Publications by authors named "Jeremy Bellien"

57 Publications

Soluble Epoxide Hydrolase Inhibition Prevents Experimental Type 4 Cardiorenal Syndrome.

Front Mol Biosci 2020 11;7:604042. Epub 2021 Mar 11.

Normandie University, UNIROUEN, INSERM U1096, FHU REMOD-VHF, Rouen, France.

Cardiovascular diseases (CVD) remain the leading cause of morbimortality in patients with chronic kidney disease (CKD). The aim of this study was to assess the cardiovascular impact of the pharmacological inhibition of soluble epoxide hydrolase (sEH), which metabolizes the endothelium-derived vasodilatory and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acid (DHETs), in the 5/6 nephrectomy (Nx) mouse model. Compared to sham-operated mice, there was decrease in EET-to-DHET ratio 3 months after surgery in vehicle-treated Nx mice but not in mice treated with the sEH inhibitor -AUCB. Nx induced an increase in plasma creatinine and in urine albumin-to-creatinine ratio as well as the development of kidney histological lesions, all of which were not modified by -AUCB. In addition, -AUCB did not oppose Nx-induced blood pressure increase. However, AUCB prevented the development of cardiac hypertrophy and fibrosis induced by Nx, as well as normalized the echocardiographic indices of diastolic and systolic function. Moreover, the reduction in endothelium-dependent flow-mediated dilatation of isolated mesenteric arteries induced by Nx was blunted by -AUCB without change in endothelium-independent dilatation to sodium nitroprusside. Inhibition of sEH reduces the cardiac remodelling, and the diastolic and systolic dysfunctions associated with CKD. These beneficial effects may be mediated by the prevention of endothelial dysfunction, independent from kidney preservation and antihypertensor effect. Thus, inhibition of sEH holds a therapeutic potential in preventing type 4 cardiorenal syndrome.
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http://dx.doi.org/10.3389/fmolb.2020.604042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991096PMC
March 2021

Preservation of epoxyeicosatrienoic acid bioavailability prevents renal allograft dysfunction and cardiovascular alterations in kidney transplant recipients.

Sci Rep 2021 Feb 12;11(1):3739. Epub 2021 Feb 12.

Department of Pharmacology, Rouen University Hospital, 76000, Rouen, France.

This study addressed the hypothesis that epoxyeicosatrienoic acids (EETs) synthesized by CYP450 and catabolized by soluble epoxide hydrolase (sEH) are involved in the maintenance of renal allograft function, either directly or through modulation of cardiovascular function. The impact of single nucleotide polymorphisms (SNPs) in the sEH gene EPHX2 and CYP450 on renal and vascular function, plasma levels of EETs and peripheral blood monuclear cell sEH activity was assessed in 79 kidney transplant recipients explored at least one year after transplantation. Additional experiments in a mouse model mimicking the ischemia-reperfusion (I/R) injury suffered by the transplanted kidney evaluated the cardiovascular and renal effects of the sEH inhibitor t-AUCB administered in drinking water (10 mg/l) during 28 days after surgery. There was a long-term protective effect of the sEH SNP rs6558004, which increased EET plasma levels, on renal allograft function and a deleterious effect of K55R, which increased sEH activity. Surprisingly, the loss-of-function CYP2C9*3 was associated with a better renal function without affecting EET levels. R287Q SNP, which decreased sEH activity, was protective against vascular dysfunction while CYP2C8*3 and 2C9*2 loss-of-function SNP, altered endothelial function by reducing flow-induced EET release. In I/R mice, sEH inhibition reduced kidney lesions, prevented cardiac fibrosis and dysfunction as well as preserved endothelial function. The preservation of EET bioavailability may prevent allograft dysfunction and improve cardiovascular disease in kidney transplant recipients. Inhibition of sEH appears thus as a novel therapeutic option but its impact on other epoxyfatty acids should be carefully evaluated.
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http://dx.doi.org/10.1038/s41598-021-83274-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881112PMC
February 2021

[Cardiovascular disorders in autosomal dominant polycystic kidney disease].

Nephrol Ther 2021 Feb 8;17(1):18-29. Epub 2021 Jan 8.

