<p> Hypertension/2006/084657.R2</p><p>Online Supplement</p><p>Methods expanded</p><p>All of the experimental procedures were approved by the Committee for Animal</p><p>Research of Sun Yat-Sen University and were in accordance with the National</p><p>Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.</p><p>Animal models</p><p>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</p><p>Tissue preparation and immunofluorescence analysis2 </p><p>The anesthetized rats were perfused with 200 ml physiologic saline through left artrium, and then fixed in 4ºC paraformaldehyde solution containing (mmol/L): 100</p><p>Na2HPO4·12H2O, 100 NaH2PO4·2H2O, 10% paraformaldehyde, pH 7.4, for 15 min.</p><p>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.</p><p>The sections were consecutively incubated with the blocking serum (20% goat serum) for 15 min, followed by incubation with monoclonal α-smooth muscle-actin</p><p>(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,</p><p>China) according to the manufacturer's instructions. All dilutions were made in PBS, pH 7.2.</p><p>All 3-µm sections were observed and captured with confocal microscope</p><p>(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,</p><p>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. </p><p>Cell isolation </p><p>Rat basilar arterial smooth muscle cells were isolated by enzymatic digestion as</p><p>3 previously described. The basilar arteries were placed in a cold (4ºC), 95% O2–5%</p><p>CO2-saturated solution containing (mmol/L): 130 NaCl, 5 KCl, 0.8 CaCl2, 1.3 MgCl2,</p><p>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</p><p>(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</p><p>(Type Ⅳ -S, 0.5 g/L) and deoxyribonuclease Ⅰ (0.1 g/L) for 1 h at room temperature.</p><p>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</p><p>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.</p><p>Measurement of cell membrane capacitance (Cm) Hypertension/2006/084657.R2</p><p>The voltage-clamp experiment was performed as previously described using an</p><p>Axopatch 200B amplifier (Axon Instrument, Foster City, CA).4 Data acquisition and command potentials were controlled by pCLAMP8.0 software (Axon Instruments).</p><p>Patch pipettes were made from borosilicate glass using a two-stage puller (pp-83,</p><p>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</p><p>PC-compatible computer. The bath solution and pipette solution were same as those described previously.4 All experiments were performed at room temperature (25ºC). </p><p>− − Measurement of the concentration of intracellular Cl ([Cl ]i ) in rat CVSM cells</p><p>− [Cl ]i in CVSM cells were measured using 6-methoxy-N-ethylquinolinium iodide</p><p>(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</p><p>(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-</p><p>− 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</p><p> and chloride concentration is given by the Stern–Volmer equation: (FO/F) – 1 = K SV</p><p>[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</p><p> quencher; and KSV is the Stern–Volmer constant. </p><p>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</p><p> different corporation of solution A (mmol/L: 25 NaCl, 120 KCl, 2 CaCl2, 2 MgSO4, 0.4</p><p>KH2PO4, 10 HEPES, 11 Glucose, pH 7.4) and solution B (25 NaNO3, 120 KNO3, 4</p><p>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. </p><p>Fluorescence quenching induced by Cl− was monitored by MetaFluor Imaging software (Universal Imaging Systems, Chester, PA) with 350-nm excitation</p><p> 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−</p><p> 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.</p><p>Background fluorescence subtracted from total fluorescence measured in experiment Hypertension/2006/084657.R2</p><p> gives the fluorescence of MEQ itself. The Stern–Volmer equation should be: (FO －</p><p>− 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</p><p>− MEQ, we used the Stern-Volmer equation to calculate [Cl ]i.