Blood vessels and human essential hypertension

International Elsevier

15

Journal of Cardiology, 20 (1988) 15-28

IJC 00710

Review

Blood vessels and human essential hypertension A.M. Heagerty *, A.S. Izzard, J.D. Ollerenshaw and S.J. Bund Department

of Medicine,

Clinical Science Building, Leicester Royal Infirmary,

(Received

Key words: Hypertension;

4 January

1988; accepted

4 January

L&ester,

LJ.K.

1988)

Arteriole structure; Vascula; proliferation

Introduction Essential hypertension as a disease entity has only been fully recognised for 100 years since Mahomed [l] began to systematically measure blood pressure and define a group of patients whose hypertension was not associated with pre-existing renal disease. Before this Schaarschmidt (cited by Backer [2]) had described the existence of “spastic constriction of the vascular bed” for which no cause could be found and noted the tendency in such subjects to “the occurrence of vascular accidents with haemorrhage”. Whilst large studies have demonstrated benefit in terms of cerebrovascular mortality and morbidity from treatment of malignant phase [3], and now mild to moderate hypertension [4], the precise cause of this disease remains uncertain. Pickering felt that the topic was characterized by prematurity of conclusion and fixation of ideas [5]. Indeed, until recently it was almost axiomatic that there must be a factor that increased arteriolar smooth muscle activity which, when found, would provide the solution to the chronic resistance rise in hypertension. Research was dominated by such doctrine which, while leading to beautiful experiments, meticulously executed, left little room for alternative hypotheses to be entertained or tested. Recent years have seen a great expansion of research aimed at the cellular level and beyond, which has moved the way we view diseases such as hypertension away from mere clinical observations and into the laboratory. New techniques permit the application of biophysics and related subjects to hypertension. It is the purpose of this article to summarise some of the findings which have been made recently in trying to unravel this disease.

Correspondence to: A.M. Heagerty, Dept. of Medicine, Clinical Sciences Infirmary, P.O. Box 65, Leicester LE2 7LX, U.K. * Dr. Heagerty is a British Heart Foundation Senior Research Fellow.

0167-5273/88/%03.50

0 1988 Elsevier Science Publishers

B.V. (Biomedical

Building,

Division)

Leicester

Royal

16

Haemodynamic

Findings

When the disease is established, essential hypertension is characterized by an increase in peripheral resistance at a time when the cardiac output is normal [6]. This increased resistance to blood flow appears to be distributed throughout all the tissues [7,8]. The exact nature of this resistance has perplexed scientists for years, and has led to intense debate. What has been known since the work of Bright and Johnson is that the walls of arteries are thickened in hypertension [9,10]. The significance of this change in vascular structure was not fully appreciated. Indeed, Pickering had noted that, even after maximal vasodilatation, resistance to blood flow remained increased in hypertensive patients but, ultimately, he ascribed the finding to a circulating factor in the blood [ll]. The pervading belief was that structural alteration of the vascular tree occurred as a consequence of whatever raised the pressure. It was left to Folkow to demonstrate that such architectural changes could influence arteriolar haemodynamics so profoundly that almost no increase of vascular myocyte activity was required further to keep high resistance to blood flow in essential hypertension [12-141. Such structural adaptation is necessary for the giraffe which stands 18-20 feet tall when erect, thereby requiring a blood pressure of 400-500 mm Hg to perfuse the brain [15]. Indeed, the minimum left ventricular pressure necessary to open the aortic valve and expel blood must exceed 250 mm Hg. It is not surprising to learn that the giraffe’s left ventricle is 7.5 cm thick and the walls of the leg arteries are enormous with consequently pinpoint lumens [15]. By analogy with man, this creature should endure massive oedema. This does not occur due to the muscular thickening in the arterioles and the taut skin overlying them [16]. Similar adaptation occurs in human neonates. The fetal pulmonary vessels are thick-walled, but demonstrate gradual regression after the first breath [17]. The problem for hypertensive man is that his circulation is unable to withstand chronic pressure to excess and as a result he is at considerable risk of premature cardiovascular death [18]. In man, research into this disease has been hindered by difficulties in studying human tissue and obtaining reliable qualitative data. Increased wall-thickness-to-lumen diameter ratio has been reported, nonetheless, by haemodynamic measurements [13,19] and by histological measurements on autopsy material [20,21]. Studies of reactivity have been undertaken although the arteries used were too large to have been of haemodynamic importance [22-251. For the most part, they have suggested that hypersensitivity to agonist stimulation is not found. Recently it has become possible to study human resistance arterioles from small biopsies of skin [26]. This technique employs a myograph [27], and allows precise measurements of vessel morphology to be made, and the recording of isometric contractions initiated by vasoconstrictor agonists or depolarizing solutions. When vessels from patients with hypertension were examined and compared to arterioles from matched control subjects, there was a 29% increase in mediathickness-to-lumen diameter [28]. This was close to the excess predicted by Folkow [14]. Moreover, in the same report, no increase in sensitivity to agonists such as noradrenaline, vasopressin or angiotensin II was observed. In the established disease, structure appears to prevail alone. Like much work in hypertension, these

