Vascular angiotensin AT1 receptor neuromodulation in fetal programming of hypertension.

Fetal stress increases the susceptibility to cardiovascular diseases in adult age, including hypertension, a process known as fetal programming of hypertension (FPH). This study intends to investigate the interplay between vascular sympathetic nervous system (SNS) and RAS, namely the neuromodulatory role exerted by Angiotensin II (Ang II) receptor-1 (AT1) in FPH, and respective contribution for hypertension. METHODS: 6-month old Sprague-Dawley offspring from mothers fed ad-libitum (CONTROL) or with 50% intake during the second half of gestation (maternal undernutrition, MUN) were used. Sympathetic neurotransmission was studied in mesenteric/tail arteries and mesenteric veins by electrically-evoked [3H]-noradrenaline release experiments using RAS drugs. AT1 receptors in sympathetic nerves of mesenteric arteries were investigated by immunohistochemistry and Laser Scanning Confocal Microscopy. RESULTS: Ang II facilitated noradrenaline release in the vessels studied from MUN and CONTROL rats. Losartan induced a tonic facilitation only in MUN vessels. Sympathetic innervation was larger in MUN versus CONTROL vessels. AT1 receptors on sympathetic nerves were present in higher amounts in MUN versus CONTROL vessels. CONCLUSIONS: Findings support that FPH is associated with a vascular hyper-sympathetic activation, involving a tonic facilitation of prejunctional AT1 receptors by endogenous Ang II, which can justify, at least in part, the development of hypertension.

Effect of fetal undernutrition and postnatal overfeeding on rat adipose tissue and organ growth at early stages of postnatal development.

Intrauterine and perinatal life are critical periods for programming of cardiometabolic diseases. However, their relative role remains controversial. We aimed to assess, at weaning, sex-dependent alterations induced by fetal or postnatal nutritional interventions on key organs for metabolic and cardiovascular control. Fetal undernutrition was induced by dam food restriction (50 % from mid-gestation to delivery) returning to ad libitum throughout lactation (Maternal Undernutrition, MUN, 12 pups/litter). Postnatal overfeeding (POF) was induced by litter size reduction from normally fed dams (4 pups/litter). Compared to control, female and male MUN offspring exhibited: 1) low birth weight and accelerated growth, reaching similar weight and tibial length by weaning, 2) increased glycemia, liver and white fat weights; 3) increased ventricular weight and tendency to reduced kidney weight (males only). Female and male POF offspring showed: 1) accelerated growth; 2) increased glycemia, liver and white fat weights; 3) unchanged heart and kidney weights. In conclusion, postnatal accelerated growth, with or without fetal undernutrition, induces early alterations relevant for metabolic disease programming, while fetal undernutrition is required for heart abnormalities. The progression of cardiac alterations and their role on hypertension development needs to be evaluated. The similarities between sexes in pre-pubertal rats suggest a role of sex-hormones in female protection against programming.

Mechanical strength of the isolated carotid artery in SHR.

We have previously reported an adaptation of arterial wall elasticity in spontaneously hypertensive rats (SHR) that involves an increase in both fibronectin/alpha5beta1-integrin complexes and smooth-muscle elastic lamellae connections. We examined the mechanical strength (MS) of the carotid artery in relation to its elastic properties, its elastin/collagen content, and the structure of the internal elastic lamina. MS was defined as the in vitro intraluminal pressure and wall stress that produces rupture of the vascular wall. Intact carotid arteries from 3-month-old normotensive rats (Wistar-Kyoto, WKY) and SHR were cannulated on a specially designed device and adjusted to their in situ length. A slowly increasing static pressure was applied until wall rupture occurred to determine the static mechanical behavior and MS. Static elasticity was similar in SHR and WKY, as were the rupture pressure (2740+/-90 versus 2740+/-40 mm Hg) and wall stress at rupture (11.5+/-1.0 versus 12.8+/-0.4 MPa), indicating equivalent MS in both groups. Histological examination showed several wall ruptures and dissociation of lamellar units that did not differ significantly between the 2 groups. Confocal microscopy showed that the size of fenestrations of the internal elastic lamina and the fraction of area occupied by them were reduced 3-fold in SHR. We have demonstrated that static elasticity of the arterial wall and mechanical strength are similar in carotid arteries from SHR and WKY.

Effect of removal of adventitia on vascular smooth muscle contraction and relaxation.

