Vascular injury: Mechanisms and manifestations

Vascular Injury: Manifestations WINIFRED G.

NAYLER,

Mechanisms and

BSc., MSc., DSc.,

Heidelberg, Australia

Abnormalities in regulatory mechanisms for calcium handling play a key role in cell death and tissue necrosis. In the cardiovascular system this applies to the vasculature and the myocardium alike. In the aged population, where hypertension is a known risk factor, manifestations of vascular injury include atherogenesis and stroke. The newly developed dihydropyridine-based calcium antagonist amlodipine was used in investigations to determine whether calcium antagonists with sustained activity, in addition to lowering blood pressure, slow the development of atherogenesis in rabbits receiving high cholesterol diets, or reduce mortality in stroke-prone hypertensive rats. To establish whether this drug protects the vasculature against excessive atheroma formation in the presence of high cholesterol intake, rabbits were given 2% cholesterol in addition to their normal food intake and either 0, 1, or 5 mg/kg/day amlodipine orally for either 8 or 12 weeks. One day after the conclusion of the treatment protocol, the thoracic aorta was excised, assayed for calcium or cholesterol concentrations, and stained to identify sudanophilic-positive lesions. Amlodipine caused a time- and dose-dependent reduction in lesion formation, calcium overload, and cholesterol level. In the second series of experiments, amlodipine (5 mg/kg/day) was added to the diets of stroke-prone hypertensive rats. Treatment was initiated at age 5 weeks and continued for 30 weeks. During the treatment period, systolic blood pressure was reduced in the amlodipinetreated rats (166 f 9 mm Hg) versus those treated with placebo (248 + 12 mm Hg) (p CO.001). A significant reduction in mortality was observed in the amlodipine-treated rats (p CO.OOl), with 93% surviving versus only 26%

From the Department of Medicine, University Heidelberg, Vtctoria, Australia. DSc., Department

48-s

of Melbourne, Austin Hospital,

of Medicine, Austin Hospital, Heidelberg, Victoria, 3084 Aus-

April 25, 1991

The American Journal of Medicine

in the placebo group at the end of the 30-week treatment period. Concomitantly, cardiac hyper-trophy was attenuated in the treated group compared with the placebo group (heart-tobody weight ratios of 4.5 + 0.01 vs 5.8 f 0.6, respectively [p CO.011). These results extend the evidence that calcium antagonists provide vascular protection in animal models. This finding may become increasingly important in the management of an aging hypertensive population.

H

ypertension, cerebrovascular accidents (including stroke), and atherosclerosis are prevalent in the aged population. Although these conditions are the end result of dissimilar pathophysiologic conditions, they share at least one common characteristic-the involvement of calcium in the underlying disease process. In patients with hypertension, there is little doubt that a raised cytosolic free calcium (Ca”) is the final mediator of increased peripheral resistance [l]. Whether this increase in cytosolic Ca” reflects enhanced Ca2’ entry in exchange for sodium (Na+) by way of the Na+:Ca2+ transporter, secondary to the activation of the Na+:hydrogen (H+) exchanger [2,3], or whether it reflects enhanced Ca2+ entry by way of the voltage-sensitive L-type Ca2’ channels [4], passive Ca2+ diffusion [5] through a leaky plasma membrane, or redistribution of internally stored Ca2’ remains a matter of debate. The causal link between the loss of homeostasis with respect to Ca2+ regulation and the steady increase in smooth muscle tone is, however, well established [l]. Added to this is the accompanying increase in vessel wall rigidity [61, due in part to calcium accumulation at sites of elastic fiber degeneration [7]. The involvement of Ca2’ in the etiology and secondary consequences of cerebrovascular accidents is well defined, irrespective of whether the cerebrovascular accident involves hemorrhage, sustained vasospasm, thrombosis, or occlusion due to plaque rupture [8,9]. The involvement of Ca2+ in the atherosclerotic process is complex but can be divided into two

Volume 90 (suppl 48)

HYPERTENSION

r-

Raised

plasma

I

Increased

IN AN AGING POPULATION / NAYLER

Hypertension’

cholesterol

I vascular

permeability’

Endothelial I

I

Macrophage infiltration*

Increased

injury’

Platelet -

p

I Lioid infiltration

Lipid “loaded2 cells I

Figure 1. Schematic representation of the involvement of calcium (Cs*‘) in the cellular events leading to the formation of atherogenic lesions. ‘The events involved in the early stages of lesion formation that are Ce2+ sensitive. PDGF = platelet-derived growth factor.

