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Involvement of transforming growth factor- in regulation of calcium transients i

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发表于 2009-4-21 13:48 |显示全部帖子
作者:Kumar Sharma, Leo Deelman, Muniswamy Madesh, Bernd Kurz, Emilio Ciccone, Senthuran Siva, Taishan Hu, Yanqing Zhu, Lewei Wang, Robert Henning, Xinliang Ma,  Gyorgy Hajnoczky作者单位:1 Dorrance Hamilton Research Laboratories, Division of Nephrology, Department of Medicine, Department of Anatomy, Pathology and Cell Biology, and Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania; and Department of Clinical Pharmacology, University of Groninge
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          【摘要】
  ?+ N9 g7 e- I6 o) B- G- U- u& ^( W+ f      Altered calcium [Ca 2  ] transients of vascular smooth muscle cells to vasoconstrictors may contribute to altered regulation of blood flow in diabetes. We postulated that diabetes-induced transforming growth factor (TGF)- production contributes to impaired ANG II response of vascular smooth muscle cells in macrovessels and microvessels. Aortic vascular smooth muscle cells isolated from diabetic rats exhibited markedly impaired ANG II-induced cytosolic calcium [Ca 2  ] signal that was completely restored by pretreatment with anti-TGF- antibodies. Similar findings were noted in microvascular smooth muscle cells isolated from preglomerular vessels and cultured in high glucose. The impact of diabetes on [Ca 2  ] transients was replicated by addition of TGF- 1 and - 2 isoforms to aortic smooth muscle cells in culture and diabetic cells had enhanced production of TGF- 2. In the in vivo condition, TGF- 1 was increased in diabetic glomeruli, whereas TGF- 2 was increased in diabetic aorta. The characteristic increase in glomerular filtration surface area found in diabetic rats was prevented by treatment with anti-TGF- antibodies, and impaired ANG II-induced aortic ring contraction in diabetic rats was completely restored by anti-TGF- antibodies. Impaired vascular dysfunction may be partly due to decreased inositol 1,4,5-trisphosphate receptor (IP 3 R), as reduced type I IP 3 R expression was found in diabetic aorta and restored by anti-TGF- antibodies. We conclude that TGF- plays an important role in the vascular dysfunction of early diabetes by inhibiting calcium transients in vascular smooth muscle cells.
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experimental diabetes; microvessels; angiotensin II; intracellular calcium; inositol 1,4,5-trisphosphate receptor; glomerular hypertrophy
0 [/ O; X/ o! b1 Q0 U8 Q          【关键词】 Involvement transforming regulation transients diabetic vascular2 }/ t% a3 P% l0 y( N  d
                  VASCULAR DYSFUNCTION IS considered to be the cardinal basis for the macrovascular and microvascular complications of diabetes ( 17, 48 ). Macrovascular complications refer to the enhanced atherosclerosis of large vessels in diabetes and lead to coronary artery disease and lower limb ischemia. Microvascular complications of diabetes primarily result in retinal, renal, and peripheral nerve involvement. Although these are late complications of diabetes, there are major disturbances of vascular function in patients with early type 1 diabetes ( 2, 30 ) that may contribute to the pathogenesis of the late complications.
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$ T  u/ G" @3 N( c3 qDuring the first 1-3 wk of early diabetes in the streptozotocin (STZ)-induced rat, the majority of studies using invasive measurements have found that the systolic blood pressure is either normal or decreased (reviewed in Refs. 21 and 61 ). Recently, blood pressure was monitored by telemetry and found to either decline ( 59 ) or remain stable ( 60 ) between week 1 and week 4 of diabetes. The blood pressure surprisingly does not increase despite a tendency for salt retention after the onset of hyperglycemia ( 63, 64 ). Numerous studies have found that infusion of vasopressors, such as ANG II and vasopressin, to the diabetic rat exhibits reduced pressor responsiveness ( 5, 21, 22, 28 ). In early diabetes, several studies have found normal or impaired isolated aortic ring contraction in response to a variety of vasoconstrictors ( 13, 39, 50 ), although there has been a recent report of increased contractility to vasoconstrictors in early and late stages of diabetes ( 38 ). Overall, there appears to be a generalized impaired response of macrovessels to the pressor effect of vasoconstrictors during the early stage of experimental diabetes. In vitro studies using aortic vascular smooth muscle cells cultured in high glucose suggest that impaired vasoconstrictor-induced calcium response may contribute to this effect ( 67, 68 ).
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With regard to the kidney, there is clear evidence that blood flow to the glomerulus of the kidney is increased in early diabetes ( 25, 26 ). The increased glomerular blood flow appears to be primarily due to an inappropriate dilation of the afferent arteriole ( 25, 26, 46 ). The enhanced glomerular blood flow has been considered to be an important pathophysiological event contributing to glomerular hypertrophy in early diabetes and shear-induced damage to vessels and glomerulosclerosis in late diabetes. An impairment in afferent arteriolar constriction in early diabetes was demonstrated by Carmines et al. ( 7 ) and found to be related to a decreased intracellular calcium rise.
