Interleukin-2 enhances angiogenesis and preserves cardiac function following myocardial infarction.

We previously demonstrated that injection of IL-2-activated natural killer (NK) cells contribute to vascular remodeling via a4b7 integrin and killer cell lectin-like receptor (KLRG) 1 and promote cardiac repair following myocardial infarction (MI). The aim of the present study is to test the hypothesis that injection of recombinant human interleukin (rhIL)-2 improves angiogenesis and preserves heart function after MI. A single IV injection of rhIL-2 two days following MI improved by 27.7% the left ventricular (LV) fractional shortening of immune competent (C57Bl6) mice, but had no effect on cardiac function of immune-deficient (NOD-SCID IL2Rγnull) mice. Immunohistochemical analysis of C57Bl6 cross sections of heart revealed that collagen deposition was reduced by 23.1% and that capillary density was enhanced in the scar area and the border zone of the infarct respectively by 22.4% and 33.6% following rhIL-2 injection. In addition, rhIL-2 enhanced 1.6-fold the in vivo endothelial cell proliferation index and 1.8-fold the number of NK cell infiltrating the infarcted heart, but had no effect on the number of cardiac CD4 and CD8 cells. In vitro, rhIL-2 activated NK cells enhanced cardiac endothelial cell proliferation by 17.2%. Here we show that a single IV injection of rhIL-2 positively impacted cardiac function by improving angiogenesis through a process involving NK cells.

Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways.

OBJECTIVE: Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs from WKY rats were studied. At low concentrations (10(-10) mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone (Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 (inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. CONCLUSIONS: Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Endothelin-1, but not Ang II, activates MAP kinases through c-Src independent Ras-Raf dependent pathways in vascular smooth muscle cells.

OBJECTIVE: Endothelin-1 (ET-1) and angiotensin II (Ang II) activate common signaling pathways to promote changes in vascular reactivity, remodeling, inflammation, and oxidative stress. Here we sought to determine whether upstream regulators of mitogen-activated protein kinases (MAPKs) are differentially regulated by ET-1 and Ang II focusing on the role of c-Src and the small GTPase Ras. METHODS AND RESULTS: Mesenteric vascular smooth muscle cells (VSMCs) from mice with different disruption levels in the c-Src gene (c-Src(+/-) and c-Src(-/-)) and wild-type (c-Src(+/+)) were used. ET-1 and Ang II induced extracellular signal-regulated kinase (ERK) 1/2, SAPK/JNK, and p38MAPK phosphorylation in c-Src(+/+) VSMCs. In VSMCs from c-Src(+/-) and c-Src(-/-), Ang II effects were blunted, whereas c-Src deficiency had no effect in ET-1-induced MAPK activation. Ang II but not ET-1 induced c-Src phosphorylation in c-Src(+/+) VSMCs. Activation of c-Raf, an effector of Ras, was significantly increased by ET-1 and Ang II in c-Src(+/+) VSMCs. Ang II but not ET-1-mediated c-Raf phosphorylation was inhibited by c-Src deficiency. Knockdown of Ras by siRNA inhibited both ET-1 and Ang II-induced MAPK phosphorylation. CONCLUSIONS: Our data indicate differential regulation of MAPKs by distinct G protein-coupled receptors. Whereas Ang II has an obligatory need for c-Src, ET-1 mediates its actions through a c-Src-independent Ras-Raf-dependent pathway for MAPK activation. These findings suggest that Ang II and ET-1 can activate similar signaling pathways through unrelated mechanisms. MAP kinases are an important point of convergence for Ang II and ET-1.

Achieving blood pressure goals globally: five core actions for health-care professionals. A worldwide call to action.

The prevalence of hypertension continues to rise across the world, and most patients who receive medical intervention are not adequately treated to goal. A Working Group including representatives of nine international health-care organizations was convened to review the barriers to more effective blood pressure control and propose actions to address them. The group concluded that tackling the global challenge of hypertension will require partnerships among multiple constituencies, including patients, health-care professionals, industry, media, health-care educators, health planners and governments. Additionally, health-care professionals will need to act locally with renewed impetus to improve blood pressure goal rates. The Working Group identified five core actions, which should be rigorously implemented by practitioners and targeted by health systems throughout the world: (1) detect and prevent high blood pressure; (2) assess total cardiovascular risk; (3) form an active partnership with the patient; (4) treat hypertension to goal and (5) create a supportive environment. These actions should be pursued with vigour in accordance with current clinical guidelines, with the details of implementation adapted to the economic and cultural setting.