Inserm U1096, FHU REMOD-VHF, UniRouen, Normandie Université, 76000 Rouen, France; Service de néphrologie, CHU de Rouen, 76000 Rouen, France. Electronic address:

Autosomal dominant polycystic kidney disease is the most frequent genetic kidney disease. Cardiovascular disorders associated with autosomal dominant polycystic kidney disease are multiple and may occur early in life. In autosomal dominant polycystic kidney disease cardiovascular morbidity and mortality are related both to the nonspecific consequences of chronic kidney disease and to the particular phenotype of autosomal dominant polycystic kidney disease. Compared to the general population, patients with autosomal dominant polycystic kidney disease present an increased prevalence of hypertension, left ventricular hypertrophy, atrial fibrillation, valvular diseases, aneurisms and arterial dissections. This review article provides an update on cardiovascular disorders associated with autosomal dominant polycystic kidney disease and recent pathophysiological developments.
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http://dx.doi.org/10.1016/j.nephro.2020.09.003DOI Listing
February 2021

Evidence for wall shear stress-dependent t-PA release in human conduit arteries: role of endothelial factors and impact of high blood pressure.

Hypertens Res 2021 Mar 18;44(3):310-317. Epub 2020 Sep 18.

Department of Pharmacology, Rouen University Hospital, 76000, Rouen, France.

Tissue plasminogen activator (t-PA) converts plasminogen into the serine protease plasmin, which in turn degrades fibrin clots. This study assessed whether an increase in shear stress is associated in humans in vivo with the release of t-PA in peripheral conduit arteries, the impact of high blood pressure and the role of NO and CYP450-derived epoxyeicosatrienoic acids (EETs). Local t-PA levels were quantified at baseline and during a sustained increase in radial artery wall shear stress induced by hand skin heating (from 34 to 44 °C) in a total of 25 subjects, among whom 8 were newly diagnosed essential hypertensive patients. The impact of the brachial infusion of NO synthase (L-NMMA) and CYP450 inhibitors (fluconazole) on t-PA release was assessed. The increase in shear stress induced by heating was associated with an increase in local t-PA release (from 3.0 ± 0.5 to 19.2 ± 5.5 ng/min, n = 25, P < 0.01). The magnitude of t-PA release was positively correlated with the increase in shear stress (r = 0.64, P < 0.001) and negatively correlated with mean blood pressure (r = -0.443, P = 0.027). These associations persisted after multiple adjustments for confounding factors. Finally, t-PA release was reduced by L-NMMA and to a larger extent by the combination of L-NMMA and fluconazole without a change in shear stress. The increase in wall shear stress in the peripheral conduit arteries induces a release of t-PA by a mechanism involving NO and EETs. The alteration of this response by high blood pressure may contribute to reducing the fibrinolytic potential and enhancing the risk of arterial thrombosis during exercise.
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http://dx.doi.org/10.1038/s41440-020-00554-5DOI Listing
March 2021

The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification.

Int J Mol Sci 2020 Jun 17;21(12). Epub 2020 Jun 17.

Department of Pharmacology, Rouen University Hospital, CEDEX 1, 76031 Rouen, France.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor -4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of -AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by -AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.
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http://dx.doi.org/10.3390/ijms21124313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352784PMC
June 2020

Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Anal Chem 2020 05 27;92(10):7334-7342. Epub 2020 Apr 27.

Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 μg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes.
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http://dx.doi.org/10.1021/acs.analchem.0c01115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744119PMC
May 2020

5/6 nephrectomy induces different renal, cardiac and vascular consequences in 129/Sv and C57BL/6JRj mice.

Sci Rep 2020 01 30;10(1):1524. Epub 2020 Jan 30.

Normandie Univ, UNIROUEN, INSERM U1096, FHU REMOD-VHF, 76000, Rouen, France.

Experimental models of cardiovascular diseases largely depend on the genetic background. Subtotal 5/6 nephrectomy (5/6 Nx) is the most frequently used model of chronic kidney disease (CKD) in rodents. However, in mice, cardiovascular consequences of 5/6 Nx are rarely reported in details and comparative results between strains are scarce. The present study detailed and compared the outcomes of 5/6 Nx in the 2 main strains of mice used in cardiovascular and kidney research, 129/Sv and C57BL/6JRj. Twelve weeks after 5/6 Nx, CKD was demonstrated by a significant increase in plasma creatinine in both 129/Sv and C57BL/6JRj male mice. Polyuria and kidney histological lesions were more pronounced in 129/Sv than in C57BL/6JRj mice. Increase in albuminuria was significant in 129/Sv but not in C57BL/6JRj mice. Both strains exhibited an increase in systolic blood pressure after 8 weeks associated with decreases in cardiac systolic and diastolic function. Heart weight increased significantly only in 129/Sv mice. Endothelium-dependent mesenteric artery relaxation to acetylcholine was altered after 5/6 Nx in C57BL/6JRj mice. Marked reduction of endothelium-dependent vasodilation to increased intraluminal flow was demonstrated in both strains after 5/6 Nx. Cardiovascular and kidney consequences of 5/6 Nx were more pronounced in 129/Sv than in C57BL/6JRj mice.
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http://dx.doi.org/10.1038/s41598-020-58393-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992698PMC
January 2020

Exploring the microvascular impact of red blood cell transfusion in intensive care unit patients.