data do not address the question of whether this structural change occurs as the cause or consequence of the high blood pressure. Indeed, herein lies the main problem inherent in research into human blood pressure: whilst there is an undoubted polygenetic component to the disease it is by no means predictable which (if any) progeny of a patient will develop phenotypic hypertension. This is, of course, in stark contrast to small mammals genetically in-bred to develop hypertension. In order to examine the problem in greater detail, the resistance vessels of a large group of normotensive first-degree offspring of hypertensives have been studied and compared to those of controls with no such family pedigree [29]. Overall, the ratio of media to lumen was not increased in the offspring, although the blood pressure was slightly higher and there was a tendency for the media to be slightly larger. The inference appears to be that blood pressure and structural alteration proceed hand in hand. Thus further questions remain: what provokes the pressure to rise in the first place and how is this stimulus transmitted to the myocyte thereby inducing growth? Will antihypertensive medication reverse the structural changes observed?

The Primary Stimulus The widely held current belief is that a stimulus slightly raises pressure initially, possibly even intermittently at first. This induces a small structural alteration in the resistance vasculature sufficient to maintain a small rise in pressure. Repeated stimulation leads to further architectural modification and, ultimately, to established hypertension [14]. This primary stimulus is often obvious in secondary forms of hypertension, for example in phaeochromocytoma or Cushing’s syndrome. It is not so clear in essential hypertension. The main candidate is excess autonomic nervous function, at least in the early phase of the disease. A number of studies in human hypertension have been reported suggesting that, at least in the early phases of the disease, sympathetic autonomic function is enhanced [30]. Both in animals genetically prone to hypertension and in hypertensive man, there is a significant correlation between change in resting blood pressure and plasma noradrenaline after ganglionic blockade, indicating that the level of pressure is in part due to increased sympathetic activity [31-331. Interference with autonomic control in rats prone to hypertension attenuates the development of hypertension [34]. Complete blockade in such mammals reduces the arterial pressure to control levels although measurements of resistance demonstrate that it is still much higher than normotensive control rats [35]. Thus, neurogenic mechanisms continue to play a role in raising pressure but structural adaptation is also contributing. In hypertensive man, particularly in younger patients, the plasma noradrenaline concentration is often elevated [30]. This has been shown to be due to increased spillover into plasma rather than due to a reduction in plasma clearance [36]. Against, recent evidence from studies of human resistance arterioles suggests that the spillover may be due to a defective mechanism for the re-uptake of neuronal amines [28]. In the presence of cocaine, sensitivity to vascular noradrenaline shifts to the left in both hypertensive patients and control subjects. But the shift is proportionally greater in the patients [28].