Synergistic vascular protective effects of combined low doses of PPARalpha and PPARgamma activators in angiotensin II-induced hypertension in rats.

BACKGROUND AND PURPOSE: Protective cardiovascular effects of peroxisome proliferator activated receptor (PPAR)alpha and PPARgamma activators have been demonstrated. If used as vasoprotective agents in high risk vascular patients rather than for their metabolic benefits, these agents could be associated with unwanted side effects. As a proof of concept to support the use of combined low doses of PPARalpha and PPARgamma as vascular protective agents in high risk vascular patients, we tested the hypothesis that combined low doses of PPARalpha (fenofibrate) and PPARgamma (rosiglitazone) activators would provide vascular protective benefits similar to full individual doses of these PPAR agonists. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats infused with Ang II (120 ng kg(-1) min(-1)) were treated with rosiglitazone (1 or 2 mg kg(-1) day(-1)) alone or concomitantly with fenofibrate (30 mg kg(-1) day(-1)) for 7 days. Thereafter, vessels was assessed on a pressurized myograph, while NAD(P)H oxidase activity was determined by lucigenin chemiluminescence. Inflammation was evaluated using ELISA for NFkappaB and Western blotting for adhesion molecules. KEY RESULTS: Ang II-induced blood pressure increase, impaired acetylcholine-induced vasorelaxation, altered vascular structure, and enhanced vascular NAD(P)H oxidase activity and inflammation were significantly reduced by low dose rosiglitazone+fenofibrate. CONCLUSIONS AND IMPLICATIONS: Combined low doses of PPARalpha and PPARgamma activators attenuated development of hypertension, corrected vascular structural abnormalities, improved endothelial function, oxidative stress, and vascular inflammation. These agents used in low-dose combination have synergistic vascular protective effects. The clinical effects of combined low-dose PPARalpha and PPARgamma activators as vascular protective therapy, potentially with reduced side-effects and drug interactions, should be assessed.

p47phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: role in NAD(P)H oxidase regulation by angiotensin II.

OBJECTIVE: We tested the hypothesis that p47phox associates with the actin cytoskeleton, enabling site-directed activation of NAD(P)H oxidase, and assessed whether these actions influence reactive oxygen species (ROS) generation and signaling by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) from human resistance and coronary arteries. METHODS AND RESULTS: Electroporation of anti-p47phox antibody into VSMCs abrogated Ang II-mediated O2 generation, establishing the requirement for p47phox in this response. Immunfluorescence confocal microscopy demonstrated a cytosolic distribution of p47phox in basal conditions. After Ang II stimulation, p47phox rearranged in a linear fashion, colocalizing with F-actin. Co-immunoprecipitation studies confirmed an association between p47phox and actin and demonstrated an interaction with the actin-binding protein cortactin. Cytoskeletal disruption with cytochalasin prevented p47phox:actin interaction and attenuated ROS formation and p38MAP kinase and Akt phosphorylation by Ang II. Intracellular ROS generation in response to LY83583 (O2 generator) or exogenous H2O2 and Ang II-induced ERK1/2 activation were unaltered by cytochalasin. CONCLUSIONS: The p47phox:actin interaction, through cortactin, plays an important role in Ang II-mediated site-directed assembly of functionally active NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAP kinase and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox signaling by Ang II in human VSMCs.

Correction of arterial structure and endothelial dysfunction in human essential hypertension by the angiotensin receptor antagonist losartan.