Crit Care 2019 Aug 30;23(1):292. Epub 2019 Aug 30.

Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Saint-Antoine, Service de Réanimation Médicale, 184 rue du Faubourg Saint-Antoine, 75571, Paris Cedex 12, France.

Background: Red blood cell (RBC) transfusion is a common treatment for hospitalized patients. However, the effects of RBC transfusion on microvascular function remain controversial.

Methods: In a medical ICU in a tertiary teaching hospital, we prospectively included anemic patients requiring RBC transfusion. Skin microvascular reactivity was measured before and 30 min after RBC transfusion. Plasma was collected to analyze intravascular hemolysis and draw the lipidomic and cytokine profiles.

Results: In a cohort of 59 patients, the median age was 66 [55-81] years and SAPS II was 38 [24-48]. After RBC transfusion, endothelium-dependent microvascular reactivity improved in 35 (59%) patients, but worsened in 24 others (41%). Comparing clinical and biological markers revealed that baseline blood leucokyte counts distinguished improving from worsening patients (10.3 [5.7; 19.7] vs. 4.6 [2.1; 7.3] × 10/L; p = 0.001) and correlated with variations of microvascular reactivity (r = 0.36, p = 0.005). Blood platelet count was also higher in improving patients (200 [97; 280] vs 160 [40; 199] × 10/mL, p = 0.03) but did not correlate with variations of microvascular reactivity. We observed no intravascular hemolysis (HbCO, heme, bilirubin, LDH), but recorded a significant increase in RBC microparticle levels specific to improving patients after transfusion (292 [108; 531] vs. 53 [34; 99] MP/μL; p = 0.03). The improvement in microvascular dilation was positively correlated with RBC microparticle levels (R = 0.83, p < 0.001) and conversion of arachidonic acid into vasodilating eicosanoids.

Conclusions: Patients displaying an improved microvascular reactivity after RBC transfusion had high blood leukocyte counts, increased RBC microparticle formation, and enhanced metabolism of arachidonic acid into vasodilating lipids. Our data suggested a contribution of recipient leukocytes to the vascular impact of RBC transfusion.
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http://dx.doi.org/10.1186/s13054-019-2572-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717366PMC
August 2019

Discovery of the First in Vivo Active Inhibitors of the Soluble Epoxide Hydrolase Phosphatase Domain.

J Med Chem 2019 09 17;62(18):8443-8460. Epub 2019 Sep 17.

Institute of Pharmaceutical Chemistry , Goethe-University Frankfurt , Max-von-Laue-Strasse 9 , 60438 Frankfurt am Main , Germany.

The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262789PMC
September 2019

Impact of the acute local inhibition of soluble epoxide hydrolase on diabetic skin microcirculatory dysfunction.

Diab Vasc Dis Res 2019 11 3;16(6):523-529. Epub 2019 Jul 3.

Department of Pharmacology, Rouen University Hospital, Rouen, France.

The impact of the local inhibition of soluble epoxide hydrolase, which metabolizes vasodilator and anti-inflammatory epoxyeicosanoids, on diabetic skin microvascular dysfunction was assessed. In diabetic db/db mice, basal skin blood flow assessed using laser Doppler imaging was similar to that of control mice, but thermal hyperemia was markedly reduced. At 2 h after the topical administration of an aqueous gel containing the soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB: 400 mg/L), the peak concentration of t-AUCB was detected in the skin of diabetic mice, which quickly decreased thereafter. In parallel, 2 h after application of t-AUCB treatment, thermal hyperemia was increased compared to the control gel. Quantification of t-AUCB in plasma of treated animals showed no or low systemic diffusion. Furthermore, haematoxylin and eosin histological staining of skin biopsies showed that skin integrity was preserved in t-AUCB-treated mice. Finally, for pig ear skin, a surrogate for human skin, using Franz diffusion cells, we observed a continuous diffusion of t-AUCB from 2 h after application to beyond 24 h. A single topical administration of a soluble epoxide hydrolase inhibitor improves microcirculatory function in the skin of db/db mice and might represent a new therapeutic approach for preventing the development of skin complications in diabetic patients.
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http://dx.doi.org/10.1177/1479164119860215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307659PMC
November 2019

Endothelium-dependent adaptation of arterial wall viscosity during blood flow increase is impaired in essential hypertension.

Atherosclerosis 2019 06 8;285:102-107. Epub 2019 Apr 8.

