Cerebral amyloid angiopathy

Amyloid angiopathy is characterized by amyloid fl-peptide (Ai) deposition and may contribute to the cerebrovascular abnormalities that precede the onset of Alzheimer's Disease bAD). That aberrant potassium (K+) channel function occurs in AD patients is supported by deleterious effects of A# on normal fibroblast K+ channels and prevention of Abeta-induced toxicity by potassium channel openers (KCOs) in neuronal cell culture. We report here that KCOs protect cerebral and peripheral vessels against the endothelial damage induced by Abeta. Pressurized posterior cerebral artery and aortic ring segments from the rat were constricted and then relaxed with the endothelium-dependent vasodilator acetylcholine before and after incubation with Af (1 6 M), or pre-treatment with KCOs before the addition of fl-amyloid. Vessels treated with A# exhibited features of endothelial dysfunction: enhanced vasoconstriction and diminished endotheliumdependent vasodilation. Pre-treatment with KCOs significantly antagonized the Af effect in both cerebral and aortic vessel segments. This protection was provided by both Kca and K^sub ATP^ channel openers. Endothelial damage by Abeta and protection by KCOs was verified by electron microscopy. The K+ channel blocker, TEA, reversed the protective effect of KCO. The results suggest that potassium channel openers protect against Abeta induced endothelial dysfunction and that KCOs may have a role in the treatment of degenerative cerebrovascular disease as seen in stroke, AD and aging. [Neurol Res 1999; 21: 345-351 ] Keywords: Alzheimer's disease; cerebrovascular disorders; endothelial function; free radicals; K+ channel; nitric oxide

INTRODUCTION

Abnormalities of the cerebral vasculature precede the neuropathology of Alzheimer's disease (AD) and several peripheral vascular diseases often parallel the onset of AD^sup 1^ . Experimental evidence points to amyloid angiopathy as a contributing factor to the degenerative changes in smooth muscle, endothelium, and basement membrane as seen in these vascular diseases. Concurrent with changes in the cerebral vasculature is an accumulation of the amyloid beta-peptide (Abeta) in the brain and cerebral vessels, a cardinal feature of AD2. Abeta is a 39-43 amino acid residue hydrophobic peptide which is a proteolytic product of the widely distrubuted amyloid precursor protein (APP) defined by a locus on chromosome 21. Abeta and other neurotoxins have been identified as possible mediators of cell damage in AD. An inflammatory response has also been observed in the AD brain3.

Abeta is normally produced in a soluble monomeric form that circulates at low levels in the blood suggesting a normal physiological role for Abeta. The observation that apolipoprotein 44 allele is a risk factor for both vascular disease and AD4 has renewed interest in the vascular abnormalities in AD. The 44 allele has been associated with Ai protein 5, senile plaques6, and a high risk for coronary heart disease7. A recent study by Kosunen et al.8 found more severe atherosclerosis of the coronary vessels among AD patients with the 44 allele than in AD patients without the zeta4 allele.

Our previous studies have shown that Abeta interacts with vascular endothelial cells causing oxygen radical production and subsequent endothelial damage with alterations in vascular tone. The results were found in both cerebra19,10 and peripheral vessel11,12. We have also found characteristics of an in vivo inflammatory response in the peripheral vasculature after i.v. injection of Abeta13. It is generally agreed that a damaged endothelium leads to vasoconstriction, hypoperfusion and ischemia in the surrounding tissue.

Kt channels participate in many important physiological functions including neurotransmission, memory and vascular responses of smooth muscle cells and endothelial cells. In normal fibroblasts soluble APi can cause the same K+ dysfunction previously found in fibroblasts from patients with AD14. Vascular endothelial cells express a variety of K+ channels including Ca++ dependent K+ (Kca) channels, inward rectifier K+ channels and ATP sensitive K+ (K^sub ATP^) channels15,16. K+ channel openers (KCOs) constitute a diverse and large group of compounds that are effective in activating one or more types of K + channels' 17,18. KCOs have been shown to protect cardiac myocytes against ischemic injury 192 and cultured neurons against A, toxicity2l. The current study investigates a possible role for K+ channels in the mechanism of Abeta-induced cerebral and peripheral endothelial dysfunction by examining the protective effects of both ICa and K^sub ATP^ channel openers against this damage.

