Hypertension/2006/084657.R2
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Methods expanded
All of the experimental procedures were approved by the Committee for Animal
Research of Sun Yat-Sen University and were in accordance with the National
Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Animal models
96 healthy male Sprague-Dawley(SD) rats were randomly divided into 3 groups: sham-operated control group (Sham, n=32), hypertensive group (Htn, n=32) and captopril-treatment group (Cap, n=32). 2-kidney, 2 clip (2k2c) stroke-prone renovascular hypertensive rats were prepared as described previously.1 Briefly, a total of 64 healthy male Sprague-Dawley rats were anaesthetized by injection of 10% chloral hydrate (3 mg·kg-1, i.p.), followed by the surgery of partial renal artery constriction with the 2k2c method.1 Both right and left renal arteries were constricted by placing ring-shaped silver clips with an inner diameter of 0.30 mm. Sham group were age-matched male SD rats underwent the same experimental procedures except for placement of renal artery clip. Systolic blood pressure measured in awaked rats by tail-cuff sphygmomanometer (SBP-1). 32 of hypertensive rats intragastrically received captopril (100 mg·kg-1·d-1) after operation and served as captopril-treatment group. All rats were allowed an ordinary life as desired. Hypertension/2006/084657.R2
Tissue preparation and immunofluorescence analysis2
The anesthetized rats were perfused with 200 ml physiologic saline through left artrium, and then fixed in 4ºC paraformaldehyde solution containing (mmol/L): 100
Na2HPO4·12H2O, 100 NaH2PO4·2H2O, 10% paraformaldehyde, pH 7.4, for 15 min.
Brain sections containing 3mm basilar artery were removed and fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS) for 1 h, washed 3 times and then cryoprotected in a graded series of sucrose solutions (5, 10 and 15% wt/vol made up in PBS) for 30 min each, and in 20% sucrose overnight at 4ºC. The fixed sections of basilar artery were then embedded by OCT compound (SAKURA, U.S.A.) and rapidly frozen. Frozen sections were cut at 3µm on a cryostat (Leica CM 1900) and store at -80ºC.
The sections were consecutively incubated with the blocking serum (20% goat serum) for 15 min, followed by incubation with monoclonal α-smooth muscle-actin
(α-SM-actin, Sigma,USA) antibody(1:500) overnight at 4ºC，biotinylated secondary antibody for 30 minutes and peroxidase-labelled ABC (Maixin.Bio, China) for 30 min at room temperature. The color reaction was developed with DAB kit (Maixin.Bio,
China) according to the manufacturer's instructions. All dilutions were made in PBS, pH 7.2.
All 3-µm sections were observed and captured with confocal microscope
(OLYMPUS, FV500-IX 81, magnification 400). For quantification of α-SM-actin staining, the images were analyzed using Image-Pro Plus 5.0(Media cybernetics,
USA). For each Sham, Htn and Cap group (all n=6 animals), a total of 10 sections Hypertension/2006/084657.R2 from the basilar artery was quantitated. The wall diameter (WD) and the lumen diameter (LD) were measured traveling across the outer and inner edges of α-SM- actin staining area, The cross-sectional area (CSA) of basilar arterial media was calculated using the following equation: CSA=[WD/2]2-(LD/2)2, the wall–to-lumen ratio (W/L, the medial thickness to the internal diameter) were also calculated.
Cell isolation
Rat basilar arterial smooth muscle cells were isolated by enzymatic digestion as
3 previously described. The basilar arteries were placed in a cold (4ºC), 95% O2–5%
CO2-saturated solution containing (mmol/L): 130 NaCl, 5 KCl, 0.8 CaCl2, 1.3 MgCl2,
10 HEPES and 5 glucose, pH 7.4. The arteries were cleaned of connective tissue and small side branches, cut into 0.2 mm rings and incubated in low Ca2+ solution
(mmol/L): 0.2 CaCl2, 130 NaCl, 5 KCl, 1.3 MgCl2, 10 HEPES and 5 glucose, pH 7.4, containing Collagenase (Type Ⅱ , 0.5 g/L), elastase (Type Ⅱ -A , 0.5 g/L), hyaluronidase
(Type Ⅳ -S, 0.5 g/L) and deoxyribonuclease Ⅰ (0.1 g/L) for 1 h at room temperature.
