Effects of dipeptidyl peptidase 4 inhibition on the endothelial control of the vascular tone.

Dipeptidyl peptidase 4 (DPP4) is a serine protease known to cleave incretin hormones, which stimulate insulin secretion after food intake, a fact that supported the development of its inhibitors (DPP4i or gliptins) for the treatment of type 2 diabetes mellitus. In addition to their glucose-lowering effects, DPP4i show benefits for the cardiovascular system that could be related, at least in part, to their protective action on vascular endothelium. DPP4i have been associated with the reversal of endothelial dysfunction, an important predictor of cardiovascular events and a hallmark of diseases such as atherosclerosis, diabetes mellitus, hypertension, and heart failure. In animal models of these diseases, DPP4i increase nitric oxide bioavailability and limits oxidative stress, thereby improving the endothelium-dependent relaxation. Similar effects on flow-mediated dilation and attenuation of endothelial dysfunction have also been noted in human studies, suggesting a value for gliptins in the clinical scenario, despite the variability of the results regarding the DPP4i used, treatment duration, and presence of comorbidities. In this mini-review, we discuss the advances in our comprehension of the DPP4i effects on endothelial regulation of vascular tone. Understanding the role of DPP4 and its involvement in the signaling mechanisms leading to endothelial dysfunction will pave the way for a broader use of DPP4i in conditions that endothelial dysfunction is a pivotal pathophysiological player.

Iron overload, oxidative stress and vascular dysfunction: Evidences from clinical studies and animal models.

Although iron is a metal involved in many vital processes due to its redox capacity, body iron overloads lead to tissue damage, including the cardiovascular system. While cardiomyopathy was the focus since the 1960s, the impact on the vasculature was comparatively neglected for about 40 years, when clinical studies correlating iron overload, oxidative stress, endothelial dysfunction, arterial stiffness and atherosclerosis reinforced an "iron hypothesis". Due to controversial results from some epidemiological studies investigating atherosclerotic events and iron levels, well-controlled trials and animal studies provided essential data about the influence of iron, per se, on the vasculature. As a result, the pathophysiology of vascular dysfunction in iron overload have been revisited. This review summarizes the knowledge obtained from epidemiological studies, animal models and "in vitro" cellular systems in recent decades, highlighting a more harmful than innocent role of iron excess for the vascular homeostasis, which supports our proposal to hereafter denominate "iron overload vasculopathy". Additionally, evidence-based potential therapeutic targets are pointed out to be tested in pre-clinical research that may be useful in cardiovascular protection for patients with iron overload syndromes.

Changes in the renal function after acute mercuric chloride exposure in the rat are associated with renal vascular endothelial dysfunction and proximal tubule NHE3 inhibition.

Mercury is an environmental pollutant and a threat to human health. Mercuric chloride (HgCl2)-induced acute renal failure has been described by several reports, but the mechanisms of renal dysfunction remain elusive. This study tested the hypothesis that HgCl2 directly impairs renal vascular reactivity. Additionally, due to the mercury toxicity on the proximal tubule, we investigated whether the HgCl2-induced natriuresis is accompanied by inhibition of Na+/H+ exchanger isoform-3 (NHE3). We found that 90-min HgCl2 infusion (6.5 µg/kg i.v.) remarkably increased urinary output, reduced GFR and renal blood flow, and increased vascular resistance in rats. "In vitro" experiments of HgCl2 infusion in isolated renal vascular bed demonstrated an elevation of perfusion pressure in a concentration- and time-dependent manner, associated with changes on the endothelium-dependent vasodilatation and the flow-pressure relationship. Moreover, by employing "in vivo" stationary microperfusion of the proximal tubule, we found that HgCl2 inhibits NHE3 activity and increases the phosphorylation of NHE3 at serine 552 in the renal cortex, in line with the HgCl2-induced diuresis. Changes in renal proximal tubular function induced by HgCl2 were parallel to increased urinary markers of proximal tubular injury. Besides, atomic spectrometry showed that mercury accumulated in the renal cortex. We conclude that acute HgCl2 exposure causes renal vasoconstriction that is associated with reduced endothelial vasodilator agonist- and flow-mediated responses and inhibition of NHE3-mediated sodium reabsorption. Thus, our data suggest that HgCl2-induced acute renal failure may be attributable at least in part by its direct effects on renal hemodynamics and NHE3 activity.

