The innervation of guinea pig epicardial coronary veins: Immunohistochemistry, ultrastructure and vasomotility

Journal of the Autonomic Nervous System, 47 (1994) 201-212

201

© 1994 Elsevier Science B.V. All rights reserved 0165-1838/94/$07.00 JANS 1500

The innervation of guinea pig epicardial coronary veins: Immunohistochemistry, ultrastructure and vasomotility S6rgio Gulbenkian

a,

Ole Saetrum O p g a a r d b, Carla P. Barroso a, John W h a r t o n c, Julia M. Polak c and Lars Edvinsson b

a Department of Cell Biology, Gulbenkian Institute of Science, Oeiras, Portugal, b Department of Internal Medicine, University of Lund, Lund, Sweden, and c Department of Histochemistry, Royal Postgraduate Medical School, London, UK (Received 1 July 1993) (Revision received and accepted 30 August 1993)

Key words: Noradrenaline; Neuropeptide; Endothelin; Serotonin; Uridine 5'-triphosphate; Coronary vein; Immunohistochemistry; Ultrastructure; In vitro pharmacology Abstract

The innervation and vasomotor responses to several vasoactive agents of guinea pig epicardial coronary veins were investigated by means of immunohistochemical, histochemical, ultrastructural and in vitro pharmacological techniques. The use of an antiserum to the general neuronal marker protein gene product 9.5 revealed that coronary veins are supplied by a network of fine varicose nerve fibres in the adventitia. The majority of the nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase immunoreactivity. Only a few nerve fibres displayed substance P, neuropeptide K (NK) and calcitonin gene-related peptide (CGRP) immunoreactivity. In double stained preparations substance P immunoreactivity was co-localized with NK and CGRP in the same nerve fibres. Nerve fibres containing vasoactive intestinal peptide (VIP) immunoreactivity or acetylcholinesterase activity were not detected. Endothelin immunoreactivity was also found in the vein endothelial cells. Ultrastructural studies revealed the presence of axon varicosities at the adventitial-medial border. In vitro pharmacological studies showed that endothelin-1 and -2 elicited a significant contractile response of epicardial vein segments. Noradrenaline, NPY, serotonin and uridine 5'-triphosphate induced only a relatively weak contractile response in the vein segments. Although vasodilatory responses were difficult to examine in these preparations, it was found that substance P, CGRP and VIP elicited a relaxation of the vein segments. These results indicate that guinea pig epicardial coronary veins are innervated by several nerve populations, however, the control of vasomotor tone of coronary veins appears to be predominantly regulated by 'non-neuronal' vasoactive agents such as endothelin and 5-HT.

Introduction

Correspondence to: Dr. S. Gulbenkian, Departamento de Biologia Celular, Instituto Gulbenkian de Ci~ncia, Apartado 14, 2781 Oeiras Codex, Portugal. Tel.: 351-1-4431454; Fax: 351-144311631.

SSD1 0 1 6 5 - 1 8 3 8 ( 9 3 ) E 0 1 17-N

It is now well known that the mammalian vascular system is supplied with nerve fibres containing various types of mediators including several neuropeptides. These neuropeptides are Io-

202

calized to specific populations of efferent and afferent nerves and are known to exert potent vasomotor effects, acting both directly via specific receptors a n d / o r indirectly by modulating the action of other transmitters (for review see Ref. 38). Recently, several studies have focused on the supply of nerve fibres storing neuropeptides in the coronary vasculature. Neuropeptides such as neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (V1P) and the tachykinins have been identified in nerves associated with both epicardial and intramyocardial coronary arteries and were suggested to have an important role in the regulation of coronary blood flow [1,3,4,17,19,32]. In contrast to the many studies that have been performed on the innervation of coronary arteries, to our knowledge, there is practically no information on the distribution and action of classical and putative transmitters in coronary veins. In the present study, the innervation of guinea-pig epicardial coronary veins was investigated using acetylcholinesterase (ACHE) histochemistry and immunofluorescence staining together with antisera against the general neuronal marker protein gene product 9.5 (PGP 9.5), neuropeptides (NPY, VIP, C G R P and tachykinins), the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) and endothelin. The ultrastructural features of perivascular nerves were examined and pharmacological studies were carried out to investigate the possible action of vasoactive agents on epicardial coronary veins.

