Ang II-induced contraction is impaired in the aortas of renovascular hypertensive animal model.

Abstract: Renin-angiotensin system plays a critical role in blood pressure control, and the abnormal activation of the AT1 receptor contributes to the development of renovascular hypertension. This study aimed to evaluate the underlying cellular signaling for AT1 receptor activation by Ang II and to compare this mechanism between aortas from 2K-1C and 2K rats. Effects of antagonists and inhibitors were investigated on Ang II-induced contractions in denuded or intact-endothelium aortas. The AT1 receptor antagonist abolished Ang II-induced contraction in 2K-1C and 2K rat aortas, while AT2 and Mas receptors antagonists had no effect. Endothelial nitric oxide synthase inhibition increased the maximal effect (Emax) of Ang II in 2K, which was not changed in 2K-1C aortas. It was associated with lower eNOS mRNA levels in 2K-1C. Endothelium removal increased the Emax of Ang II in 2K-1C and mainly in 2K rat aortas. Nox and COX inhibition did not alter Ang II-induced contraction in 2K and 2K-1C rat aortas. However, AT1 expression was higher in 2K-1C compared to 2K rat aortic rings, whereas expression of phosphorylated (active) IP3 receptors was lower in 2K-1C than in 2K rats. These results demonstrate that endothelium removal impairs Ang II-stimulated contraction in the aorta of 2K-1C rats, which is associated with the reduction of IP3 receptor phosphorylation and activation. In addition, eNOS plays a critical role in Ang II-induced contraction in 2K rat aortas. It is possible that the high Ang II plasma levels could desensitize AT1 receptor in 2K-1C rats, leading to impaired IP3 receptors activation.

Influence of nitro ruthenium isomerization on photochemically induced nitric oxide release: Vasorelaxant activities.

We have synthesized cis-[Ru(bpy)2(NO2-κN)Ln-](n-1) and cis-[Ru(bpy)2(NO2-κO)L n-](n-1) (bpy = 2,2'-bipyridine; k = indication of the coordinated center to Ruthenium; L = pyridine type ligand) by reacting cis-[Ru(bpy)2(H2O)Ln-](n-2) with sodium nitrite or conducting basic cis-[Ru(bpy)2NO(Ln-)](n-3) hydrolysis. Photolysis at the metal-ligand charge transfer band (MLCT) of the isomers yielded nitric oxide (NO) as determined by NO measurement. The NO photorelease rates obtained upon 447 nm laser irradiation of the ruthenium complexes showed that cis-[Ru(bpy)2(NO2-κO)Ln-](n-1) released NO three times faster than cis-[Ru(bpy)2(NO2-κN)Ln-](n-1). We investigated endothelium-dependent vasodilation induced by cis-[Ru(bpy)2(4-pic)(NO2-κN)]+ and cis-[Ru(bpy)2(4-pic)(NO2-κO)]+ (4-pic = 4-picoline) in isolated 3 mm aortic rings precontracted with L-phenylephrine. Maximum vasodilation was achieved under 447 nm laser irradiation of 0.5 µMol.L-1 ruthenium complexes for 100 s.

Glypican 1 and syndecan 1 differently regulate noradrenergic hypertension development: Focus on IP3R and calcium.

BACKGROUND: Vascular dysfunction is a checkpoint to the development of hypertension. Heparan sulfate proteoglycans (HSPG) participate in nitric oxide (NO) and calcium signaling, key regulators of vascular function. The relationship between HSPG-mediated NO and calcium signaling and vascular dysfunction has not been explored. Likewise, the role of HSPG on the control of systemic blood arterial pressure is unknown. Herein, we sought to determine if the HSPG syndecan 1 and glypican 1 control systemic blood pressure and the progression of hypertension. PURPOSE: To determine the mechanisms whereby glypican 1 and syndecan 1 regulate vascular tone and contribute to the development of noradrenergic hypertension. EXPERIMENTAL APPROACH AND KEY RESULTS: By assessing systemic arterial blood pressure we observed that syndecan 1 (Sdc1-/-) and glypican 1 (Gpc1-/-) knockout mice show a similar phenotype of decreased systolic blood pressure that is presented in a striking manner in the Gpc1-/- strain. Gpc1-/- mice are also uniquely protected from a norepinephrine hypertensive challenge failing to become hypertensive. This phenotype was associated with impaired calcium-dependent vasoconstriction and altered expression of calcium-sensitive proteins including SERCA and calmodulin. In addition, Gpc1-/- distinctively showed decreased IP3R activity and increased calcium storage in the endoplasmic reticulum. CONCLUSIONS AND IMPLICATIONS: Glypican 1 is a trigger for the development of noradrenergic hypertension that acts via IP3R- and calcium-dependent signaling pathways. Glypican 1 may be a potential target for the development of new therapies for resistant hypertension or conditions where norepinephrine levels are increased.