BACKGROUND: Structural and functional alterations of the vasculature may contribute to complications of hypertension. Because angiotensin II may be pivotal in some of these vascular abnormalities, we tested the hypothesis that the angiotensin type 1 (AT(1)) receptor antagonist losartan, in contrast to the beta-blocker atenolol, would correct resistance artery abnormalities in patients with essential hypertension. METHODS AND RESULTS: Nineteen untreated patients with mild essential hypertension (47+/-2 years, range 30 to 65 years; 57% male) were randomly assigned in double-blind fashion to losartan or atenolol treatment for 1 year. Nine age/sex-matched normotensive subjects were also studied. Both treatments reduced blood pressure to a comparable degree (losartan, from 149+/-4.1/101+/-1.6 to 128+/-3.6/86+/-2.2 mm Hg, P<0.01; atenolol, from 150+/-4.0/99+/-1.2 to 130+/-3.2/84+/-1.4 mm Hg, P<0.01). Resistance arteries (luminal diameter 150 to 350 microm) dissected from gluteal subcutaneous biopsies were studied on a pressurized myograph. After 1 year of treatment, the ratio of the media width to lumen diameter of arteries from losartan-treated patients was significantly reduced (from 8.4+/-0.4% to 6.7+/-0.3%, P<0.01). Arteries from atenolol-treated patients exhibited no significant change (from 8. 3+/-0.3% to 8.8+/-0.5% after treatment). Endothelium-dependent relaxation (acetylcholine-induced) was normalized by losartan (from 82.1+/-4.9% to 94.7+/-1.1%, P<0.01) but not by atenolol (from 80. 4+/-2.7% to 81.7+/-4.6%). Endothelium-independent relaxation (by sodium nitroprusside) was unchanged after treatment. CONCLUSIONS: The AT(1) antagonist losartan corrected the altered structure and endothelial dysfunction of resistance arteries from patients with essential hypertension, whereas the beta-blocker atenolol had no effect.

Resistance artery mechanics, structure, and extracellular components in spontaneously hypertensive rats : effects of angiotensin receptor antagonism and converting enzyme inhibition.

BACKGROUND: Altered vascular mechanics resulting from changes in collagen and integrins may influence resistance artery structure and function and, therefore, peripheral resistance and blood pressure in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: Effects of age, angiotensin-converting enzyme inhibition (fosinopril, 10 to 30 mg/kg per day), and AT(1)-receptor antagonism (irbesartan, 50 mg/kg per day) on vascular structure, mechanics, and composition were assessed in SHR. Systolic blood pressure was elevated in young SHR (130+/-2 mm Hg) compared with Wistar-Kyoto (WKY) rats (106+/-2 mm Hg). In adult SHR, the rise in systolic blood pressure (44+/-3 mm Hg) was blunted by fosinopril (18+/-1 mm Hg) and irbesartan (9+/-3 mm Hg). Lumen diameter of mesenteric resistance arteries was smaller and media/lumen ratio was greater in young and adult SHR versus WKY rats. Growth index was 24% in untreated adult SHR versus WKY rats; these values were -35% for fosinopril-treated and -29% for irbesartan-treated SHR versus untreated SHR. Isobaric wall stiffness was normal despite increased stiffness of wall components in adult SHR vessels. Irbesartan partially prevented stiffening of wall components in SHR. The collagen/elastin ratio was greater in adult SHR vessels (6.5+/-1.3) than in WKY (3.2+/-0.4) vessels. Expression of alpha(v)beta(3) and alpha(5)beta(1) integrins was increased in SHR aged 20 versus 6 weeks. Expression of alpha(5)beta(1) integrins was lower in young SHR, and alpha(v)beta(3) integrins were overexpressed in adult SHR versus WKY rats. Irbesartan and fosinopril attenuated differences in the collagen/elastin ratio and integrin expression. CONCLUSIONS: Wall components of mesenteric resistance arteries stiffen with age in SHR. Interrupting the renin-angiotensin system has normalizing effects on integrin expression and composition, stiffness, and growth of the arterial wall.

c-Src induces phosphorylation and translocation of p47phox: role in superoxide generation by angiotensin II in human vascular smooth muscle cells.

