C.J. Daly

Role of Elastin in Spontaneously Hypertensive Rat Small Mesenteric Artery Remodelling

Chronic hypertension is associated with resistance artery remodelling and mechanical alterations. However, the contribution of elastin has not been thoroughly studied. Our objective was to evaluate the role of elastin in vascular remodelling of mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR). MRA segments from Wistar Kyoto rats (WKY) and SHR were pressurised under passive conditions at a range of physiological pressures with pressure myography. Confocal microscopy was used to determine differences in the quantity and organisation of elastin in intact pressure‐fixed arteries. To assess the contribution of elastin to MRA structure and mechanics, myograph‐mounted vessels were studied before and after elastase incubation. When compared with WKY, MRA from SHR showed: (1) a smaller lumen, (2) decreased distensibility at low pressures, (3) a leftward shift of the stress‐strain relationship, (4) redistribution of elastin within the internal elastic lamina (IEL) leading to smaller fenestrae but no change in fenestrae number or elastin amount. Elastase incubation (1) fragmented the structure of IEL in a concentration‐dependent fashion, (2) abolished all the structural and mechanical differences between strains, and (3) decreased distensibility at low pressures. The study shows the overriding role of elastin in determining vascular dimensions and mechanical properties in a resistance artery. In addition, it informs hypertensive remodelling. MRA remodelling and increased stiffness are accompanied by elastin restructuring within the IEL and elastin degradation reverses structural and mechanical alterations of SHR MRA. Differences in elastin organisation are, therefore, a central element in small artery remodelling in hypertension.

Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and ‘cannabinoid-like’ receptors in small arteries

Background and purpose:  Pharmacological analysis of synergism or functional antagonism between different receptors commonly assumes that interacting receptors are located in the same cells. We have now investigated the distribution of α‐adrenoceptors, β‐adrenoceptors and cannabinoid‐like (GPR55) receptors in the mouse arteries.

New aspects of vascular remodelling: the involvement of all vascular cell types.

Conventionally, the architecture of arteries is based around the close‐packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self‐maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re‐arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.

Direct demonstration of β1‐ and evidence against β2‐ and β3‐adrenoceptors, in smooth muscle cells of rat small mesenteric arteries

Recent evidence supports additional subtypes of vasodilator β‐adrenoceptor (β‐AR) besides the ‘classical’ β2. The aim of this study was to investigate the distribution of β‐ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of β‐ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. We first examined the vasodilator β‐AR subtype using ‘subtype‐selective’ agonists against the, commonly employed, phenylephrine‐induced tone. Concentration‐related relaxation was produced by isoprenaline (pEC50: 7.70±0.1) (β1 and β2). Salbutamol (β2), BRL 37344 (β3) and CGP 12177 (atypical β) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the ‘β3‐adrenoceptor agonist’ CL 316243 was ineffective. In arteries precontracted with 5‐HT or U 46619, isoprenaline produced concentration‐related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration–response curve to phenylephrine indicating competitive α1‐AR antagonism, explaining the false‐positive ‘vasodilator’ action against phenylephrine‐induced tone. Endothelial denudation but not L‐NAME slightly attenuated isoprenaline‐mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. The β‐AR fluorescent ligand BODIPY TMR‐CGP 12177 behaved as an irreversible β1‐AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. β‐ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. Binding of BODIPY TMR‐CGP 12177 was inhibited by BAAM (1 μM) in all three vascular tunics, confirming the presence of β‐ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non‐neuronal cell types. Lack of co‐localisation with a fluorescent ligand for α‐ARs confirms the selectivity of BODIPY TMR‐CGP 12177 for β‐ARs over α‐ARs. Our results support the presence of functional vasodilator β1‐ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR‐CGP 12177 for identifying β‐AR distribution in the ‘living’ vascular wall.

Pharmacological analysis of postjunctional α-adrenoceptors mediating contractions to (−)-noradrenaline in the rabbit isolated lateral saphenous vein can be explained by interacting responses to simultaneous activation of α1-and α2-adrenoceptors

1 The pharmacological characteristics of the α‐adrenoceptor population in the rabbit isolated saphenous vein has been examined with (–)‐noradrenaline (NA), as principal agonist, and a number of antagonists with selectivity for either α1‐ or α2‐adrenoceptors. 2 The rank order of potency of various agonists is consistent with a population of α2‐adrenoceptors; UK‐14304 > (—)‐noradrenaline = (—)‐adrenaline > B‐HT 920 = cirazoline > phenylephrine > amidephrine, but the rank order of pA2 values for the antagonists against (—)− noradrenaline: BDF‐6143 > rauwolscine = prazosin > CH‐38083 = YM‐12617 > Wy‐26703 = phentolamine > corynanthine, is indicative of a mixed population of α1‐ and α2‐adrenoceptors or, alternatively, a new subtype with characteristics of both the α1‐ and α2‐subtypes. 3 Further evidence for two discrete populations of α‐adrenoceptors is provided by, (a) the potent but non‐competitive effect of prazosin against (—)‐noradrenaline, (b) the presence of a component of the contractions elicited by NA and phenylephrine which is resistant to the selective α2‐adrenoceptor antagonists rauwolscine and CH‐38083: these responses were inhibited by the selective α1‐adrenoceptor antagonists prazosin and YM‐12617, but not by the selective α2‐adrenoceptor antagonist BDF‐6143 and, (c) the relative potency of the yohimbine diastereoisomers rauwolscine and corynanthine against NA, phenylephrine and UK‐14304. 4 In spite of the overwhelming evidence for a population of postjunctional α2‐adrenoceptors, prazosin was similarly effective against all agonists and failed to discriminate between those with putative selectivity for α1‐ and α2‐adrenoceptors. This suggests an interaction of the effects of agonists at the two α‐adrenoceptor subtypes. 5 An attempt has been made to reconcile a number of paradoxical observations with regard to the identification of postjunctional α2‐adrenoceptors in vitro, and it is suggested that in many of the isolated blood vessels presently available for examination both subtypes reside on the same smooth muscle cell. The pharmacological consequences of multiple subtypes of receptors mediating the same response is considered.