Materials and methods

lmmunohistochemistry Tissues for immunostaining were collected from male guinea pigs (n = 5; Dunkin Hartley strain, weight 250-350 g) and fixed by immersion in Zamboni's fixative [35] for 16 h at 4°C. Following thorough rinsing in phosphate-buffered saline (PBS) containing 15% sucrose (w/v) and 0.01% (w/v) sodium azide the heart was either processed for cryostat sectioning or dissected to obtain whole mount preparations of large superfi-

TABLE 1 Source and characterization of" the primary antisera Antigen

Donor Code species

Source

PGP 9.5 TH NPY CGRP Substance P Neuropeptide K VIP Endothelin-I

Rabbit Rabbit Rabbit Rabbit Rat

Ultraclone, UK Eugene Tech, USA Hammersmith Hospital Hammersrnith Hospital Sera Lab, UK

RA95103 TEl01 1086 1208 MAS035b

Rabbit 15-36 R2 Hammersmith Hospital Rabbit 652 Hammersmith Hospital Rabbit RAS6901-N Peninsula Lab., UK

References for characterization of the antisera: [11,16,17].

cial epicardial coronary veins (great and middle cardiac veins and marginal left ventricular vein). Cryostat sections (15 /xm thick) were immunostained by an indirect immunofluorescence method. A modified indirect immunofluorescence method was performed on whole mount preparations of the veins as previously described [16]. Briefly, after pretreatment with a solution containing 0.2% Triton X-100 in PBS for 2 h at room temperature and impregnation with the dye pontamine sky blue for 30 min, blood vessels were incubated in diluted primary antisera (Table 1) overnight at room temperature. The preparations were then washed in PBS and incubated with fluorescein isothiocyanate conjugated goat antirabbit IgG (1:100 dilution; Sigma) for 1 h at room temperature. For the simultaneous localization of two antigens, preparations were first exposed to a primary antiserum raised in rabbit which was visualized by a rhodamine-labelled goat anti-rabbit IgG (1 : 100 dilution; Sigma) and then to a second primary antiserum raised in rat which was visualized by a fluorescein isothiocyanatelabelled goat anti-rat IgG (1 : 100 dilution; Sigma). The preparations were finally examined using an Olympus BH-2 microscope equipped for epi-illumination with filters selective for fluorescein and rhodamine fluorescence.

Acetylcholinesterase (ACHE) staining For the histochemical demonstration of AChE activity in cryostat sections a staining procedure

203 was performed following a method adapted from Gulbenkian et al. [18]. Briefly, cryostat sections were immersed in incubation medium (consisting of 10 mg acetylthiocholine iodide, 12.64 ml 0.1 M sodium acetate, 0.4 ml 0.1 M acetic acid, 0.96 ml 0.1 M sodium citrate, 2 ml 30 mM cupric sulphate, 2 ml 5 mM potassium ferricyanide and 382 ml distilled water) for 30 min at 37°C. After rinsing in distilled water the AChE activity was visualized by immersing the preparations for 5 min in Tris-HCl buffer (50 mM, pH 7.6) containing 0.04% 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0.3% nickel ammonium sulphate and then for a further 5 min with the addition of 0.003% hydrogen peroxide. After a brief wash in distilled water, cryostat sections were dehydrated in an ascending series of acetone concentrations (70-95% for 2 min each change, then 100% for two changes of 5 min each) cleared with xylene (two changes of 5 min each) and mounted in DPX. The preparations were finally observed and photographed with an Olympus BH-2 microscope using transmitted bright field illumination.

Immunohistochemical and histochemical controls In control experiments no immunostaining was observed when primary antisera (Table 1) were omitted, replaced with non-immune serum or pre-absorbed with their corresponding antigens (10-5-10 -6 M) for 24 h at 4°C. Labelled secondary antisera also exhibited no cross-reactivity with IgG from inappropriate species. For the histochemical demonstration of AChE activity the following control incubation media were used: (i) 10-4-10 -6 M tetraisopropyl-pyrophosphoramine (iso-OMPA, Sigma) was added to the incubation medium as an inhibitor for nonspecific cholinesterase; (ii) preparations were incubated in a substrate-free medium omitting acetylthiocholine.

