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.
Cytochrome-derived epoxyeicosatrienoic acids may be important endothelium-derived hyperpolarizing factors, opening calcium-activated potassium channels, but their involvement in the regulation of ...arterial stiffness during changes in blood flow in humans is unknown. In healthy volunteers, we measured arterial pressure, radial artery diameter, wall thickness, and flow (NIUS02) during hand skin heating in the presence of saline or inhibitors of NO synthase (N(G)-monomethyl-L-arginine), calcium-activated potassium channels (tetraethylammonium), and cytochrome epoxygenases (fluconazole). Arterial compliance and elastic modulus were calculated and fitted as functions of midwall stress to suppress the confounding influence of geometric changes. Under saline infusion, heating induced an upward shift of the compliance-midwall stress curve and a downward shift of the modulus-midwall stress curve demonstrating a decrease in arterial tone and stiffness when blood flow increases. These shifts were reduced by N(G)-monomethyl-L-arginine and abolished by the combinations of N(G)-monomethyl-L-arginine+tetraethylammonium and N(G)-monomethyl arginine+fluconazole. In parallel, in isolated mice coronary arteries, fluconazole and tetraethylammonium reduced the relaxations to acetylcholine. However, fluconazole did not affect the relaxations to the openers of calcium-activated potassium channels of small- and intermediate-conductance NS309 and of large-conductance NS1619 excluding a direct effect on these channels. Moreover, tetraethylammonium reduced the relaxations to NS1619 but not to NS309, suggesting that the endothelium-derived hyperpolarizing factor involved mainly acts on large-conductance calcium-activated potassium channels. These results show in humans that, during flow variations, arterial stiffness is regulated by the endothelium through the release of both NO and cytochrome-related endothelium-derived hyperpolarizing factor.
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.
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.
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.
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.
Red blood cell (RBC) transfusion is a common treatment for hospitalized patients. However, the effects of RBC transfusion on microvascular function remain controversial.
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.
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.
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.
Mainly constituted of glycosaminoglycans and proteoglycans, the glycocalyx is anchored in the plasma membrane, covering, in particular, the extracellular face of the arterial endothelium. Due to its ...complex three-dimensional (3D) architecture, the glycocalyx interacts with a wide variety of proteins, contributing to vascular permeability, the flow of mechanotransduction, and the modulation of local inflammatory processes. Alterations of glycocalyx structure mediate the endothelial dysfunction and contribute to the aggravation of peripheral vascular diseases. Therefore, the exploration of its ultrastructure becomes a priority to evaluate the degree of injury under physiopathological conditions and to assess the impact of therapeutic approaches. The objective of this study was to develop innovative approaches in electron microscopy to visualize the glycocalyx at the subcellular scale. Intravenous perfusion on rats with a fixing solution containing aldehyde fixatives enriched with lanthanum ions was performed to prepare arterial samples. The addition of lanthanum nitrate in the fixing solution allowed the enhancement of the staining of the glycocalyx for transmission electron microscopy (TEM) and to detect elastic and inelastic scattered electrons, providing complementary qualitative information. The strength of scanning electron microscopy (SEM) was used on resin-embedded serial sections, allowing rapid and efficient large field imaging and previous correlative TEM observations for ultrastructural fine details. To demonstrate the dynamic feature of the glycocalyx, 3D tomography was provided by dual-beam focus-ion-beam-SEM (FIB-SEM). These approaches allowed us to visualize and characterize the ultrastructure of the pulmonary artery glycocalyx under physiological conditions and in a rat pulmonary ischemia-reperfusion model, known to induce endothelial dysfunction. This study demonstrates the feasibility of combined SEM, TEM, and FIB-SEM tomography approaches on the same sample as the multiscale visualization and the identification of structural indicators of arterial endothelial glycocalyx integrity.
Endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive factors. Besides nitric oxide (NO) and prostacyclin, increasing evidences show that ...endothelium‐derived hyperpolarizing factors (EDHF) participate in the control of vasomotor tone through the activation of calcium‐activated potassium channels. In humans, the role of EDHF has been demonstrated in various vascular beds including coronary, peripheral, skin and venous vessels. The mechanisms of EDHF‐type relaxations identified in humans involved the release by the endothelium of hydrogen peroxide, epoxyeicosatrienoic acids (EETs), potassium ions and electronical communication through the gap junctions. The role of EETs could be particularly important because, in addition contributing to the maintenance of the basal tone and endothelium‐dependent dilation of conduit arteries, these factors share many vascular protective properties of NO. The alteration of which might be involved in the physiopathology of cardiovascular diseases. The evolution of EDHF availability in human pathology is currently under investigation with some results demonstrating an increase in EDHF release to compensate the loss of NO synthesis and to maintain the endothelial vasomotor function whereas others reported a parallel decrease in NO and EDHF‐mediated relaxations. Thus, the modulation of EDHF activity emerges as a new pharmacological target and some existing therapies in particular those affecting the renin–angiotensin system have already been shown to improve endothelial function through hyperpolarizing mechanisms. In this context, the development of new specific pharmacological agents especially those increasing EETs availability may help to prevent endothelial dysfunction and therefore enhance cardiovascular protection in patients.
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