The aim of the present study was to determine whether the adventitia of large arteries modulates vascular function. We developed a method to obtain functional vascular rings devoid of adventitia. Carotid and iliac arteries from 3-mo-old Sprague-Dawley rats were denuded from adventitia after treatment with collagenase followed by gentle peeling. Adventitia removal and integrity of the media was demonstrated by optical and confocal microscopy. Arterial rings with or without adventitia and with or without endothelium were mounted in an organ bath for isometric tension recording. Responses to 75 mM KCl or norepinephrine (0.1 nM-1 microM) were significantly reduced in segments without adventitia. Acetylcholine-induced relaxation (0.1 microM-0.1 mM) was enhanced in arteries without adventitia, whereas sodium nitroprusside-induced responses were not modified. These results demonstrate that the combination of stripping with a previous collagenase treatment allows us to obtain functional rings devoid of adventitia and that this layer plays a role in contractile capacity and in endothelium-modulated responses.

Fenestrations of the carotid internal elastic lamina and structural adaptation in stroke-prone spontaneously hypertensive rats.

Our aim was to determine the structural factors that determine the mechanical adaptation of the carotid arterial wall in stroke-prone hypertensive rats (SHRSP). Distensibility-pressure and elastic modulus-stress curves assessed by in vivo echo-tracking measurements indicated a reduction in arterial stiffness in 13-week-old SHRSP compared with Wistar-Kyoto rats (WKY). Elastin and collagen contents determined biochemically were not different between SHRSP and WKY. Confocal microscopy showed that the mean area of fenestrations and fraction of area occupied by fenestrations of the internal elastic lamina (IEL) were smaller in SHRSP than in WKY, which indicated a reduction in stress-concentration effects within the IEL. Immunohistologic staining of EIIIA fibronectin isoform and total fibronectin (also as determined by Western blot) was greater in SHRSP, which suggested increased cell-matrix interactions. We suggest that these structural modifications of the vascular wall play a synergistic role in the mechanical adaptation to a high level of stress in SHRSP.

Measurements of vascular remodeling by confocal microscopy.

The introduction of myographs was crucial for the study of structure and function of resistance arteries. The ability to support and maintain small blood vessels paved the way for true microscopic studies of the vascular cells. However, even after decades of study, we still do not know very much about the "normal" arrangement of smooth muscle cells in the vascular wall and how their distribution affects function. It was clearly time for the next technological step forward. We have shown here how the combination of myography and confocal microscopy creates a platform for the study of vascular structure at the cellular level and in 3D. In addition, the possibility of using live myograph-mounted vessels in combination with LSCM opens a new field of research to assess vascular remodeling from a physiological point of view and to study vascular function at a level not achieved by any other method at present. Now that the hardware is in place it is time to concentrate on the software and improve the methods of analysis. We have used 2D analysis of 3D data sets to describe differences in vascular structure and, at the same time, developed methods to semiautomate the process. The success of the 3D methods will ultimately depend on the reliability and accuracy of the analysis routines. There are still problems to overcome en route to finding a complete solution. However, we believe that the search for a robust fully (or semi-) automated method of 3D analysis will be more than worthwhile. We have defined vascular remodeling to include any changes in cellular arrangement or morphology. However, on a more subtle level, changes in receptors, enzymes, and proteins leading to altered functionality could also be regarded as remodeling. In that respect it may be interesting to map the distribution of the many receptors, channels, and proteins that regulate vascular growth, death, and function. Currently, there is a growing list of fluorescent ligands and antibodies that can be used in conjunction with confocal microscopy. It is possible that multiple stains could be used and imaged at different wavelengths with a view to constructing full 3D models of various structures and their colocalization. It is our belief that the confocal approach will prove to be a major tool in unraveling the complexities of cell-cell interactions and arrangements and will allow a better understanding of the process of vascular remodeling and function.