The rings were washed in fresh low Ca2+ solution containing trypsin inhibitor (0.5 g/L), deoxyribonucleaseⅠ (0.1 g/L) and then triturated gently for 15~20 times. The isolated cells were plated on glass coverslips in the above buffer solution containing
0.8mM CaCl2 and fatty acid-free bovine serum albumin (BSA, 2 g/L). The freshly isolated CVSM cells should be used for experiments within 10 hours.
Measurement of cell membrane capacitance (Cm) Hypertension/2006/084657.R2
The voltage-clamp experiment was performed as previously described using an
Axopatch 200B amplifier (Axon Instrument, Foster City, CA).4 Data acquisition and command potentials were controlled by pCLAMP8.0 software (Axon Instruments).
Patch pipettes were made from borosilicate glass using a two-stage puller (pp-83,
Narishige, Tokyo, Japan) and had the resistances of 3–5Ω when the pipettes were filled with the pipette solution. The Cm was calculated by integrating the area under an uncompensated capacitive transient elicited by a 5-mV hyperpolarizing pulse from a holding potential of 0 mV.5 The data were directly entered into the hard drive of a
PC-compatible computer. The bath solution and pipette solution were same as those described previously.4 All experiments were performed at room temperature (25ºC).
− − Measurement of the concentration of intracellular Cl ([Cl ]i ) in rat CVSM cells
− [Cl ]i in CVSM cells were measured using 6-methoxy-N-ethylquinolinium iodide
(MEQ) as previously described.4 Briefly, MEQ was reduced to its cell-permeable derivative 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ). Cells were incubated with 100 ～ 150 μM diH-MEQ in a Ringer’s buffer solution containing
(mM): 119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 1.3 MgSO4, 2.5 CaCl2, 26 NaHCO3, and 11 glucose, pH 7.4 at room temperature in the dark for 30 min. In cytoplasm, dihydro-
− MEQ is quickly oxidized to MEQ, which is sensitive to [Cl ]i. Fluorescence of MEQ is quenched collisionally by Cl−. Relationship between fluorescence intensity of MEQ
and chloride concentration is given by the Stern–Volmer equation: (FO/F) – 1 = K SV
[Q]. Where FO is the fluorescence intensity without halide or other quenching ions; F Hypertension/2006/084657.R2 is the fluorescence intensity in the presence of quencher; [Q] is the concentration of
quencher; and KSV is the Stern–Volmer constant.
For the calibration of intracellular Cl− concentration, double ionophore technique was used here according to the manual of Molecular Probes Company and method used by Verkman et al.6 Solutions containing different concentration of Cl− ranging from 0 to 150 mmol/L (usually 0, 15, 30, 60, 150 mmol/L) were made by mix of
different corporation of solution A (mmol/L: 25 NaCl, 120 KCl, 2 CaCl2, 2 MgSO4, 0.4
KH2PO4, 10 HEPES, 11 Glucose, pH 7.4) and solution B (25 NaNO3, 120 KNO3, 4
Ca(NO3)2, 2 MgSO4, 0.4 KH2PO4, 10 HEPES, 1 Glucose, pH 7.4). Both 10μmol/L nigericin and 100μmol/L tributyltin were added into solutions. Nigericin is an exchanger of K ＋ /H ＋ , was used here to clamp intracellular pH. Tributyltin is an exchanger of Cl−/OH−, was used here to remove the Cl− concentration gradient between both sides of cell membrane.
Fluorescence quenching induced by Cl− was monitored by MetaFluor Imaging software (Universal Imaging Systems, Chester, PA) with 350-nm excitation
wavelength and 435-nm emission wavelength. Firstly we got FO, which is the fluorescence intensity in the solution containing 0 mmol/L Cl− concentration. Then we got different fluorescence (F) under solutions containing 15, 30, 60, 150 mmol/L Cl−
concentration. Finally, we got background fluorescence (FB) after 170 mmol/L KSCN and 5μmol/L Valinomycin were added into the last solution. KSCN can quench the fluorescence of MEQ thoroughly. Valinomycin could promote K+ into cells.
Background fluorescence subtracted from total fluorescence measured in experiment Hypertension/2006/084657.R2
gives the fluorescence of MEQ itself. The Stern–Volmer equation should be: (FO －
− FB)/(F－FB) – 1 = Kq[Cl−]. Kq was got by linear regression through the known [Cl ] and the corresponding fluorescence of MEQ. Based on the fluorescence quenching of
− MEQ, we used the Stern-Volmer equation to calculate [Cl ]i.