OBJECTIVE: The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived *O2- in human vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs from human small arteries were studied. Ang II increased NAD(P)H oxidase-mediated generation of *O2- and H2O2 (P<0.01). PP2, c-Src inhibitor, attenuated these effects by 70% to 80%. Immunoprecipitation of p47phox, followed by immunoblotting with antiphosphoserine antibody, demonstrated a rapid increase (1.5- to 2-fold) in p47phox phosphorylation in Ang II-stimulated cells. This was associated with p47phox translocation from cytosol to membrane, as assessed by immunoblotting and immunofluorescence. PP2 abrogated these effects. Long-term Ang II stimulation (6 to 24 hours) increased NAD(P)H oxidase subunit expression. c-Src inhibition decreased abundance of gp91phox, p22phox, and p47phox. Confirmation of c-Src-dependent regulation of NAD(P)H oxidase was tested in VSMCs from c-Src-/- mice. Ang II-induced *O2- generation was lower in c-Src-/- than c-Src+/+ counterparts. This was associated with decreased p47phox phosphorylation, blunted Ang II-stimulated NAD(P)H oxidase activation, and failure of Ang II to increase subunit expression. CONCLUSIONS: c-Src regulates NAD(P)H oxidase-derived *O2- generation acutely by stimulating p47phox phosphorylation and translocation and chronically by increasing protein content of gp91phox, p22phox, and p47phox in Ang II-stimulated cells. These novel findings identify NAD(P)H oxidase subunits, particularly p47phox, as downstream targets of c-Src.

Endothelin receptor subtypes in resistance arteries from humans and rats.

OBJECTIVE: Endothelins bind to different receptor subtypes identified as ETA and ETB receptors, ETA receptors are present on smooth muscle cells of blood vessels. ETB receptors seem to predominate on endothelial cells of blood vessels, but may also be present on smooth muscle in some vascular beds. The aim of this study was to investigate the distribution of ETA and ETB receptors in human and in rat resistance arteries. METHODS: Contractile responses to endothelin-1 and to the ETB agonist sarafotoxin S6c were investigated in human subcutaneous resistance arteries and in rat mesenteric resistance arteries mounted on a wire myograph. RESULTS: Contractile responses to endothelin-1 were significantly reduced by the ETA selective antagonists BQ123 or BQ610 in human resistance arteries, and were almost abolished by the antagonists in rat arteries. The ETB agonist sarafotoxin S6c induced smaller responses than endothelin-1. When responses to sarafotoxin S6c were obtained in the presence of BQ610, the relatively small contractile response remained. It was slightly enhanced if the responses were obtained in the presence of the nitric oxide synthase inhibitor L-NAME or after removal of the endothelium. CONCLUSIONS: Vasoconstrictor ETB receptors appear to be present in resistance vessels isolated from subcutaneous fat in humans and from the mesentery in rats, but their contribution to endothelin mediated constriction is small, particularly in rat resistance arteries. ETA receptors appear to be the more important endothelin receptor subtype in the human and rat resistance arteries examined.

Glomerular and vascular atrial natriuretic factor receptors in cardiomyopathic hamsters: correlation with the peptide biological effects.

STUDY OBJECTIVE: The aim was to study glomerular and vascular atrial natriuretic factor (ANF) receptors and their relationship with the post-receptor effects of the peptide in experimental heart failure. DESIGN: Binding sites ANF were studied in renal glomerular and mesenteric artery membranes. The natriuretic and relaxing effects of ANF were evaluated in the intact animal and in noradrenaline precontracted aortic strips respectively. Plasma and tissue ANF levels were also assessed. EXPERIMENTAL MATERIAL: The study was performed on cardiomyopathic (UM-X7.1) hamsters (n = 15) with a moderate degree of heart failure. Age matched Golden Syriam hamsters (n = 15) were used as controls. MEASUREMENTS AND MAIN RESULTS: Cardiomyopathic hamsters presented lower blood pressure, body weight, and plasma Na+, and higher heart weight than normal hamsters. Plasma ANF (1-98) and (99-126) levels and ventricular ANF content were higher in cardiomyopathic hamster than in controls. ANF and frusemide decreased blood pressure, and increased diuresis and natriuresis in normal hamsters. The blood pressure reduction by ANF in cardiomyopathic hamsters was approximately of the same magnitude as in normal hamsters but their renal response was blunted. The blood pressure lowering effect of frusemide was similar in both cardiomyopathic and normal hamsters, but the diuretic and natriuretic responses were greatly reduced in the former. Glomerular ANF receptor density was higher and receptor affinity was lower in cardiomyopathic hamsters than in controls. Noradrenaline precontracted vascular strips from cardiomyopathic hamster were more sensitive to the relaxant effect of ANF than those from controls. No differences in either density or affinity of vascular receptor were observed. CONCLUSIONS: The results suggest that the renal hyporesponsiveness of cardiomyopathic hamsters to ANF is not due to a down regulation of glomerular ANF receptors. The fact that the natriuretic response to frusemide is also blunted suggest the involvement of other factors.