Rouen University Hospital, Department of Pharmacology, F 76000, Rouen, France; Normandie Univ, UNIROUEN, Inserm U1096, F 76000, Rouen, France; University of Rouen, Institute for Research and Innovation in Biomedicine, Rouen, France; Clinical Investigation Center CIC-CRB 1404, Rouen University Hospital, Rouen, France.

Background And Aims: Arterial wall viscosity (AWV) is regulated by endothelium-derived NO and epoxyeicosatrienoic acids (EETs) under baseline physiological conditions. Whether these factors regulate AWV during blood flow increase and whether this mechanism is affected in essential hypertensive patients (HT) remain unknown.

Methods: The evolution of radial artery diameter, wall thickness and arterial pressure in response to an increase in flow induced by hand skin heating were measured in 18 untreated HT and 14 normotensive controls (NT) during local infusion of saline and the respective pharmacological inhibitors of NO-synthase and EETs synthesis by cytochrome P450, L-NMMA and/or fluconazole. AWV was estimated by the ratio of the viscous energy dissipated (W) to the elastic energy stored (W) obtained from the pressure-diameter relationship. Concomitant changes in operating conditions, which influence the AWV, were taken into account by calculating the midwall stress.

Results: Baseline W and W were higher in HT than in NT but W/W was similar. In saline condition, W/W increased in HT during heating but not in NT. In the presence of L-NMMA and/or fluconazole, W/W increased during heating in NT. In contrast, these inhibitors did not modify the increase in W/W during heating in HT compared to saline. In all conditions, a larger increase in W than W was responsible for the increase in W/W.

Conclusions: The release of NO and EETs maintains a stable AWV during flow increase and this endothelial adaptive regulation is lost during essential hypertension, which may promote excessive viscous energy dissipation and cardiovascular uncoupling. Restoration of EETs availability with inhibitors of soluble epoxide hydrolase could thus constitute a promising pharmacological approach to restore the endothelial adaptive regulation of AWV.
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http://dx.doi.org/10.1016/j.atherosclerosis.2019.04.208DOI Listing
June 2019

[Diuretic therapy].

Rev Prat 2019 Jan;69(1):e19-e27

Service de pharmacologie, unité de pharmacologie clinique, CHU-Hôpitaux de Rouen, UMR Inserm 1096, université de Rouen-Normandie, 76000 Rouen, France.

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January 2019

Altered bioavailability of epoxyeicosatrienoic acids is associated with conduit artery endothelial dysfunction in type 2 diabetic patients.

Cardiovasc Diabetol 2019 03 18;18(1):35. Epub 2019 Mar 18.

Department of Pharmacology, Rouen University Hospital, 76000, Rouen Cedex, France.

Background: This pathophysiological study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetes.

Methods And Results: Radial artery endothelium-dependent flow-mediated dilatation in response to hand skin heating was reduced in essential hypertensive patients (n = 9) and type 2 diabetic subjects with (n = 19) or without hypertension (n = 10) compared to healthy subjects (n = 36), taking into consideration cardiovascular risk factors, flow stimulus and endothelium-independent dilatation to glyceryl trinitrate. Diabetic patients but not non-diabetic hypertensive subjects displayed elevated whole blood reactive oxygen species levels and loss of NO release during heating, assessed by measuring local plasma nitrite variation. Moreover, plasma levels of EET regioisomers increased during heating in healthy subjects, did not change in hypertensive patients and decreased in diabetic patients. Correlation analysis showed in the overall population that the less NO and EETs bioavailability increases during heating, the more flow-mediated dilatation is reduced. The expression and activity of sEH, measured in isolated peripheral blood mononuclear cells, was elevated in diabetic but not hypertensive patients, leading to increased EETs conversion to DHETs. Finally, hyperglycemic and hyperinsulinemic euglycemic clamps induced a decrease in flow-mediated dilatation in healthy subjects and this was associated with an altered EETs release during heating.

Conclusions: These results demonstrate that an increased EETs degradation by sEH and altered NO bioavailability are associated with conduit artery endothelial dysfunction in type 2 diabetic patients independently from their hypertensive status. The hyperinsulinemic and hyperglycemic state in these patients may contribute to these alterations. Trial registration NCT02311075. Registered December 8, 2014.
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http://dx.doi.org/10.1186/s12933-019-0843-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423843PMC
March 2019

Endothelium structure and function in kidney health and disease.

Nat Rev Nephrol 2019 02;15(2):87-108

INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.