− − The hypotonic-induced decrease in [Cl ]i (Δ[Cl ]i hypo) was calculated using the
− − − following equation: Δ[Cl ]i hypo= [Cl ]i hypo-[Cl ]i iso. The percentage inhibition of
− reduction in [Cl ]i. by genistein was calculated using the following equation:
− − − − − Inhibition % = {[Cl ]i.hypo+gen[Cl ]i-[Cl ]i.hypo}/{[Cl ]i.iso-[Cl ]i.hypo}×100%. Where
− − − [Cl ]i.iso, [Cl ]i.hypo, [Cl ]i.hypo+gen is the concentration of intracellular chloride under isotonic, hypotonic and hypotonic+genistein conditions. The percentage increase of
− hypotonic-induced reduction in [Cl ]i. by orthovandate was calculated using the
− − − following equation: Increase % = /{[Cl ]i.iso-[Cl ]i.hypo}×100%. Where [Cl ]i.iso,
− − [Cl ]i.hypo, [Cl ]i.hypo+orthovandate is the concentration of intracellular chloride under isotonic, hypotonic and hypotonic conditions.
Solutions
The hypotonic bath solution contained (mmol/L): 111 NaCl, 0.5 MgCl2, 2.5 KCl,
4 1.8 CaCl2, 5 glucose and 10 Hepes, pH 7.4. The solution osmolarity measured by a freezing point depression osmometer (OSMOMAT030, Germany) was 230
mosmol/kg·H2O. A 300 mosmol /kg·H2O isotonic bath solution was prepared by
adding 70 mmol/L sucrose to the hypotonic solution. A 370 mosmol/kg·H2O hypertonic bath solution was prepared by adding 140 mmol/L sucrose to the Hypertension/2006/084657.R2 hypotonic solution. All chemicals were purchased from Sigma (Sigma, St Louis, MO,
U.S.A.).
Results
The supplementary tables and related descriptive text is introduced in the same order as they are mentioned in the text of the article.
Development of hypertension and morphological change in basilar arterial smooth muscle media in 2k2c models
The mean BP in sham-operated rats at the end of 1 week, 4 weeks, 8 weeks and
12 weeks after renal artery constriction was 99.0±9.9, 102.5±8.9, 105.5±8.6 and
106.5±9.4 mm Hg respectively (n=8, P>0.05). In 2k2c group, the mean BP rose to
107.0±11.6 mm Hg at the end of 1 week after operation (n=8, P>0.05). Afterwards, the 2k2c group had higher mean BPs than sham-operated group with the mean values of 146.0±15.8, 172.5±14.7 and 200.5±15.3 mm Hg at 4 weeks, 8 weeks and 12 weeks postoperatively, respectively (n=8, P<0.01). In 2k2c group, BP tended to rise with time after the 2-kindey 2-clip renal artery constriction (n=8, r= 0.8664, P<0.001). The above results are consistent with previous report.1 Development of hypertension in
2k2c group could be prevented by chronic captopril treatment. The mean values of BP in 2k2c group receiving captopril were similar to those of sham-operated groups, which were 98.5±6.7, 99.0±8.8, 98.0±7.5 and 97.5±10.3 mm Hg at 1 week, 4 weeks,
8 weeks and 12 weeks after operation, respectively (n=8, P >0.05). Hypertension/2006/084657.R2
It is well accepted that small resistance arteries (including cerebral basilar arteries) in spontaneously hypertensive rats (SHR) undergo a combination of hypertrophic (increased media thickness and crossectional area) and eutrophic
(decreased lumen and external diameters with unaltered media cross-sectional area) remodeling.7-9 However, the change in structure of cerebral arteries in 2k2c renal hypertensive rats has not been reported. As an indicator of smooth muscle cells, the increased amount of α-SM actin staining suggests the increases in numbers of SMCs or SM-like cells. Immunostaining demonstrated a time-dependent increase in α-SM actin staining in basilar arteries from 2k2c group as BP increased. At 4 weeks postoperatively, there was no significant difference in the structure parameters among all groups. However, at the end of week 8 and 12, the mean values of CSA, WD, LD and W/L in hypertensive groups were significant higher than those in sham-operated group, which could be reversed by captopril treatment (Table II). Hypertension/2006/084657.R2
References:
1. Zeng J, Zhang Y, Mo J, Su Z, Huang R. Two-kidney, two clip renovascular
hypertensive rats can be used as stroke-prone rats. Stroke. 1998; 29: 1708-
1713.
