Resistance Arteries Of Hypertensive Patients Research Articles

Abstract 064: Transactivation of Egfr by Tgfβ Induces Hypertension by Increasing Arteriolar Myogenic Response

Emilin1 (E1) is a protein of the extracellular matrix regulating TGFβ bioavailability through proTGFβ proteolysis. E1 KO mice are hypertensive with increased TGFβ activation. As E1 is expressed in vessels from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting inactivating E1 in smooth muscle cells (SMCs) of adult mice, by the use of E1flox/flox and tamoxifen (TAM) inducible Cre recombinase specific for SMCs. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (SBP: 123±2 vs basal condition 104±3;***p< 0.001) as well as myogenic tone (MT) of resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). How increased TGFβ signaling in SMCs could determine an increased MT is still unknown. Relevant to this, we found that the higher TGFβ signaling in E1 KO SMCs stimulates heparin binding epidermal growth factor (HB-EGF) and subsequent transactivation of the EGF receptor, a mechanism typically implied in potentiating MT. When mesenteric resistance arteries from E1 were subjected to step-increases in intraluminal pressure, EGFR inhibition rescued the increased MT. At the molecular level, TGFβ-induced EGFR transactivation resulted into the activation of transient receptor potential classical type 6 (TRPC6) and melastatin type 4 (TRPM4) channels. To put our data into translational perspective, we measured MT of resistance arteries isolated from hypertensive patients and normotensive subjects, finding increased MT (HT 16.4±0.7; NT 11±0.4;***p< 0.001) and TGFβ signaling in the former group. By using a neutralizing anti-TGFβ or an anti-EGFR we found a normalization of the increased MT (HT+anti-TGFβ 11±1.3; NT+anti-TGFβ 11.2±1.2;***p< 0.001 and HT+anti-EGFR 9±0.6; NT+anti-EGFR 10.8±1.1;***p< 0.001), thus confirming the relevance of TGFβ-EGFR pathway in humans. Taken together these data suggest that primary increase of MT induced by TGFβ-EGFR transactivation can cause hypertension and that higher TGFβ signaling and MT are common alterations of resistance arteries of hypertensive patients.

Abstract P159: MicroRNA Profiling in Small Resistance Arteries of Hypertensive Patients With or Without Chronic Kidney Disease

Background: Hypertension (HTN) and chronic kidney disease (CKD) are global health disorders that are epidemiologically associated. Vascular injury is an early manifestation in HTN and contributes to CKD. It is characterized by endothelial dysfunction and vascular remodeling that are accompanied by gene expression changes. MicroRNAs (miRs) are important non-coding RNA regulators of gene expression. Dysregulation of miRs has been shown in HTN and CKD, but their implication in vascular injury remains unclear. We aimed to identify differentially expressed (DE) miRs in small arteries of HTN and CKD human subjects to get further insight into pathophysiological molecular mechanisms in these conditions. Methods and Results: Normotensive, HTN (systolic blood pressure (BP) > 135 mm Hg or diastolic BP of 85-115 mm Hg with BpTRU) and CKD subjects (estimated glomerular filtration rate <60mL/min/m 2 ) (n=15-16) were studied. Small arteries were dissected from a subcutaneous gluteal biopsy under RNAse free condition and used for total RNA extraction with the mirVana miR isolation kit. cDNA libraries were prepared using the TruSeq small RNA prep kit and the TruSeq stranded total RNA prep kit, and sequenced by Illumina HiSeq 2500. DE miRs and DE mRNAs ( P <0.05) were identified using EdgeR, which found 3 up- and 6 down-regulated miRs, as well as 134 up- and 149 down-regulated mRNAs uniquely associated with HTN, 42 up- and 39 down-regulated miRs, as well as 743 up- and 348 down-regulated mRNAs uniquely associated with CKD, while 2 up-regulated miRs and 101 up- and 75 down-regulated mRNAs were found in both groups. Target Scan was used to predict DE miR targets in the DE mRNAs. Enrichment analysis showed that the HTN-associated DE miR-targeting DE mRNAs were highly enriched in gene ontology (GO) terms involved in peptidase activity, mitochondrial activity and immune response ( q <0.01), while the CKD-associated DE miR-targeting DE genes were highly enriched in GO terms involved in tube formation, fibroblast proliferation and EGF response ( q <0.001). Conclusions: DE miRs were identified in small arteries of HTN and CKD patients. Enrichment analysis in DE miR-targeting DE mRNAs revealed GO terms that could be linked to different degrees of vascular changes in HTN and CKD.