The kidney harbours different types of endothelia, each with specific structural and functional characteristics. The glomerular endothelium, which is highly fenestrated and covered by a rich glycocalyx, participates in the sieving properties of the glomerular filtration barrier and in the maintenance of podocyte structure. The microvascular endothelium in peritubular capillaries, which is also fenestrated, transports reabsorbed components and participates in epithelial cell function. The endothelium of large and small vessels supports the renal vasculature. These renal endothelia are protected by regulators of thrombosis, inflammation and complement, but endothelial injury (for example, induced by toxins, antibodies, immune cells or inflammatory cytokines) or defects in factors that provide endothelial protection (for example, regulators of complement or angiogenesis) can lead to acute or chronic renal injury. Moreover, renal endothelial cells can transition towards a mesenchymal phenotype, favouring renal fibrosis and the development of chronic kidney disease. Thus, the renal endothelium is both a target and a driver of kidney and systemic cardiovascular complications. Emerging therapeutic strategies that target the renal endothelium may lead to improved outcomes for both rare and common renal diseases.
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http://dx.doi.org/10.1038/s41581-018-0098-zDOI Listing
February 2019

Alteration in the availability of epoxyeicosatrienoic acids contributes with NO to the development of endothelial dysfunction in conduit arteries during aging.

Atherosclerosis 2018 08 19;275:239-245. Epub 2018 Jun 19.

Department of Pharmacology, CHU de Rouen, 76000, Rouen, France; INSERM U1096, Normandie University, UNIROUEN, 76000, Rouen, France; Centre d'Investigation Clinique (CIC)-INSERM 1404, CHU de Rouen, 76000, Rouen, France. Electronic address:

Background And Aims: The mechanisms involved in endothelial dysfunction in humans during aging are largely unknown at the level of conduit arteries. We aimed to asses the role of NO and CYP450 epoxygenases-derived epoxyeicosatrienoic acids (EETs) in the regulation of endothelium-dependent flow-mediated dilatation of conduit arteries during aging.

Methods: Radial artery diameter and mean wall shear stress were determined by echotracking coupled with Doppler in 83 subjects (19-71 years old) during a sustained flow increase induced by hand skin heating, with the brachial infusion of saline or NO-synthase and cytochrome P450 epoxygenase inhibitors (L-NNMA and fluconazole respectively). Local blood sampling was performed for the quantification of NO metabolite nitrite and EETs.

Results: The magnitude of flow-mediated dilatation was independently and negatively correlated with age, baseline artery diameter and systolic blood pressure, and positively correlated with the increase in shear stress induced by heating. There was an increase in nitrite level during heating until the age of 35-40 years, which declined thereafter. However, the inhibitory effect of L-NMMA on flow-mediated dilatation progressively decreased during aging, demonstrating a decrease in functional NO availability. Moreover, aging progressively reduced the increase in EET level during heating as well as the inhibitory effect of fluconazole on flow-mediated dilatation.

Conclusions: These results show that aging impairs the availability of EETs and NO and epoxyeicosatrienoic acids in peripheral conduit arteries, contributing to the development of endothelial dysfunction.
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http://dx.doi.org/10.1016/j.atherosclerosis.2018.06.865DOI Listing
August 2018

Possible role of CYP2B6 genetic polymorphisms in ifosfamide-induced encephalopathy: report of three cases.

Fundam Clin Pharmacol 2018 Jun 25;32(3):337-342. Epub 2018 Jan 25.

Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, F 76000, Rouen, France.

Ifosfamide (IFA) is a potent alkylating antitumoral agent, but its use is limited by neurological side effects. IFA is a racemic mixture of two enantiomeric forms, R-IFA and S-IFA with a stereoselective metabolism by CYP3A4 and CYP2B6, leading either to bioactive or to toxic pathways. In three consecutive cases of pediatric patients who exhibited IFA-induced encephalopathy (IIE), genotyping of clinically relevant single-nucleotide polymorphisms associated with decreased CYP3A4 and CYP2B6 activities was performed. Genetic investigations revealed the presence of CYP2B6 rs4803419 (C>T) in one patient while the two others carried the CYP2B6*6 allelic variant. All patients carried CYP3A4 wild-type genotype (CYP3A4*1/*1). Because CYP2B6-deficient alleles may be responsible for an increased conversion of S-IFA into neurotoxic metabolites, screening for CYP2B6 polymorphisms may help to avoid IIE and improve clinical outcomes.
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http://dx.doi.org/10.1111/fcp.12345DOI Listing
June 2018

Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation.

Sci Rep 2017 11 29;7(1):16554. Epub 2017 Nov 29.

Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Université de Nantes, Nantes, France.

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.
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http://dx.doi.org/10.1038/s41598-017-16328-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707420PMC
November 2017

Evidence for a Role of Vascular Endothelium in the Control of Arterial Wall Viscosity in Humans.

Hypertension 2018 01 20;71(1):143-150. Epub 2017 Nov 20.