2. Lee RM, Forrest JB, Garfield RE, Daniel EE. Comparison of blood vessel wall
dimensions in normotensive hypertensive rats by histometric and
morphometric methods. Blood Vessels. 1983; 20: 245-254.
3. Guan YY, Weir BK, Marton LS, Macdonald RL, Zhang H. Effects of
erythrocyte lysate of different incubation times on intracellular free calcium in
rat basilar artery smooth-muscle cells. J Neurosurg. 1998; 89: 1007-1014.
4. Zhou JG, Ren JL, Qiu QY, He H, Guan YY. Regulation of intracellular Cl-
concentration through volume-regulated ClC-3 chloride channels in A10
vascular smooth muscle cells. J Biol Chem. 2005; 280: 7301-7308.
5. Wang GL, Wang GX, Yamamoto S, Ye L, Baxter H, Hume JR, Duan D.
Molecular mechanisms of regulation of fast-inactivating voltage-dependent
transient outward K+ current in mouse heart by cell volume changes. J
Physiol. 2005; 568: 423-443. Hypertension/2006/084657.R2
6. Biwersi J, Verkman AS. Cell-permeable fluorescent indicator for cytosolic
chloride. Biochemistry. 1991; 30: 7879-7883.
7. Izzard AS, Graham D, Burnham MP, Heerkens EH, Dominiczak AF, Heagerty
AM. Myogenic and structural properties of cerebral arteries from the stroke-
prone spontaneously hypertensive rat. Am J Physiol Heart Circ Physiol. 2003;
285: H1489-H1494.
8. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic
hypertension. Hypertension. 1989; 13: 968-972.
9. Baumbach GL, Sigmund CD, Faraci FM. Cerebral arteriolar structure in mice
overexpressing human renin and angiotensinogen. Hypertension. 2003; 41: 50-
55. Hypertension/2006/084657.R2
Table I.
Comparison of Cm of CVSM cells (pF)
Groups 1 week 4 weeks 8 weeks 12 weeks Sham 11.8±2.3 11.8±2.6 12±3.5 13.6±3.5 Htn 15.0±2.8* 18.2±1.8† 24.1±4.9†,‡ 26.4±5.4†,‡ * P<0.05 vs corresponding sham-operated group, †P<0.01 vs corresponding sham-operated group，‡ P<0.01 vs 1-week hypertensive group (n=10) Hypertension/2006/084657.R2
Table II
Structure parameters of cerebral basilar arteries in hypertension
Parameters 1 week 4 weeks 8 weeks 12 weeks Sham
WD (μm) 336.8±16.8 334.1±6.8 337.3±14.0 349.5±14.4 LD (μm) 307.6±15.5 302.4±6.0 304.3±13.6 309.9±12.3 WT/LD (%) 8.7±0.3 9.5±0.6 9.8±1.3 11.3±0.7 CSA(μm2) 14840.7±1530.6 15284.1±1233.6 16622.9±2395.4 20514.5±2271.9 Htn WD (μm) 336.7±22.7 328.4±24.8 312.3±14.8* 307.9±5.3‡ LD (μm) 305.6±20.4 290.6±26.4 271.7±19.0* 257.4±8.9‡ WT/LD (%) 9.2±1.4 11.6±1.8* 13.1±2.6‡ 16.4±3.4‡ CSA(μm2) 15756.9±3428.4 18348.2±1925.6*18535.1±2746.7 22375.0±4622.7* Cap WD (μm) 335.2±9.1 334.5±12.3 328.1±14.1 343.5±33.0† LD (μm) 303.8±8.2 301.9±12.0 292.8±11.8* 303.8±32.2§ WT/LD (%) 9.4±1.4 9.7±0.6† 10.7±0.7 11.6±1.4§ CSA(μm2) 15768.4±2510.3 16282.8±1182.9† 17234.9±2174.7 20238.5±3084.4† WD, wall diameter; LD, lumen diameter; W/L, wall–to-lumen ratio; CSA, cross sectional area; * P<0.05 vs corresponding sham group, †P<0.05 vs corresponding hypertensive group, ‡ P<0.01 vs corresponding sham group, §P<0.01 vs corresponding hypertensive group (n=6)