Abstract 615: Transactivation of EGFR by TGFβ Induces Hypertension by Increasing Arteriolar Myogenic Response

Myogenic tone (MT) is an intrinsic property of arteriolar smooth muscle cells (SMCs) that contract when intraluminal pressure is increased. MT is a relevant physiological mechanism to maintain stable blood flow through important organs like kidney and brain, thus preventing organ damage that could derive from increase in blood pressure (BP). On the other hand, enhancement of the basal MT could raise BP by increasing systemic vascular resistance, thus further contributing to hypertension. Several studies in animal models suggest that primary dysfunctions in SMCs can directly cause abnormalities in BP. However, in hypertensive animal models investigated so far, the increment of MT is accompanied by a stronger response to G protein coupled receptor (GPCR) agonists, thus making it difficult to distinguish whether increased vascular resistance and BP are primary effects of a more robust MT or the consequence of increased reactivity to agonists. Here we show an increased MT in mice deficient of Emilin1 , an extracellular inhibitor of TGFβ signaling, displaying a phenotype of spontaneous hypertension. We also found that the higher TGFβ signaling in Emilin1 deficient SMCs stimulates heparin binding epidermal growth factor (HB-EGF) expression and subsequent transactivation of the EGF receptor. When step-increases in intraluminal pressure are applied to mesenteric resistance arteries (MRA), the combined stimulation of mechanosensor and EGF transactivation results into activation of transient receptor potential classical type 6 (TRPC6) and melastatin type 4 (TRPM4) channels, stimulation of voltage dependent calcium channels (VDCC). To put our data into translational perspective, we measured MT on resistance arteries isolated from hypertensive patients and untreated normotensives, finding increased MT and TGFβ signaling in the former group. In addition, by using an antibody neutralizing EGFR signaling we found a normalization of the increased MT, thus confirming the relevance of TGFβ-EGFR pathway in humans. Overall our results suggest that primary increase of MT induced by TGFβ-EGFR transactivation can cause hypertension and that higher TGFβ-EGFR signaling and MT are common alterations of resistance arteries of hypertensive patients.

Abstract P255: Age and Structural Alterations in Subcutaneous Small Resistance Arteries of Hypertensive Patients

Background: It was proposed that early vascular ageing may be an important mechanism of vascular damage in large conductance arteries. However it is not known whether aging may also affect small resistance artery morphology. Patients and Methods: For this reason, we investigated 100 patients with essential hypertension. Secondary forms of hypertension were excluded according to standard clinical evaluations and biochemical or instrumental assessments. In all patients, an evaluation of small resistance arteries morphology was performed by a wire micromyographic approach (Mulvany’s technique). A small amount of subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured. Results: The age range of our populaton was 22-81 years, with a mean value of 57±12 years; 14% of them were current smokers, 32% had alterations in lipid patterns, none of them had diabetes mellitus, 58 were males and average blood pressure values were 156/95±19/12 mmHg. We found a significant correlation between M/L and age (r=0.30, p=0.002): the statistical significance of the correlation persisted after correction for counfounding variables (gender, serum cholesterol, smoking status, serum glucose, systolic or diastolic blood pressure values). A statistically significant inverse correlation was also observed between internal diameter and age (r=-0.20, p=0.046), while the correlation beween age and media thickness did not reach statistical significance (r=0.09, p=0.37). Conclusion: Our data suggest that age may affect microvascular structure in hypertensive patients. It is also possible that hypertension may anticipate the effects of physiological aging, and this should be explored in a relatively large population of normotensive subjects.