1

shear

stress*

$

aggregation’

I Release

of PDGF’

Smooth muscle cell proliferation and migration’ I

w Ca*+ d Plaque

stages-early and late. The early stage encompasses the conditions and processes that contribute to lesion formation, including increased shear stress associated with hypertension, cell proliferation and migration, macrophage infiltration, excess secretion of extracellular matrix proteins, release of platelet-derived growth factors, and cholesterolinduced changes in membrane Ca2’ permeability. Many of these events, including the excess secretion of matrix protein [lo], cell proliferation [ll], smooth muscle cell migration and macrophage infiltration [12,13], cell necrosis [8], and the excess secretion of extracellular matrix materials such as glucosaminoglycans, are Ca2+-mediated events (Figure 1). The second or late stage of lesion formation involves the accumulation of calcium (as calcium apatite) in the newly formed plaques, where the calcium contributes to the formation of the fibrous cap. This fibrous cap is the prominent feature of the mature plaques [14] seen by pathologists. Studies documenting the involvement of calcium in the atherosclerotic process have detected substantial increases in total aortic calcium content in the cholesterol-fed animals (usually rabbits) used to study this phenomenon [15]. Some of this calcium is bound to extracellular proteins [16], but a substantial proportion accumulates intracellularly [ 171. In the cholesterol-fed rabbit model of atherosclerosis, this increase in cytosolic Ca2’ is accompanied by a fivefold increase in plasma membrane calcium permeability over a treatment period of 13 to 17 weeks

Proliferation of ground substance

[17]. Under these same conditions, aortic cholesterol increases [ 151. Since increased membrane cholesterol is often associated with an enhanced rate of Ca2’ influx [1’7], the sequence of events that ultimately results in the formation of atherosclerotic lesions may be described as follows: increased plasma cholesterol + altered membrane permeability + enhanced cytosolic Ca”+ + stimulation of smooth muscle cell proliferation and migration, macrophage infiltration and matrix formation -+ lesion development [16]. Other factors must be added to this scheme, including endothelial injury, platelet aggregation, and the local release or production of mitogenic agents (e.g., endothelin) [HI. Risk factors such as hypertension [19] must also be included because of the likelihood of an increase in peripheral vascular resistance being due to a raised cytosolic Ca 2+ level [l] and an altered membrane permeability to Ca” [4]. Inasmuch as the processes involved in hypertension, atherosclerosis, and cerebrovascular accidents, including stroke, involve Ca2+, it is not unreasonable to assume that interventions that reduce Ca2’ availability at the cellular level may be beneficial. The calcium antagonists theoretically meet this criterion because they modulate Ca”+ influx at the level of the voltage-operated Ca2+ channels. The following experiments were undertaken to investigate whether the long-acting calcium antagonist amlodipine [20,21] is effective under these conditions. Specifically, experiments were de-

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TABLE I. Effect of 5 mg/kg/day Amlodipine on Ca2+ and Cholesterol Content of Control and Cholesterol-Fed Rabbits Aottic Concentration* Ca2+ (Ir%g ProtWl

Treatment Group Control diet t placebo Cholesterol diet t placebo Control diet t amlodlplne Cholesterol diet t amlodipme

190? 20 310k 28 180t 15 220r 2045

Cholesterol hdg dry weight)

21.22 0.6 61.2it 1.5 22.6? 3.2 40.6A 3.2$

*Each result represents the mean ? standard error ot results tram SIXrabbits. Amlodlplne or placebo treatment (mixed with food) was maintained throughout the 12week study. tProteln was determined by the Lowry methodF4 sp < 0.1 versus cholesterol diet + placebo group. §p < 0.01 versus control diet + amlodipine group.

signed to determine whether amlodipine protects the vasculature against excessive atheroma formation in the presence of a high cholesterol intake, and whether the drug in stroke-prone hypertensive rats reduces blood pressure, increases survival, and attenuates the development of cardiac hypertrophy.