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' K2 Y' r* l, Z% f* `' EIn prior studies, we identified the prosclerotic cytokine transforming growth factor (TGF)- as a critical player in mediating the increase in diabetic mesangial matrix expansion ( 54, 74, 75 ), which is the hallmark of progressive diabetic nephropathy ( 58 ). Apart from enhancing matrix formation, TGF- may play a role in vascular responsiveness by interfering with the calcium-mobilizing potential of cells involved in vasoconstriction ( 4, 55, 73 ). TGF- was found to inhibit calcium transients to agonists that increase inositol 1,4,5-trisphosphate (IP 3 ) production in both vascular smooth muscle cells ( 73 ) and in mesangial cells ( 4 ). This effect of TGF- is likely due to downregulation of IP 3 receptors as TGF- decreases the expression of IP 3 R1 ( 56 ) and inhibits IP 3 sensitivity in permeabilized mesangial cells ( 55 ). As an increase in intracellular calcium is a critical initial step stimulating the vascular smooth muscle cell to contract, we postulated that local production of TGF- may contribute to the impaired contractile response of macrovascular and microvascular smooth muscle cells in diabetes.
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6 l$ o- o( }- S1 {EXPERIMENTAL PROCEDURES
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Animal studies. Sprague-Dawley rats weighing 134-151 g were made diabetic by a single intraperitoneal injection of STZ (65 mg/kg body wt) in 10 mmol/l sodium citrate, pH 5.5. Controls were injected with buffer alone. The levels of blood glucose were determined 2 days after injection, and rats with 300 mg/dl were used as diabetic rats. The diabetic rats were treated with 1-2 U of Humulin (70/30 preparation) subcutaneously each day to maintain glycemic levels between 300 and 500 mg/dl and avoid ketosis and muscle wasting. After 14 days of diabetes, aortic smooth muscle cells (aSMC) were isolated as described below. To study the in vivo role of TGF-, a separate group of diabetic rats received neutralizing anti-TGF- antibody (anti-T) before cell isolation, aortic ring studies, and glomerular morphometry. Anti-T was administered intraperitoneally at a dose of 15 mg/kg on alternate days, as previously described ( 27 ), over a 14-day period. Isotype-specific control IgG was administered to diabetic rats in the same manner. Diabetic and nondiabetic rats were given standard rodent diet and water ad libitum. Animals were housed separately and maintained in a temperature- and light-controlled environment. All protocols in this study were approved by the Institutional Animal Care and Use Committee.3 O) H- i1 D/ `& H: K7 p

( z6 }- Y8 F/ L) c* B$ aSmooth muscle cell isolation. Aortic tissue was obtained from three normal rats and three diabetic rats. The tissues were pooled and labeled as normal and diabetic aSMC. Tissue was washed twice, and aSMC were isolated using established methods ( 16, 35 ). Cells were plated on 35-mm culture dishes with 10 ml RPMI containing either 5.5 mM D -glucose or 25 mM D -glucose with 10% FCS. Cells were assessed for smooth muscle-specific -actin and myosin (Sigma, St. Louis, MO) by FACS analysis to establish smooth muscle cell origin. Experiments were performed on aSMC between passages 3 and 7. In an additional set of studies, aSMC were freshly isolated from diabetic rats treated with nonspecific IgG or anti-T and plated immediately on coverslips containing RPMI with 25 mM D -glucose/10% FCS and either IgG or anti-T, respectively. Calcium imaging was performed during the primary culture period.1 D3 V& J2 ]2 f( G
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To isolate microvascular smooth muscle cells (mSMC) from renal resistance vessels, we used a technique previously described by Zhu and Arendshorst ( 72 ) for the rat kidney. For establishing mSMC in culture, three normal Sprague-Dawley male rats (4 wk of age) were anesthetized with pentobarbital sodium (60 mg/kg ip), and the abdominal aorta was cannulated below the renal arteries. The kidneys were perfused with ice-cold PBS, followed by 5 ml of a magnetized iron oxide suspension (1% Fe 3 O 4 in PBS), excised, and placed in fresh cold PBS. The cortical tissue was gently minced with a razor blade, and the crude homogenate was then resuspended in PBS, passed through needles of decreasing size (22- and 23-gauge), and filtered through a 120-µm sieve. The microvessels were recovered from the retentate and purified by magnetic separation. The final preparation was digested with collagenase (8 mg/10 ml, type 1A; Worthington Biochemical, Lakewood, NJ) for 30 min with constant shaking at 37°C to disperse the cells and iron oxide. Cells of the digested microvessels were collected by brief centrifugation, washed once with PBS, and plated in either normal glucose (5.5 mM, RPMI, 10% FCS) or high glucose (25 mM D -glucose RPMI, 10% FCS). Cells were studied between passages 3 and 7. Cells were characterized as smooth muscle in origin by FACS analysis with antibodies against smooth muscle -actin and myosin.