Abstract 024: The Direct Renin Inhibitor Aliskiren Improves Vasodilation and Endothelial Function in Resistance Arteries from Diabetic and Hypertensive Patients

We previously demonstrated that the direct renin inhibitor aliskiren (ALK) significantly reduced the remodeling of subcutaneous resistance arteries of hypertensive patients as compared to the angiotensin-converting enzyme inhibitor ramipril (RAM). Here we questioned whether endothelial function of resistance arteries would improve after 1 year of blood pressure (BP) control with ALK or RAM. Sixteen diabetic patients with mild essential hypertension were randomized to ALK (150-300 mg once daily, n=9) or RAM (5-10 mg once daily, n=7). Subcutaneous resistance arteries were dissected from gluteal biopsy and mounted on a pressurized micromyograph. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) and sodium nitroprusside (10 nM to 1 mM) respectively, in arteries pre-contracted with norepinephrine (10 μM). Carotid-femoral pulse wave velocity (PWV) was assessed by applantion tonometry. Forearm flow mediated dilation (FMD) was assessed by ultrasounds. The expression of P-eNOS/e-NOS and the markers of oxidative stress nitrotyrosine and LOX-1 were assessed by immunohistochemistry in resistance arteries. Patients were similar for age, sex, BMI and glycemic and metabolic control. Systolic BP was significantly and equally reduced by both ALK and RAM (153±8.9 mmHg reduced to 128±7.3 mmHg and 151±10.6 mmHg reduced to 121±12.1, respectively, P<0.01) whereas diastolic BP was significantly reduced only in ALK-treated but not in RAM-treated patients (94.2±7.17 mmHg reduced to 81.4±6.31 mmHg, P<0.01; and 84.7±12.22 reduced to 78.6±7.48, NS, respectively). PWV and FMD were similar in both groups before and after treatment. Endothelium-dependent vasodilation was improved only by ALK (max dilation 92.5±3.8% vs 50.5±14%, P<0.05) but not by RAM (max dilation 72.9±5.6% vs 61.8±14.5%, NS). Endothelium-independent vasodilation was similar in all the groups. Only ALK increased P-eNOS/eNOS expression (+44% vs before treatment, p<0.05). The markers of oxidative stress were similar in both groups before and after treatment. In conclusion ALK improved endothelial function and induced vasodilation in resistance arteries from diabetic and hypertensive patients.

Interrelationships between macro and microvascular structure and function

Macrovasculature and microvasculature are interrelated, and may influence each other. Microvascular structure is not only the site of vascular resistance but probably also the origin of most of the wave reflections generating increased central systolic blood pressure. Some index of large artery stiffness seems to be related with the media to lumen ratio of subcutaneous small resistance arteries of hypertensive patients. In addition, both microvascular structural alterations and changes in the mechanical properties of large arteries represent potent predictors of prognosis. In addition, drugs that may improve microvascular structure may also correct alterations in the mechanical properties of large arteries In fact, while β-blocking agents and diuretics have a negligible effect on microvascular structure, renin-angiotensin system antagonists and calcium entry blockers have favorable actions, improving also large artery mechanics and possibly reducing central wave reflections.

Structural alterations in subcutaneous small resistance arteries predict changes in the renal function of hypertensive patients

We have previously demonstrated that structural alterations in subcutaneous small resistance arteries of hypertensive patients, as indicated by an increased media to lumen ratio (M/L), are a potent predictor of cardiovascular events, and that a close correlation exists between serum creatinine and M/L. The aim of the present study was to assess whether M/L of subcutaneous small resistance arteries may predict subsequent changes in renal function in hypertensive patients. Sixty participants (13 normotensive participants and 47 hypertensive patients) underwent a biopsy of subcutaneous fat. Resistance-sized arteries were dissected and mounted on a wire myograph, and M/L was measured. Patients were re-evaluated after a mean follow-up period of 8.6 years. Serum creatinine, blood urea nitrogen, and uric acid were measured; glomerular filtration rate (eGFR) was estimated according to Modification of Diet in Renal Disease formula. At baseline, we observed significant correlations between M/L and serum creatinine, eGFR, blood urea nitrogen, systolic, diastolic, mean, and pulse pressure. In addition, we observed significant correlations between M/L and serum creatinine at follow-up (r = 0.57; P < 0.001), percentage changes in serum creatinine (r = 0.46; P < 0.001), eGFR at follow-up (r = -0.43; P < 0.001); percentage changes in eGFR, yearly changes in eGFR, blood urea nitrogen at follow-up, and uric acid at follow-up. A multivariate analysis in which all common cardiovascular risk factors were included showed that M/L ratio is the most potent predictor of changes in renal function. Our data suggest that structural alterations in subcutaneous small arteries may predict the time course of changes in renal function during a follow-up period of about 9 years.