18

Moreover, this phenomenon is also found when the blood pressure is normal in the vessels from first-degree offspring of hypertensive patients [29]. This abnormality, therefore, precedes the rise in pressure. Whall et al. [37] have previously reported the results of a similar experiment in the spontaneously hypertensive rat where the vessels were denervated using 6-hydroxydopamine. Before denervation, noradrenaline sensitivity was similar in spontaneously hypertensive and control rats, but afterwards the arterioles from the hypertensive rats showed a two-fold increase in sensitivity. Experiments using cocaine have also been performed in spontaneously hypertensive rats which revealed increased sensitivity to noradrenaline after cocaine compared to control animals [38]. Denervation with 6-hydroxydopamine abolished the effect in the control but not completely in the hypertensive animals [39]. The evidence, therefore, suggests that sympathetic nervous activity is deranged at the neuroeffector junction. In human hypertension, nonetheless, the exact nature of the problem has not been defined. In the human arteriole responsible for resistance there is no information concerning nerve firing rate, nerve density or synaptic cleft width, all of which may influence neuronal activity. The experiments with cocaine suggest that there may be a fundamental defect in neuronal amine uptake. Cocaine, however, can induce an increase in smooth muscle sensitivity itself [40]. Reiffenstein and Triggle have investigated the effects of cocaine upon the uptake of noradrenaline in the human umbilical artery [41]. This preparation does not receive sympathetic innervation and these workers demonstrated that cocaine potentiated the mean effective dose responses to noradrenaline and serotonin. Thus, caution must be exercised in our interpretation of the data so far gathered in essential hypertension. We await eagerly the results of current histological studies of skin vessels from hypertensive patients. Moreover, experiments are in hand to repeat dose-response curves to noradrenaline in the presence of desipramine, which appears to be a more specific blocker of amine uptake mechanisms [42,43] although it may have cu-adrenoceptor antagonist effects. From what we already know in essential hypertension, however, the activity of the sympathetic nervous system is overactive, especially in young patients [44]. If the data from human arterioles in the presence of cocaine are substantiated, it can be concluded that uptake of neuronal amine is defective and that this again is present in the younger subjects even before any rise in pressure is observed. The resistance vessels are presented with an excess of a vasoconstrictor hormone despite an overactive amine pump. The implications of this are discussed below. The Mechanisms of Cellular Proliferation When cells proliferate, an ordered process is pursued resulting in synthesis of DNA and mitosis [45]. Two major signalling systems have been described in the control of growth. Some mitogens act on receptors which initiate tyrosine kinase activity [46]. Indeed, two factors that may play important roles in modulating growth, platelet derived growth factor and epidermal growth factor, both work through this system [47]. The alternative pathway is utilised by factors that provoke the hydrolysis of inositol lipids. It is important to remember that both mechanisms may not be mutually exclusive, with both contributing to cellular proliferation. The

19

subject of the metabolism of phosphoinositide lipids has recently been extensively reviewed, both in respect of our current biochemical knowledge of the system [48-521, and with regard to its possible role in hypertension [53]. It has now been demonstrated that the vasoconstrictor hormones angiotensin, noradrenaline and vasopressin stimulate the production of inositol 1,4,5-trisphosphate (Ins 1,4,5,P,) and l,Zdiacylglycerol(1,2-DG) [54-561. Ins 1,4,5, Ps has now been shown to release calcium from non-mitochondrial organelles [57,58]. In these agonist-receptor actions, the released calcium initiates the pressor response which is fortified and maintained by calcium movements from without mediated by 1,2-DG activation of protein kinase C [59]. The interesting feature about this cell signalling system is that its functions appear two-fold. Vasoconstriction provoked by a pressor agent is relatively short-lived, whereas the same stimulus can influence cell growth [45] and yet cell division does not occur every time an arteriole is made to contract. Berridge [45] surmises that the reason this does not occur is because the constrictor stimulus is short-term and sufficient to initiate contraction but not proliferation. Cellular growth only occurs when the tissue is stimulated excessively for a long time. The interest in these polyphosphoinositide lipids in hypertension now becomes clearer in the knowledge of the evidence for autonomic excess in the early phases of the disease. It has been known for some time that the application of mitogens to quiescent cells in culture causes a rapid increase in sodium influx and activation of Na+/K+ATPase activity [60] (phenomena noted in spontaneously hypertensive rats and some hypertensive human tissues) [61]. The influx of sodium occurs via a sodium-hydrogen exchange system [62], and mitogens effect this by activation of either tyrosine kinase [47] or the phosphoinositide system [63]; both 1,2-DG/protein kinase C and tyrosine kinase activation influence exchange of sodium and hydrogen ions [64]. An alkaline change in pH in the cell cytosol appears to be the cell signal to provoke the nucleus to initiate protein synthesis and division [60,63]. What is less certain is whether this exchanger is the mechanism by which this alkalinisation is brought about. Mutant Escherichia coli have been isolated with no significant activity of sodium-hydrogen exchange but normal activity on the exchange of hydrogen ions with calcium and potassium, respectively. This strain could not grow at alkaline pH [66], but did survive, suggesting that exchange of sodium and hydrogen is an integral part but not the whole mechanism for pH control. Moreover, there are data from studies on lymphocytes that activation of this antiport is not a prerequisite for proliferation in this tissue [67]. In most cell systems, exchange of sodium and hydrogen is important in maintenance of pH. but so also is counter transport of chloride and bicarbonate ions in some cell lines, where influx of sodium is coupled to this exchange mechanism [68]. Which system is present or predominates depends on the species and tissue under test. The situation, therefore, is not as clear as some would suggest. It is conceivable that pH homeostasis inside cells is a function of a series of antiports. Many data, however, suggest that exchange of sodium and hydrogen is the dominant modulator in vertebrate cells but with contributions from exchange of chloride and bicarbonate [69]. What has become clear recently is that a change in internal pH is one of the early events in fusion of the sperm and the egg, and experiments have demonstrated