</p><p>− − The hypotonic-induced decrease in [Cl ]i (Δ[Cl ]i hypo) was calculated using the</p><p>− − − following equation: Δ[Cl ]i hypo= [Cl ]i hypo-[Cl ]i iso. The percentage inhibition of</p><p>− reduction in [Cl ]i. by genistein was calculated using the following equation:</p><p>− − − − − Inhibition % = {[Cl ]i.hypo+gen[Cl ]i-[Cl ]i.hypo}/{[Cl ]i.iso-[Cl ]i.hypo}×100%. Where</p><p>− − − [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</p><p>− hypotonic-induced reduction in [Cl ]i. by orthovandate was calculated using the</p><p>− − − following equation: Increase % = /{[Cl ]i.iso-[Cl ]i.hypo}×100%. Where [Cl ]i.iso,</p><p>− − [Cl ]i.hypo, [Cl ]i.hypo+orthovandate is the concentration of intracellular chloride under isotonic, hypotonic and hypotonic conditions.</p><p>Solutions</p><p>The hypotonic bath solution contained (mmol/L): 111 NaCl, 0.5 MgCl2, 2.5 KCl,</p><p>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</p><p> mosmol/kg·H2O. A 300 mosmol /kg·H2O isotonic bath solution was prepared by</p><p> 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,</p><p>U.S.A.).</p><p>Results</p><p>The supplementary tables and related descriptive text is introduced in the same order as they are mentioned in the text of the article.</p><p>Development of hypertension and morphological change in basilar arterial smooth muscle media in 2k2c models </p><p>The mean BP in sham-operated rats at the end of 1 week, 4 weeks, 8 weeks and</p><p>12 weeks after renal artery constriction was 99.0±9.9, 102.5±8.9, 105.5±8.6 and</p><p>106.5±9.4 mm Hg respectively (n=8, P>0.05). In 2k2c group, the mean BP rose to</p><p>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</p><p>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,</p><p>8 weeks and 12 weeks after operation, respectively (n=8, P >0.05). Hypertension/2006/084657.R2</p><p>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</p><p>(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</p><p>References:</p><p>1. Zeng J, Zhang Y, Mo J, Su Z, Huang R. Two-kidney, two clip renovascular</p><p> hypertensive rats can be used as stroke-prone rats. Stroke. 1998; 29: 1708-</p><p>1713.</p><p>2. Lee RM, Forrest JB, Garfield RE, Daniel EE. Comparison of blood vessel wall</p><p> dimensions in normotensive hypertensive rats by histometric and</p><p> morphometric methods. Blood Vessels. 1983; 20: 245-254.</p><p>3. Guan YY, Weir BK, Marton LS, Macdonald RL, Zhang H. Effects of</p><p> erythrocyte lysate of different incubation times on intracellular free calcium in</p><p> rat basilar artery smooth-muscle cells. J Neurosurg. 1998; 89: 1007-1014.</p><p>4. Zhou JG, Ren JL, Qiu QY, He H, Guan YY. Regulation of intracellular Cl-</p><p> concentration through volume-regulated ClC-3 chloride channels in A10</p><p> vascular smooth muscle cells. J Biol Chem. 2005; 280: 7301-7308.</p><p>5. Wang GL, Wang GX, Yamamoto S, Ye L, Baxter H, Hume JR, Duan D.</p><p>Molecular mechanisms of regulation of fast-inactivating voltage-dependent</p><p> transient outward K+ current in mouse heart by cell volume changes. J</p><p>Physiol. 2005; 568: 423-443. Hypertension/2006/084657.R2</p><p>6. Biwersi J, Verkman AS. Cell-permeable fluorescent indicator for cytosolic</p><p> chloride. Biochemistry. 1991; 30: 7879-7883.</p><p>7. Izzard AS, Graham D, Burnham MP, Heerkens EH, Dominiczak AF, Heagerty</p><p>AM. Myogenic and structural properties of cerebral arteries from the stroke-</p><p> prone spontaneously hypertensive rat. Am J Physiol Heart Circ Physiol. 2003;</p><p>285: H1489-H1494.</p><p>8. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic</p><p> hypertension. Hypertension. 1989; 13: 968-972.</p><p>9. Baumbach GL, Sigmund CD, Faraci FM. Cerebral arteriolar structure in mice</p><p> overexpressing human renin and angiotensinogen. Hypertension. 2003; 41: 50-</p><p>55. Hypertension/2006/084657.R2</p><p>Table I. </p><p>Comparison of Cm of CVSM cells (pF) </p><p>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</p><p>Table II </p><p>Structure parameters of cerebral basilar arteries in hypertension </p><p>Parameters 1 week 4 weeks 8 weeks 12 weeks Sham</p><p>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)</p>