Abstract 5152: Normotensive and Hypertensive Obese Patients Show the Presence of Structural and Functional Alterations in Subcutaneous Small Resistance Arteries

Structural alterations of subcutaneous small resistance arteries of hypertensive patients, as indicated by an increased media to lumen (M/L) ratio, are frequently present in hypertensive and or/diabetic patients, and may represent the earliest alterations that may be observed. In addition, M/L of small arteries have a strong prognostic significance. However, no data are available about the structure and endothelial function of small resistance arteries of obese patients. Therefore, we have investigated 16 patients with severe obesity. Six of them were normotensive, and 10 hypertensive. We compared results obtained with those observed in 12 normotensive lean controls and in 12 hypertensive lean patients. Systolic and diastolic blood pressure were evaluated by a standard sphygmomanometric approach, using appropriate cuff for obese patients. All patients underwent a biopsy of subcutaneous fat before or during surgical intervention of gastric banding or intestinal derivation. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph, according to Mulvany-Halpern technique, and M/L, media thickness, media cross-sectional area were measured. A concentration-response curve to acetylcholine (10 –9 –10 –5 Mol/l) was performed, in order to evaluate endothelial function. The results are summarized in the Table . Obese patients, independently from the presence of hypertension, diabetes and dyslipidemia, show the presence of an increased M/L and of an increased media cross-sectional area. Preliminary data suggest the presence of endothelial dysfunction, as indicated by a reduced endothelium-dependent vasodilatation, especially in hypertensive obese patients. In conclusion, our data suggest that the presence of obesity is associated to structural alterations of subcutaneous small resistance arteries, mainly characterized by hypertrophic remodeling. Table

Abstract 5418: Structural Alterations in Subcutaneous Small Resistance Arteries May Predict Changes of the Renal Function in Hypertensive Patients

We have previously demonstrated that structural alteration of subcutaneous small resistance arteries of hypertensive patients, as indicated by an increased media to lumen (M/L) ratio, has a strong prognostic significance. Among the predictors of M/L indicators of renal function are those more closely correlated. Aim of the present study was to assess whether M/L ratio can predict subsequent changes in renal function in hypertensive patients. Sixty patients (33 males; 27 females; 15 normotensives and 45 hypertensives) underwent a biopsy of subcutaneous fat at a mean age of 56 years. Resistance-sized arteries were dissected and mounted on a wire myograph according to Mulvany-Halpern technique, and M/L was measured. Patients were re-evaluated after a mean time period of 8.5 years; serum creatinine, blood urinary nitrogen (BUN) and uric acid were measured, and glomerular filtration rate (GFR) was calculated according to MDRD formula. At baseline, we observed significant correlations between M/L and serum creatinine (r=0.32; p=0.013), GFR (r= −0.26; p=0.047), BUN (r=0.37; p=0.009), systolic (r=0.45, p&lt;0.001), diastolic (r=0.29, p=0.02), mean blood pressure (r=0.38, p=0.002) and pulse pressure (r=0.40, p=0.002). In addition, we observed significant correlations between M/L and creatinine at follow up (r=0.57; p&lt;0.001), % changes in serum creatinine (r=0.46; p&lt;0.001), GFR at follow up (r= −0.43; p&lt;0.001); % changes in GFR (as ml/min r= −0.33; p=0.008), as well as yearly changes (r= −0.34, p=0.007), BUN at follow up, (r=0.55; p&lt;0.001), as well as uric acid at follow up (r=0.35; p=0.026). A multivariate analysis in which all common cardiovascular risk factors were included, showed that M/L is the most potent predictor of changes in renal function. In conclusion, our data suggest that structural alterations of subcutaneous small resistance arteries (which may be present also in other vascular beds), may predict the time-course of changes of renal function during a follow-up period of about 9 years.