Blockade of angiotensin AT1 receptors prevents arterial remodelling and stiffening in iron-overloaded rats.

BACKGROUND AND PURPOSE: Damage to the vasculature caused by chronic iron-overload in both humans and animal models, is characterized by endothelial dysfunction and reduced compliance. In vitro, blockade of the angiotensin II AT1 receptors reversed functional vascular changes induced by chronic iron-overload. In this study, the effect of chronic AT1 receptor blockade on aorta stiffening was assessed in iron-overloaded rats. EXPERIMENTAL APPROACH: Male Wistar rats were treated for 15 days with saline as control group, iron dextran 200 mg·kg-1 ·day-1 , 5 days a week (iron-overload group), losartan (20 mg·kg-1 ·day-1 in drinking water), and iron dextran plus losartan. Mechanical properties of the aorta were assessed in vivo. In vitro, aortic geometry and biochemical composition were assessed with morphometric and histological methods. KEY RESULTS: Thoracoabdominal aortic pulse wave velocity (PWV) increased significantly, indicating a decrease in aortic compliance. Co-treatment with losartan prevented changes on PWV, ß-index, and elastic modulus in iron-overloaded rats. This iron-related increase in PWV was not related to changes in aortic geometry and wall stress. but to increased elastic modulus/wall stress ratio, suggesting that a change in the composition of the wall was responsible for the stiffness. Losartan treatment also ameliorated the increase in aorta collagen content of the iron-overload group, without affecting circulating iron or vascular deposits. CONCLUSIONS AND IMPLICATIONS: Losartan prevented the structural and functional indices of aortic stiffness in iron-overloaded rats, implying that inhibition of the renin-angiotensin system would limit the vascular remodelling in chronic iron-overload.

Sildenafil reduces aortic endothelial dysfunction and structural damage in spontaneously hypertensive rats: Role of NO, NADPH and COX-1 pathways.

Arterial hypertension is a condition associated with endothelial dysfunction, accompanied by an imbalance in the production of reactive oxygen species (ROS) and NO. The aim of this study was to investigate and elucidate the possible mechanisms of sildenafil, a selective phosphodiesterase-5 inhibitor, actions on endothelial function in aortas from spontaneously hypertensive rats (SHR). SHR treated with sildenafil (40 mg/kg/day, p.o., 3 weeks) were compared to untreated SHR and Wistar-Kyoto (WKY) rats. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and vascular reactivity was determined in isolated rat aortic rings. Circulating endothelial progenitor cells and systemic ROS were measured by flow cytometry. Plasmatic total antioxidant capacity, NO production and aorta lipid peroxidation were determined by spectrophotometry. Scanning electron microscopy was used for structural analysis of the endothelial surface. Sildenafil reduced high SBP and partially restored the vasodilator response to acetylcholine and sodium nitroprusside in SHR aortic rings. Using selective inhibitors, our experiments revealed an augmented participation of NO, with a simultaneous decrease of oxidative stress and of cyclooxygenase-1 (COX-1)-derived prostanoids contribution in the endothelium-dependent vasodilation in sildenafil-treated SHR compared to non-treated SHR. Also, the relaxant responses to sildenafil and 8-Br-cGMP were normalized in sildenafil-treated SHR and sildenafil restored the pro-oxidant/antioxidant balance and the endothelial architecture. In conclusion, sildenafil reverses endothelial dysfunction in SHR by improving vascular relaxation to acetylcholine with increased NO bioavailability, reducing the oxidative stress and COX-1 prostanoids, and improving cGMP/PKG signaling. Also, sildenafil reduces structural endothelial damage. Thus, sildenafil is a promising novel pharmacologic strategy to treat endothelial dysfunction in hypertensive states reinforcing its potential role as adjuvant in the pharmacotherapy of cardiovascular diseases.

Chronic iron overload intensifies atherosclerosis in apolipoprotein E deficient mice: Role of oxidative stress and endothelial dysfunction.