Functional reduction and associated cellular rearrangement in SHRSP rat basilar arteries are affected by salt load and calcium antagonist treatment.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a strain with high incidence of cerebrovascular accidents increased by salt-rich diet and decreased by calcium-antagonist treatment. In the SHRSP rat basilar artery the authors have previously shown reduced contractility and altered structure including regions of smooth muscle cell (SMC) disorganization. The aims of this study have been to analyze (1) the morphology of these abnormal regions, (2) the structural modifications responsible for the reduced function, and (3) the effect of salt and calcium-antagonist treatment on vascular structure and function. Wistar Kyoto and SHRSP rats, untreated or treated from week 8 through 14 with 1% NaCl or 1% NaCl + 1 mg x kg(-1) x d(-1) lacidipine, were used. Function was studied with wire myography. Structure was analyzed in fixed intact arteries with confocal microscopy. Basilar arteries from SHRSP rat showed (1) reduced contractility, (2) discrete foci of SMC disarray with altered proportion of adventitia to SMC, and (3) decreased SMC and increased adventitial cell number. Arteries from salt-loaded SHRSP rats showed a higher degree of SMC disarray and further reduction in contractility. Lacidipine treatment of salt-loaded rats significantly improved structure and function. These data suggest that vascular remodeling can provide an explanation for the observed reduction in vascular contractility of SHRSP rat basilar arteries and might show light on the effects of salt load and calcium-channel blockers in life span and the incidence of cerebrovascular accidents in SHRSP rats.

Genes encoding atrial and brain natriuretic peptides as candidates for sensitivity to brain ischemia in stroke-prone hypertensive rats.

-Previous studies suggested that atrial natriuretic peptide gene (Anp) and brain natriuretic peptide gene (Bnp) are plausible candidate genes for susceptibility to stroke and for sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). We performed structural and functional analyses of these 2 genes in SHRSP from Glasgow colonies (SHRSPGla) and Wistar-Kyoto rats from Glasgow colonies (WKYGla) and developed a radiation hybrid map of the relevant region of rat chromosome 5. Sequencing of the coding regions of the Anp and Bnp genes revealed no difference between the 2 strains. Expression studies in brain tissue showed no differences at baseline and at 24 hours after middle cerebral artery occlusion. Plasma concentrations of atrial natriuretic peptide (ANP) did not differ between the SHRSPGla and WKYGla, whereas concentrations of brain natriuretic peptide were significantly higher in the SHRSPGla as compared with the WKYGla (n=11 to 14; 163+/-21 pg/mL and 78+/-14 pg/mL; 95% confidence interval 31 to 138, P=0.003). We did not detect any attenuation of endothelium-dependent relaxations to bradykinin or ANP in middle cerebral arteries from the SHRSPGla; indeed the sensitivity to ANP was significantly increased in arteries harvested from this strain (WKYGla: n=8; pD2=7. 3+/-0.2 and SHRSPGla: n=8; pD2=8.2+/-0.15; P<0.01). Moreover, radiation hybrid mapping and fluorescence in situ hybridization allowed us to map the Anf marker in the telomeric position of rat chromosome 5 in close proximity to D5Rat48, D5Rat47, D5Mgh15, and D5Mgh16. These results exclude Anp and Bnp as candidate genes for the sensitivity to brain ischemia and pave the way to further congenic and physical mapping strategies.

Cellular aspects of vascular remodeling in hypertension revealed by confocal microscopy.

Cellular aspects of remodeling in intact arteries have not been fully investigated, mainly due to the lack of an appropriate methodology that allows for simple measurements. The aim of this study was to develop a method based on laser scanning confocal microscopy (LSCM), compare it with previous methodology, and apply it to the study of remodeling in hypertension. The morphology of mesenteric resistance arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) was determined with wire myography on one segment with a standardized diameter setting (0.9[d100]) and with perfusion myography on a second segment from the same artery at the calculated equivalent pressure. The second segments were stained with the nuclear dye Hoechst 33342 (live tissue) or propidium iodide (fixed tissue) and measured with LSCM and MetaMorph software. Compared with wire myography, perfusion myography showed similar differences from those previously reported. Compared with LSCM, perfusion myography showed a similar lumen but significantly smaller wall thickness in both live and fixed tissue, probably due to measurement underestimation. In the study with LSCM, arteries from SHRSP compared with those from WKY showed (1) reduced lumen, (2) altered cell density that was significantly increased in the adventitia, decreased in the media, and unchanged in the intima, (3) significantly increased medial volume, (4) significantly smaller endothelial cell nuclei, and (5) adventitial-like cells in the media. We conclude that (1) LSCM is a reliable and straightforward method to study morphology in intact vessels, (2) it provides new information on the cellular changes in remodeling, (3) adventitia might play an active role in the process of remodeling in hypertension, and (4) endothelium "remodels" in hypertension.

Impairment of vasodilator function in basilar arteries from aged rats.