Vascular remodeling, macro- and microvessels: Therapeutic implications

Macrovasculature and microvasculature are deeply interrelated, since microvascular structure is not only the site of vascular resistance but probably also the origin of most of the wave reflections generating increased central systolic blood pressure. In fact, preliminary data suggest that some index of large artery stiffness is related with the media to lumen ratio of subcutaneous small resistance arteries of hypertensive patients. Microvascular structural alterations and changes in the mechanical properties of the macrovessels represent potent predictors of prognosis. Hypertension-related damage to the micro- and macrovascular system may be corrected by pharmacological agents. Among them, beta-blocking agents and diuretics have a negligible effect on microvascular structure, while renin–angiotensin system antagonists and calcium entry blockers have favorable actions, improving large artery mechanics and possibly reducing central wave reflections.

Vascular remodeling, macro- and microvessels: Therapeutic implications

Macrovasculature and microvasculature are deeply interrelated, since microvascular structure is not only the site of vascular resistance but probably also the origin of most of the wave reflections generating increased central systolic blood pressure. In fact, preliminary data suggest that some index of large artery stiffness is related with the media to lumen ratio of subcutaneous small resistance arteries of hypertensive patients. Microvascular structural alterations and changes in the mechanical properties of the macrovessels represent potent predictors of prognosis. Hypertension-related damage to the micro- and macrovascular system may be corrected by pharmacological agents. Among them, beta-blocking agents and diuretics have a negligible effect on microvascular structure, while renin–angiotensin system antagonists and calcium entry blockers have favorable actions, improving large artery mechanics and possibly reducing central wave reflections.

Determinants of the structure of resistance‐sized arteries in hypertensive patients

Objective. It has been previously demonstrated that structural alterations of subcutaneous small resistance arteries of hypertensive patients, as indicated by an increased media to lumen (M/L) ratio, is the most potent predictor of cardiovascular events. The aim of the present study was to identify possible determinants of small resistance artery structure that may be evaluated with non‐invasive approaches. Materials and methods. One hundred and ninety‐nine subjects (normotensives, essential hypertensives and patients with secondary hypertension) were included in the present study. All subjects were submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Small resistance arteries were dissected and mounted on an isometric myograph, and M/L ratio was measured. All patients underwent standard biochemical tests, clinic blood pressure measurement, standard echocardiography and 24‐h ambulatory blood pressure measurement. Glomerular filtration rate (GFR) was calculated according to MDRD study formula and Cockroft's formula. Results. Significant correlation was found between M/L ratio and, respectively: GFR calculated both with MDRD study formula and Cockroft–Gault formula, creatinine serum, blood urea nitrogen, glycaemia, circulating sodium, clinical pulse pressure, stroke volume to pulse pressure ratio, clinical systolic, diastolic and mean arterial pressure, daytime pulse pressure. However, in a multivariate regression analysis, only serum creatinine remained in the model, and proved to be an independent predictor of small artery structure. Conclusions. Indices of renal function and, probably, of large artery distensibility may be related to small arteries remodelling in hypertension.

Mechanism(s) of Systolic Blood Pressure Reduction and Drug Therapy in Hypertension