MATERIALS AND METHODS

Cerebral vessel preparation and protocol

Male Sprague-Dawley rats (Zivic Miller, 227-375 g) were anesthetized with a 50 mg kg ' phenobarbital (i.p.) and decapitated. Segments from the posterior cerebral artery (internal basal diameter 219 +/- 5 pm, n= 31 ) were dissected and cannulated with glass pipettes. The segment was mounted horizontally in a 3 ml arteriograph as a blind pressurized vessel at 60 mmHg9,12 and equilibrated for 1 h in a physiological saline solution (PSS). The solution containing (in mM) 115 NaCI, 5 KCI, 1.4 NaH2PO4, 1.2 MgSO4, 1.7 CaCI2, 11 glucose, 0.025 EDTA and 10 MOPS was circulated by a pump through the bath from a flask heated to 370degC, bubbled with 95% OZ and 5% CO2 at pH 7.4. The bath was positioned on the stage of a microscope (Leitz Dialux 22, Germany). Dimensional analysis of the vessel image was provided by a video measuring system, VDAS-1 (Living Systems Instrumentation, Inc., Burlington, VT, USA). Pressure was adjusted by a servo driven 20 cc syringe filled with PSS and connected to the cannula via PTE tubing. Diameter measurements from the video amplifier and pressure measurements were recorded. The technique allowed the vessel to experience a transmural pressure and preserved the endothelium and the in vivo cylindrical shape of the vessel. For each experimental protocol the vessels were constricted with 10^sup -8^M serotonin (5HT) and relaxed with acetylcholine (2x10-5M). Equilibration time for incubation in Abeta (10-6 M) was 30 min.

Aortic ring preparation and protocol

Thoracic aortic segments were excised taking care to avoid damage to the intimal surface and then cut into rings of 2 mm. A ring segment was mounted on stainless steel hooks, attached to force displacement transducers and equilibrated under optimum tension of 2 g. The 8 ml tissue bath was filled with oxygenated (5% CO2 and 95% O2) saline solution at 37degC. The solution consisted of (in mM): NaCl 118.2, KCI 4.7, MgSO4 1.2, CaCI2 2.5, NaH2PO4 1.2, NaHCO3 23, and glucose 11.2. All vessels were equilibrated for 1 h preconstricted with 5xl0-8M phenylephrine (PE) and relaxed with increasing concentrations of ACh (10-9 M, 5 x 10-8, 10-8, 5x10-7 10-7, 10-6,10-5, 10-4). Aortic responses were obtained during control conditions or following 30 min incubation with 10-6 M Abeta, or pre-treatment with KCOs (diazoxide or NS1619 at 10-5 M) 60 sec before the addition of Abeta. Experiments were also performed on aortic rings where a Kca channel blocker (TEA, 10-3 M) was added 30 sec before adding NS1619 and then A#. Control experiments were also done with treatment by KCO only and TEA only.

Electron microscopy

Segments of rat posterior cerebral artery and thoracic aorta were processed for electron microscopy as follows. Vessel segments were incubated as previously described9-1 at 37degC for 30 min either alone or in the presence of Abeta (10-6 M), or KCO (10-5 M) plus Abeta. The tissue samples were fixed overnight in glutaraldehyde solution at 4C and then rinsed with cacodylate buffer. The tissues were post-fixed with 1% buffered osmium tetroxide, dehydrated with graded ethanol solutions, and embedded in a mixture of eponaraldite. Ultra-thin sections were stained with uranyl acetate and lead citrate and viewed on an electron microscope (Hitachi 7000, Hitachi Instruments, San Jose, CA, USA). The experiment was repeated three or more times with blood vessels from different animals.

Data analysis and materials

Differences in relaxation and constriction between control and treatment trials were considered significant at p

RESULTS

Effect of KCOs on Ap induced endothelial dysfunction in cerebral vessels

Real time recordings of the diameter response to ACh in cerebral vessels are shown in Figure 1. Treatment with Abeta decreased the response to ACh (Panel A) and the addition of diazoxide eliminated the effect of Abeta (Panel B). The effect of KCOs on the reduced response to ACh in cerebral vessels after A/ treatment (n=5) is shown in Figure 2. Abeta severely depressed the response to ACh (2x10-5M), and ATP-dependent K+ channel (K^sub ATP^) opener diazoxide (n=4) and Ca++-dependent K+ channel (Kca) opener NS1619 (n=4) significantly antagonized the Abeta effect. The effect of KCOs on the enhanced response to 5HT is shown in Figure 3. Abeta significantly increased the response to 5HT. Serotonin responses after treatment with KCO and AP were not different from 5HT responses after treatment with KCO only. This indicates a protective effect by the KCOs against APi induced damage. Three series of control experiments were performed on cerebral vessels without Abeta:

1. Treatment with diazoxide and buffer. 2. Treatment with NS1619 and buffer. 3. Treatment with buffer only.

There were no significant differences in the 5HT response or in the ACh response before and after treatment with any of the control solutions.