BACKGROUND AND PURPOSE: Aging is associated with a reduction in cerebral perfusion. Impaired vasodilatation in large brain arteries could be implicated. This study sought age-related changes in vasodilator responses to norepinephrine in rat basilar artery and investigated which aspects of norepinephrine's action are responsible. To study the effect of aging per se, we used the rat, an animal with resistance to development of age-related pathologies. METHODS: Vascular responses were studied in basilar arteries from young (3 to 4 months old) and old (20 to 22 months old) normotensive Sprague-Dawley rats with wire myography. Endothelial structure was assessed with confocal microscopy. RESULTS: There was no age-related difference in blood pressure and in KCl or 5-hydroxytryptamine (5-HT) contractions. Relaxation to bradykinin or its absence predicted an intact or denuded endothelium, confirmed by confocal microscopy. Norepinephrine produced concentration-dependent relaxation that was significantly smaller in old rats, with or without endothelium. This response was significantly smaller in endothelium-denuded vessels, or after preincubation with NG-nitro-L-arginine methyl ester or propranolol, but not with rauwolscine. CONCLUSIONS: In old and young rats the vasodilator action of norepinephrine in basilar artery is dependent on beta-adrenoceptors and nitric oxide. The impaired vasodilatation to norepinephrine found in the basilar artery from old rats might be caused by (1) a reduction in nitric oxide production and/or release or (2) beta-adrenoceptor alteration at the endothelium and/or the vascular smooth muscle. This impairment of vasodilator function can be ascribed to the aging process per se and not to other age-related alterations, such as hypertension.

Alpha 1-adrenoceptor vasoconstriction in the tail artery during ageing.

1. We have studied the alpha 1-adrenoceptor-mediated responses in intact tail artery rings from 3-4 and 20-22 months old Sprague-Dawley rats, focusing on possible endothelial alterations. The influence of nitric oxide released by the endothelium, the number of alpha 1-adrenoceptors and the functional receptor reserve were evaluated to determine their contribution to the contractile response mediated by this receptor. The state of the endothelial layer was assessed by confocal microscopy. 2. Noradrenaline (1 nM-100 microM) induced concentration-dependent vasoconstriction. The maximum contractions to noradrenaline (P < 0.05) and to 75 mM KCl (P < 0.01) were higher in young than in old animals. 3. The density (Bmax) of alpha 1-adrenoceptors and the dissociation constant (KD) obtained in [3H]-prazosin binding experiments were unchanged by age. 4. The apparent affinity (pKA) and the percentage of functional receptors (qx 100) remaining after phenoxybenzamine (0.03 microM) were similar in both age groups. 5. After partial alpha 1-adrenoceptor inactivation with phenoxybenzamine, NG-nitro-L-arginine methylester (30 microM) significantly potentiated the E/[A] curve to noradrenaline in young rats. However, only responses to 0.1 to 1 microM noradrenaline were significantly potentiated in old animals. In addition, 94% of the vessels from young, but only 52% from old rats were relaxed by 80-100% of the noradrenaline (0.03 microM) contraction, with 1 microM acetylcholine. 6. No modifications in the area (micron2) or in the number of endothelial nuclei (per mm2) were observed between age groups. An elongation of the nuclei of endothelial cells was observed in the old animals. 7. These data suggest that the noradrenaline-induced contraction is decreased in old rats probably due to differences in either the contractile machinary or postreceptor mechanisms. These alterations may be accompanied by an impairment of the release or production of NO from endothelial cells.

Cellular changes induced by chronic nitric oxide inhibition in intact rat basilar arteries revealed by confocal microscopy.

BACKGROUND: Cellular aspects of remodelling have not been investigated fully in intact vessels due to lack of appropriate methodology. OBJECTIVE: To determine the cellular alterations induced by chronic inhibition of nitric oxide (NO) production in intact rat basilar arteries by combined use of perfusion myography and a laser scanning confocal microscope. METHODS: Wistar-Kyoto rats were treated with 10 mg/kg per day NG-nitro-L-arginine methyl ester (L-NAME) for 3 weeks. Basilar arteries from treated and age-matched Wistar-Kyoto rat controls were mounted on a perfusion myograph, stained with the nuclear dye Hoechst 33342 and fixed under pressure. The segments were mounted on a slide and visualized using the 364 nm line of a laser scanning confocal microscope. MetaMorph software was used to obtain optical sections from the vessel and for morphology determinations. RESULTS: L-NAME treatment induced hypertension (systolic blood pressure control 129.2+/-2.7 mmHg and SBP L-NAME treatment 176.3+/-5.2 mmHg, P< 0.001). Compared with control rat arteries, arteries from treated rats had a reduced lumen diameter, similar wall thickness and an increased wall: lumen ratio. L-NAME treatment induced specific changes in adventitia, media and intima, namely an increase in number of adventitial cells and in adventitia thickness, a reduction in number of smooth muscle cells with no change in media thickness and reductions in number of endothelial cells, size of nuclei and luminal surface area. CONCLUSIONS: Hypertension induced by chronic inhibition of NO production is associated with eutrophic remodelling of rat basilar artery. However, within this overall maintenance of constant volume, there are marked cellular changes in adventitia, media and intima. The separate contributions of inhibition of NO production and hypertension to the remodelling process need to be elucidated.