Transmission electron microscopy Tissues for electron microscopy were fixed by immersion in a solution containing 2.5% glutaraldehyde (v/v) in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4°C. The tissues were then washed

in buffer containing 0.1 M sucrose, post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4°C, dehydrated in a graded series of ethanol concentrations, cleared in propylene oxide and infiltrated with Epon resin. Ultrathin sections of silver interference colour (70-90 nm) were collected onto 300-mesh formvar-coated grids. Sections were finally counterstained with uranyl acetate and lead citrate and examined using a Jeol CX electron microscope operating at 60 kV.

In vitro pharmacology Male guinea pigs were killed by decapitation after being anaesthetised with approximately 30 mg pentobarbital (Mebumal R) i.p. The heart was quickly removed and placed in a solution containing 119 mM NaC1; 15 mM NaHCO3; 4.6 mM KC1; 1.5 mM CaCI2; 1.2 mM NaH2PO4; 1.2 mM MgC12; and 11 mM glucose. Large superficial epicardial segments (1-2 mm in lenght) from the great cardiac vein and its branches were excised and mounted in a temperature-controlled tissue bath (37°C) containing the previously described buffer solution, which was continuously bubbled with a mixture of 5% COz/95% 02, giving a pH of approximately 7.4. For further details see Ref. 21. To measure the isometric circular wall tension of the vessels, each vessel segment was suspended between two L-shaped metal holders (0.05 to 0.1 mm in diameter). One of the metal holders was connected to a Grass FT-03 transducer for continuous recording of isometric tension on a Grass polygraph. The resting tension of the vessel segments was adjusted by varying the distance between the holders and depending on the vessel size, a tension of 0.2-0.5 mN was appfied. Due to initial spontaneous relaxations of the vessel segments, it was necessary to make several adjustments to the metal holders in order to maintain a stable resting tension. After approximately 1 h, when the tension had stabilized at the desired level, the vessel was exposed to a buffer solution containing 60 mM KCI, obtained by substituting equimolar concentrations of NaC1 for KCI in the previously described buffer solution. Only those

204 vessels which responded with a K+-induced contraction, that was reproducible a second time after washing out with the normal buffer were used for investigation. The K+-induced tension for these vessels was 0.46 _+ 0.09 m N (mean _+ SEM). Because the lenght and thickness of the vessel segments varied, the tension induced by K + was set arbitrarily at 100% and used as an internal standard to which contractile responses were compared. In relaxant trials, the relaxation was compared to the precontraction induced by (3 × 10 -5 M) prostaglandin F2,~ (PGF2~), which was set at 100%.

TABLE I1 Contractions induced by endothelin-l, endothelin-2, serotonin, NPY, noradrenaline and UTP on guinea pig epicardial coronary L,eins. Ema x values represent percentage o f K +-induced contraction. E,,,, x and pD 2 values are git~en in means + S E M and were calculated only from responding t,essel segments, n = number of vessel segments tested, n R = responding cessel segments

Agonist Endothelin-1 Endothelin-2 Serotonin NPY Noradrenaline UTP

n

I1R

Emax

pD 2

7

7

197.3_+50.6

8.21 _+0.16

5 7 5 7 6

5 5 3 3 3

226.8 + 38.5 35.8_+ 14.2 25.3_+ 11.6 25.2_+ 5.9 7.1 + 1.9

8.36 _+0.03 6.11 _+0.40 6.91 _+0.06 5.15_+0.94 5.62_+0.03

A n a l y s i s o f in v i t r o d a t a

For each vessel segment, the contraction induced by different concentrations of agonist was calculated as a percentage of the potassium-induced contraction of the same segment, using an Excel computer program. Separate vessel segments were used for the different drugs tested. Emax (maximum effect obtained with an agonist) and pD 2 (negative logarithm of the concentration of agonist that elicited half maximum effect) were calculated. The Emax values are given as a percentage of the potassium-induced contraction. Values are given as mean _+ SEM. The Emax values seen in Table 2 express the mean of the maximum response only from those vessels where agonists induced a contraction, whereas, when calculating the concentration-response curves seen in Fig. 7, the non-responding vessels were also taken into the calculations. A Mann-Whitney U-test was used to determine statistical significance with respect to differences in pD 2 and Emax values, and statistical significance was assumed when P < 0.05. Drugs