AIMS: We previously demonstrated that iron overload induces endothelial dysfunction and oxidative stress, which could increase the risk for atherosclerosis. However, the iron-related harmfulness under a genetic predisposition to atherosclerosis is still unclear. Here, we have tested the hypothesis that chronic iron overload may change vascular reactivity associated with worsening of the atherosclerotic process in apolipoprotein E knockout (apoE(-/-)) mice. MAIN METHODS: Serum and aortas of wild-type (WT) and apoE(-/-) mice injected with iron-dextran (IO, 10 mg/mouse/day, ip) or saline 5 times a week for 4 weeks, were used. KEY FINDINGS: Iron overload increased serum levels of iron and biomarkers of liver injury and oxidative stress, and iron deposition in the aorta in both lines, but only apoE(-/-) IO mice had intensified hypercholesterolemia and atherosclerosis. By scanning electron microscopy, the small endothelial structural damage caused by iron in WT was worsened in the apoE(-/-) group. However, endothelial dysfunction was found only in the apoE(-/-) IO group, identified by impaired relaxation to acetylcholine and hyperreactivity to phenylephrine associated with reduced nitric oxide modulation. Moreover, tiron and indomethacin attenuated reactivity to phenylephrine with greater magnitude in aortas of the apoE(-/-) IO group. Confirming, there were changes in the antioxidant (superoxide dismutase and catalase) activity, increased expression of cyclooxygenase-2 in the aorta and elevated levels of thromboxane A2 and prostacyclin metabolites in the urine of apoE(-/-) IO. SIGNIFICANCE: Our results showed that chronic iron overload intensifies the atherosclerotic process and induces endothelial dysfunction in atherosclerotic mice, probably due to the oxidative stress and the imbalance between the relaxing and contractile factors synthesized by the damaged endothelium.

Dipeptidyl peptidase-4 inhibition prevents vascular dysfunction induced by ß-adrenergic hyperactivity.

Chronic stimulation of the ß-adrenergic sympathetic system induces vascular dysfunction which is associated with increased inflammatory cytokines production. A recently proposed therapy to control vascular injury through inflammatory processes involves inhibition of the enzyme dipeptidyl peptidase-IV (DPP4). The present study investigates whether the inhibition of DPP4 prevents the increase in inflammatory markers induced by isoproterenol and restores endothelial function in vivo and in vitro. Male Wistar rats were divided into four groups: vehicle (VHC), an isoproterenol-treated group (ISO), a sitagliptin-treated group (SITA), and an isoproterenol and sitagliptin-treated group (ISO + SITA). The ISO group exhibited significantly increased contractile responses to phenylephrine associated with reduced endothelial participation, which was totally prevented by DPP4 inhibition. In vitro incubation with isoproterenol had no effect on vascular smooth muscle cells, however isoproterenol increased the activity of DPP4 and the expression of inflammatory cytokines in endothelial cells, while sitagliptin reduced the level of cytokines to basal level. In conclusion, we have shown that beta-adrenergic receptor activation can increase DPP4 activity, which was associated with vascular dysfunction and cytokine expression in endothelial cells. The important role of DPP4 was further supported by sitagliptin, which reversed vascular changes induced by isoproterenol in vivo and in vitro.

Linoleic acid reduces vascular reactivity and improves the vascular dysfunction of the small mesentery in hypertension.

We aimed to investigate the effect of linoleic acid (LA) treatment on the blood pressure and function of mesenteric resistance arteries (MRA) in spontaneous hypertensive rats (SHR). Male SHR were treated daily with LA (15 mg/kg) or vehicle (control) for 15 days. Compared with controls, LA treatment decreased blood pressure and showed the following in MRA: (1) increased lumen and external diameter, (2) decreased wall:lumen ratio and wall thickness, (3) decreased stiffness and (4) less collagen deposition. LA treatment reduced the contractile response to phenylephrine, although there were no changes observed in MRA in regard to the acetylcholine or sodium nitroprusside responses. Incubation with L-NAME left-shifted the reactivity to phenylephrine only in the MRA treated group, suggesting that LA treatment can improve NO bioavailability. This result was accompanied by an increase "in situ" NO production. Incubation with tiron decreased vascular reactivity to phenylephrine in MRA in LA rats, which was accompanied by decreased superoxide anion production. Moreover, incubation with indomethacin (non-selective COX inhibitor, 10 µM), NS 398 (COX-2 specific inhibitor, 1 µM), furegrelate (TXA2 synthase inhibitor, 1 µM), SQ 29.548 (TP receptor antagonist, 1 µM) and SC 19220 (EP1 receptor antagonist, 10 µM) reduced the vasoconstrictor responses to phenylephrine in MRA in the treated group. These results were accompanied by a reduction in COX-2 protein expression. In conclusion, these findings show that LA treatment decreases blood pressure. In addition, the improvement of endothelial dysfunction and structural changes in this hypertension model may be responsible for the reduction in blood pressure.

