Paul Michel Vanhoutte

Lipocalin-2 is not only a sensitive biomarker, but it also contributes to the pathogenesis of renal injuries. The present study demonstrates that adipose tissue–derived lipocalin-2 plays a critical role in causing both chronic and acute renal injuries. Four-week treatment with aldosterone and high salt after uninephrectomy (ANS) significantly increased both circulating and urinary lipocalin-2, and it induced glomerular and tubular injuries in kidneys of WT mice. Despite increased renal expression of lcn2 and urinary excretion of lipocalin-2, mice with selective deletion of lcn2 alleles in adipose tissue (Adipo-LKO) are protected from ANS- or aldosterone-induced renal injuries. By contrast, selective deletion of lcn2 alleles in kidney did not prevent aldosterone- or ANS-induced renal injuries. Transplantation of fat pads from WT donors increased the sensitivity of mice with complete deletion of Lcn2 alleles (LKO) to aldosterone-induced renal injuries. Aldosterone promoted the urinary excretion of a human lipocalin-2 variant, R81E, in turn causing renal injuries in LKO mice. Chronic treatment with R81E triggered significant renal injuries in LKO, resembling those observed in WT mice following ANS challenge. Taken in conjunction, the present results demonstrate that lipocalin-2 derived from adipose tissue causes acute and chronic renal injuries, largely independent of local lcn2 expression in kidney.

Synthetic ion channels may have potential therapeutic applications, provided they possess appropriate biological activities. The present study was designed to examine the ability of small molecule-based synthetic Cl^– channels to modulate airway smooth muscle responsiveness. Changes in isometric tension were measured in rat tracheal rings. Relaxations to the synthetic chloride channel SCC-1 were obtained during sustained contractions to KCl. The anion dependency of the effect of SCC-1 was evaluated by ion substitution experiments. The sensitivity to conventional Cl^– transport inhibitors was also tested. SCC-1 caused concentration-dependent relaxations during sustained contractions to potassium chloride. This relaxing effect was dependent on the presence of extracellular Cl^– and HCO₃⁻. It was insensitive to conventional Cl^– channels/transport inhibitors that blocked the cystic fibrosis transmembrane conductance regulator and calcium-activated Cl^– channels. SCC-1 did not inhibit contractions induced by carbachol, endothelin-1, 5-hydroxytryptamine or the calcium ionophore A23187. SCC-1 relaxes airway smooth muscle during contractions evoked by depolarizing solutions. The Cl^– conductance conferred by this synthetic compound is distinct from the endogenous transport systems for chloride anions.

Vascular complications are an important pathological issue in diabetes that lead to the further functional deterioration of several organs. The balance between endothelium‐dependent relaxing factors and endothelium‐dependent contracting factors (EDCFs) is crucial in controlling local vascular tone and function under normal conditions. Diabetic endothelial dysfunction is characterized by reduced endothelium‐dependent relaxations and/or enhanced endothelium‐dependent contractions. Elevated levels of oxygen‐derived free radicals are the initial source of endothelial dysfunction in diabetes. Oxygen‐derived free radicals not only reduce nitric oxide bioavailability, but also facilitate the production and/or action of EDCFs. Thus, the endothelial balance tips towards vasoconstrictor responses over the course of diabetes.

Genetic and dietary effects on LDL phenotypes, including predominant LDL particle diameter, LDL size distribution, and non–HDL cholesterol and apoB concentrations, were investigated in 150 pedigreed baboons that are members of 19 sire groups. Baboons were fed a sequence of three defined diets differing in levels of fat and cholesterol. Increasing dietary fat had relatively little effect on two measures of LDL particle size. However, increasing the level of cholesterol in the diet resulted in larger increases of the predominant LDL particle diameters and in the proportion of stain on LDLs >28 nm. As expected, apoB and non–HDL cholesterol concentrations significantly increased when levels of dietary fat and cholesterol were increased. Correlations among the LDL phenotypes suggested that several different aspects of the LDL phenotype were captured by the four LDL measures across the three diets. Genetic effects indicated by sire group membership were significant both for expression of the LDL phenotypes and for response to changes in diet.