Vascular atrial natriuretic factor receptors in spontaneously hypertensive rats.

OBJECTIVE: The aim was to investigate vascular receptors for atrial natriuretic factor (ANF) in spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats (WR) at different ages. METHODS: Relaxation and guanylate cyclase responses of blood vessels to atrial natriuretic factor were investigated, as was the binding of 125I-ANF to vascular membranes and ANF receptor subtypes, using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in reducing conditions, after solubilisation and irreversible binding of 125I-ANF. RESULTS: Vascular relaxation responses of aorta showed an increased sensitivity to ANF in four week old SHR [pD2 = 8.9 (SEM 0.1) v 8.5(0.1) in WKY rats, p < 0.05] while sensitivity was similar for the three strains at older ages. Production of cyclic GMP in mesenteric arteries in response to 100 nmol.litre-1 ANF was greater (p < 0.05) in SHR than in WKY rats at four weeks of age, but was similar in older rats. The density of binding sites for ANF in mesenteric arteries, however, was lower in SHR at four weeks (p < 0.01), and increased in older rats, becoming similar to that of normotensive rats at 12 weeks of age. Affinity of ANF sites was similar in all strains. The proportion of high and low molecular weight ANF binding peptides in solubilised blood vessel membranes on SDS-PAGE was similar in all strains except in four week old SHR, in which binding to the high molecular weight band (presumably the guanylate cyclase containing receptor) was increased relative to the low molecular weight band (non-cyclase-coupled receptor) in comparison to other strains and ages. CONCLUSIONS: Activity of guanylate cyclase in response to occupancy of ANF receptors may be increased in young SHR. Normal relaxation of blood vessels in response to ANF in older SHR could result in failure to counteract the increased vasoconstrictor activity present in these rats, which could play a role in the increase in blood pressure.

3H-vasopressin binding to the rat mesenteric artery.

We investigated the binding of 3H-Arg8-vasopressin to membranes from rat mesenteric arteries. Specific binding of 3H-vasopressin was 60-75% of total binding. Binding at 22 C achieved a plateau at 30 min whereas at 4 C binding was significantly slower. Binding was reversible upon addition of 1 microM Arg8-vasopressin after 30 min of incubation. Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant of 5.1 +/- 0.6 nM and a total binding capacity of 91 +/- 12 fmol/mg protein. Competitive inhibition of 3H-Arg8-vasopressin binding showed an IC50 of 3 nM for Arg8-vasopressin, 14 nM for [I-(beta-mercapto-beta-beta-cyclopentamethylene-propionic, 4-valine, 8-D-arginine]-vasopressin, 31 nM for oxytocin, 52 nM for I-deamino-8-D-arginine-vasopressin, 0.1 microM for [I-deamino-penicillamine, 4-valine, 8-D-arginine]-vasopressin, and 0.8 microM for desglycinamide-deamino-Arg8-vasopressin. Unrelated peptides did not displace 3H-Arg8-vasopressin. We conclude that these binding sites possess characteristics of physiologically relevant vasopressin receptors in vascular smooth muscle of a resistance type vessel.

Endothelin: potential role in hypertension and vascular hypertrophy.

