity for all three dipeptides but no correlation to serum concentrations although anserine and homocarnosine inhibited carnosinase activity. Patients with liver cirrhosis have low CN1 activity (0.24 ± 0.17 μmol/ml/h, n=7 males; normal range: 3.2 ± 1.1, n=104; p<0.05) and CN1 concentrations (2.3 ± 1.5 μg/ml; normal range: 24.9 ± 8.9, p<0.05) but surprisingly, histidine dipeptide concentrations in serum are not increased compared to controls. CONCLUSIONS Serum histidine dipeptide concentrations are not correlated to CN1 activity. The protective effect of low CN1 activity might be related either to turnover of CN1 substrates or a protective function of dipeptides might be localized in other tissues.

N-Octanoyl Dopamine Inhibits the Expression of a Subset of κB Regulated Genes: Potential Role of p65 Ser276 Phosphorylation

Background and Purpose Catechol containing compounds have anti-inflammatory properties, yet for catecholamines these properties are modest. Since we have previously demonstrated that the synthetic dopamine derivative N-octanoyl dopamine (NOD) has superior anti-inflammatory properties compared to dopamine, we tested NOD in more detail and sought to elucidate the molecular entities and underlying mechanism by which NOD down-regulates inflammation. Experimental Approach Genome wide gene expression profiling of human umbilical vein endothelial cells (HUVECs) was performed after stimulation with TNF-α or in the combination with NOD. Confirmation of these differences, NFκB activation and the molecular entities that were required for the anti-inflammatory properties were assessed in subsequent experiments. Key Results Down regulation of inflammatory genes by NOD occurred predominantly for κB regulated genes, however not all κB regulated genes were affected. These findings were explained by inhibition of RelA phosphorylation at Ser276. Leukocyte adherence to TNF-α stimulated HUVECs was inhibited by NOD and was reflected by a diminished expression of adhesion molecules on HUVECs. NOD induced HO-1 expression, but this was not required for inhibition of NFκB. The anti-inflammatory effect of NOD seems to involve the redox active catechol structure, although the redox active para-dihydroxy benzene containing compounds also displayed anti-inflammatory effects, provided that they were sufficiently hydrophobic. Conclusions and Implications The present study highlighted important mechanisms and molecular entities by which dihydroxy benzene compounds exert their potential anti-inflammatory action. Since NOD does not have hemodynamic properties, NOD seems to be a promising candidate drug for the treatment of inflammatory diseases.

Chronic allograft rejection: a fresh look.

Purpose of reviewNew developments suggest that the graft itself and molecules expressed within the graft microenvironment dictate the phenotype and evolution of chronic rejection. Recent findingsOnce ischemia-reperfusion injury, cellular and humoral immune responses target the microvasculature, the associated local tissue hypoxia results in hypoxia-inducible factor 1&agr;-dependent expression of pro-inflammatory and proangiogenic growth factors including vascular endothelial growth factor (VEGF) as a physiological response to injury. Local expression of VEGF can promote the recruitment of alloimmune T cells into the graft. mTOR/Akt signaling within endothelial cells regulates cytokine- and alloantibody-induced activation and proliferation and their proinflammatory phenotype. Inhibition of mTOR and/or Akt results in an anti-inflammatory phenotype and enables the expression of coinhibitory molecules that limit local T cell reactivation and promotes immunoregulation. Semaphorin family molecules may bind to neuropilin-1 on regulatory T cell subsets to stabilize functional responses. Ligation of neuropilin-1 on Tregs also inhibits Akt-induced responses suggesting common theme for enhancing local immunoregulation and long-term graft survival. SummaryEvents within the graft initiated by mTOR/Akt-induced signaling promote the development of chronic rejection. Semaphorin–neuropilin biology represents a novel avenue for targeting this biology and warrants further investigation.

Vitamin D inhibits lymphangiogenesis through VDR-dependent mechanisms

Excessive lymphangiogenesis is associated with cancer progression and renal disease. Attenuation of lymphangiogenesis might represent a novel strategy to target disease progression although clinically approved anti-lymphangiogenic drugs are not available yet. VitaminD(VitD)-deficiency is associated with increased cancer risk and chronic kidney disease. Presently, effects of VitD on lymphangiogenesis are unknown. Given the apparently protective effects of VitD and the deleterious associations of lymphangiogenesis with renal disease, we here tested the hypothesis that VitD has direct anti-lymphangiogenic effects in vitro and is able to attenuate lymphangiogenesis in vivo. In vitro cultured mouse lymphatic endothelial cells (LECs) expressed VitD Receptor (VDR), both on mRNA and protein levels. Active VitD (calcitriol) blocked LEC tube formation, reduced LEC proliferation, and induced LEC apoptosis. siRNA-mediated VDR knock-down reversed the inhibitory effect of calcitriol on LEC tube formation, demonstrating how such inhibition is VDR-dependent. In vivo, proteinuric rats were treated with vehicle or paricalcitol for 6 consecutive weeks. Compared with vehicle-treated proteinuric rats, paricalcitol showed markedly reduced renal lymphangiogenesis. In conclusion, our data show that VitD is anti-lymphangiogenic through VDR-dependent anti-proliferative and pro-apoptotic mechanisms. Our findings highlight an important novel function of VitD demonstrating how it may have therapeutic value in diseases accompanied by pathological lymphangiogenesis.

