Saiprasad M. Zemse

Interleukin-10 inhibits the in vivo and in vitro adverse effects of TNF-α on the endothelium of murine aorta

TNF-α is a proinflammatory cytokine and is an important mediator of maternal endothelial dysfunction leading to preeclampsia. In this study, we tested whether IL-10 protects against TNF-α-induced endothelial dysfunction in murine aorta. In in vitro experiments, aortic rings of C57BL/6 female mice were incubated in Dulbeccos modified Eagles medium in the presence of either vehicle (distilled H(2)O), TNF-α (4 nmol/l), or recombinant mouse IL-10 (300 ng/ml) or in the presence of both TNF-α and IL-10 for 22 h at 37°C. In in vivo experiments C57BL6/IL-10 knockout female mice were treated with saline or TNF-α (220 ng·kg(-1)·day(-1)) for 14 days. Aortic rings were isolated from in vitro and in vivo experiments and mounted in a wire myograph (Danish Myotech) and stretched to a tension of 5 mN. Endothelium-dependent relaxation was assessed by constructing cumulative concentration-response curves to acetylcholine (ACh, 0.001-10 μmol/l) during phenylephrine (10 μmol/l)-induced contraction. As a result, overnight exposure of aortic rings to TNF-α resulted in significant blunted maximal relaxing responses (E(max)) to ACh compared with untreated rings (22 ± 4 vs. 82 ± 3%, respectively). IL-10 knockout mice treated with TNF-α showed significant impairment in ACh responses (E(max)) compared with C57BL/6 mice treated with TNF-α (51 ± 3 vs. 72 ± 3%, respectively). Western blot analysis showed that endothelial nitric oxide synthase (eNOS) expression was reduced by TNF-α in in vitro and in vivo experiments, whereas IL-10 restored the eNOS expression. In conclusion, the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation caused by TNF-α by protecting eNOS expression.

Oxidative Stress Impairs Vasorelaxation Induced by the Soluble Guanylyl Cyclase Activator BAY 41-2272 in Spontaneously Hypertensive Rats

BACKGROUND BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) relaxes mesenteric arteries (MA) in a synergistic fashion with nitric oxide (NO). We hypothesized that the relaxation to BAY 41-2272 is decreased in spontaneously hypertensive rats (SHR) because of the reduced NO bioavailability in this strain and that relaxation would be improved by inhibiting the oxidative stress. We aimed to evaluate the influence of oxidative stress in BAY 41-2272-induced vasorelaxation in isolated MA from SHR. METHODS MA function was evaluated by concentration-response curves to BAY 41-2272. We measured protein expression of endothelial NO synthase (eNOS), soluble guanylyl cyclase (sGC) and human-antigen R (HuR) (sGC mRNA-stabilizing protein), sGC activity and plasma levels of superoxide dismutase (SOD), and total antioxidant status (TAS). RESULTS Cyclic guanosine monophosphate (cGMP)-dependent and -independent relaxation induced by BAY 41-2272 (0.0001-1 micromol/l) was impaired in SHR compared with Wistar-Kyoto (WKY). We observed reduced expression of eNOS, sGC and HuR, and decreased sGC activity in SHR. Plasma levels of SOD and TAS were also diminished in SHR. Incubation with SOD or indomethacin increased relaxation to BAY 41-2272 in SHR. Furthermore, acetylcholine (ACh)-induced relaxation was increased in the presence of BAY 41-2272 or SOD, apocynin, or indomethacin. CONCLUSION Augmented oxidative stress in SHR impaired cGMP-dependent and -independent relaxation induced by BAY 41-2272, by decreasing NO bioavailability and sGC expression and by increasing contractile activity. Inhibiton of oxidative stress improved the relaxation of BAY 41-2272 in SHR. BAY 41-2272 might be an alternative therapeutic tool for hypertension if administrated with antioxidant compounds.

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin (ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng x kg(-1) x day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ET(A)) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ET(A) receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ET(A) receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.

Restoration of endothelin-1-induced impairment in endothelium-dependent relaxation by interleukin-10 in murine aortic rings

Endothelin-1 (ET-1) is implicated in the development of endothelial dysfunction through the generation of reactive oxygen species by NADPH oxidase activation. Interleukin-10 (IL-10) is an antiinflammatory cytokine that stimulates nitric oxide production, decreases superoxide production, and restores endothelial integrity after vascular injury. In this study, we tested whether IL-10 attenuates ET-1-induced endothelial dysfunction by improving acetylcholine (ACh)-induced relaxation of cultured murine aortic rings. Aortic rings (2 mm long) of C57BL/6 mice were incubated in 2 mL DMEM containing 120 U/mL penicillin and 120 microg/mL streptomycin in the presence of one of 4 treatments: vehicle (deionized water), ET-1 (100 nmol/L), recombinant mouse IL-10 (300 ng/mL), or a combination of both ET-1 and IL-10. After incubation at 37 degrees C for either 1 or 6 h (short-term exposure) or 22 h (overnight exposure), rings were mounted in a wire myograph and stretched to a passive force of 5 mN. Endothelium-dependent vasorelaxation was assessed by constructing cumulative concentration-response curves to ACh (0.001-10 micromol/L) during 10 mumol/L phenylephrine (PE)-induced contraction. Short-term exposure of ET-1 did not result in an impairment of ACh-induced relaxation. Overnight exposure of aortic rings to ET-1 resulted in a statistically significant endothelial dysfunction characterized by a reduced maximal relaxation response to ACh compared with that of untreated rings (Emax 57% +/- 3% versus 82% +/- 4%). IL-10 treatment restored ACh-induced relaxation (Emax 77% +/- 3%). Western blotting showed decreased eNOS expression in response to ET-1, whereas vessels treated with a combination of ET-1 and IL-10 showed increased expression of eNOS. Immunohistochemical analysis showed decreased eNOS expression in ET-1-treated vessels compared with those treated with both ET-1 and IL-10. We conclude that, in murine aorta, the antiinflammatory cytokine IL-10 prevents impairment in endothelium-dependent relaxation induced in response to long-term incubation with ET-1 via normalization of eNOS expression.