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2 {: U, I, d; t1 \+ }" e7 [Fluorescence imaging measurements of cytosolic [Ca 2   ]. For calcium imaging, the normal and diabetic aSMC were plated onto lysine-coated coverslips with RPMI/10% FCS with 5.5 or 25 mM D -glucose, as appropriate. In experiments where antibodies were used, cells were exposed to either control isotype-specific IgG or anti-T. After 3 days in culture on coverslips, the medium was replaced with an extracellular medium (2% BSA/ECM) consisting of 121 mM NaCl, 5 mM NaHCO 3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH 2 PO 4, 1.2 mM MgSO 4, 2 mM CaCl 2, 10 mM glucose, and 2% BSA, pH 7.4. A similar protocol was employed with primary passage aSMC from normal and diabetic rats treated with control IgG and anti-T and with mSMC cultured in normal (5.5 mM) or high glucose (25 mM). To monitor cytosolic [Ca 2  ] (Ca 2  c ), cells were loaded with 5 µM fura-2 AM for 30 min in the presence of 100 µM sulfinpyrazone and 0.3% pluronic acid at room temperature. Sulfinpyrazone was also present during the imaging measurements to minimize dye loss. Dye-loaded cells were washed two to three times with 2% BSA/ECM. Imaging measurements were performed in ECM containing 0.25% BSA (0.25% BSA/ECM) at 35°C. Fluorescence images were acquired using an Olympus IX70 inverted microscope fitted with a x 40 (UApo, NA 1.35) oil immersion objective and a cooled CCD camera (PXL, Photometrics) under computer control. The computer also controlled a scanning monochromator (DeltaRam, PTI) to select the excitation wavelength ( 11 ). Time courses of [Ca 2  ] c in individual cells were calculated from fluorescence image pairs obtained using 340- and 380-nm excitation (10-nm bandwidth) with a broadband emission filter passing 460-600 nm. The lag time was calculated as the time in seconds to attain half-maximal [Ca 2  ] c peak following agonist stimulation. Experiments were carried out with three different cell cultures, at least three parallel experiments on each occasion and 50-100 cells were monitored in each experiment.
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% }3 v7 S5 ~. s9 }7 b) R) ZAortic ring preparation and isometric contraction measurement. For the in vivo studies, after 2 wk of diabetes normal and diabetic rats were killed by cervical dislocation. After excision, rat thoracic aortas cut into rings were used for contraction measurements. Aortic rings of 3-mm width were mounted in a 10 ml organ bath containing Krebs buffer. Buffer was maintained at 37°C and continuously gassed with 95% O 2 -5% CO 2. Rings were equilibrated for 30 min using a resting tension of 1 g, during which the buffer was changed every 10 min. Dose-response curves to ANG II (Sigma) were constructed by cumulative addition of small volumes of stock solution. Rings were maximally contracted with the thromboxane A2 analog U-46619 (1 µM) (Sigma).
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: F# U" x; b) r- x$ ZGlomerular histology and morphometry. A portion of the renal cortex was fixed in 10% neutral buffered formalin and embedded in paraffin. Renal sections (3-µm thick) were stained with PAS. For quantitation of glomerular volumes, sections were coded and read by an observer unaware of the experimental protocol. Glomerular surface area was measured by an independent pathologist who was blinded to the experiment. The technique used the SAMPA 4000, Cell Image program using measurements of glomerular tuft surface area. The results represent the mean surface area of at least 20 cortical glomeruli. Glomerular volumes (V) were derived by the method of Weibel ( 65 ): V = b/ k x ( AG )3/2 x (m 3 ), where A is the glomerular surface area, G = 1.38 is the shape coefficient for spheres, and k = 1.1 is a size distribution coefficient. Glomerular PAS-positive material and open capillary loop surface area were quantified by ImagePro Plus, version 4.5 (Media Cybernetics, Silver Spring, MD).3 l0 K. T) z1 O7 W7 B. ?

/ O* z4 m, C4 d, K& V5 ATGF- and IP 3 R protein analysis. Glomerular TGF- 1 measurements were performed from isolated glomeruli obtained from renal cortex. Isolated glomeruli were obtained from differential sieving using standard techniques. The glomerular sample was placed into 1 ml of RD-51 diluent buffer (R & D Systems, Minneapolis, MN), homogenized, acid-activated, and TGF- 1 was measured by ELISA, as previously described ( 27 ). One of the aliquots from the glomerular tissue homogenate was assayed for total protein using a Bio-Rad protein assay kit. Conditioned media from smooth muscle cells cultured in normal- and high-glucose concentrations were assessed for TGF- 1 and - 2 using the Quantikine ELISA kit (R & D Systems) and standardized for total cell protein.$ y5 B0 X) b( p; V) N* w8 z" g9 A
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Immunostaining for TGF- 1 and - 2 was performed with 3-µm paraffin-embedded sections from aorta. Slides of the aortic tissue were incubated at 60°C for 30 min and immediately placed in xylene. Tissue was rehydrated and endogenous peroxidase was blocked with 3% H 2 O 2 /methanol bath. Antigen retrieval was performed by placing slides in microwave at high power in Citra Plus solution (BioGenex, San Ramon, CA) for 10 min. Biotin was blocked with the DAKO Biotin Blocking System using PBS/Ringers. Blocking of non-specific antibody binding was performed with DAKO Protein Block, Serum Free, and TGF- 1 antibody (1:10 dilution; Santa Cruz Biotech, Santa Cruz, CA) or TGF- 2 antibody (1:100 dilution; Santa Cruz) was applied to each tissue section at room temperature for 1 h. Peptide blocking to verify specificity of binding was performed with preadsorption of antibody with excess TGF- 1 or - 2 peptide (Santa Cruz). Subsequent staining steps were done using DAKO LSAB System, HRP. Tissues were counterstained with Harris Hematoxylin (Sigma) before mounting of the slides in Permount (Fisher)./ B8 U$ ^8 j3 [0 H
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Immunoblotting of aortic tissue was performed by homogenizing aortic tissue from normal and diabetic rats in lysis buffer containing 50 mM Tris·HCl (pH 7.2), 150 mM NaCl, 1% (wt/vol) Triton X-100, 1 mM EDTA, 1 mM PMSF, and 5 µg/ml each of aprotonin and leupeptin. Protein concentration of samples was quantitated (Bio-Rad DC, Hercules, CA), and equal amounts of protein were run on a 7% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with an antibody raised to the COOH terminus of the type I IP 3 R from brain, as previously described ( 57 ). For standardization, the blots were stripped and immunoblotted with a monclonal antibody to -actin (Sigma).