The endothelins are a family of 21-amino acid peptides that are powerful vasoconstrictors. They may also induce vascular hypertrophy. These peptides may participate through these two mechanisms in the pathogenesis of the elevation of blood pressure and/or in the maintenance of hypertension in both experimental animal models and human essential hypertension. This review presents evidence both in favor and against the involvement of endothelins in hypertension. Plasma levels of endothelin-1 are either normal or slightly elevated in experimental and human essential hypertension. Responses of blood vessels to endothelin-1 may be normal or depressed in many models of experimental hypertension and also in essential hypertension in humans. It has recently been demonstrated that endothelin content and mRNA are increased in blood vessels of deoxycorticosterone acetate-salt hypertensive rats. When endothelin receptor antagonists are administered chronically, elevation of blood pressure and development of vascular hypertrophy are blunted in this experimental model of hypertension. In contrast, spontaneously hypertensive rats do not exhibit any increase in either endothelin-1 mRNA or immunoreactive endothelin in blood vessels and fail to respond with lowering of blood pressure to longterm treatment with endothelin receptor antagonists. Blood pressure development in young spontaneously hypertensive rats is also unaffected by long-term administration of endothelin antagonists. Molecular genetic studies appear to support a genetic role of components of the endothelin system in Dahl salt-sensitive rats. In human essential hypertension, there is some evidence of activation of the endothelin system despite depressed responses of small arteries to endothelin-1 and normal circulating levels of endothelin-1 in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivity of small blood vessels in hypertension: relation with structural changes. State of the art lecture.

Small blood vessels, particularly small arteries of 150-300 microns in lumen diameter and larger arterioles of 50-150 microns in lumen diameter, are the most important location of the arterial bed that undergoes changes resulting in the increased peripheral resistance that characterizes elevated blood pressure. This article reviews these morphological and functional alterations of small blood vessels. Study of mesenteric small arteries with a lumen diameter of 220-260 microns revealed consistently a reduced external and lumen diameter in renal and deoxycorticosterone acetate-salt hypertensive rats early in the evolution of hypertension. The media of the vessel wall was significantly thickened in the hypertensive rats, and the media/lumen ratio was increased. Although the tension that developed in response to different vasoconstrictors was not elevated or was even decreased in hypertensive rats, exaggerated transmural pressures resulted in the isolated blood vessels as a consequence of the law of Laplace because of the narrowed lumen. Similar findings were obtained in small resistance arteries from subcutaneous gluteal biopsies of hypertensive humans. In arterioles, on the other hand, decreases in the density of blood vessels (rarefaction) and in vasomotion amplitude may play a more important role than reductions in lumen diameter. As a consequence of the design of small resistance blood vessels and as a result of functional and structural alterations, which may be primary to or a consequence of high blood pressure, the pressor effect of vasoconstrictors is amplified and interacts with other factors to contribute to the maintenance of elevated blood pressure even if the intrinsic response of vascular smooth muscle to these agents is not exaggerated.

State-of-the-Art lecture. Role of endothelin-1 in hypertension.

Endothelin-1 (ET-1) is overexpressed in the vascular wall in certain models of experimental hypertension: deoxycorticosterone acetate salt-treated rats, deoxycorticosterone acetate salt-treated spontaneously hypertensive rats (SHR), stroke-prone SHR, Dahl salt-sensitive rats, angiotensin II-infused rats, and 1-kidney 1 clip Goldblatt rats; it is not overexpressed in SHR, 2-kidney 1-clip hypertensive rats, or L-NAME-treated rats. In hypertensive rats without generalized vascular overexpression, however, expression of ET-1 was often enhanced in intramyocardial coronary arteries, suggesting a role of endothelin in myocardial ischemia in hypertension. In rats overexpressing ET-1, ET(A/B) and ET(A)-selective receptor antagonists lowered blood pressure and reduced vascular growth, particularly in small arteries, beyond what could be attributed to blood pressure lowering, suggesting a direct effect of ET-1 on growth. Hypertensive rats treated with endothelin antagonists are protected from stroke and renal injury. The ET(A/B) antagonist bosentan induced blood-pressure reductions in mildly hypertensive patients similar to those achieved with an angiotensin-converting enzyme inhibitor. Moderately to severely hypertensive patients presented with enhanced expression of prepro-ET-1 mRNA in the endothelium of subcutaneous resistance arteries, suggesting that these stages of hypertension may respond particularly well to endothelin antagonism. Hypertensive patients with coronary artery disease have increased arterial expression of ET-1, and increased plasma levels of immunoreactive endothelin have been described in black patients. ET-1 plays an important role in atherosclerosis, for which hypertension is an important risk factor. Thus, ET-1 may be involved in experimental and human hypertension. Endothelin antagonists may prove effective as disease-modifying agents if they are shown clinically, as they are experimentally, to offer target organ protection and reduce long-term complications of hypertension. This remains to be demonstrated in humans.