Thromboxane-prostanoid receptor activation blocks ATP-sensitive potassium channels in rat aortas.

K+ channel activation is one of the major mechanisms involved in vasodilation. Vasoconstrictor agonists such as angiotensin II promote ATP-dependent potassium channels (KATP ) dysfunction. This study evaluates whether thromboxane-prostanoid (TP receptor) activation by the agonist U46619 increases reactive oxygen species (ROS) production in rat aortas, which could contribute to KATP channel dysfunction and impaired NO-dependent vasodilation. TP receptor activation with the selective agonist U46619 increased ROS in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but the TP receptor antagonist SQ29548 abolished this effect. ECs and VSMCs incubation with ROS scavengers like Tiron or PEG-Catalase impaired U46619-induced ROS production. U46619 at the concentrations of 0.1 and 1 µmol/L induced contractions with similar amplitude. KATP channel activation with pinacidil-induced relaxation was lower for the contractions induced with 0.1 or 1 µmol/L U46619 than with 10 nmol/L U46619. Acetylcholine-induced relaxation provided similar results. In aortas pre-contracted with 10 nmol/L U46619, neither Tiron (100 µmol/L) nor catalase (300 U/mL) affected pinacidil-induced relaxation. However, in aortas pre-contracted with 0.1 µmol/L U46619, catalase potentiated pinacidil-induced relaxation. Pinacidil potentiated acetylcholine-induced relaxation in aortas pre-contracted with 0.1 and 1 µmol/L U46619. Incubation with 10 nmol/L U46619 increased NO concentration in ECs. Taken together, these results show that high concentrations of the TP receptor agonist U46619 impair KATP channels, which is probably due to ROS production. It is likely that hydrogen peroxide is the ROS.

Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats.

This study aimed to investigate the antiproteinuric and hyperkalemic mechanisms activated by dual renin-angiotensin system (RAS) blockade in renovascular hypertensive rats (2-kidney 1-clip model [2K-1C]). Six weeks after clipping the left renal artery or sham operation (2K), rats were treated with losartan, enalapril, or both drugs for two weeks. We found that 2K-1C rats displayed higher tail-cuff blood pressure (BP), increased non-clipped kidney Ang II concentration, and more pronounced urinary albumin excretion than 2K. BP was decreased by the treatment with either enalapril or losartan, and the combination of both drugs promoted an additional antihypertensive effect in 2K-1C rats. Renal Ang II content and albuminuria were reduced by either enalapril or losartan in monotherapy and restored to control levels by dual RAS blockade. Albuminuria in 2K-1C rats was accompanied by downregulation of the glomerular slit protein podocin, reduction of the endocytic receptors megalin and cubilin, and a marked decrease in the expression of the ClC-5 chloride channel, compared to 2K animals. Treatment with losartan and enalapril in monotherapy or combination increased the expression of podocin, cubilin, and ClC-5. However, only the combined therapy normalized podocin, cubilin, and ClC-5 protein abundance in the non-clipped kidney of 2K-1C rats. Renovascular hypertensive 2K-1C rats had a lower concentration of plasma potassium compared to 2K rats. Single RAS blockade normalized potassium plasma concentration, whereas 2K-1C rats treated with dual RAS blockade exhibited hyperkalemia. Hypokalemia in 2K-1C rats was accompanied by an increase in the cleaved activated forms of α-ENaC and γ-ENaC and the expression of ß-ENaC. Combined RAS blockade but not monotherapy significantly reduced the expression of these ENaC subunits in 2K-1C rats. Indeed, double RAS blockade reduced the abundance of cleaved-α-ENaC to levels lower than those of 2K rats. Collectively, these results demonstrate that the antiproteinuric effect of dual RAS blockade in 2K-1C rats is associated with the restored abundance of podocin and cubilin, and ClC-5. Moreover, double RAS blockade-induced hyperkalemia may be due, at least partially, to an exaggerated downregulation of cleaved α-ENaC in the non-clipped kidney of renovascular hypertensive rats.