Chronic iron overload induces vascular dysfunction in resistance pulmonary arteries associated with right ventricular remodeling in rats.

Although iron excess is toxic to the vasculature and even that pulmonary hypertension has been reported in this scenario, the role of iron overload per se remains to be clarified. This study aimed to test the effects of chronic iron-overload in rats on the morphophysiology of resistance pulmonary arteries (RPA) and right ventricle (RV) remodeling. Rats were injected with saline or iron-dextran (10, 100 and 200 mg/kg/day i.p.) for 28 days. Our results indicated increased circulating iron with significant lung deposits. Moreover, rats treated with the highest dose exhibited RV dysfunction and hypertrophy; inward remodeling and increased vasoconstriction of the RPA. Vascular hyperreactivity was accompanied by reduced nitric oxide (NO), and was reversed by incubation with Dimethylsulfoxide, Catalase and Tempol. The NADPH oxidase subunit gp91phox was increased due to iron-overload, and incubation with angiotensin II type-1 receptor (AT1) antagonist losartan not only reduced oxidative stress but also restored vascular function. Thus, we concluded that AT1 pathway plays a role in pulmonary vascular dysfunction by increasing oxidative stress and reducing NO bioavailability, thereby contributing to vascular remodeling and pulmonary hypertension of iron-overload. This finding should instigate future studies on the beneficial impacts of in vivo blockade of AT1 receptor under iron overload.

Overview of the Pathophysiological Implications of Organotins on the Endocrine System.

Organotins (OTs) are pollutants that are used widely by industry as disinfectants, pesticides, and most frequently as biocides in antifouling paints. This mini-review presents the main evidences from the literature about morphophysiological changes induced by OTs in the mammal endocrine system, focusing on the metabolism and reproductive control. Similar to other toxic compounds, the main effects with potential health risks to humans and experimental animals are not only related to dose and time of exposure but also to age, gender, and tissue/cell exposed. Regarding the underlying mechanisms, current literature indicates that OTs can directly damage endocrine glands, as well as interfere with neurohormonal control of endocrine function (i.e., in the hypothalamic-pituitary axis), altering hormone synthesis and/or bioavailability or activity of hormone receptors in the target cells. Importantly, OTs induces biochemical and morphological changes in gonads, abnormal steroidogenesis, both associated with reproductive dysfunctions such as irregular estrous cyclicity in female or spermatogenic disorders in male animals. Additionally, due to their role on endocrine systems predisposing to obesity, OTs are also included in the metabolism disrupting chemical hypothesis, either by central (e.g., accurate nucleus and lateral hypothalamus) or peripheral (e.g., adipose tissue) mechanisms. Thus, OTs should be indeed considered a major endocrine disruptor, being indispensable to understand the main toxic effects on the different tissues and its causative role for endocrine, metabolic, and reproductive dysfunctions observed.

Acute copper overload induces vascular dysfunction in aortic rings due to endothelial oxidative stress and increased nitric oxide production.

The mechanisms involved in vascular reactivity alterations promoted by copper (Cu) overload were investigated. Thoracic aorta obtained from male Wistar rats were cut into rings and exposed for 1 h to 10 µg/ml Cu. Exposure to Cu decreased the contractile responses of aortic rings to phenylephrine (PHE). Removal of endothelium and subsequent administration of N-nitro-L arginine methyl ester (L-NAME), tetrahydrobiopterin, aminoguanidine, diethyldithiocarbamic acid, catalase, or tetraethylammonium increased contractile responses. Incubation with apocinyn and tiron enhanced the sensitivity to PHE. Data demonstrated that high concentrations of Cu reduced PHE-mediated vascular reactivity which was associated with elevated production of nitric oxide (NO), which was attributed to activation of inducible NO synthase, and elevated levels of hydrogen peroxide probably related to a rise in superoxide dismutase activity and reactive oxygen species generation.

Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogen-deficient female rats.

The goal of the present study was to evaluate vascular potassium channels and Na+-K+-ATPase activity in estrogen deficient female rats. Female rats that underwent ovariectomy were assigned to receive daily treatment with placebo (OVX) or estrogen replacement (OVX+E2, 1mg/kg, once a week, i.m.). Aortic rings were used to examine the involvement of K+ channels and Na+-K+-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100µM) and K+ channels blockers: tetraethylammonium (TEA, 5mM), 4-aminopyridine (4-AP, 5mM), iberiotoxin (IbTX, 30nM), apamin (0.5mM), charybdotoxin (ChTX, 0.1mM) and iberiotoxin plus apamin. When aortic rings were pre-contracted with KCl (60mM) or pre-incubated with TEA (5mM), 4-aminopyridine (4-AP, 5mM) and iberiotoxin (IbTX, 30nM) plus apamin (0.5µM), the ACh-induced relaxation was less effective in the ovariectomized group. Additionally, 4-AP and IbTX decreased the relaxation by sodium nitroprusside in all groups but this reduction was greater in the ovariectomized group. Estrogen deficiency also increased aortic functional Na+-K+ ATPase activity evaluated by K+-induced relaxation. L-NAME or endothelium removal were not able to block the increase in aortic functional Na+-K+ ATPase activity, however, TEA (5mM) restored this increase to the control level. We also found that estrogen deficiency increased superoxide anion production and reduced nitric oxide release in aortic ring from ovariectomized animals. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals due to the activation of K+ channels and increased Na+-K+ ATPase activity.

Chronic iron overload induces functional and structural vascular changes in small resistance arteries via NADPH oxidase-dependent O2- production.

Iron overload leads to excessive free radical formation and induces cardiovascular dysfunction. Thus, our aim was to investigate the structural and endothelial modulation of vascular tone induced by chronic iron overload in mesenteric arteries. Rats were divided into two groups: the control (vehicle) group and the group treated with iron dextran for 28days (100mg/kg, 5days a week). Chronic iron overload altered the following morpho-physiological parameters of third-order mesenteric resistance arteries: decreased lumen and external diameters; increased wall/lumen ratio and wall thickness; decreased distensibility and increased stiffness; and increased pulse wave velocity. Additionally, iron overload increased the vasoconstrictor response in mesenteric arterial rings in vitro but did not affect the relaxation induced by acetylcholine and sodium nitroprusside. It is suggested that iron overload reduces nitric oxide bioavailability by increasing free radicals, because L-NAME did not shift the concentration-response curve to phenylephrine, but L-NAME plus superoxide dismutase shifted the curve to the left. In vitro assays with DAF-2 and DHE indicated reduced NO production and increased superoxide anion (O2-) generation in the iron-overloaded group. Furthermore, tiron, catalase, apocynin and losartan induced reduced reactivity only in iron-overloaded rats. Moreover, increased ACE activity was observed in the mesenteric resistance arteries of iron-overloaded rats accompanied by an increase in gp91phox, catalase, ERK1/2 and eNOS protein expression. In conclusion, these findings show that chronic iron overload induces structural and functional changes in resistance arteries, most likely due to a decrease in NO bioavailability resulting from an increase in O2- production by NADPH oxidase.

Low-level lead exposure changes endothelial modulation in rat resistance pulmonary arteries.