Antihypertensive drug therapy is a major modality of cardiovascular (CV) disease prevention, especially for stroke, congestive heart failure, and renal insufficiency. Coronary risk is also consistently prevented by antihypertensive treatment, but to a lesser extent.1–4 The Prime Study2 has shown that drug-induced blood pressure (BP) reduction in hypertensive subjects is associated with a significant decrease of coronary risk. However, when treated and untreated subjects are compared, at any given value of systolic BP (SBP), this risk remains higher in treated than in untreated subjects, suggesting the persistence of residual coronary complications. Results of therapeutic trials also indicate that coronary risk is associated with a particular hemodynamic pattern in treated hypertensive subjects.3 Although diastolic BP (DBP) is significantly lowered by drug treatment to <90 mm Hg (80% of the patients), SBP remains above 140 mm Hg in 60%, indicating an increase of pulse pressure (PP), which is the difference between SBP and DBP.4 It is widely accepted that SBP and PP rise sharply with age and that this increase is a major manifestation of vascular aging and stiffening, which are now considered major predictors of CV risk, independent of the mean BP (MBP) level.4 The purpose of this review is to analyze the mechanism(s) of SBP in untreated and chronically treated hypertensive subjects and to determine which pathophysiological factors are susceptible to attenuating consistently coronary and CV complications in hypertensive populations. Ventricular ejection in humans is associated with 2 principal events. First, the coronary circulation is transiently interrupted, as the principal consequence of cardiac contraction, and, second, an acute shock of stroke volume against the aortic wall is observed (Figure 1). Then, the BP curve may be considered as a wave, which travels along the arterial tree at a given speed, the pulse wave velocity (PWV). …

Lack of prognostic role of endothelial dysfunction in subcutaneous small resistance arteries of hypertensive patients

The presence of endothelial dysfunction in the coronary circulation or in the brachial artery has been found to be associated with a greater incidence of cardiovascular events. However, no data are presently available about the prognostic role of endothelial dysfunction in human small resistance arteries. Ninety subjects were included in the present study. They were: 10 normotensive subjects, 36 patients with essential hypertension, 10 patients with phaeochromocytoma, 11 patients with primary aldosteronism, 10 patients with renovascular hypertension, and 13 normotensive patients with non-insulin-dependent diabetes mellitus (NIDDM). All subjects were submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Small resistance arteries were dissected and mounted on an isometric myograph, and the concentration-response curves to acetylcholine (from 10 to 10 mol/l) (endothelium-dependent vasodilatation) and sodium nitroprusside (from 10 to 10 mol/l) (endothelium-independent vasodilatation) after precontraction of the vessels with norepinephrine were evaluated. The subjects were re-evaluated (by clinical visits or telephone interviews) after an average follow-up time of 5.5 years. Twenty-nine subjects had a documented fatal or non-fatal cardiovascular event (5.87%/year). The endothelium-dependent vasodilatation in the subcutaneous small arteries was similar in subjects with or without cardiovascular events. Also, endothelium-independent vasodilatation to sodium nitroprusside was similar in the two groups. Similar results were obtained by subdividing patients in the different subgroups (essential hypertension, secondary hypertension, etc.). Our results indicate that endothelial dysfunction in the microcirculation does not predict cardiovascular events. It is possible that a prognostic role of endothelial dysfunction may be observed when other vascular districts prone to atherosclerosis are evaluated, or it might be detected only in patients at low to medium cardiovascular risk, in whom endothelial dysfunction is less advanced.

Lack of Prognostic Role of Endothelial Dysfunction in Subcutaneous Small Resistance Arteries of Hypertensive Patients

Lack of Prognostic Role of Endothelial Dysfunction in Subcutaneous Small Resistance Arteries of Hypertensive Patients

Redox-dependent signalling by angiotensin II and vascular remodelling in hypertension.

1. Hypertension is associated with structural alterations of resistance arteries, a process known as remodelling (increased media-to-lumen ratio). 2. At the cellular level, vascular remodelling involves changes in vascular smooth muscle cell (VSMC) growth, cell migration, inflammation and fibrosis. These processes are mediated via multiple factors, of which angiotensin (Ang) II appears to be one of the most important in hypertension. 3. Angiotensin II signalling, via AT1 receptors, is upregulated in VSMC from resistance arteries of hypertensive patients and rats. This is associated with hyperactivation of vascular NADPH oxidase, leading to increased generation of reactive oxygen species (ROS), particularly O2- and H2O2. 4. Reactive oxygen species function as important intracellular second messengers to activate many downstream signalling molecules, such as mitogen-activated protein kinase, protein tyrosine phosphatases, protein tyrosine kinases and transcription factors. Activation of these signalling cascades leads to VSMC growth and migration, modulation of endothelial function, expression of pro-inflammatory mediators and modification of extracellular matrix. 5. Furthermore, ROS increase intracellular free Ca2+ concentration ([Ca2+]i), a major determinant of vascular reactivity. 6. All these processes play major roles in vascular injury associated with hypertension. Accordingly, ROS and the signalling pathways that they modulate provide new targets to regress vascular remodelling, reduce peripheral resistance and prevent hypertensive end-organ damage. 7. In the present review, we discuss the role of ROS as second messengers in AngII signalling and focus on the implications of these events in the processes underlying vascular remodelling in hypertension.

Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients.

The role of c-Src in growth signaling by angiotensin (Ang) II was investigated in vascular smooth muscle cells (VSMCs) from arteries of hypertensive patients. c-Src and extracellular signal-regulated kinase 1/2 (ERK1/2) activity, proto-oncogene expression, activating protein-1 (AP-1) DNA-binding activity, and DNA and protein synthesis were studied in Ang II-stimulated VSMCs derived from small peripheral resistance arteries of normotensive subjects (NTs, n=5) and age-matched untreated hypertensive patients (HTs, n=10). Ang II type 1 (AT(1)) and type 2 (AT(2)) receptor status was also assessed. Ang II dose-dependently increased the synthesis of DNA and protein, with enhanced effects in VSMCs from HTs. PD 098,059, a selective inhibitor of the ERK1/2 pathway, attenuated Ang II-stimulated growth in HTs. The effects of PD 098,059 were greater in HTs than in NTs. In NTs, Ang II transiently increased ERK1/2 phosphorylation, whereas in HTs, Ang II-stimulated actions were augmented and sustained. PP2, a selective Src inhibitor, reduced ERK1/2 activity and normalized ERK1/2 responses in HTs. Ang II-induced c-Src phosphorylation was 2- to 3-fold greater in HTs than in NTs. In HTs but not NTs, kinase activation was followed by overexpression of c-fos and enhanced AP-1 DNA-binding activity. PD 098,059 and PP2 attenuated these responses. AT(1) receptor expression was similar in NTs and HTs. In HT cells transfected with c-fos antisense oligodeoxynucleotide, Ang II-stimulated growth was reduced compared with sense oligodeoxynucleotide. Our findings suggest that augmented Ang II-stimulated VSMC growth is mediated via hyperactivation of c-Src-regulated ERK1/2-dependent pathways, leading to overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. Because AT(1) receptor expression was unaltered in HTs, increased Ang II signaling may be a postreceptor phenomenon. These data define a signal transduction pathway whereby Ang II mediates exaggerated growth in VSMCs from HTs.

Increased generation of superoxide by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients: role of phospholipase D-dependent NAD(P)H oxidase-sensitive pathways.

We tested the hypothesis that increased responsiveness of phospholipase D (PLD) to angiotensin II (Ang II) is associated with increased oxidative stress and exaggerated growth responses in vascular smooth muscle cells (VSMC) from untreated essential hypertensive patients. VSMCs from peripheral resistance arteries of normotensive and hypertensive subjects were studied. Production of reactive oxygen species (ROS) was measured with the fluoroprobe 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). PLD and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase were assessed with the inhibitors, dihydro-D-erythro-sphingosine (sphinganine) and diphenylene iodinium (DPI), respectively, and protein kinase C (PKC) effects were determined using chelerythrine chloride and calphostin C. PLD activity was measured by the transphosphatidylation assay. Ang II increased the CM-H2DCFDA fluorescence signal, derived predominantly from H2O2. Ang II-induced generation of DPI-inhibitable ROS was significantly enhanced in cells from hypertensives compared with normotensives (Emax = 72 +/- 2 versus 56.9 +/- 1.8 fluorescence units, P< 0.01). PLD inhibition attenuated Ang II-induced ROS generation, with greater effects in the hypertensive group than the normotensive group (delta = 42 +/- 3.3 versus 21 +/- 2 units). PKC inhibition partially decreased Ang II-elicited signals. Ang II-stimulated PLD activity and DNA and protein synthesis were significantly greater in cells from hypertensives than normotensives. These effects were normalized by DPI and sphinganine. Our results suggest that in essential hypertension enhanced oxidative stress and augmented growth-promoting actions of Ang II are associated with increased activation of PLD-dependent pathways. These processes may contribute to vascular remodeling in hypertension.