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8 [0 @; T2 g  s% g4 kQuantitative real-time PCR. For quantitating aortic mRNA levels using real-time PCR, TGF- 1, TGF- 2, IP 3 RI, and angiotensin type 1 receptor (AT 1 R) mRNA expression in aortic sections were analyzed using real-time two-step quantitative RT-PCR. Total RNA was isolated from frozen aortic tissue, and cDNA was prepared using random priming. Quantitation was performed with SYBR Green PCR reagents (Molecular Probes Europe, Leiden, The Netherlands) and an ABI PRISM 5700 Sequence Detection System (Applied Biosystems, Nieuwerkerk a/d Ijssel, The Netherlands). A 50-µl PCR reaction mixture contained 0.5 U Taq polymerase (Eurogentec, Seraing, Belgium), 5 µl of the supplied reaction buffer, 250 nM dATP, 250 nM dCTP, 250 nM dGTP, 500 nM dUTP, 2 mM MgCl 2, 50 ng cDNA, 500 nM of each gene-specific primer, 1 µl of 50 ROX reference dye (Invitrogen, Breda, The Netherlands), and 1 µl of 10 Sybr Green I (Molecular Probes Europe). The PCR profile consisted of 5 min at 95°C, followed by 40 cycles with heating to 95°C for 15 s and cooling to 60°C for 1 min. PCR product specificity and purity were evaluated by gel electrophoresis and by generating a dissociation curve following the manufacturer's recommendations. Sample CT values were normalized to CT values for 18S RNA. Sequence-specific PCR primers were purchased from Eurogentec. The PCR primers used were as follows: TGF- 1 : sense, 5'-AAAGAAGTCACCCGCGTGCTA-3'; antisense, 5'-CCCGAATGTCTGACGTATTGAA-3'; TGF- 2 : sense, 5'-ATGGCTCTCCTTCGACGTGA-3'; antisense, 5'-TTGTGGTGAAGCCACTCGTG-3'; IP 3 R-I: sense, 5'-GGATGCCCCATCCCGA-3'; antisense, 5-GCTGATCCCGGACCTCTTCT-3'; AT 1 R: sense, 5'-CACCAATATCACAGTGTGCGC-3'; antisense, 5'-AGCGTCGAATTCCGAGACTC-3'; and 18S: sense, 5'-CATTCGAACGTCTGCCCTATC-3'; antisense, 5'-CCTGCTGCCTTCCTTGGA-3'.& x: d4 `/ A. q, B  J. x
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Statistics. Results are presented as means ± SE unless indicated otherwise. Differences between concentration-response curves were analyzed using repetitive-measurement ANOVA (SigmaStat 1.01, Jandel Scientific) and considered significant at P 0.05. Differences in other variables were tested using Student's t -test or 2 -test as indicated.) F/ b7 p: w) R  n2 D* L: S! k

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1 k1 X# N" ~+ ?. M& cImpaired calcium response to ANG II in aortic vascular smooth muscle cells from diabetic rats. To determine whether vascular smooth muscle cells exhibited impaired response to vasoconstrictors, aortic vascular smooth muscle cells were isolated from normal and diabetic rats of 2-wk duration of diabetes. The aSMC from the normal rats were cultured in normal-glucose conditions (5.5 mM D -glucose, RPMI) and the diabetic aSMC were cultured in high glucose (25 mM D -glucose, RPMI) for passages 3 - 7 before being studied. Cells were loaded with fura-2-AM and exposed to a low and high dose of ANG II. aSMC from normal rats had a rapid and marked increase in [Ca 2  ] c in response to low-dose (2 nM) and high-dose ANG II (100 nM; Fig. 1 ). In contrast, the [Ca 2  ] c response of diabetic aSMC to a low dose of ANG II was markedly altered. The diabetic aSMC had a prolonged delay before attaining peak increase in [Ca 2  ] c and the amplitude of the [Ca 2  ] c response was substantially diminished to the low dose of ANG II. At higher doses of ANG II, the duration until peak [Ca 2  ] c remained delayed and the amplitude of response was reduced in the diabetic aSMC, although the majority of cells had some response.