METHODS Effect of Amlodip/ne on Atheroma Formation in Cholesterol-Fed Rabbits White New Zealand male rabbits weighing 2.2 to 2.5 kg were used for these experiments. They were housed individually under temperature-controlled conditions. After stabilization, during which time they were fed standard rabbit pellets and water ad libitum, the rabbits were assigned to one of the following regimens: (1) standard pellets plus placebo; (2) standard pellets plus amlodipine (1 or 5 mgikg/ day); (3) cholesterol-enriched pellets (2% cholesterol plus 1% peanut oil, by weight) plus placebo; or (4) cholesterol-enriched pellets, as previously mentioned, plus amlodipine (1 or 5 mg/kg/day). The cholesterol-enriched pellets were prepared by suspending the cholesterol in peanut oil and ether, and then adding the resultant suspension to the homogenized standard pellets. The ether was allowed to evaporate overnight before the food was repelleted for use. The amlodipine was dissolved in ethanol and added to the homogenized food. For the placebo group, only ethanol was added to the homogenized food. Food intake was matched on a daily basis. After the required period of treatment, the rabbits were killed by cervical dislocation. The thoracic aorta was then removed and either stained with Sudan III for cholesterol or assayed for tissue cholesterol [22] or calcium content. Calcium content was determined by atomic absorption spectrometry as previously described [23] using nitric acid digests of aorta dried to constant weight. Protein 4510s

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1991 The American Journal of Medicine

concentration was determined by the method of Lowry et al [24]. The percentage of aortic surface area occupied by sudanophilic-positive lesions was determined by computer-assisted analysis. Effect of Amlodipine on Blood Pressure, Survival, and Cardiac Hypertrophy in Stroke-Prone Hypertensive Rats In this series of experiments, rats from the stroke-prone colony of hypertensive rats were used. Treatment was initiated at age 5 weeks and continued for 30 weeks until the animals were 35 weeks of age. Amlodipine was added to the diet to provide a daily intake of 5 mglkglday. Blood pressure was recorded at weekly intervals, using the tail cuff method and accepting the measurement obtained on the third occlusion. When required, the rats were weighed, anesthetized, and their hearts excised and weighed to establish heart-to-body weight ratios. Only rats that survived the required treatment period (10, 20, or 30 weeks) were used. Statistical Analysis Results were analyzed for significance by analysis of variance with the Bonferroni adjustment for multiple comparisons. Unless otherwise stated, each result is presented as the mean * standard error of the indicated number of experiments.

RESULTS Effect of Amlodipine on Atheroma Formation in Cholesterol-Fed Rabbits Thoracic aorta excised from rabbits receiving a control diet for 12 weeks and given placebo contained 190 ? 20 pglg protein Ca” and 21.2 ? 0.6 mg cholesterol per gram dry weight (Table I). These levels were significantly lower (p ~0.01) than values obtained for the cholesterol-fed rabbits treated with placebo, in which aortic Ca2’ increased to 310 ? 28 pglg protein and cholesterol reached levels slightly above 60 mg/g dry weight (61.2 ? 1.5 mg/g dry weight, n = 6). Adding amlodipine (5 mg/kg/day) to the control diet failed to alter either the Ca2+ (180 & 15 pg/g protein) or cholesterol (22.6 t 3.2 mglg dry weight) content of the aorta. However, adding amlodipine to the cholesterol-enriched diet reduced the cholesterol-induced gain in Ca2+ (p
Volume 90 (suppl 48)

1

120-

80 -

0 1 mg/kg amlodipine t7d 5mg/kg amlodipme

75 .g

60 -

zl z

40. 20 -

F0

PC.01

60 1

O10

Figure 2. Effect of 1 and 5 mg/kg/day amlodipine (given orally with food) on incidence of sudanophilic-positive lesions in the thoracic aortas of rabbits maintained on a 2% cholesterol diet for 12 weeks. Note that amlodipine caused a dose-dependent reduction in the incidence of lesion formation. In the untreated cholesterol-fed rabbits, 38 2 7% of the aorta stained positive for sudanophilic lesions after 12 weeks of treatment. Each bar represents the mean results of six animals. The difference between the reduction caused by 1 and 5 mg/kg/day amlodipine was significant (p < 0.01). Each treatment

group contained

six rabbits.