From the Department of Pharmacology, Rouen University Hospital, France (F.R., M.I., J.B., R.J.); Inserm U1096, Normandie Univ, UNIROUEN, France (F.R., M.I., I.R.-J., J.B., R.J.); Institute for Research and Innovation in Biomedicine, University of Rouen, France (F.R., I.R.-J., J.B., R.J.); and Clinical Investigation Center CIC-CRB 1404, Rouen University Hospital, France (F.R., J.B., R.J.).

Arterial wall viscosity (AWV) is a major cause of energy dissipation along the arterial tree. Its determinants remain controversial but an active endothelial regulation has been suggested. Our objective was to assess in humans the physiological role of endothelium-derived nitric oxide (NO), epoxyeicosatrienoic acids and the effect of modulating smooth muscle tone in the regulation of AWV. We simultaneously measured radial artery diameter, wall thickness, and arterial pressure in healthy volunteers during the local infusion of inhibitors of NO-synthase (-monomethyl-l-arginine), epoxyeicosatrienoic acids synthesis by cytochrome P450 (fluconazole), the epoxyeicosatrienoic acids cellular targets calcium-activated potassium channels (tetraethylammonium), alone and in combination. AWV was estimated from the relative viscosity expressed as the ratio of the area of the hysteresis loop of the pressure-diameter relationship to the area under the loading phase. Arterial tone was assessed by measuring change in wall stiffness and midwall stress. -monomethyl-l-arginine paradoxically reduced relative viscosity (34.9±8.9%-28.9±8.6%). Conversely, relative viscosity was not modified by fluconazole (33.5±15.5%-32.0±13.6%) but increased by tetraethylammonium (31.7±6.6%-35.7±8.0%). This increase was more marked with -monomethyl-l-arginine+fluconazole (31.1±10.7%-43.3±13.2%) and -monomethyl-l-arginine+tetraethylammonium (29.5±2.3%-41.5±11.1%) compared with inhibitors alone. Sodium nitroprusside decreased AWV (35.4±2.9%-28.7±2.0%). These effects were associated with parallel change in tone but of different magnitude for similar variations in viscosity, suggesting tone-dependent and independent mechanisms. In conclusion, this is the first demonstration that the endothelial factors, NO and epoxyeicosatrienoic acids, regulate AWV in humans and support the role of arterial tone in this regulation.

Clinical Trial Registration: URL: https://eudract.ema.europa.eu. Unique identifier: RCB2007-A001-10-53.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.117.09870DOI Listing
January 2018

Letter by Bellien and Kamel Regarding Article, "Dipeptidyl Peptidase-4 Induces Aortic Valve Calcification by Inhibiting Insulin-Like Growth Factor-1 Signaling in Valvular Interstitial Cells".

Circulation 2017 10;136(17):1670-1671

From Institut National de la Santé et de la Recherche Biomédicale (INSERM) U1096, FHU-REMOD-VHF, Normandy University, UNIROUEN, Rouen, France (J.B.); Department of Pharmacology, Rouen University Hospital, France (J.B.); Centre de Recherche Universitaire en Santé, INSERM U1088, FHU-REMOD-VHF, Université de Picardie Jules Verne, Amiens, France (S.K.); and Centre de Biologie Humaine, Centre Hospitalier Universitaire Sud Amiens-Picardie, France (S.K.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.029883DOI Listing
October 2017

Impact of CYP3A4 Genotype on Voriconazole Exposure.

Clin Pharmacol Ther 2018 02 29;103(2):185-186. Epub 2017 Aug 29.

Rouen University Hospital, Department of Pharmacology - Toxicology and Pharmacogenetics, Rouen, France.

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http://dx.doi.org/10.1002/cpt.809DOI Listing
February 2018

Multiple and Opposite Effects of Angiotensin II Receptor Blockers on the Bioavailability of Epoxyeicosatrienoic Acids.

Basic Clin Pharmacol Toxicol 2017 10 22;121(4):213-214. Epub 2017 Jun 22.

Department of Pharmacology & INSERM U1096, Rouen University Hospital, Normandie University, Rouen, France.

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http://dx.doi.org/10.1111/bcpt.12810DOI Listing
October 2017

Physiological role of endothelin-1 in flow-mediated vasodilatation in humans and impact of cardiovascular risk factors.

J Hypertens 2017 06;35(6):1204-1212

aDepartment of Pharmacology, Rouen University HospitalbInstitut National de la Santé et de la Recherche Médicale (INSERM) U1096cInstitute for Research and Innovation in Biomedicine, Normandy University, University of RouendCentre d'Investigation Clinique (CIC)-INSERM 1404, Rouen University HospitaleEquipe d'Accueil (EA) 4651, Rouen, France.