20

that this process is sodium-dependent and probably effected through the antiport for exchange of sodium and hydrogen [69]. Furthermore, when quiescent cells in culture are stimulated to grow, sodium influx increases, a process which can be blocked by amiloride [70], which is used as an inhibitor of exchange of sodium and hydrogen. It is clear that amiloride is not a specific blocker of the sodium/ hydrogen exchange, also having effects on sodium channels, on exchange of sodium and calcium, a-adrenoreceptors, protein synthesis and the ATPase pump for sodium and potassium [71,72]. Nevertheless, the alkalinisation observed following agonist or mitogen stimulation via the polyphosphoinositide system can be blocked with this compound suggesting, if far from proving, that exchange of sodium and hydrogen is important in controlling this parameter. The induction of an alkaline pH causes the phosphorylation of proteins [73] and appears to initiate protein synthesis [74]. Thus, in a disease such as hypertension where abnormal medial thickening is observed, the process of activation of this system such as by autonomic excess, and the possibility of a genetically inherited abnormal smooth muscle cell reinforcing the stimulus by proliferating more avidly, might hold the key. It is already established that the vasoconstrictor hormones (noradrenaline, vasopressin and angiotensin II) activate the polyphosphoinositide system and liberate diacylglycerol [54-561, which is the essential co-factor for protein kinase C. The production of 1,2-DG may not be solely from phosphatidylinositide metabolism [75], but it is essential for activation of protein kinase C. The important question left to answer is whether this leads to cytoplasmic alkalinisation? Much of the research has been performed upon cultured smooth muscle cells. Hatori et al. [76] and Berk et al. [77] have reported brief acidification followed by prolonged alkalinisation with angiotensin II. The acidification is believed to be due to the initial mobilisation of intracellular calcium because phorbol esters inhibit calcium mobilisation mediated via angiotensin II [78] and the acidification [79]. Owen has reported the effects of catecholamines upon cultured smooth muscle sodium influx and pH [80] in common with other workers [81]. She found that benzamil and ethylisopropylamiloride, putative inhibitors of sodium/ hydrogen exchange, were inactive in cultured cells if the cells were unstimulated. Alpha adrenoreceptor activation stimulated phosphoinositide breakdown, an increase in benzamil-sensitive sodium influx and intracellular alkalinization occurred [80]. The recent report of these amiloride analogues having a-receptor blocking effects in kidney cells [71] means that all attempts to block selectively exchange of sodium and hydrogen are at present unsatisfactory. Nevertheless, these data and other recent reports suggest that this antiport is important in regulation of pH in vascular smooth muscle [82,83]. Beyond this, the role of cultured cells in dissecting out possible causes of hypertension must be viewed with scepticism. If, as some have reported [82], the mechanism for exchange of sodium and hydrogen is active under basal conditions, one must question whether the cells are not losing their contractile phenotype and switching to a synthetic role. Reports of measurements of pH in intact resistance arterioles are only just beginning to emerge. The use of pH-sensitive fluorescent dyes in such tissues suggests that the dramatic alkalinisation induced by agonists in dispersed or cultured cells is hardly seen at all [84] although the stimulation of the arterioles has only occurred for a few minutes. Amiloride and