Nitric Oxide Donors as Potential Drugs for the Treatment of Vascular Diseases Due to Endothelium Dysfunction.

Endothelial dysfunction and consequent vasoconstriction are a common condition in patients with hypertension and other cardiovascular diseases. Endothelial cells produce and release vasodilator substances that play a pivotal role in normal vascular tone. The mechanisms underlying endothelial dysfunction are multifactorial. However, enhanced reactive oxygen species (ROS) production and consequent vasoconstriction instead of endothelium-derived relaxant generation and consequent vasodilatation contribute to this dysfunction considerably. The main targets of the drugs that are currently used to treat vascular diseases concerning enzyme activities and protein functions that are impaired by endothelial nitric oxide synthase (eNOS) uncoupling and ROS production. Nitric oxide (NO) bioavailability can decrease due to deficient NO production by eNOS and/or NO release to vascular smooth muscle cells, which impairs endothelial function. Considering the NO cellular mechanisms, tackling the issue of eNOS uncoupling could avoid endothelial dysfunction: provision of the enzyme cofactor tetrahydrobiopterin (BH4) should elicit NO release from NO donors, to activate soluble guanylyl cyclase. This should increase cyclic guanosine-monophosphate (cGMP) generation and inhibit phosphodiesterases (especially PDE5) that selectively degrade cGMP. Consequently, protein kinase-G should be activated, and K+ channels should be phosphorylated and activated, which is crucial for cell membrane hyperpolarization and vasodilation and/or inhibition of ROS production. The present review summarizes the current concepts about the vascular cellular mechanisms that underlie endothelial dysfunction and which could be the target of drugs for the treatment of patients with cardiovascular disease.

Reduced caveolae density in arteries of SHR contributes to endothelial dysfunction and ROS production.

Caveolae are plasma membrane invaginations enriched with high cholesterol and sphingolipid content; they also contain caveolin proteins in their structure. Endothelial nitric oxide synthase (eNOS), an enzyme that synthesizes nitric oxide (NO) by converting L-arginine to L-citrulline, is highly concentrated in plasma membrane caveolae. Hypertension is associated with decreased NO production and impaired endothelium-dependent relaxation. Understanding the molecular mechanisms that follow hypertension is important. For this study, we hypothesized that spontaneously hypertensive rat (SHR) vessels should have a smaller number of caveolae, and that the caveolae structure should be disrupted in these vessels. This should impair the eNOS function and diminish NO bioavailability. Therefore, we aimed to investigate caveolae integrity and density in SHR aortas and mesenteric arteries and the role played by caveolae in endothelium-dependent relaxation. We have been able to show the presence of caveolae-like structures in SHR aortas and mesenteric arteries. Increased phenylephrine-induced contractile response after treatment with dextrin was related to lower NO release. In addition, impaired acetylcholine-induced endothelium-dependent relaxation could be related to decreased caveolae density in SHR vessels. The most important finding of this study was that cholesterol depletion with dextrin induced eNOS phosphorylation at Serine1177 (Ser1177) and boosted reactive oxygen species (ROS) production in normotensive rat and SHR vessels, which suggested eNOS uncoupling. Dextrin plus L-NAME or BH4 decreased ROS production in aorta and mesenteric arteries supernatant's of both SHR and normotensive groups. Human umbilical vein endothelial cells (HUVECs) treated with dextrin confirmed eNOS uncoupling, as verified by the reduced eNOS dimer/monomer ratio. BH4, L-arginine, or BH4 plus L-arginine inhibited eNOS monomerization. All these results showed that caveolae structure and integrity are essential for endothelium-dependent relaxation. Additionally, a smaller number of caveolae is associated with hypertension. Finally, caveolae disruption promotes eNOS uncoupling in normotensive and hypertensive rat vessels and in HUVECs.

NO donors induce vascular relaxation by different cellular mechanisms in hypertensive and normotensive rats.