Confocal microscopic characterization of a lesion in a cerebral vessel of the stroke-prone spontaneously hypertensive rat.

BACKGROUND AND PURPOSE: Hypertension is a major risk factor for stroke and is associated with alterations in vascular structure and function. The aim of this study was to determine vascular function, wall morphology, and vascular smooth muscle cell (VSMC) arrangement in basilar arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control strain Wistar-Kyoto rats (WKY). The effect of perindopril treatment on SHRSP structure and function was also assessed. METHODS: VSMC orientation was determined with laser-scanning confocal microscopy and computer-assisted image processing in basilar arteries stained with 5(6)-carboxyfluorescein (wavelengths: excitation, 488; emission, 515) or propidium iodide (excitation, 529; emission, 550). Measurements of wall morphology and functional responses to serotonin and KCl were assessed with wire myography. RESULTS: In the WKY basilar arteries, VSMCs were uniformly oriented perpendicular to the longitudinal axis of the vessel, whereas in the SHRSP there were localized foci of VSMC geometric disorganization, with a significant deviation from 90 degrees. The SHRSP basilar arteries also showed structural remodeling and reduced contractile responses to serotonin and KCl. Perindopril treatment normalized blood pressure, prevented wall morphology alterations, and improved function but had no effect on VSMC disorganization. CONCLUSIONS: This is the first demonstration of lesions of VSMC geometric disorganization in a cerebral artery from a stroke-prone genetically hypertensive rat strain. These structural abnormalities are independent of blood pressure. Their functional sequel may play a role in the pathogenesis of stroke in this model.

Noradrenergic transmission in the tail artery of hypertensive rats transgenic for the mouse renin gene Ren-2.

1. The aim of the present study was to analyse the noradrenergic transmission in the tail artery of hypertensive rats transgenic for the mouse renin gene Ren-2 (TGR) in comparison with its control, the Sprague-Dawley (SD) rat. 2. Electrical field stimulation (EFS) of vascular segments produced frequency-dependent vasoconstrictions that were significantly greater in TGR arteries. 3. These contractions were abolished by tetrodotoxin (0.1 microM). Phentolamine (50 nM) and prazosin (1 - 10 nM) produced an inhibition of these responses that was significantly greater in SD arteries, whereas that produced by yohimbine (0.5-1 microM) was higher in TGR arteries. In both strains, propranolol (1 microM) potentiated the responses to EFS, and this increase was observed at lower frequencies in TGR arteries. 4. The EFS-evoked [3H]-noradrenaline (NA) release was significantly greater in TGR than in SD rats. However, NA (10 nM-10 microM) reduced and yohimbine and phentolamine (10 nM-10 microM) increased the tritium outflow to a similar degree in both strains. 5. Exogenous NA also induced greater vasoconstriction in TGR arteries. 6. These results suggest the existence in TGR tail artery of an increase in: (a) NA-release and alpha 2-adrenoceptor-mediated contractions, which could contribute to the elevated blood pressure in these rats; and (b) beta-adrenoceptor-mediated vasodilatations, which may be a mechanism to counteract high blood pressure.

Confocal microscopy for structure and real-time pharmacology in blood vessels.

The structure and composition of living small blood vessels can be studied in great detail in three dimensions using confocal microscopy. Individual cells and their components can be visualised by vital dyes for the nucleus, cytoplasm or extracellular space. Specific ligands can then localise individual components such as membrane receptors with great precision. Cell function is unaffected, allowing the study, in real time, of the changing relation and contribution to vascular contraction or dilatation of different cell types particularly smooth muscle, endothelium and adventitia. This allows not only visualisation but quantification, using image analysis software. These techniques will be of particular value in assessing the contribution of form to function in pathological situations such as vascular remodelling in hypertension.