Effect of KCOs on Abetainduced endothelial dysfunction in the rat aorta

Real time recordings of an aorta ring segment are shown in Figure 4. Treatment with a single-dose of Abeta (10-6 M) enhanced the vasoconstriction response to phenylephrine (PE) (5 x 10-8 M) and reduced the response to the endothelium-dependent vasodilator acetylcholine (ACh). Pre-treatment with diazoxide (lower panel) eliminated the effect of Abeta on the responses to PE and ACh. The effect of Abeta on dose-dependent relaxation of ACh and the inhibition of this effect by KCOs is shown in Figure 5. In the presence of AP the ACh relaxation response curve was shifted to the right, but maintained its sigmoidal form, indicating reduced sensitivity to AChinduced relaxation. The presence of NS1619 (Figure 5) or diazoxide (Figure 6) significantly antagonized the effect of beta-amyloid on aorta sensitivity to ACh. Pretreatment with the KCa channel blocker, TEA, reversed the protective effect of NS1619 (Figure 6). Treatment with TEA alone reduced sensitivity to ACh which is similar to the effect of Af alone (Figure 6). There was no difference in the maximum ACh-induced relaxation between control, treatment with A,, and treatment with Abeta+KCO. Pre-treatment with diazoxide (n=5) or NS1619 (n=5) significantly reduced the enhancement of contraction by A# in aorta (data not shown). The constriction response to PE was not significantly different in the presence of KCOs alone (vehicle (no Ap)). The presence of KCOs alone did not alter the relaxation response to ACh (data not shown).

Electron microscopic observations

Untreated cerebral vessels (Figure 7A) and aorta (Figure 8A) show intact endothelial cells adhering to the internal elastic lamina of vessel wall. Organelles including mitochondria and Golgi apparatus are also clearly visible in the cytoplasm of the untreated vessels. Cerebral vessels (Figure 7B) and aorta (Figure 8B) treated with beta-amyloid show remarkable endothelial cell destruction, a discontinuous internal elastic lamina and visible cytoplasmic and membrane disruption. The effect of pre-treatment with diazoxide followed by Abeta treatment of cerebral and aorta vessels is shown in Figures 7C and 8C respectively. Effect of pre-treatment with NS1619 is shown in Figures 7D and 8D. In the presence of KCOs, the toxic effect of Af is absent and the endothelial cells appear normal with an intact internal elastic lamina.

DISCUSSION

The main finding of this study is that potassium channel openers (KCOs) significantly antagonized the toxic effect of the Alzheimer's peptide, beta-amyloid, on the vascular endothelium. KCOs for both Kca and K^sub ATP^ channels were effective in preventing the Abeta induced damage in cerebral microvessels and the aorta. The loss of endothelial function induced by Abeta and its prevention by KCOs was supported by results from electron microscopy showing Abeta damage to the structure of the endothelial cells and a protective effect by KCOs. The results suggest that K+ channels may be involved in the mechanism of Abeta induced endothelial dysfunction and may play a role in cerebral angiopathy and peripheral artery disease that is associated with Alzheimer's disease and with aging.

A number of studies1,22,23 that show endothelial disruption and vascular dysfunction are increasing evidence suggesting a role for Af deposition in vascular disease. Abnormalities in the microvasculature have been shown to precede other neuropathological features of AD24. Prominent changes include distortions of small cerebral vessels24,25, white matter lesions with associated beta-amyloid deposits, reduced lumen diameter of small cerebral vessels26, and endothelial cell dysfunction27. Endothelial abnormalities include cells without basement membrane28, an increased number of endothelial gap junctions29 and altered and decreased numbers of mitochondria30,31. Af toxicity has been associated with the formation of free radicals in neurons32 and our previous findings show that flamyloid deposition induces endothelial damage (within minutes) in the peripheral""z and cerebral vessels910 through the production of reactive oxygen species. Interaction between beta-amyloid and endothelial cells is supported by the recent finding of an Abetabinding cell surface receptor (receptor for advanced glycations end products or RAGE) in endothelial cells, and the upregulation of these receptors in AD33. We have shown that AP deposition ultimately (within 5 h) results in necrotic cell death in cultured endothelial cells34. The main source of O2 in blood vessels is the membrane associated NADH dependent oxidoreductase which can be triggered by peptide binding to a Gprotein coupled receptor35. Other sources of oxidants include the mitochondria, xanthine oxidase, cyclooxygenase, and lipoxygenase. The threat of O2 induced toxicity is normally countered by the presence of superoxide dismutase (SOD) on the cell membrane. Our previous finding9-13,34 that exogenous SOD protects against Abeta related damage places the source of Abeta induced OZ release in or at the plasma membrane. This is supported by reports that f-amyloid activates membrane associated NADPH oxidase and its neurotoxicity is mediated by the generation of Oz/H20235. Normally the intact endothelium provides a relative excess of NO over Oz due to the presence of SOD. Physiological levels of NO are advantageous for various functions including smooth muscle relaxation and nerve-effector transmission. An excess of OZ relative to NO levels would lead to toxicity if the antioxidant mechanisms of the cell are overwhelmed.