TABLE Ill

Effect of 5 mg/kg/day Amlodipine on Mean Systolic Blood Pressure in Stroke-Prone Hypertensive Rats

Effect of 5 mgIkg/day Amlodipine on Heart-to-Body Weight Ratio in Stroke-Prone Hypertensive Rats After 30.Week Treatment*

Systolic Blood Pressure (mm Hg)

5* :i: 35

Placebo

(II = 24)

TreatmentGroup

P Value

Normotenslve Sprague-Dawleyrats Placebo (n = 24)

158 ? 4 (24)

154 2 6 (24)

NS

254*2 8(18) 210 12(10)

164kt 85(23) 159 (24) 166+9(21)


248+ 26(6)

25

of treatment

lgure 3. Effect of oral amlodipine (5 mg/kg/day) on survival in stroke-prone hypertensive rats. Treatment was initiated at age 5 weeks and continued until the animals were 35 weeks old (up to 30 weeks of treatment). The difference in survival was significant (p < 0.01 after 15 weeks; p < 0.001 after 25 and 30 weeks of treat. ment). Twenty-four rats were entered into the placebo and 24 into the amlodipine group. The survival rate is listed in Table II

TABLE II

Age (weeks)

15

Weeks

Amlodipine (n = 24) Stroke+rone hvuertensive rats Placebo (n i.24)

I

*Treatment (either placebo or 5 mg/kg/day amlodipine given orally in food) was initiated at age 5 weeks. The numbers in parentheses equal the number of survivors after 10,20, or 30 weeks of amlodipine treatment. NS = not significant.

lodipine added to the cholesterol diet from the start of the cholesterol feeding resulted in a significant reduction in sudanophilic lesion formation (p
Amlodipine (n = 24)

IO3 x Heart/BodyWeight 4.682 0.60

(24)

4.65+_0.9Ot

(24)

5.8+ 0.60

(6)

4.75t 0.80t

(211

NP

“p<

0.01

*Treatment was initiated at 5 weeks of age and continued for 30 weeks. TAmlodipine treatment (5 mg/kg/day added to food) had no effect on the heartto-body weight ratio of normotensive rats. The numbers in parentheses equal the number of survivors after 30 weeks of treatment. NS = not significant at p = 0.05, but reduced (p -C 0.01) the increase in hear&to-body weight ratio in the hypertensive rats.

feet was maintained throughout 30 weeks of treatment, starting at 5 weeks of age. At the same time, the survival rate increased (Figure 3). For example, after 25 weeks of treatment, 98% of the amlodipine-treated rats survived, compared with only 40% survival in the placebo group (p
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COMMENTS These results show that amlodipine has a vascular protective effect. Evidence of this protection included reduced atheroma formation and prolonged survival in stroke-prone rats (a strain of rats in which death is commonly due to cerebral hemorrhage and stroke). This prolonged survival was accompanied by a reduction in hypertension-induced hypertrophy, an effect that may have contributed to the improved survival of the stroke-prone animals. Other factors, however, also may have contributed to survival, including the ability of calcium antagonists to protect against excess Ca2+, as well as the ability to dilate endothelin-constricted cerebral arteries. Plasma endothelin levels increase in response to subarachnoid hemorrhage [25]. Although results showed that amlodipine reduced atheroma in a dose-dependent manner, the mechanisms involved require further investigation. The calcium antagonists, as a group, exhibit other properties in addition to that of inhibiting Ca2’ influx at the level of the L-type Ca2+ channel. These effects include inhibition of smooth muscle cell proliferation [26,27], inhibition of neutrophil and macrophage chemotaxis, and interference with polymorphonuclear leukocyte function [28]. Stimulation of low-density lipoprotein receptor synthesis [29], enhanced receptor-mediated lipoprotein uptake [30], inhibition of lysosomal hydrolysis of lipoproteins [31], inhibition of cholesterol ester synthesis [32], increased cholesterol ester hydrolysis [33], inhibition of matrix synthesis [34], and inhibition of very low-density lipoprotein secretion by hepatocytes [35] have also been demonstrated as effects of calcium antagonists. Whether any of these other actions occur in vivo and at therapeutically relevant concentrations and thus contribute to the antiatherosclerotic activity of amlodipine requires further study. The relevance of the rabbit model may also need to be considered, although it is worth noting that the atheromatous lesions formed in this model are new lesions. In clinical trials investigating the effectiveness of calcium antagonist therapy in controlling atheroma formation in humans [36], the results showed a reduction in new plaque formation, without regression of preexisting plaques. Such an outcome is completely in accord with the results presented here. These results provide evidence to support the hypothesis that amlodipine has vascular protective properties. Such an effect, if it occurs in humans, should be useful in the management of the elderly population, in whom hypertension, cerebrovascular accidents (including stroke), and atherosclerosis are prevalent. Under these circumstances, am-