Background and purpose: To investigate the dysfunction of vascular smooth muscle in streptozotocin‐induced diabetic rats.Experimental approach: Rings without endothelium of femoral arteries were suspended in organ chambers for isometric tension recording. The production of oxygen‐derived free radicals was measured with 2′,7′‐dichlorodihydrofluorescein diacetate using confocal microscopy. The protein expressions were measured by western blotting.Key results: The concentration–response curves to U46619 and phenylephrine, but not that to KCl, were shifted to the left, suggesting a hypersensitivity of cell membrane receptors in diabetes. Exogenous oxygen‐derived free radicals induced greater vasoconstrictions in the femoral artery from diabetic rats. Chronic treatment with apocynin (inhibitor of NADPH oxidase) and acute exposure to MnTMPyP (SOD/catalase mimetic) normalized the response. The catalase activity and the total glutathione level were reduced in arteries from streptozotocin‐treated rats, confirming a redox abnormality. The basal oxidative state was higher in arteries from streptozotocin‐treated rats and reduced in arteries from apocynin‐ and streptozotocin‐treated rats, suggesting that the functional changes in diabetes are due to a chronic increase in oxidative stress. In the arteries of streptozotocin‐treated rats, inhibitors of COX‐1 and/or COX‐2 prevented the hypersensitivity and reduced the increase in oxidative stress caused by phenylephrine and U46619, suggesting that both isoforms contribute to the smooth muscle dysfunction. The expression of proteins for COX‐1 and COX‐2 was increased in arteries of streptozotocin‐treated rats and reduced in preparations of apocynin‐ and streptozotocin‐treated rats.Conclusions and implications: Chronic diabetes and the resulting increased oxidative stress activate the production of COX‐derived vasoconstrictor prostanoids causing hypersensitivity of vascular smooth muscle.British Journal of Pharmacology (2008) 154, 639–651; doi:10.1038/bjp.2008.110; published online 14 April 2008

The present study examined the hypothesis that prostaglandin E₂ (PGE₂) through activation of prostaglandin E (EP) receptor contributes to endothelium-dependent contractions. Western blotting revealed that the protein expression of EP1 receptor was significantly down-regulated in the aorta of the spontaneously hypertensive rat (SHR), but there was no significant difference in the expression of EP2, EP4, and total EP3 receptors between preparations of Wistar Kyoto rats (WKY) and SHR. Isometric tension studies showed that low concentrations of PGE₂ caused endothelium-dependent relaxations in WKY but not in aortas of the SHR. High concentrations of PGE₂ evoked contractions predominately through the activation of thromboxane-prostanoid (TP) receptors in the WKY, but involves the dual activation EP and TP receptors in the SHR. SQ29,548, BAYu3405 and Terutroban (TP receptor antagonists), and AH6809 (non-selective EP receptor antagonist) abolished, while SC19220 (preferential EP1 receptor antagonist) did not inhibit endothelium-dependent contractions. Both SC19220 and AH6809 significantly inhibited contractions to U46619 (TP receptor agonist). The present study demonstrates that the contraction caused by PGE₂ in the SHR aorta is dependent on the activation of EP1 and TP receptors, but that endothelium-dependent contractions do not require the former. Thus, PGE₂ is unlikely to be an endothelium-derived contracting factor in this artery. The ability of AH6809 to inhibit endothelium-dependent contractions can be attributed to its partial antagonism at TP receptors. Nevertheless, the impairment of PGE₂-mediated relaxation may contribute to endothelial dysfunction in the aorta of the SHR.