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its analogues, however, will relax arterioles preconstricted with noradrenaline [85]. Again this may be due in part to a-receptor antagonism, although it is known that external alkalinisation will induce vasoconstriction and acidification will cause vasodilatation [68,78]. Thus, evidence is beginning to emerge of how an initial pressor stimulus might be transmitted into a proliferative response in arteriolar tissue. Indeed, catecholamines have been shown to stimulate DNA synthesis in cultured monocytes, and vasopressin and angiotensin II can also act as trophic factors without exerting a vasoconstrictor effect [86,87]. As Folkow has indicated [14], subjects with a genetic predisposition to developing the disease may have cells with the propensity to mount an abnormal proliferative response when faced with such a trophic challenge. Hypertension

and Polyphosphoinositide

Metabolism

Recently it has become possible to label radioactively the phosphoinositide lipids and measure inositol phosphate production in the presence of lithium. Lithium chloride prevents the enzymic breakdown of inositol monophosphate to inositol and, therefore, allows the rate of inositol phosphate production to be examined in the basal and agonist stimulated state. Such experiments have yet to be reported in human arterial specimens from hypertensive patients. In the aorta of mature spontaneously hypertensive rats, nonetheless, production of inositol phosphate was normal when unstimulated and reduced in the presence of increasing doses of noradrenaline when compared with the responses observed in tissue from control animals [Ml. At 5 weeks, however, inositol phosphate production was greater in hypertensive animals either unstimulated or activated by noradrenaline. In the early stages of the disease, therefore, the system appears to be overactive (at least in the aorta). In the resistance arterioles in contrast, even in the early phase of hypertension, overall inositol phosphate production does not appear to be different [89]. But it is now possible to use high performance liquid chromatography to analyse production of separate inositol phosphates [90]. Preliminary observations suggest that Ins 1,4,5,P, is produced in excess in spontaneously hypertensive animals. These measurements must reflect an indirect assessment of 1,2-DG production which therefore will also be produced in excess. Thus, it is conceivable that an overactive phosphoinositide system could underlie the structural changes in hypertension. Indeed, a variety of stimuli acting either through this system or via tyrosine kinase activation could generate smooth muscle hypertrophy in various models of hypertension. In a recent experiment, inositol phosphate production was assessed in segments of aorta above and below a ligature placed between the renal arteries of Wistar rats. Increased phosphoinositide hydrolysis was observed proximal to the constriction 72 hours after operation and the raised levels were sustained 17 days later [91]. This effect preceded the rise in blood pressure and significant changes in structure. No such increase was observed below the coarctation. The recorded changes in hydrolysis persist after the aorta has been removed from the rat. Therefore it is unlikely that the high levels of angiotensin II in this model contribute significantly to this finding. It is possible that load-mediated endothelial damage

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allows the angiotensin II to stimulate the system [92], and evidence is available that this may happen [93]. This was not observed in resistance arterioles in the same model, but structural changes did not occur in these smaller vessels either [94]. In similar experiments on aorta, Loeb et al. have demonstrated that DNA synthesis increased at 4 days in rats made hypertensive by clipping one of two kidneys. This could be attenuated either with antihypertensive medication or with cytosine arabinoside [95,96]. Thus, in these models, the temporal association would suggest that production of inositol phosphate and synthesis of DNA are closely related. The precise mitogenic stimulus remains uncertain: It may be triggered by the act of stretch induced by the load excess placed upon the aorta. Stretch has certainly been shown to influence calcium influx [97] which itself may ‘alter inositol phosphate production or protein kinase C activity [98]. The identity of the local mitogen at work here or, indeed in all forms of hypertension, is unknown. Well recognised tumour promoters such as phorbol esters are known to induce cellular alkalinisation [99,100], although a role for phosphoinositide metabolism and ultimately protein kinase C activation is not always required [loll. Other pathways using tyrosine kinase may be at work. In pulmonary hypertension induced in newborn calves, the pulmonary arteries demonstrate a two to fourfold increase in elastin production. This is accompanied by a corresponding increase in elastin messenger RNA consistent with regulation at the transcriptional level [loll. In addition, conditional serum from cultures of pulmonary vessels from the hypertensive animals contained one or more mitogens. These altered both the secretory phenotype and responsive properties of the cells. Similar experiments may allow local mitogens at work in essential hypertension to be identified.