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0 w# q/ ^- C" R$ \Fig. 1. Aortic smooth muscle cells (aSMC) from diabetic rats exhibit impaired cytosolic [Ca 2  ] (Ca 2  c) mobilization in response to ANG II. A representative group of aortic vascular smooth muscle cells from normal rats, cultured in normal glucose (5.5 mM; normal), and aortic vascular smooth muscle cells from diabetic rats, cultured in high glucose (25 mM; diabetic), are depicted. Cells were loaded with fura-2-AM and exposed to ANG II (2 nM followed by 100 nM) for the indicated time points. The [Ca 2  ] c concentration was calculated based on F fura2 340/380-nm ratios for individual cells 1, 2, and 3 from the normal and diabetic condition, respectively ( bottom ).2 C! J9 R, l5 Y8 L

7 L- |% L8 u5 c2 zComplete reversal of diabetic-induced impaired calcium response in aSMC by anti-TGF- antibodies. As TGF- may mediate a variety of effects of cells cultured in high glucose or derived from the diabetic milieu, anti-T was added to the aSMC in culture before measuring [Ca 2  ] c in response to ANG II ( Fig. 2 A ). The effect of diabetes on low- and high-dose ANG II-induced maximal stimulation of [Ca 2  ] c was completely prevented by anti-T. The lag phase before reaching half-maximal [Ca 2  ] c to ANG II (2 nM) in the diabetic aSMC was increased by sixfold; however, in the presence of anti-T, the lag phase was restored to control levels ( Fig. 2 B ). The antibody had virtually no effect on normal aSMC ( Fig. 2 B ), suggesting that only in the diabetic condition is the TGF- system involved in ANG II-induced [Ca 2  ] c stimulation. Control IgG had no effect on either normal or diabetic aSMC ANG II-induced [Ca 2  ] c stimulation (data not shown).4 s3 H+ t1 o: w( R" w, G8 p+ p
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Fig. 2. Impaired ANG II-induced [Ca 2  ] c mobilization in diabetic aSMC is prevented by anti-transforming growth factor (TGF)- antibodies (TGF Ab). A : mean tracing of [Ca 2  ] c in response to low- and high-dose ANG II in normal aSMC and diabetic aSMC showing a decreased amplitude and delay in onset of [Ca 2  ] c rise. Treatment of the diabetic aSMC with anti-T completely restored ANG II-induced [Ca 2  ] c mobilization with respect to onset of the [Ca 2  ] c increase and the amplitude of the [Ca 2  ] c increase. B : lag time until half-maximal [Ca 2  ] c rise was markedly increased in diabetic aSMC and restored to normal by anti-T. Results are presented as means ± SE from 3 independent experiments with 50-100 cells monitored in each experiment for each condition.
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To determine whether the altered ANG II-induced [Ca 2  ] c response in diabetic aSMC is present in primary aSMC, studies were repeated with primary passage cells obtained from diabetic rats treated with control IgG or anti-T for 2 wk. There were no effects of IgG or anti-T to affect body weight or blood glucose (data not shown). In primary culture aSMC from diabetic rats treated with nonspecific IgG, the mean increase in [Ca 2  ] c with 2 nM ANG II was impaired (basal [Ca 2  ] c 113.69 ± 18.50 ANG II stimulated max [Ca 2  ] c 144.50 ± 19.30 nM), whereas anti-T-treated aSMC exhibited a marked increase of 482% in ANG II-induced [Ca 2  ] c rise (basal [Ca 2  ] c 92.45 ± 6.47 ANG II stimulated max [Ca 2  ] c 445.70 ± 46.30 nM, P of diabetes to impair ANG II-induced [Ca 2  ] c was noted in primary passage aSMC from diabetic rats as well as in diabetic aSMC maintained in prolonged culture with high glucose; in both cases, the impaired ANG II-induced [Ca 2  ] c response was completely prevented by anti-TGF- antibodies.: e; B  ^' C4 Z. Q% l( Z% p
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Impaired [Ca 2   ] c response to ANG II in diabetic preglomerular mSMC is reversed by anti-TGF- antibodies. To evaluate the effects of diabetes and TGF- on mSMC, preglomerular mSMC from normal rats were isolated and cultured in normal or high glucose for 6 days before assessment. Periods of culture in high glucose for 3 days or less did not affect ANG II-induced [Ca 2  ] c (data not shown). Addition of either the low dose or high dose of ANG II to mSMC cultured in normal glucose stimulated a brisk rise in [Ca 2  ] c that was maximal within 30 s ( Fig. 3 A ). In mSMC cultured in high glucose, there was a delay of greater than 60 s before the maximal rise in [Ca 2  ] c in response to low-dose ANG II (2 nM). Although no delay in [Ca 2  ] c rise was noted to high-dose ANG II (100 nM; Fig. 3 B ), the amplitude of [Ca 2  ] c increase was reduced with both the low dose as well as the high dose of ANG II. The effects of high glucose on ANG II-induced [Ca 2  ] c increase were completely prevented when mSMC were cultured in the presence of anti-TGF- antibodies ( Fig. 3, B and C ). There was no effect of anti-TGF- antibodies on mSMC cultured in normal glucose ( Fig. 3 A ) and no effect of nonspecific IgG in either glucose concentration, suggesting that basal levels of TGF- were not affecting normal ANG II-induced [Ca 2  ] c response and the effect of anti-T was specific.