Background and Purpose: Experiments were designed to assess whether or not the intracellular concentration of calcium and reactive oxygen species (ROS) increase in endothelial cells of the rat thoracic aorta in response to releasers of endothelium‐derived contracting factor (EDCF) and if so, whether or not a difference exists between spontaneously hypertensive (SHR) and normotensive (WKY) rats.Experimental approach: Calcium and ROS were measured by confocal microscopy, using Fura‐red in combination with Fluo‐4 and dichlorodihydrofluorescein diacetate, respectively.Key results: Acetylcholine caused a rapid increase in cytosolic calcium concentration in endothelial cells of both SHR and WKY, which was significantly more pronounced in aortae of the former strain. This rise of calcium was not affected by indomethacin (an inhibitor of cyclooxygenase) or Tiron plus diethyldithiocarbamate acid (DETCA) (membrane permeable antioxidants). In the presence of a nitric oxide synthase blocker, acetylcholine also caused a rapid increase in ROS in endothelial cells of SHR but not in those of WKY. The burst of ROS was prevented by indomethacin or Tiron plus DETCA.Conclusions and implications: These experiments show that endothelial cells of SHR are more prone to calcium and ROS overload upon stimulation with acetylcholine. The abnormal accumulation of calcium is a prerequisite to initiate the release of EDCF and can be mimicked using the calcium ionophore A23187. The sequence of events occurring during endothelium‐dependent contractions firstly requires the accumulation of calcium, which then activates cyclooxygenase and produces ROS along with EDCF that in turn stimulates TP‐receptors, resulting in EDCF‐mediated contractions.British Journal of Pharmacology (2007) 151, 15–23. doi:10.1038/sj.bjp.0707190

Background and purpose: To study the importance of endothelium‐derived contracting factors (EDCFs) in arteries of rats with type I diabetes.Experimental approach: Rat femoral arteries were collected four or twelve weeks after induction of diabetes with streptozotocin. Rings, with or without endothelium, were suspended in organ chambers for isometric tension measurement. COX protein levels were determined by Western blotting.Key results: Four weeks after the injection of streptozotocin, the endothelium‐dependent relaxations (during contractions to phenylephrine) to A23817 were attenuated, but the endothelium‐dependent contractions (quiescent preparations) to the ionophore were augmented. Indomethacin and S18886 prevented the endothelium‐dependent contractions, while dazoxiben reduced them in rings from streptozotocin‐treated rats, suggesting that thromboxane A₂, activating TP‐ receptors, is involved. Twelve weeks after the injection of streptozotocin, the changes in endothelium‐dependent relaxations and contractions to A23187 were even more noticeable. The protein expression of COX‐1 was increased in femoral arteries of the diabetic rats. Valeryl salicylate and SC560 inhibited the contractions, suggesting that the EDCFs are produced by COX‐1. At that time, a combination of S18886 with EP1‐blockers was required to abolish the contractions, suggesting that the EDCFs involved act at both TP‐ and EP‐receptors. Rings without endothelium from streptozotocin‐treated rats exhibited a reduced maximal contraction to potassium chloride and U46619, combined with hyper‐responsiveness to the latter, suggesting that more prolonged diabetes also alters the responsiveness of vascular smooth muscle.Conclusion and Implications: The production of EDCFs is progressively increased in the course of type I diabetes. Eventually, the disease also damages vascular smooth muscle.British Journal of Pharmacology (2007) 150, 624–632. doi:10.1038/sj.bjp.0706999

Preliminary studies suggested that previous exposure to acetylcholine (ACh) exerts a delayed inhibition of subsequent contractions mediated by endothelium-derived contracting factor (EDCF). To confirm this long-term inhibitory effect of ACh and to determine whether nitric oxide (NO) mediates the phenomenon, we suspended rings of spontaneously hypertensive rat (SHR) aortas in organ chambers for the recording of isometric force. The rings were incubated in the absence or presence of N^ω-nitro-l-arginine methyl ester (l-NAME; inhibitor of NO synthases) or 1 H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ; inhibitor of soluble guanylyl cyclase) before exposure to increasing concentrations of ACh or sodium nitroprusside (SNP) during contractions to phenylephrine. Thereafter, EDCF-mediated contractions to ACh or the calcium ionophore A-23187 were elicited. If the rings were preexposed to ACh or SNP, the subsequent ACh-induced EDCF-mediated contractions were reduced compared with those obtained in rings of the same arteries not previously exposed to either agent. ODQ did not affect the inhibition caused by preexposure to ACh but significantly reduced that caused by preexposure to SNP. Previous exposure to SNP reduced, whereas previous exposure to ACh did not affect, endothelium-dependent contractions to A-23187. Previous exposure to either ACh or SNP did not affect the contractions to the thromboxane mimetic U-46619. Thus ACh and SNP exert delayed inhibition of EDCF-mediated contractions via distinct pathways. The effect of ACh is NO independent and upstream of the increase in calcium concentration that triggers the release of EDCF. The effect of SNP is downstream of the calcium rise and is mainly NO dependent.