The following pharmacological agents (obtained from Sigma) were used: endothelin-1 (ET1), endothelin-2 (ET-2), neuropeptide Y (NPY), noradrenaline (NA), serotonin (5-HT), vasoactive intestinal polypeptide (VIP), substance P (SP), uridine 5'-triphosphate (UTP). Prostaglandin F2. (PGF2.; Amoglandin R, Astra, Sweden) was also

used. All agents were dissolved and diluted in saline containing 10 -4 M ascorbic acid to minimize oxidation. The saline used to dilute peptides also contained 1% bovine serum albumin to prevent peptides sticking to solid surfaces.

Results Light

microscopical

immunohistochemistry

and

histochemistry

The use of the antiserum to the general neuronal marker P G P 9.5 demonstrated that the guinea-pig major epicardial coronary veins (great and middle cardiac veins and marginal left ventricular vein) were supplied by numerous varicose nerve fibres forming a loose network in the adventitia (Fig. la and b). The distribution of this perivascular innervation was found similar in all the vessels examined. The majority of the nerve fibres in the epicardial veins displayed immunoreactivity for NPY and T H (Fig. lc and d). It was also observed that the number and distribution pattern of NPY-containing nerves was similar to that of nerves displaying T H immunoreactivity (Fig. lc and d). In contrast, relatively few nerve fibres displaying CGRP, substance P and neuropeptide K immunoreactivity were identified in the veins (Figs. 2 and 3). Double immunostaining for either C G R P and substance P (Fig. 2a and b)

205

Figs. 1-3. Cryostat section (la) and whole mount preparations (lb-d; 2a-b; 3a-b) of a guinea pig great cardiac vein immunostained for PGP 9.5 (Fig. 1a-b), NPY (Fig. le), TH (Fig. ld), CGRP (Fig. 2a), substance P (SP, Figs. 2b, 3b) and neuropeptide K (NK, Fig. 3a). The co-localization of SP with CGRP (Fig. 2a-b) as well as with NK (Fig. 3a-b) was determined by double immunostaining the same preparations. (la) shows PGP-immunoreactive nerve fibres (arrows) in the adventitia. Bar = 50/zm.

206

207 or substance P and neuropeptide K (Fig. 3a and b) revealed that the peptide immunoreactivities coexisted in the same varicose nerve fibres. Nerve fibres containing VIP immunoreactivity were not detected. The histochemical demonstration of A C h E activity in cryostat sections of left ventricle showed that guinea-pig epicardial coronary veins are not supplied with AChE-containing nerve fibres (Fig. 4). In marked contrast to the distribution of the neuropeptides, endothelin immunoreactivity was only detected in the cytoplasm of endothelial cells (Fig. 5).

Transmission electron microscopy Electron microscopical examination of guineapig epicardial coronary veins revealed that the wall of these vessels consisted of a tunica intima, a thin tunica media, formed by one or two layers of smooth muscle cells circularly arranged and a large tunica adventitia (Fig. 6). In the adventitia numerous unmyelinated axons were observed. At the adventitial-medial border axon varicosities were usually separated from smooth muscle cell membranes by a cleft 500 to 800 nm wide (Fig. 6).

In vitro pharmacology In the present study we used: ET-1 and ET-2 ( 1 0 -12 t o 3 x 10 - 7 M), serotonin ( 1 0 -11 to 10 - 4 M), NPY (10 -11 to 3 x 10 -7 M), noradrenaline

(10 -11 to 3 X 10 -4 M), and U T P (10 - t ° to 10 -4 M). ET-1 and ET-2 showed no significant difference in potency (pD 2 value) and maximum effect (Emax value), and were the strongest and most

potent vasoconstrictor agents. Serotonin, NPY, noradrenaline and UTP induced only weak contractions in some of the tested vessel segments. Emax and pD 2 values are listed in Table 2 and concentration-response curves are given in Fig. 7. To test the responses induced by the vasodilator peptides, substance P, C G R P and VIP, vessel segments were precontracted with 3 x 10 -5 M PGF2,. Due to the fact that the precontractile response induced by PGF2, , was relatively weak and unstable it was not possible to accurately determine the magnitude of relaxant responses. Nevertheless, a relaxant response was noted in most of the tested vessel segments, ranging from approximately 15-25% of the PGF2~-induced contraction for substance P, and 10-20% for C G R P and VIP (n = 4 - 6 vessel segments in each group).