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lodipine may be particularly useful because its sustained duration of action is due, in part, to its slow dissociation from its receptor [21]. Hence, its availability to act as a calcium antagonist within the cardiovascular system is not based solely on its bioavailability as determined by its hepatic and renal excretion.

REFERENCES 1. Dominiczak AF, Bohr DF: Vascular smooth muscle in hypertension, J Hypertens 1989; 7(suppl 4): S107-S115. 2. Aviv A, Livne A: The Na+/H+ antiport, cytosolic free Ca*+ and essential hypertension: A hypothesis. Am J Hypertens 1988; 1: 410-413. 3. Aviv A: The link between cytosolic Ca’+ and the Na+/H’ antiport A unifymg factor for essential hypertension. J Hypertens 1988; 6: 685-691. 4. Van Breemen C, Cauvin C, Johns A, et al: Ca*’ regulation of vascular smooth muscle. Fed Proc 1986; 45: 2746-2751. 5. Bohr DR, Webb RC: Vascular smooth muscle membrane in hypertension, Annu Rev Pharmacol Toxicol 1988; 28: 389-409. 6. O’Rourke M: Arterial stiffness, systolic blood pressure, and logrcal treatment of arterral hypertension. Hypertension 1990; 15: 339-347. 7. Yu S, Blumenthal H: The calcification of elastic fiber. J Atheroscleros Res 1965; 5: 159-173. 8. Schanne R, Kane A, Young E, et at Calcium dependence of toxic cell death. A final common pathway. Science 1979; 206: 700-702. 9. Strandgaard S, Paulson OB: Pathophysiology of stroke. J Cardiovasc Pharmacol 1990; 15(suppl 1): S38-S42. 10. Berridge MJ: The interaction of cyclic nucleotides and calcium rn the control of cellular activity. Adv Cyclic Nucleotide Res 1975; 6: 1-198. 11. D’Amore P, Shepro D: Stimulation of growth and calcium Influx rn cultured, bovine, aortic endothelial cells by platelets and vasoactive substances. J Cell Physrol 19n; 92: 177-184. 12. Bowcek M, Snyderman R: Calcium influx requirement for human neutrophil chemotaxis: Inhibition by lanthanum chloride. Science 1976; 193: 905-907. 13. Nakao J, Ito H, Ooyama T, et a6 Calcium dependency of aortic smooth muscle cell migration induced by 12.l-hydroxy-5,8,10,14-eicosatetraenoic acid. Atherosclerosis 1983; 46: 309-319. 14. Schmid K, McSharry WO, Pameijer CH, et al: Chemical and physiochemical studies on mineral deposits of the human atherosclerotic aorta. Atherosclerosis 1980; 37: 199-210. 15. Henry P, Bentley K: Suppression of atherogenesis in cholesterol-fed rabbits treated with nifedipine. J Clin Invest 1981; 68: 1366-1369. 16. Yu S, Blumenthal H: The calcification of elastic fiber. Part 4. Epinephrine and beta-aminopropionitrile-Induced calcification in animal aorta. J Atheroscleros Res 1965; 5: 159-173. 17. Strickberger SA, Russek LN, Phair RD: Evidence for increased aortic plasma membrane calcium transport caused by experimental atherosclerosis in rabbds. Crrc Res 1988; 62: 75-80. 18. Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoconstrictor peptide produced by vascular endothekal cells. Nature 1988; 332: 411-415. 19. Debakey ME, Gotto AM: Foreword. In: Paoletti R, Gotta A (eds). Atherosclerotic Reviews. New York: Raven Press, 1976, vol 4; 9-11. 20. Burges RA, Carter Al, Gardiner DG, et at Amlodrprne, a new dihydropyridine calcrum channel blocker with slow onset and long duration of action (abstr). Br J Pharmacol 1985; 85: 21P. 21. Nayler WG, Gu XH: (-1 [3H] amlodiprne bindrng to rat cardiac membranes, J Cardiovasc Pharmacol 1990 (in press). 22. Chan CT, Wells H, Kramsch DM: Suppression of calcific fibrous-fatty plaque formation in rabbits by agents not affecting elevated serum cholesterol levels. The effect of thiophene compounds. Circ Res 1978; 43: 115-125. 23. Daly MJ, Elz JS, Nayler WG: Contracture and the calcium paradox in the rat heart. Circ Res 1987; 61: 560-569. 24. Lowry OH, Rosebrough NJ, Farr AL, et a/: Protein measurement with Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 25. Masaoka H, Suzuki R, Hirata Y, et at Raised plasma endothekn rn aneurysmal