Objectives: The current study addressed the hypothesis that the local decrease in endothelin-1 (ET-1) bioavailability during sustained flow increases contributes to endothelium-dependent, flow-mediated dilatation (FMD) of conduit arteries and is altered in presence of cardiovascular risk factors.

Methods And Results: In nine young healthy individuals, the decrease in local ET-1 plasma levels and radial artery FMD in response to hand skin heating (from 34 to 44 °C) was not affected by endothelin type A (ETA) receptor blockade, achieved using the brachial infusion of BQ-123 (100 nmol/min per l of forearm), as compared with physiological saline (0.9% NaCl) infusion. In contrast, endothelin type B (ETB) receptor blockade with BQ-788 (10 nmol/min per l) suppressed the decrease in plasma ET-1 during heating and reduced FMD, without altering nitric oxide release. The coinfusion of BQ-123 did not affect the inhibitory effect of ETB receptor blockade on the decrease in ET-1 plasma levels during heating but prevented the reduction in FMD. Basal radial artery parameters, systemic hemodynamics, and endothelium-independent dilatation to glyceryl trinitrate were not modified by ETA and/or ETB blockade. In a general population of 40 participants without treatment or major cardiovascular diseases, including the nine healthy individuals, the reduction in endothelin-1 level during heating was correlated with FMD (r = -0.55, P < 0.001) and decreased with increased age (r = 0.49, P = 0.001), mean arterial blood pressure (r = 0.48, P = 0.002), and total cholesterol level (r = 0.37, P = 0.024).

Conclusion: The uptake of endothelin-1 by ETB receptors contributes to conduit artery FMD, preventing its vasoconstrictor action mediated by ETA receptors. The alteration of this mechanism by cardiovascular risk factors may contribute to endothelial dysfunction.
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http://dx.doi.org/10.1097/HJH.0000000000001307DOI Listing
June 2017

A sensitive LC-MS/MS method for the quantification of regioisomers of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in human plasma during endothelial stimulation.

Anal Bioanal Chem 2017 Mar 15;409(7):1845-1855. Epub 2016 Dec 15.

Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.

Epoxyeicosatrienoic acids (EETs) are vasodilating lipid mediators metabolized into dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. We aimed to develop a LC-MS/MS method to quantify EETs and DHETs in human plasma and monitored their levels during vascular endothelial stimulation. Plasma samples, collected from 14 healthy and five hypertensive subjects at baseline and during radial artery endothelium-dependent flow-mediated dilatation, were spiked with internal standards. Lipids were then extracted by a modified Bligh and Dyer method and saponified to release bound EETs and DHETs. Samples were purified by a second liquid-liquid extraction and analyzed by LC-MS/MS. The assay allowed identification of (±)8(9)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-EET); (±)11(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-EET); (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET); (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DHET); (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DHET); and (±)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DHET). (±)5(6)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (5,6-EET) was virtually undetectable due to its chemical instability. The limits of quantification were 0.25 ng/mL for DHETs and 0.5 ng/mL for EETs. Intra- and inter-assay variations ranged from 1.6 to 13.2%. Heating induced a similar increase in 8,9-EET, 11,12-EET, and 14,15-EET levels and in corresponding DHET levels in healthy but not in hypertensive subjects. We validated a sensitive LC-MS/MS method for measuring simultaneously plasma EET and DHET regioisomers in human plasma and showed its interest for assessing endothelial function.
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http://dx.doi.org/10.1007/s00216-016-0129-1DOI Listing
March 2017

Modulating putative endothelial progenitor cells for the treatment of endothelial dysfunction and cardiovascular complications in diabetes.

Pharmacol Ther 2017 02 20;170:98-115. Epub 2016 Oct 20.

Department of Pharmacology, Rouen University Hospital, Rouen, France; Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, Rouen, France; University of Rouen, Institute for Research and Innovation in Biomedicine, Rouen, France. Electronic address:

Diabetes induces a decrease in the number and function of different pro-angiogenic cell types generically designated as putative endothelial progenitor cells (EPC), which encompasses cells from myeloid origin that act in a paracrine fashion to promote angiogenesis and putative "true" EPC that contribute to endothelial replacement. This not only compromises neovasculogenesis in ischemic tissues but also impairs, at an early stage, the reendotheliziation process at sites of injury, contributing to the development of endothelial dysfunction and cardiovascular complications. Hyperglycemia, insulin resistance and dyslipidemia promote putative EPC dysregulation by affecting the SDF-1/CXCR-4 and NO pathways and the p53/SIRT1/p66Shc axis that contribute to their mobilization, migration, homing and vasculogenic properties. To optimize the clinical management of patients with hypoglycemic agents, statins and renin-angiotensin system inhibitors, which display pleiotropic effects on putative EPC, is a first step to improve their number and angiogenic potential but specific strategies are needed. Among them, mobilizing therapies based on G-CSF, erythropoietin or CXCR-4 antagonism have been developed to increase putative EPC number to treat ischemic diseases with or without prior cell isolation and transplantation. Growth factors, genetic and pharmacological strategies are also evaluated to improve ex vivo cultured EPC function before transplantation. Moreover, pharmacological agents increasing in vivo the bioavailability of NO and other endothelial factors demonstrated beneficial effects on neovascularization in diabetic ischemic models but their effects on endothelial dysfunction remain poorly evaluated. More experiments are warranted to develop orally available drugs and specific agents targeting p66Shc to reverse putative EPC dysfunction in the expected goal of preventing endothelial dysfunction and diabetic cardiovascular complications.
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http://dx.doi.org/10.1016/j.pharmthera.2016.10.014DOI Listing
February 2017

Impact of soluble epoxide hydrolase inhibition on early kidney damage in hyperglycemic overweight mice.

Prostaglandins Other Lipid Mediat 2015 Jul 27;120:148-54. Epub 2015 May 27.

Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, Rouen, France; University of Rouen, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France; Department of Pharmacology, Rouen University Hospital, Rouen, France. Electronic address:

This study addressed the hypothesis that inhibition of the EETs degrading enzyme soluble epoxide hydrolase affords renal protection in the early stage of diabetic nephropathy. The renal effects of the sEH inhibitor t-AUCB (10mg/l in drinking water) were compared to those of the sulfonylurea glibenclamide (80mg/l), both administered for 8 weeks in FVB mice subjected to a high-fat diet (HFD, 60% fat) for 16 weeks. Mice on control chow diet (10% fat) and non-treated HFD mice served as controls. Compared with non-treated HFD mice, HFD mice treated with t-AUCB had a decreased EET degradation, as shown by their higher plasma EETs-to-DHETs ratio, and an increased EET production, as shown by the increase in EETs+DHETs levels, which was associated with induction of CYP450 epoxygenase expression. Both agents similarly reduced fasting glycemia but only t-AUCB prevented the increase in the urinary albumine-to-creatinine ratio in HFD mice. Histopathological analysis showed that t-AUCB reduced renal inflammation, which was associated with an increased mRNA expression of the NFκB inhibitor Iκ≡ and related decrease in MCP-1, COX2 and VCAM-1 expressions. Finally, there was a marginally significant increase in reactive oxygen species production in HFD mice, together with an enhanced NOX2 expression. Both agents did not modify these parameters but t-AUCB increased the expression of the antioxidant enzyme superoxide dismutase 1. These results demonstrate that, independently from its glucose-lowering effect, sEH inhibition prevents microalbuminuria and renal inflammation in overweight hyperglycemic mice, suggesting that this pharmacological strategy could be useful in the management of diabetic nephropathy.
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http://dx.doi.org/10.1016/j.prostaglandins.2015.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575616PMC
July 2015

The authors reply.

Kidney Int 2015 Apr;87(4):857

Department of Pharmacology & INSERM U1096, Rouen University Hospital, Rouen, France.

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http://dx.doi.org/10.1038/ki.2014.421DOI Listing
April 2015

Soluble epoxide hydrolase inhibition improves coronary endothelial function and prevents the development of cardiac alterations in obese insulin-resistant mice.

Am J Physiol Heart Circ Physiol 2015 May 27;308(9):H1020-9. Epub 2015 Feb 27.

Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, Rouen, France; University of Rouen, Institute for Research and Innovation in Biomedicine, Rouen, France; Department of Pharmacology, Rouen University Hospital, Rouen, France

This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in insulin resistance, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 10 mg/l in drinking water) were compared with those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 wk in FVB mice subjected to a high-fat diet (HFD; 60% fat) for 16 wk. Mice on control chow diet (10% fat) and nontreated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice, but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids, and LDL cholesterol and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the nitric oxide synthase and cytochrome P-450 epoxygenase inhibitors l-NA and MSPPOH on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis, and inflammation and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the end-diastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that sEH inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese insulin-resistant mice.
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http://dx.doi.org/10.1152/ajpheart.00465.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551118PMC
May 2015

Editorial: renal endothelial dysfunction: evolving concepts and perspectives.

Cardiovasc Hematol Disord Drug Targets 2014 ;14(1):1-2

Department of Nephrology Rouen University Hospital 1 rue de Germont, 76031 Rouen France.

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http://dx.doi.org/10.2174/1871529x1401140724093325DOI Listing
March 2015