Discussion The present data show that the guinea pig major epicardial coronary veins are supplied with peptide-containing nerve fibers in the adventitia and at the adventitial-medial border, although the innervation is less dense than that previously demonstrated around coronary arteries [17,19]. NPY-immunoreactive nerves represent the main peptide-containing nerve population identified in epicardial coronary veins and display a similar distribution p a t t e r n to T H - i m m u n o r e a c t i v e nerves. These observations are consistent with the general assumption that the majority of NPY-immunoreactive nerves in the mammalian cardiovascular system represent postganglionic sympathetic neurones [12,28].

Fig. 4. Cryostat section of guinea pig left ventricle histochemically stained for AChE activity. AChE-containing nerve fibres (arrows) are not found associated with the great cardiac vein (V). E: epicardium. Bar = 50/~m. Fig. 5. Cryostat section of a guinea pig great cardiac vein immunostained for endothelin. (a) Endothelial cells display a strong immunofluorescence staining (arrows). (b) Control micrograph of an adjacent section of the same vessel in which the primary antiserum was replaced with non-immune serum, demonstrating no staining. Bar = 50/~m. Fig. 6. Electron micrograph of nerve varicosities (asterisks) at the adventitial-medial border of a guinea pig great cardiac vein. The cleft, between the varicosities and the muscle cell membrane (double-headed arrow) is generally greater than 500 nm wide. A: adventitia; SM: smooth muscle; E: endothelium. Bar = 500 nm.

208

A

B

16

300 250

""61 2

"6 200

i

~8

15o

34 "E 100 0

0

5O 10 -8

10-7

10 -6

10 -5

10 -4

10 -3

Noradrenaline conc (M)

10-10

10 -9

10-6 10-7 Endothelin conc (M)

10-6

D

35 3O

C "6

25

25 20

+.-. 20

15

"E o 0 10

v

"6 15 tO O

10

tO

10 -9

10-8

10-7

10-6

10-5

10 "4

Serotonin conc (M)

O

E 6 10-9

10-8

10 -7

Neuropeptide Y conc (M)

10 -6

TJ

+.-. v

"6 4 tO

g 2, O

10-6

10-5 UTP conc (M)

10-4

209 In the present study it was observed that N A induced a weak vasoconstriction at high concentrations in only 3 out of 7 vein segments tested. It is well known that responses to N A are complex and vary greatly between arteries and veins as well as between different vascular beds and different species. The relaxing or contracting action of N A in the coronary vascular bed has been attributed to segmental variation in the distribution of alpha- and beta-adrenoceptors [9]. It has also been reported that N A could induce the release of an endothelium relaxing factor [6]. In isolated rat coronary arteries it has been shown that N A induced the relaxation of small intramural coronary arteries but had either no effect or induced only a small contraction in epicardial segments [31]. In the same study it was reported that N A induced a contraction in both proximal and distal coronary arteries after blockade of beta-adrenoceptors with propranolol. In our study, the relatively poor contractile effect induced by N A suggests that the a - a d r e n o c e p t o r constrictive mediated responses in guinea pig coronary veins are of minor importance for the regulation of epicardial coronary venous tone. In the present study, NPY induced a weak constriction in 3 out of 5 vessel segments tested, the vasoconstrictory effect starting at lower concentrations than those necessary for the NA-induced contraction. Although, little work has been performed on coronary veins, our findings are in concert with the general view that N P Y is a vasoconstrictor peptide [10,36] and suggest that this neuropeptide may have a role in the regulation of epicardial coronary venous tone. The action of N P Y has been shown to be mediated via specific Y1 and Y2 receptors [34]. In experiments using isolated coronary arteries from man [13] and guinea pig [17] it appears that NPY induces a constrictory effect on intramyocardial arteries rather than epicardial arteries. It should be noted, however, that the effects of NPY on cardiovascu-