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Fig. 3. Microvascular SMC (mSMC) cultured in high glucose have impaired ANG II-induced [Ca 2  ] c responses: reversal by anti-T. Preglomerular mSMC were cultured in normal glucose (5.5 mM; A ) or in high glucose (25 mM, 6 days; B ) and loaded with fura-2 AM (thin lines). [Ca 2  ] c response to low-dose ANG II is delayed and attenuated, and [Ca 2  ] c response to high-dose ANG II (100 nM) is attenuated but not delayed. B : both the lag time and amplitude of [Ca 2  ] c responses to low- and high-dose ANG II are restored to control values by anti-T in mSMC cultured in high glucose (thick lines). C : amplitude of ANG II-induced [Ca 2  ] c response is significantly decreased in cells cultured in high glucose. The effect of high glucose is not affected by control IgG but completely restored to control values by anti-T. Basal levels of [Ca 2  ] c (open bars). ANG II (2 nM) stimulated [Ca 2  ] c levels (filled bars). Results are presented as means ± SE from 3 independent experiments with at least 30 cells monitored in each experiment for each condition. * P
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. k1 e: g3 u4 DTGF- 1 or TGF- 2 reproduces the effect of diabetes on aSMC response to ANG II. As the anti-TGF- antibody used is a pan-neutralizing antibody, it is conceivable that TGF- 1, TGF- 2, or TGF- 3 may mediate the effect of diabetes. We first determined whether either TGF- 1 or - 2 could replicate the effect of diabetes by adding TGF- 1 or - 2 to normal aSMC for 24 h and then determining ANG II-induced [Ca 2  ] c release ( Fig. 4 ). Compared with normal aSMC that received vehicle, both TGF- 1 and - 2 blocked ANG II-induced [Ca 2  ] c mobilization. The effect of TGF- was more marked than the diabetic effect, as virtually 100% of cells pretreated with either TGF- isoform failed to elicit a rise in [Ca 2  ] c with the low dose of ANG II. At the high dose of ANG II, there was a marked delay in [Ca 2  ] c rise. A high dose of ATP elicited a marked increase in [Ca 2  ] c in TGF- -pretreated cells, suggesting that [Ca 2  ] c stores were not depleted (data not shown). The addition of TGF- 1 and - 2 had a similar effect in normal as well as diabetic aSMC, although the effect of TGF- 2 appeared to be more pronounced compared with TGF- 1 ( Fig. 4 ).
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, @0 L2 D# k9 @6 I4 {8 ^6 @Fig. 4. Effect of TGF- 1 and TGF- 2 on normal and diabetic aSMC. The effects of TGF- 1 (10 ng/ml, 24 h; A and B ) and TGF- 2 (10 ng/ml, 24 h; C and D ) were tested on both normal and diabetic aSMC with respect to ANG II-induced [Ca 2  ] c increase. There was a similar inhibition of ANG II-induced [Ca 2  ] c increase by both TGF- 1 and TGF- 2 in normal and diabetic aSMC. Results are presented as mean tracings from 3 independent experiments with at least 50 cells monitored in each experiment for each condition.0 D; u8 g9 h1 {# u1 n4 S- s! Y
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A dose-response relationship was examined with both TGF- 1 and - 2 in normal aSMC ( Fig. 5 ). Both isoforms led to a significant delay in ANG II-induced [Ca 2  ] c rise as well as the amplitude of ANG II-induced [Ca 2  ] c mobilization in normal cells at a dose as low as 0.1 ng/ml. However, TGF- 2 exhibited a more potent effect in delaying and decreasing the magnitude of [Ca 2  ] c mobilization at concentrations of 1 and 10 ng/ml compared with TGF- 1 ( Fig. 5 B ).
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Fig. 5. Dose response of TGF- 1 and TGF- 2 to impair ANG II-induced [Ca 2  ] c increase. Differential production of TGF- 2 by diabetic aSMC. Dose-response relationships with TGF- 1 and TGF- 2 indicated impaired ANG II [Ca 2  ] c increase noted at 0.1, 1.0, and 10 ng/ml with both TGF- 1 ( A ) and TGF- 2 ( B ). C : there was a further impairment of TGF- 2 with doses of 1.0 and 10 ng/ml compared with TGF- 1. Results are presented as mean tracings from 3 independent experiments with 50 cells monitored in each experiment for each condition. Normal and diabetic aSMC were cultured in normal (5.5 mM) or high glucose (25 mM), respectively, and TGF- 1 and TGF- 2 concentrations were measured by ELISA specific for each isoform ( D ). TGF- levels were standardized per mg total protein of cell monolayer. Only TGF- 2 was found to be increased by high glucose. Results are presented as means ± SE from 3 independent experiments with triplicates for each condition. * P 1 at corresponding dose ( C ). * P D ).! f: U( `; _3 K1 I

( t( b. [6 K! L. i- O6 jTo establish whether either the TGF- 1 or TGF- 2 isoform was stimulated in the diabetic milieu, we measured TGF- 1 and TGF- 2 in the conditioned media from normal and diabetic aSMC ( Fig. 5 ). Interestingly, we were unable to detect an increase in TGF- 1 levels in the diabetic aSMC compared with the normal aSMC. In contrast, there was a fourfold increase in TGF- 2 concentrations in the diabetic condition (TGF- 2 ng/mg protein: 4.3 ± 0.06 in normal aSMC vs. 19.6 ± 1.31 in diabetic aSMC, P Thus TGF- 2 appears to be the primary isoform responsible for inhibiting [Ca 2  ] c mobilization in diabetic aSMC in culture.3 T6 Q% J, M7 g/ G0 j( n! E* k3 l

/ e6 m) Z4 ]$ ~' @$ XIn vivo neutralization of TGF- in diabetic rats prevents diabetic glomerular hypertrophy. To examine for in vivo relevance to the findings in isolated smooth muscle cells, the effect of anti-T on diabetic glomerular hypertrophy and aortic vascular function was assessed. Rats were made diabetic and treated with anti-T. Diabetic rats treated with anti-T had the same glycemic levels as untreated diabetic rats ( Table 1 ). The diabetic rats did not gain as much weight as the control rats but were not catabolic as they gained weight from prediabetic levels. Kidney hypertrophy, as defined by kidney weight-to-body weight ratios, was noted in both diabetic groups and not affected by anti-T. There was no effect of control antibodies in diabetic rats with respect to body weight and degree of hyperglycemia.