Correlation of endothelial function in large and small arteries in human essential hypertension.

The structure and function of blood vessels varies along the vascular tree, and alterations found in hypertension are also different. The aim of this study was to determine whether non-invasive measurement of endothelial function in conduit arteries reflects that of subcutaneous resistance arteries measured in vitro. Sixteen essential hypertensive patients (aged 50 +/- 2 years) were studied. Flow-mediated dilation (FMD) during reactive hyperemia (endothelium-dependent) and sublingual nitroglycerin (NTG)-induced dilatation (endothelium-independent) were assessed in brachial arteries by ultrasound. Structure, and acetylcholine (10(-9) to 10(-4) mol/l) and sodium nitroprusside (SNP, 10(-8) to 10(-3) mol/l)-induced vasorelaxation of resistance arteries dissected from gluteal subcutaneous biopsies were measured in vitro using a pressurized myograph. Brachial artery FMD and NTG-induced dilatation were 8.4 +/- 1.0 and 18.1 +/- 1.4%, respectively. Resistance arteries of hypertensive patients showed greater media:lumen ratio (8.6 +/- 0.4 versus 5.9 +/- 0.3% in normotensive subjects, P< 0.01), and maximal acetylcholine responses was diminished to 75 +/- 6% compared to normotensive subjects (97 +/- 2%, P< 0.01). FMD correlated with maximal acetylcholine responses (r2 = 0.57, P< 0.001). FMD did not correlate significantly with the media: lumen ratio of resistance arteries (r2 = -0.22, P= 0.07). By multivariate analysis, FMD predicted resistance artery endothelial function independently of age, sex, body mass index, blood lipid status and lumen diameter of brachial artery (beta = 0.81, P< 0.001). Endothelial dilatory responses are similar in large and small arteries in hypertensive patients. Abnormal FMD in the brachial artery predicts the presence of endothelial dysfunction in human resistance arteries, suggesting that impairment of endothelial function is a generalized alteration in hypertension. Ultrasound measurement of endothelial dysfunction in the brachial artery appears to be less sensitive than in-vitro measurement in resistance arteries.

Effects of long-term antihypertensive treatment with lisinopril on resistance arteries in hypertensive patients with left ventricular hypertrophy.

To evaluate the effects of long-term antihypertensive therapy with the angiotensin converting enzyme inhibitor lisinopril on structural alterations and the endothelial function of small resistance arteries in hypertensive patients with left ventricular hypertrophy. Fourteen patients with left ventricular hypertrophy were treated for 3 years with a lisinopril-based regimen. Patients underwent an echocardiographic evaluation of left ventricular mass index at baseline, during the first and third years of treatment. At the end of the treatment period, subcutaneous small resistance arteries (obtained by biopsy of the subcutaneous fat from the gluteal region) were dissected and mounted on a micromyograph (Mulvany's technique); the media : lumen ratio was then calculated. Data obtained were compared with those observed for 14 untreated essential hypertensive patients and 14 normotensive subjects, age- and sex-matched. In the present study, a significantly lower media : lumen ratio was observed in treated compared with untreated hypertensive patients, although it remained significantly higher than that in normotensive subjects. In treated hypertensive patients a significant reduction in clinic blood pressure was observed. However, their blood pressure remained significantly higher than that in normotensive subjects. Significant correlations between the media : lumen ratio and blood pressure, left ventricular mass index or changes in left ventricular mass index during treatment were observed. The response to acetylcholine administration was reduced in untreated hypertensives compared with that in normotensives. In patients treated with lisinopril, the vasodilatation obtained with the two higher doses of acetylcholine was greater than that in untreated hypertensives, thus suggesting an improvement of endothelial function. Long-term therapy based on lisinopril was associated with a smaller media : lumen ratio in the subcutaneous small resistance arteries of hypertensive patients with left ventricular hypertrophy. Our retrospective study confirms previous findings obtained in prospective studies with other angiotensin converting enzyme inhibitors. Endothelial function was probably improved by lisinopril therapy.