lar tissues are complex, including the presynaptic inhibition of N A release [14], the potentiation of NA-induced vasoconstrictor responses [20,30] as well as the modulation of the action of vasodilator agents [19]. It is now well known that endothelin is a 21 amino-acid peptide produced by endothelial cells, and it has a very potent vasoconstrictor effect [5,39]. Three endothelin-isopeptides, produced by three separate genes, have been identified; endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) [23]. ET-1 has been shown to induce contraction of the guinea pig portal vein and coronary arteries [27]. In vitro studies on dog coronary arteries and veins have revealed a 5 - 1 0 times higher sensitivity for ET-1 (higher pD 2 value) in large veins compared to large arteries [7,8]. Our finding that ET-1 and ET-2 evoke strong contractions of guinea pig coronary veins together with the finding of E T immunoreactivity in endothelial cells indicate a possible role for this peptide in modulating the coronary venous blood flow. Serotonin (5-HT) has been shown to induce vasoconstriction as well as endothelium-dependent vasodilation in coronary arteries of different species [2,22,25]. Since the 5-HT receptor group consists of at least three main classes termed 5-HT 1, 5-HT 2 and 5-HT 3 as well as many subclasses [15,26] the actions of 5-HT in different vascular beds may be the consequence of activation of different subtypes of 5-HT receptors which mediate different responses. The moderate constrictor effect elicited by 5-HT in the present study, suggests that guinea pig epicardial coronary veins are supplied with 5-HT receptors mediating constriction. The nucleotide U T P has been described as an inductor of both vasoconstriction and vasodilation in different vascular beds [24]. U T P induced a sustained contraction of rat tail and femoral arteries as well as the dog saphenous vein in vitro

Fig. 7. Contraction elicited by ET-1 (A; m), ET-2 (A; A), noradrenaline (B), neuropeptide Y (C), serotonin (D) and uridine triphosphate (UTP); E in guinea pig epicardial coronary veins. The contraction of each vessel segment tested was calculated as a percentage of the contraction induced by potassium (60 mM) in the same segment and each point represent the mean values _+S.E.M.

210

[33]. UTP has also been shown to elicit an increase in prostacyclin (PGI2), a potent vasodilator, probably via an increase in intracellular calcium levels [29]. The present finding showing only a weak contractile response in some of the vein segments tested does not indicate any important vasoconstrictory function of UTP in the guinea pig epicardial coronary veins. When compared to the NPY and TH-immunoreactive innervation, guinea pig epicardial coronary veins received a relatively sparce CGRP-, substance P- and neuropeptide K-immunoreactive nerve supply. It was also observed that substance P immunoreactivity was co-localised with CGRP and neuropeptide K in the same varicose nerve fibres. These observations are in accordance with previous immunohistochemical studies which have established that these neuropeptides often occur together in afferent nerve fibres supplying the guinea pig cardiovascular system [17,37]. CGRP, substance P and VIP have been shown to elicit a concentration-dependent relaxation of both guinea pig epicardial and intramyocardial arteries [17]. In the present study, substance P and CGRP were also found to induce relaxant responses in epicardial coronary vein segments. It should be noted, however, that due to the fact that the PGF2,~-precontractile response of coronary vein segments was weak and unstable the vasodilator activity of these neuropeptides remains to be clearly demonstrated. VIP was also found to elicit relaxant responses; however, in view of our morphological observations showing a lack of VIP-immunoreactive fibres, the functional role of VIP in guinea pig epicardial coronary veins is still uncertain. In conclusion, the results indicate that the guinea pig epicardial coronary veins are supplied by several peptide-containing nerve populations. Our results also show that endothelin and 5-HT are the most potent vasoconstrictor agents, therefore suggesting that a 'non-neuronal' control of vasomotor tone may be the most relevant mechanism of regulation of the guinea-pig epicardial coronary venous tone.

Acknowledgements Antisera raised to regulatory peptides at the Hammersmith Hospital were produced in conjunction with Prof. S.R. Bloom. We thank Mrs. M.R. Alpiar~a for the excellent technical assistance. C.P. Barroso was supported by a fellowship from Junta Nacional de Investiga§fio Cientlfica e Tecnol6gica (BIC 673). This work was supported in part by grants from the Swedish Medical Research Council (05958) and the Faculty of Medicine, Lund University, Sweden.

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