Erythropoietin-induced hypertension and vascular injury in mice overexpressing human endothelin-1: exercise attenuated hypertension, oxidative stress, inflammation and immune response

OBJECTIVE: Erythropoietin used to correct anaemia in chronic kidney disease (CKD) has been shown to increase blood pressure (BP) in CKD patients and experimental animals. Endothelin (ET)-1 expression is increased in CKD animals and patients, and enhanced by erythropoietin. Erythropoietin-induced BP rise was blunted by ETA receptor blockers. This study was designed to determine whether preexisting endothelin (ET)-1 overexpression is required for erythropoietin to cause adverse vascular effects and whether this could be prevented by exercise training. METHODS: Eight to 10-week old male wild-type mice and mice with endothelial-specific ET-1 overexpression (eET-1) were treated or not with EPO (100 IU/kg, SC, 3 times/week). eET-1 was subjected or not to swimming exercise training (1 h/day, 6 days/week) for 8 weeks. SBP, mesenteric artery endothelial function and remodelling, NADPH oxidase activity, reactive oxygen species (ROS) generation, vascular cell adhesion protein (VCAM)-1, monocyte/macrophage infiltration, T regulatory cells (Tregs) and tissue ET-1 and plasma endothelin were determined. RESULTS: Erythropoietin increased SBP by 24 mmHg (P < 0.05) and decreased by 25% vasodilatory responses to acetylcholine (P < 0.01) in eET-1 mice. Erythropoietin enhanced ET-1 induced increase in resistance artery media/lumen ratio (31%, P < 0.05), aortic NADPH oxidase activity (50%, P < 0.05), ROS generation (93%, P < 0.001), VCAM-1 (80%, P < 0.01) and monocyte/macrophage infiltration (159%, P < 0.001), and raised plasma and aortic ET-1 levels (>/=130%, P < 0.05). EPO had no effect in wild-type mice. Exercise training prevented all of the above (P < 0.05). CONCLUSION: Erythropoietin-induced adverse vascular effects are dependent on preexisting elevated ET-1 expression. Exercise training prevented erythropoietin-induced adverse vascular effects in part by inhibiting ET-1 overexpression-induced oxidative stress, inflammation and immune activation

Vascular Remodeling in Health and Disease

The term vascular remodeling is commonly used to define the structural changes in blood vessel geometry that occur in response to long-term physiologic alterations in blood flow or in response to vessel wall injury brought about by trauma or underlying cardiovascular diseases.1, 2, 3, 4 The process of remodeling, which begins as an adaptive response to long-term hemodynamic alterations such as elevated shear stress or increased intravascular pressure, may eventually become maladaptive, leading to impaired vascular function. The vascular endothelium, owing to its location lining the lumen of blood vessels, plays a pivotal role in regulation of all aspects of vascular function and homeostasis.5 Thus, not surprisingly, endothelial dysfunction has been recognized as the harbinger of all major cardiovascular diseases such as hypertension, atherosclerosis, and diabetes.6, 7, 8 The endothelium elaborates a variety of substances that influence vascular tone and protect the vessel wall against inflammatory cell adhesion, thrombus formation, and vascular cell proliferation.8, 9, 10 Among the primary biologic mediators emanating from the endothelium is nitric oxide (NO) and the arachidonic acid metabolite prostacyclin [prostaglandin I2 (PGI2)], which exert powerful vasodilatory, antiadhesive, and antiproliferative effects in the vessel wall

Activation of the Na+-H+ exchanger modulates angiotensin II stimulated Na+-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension

This study investigated the role of the Na+-H+ exchanger (NHE) on angiotensin II (Ang II)–induced activation of Na+-dependent Mg2+ transport in vascular smooth muscle cells (VSMCs) from Wistar-Kyoto rats (WKY; n=20) and spontaneously hypertensive rats (SHR; n=20). Intracellular free concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) and intracellular pH (pHi) were measured with the specific fluorescent probes mag–fura 2-AM, SBFI-AM, and BCECF-AM, respectively. Na+ dependency of Mg2+ transport was assessed in Na+-free buffer, and the role of the NHE was determined with the highly selective NHE blocker 5-(N-methyl-N-isobutyl) amiloride (MIA). Basal [Mg2+]i was lower in SHR than WKY (0.59±0.01 versus 0.71±0.01 mmol/L, P<0.05). Basal pHi and [Na+]i were not different between the 2 groups. Ang II dose dependently increased [Na+]i and pHi and decreased [Mg2+]i. Responses were significantly greater (P<0.05) in SHR versus WKY ([Na+]i Emax=37.5±1.1 versus 33.7±1.9 mmol/L; pHi Emax=7.35±0.04 versus 7.20±0.01; [Mg2+]i Emin=0.28±0.09 versus 0.53±0.02 mmol/L, SHR versus WKY). In Na+-free buffer, Ang II–elicited [Mg2+]i responses were inhibited. MIA (1 μmol/L) inhibited Ang II–stimulated responses in WKY and normalized responses in SHR ([Mg2+]i Emin=0.49±0.02). Ang II–stimulated activation of NHE was significantly increased (P<0.05) in SHR (0.07±0.002 ΔpHi/s) compared with WKY (0.05±0.004 ΔpHi/s). These data demonstrate that in VSMCs [Mg2+]i regulation is Na+ dependent, that activation of NHE modulates Na+-Mg2+ transport, and that increased activity of NHE may play a role in altered Na+-dependent regulation of [Mg2+]i in SHR

Role of protein kinase C in the antiaggregatory effects of endothelin-l on human platelets

1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10(-11) to 10(-6) mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD2 for inhibition by endothelin = 8.1 +/- 0.12). 3. Endothelin-1 at 10(-9) mol/l significantly decreased (P < 0.01) thrombin-stimulated aggregation from 81.4 +/- 1.5% (in the absence of endothelin-1) to 53.5 +/- 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 +/- 1.7 nmol/l to 140 +/- 1.8 nmol/l. 4. Preincubation of platelets with 10(-7) mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na(+)-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration

Angiotensin II regulates vascular smooth muscle cell ph, contraction, and growth via tyrosine kinase dependent signaling pathways

Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca2+ concentration ([Ca2+]i) and pH (pHi). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II–stimulated pHi, and in simultaneously measured contractile and [Ca2+]i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pHi was determined by fluorescent digital imaging using 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca2+]i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. The Ang II receptor subtypes (AT1 or AT2) through which Ang II mediates effects were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD 123319 (a selective AT2 antagonist). To determine whether tyrosine kinases influence Ang II–stimulated responses, cells were pretreated with 10−5 mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pHi in a dose-dependent manner (pD2, 9.2±0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca2+]i (pD2, 7.4±0.1). Ang II (10−7 mol/L) increased DNA ([3H]thymidine incorporation) and protein synthesis ([3H]leucine incorporation). [Sar1,Ile8]Ang II and losartan but not PD 123319 abolished Ang II–elicited responses. Tyrphostin A-23 significantly attenuated Ang II–stimulated pHi responses; it also inhibited [Ca2+]i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II–stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II–mediated pHi responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT1 receptor subtype–mediated contraction and growth