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, ^3 T% T; o, w9 {Table 1. Clinical parameters in the control, diabetes, and diabetes/anti-T groups
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9 C, d$ {' a) \8 xGlomerular volumes were measured in the outer cortex of the rats in each of the groups. Glomerular volumes were increased by 43% in the diabetic rats compared with control; however, the glomerular volume was increased by only 15% in the anti-TGF- -treated diabetic group ( Table 2 ). Assessment of the relative contribution of the open capillary luminal compartment and the cellular compartment revealed that the increase in glomerular volume in the diabetic group is almost exclusively due to an increase in the capillary luminal compartment ( Fig. 6 and Table 2 ). Although there is an increase in PAS-positive material in the diabetic glomeruli, the relative contribution of PAS-positive material to overall glomerular volume increase is minimal at this stage of disease ( Table 2 ). There was also no contribution of cell hypertrophy to overall diabetic glomerular hypertrophy. The effect of anti-T treatment to reduce glomerular volume was primarily due to reduction of the open capillary luminal compartment.
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Table 2. Glomerular compartments and TGF- 1 levels in the control, diabetes, and diabetes/anti-T groups6 R0 s8 B. p2 }- v8 S: f4 a! V
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Fig. 6. Increase in glomerular capillary filtration area in diabetic rats is reduced by anti-T. Image analysis was performed to quantitate the relative contributions of the PAS-positive area, glomerular filtration surface area, and the overall cell compartment in normal and diabetic rats. A representative view of a glomerulus from normal ( A ), diabetic ( B ), and diabetic anti-T ( C ) groups is depicted for regions of PAS-positive area (red shaded area at left ) and open capillary lumen area (dark blue shaded area at right ). Right : gray region of the glomerulus denotes the cellular fraction of the glomerulus.+ O/ q" F/ G* E0 d, v/ D) N& N4 S

3 S5 q0 L9 B. }8 K3 E: dGlomeruli were isolated from each of the groups, and the glomerular lysate was assessed for TGF- 1 content and standardized for total glomerular protein. As noted in Table 2, TGF- 1 levels were increased by 3.7-fold in diabetic rats. Anti-TGF- antibody treatment in the diabetic rats prevented the increase in glomerular TGF- 1 levels, confirming a positive autofeedback loop on TGF- production. There was a significant correlation between glomerular TGF- 1 values and glomerular volumes in all the groups studied ( r = 0.70, P
+ M; r8 t8 C7 @7 ?+ g# J0 g: t* T
Impaired ANG II-induced aortic ring contraction in diabetic rats is restored by anti-T treatment. Cumulative dose-response curves to ANG II were constructed ( Fig. 7 ) to investigate the effect of short-term diabetes on aortic ring responsiveness to ANG II. Dose-response curves to ANG II were significantly decreased in diabetic animals. Aortic rings from diabetic rats exhibited a 50% reduction of E max, without shift in EC 50 of ANG II. Anti-T treatment of diabetic animals restored the ANG II-induced aortic contractions to control levels.# V, X) \, _  B# H; c$ d5 R
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Fig. 7. Hyporeactivity to ANG II (AII) of aortic rings from diabetic rats is restored by anti-T treatment. Dose-response curves of aortic rings of control animals ( ), diabetic rats ( ), and anti-T-treated diabetic rats ( ) to ANG II. Relative maximal contractions for control animals, diabetic rats, and anti-T-treated diabetic rats were 14.5 ± 2.1, 7.4 ± 1.3, and 12.6 ± 1.9%, respectively. Results are expressed as a percentage of maximal contraction to the thromboxane A 2 analog U-44619 (1 µM) and presented as means ± SE. * P
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TGF- isoform mRNA levels and protein distribution were assessed in normal and diabetic aortic tissue ( Fig. 8, A and B ). With the use of real-time quantitative PCR, there was no increase in TGF- 1 mRNA levels in the diabetic aorta; however, there was a 62% increase of TGF- 2 in the diabetic aorta compared with normal aortic tissue ( Fig. 8 A ). By immunostaining, TGF- 2 was present primarily in the smooth muscle layers of diabetic aorta and appeared more intense in the luminal and adventititial regions compared with normal aortic tissue ( Fig. 8 B ). TGF- 1 was present in a similar distribution in normal aorta and not altered in diabetic aorta (data not shown).% G, ^# v$ ^. D8 \" Z2 `
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Fig. 8. Differential pattern of upregulation of TGF- 2 and downregulation of type I inositol 1,4,5-trisphosphate receptor (IP 3 R) in aortic tissue from diabetic rats. A : aortic tissues from normal and diabetic rats were assessed for TGF- 1 and TGF- 2 mRNA levels by quantitative RT-PCR. The diabetic aortic tissue had an increase in TGF- 2 mRNA levels but not TGF- 1 mRNA levels compared with the aortic tissue from normal rats. Immunostaining for TGF- 2 revealed that distribution of TGF- 2 is increased in the aSMC layers in the diabetic rat ( B, middle ) compared with the normal rat ( B, top ). There was complete loss of immunostaining when the diabetic aortic tissue was preincubated with TGF- 2 peptide ( B, bottom ). C : type I IP 3 R mRNA was reduced in diabetic aortic tissue as measured by quantitative real-time PCR and restored by anti-T treatment. Western blot analysis of aortic tissue revealed decreased type I IP 3 R in the diabetic samples and restoration with anti-T treatment ( D ). Protein was resolved on a 7% SDS-PAGE and immunoblotted with antibody to IP 3 R1 and -actin, for standardization. Results are presented as means ± SE from n = 4-6 for each group. * P