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HYPERTENSION subarachnoid haemorrhage. Lancet 1989; 2: 1402. 26. Orekhov AN, Tertov W, Khashimov KA, et al: Evidence of atherosclerotic action of verapamil from direct effects in arterial ceils. Am J Cardiol 1987; 59: 495-496. 27. Stein 0, Halpenrn G, Stein Y: Long-term effects of verapamil on aorhc smooth muscle ceils cultured rn the presence of hypercholesterolemic serum. Atherosclerosis 1987; 7: 585-592. 28. Pennrngton JE, Kammerich B. Kazanjian PH, et al: Verapamil impairs human neutrophil chemotaxis by a non-calcium mediated mechanism. J Lab Clin Med 1986; 108: 44-52. 29. Fikpovic I, Buddecke E: Calcium channel blockers stimulate LDL receptor synthesis in human skin fibroblasts. Blochem Biophys Res Commun 1985; 136: 845850. 30. Stein 0, Lettersdorf E, Stein Y: Verapamil enhances receptor-medrated endocy tosis of low density lipoproteins by aortic cells in culture. Atherosclerosis 1985; 5: 35-44. 31. Ranganthan S, Jackson RL: Effect of calcium channel blocking drugs on lysosomal function in human skin fibroblasts. Brochem Pharmacol 1984; 33: 2377-

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2382. 32. Stein 0, Stein Y: Effect of verapamil on cholesteryl ester hydrolysis and re-esterification in macrophages. Atherosclerosis 1987; 7: 578-584. 33. Yatsu FM, Alam R, Alam SS: Enhancement of cholesteryl ester metabolism in cultured human myocyte-derived macrophages by verapamil. Biochem Brophys Acta 1985; 847: 77-81. 34. Heider JG, Weinstein DB, Pickens CE, et at Antratherogenic activity of the calcium channel blocker isradipine (PN200-110): A novel effect on matrix synthesis independent of calcium channel blockade. Transplant Proc 1987; 29(suppl 5): 96101. 35. Rustan AC, Nossen JO, Tefre T, et al: Inhibition of very low densrty kpoprotein secretion by chloroquine, verapamil, and monensin takes place in the Golgr complex. Biochem Biophys Acta 1987; 930: 311-319. 36. Lichtlen PR, Hugenholtz PG, Rafflenbeul W, et al: Retardation of the angiographic progression of coronary artery disease in man by the calcium channel blocker, nifedipine-Results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT). Lancet 1990; 335: 1109-1113.

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