The Effects of Treating Hypertension There are few data concerning direct measurements of resistance arteriolar morphology and reactivity after a period of effective antihypertensive therapy. Jennings et al. [102] studied the haemodynamics of patients with hypertension before and after 1 year’s antihypertensive medication. They concluded that there was complete restoration of “non-autonomic” total peripheral resistance, which would suggest that medial hypertrophy had regressed. In each patient, however, blood pressure rose and returned to previous levels in 4-20 weeks in those from whom medication was withheld [103]. Similar findings have been reported in a sub-group of patients studied in the MRC blood pressure study who completed the experiment before the entire study was concluded. Again, blood pressure rose to pretreatment levels within 12 months [104]. Thus, the primary stimulus is still present in the individual patient, and rapidly reactivated when medication is withdrawn. Data from the spontaneously hypertensive rat suggest that antihypertensive drugs may have varying effects upon vascular structure. Long-term therapy with B-blockers caused good regression and when treatment was withdrawn only a slow rise of blood pressure occurred [105]. Two studies in spontaneously hypertensive rats suggest that captopril is extremely effective in altering the vascular architecture [106,107]. In the work by Loeb and Bean [107], captopril, hydralazine

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and verapamil were each able to prevent the increase in synthesis of aortic DNA observed during the early development of renal hypertension induced in the rat using the “two kidney-one clip” model, although the authors advise caution in interpreting the results because the change seen in DNA synthesis could not be due entirely to changes in blood pressure [107]. Owens [108] administered captopril, hydralazine and propranolol to spontaneously hypertensive animals and noted that all drugs significantly lowered blood pressure but only captopril was effective in regressing aortic medial hypertrophy [log]. It is concluded that the hypertrophy observed in the aorta was simply a response to increased blood pressure or wall stress. Indeed the data of Owens [108] are similar to those of Freslon and Giudicelli [106], who also found captopril superior to hydralazine in preventing aortic hypertrophy. Similarly, Jespersen et al. [109] found that hydralazine reduced blood pressure in spontaneously hypertensive rats but was not effective in averting structural alteration in mesenteric arteries [109]. Again, the attractive possibility is raised that angiotensin II might behave as a trophic factor in itself [llO]. The failure of hydralazine to reverse hypertrophy may be attributable to increased sympathetic discharge [ill]. Various treatment regimes have been used in man. Indirect measurements of peripheral resistance suggest that thiazide diuresis, B-blockers and methyldopa all produce regression of structural changes [103]. Recently, we have rebiopsied patients after 12 months’ antihypertensive medication and a variety of treatments appeared to have been effective in the regression of medial hypertrophy in resistance vasculature [112]. We eagerly await larger numbers to investigate whether one drug is superior to another. Conclusions It is now 12 years since Folkow described hypertension as an “irritatingly elusive disorder of regulation” [35], and over 30 years since he alerted us to the possible haemodynamic consequences of structural alterations in vascular architecture. Increasingly sophisticated methods are now being developed to allow students to investigate the effects of flow and distending pressure upon vascular metabolism. Perhaps we are beginning to understand the mechanism of hypertrophy of myocytes. The search is on for putative mitogens that may be stimulating the growth response. We look forward with eagerness to the next few years which may well allow us to unlock the secrets of how such a crippling disease occurs and, perhaps, how better to attack it. Acknowledgements We thank the British Heart Foundation which supports our work, as well as Trent Research and Leicester Research Funds which provide financial support and the Mason Trust which provides equipment for our studies. Our thanks are also extended to Miss Denise Huckerby who typed this article.

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