N-Octanoyl Dopamine Treatment of Endothelial Cells Induces the Unfolded Protein Response and Results in Hypometabolism and Tolerance to Hypothermia

Aim N-acyl dopamines (NADD) are gaining attention in the field of inflammatory and neurological disorders. Due to their hydrophobicity, NADD may have access to the endoplasmic reticulum (ER). We therefore investigated if NADD induce the unfolded protein response (UPR) and if this in turn influences cell behaviour. Methods Genome wide gene expression profiling, confirmatory qPCR and reporter assays were employed on human umbilical vein endothelial cells (HUVEC) to validate induction of UPR target genes and UPR sensor activation by N-octanoyl dopamine (NOD). Intracellular ATP, apoptosis and induction of thermotolerance were used as functional parameters to assess adaptation of HUVEC. Results NOD, but not dopamine dose dependently induces the UPR. This was also found for other synthetic NADD. Induction of the UPR was dependent on the redox activity of NADD and was not caused by selective activation of a particular UPR sensor. UPR induction did not result in cell apoptosis, yet NOD strongly impaired cell proliferation by attenuation of cells in the S-G2/M phase. Long-term treatment of HUVEC with low NOD concentration showed decreased intracellular ATP concentration paralleled with activation of AMPK. These cells were significantly more resistant to cold inflicted injury. Conclusions We provide for the first time evidence that NADD induce the UPR in vitro. It remains to be assessed if UPR induction is causally associated with hypometabolism and thermotolerance. Further pharmacokinetic studies are warranted to address if the NADD concentrations used in vitro can be obtained in vivo and if this in turn shows therapeutic efficacy.

N-Octanoyl dopamine transiently inhibits T cell proliferation via G1 cell-cycle arrest and inhibition of redox-dependent transcription factors

Recently, we developed a nonhemodynamic dopamine derivative, NOD, which has profound anti‐inflammatory effects in vitro. As NOD also protects rats from ischemic AKI, the present study tested whether NOD is able to modulate cellular immunity for potential use as a T cell‐suppressive agent. To this end, T cells were stimulated by anti‐CD3/CD28 or PMA/ionomycin in the presence or absence of different concentrations of NOD. T cell proliferation, activation markers, intracellular cytokine expression, and activation of transcription factors were assessed. Whereas T cell proliferation was inhibited significantly by NOD at Day 3, proliferation was restored at Day 7 or later depending on the NOD concentration used. Inhibition of proliferation was reflected by a diminished CD25 expression and switch from naive to memory T cells. Early TCR activation events were unaffected, yet NF‐κB and AP‐1 were strongly inhibited by NOD. The inhibitory effect of NOD seemed to be dependent on its redox activity, as NOT, a redox‐inactive NOD derivate, did not influence proliferation. NOD displayed synergistic effects with CNIs on T cell proliferation. Our data demonstrate that NOD displays T cell‐suppressive activity. In keeping with its anti‐inflammatory action and its beneficial effect on ischemia‐induced AKI, NOD may be an interesting drug candidate to prevent CNI‐related side‐effects.

N-octanoyl-dopamine is a potent inhibitor of platelet function

Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5′-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.

Simultaneous subcutaneous implantation of two osmotic minipumps connected to a jugular vein catheter in the rat

Subcutaneous osmotic pump implantation connected to a venous catheter is a well-established method for delivering compounds intravenously for an intermediate duration (approximately two weeks). When prolonged release is desired (approximately four weeks) reduced flow rate is needed with a similar pump volume. With a fixed intra-pump compound concentration, reduced flow rate results in unwanted reduced bioavailability of the compound. Prolonged intravenous delivery would therefore need a pump replacement, resulting in increased discomfort and confounding effects on experimental outcome. To overcome this, we describe a method to double the compound infusion rate for four weeks by implanting two low-flow rate osmotic pumps (2.5 µL/h for 28 days) connected to a jugular vein catheter in a single rat. Rats implanted with a single high-flow rate pump (5 µL/h for 14 days) served as controls. Double pump-implanted rats displayed similar post-operative weight gain and physical activity indicating similar levels of discomfort when compared with single pump-implanted rats. Double pump-implanted rats had an increased risk of pump-related complications (four delivery failures [double pump] versus one delivery failure [single pump]). Our data show that double pump implantation is a feasible alternative to changing pumps or the use of extracorporeal pump systems connected via a long wire to partly restrained animals.