Interleukin-10 limits increased blood pressure and vascular RhoA/Rho-kinase signaling in angiotensin II-infused mice

AIMS Interleukin-10 (IL-10) is a multi-functional cytokine with potent anti-inflammatory properties. We hypothesized that IL-10 limits increased RhoA/Rho-kinase signaling and vascular reactivity in arteries from angiotensin II (Ang II) hypertensive mice. MAIN METHODS Wild type (WT) and IL-10 knockout ((-/-)) mice were infused with Ang II (90ng/min) for 14days. Additionally, WT mice were infused with Ang II and simultaneously infused with exogenous IL-10 (0.5ηg/min, 14days). Aortic rings were mounted in a myograph and concentration-response curve to phenylephrine (PE) were evaluated. KEY FINDINGS After Ang II infusion, blood pressure responses, but not maximal contraction to PE, was greater in IL-10(-/-) mice, compared to WT. Rho-kinase inhibition (Y-27632; 10μM) resulted in a more evident reduction of PE-induced contraction in WT hypertensive mice, when compared to IL-10(-/-) hypertensive mice. IL-10 exogenous infusion prevented the blood pressure increase in Ang II-infused WT mice. The augmented PE-contraction observed in aorta from WT mice infused with Ang II was also prevented by exogenous infusion of IL-10. Additionally, Rho-kinase inhibition (Y-27632; 10μM) abolished the differences in the contractile response to PE between these groups. SIGNIFICANCE These results demonstrate that IL-10 counteracts both the pressoric activity of Ang II as well as vascular dysfunction associated with hypertension, partially, modulating the RhoA-Rho kinase pathway. Strategies to enhance IL-10 levels during hypertension may enhance the benefits provided by regular treatments.

EFFECT OF THE PHOSPHODIESTERASE 5 INHIBITORS SILDENAFIL, TADALAFIL AND VARDENAFIL ON RAT ANOCOCCYGEUS MUSCLE: FUNCTIONAL AND BIOCHEMICAL ASPECTS

1 The anococcygeus muscle is part of the erectile machinery in male rodents. Phosphodiesterase (PDE) 5 inhibitors enhance and prolong the effects of cGMP, which has a key role in penile erection. The aim of the present study was to provide a functional and biochemical comparison of the three PDE5 inhibitors, namely sildenafil, tadalafil and vardenafil, in the rat anococcygeus muscle. 2 Muscle strips were mounted in 4 mL organ baths and isometric force recorded. Levels of cGMP were measured using an enzyme immunoassay kit. Western blots were used to determine PDE5 protein expression. 3 The PDE5 inhibitors concentration‐dependently relaxed carbachol‐precontracted anococcygeus muscle; however, vardenafil was more potent (pEC50 = 8.11 ± 0.05) than sildenafil (7.72 ± 0.06) or tadalafil (7.69 ± 0.05). Addition of NG‐nitro‐l‐arginine methyl ester (100 µmol/L) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (10 µmol/L) to the organ baths caused significant rightward shifts in concentration–response curves for all PDE5 inhibitors. 4 Sildenafil, tadalafil and vardenafil (all at 0.1 µmol/L) caused leftward shifts in the glyceryl trinitrate (GTN) concentration‐response curves (by 4.0‐, 3.7‐ and 5.5‐fold, respectively). In addition, all three PDE5 inhibitors significantly potentiated relaxation responses to both GTN (0.01–10 µmol/L) and electrical field stimulation (EFS; 1–32 Hz), with vardenafil having more pronounced effects. 5 All three PDE5 inhibitors reduced EFS‐evoked contractions in a concentration‐dependent manner over the concentration range 0.001–1 µmol/L. There were no significant differences between the effects of the three PDE5 inhibitors. 6 Vardenafil (0.01–0.1 µmol/L) was more potent in preventing cGMP degradation in vitro than sildenafil (0.01–0.1 µmol/L) and tadalafil (0.01–0.1 µmol/L). 7 Under control conditions, the expression of PDE5 was higher in the anococcygeus muscle than in the corpus cavernosum. 8 In conclusion, PDE5 inhibitors enhance exogenous and endogenous nitric oxide‐mediated relaxation in the rat anococcygeus muscle. The potency of vardenafil was greater than that of either sildenafil or tadalafil.