Tyrosine kinase signaling pathways modulate angiotensin II-induced calcium ([Ca2+]i) transients in vascular smooth muscle cells.

Tyrosine kinases have been implicated in vascular smooth muscle cell proliferation and contraction. Underlying mechanisms may involve C(a2+) -dependent pathways. This study assesses relationships between angiotensin II (Ang II)-stimulated phospholipase C-mediated Ca2+ transients and tyrosine kinase-dependent pathways in vascular smooth muscle cells. Intracellular free Ca2+ concentration ([Ca2+]i) was measured in primary cultured unpassaged vascular smooth muscle cells derived from mesenteric resistance vessels of Wistar-Kyoto rats with the use of fura 2 methodology. [Ca2+]i effects of Ang II (1 nmol/L) were determined in vascular smooth muscle cells in which tyrosine kinase pathways were stimulated by insulin (70 muU/mL; 0.5 nmol/L), insulin-like growth factor-I (1 ng/mL; 0.13 nmol/L), or platelet-derived growth factor-BB (1 ng/mL; 0.04 nmol/L) and in cells in which tyrosine kinase was inhibited by specific inhibitors (1 mumol/L tyrphostin A-23 and genistein). Ang II elicited a rapid and transient [Ca2+]i response (from 94 +/- 8 to 239 +/- 5.8 nmol/L). Activation of the receptor tyrosine kinase by insulin, platelet-derived growth factor, and insulin-like growth factor-I significantly reduced (P < .01) Ang II-induced [Ca2+]i to 161 +/- 7, 189 +/- 3.7, and 183 +/- 5 nmol/L, respectively. In the presence of tyrphostin A-23 and genistein, Ang II-stimulated [Ca2+]i remained persistently elevated and failed to return to basal levels. Tyrphostin A-1, the inactive tyrphostin analogue, had not significant effect on Ang II-induced [Ca2+]i. This study demonstrates that activation of tyrosine kinase pathways reduces Ang II-elicited [Ca2+]i responses, whereas tyrosine kinase inhibition prevents [Ca2+]i recovery after agonist stimulation. Interaction between tyrosine kinase- and phospholipase C-dependent signaling pathways modulates vascular smooth muscle cell [Ca2+]i responses to Ang II.

Effects of angiotensin II and endothelin-1 on platelet aggregation and cytosolic pH and free Ca2+ concentrations in essential hypertension.

The aims of this study were to determine the relations between platelet free calcium concentrations ([Ca2+]i), intracellular pH (pHi), and aggregation and to assess the effects of angiotensin II (Ang II) and endothelin-1 on these platelet parameters in normotensive subjects and hypertensive patients. Seventeen normotensive subjects, 25 untreated hypertensive patients, and 34 treated hypertensive patients were studied. Platelet cytosolic free [Ca2+]i and pHi were measured spectrofluorometrically using specific fluorescent probes (fura 2-AM and BCECF-AM, respectively) in unstimulated and Ang II- and endothelin-1-stimulated platelets. Aggregation was measured by a turbidometric technique. Basal [Ca2+]i (141 +/- 11 nmol/L) and pH (7.16 +/- 0.01) were higher (P < .05) in the untreated hypertensive group compared with the normotensive (118 +/- 9 nmol/L, 7.11 +/- 0.01, respectively) and treated hypertensive (121 +/- 11 nmol/L, 7.12 +/- 0.01, respectively) groups. In the combined normotensive and hypertensive groups, there were significant correlations between [Ca2+]i and mean arterial pressure (r = .75, P < .01), pHi and mean arterial pressure (r = .72, P < .01), [Ca2+]i and pHi (r = .71, P < .01), [Ca2+]i and aggregation (r = .69, P < .02), and pHi and aggregation (r = .56, P < .05). Ang II stimulation significantly increased [Ca2+]i and pHi in the untreated hypertensive and normotensive groups. The net change in [Ca2+]i induced by Ang II was significantly higher (P < .05) in the untreated hypertensive group compared with the other groups (67 +/- 6 nmol/L for the untreated hypertensive group versus 54 +/- 5 and 29 +/- 8 nmol/L for the normotensive and treated hypertensive groups, respectively). In the presence of Ang II, thrombin-induced aggregatory responses were increased in all three groups, but the maximal response was significantly higher in the untreated hypertensive group compared with the other groups (P < .05). Endothelin-1 increased pHi through endothelin A-receptors (effect blocked by the specific antagonist BQ-123) but had no significant effect on [Ca2+]i or aggregation. However, endothelin-1 blunted thrombin-induced platelet aggregation in normotensive subjects but not in hypertensive patients. In conclusion, increased Ang II-stimulated [Ca2+]i and pHi in platelets of essential hypertensive patients may be associated with increased aggregatory responses. The stimulatory effect of endothelin-1 on pHi but not on [Ca2+]i or aggregation suggests that in platelets endothelin-induced signaling pathways other than phospholipase C may be involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of chronic treatment of adult spontaneously hypertensive rats with an endothelin receptor antagonist.