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.

The use of fluorescent nuclear dyes for the study of blood vessel structure and function: novel applications of existing techniques

We have used nuclear fluorescent dyes to develop a technique for the study of vascular structure and function. Nuclear stained blood vessels, viewed with the appropriate filter sets, can be studied in great detail. Only the nuclei of the cells which form the walls are visible and so their positions relative to one another as well as their viability can be quickly assessed. The dyes are not toxic, therefore when the vessel contracts or relaxes, the changes in position of the nuclei can be monitored. In this paper we describe two original applications of fluorescent nuclear dyes in vascular research.

Influence of elastin on rat small artery mechanical properties

We have previously developed a method for estimating elastin content and organization in resistance arteries, where it is a minor component. The aim of the present study was to validate the method against a quantitative assay and to determine the relative importance of elastin content and organization for intrinsic elasticity of small arteries. Mesenteric third order branches (from 10‐day‐old, 1‐ and 6‐month‐old rats) and middle cerebral arteries (from 6‐month‐old rats) were pressurized. β‐Values were calculated from stress–strain relationships and used as indicators of intrinsic stiffness. The same pressure‐fixed arteries were used to estimate elastin content and organization in the internal elastic lamina with confocal microscopy. Collagen and elastin contents were determined by Picrosirius Red staining and radioimmunoassay for desmosine, respectively. Confocal and desmosine assays gave similar results: no difference in elastin content of mesenteric vessels from 1‐ and 6‐month‐old rats, and a significant reduction in cerebral compared to mesenteric arteries. For all parameters (elastin and collagen content, fenestrae area and internal elastic lamina thickness) the best correlation was found between β‐values and fenestrae size. These data suggest that in small arteries: (1) confocal microscopy can be used as a method for the simultaneous study of changes in elastin content and organization; and (2) elastin organization might be a key determinant of intrinsic elastic properties.

An examination of the sources of calcium for contractions mediated by postjunctional α1‐ and α2‐adrenoceptors in several blood vessels isolated from the rabbit

1 The roles of intracellular and extracellular‐derived Ca2+ in α‐adrenoceptor‐mediated contractions to noradrenaline (NA) have been investigated in several isolated blood vessels from the rabbit by examining responses in the presence of a modified Krebs‐Henseleit saline with 2.5 mm Ca2+ and a Ca2+‐buffered saline with 0.1 μm free Ca2+. 2 NA was tested in preparations of the abdominal aorta, distal saphenous artery, renal vein, lateral saphenous vein, plantaris vein and ear vein exposed to a Ca2+‐buffered saline with 0.1 μm [Ca2+]. A concentration of NA which was maximally effective in modified Krebs‐Henseleit saline, produced an initial transient contraction (ITC) followed by a relaxation towards baseline. This is evidence that α‐adrenoceptor‐mediated responses in all these blood vessels depend upon calcium from both sources. 3 The ITC was particularly pronounced in the arteries and was associated more closely with the α1‐receptor subtype. In the abdominal aorta, distal saphenous artery and renal vein the ITC can almost exclusively be attributed to an α1‐adrenoceptor (prazosin‐sensitive, rauwolscine‐resistant). In the ear vein, and to a lesser extent the plantaris vein, the ITC was mediated in part by an α2‐adrenoceptor (prazosin‐resistant, rauwolscine‐sensitive). 4 α2‐Adrenoceptors in the lateral saphenous vein largely account for the response to NA in modified Krebs‐Henseleit saline, but α1‐adrenoceptors mediate the ITC in Ca2+‐buffered saline. After selective inactivation of α1‐adrenoceptors with a combination of phenoxybenzamine and rauwolscine, responses to NA in modified Krebs‐Henseleit saline are slow in onset and there is no ITC in Ca2+‐buffered saline. 5 The possible significance of the coupling of postjunctional α2‐adrenoceptors to dual sources of Ca2+ is discussed in relation to the interaction between α‐adrenoceptor subtypes and the ease of demonstrating functional α2‐adrenoceptors in isolated blood vessels.

Imaging the vascular wall using confocal microscopy

Blood vessels are capable of structural changes in a dynamic process called ‘vascular remodelling’, which involves cell growth, death, phenotypic change and migration, as well as extracellular matrix synthesis and degradation. An integrated view of the interrelationships of the different elements of the arterial wall is made possible by fluorescence confocal microscopy which enables collection of serial optical sections of relatively thick specimens without the need to cut them as with conventional histology. With the aid of image analysis software, these serial sections can be further reconstructed to obtain 3‐D images, where the structures of interest are localized and quantified. Confocal microscopy can be combined with pressure myography to obtain, simultaneously, information on vascular function and 3‐D structure at near‐to‐physiological conditions. There are a vast number of fluorescent compounds useful for imaging vessel structure and function. Nuclear dyes allow the identification of the different types of vascular cells and the quantification of their number, shape and orientation. The speed of confocal image acquisition and processing makes it possible to scan entire intact arteries stained with fluorescent kits or antibodies to locate infrequent events such as cell apoptosis, proliferation or migration. Confocal microscopy is not only useful for imaging vascular wall structure, but also to visualize and quantify, by the intensity of fluorescence, the generation of vascular cell factors such as nitric oxide or superoxide anion. In conclusion, confocal microscopy and image analysis software provide insight into vascular wall structure and function and the active process of vascular remodelling in physiological and pathological situations.