One mechanism for the protective effect of KCOs against Af/ toxicity in endothelial cells can be explained by their ability to activate K+ channels and may involve an imbalance between NO and OZ . Several types of K+ channels, including K^sub ATP^ and Kca channels, have been found in cardiac, smooth muscle, endothelial and neuron cells17-26. KCOs such as diazoxide have been shown to protect cardiac myocytes against ischemic injury19,20 . Other reports have described the protective effect of KCOs in neurons against oxidative injury induced by beta-amyloid21. It is now well established that endothelium cells release EDRF in response to an increase in intracellular Ca++36. Activation of K+ conductance has been shown to increase intracellular Ca++ stimulating the secretion of EDRFs37,38. These studies suggest a mechanism for the synthesis and release of vascular endothelium derived relaxing factors (EDRF) that involves Ca+ efflux from internal stores to open K+ channels and hyperpolarize the endothelial cell. Hyperpolarization is believed to increase the electrochemical gradient for maintained Ca++ entry during stimulation resulting in greater activation of NO synthesis39. beta-amyloid could induce K+ channel dysfunction in endothelial cells resulting in membrane depolarization, reduced Ca++ influx and lower NO synthesis. KCOs could protect against Af toxicity by opening K+ channels and maintaining Ca++ influx and NO production. Reversal of the protective effect of KCOs in our aorta ring segments by TEA suggest that the mechanism for KCO protection does involve K+ channels. However, the reversal effect by TEA is not definitive since TEA alone decreased the sensitivity to ACh which is similar to the effect of Ai alone. The effect of TEA alone does indicate that sufficient impairment of endothelial K+ channels alters the response to ACh.

KCOs may also provide a protective effect by mechanisms independent of K+ channel activation. In neurons Goodman and Mattson21 found that KCOs provide partial protection against the oxidative insult of beta-amyloid in the presence of K+ channel blockers indicating that KCOs exert some protective effects independent of K+ channel activation. They concluded that KCOs can both suppress excitability and act as antioxidants when protecting against Ai toxicity in neurons. They also acknowledged that experiments in the presence of a K+ channel blocker were complicated by the fact that exposure to the K+ channel blocker alone produced results similar to Abeta toxicity (i.e. reduced neuronal survival). Other studies of K+ channel-independent actions of KCOs have found that diazoxide suppressed Na+ and Ca++ currents in neurons4. Some studies of vascular smooth muscle suggest that KCOs can produce vasorelaxation independently of their hyperpolarizing effect41 by inhibiting the refilling of intracellular calcium stores.

The results here and in our previous work suggest that damage to endothelial function by Af could lead to peripheral arterial disease such as coronary artery disease, cerebral ischemia, stroke and impaired cognitive function as seen in Alzheimer's disease. The development of Alzheimer's disease, atherosclerosis, or hypertension could take years. We propose that amyloid mediated vascular damage is an early event relative to the development of cerebrovascular disease. The initial damage by Abetacould occur at limited locations in the vascular endothelium. Subsequent damage would escalate to surrounding tissues if antioxidant defense mechanisms are compromised due to aging, environmental factors or genetics. KCOs can oppose this process and protect endothelial cells against beta-amyloid toxicity.

The possibility that cerebrovascular disorders associated with deposition of beta-amyloid may involve an NO/ 02 imbalance and endothelial dysfunction has potential therapeutic implication. The protective effect of KCOs against beta-amyloid-mediated endothelial dysfunction shown here, together with its cardioprotective and neuroprotective effects, suggests a promising therapeutic value of KCOs in stroke and other vascular disorders that involve oxidative injury.

ACKNOWLEDGEMENTS

This study was supported by a Grant-in-Aid from the American Heart Association, Florida Affiliate.

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Xuedong Chi, E. Truitt Sutton, Tom Thomasbeta and Joel M. Price

*Department of Physiology and Biophysics, *Woodlands Medical Center and Department of Anatomy University of South Florida, Tampa, FL, USA

Correspondence and reprint requests to: Joel M. Price PhD, University of South Florida, Department of Physiology and Biophysics, College of Medicine, Box 8, 12901 Bruce B. Downs Blvd., Tampa, FL 336124799, USA. Accepted for publication December 1998.

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