PURPOSE: This study investigated the intracellular mechanisms involved in the vasodilatation induced by the classic NO donor SNP and the non-classic NO donor cis-[Ru(bpy)2(py)(NO2)](PF6) (or RuBPY) in mesenteric resistance arteries obtained from renal hypertensive (2K-1C) and normotensive (2K) rats. METHODS: On the basis of fluorimetric assays in cultured vascular smooth muscle cells (VSMCs) isolated from 2K-1C and 2K rats, we measured NO release from SNP and RuBPY, cytosolic Ca2+ concentration ([Ca2+]c), and reactive oxygen species (ROS) with the selective probes DAF-2DA, Fluo-3AM and the more selective probe for peroxynitrite (7-CBA), respectively. We determined isometric tension in mesenteric arteries to assess SNP- and RuBPY-induced relaxation. RESULTS: SNP and RuBPY released NO in comparable amounts in cultured aortic VSMCs from hypertensive 2K-1C and normotensive 2K rats. The NO0 scavenger hydroxocobalamin blunted NO release. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibition with thapsigargin reduced [Ca2+]c in normotensive 2K rat VSMCs only. ROS amounts were greater in hypertensive 2K-1C than in normotensive 2K rat VSMCs, but neither SNP nor RuBPY altered ROS concentrations in any of the groups. SNP and RuBPY induced similar relaxation in hypertensive 2K-1C and normotensive 2K rat mesenteric resistance arteries. The SNP and RuBPY-induced relaxation involves sGC and PKG activation. On the other hand, SNP but not RuBPY activates K+ channels. Interestingly, SERCA inhibition reduces SNP induced relaxation only in normotensive 2K rat mesenteric arteries whereas RuBPY-induced relaxation does not involve SERCA activation in both normotensive and hypertensive arteries. CONCLUSION: Our results indicate that SNP and RuBPY-induced mesenteric resistance artery relaxation involves NO/sGC/cGMP/PKG pathway activation. K+ channels and SERCA activation is required to SNP but not for RuBPY-induced relaxation. Moreover, SERCA seems to be impaired in hypertensive 2K-1C rat mesenteric resistance arteries although it does not impact SNP- or RuBPY-induced relaxation.

Apocynin alters redox signaling in conductance and resistance vessels of spontaneously hypertensive rats.

Chronic treatment with apocynin reduces blood pressure and prevents endothelial dysfunction development in spontaneously hypertensive rats (SHR). Mechanisms underlying apocynin effects on SHR remain unclear. Compared to diapocynin and other drugs, apocynin is a weak antioxidant, which suggests that its effects on SHR are associated with other mechanisms besides its antioxidant capacity. Angiotensin (Ang) II regulates NOX, the major reactive oxygen species (ROS) source in the cardiovascular system. We hypothesized that, by inhibiting NOX, apocynin could alter Ang II pressor and vasoconstrictor effects on SHR. We analyzed how Ang II affects blood pressure and vascular reactivity in aorta and mesenteric resistance arteries and evaluated plasma antioxidant capacity, NOX isoforms and subunits, NOS isoforms, AT1 and AT2 receptors expression, ROS production, and NOS activity in apocynin-treated SHR blood vessels (30 mg/Kg/day, p.o.). In SHR, apocynin reduced Ang II pressor effects, increased plasmatic antioxidant capacity, and blunted aortic and mesenteric NOX-dependent oxidants production and NOX2 and p47phox overexpression, which demonstrated that apocynin inhibits NOX in SHR blood vessels. Moreover, apocynin raised plasmatic and aortic nitrate/nitrite levels, maintained NOS activity and eNOS, p-eNOS, nNOS, iNOS, sGC-α, and sGC-ß expression in mesenteric bed, diminished AT1 expression in aorta and mesenteric bed, and elevated AT2 expression in SHR aorta. Apocynin increased Ang II vasoconstriction endothelial modulation in SHR resistance arteries. All these results showed that in vivo treatment with apocynin alters several mechanisms that reduce Ang II pressor and vasoconstrictor effects on SHR. Such apocynin effects involve other mechanisms besides vascular ROS modulation, which improves NO availability in SHR vascular cells. These integrated data could help us to understand the promising apocynin activity as an antihypertensive drug that acts differently from the drugs that are currently being used in the clinical setting.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction.

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.