We previously showed that endothelin-1 expression was increased in vascular endothelium of deoxycorticosterone acetate-salt hypertensive rats, whereas in spontaneously hypertensive rats (SHR) it is similar to or less than that in normotensive rats. Treatment with the combined endothelin type A/endothelin type B receptor antagonist bosentan moderately reduced blood pressure rise and nearly completely blunted the development of vascular hypertrophy, particularly in small arteries, in the deoxycorticosterone acetate-salt hypertensive model, suggesting a paracrine role for vascular endothelin-1 in the induction of blood vessel hypertrophy in some forms of experimental hypertension. In the present study we examined the effect of chronic oral treatment for 4 weeks of 12-week-old SHR and Wistar-Kyoto rats (WKY) with 100 mg/kg per day bosentan. Blood pressure rose to hypertensive levels similarly in bosentan-treated and untreated SHR; systolic pressure of WKY was also unaffected. The wet weights of the heart, of aortic segments, and of the mesenteric arterial bed were similar in treated and untreated SHR. When coronary, renal arcuate, mesenteric, and femoral small arteries were evaluated on a wire myograph, the media width and media-to-lumen ratio were greater and the lumen diameter was smaller in vessels from SHR relative to those from WKY, except in small arteries from the renal cortex, in which the lumen was not significantly different in both strains. The media cross-sectional area of small arteries fom the four vascular beds was similar in both strains. Identical morphometric parameters were found in the four vascular beds in bosentan-treated and untreated rats of eh strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of effects of angiotensin I-converting enzyme inhibition and beta-blockade for 2 years on function of small arteries from hypertensive patients.

The effect of treatment with two different antihypertensive agents on the function of small arteries from 17 patients with essential hypertension randomly assigned to receive either the angiotensin I-converting enzyme inhibitor cilazapril or the beta-blocker atenolol was investigated. Subcutaneous small arteries obtained from gluteal fat biopsies were studied on a wire myograph before treatment and at 1 and 2 years of treatment. Blood pressure was mildly elevated in both groups of patients (mean, 150/100 mm Hg) and was well controlled throughout the 2 years of treatment (mean, 130/85 mm Hg). We previously reported, in arteries from patients treated with cilazapril, an improvement at 1 year of treatment of the vasoconstrictor effect of endothelin-1, which had been significantly attenuated in the untreated hypertensive patients compared with normotensive subjects. After 2 years of treatment, this normalization of endothelin-1 response was still present in small arteries of patients treated with the angiotensin I-converting enzyme inhibitor, whereas in patients treated with atenolol, responses were still unchanged after 2 years of treatment. Endothelial function was tested by examining the response of norepinephrine-precontracted arteries to acetylcholine. Untreated hypertensive patients exhibited a slightly but significantly blunted vasorelaxation in response to 10 mumol/L acetylcholine compared with normotensive subjects. After 1 and 2 years of effective antihypertensive treatment, cilazapril-treated patients exhibited responses to acetylcholine that were not different from those of normotensive subjects, whereas atenolol-treated patients still had impaired responses.(ABSTRACT TRUNCATED AT 250 WORDS)