Lead exposure induces hypertension and endothelial dysfunction. However, the effects on the pulmonary vasculature have not been explored. In this study, rats exposed to lead acetate for seven days (4µg/100g on the 1st day and 0.05µg/100g/day i.m. subsequently) had lead blood level of 3.9±0.7µg/dL and increased right ventricular pressures. There was an increased Pb deposition and superoxide anions production in the pulmonary arteries, associated with reduced vasoconstriction but unchanged endothelium-dependent vasodilatation to acetylcholine (ACh). In both groups, inhibition of the nitric oxide (NO) synthase with L-NAME blocked the response to ACh, while indomethacin (cycloxygenase inhibitor) had no effect. Incubation with nonspecific potassium channel blocker (tetraethylammonium) reduced the ACh-induced vasodilatation only in the Pb group. Apamin (SKCa channel blocker) and 4-aminopyridine (Kv channel blocker), but not iberiotoxin (BKCa channel blocker), also inhibited this response in the Pb group. The vasodilatation to exogenous NO was reduced by Pb, while relaxation to the cGMP analogue was similar between groups. Concordantly, the protein level of soluble guanylate cyclase (sGC) was reduced. In conclusion, short-term and low-level exposure to Pb changes pulmonary haemodynamic and increases oxidative stress. The pulmonary vasculature exhibited increased hyperpolarization by the Kv and SKCa channels, probably as a compensatory mechanism to the decreased responsiveness to NO.

Tributyltin chloride increases phenylephrine-induced contraction and vascular stiffness in mesenteric resistance arteries from female rats.

Tributyltin chloride (TBT) is an organotin compound that reduces estrogen levels in female rats. We aimed to investigate the effects of TBT exposure on vascular tonus and vascular remodelling in the resistance arteries of female rats. Rats were treated daily with TBT (500 ng/kg) for 15 days. TBT did not change arterial blood pressure but did modify some morpho-physiological parameters of third-order mesenteric resistance arteries in the following ways: (1) decreased lumen and external diameters; (2) increased wall/lm ratio and wall thickness; (3) decreased distensibility and increased stiffness; (4) increased collagen deposition; and (5) increased pulse wave velocity. TBT exposure increased the phenylephrine-induced contractile response in mesenteric resistance arteries. However, vasodilatation responses induced by acetylcholine and sodium nitroprusside were not modified by TBT. It is suggested that TBT exposure reduces vascular nitric oxide (NO) production, because:(1) L-NAME incubation did not cause a leftward shift in the concentration-response curve for phenylephrine; (2) both eNOS protein expression; (3) in situ NO production were reduced. Incubation with L-NAME; and (4) SOD shifted the phenylephrine response curve to the left in TBT rats. Tiron, catalase, ML-171 and VAS2870 decreased vascular reactivity to phenylephrine only in TBT rats. Moreover, increased superoxide anion production was observed in the mesenteric resistance arteries of TBT rats accompanied by an increase in gp91phox, catalase, AT1 receptor and total ERK1/2 protein expression. In conclusion, these findings show that TBT induced alterations are most likely due to a reduction of NO production combined with increased O2(-) production derived from NADPH oxidase and ERK1/2 activation. These findings offer further evidence that TBT is an environmental risk factor for cardiovascular disease.

Chronic iron overload in rats increases vascular reactivity by increasing oxidative stress and reducing nitric oxide bioavailability.

AIMS: Iron overload in animal models and humans increases oxidative stress and induces cardiomyopathy. It has been suggested that the vasculature is also damaged, but the impacts on vascular reactivity and the underlying mechanisms remain poorly understood. In this study, we aimed to identify possible changes in the vascular reactivity of aortas from iron overloaded rats and investigate the underlying mechanisms. MAIN METHODS: Rats were treated with 100mg/kg/day iron-dextran, ip, five days a week for four weeks and compared to a saline-injected group. KEY FINDINGS: Chronic iron administration increased serum iron and transferrin saturation with significant deposition in the liver. Additionally, iron overload significantly increased the vasoconstrictor response in aortic rings as assessed in vitro, with reduced influence of endothelial denudation or l-NAME incubation on the vascular reactivity. In vitro assay with DAF-2 indicated reduced NO production in the iron overload group. Iron overload-induced vascular hyperactivity was reversed by incubation with tiron, catalase, apocynin, allopurinol and losartan. Moreover, malondialdehyde was elevated in the plasma, and O2(•-) generation and NADPH oxidase subunit (p22phox) expression were increased in the aortas of iron-loaded rats. SIGNIFICANCE: Our results demonstrated that chronic iron overload is associated with altered vascular reactivity and the loss of endothelial modulation of the vascular tone. This iron loading-induced endothelial dysfunction and reduced nitric oxide bioavailability may be a result of increased production of reactive oxygen species and local renin-angiotensin system activation.