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' @& D, h+ ~5 k% w0 QRegulation of aortic IP 3 RI and AT 1 R by diabetes and TGF-. We previously found that diabetic glomeruli had reduced type I IP 3 R levels ( 57 ) and that TGF- reduces IP 3 RI levels in glomerular cells ( 55, 56 ). In addition, several studies found that high glucose and TGF- reduce AT 1 receptors ( 47, 68 ). As a possible explanation for the impaired contractile response in aortic rings of diabetic rats, IP 3 R1 and AT 1 R levels were assessed in aortic tissue. By quantitative real-time PCR, there was a reduction of type I IP 3 R in the diabetic aorta compared with control aorta ( Fig. 8 C ). The reduction in the diabetic aorta was prevented by anti-T ( Fig. 8 C ). Correspondingly, at the protein level there was a decrease in IP 3 RI protein levels in diabetic aorta and restoration with anti-T ( Fig. 8 D ). AT 1 R mRNA levels were significantly increased in aortic tissue of diabetic rats and further increased with anti-T treatment [normal 100 ± 19.1; diabetic 143 ± 15.3 ( P   anti-T 199 ± 9.9 ( P, p& u& m3 `  v

0 O3 `; p3 M4 Y( eDISCUSSION; U- u3 F8 g( `7 D

8 K: g- K  d) Q% eThe role of TGF- in vascular smooth muscle cells has primarily been considered to alter its growth characteristics ( 1, 15 ), enhance matrix accumulation ( 8, 32 ), and may enhance transdifferentiation ( 19 ). Our present study introduces a novel aspect of TGF- by demonstrating that TGF- contributes to macrovascular and microvascular smooth muscle cell dysfunction in the diabetic milieu. An important implication is that in diabetes, localized increases of TGF- in the vascular compartment regulate smooth muscle cell response by interfering with normal intracellular calcium regulation. This would result in altered vascular cell function and dysregulated blood flow.$ k$ y* n4 q0 i( Y1 }3 X" a

: a7 i+ ?% z. q) d$ ^A vascular role for TGF- is supported by recent studies that demonstrate profound vascular changes in the kidney of mice and rats exposed to high levels of exogenous TGF- 2 ( 34, 37 ). A recent clinical study found that administration of TGF- 2 to patients with multiple sclerosis led to acute renal failure in some patients as quickly as 1 wk after administration ( 6 ). The rapid onset of renal failure would suggest a hemodynamic or acute toxic effect of TGF- on vascular smooth muscle cells or renal cells. The effect of TGF- to cause renovascular dysfunction may be especially prominent in the setting of preexisting alteration of renal function or vascular hemodynamics. In the setting of diabetes, the effect of local production of TGF- on macrovascular smooth muscle cells could contribute to impaired contractile function and the depressed systemic blood pressure that has been found in early experimental diabetes ( 51, 59 ). In the clinical state, there have been reports of increased basal forearm blood flow ( 10, 18 ) and impaired vasoconstrictor response in type 1 and type 2 diabetic patients to a variety of stimuli ( 2, 30, 45 ). Increased basal blood flow, likely due to altered vascular smooth muscle cell function, would lead to shear stress on endothelial cells with consequent endothelial cell dysfunction ( 62 ). The initial inappropriate increase in basal blood flow may be contributed by local TGF- production.
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Our study is the first to demonstrate the effects of high glucose on ANG II-induced [Ca 2  ] c rise in preglomerular vascular smooth muscle cells. As this is the critical cell type regulating afferent arteriolar tone, it is likely that the impaired ANG II-induced [Ca 2  ] c rise contributes to the characteristic increased glomerular blood flow of early diabetes ( 26 ). As increased glomerular blood flow may be due to decreased responsiveness to vasoconstrictors ( 38 ) and is closely associated with glomerular hypertrophy ( 24, 26 ), our finding that anti-TGF- antibodies attenuate diabetic glomerular hypertrophy may be explained by a restoration of vascular smooth muscle cell responsiveness. Early diabetic glomerular hypertrophy in humans and in experimental diabetes is primarily characterized by increased capillary loop surface area rather than increased mesangial matrix accumulation ( 23, 38, 49 ). Therefore, it is unlikely that the explanation for the anti-TGF- effect to reduce early diabetic glomerular hypertrophy can be explained by inhibiting mesangial matrix accumulation. Mesangial cell hypertrophy is stimulated by TGF- in cell culture ( 9 ) and related to alteration of the cyclin-dependent kinase inhibitors p21 and p27 ( 42, 69 ). Although mesangial cell hypertrophy has been linked to progressive mesangial matrix expansion in later stages of diabetes ( 3 ), it is presently unclear what the role of mesangial cell hypertrophy is in early diabetic glomerular hypertrophy. Therefore, based on our results, the role of TGF- in early diabetic glomerular hypertrophy appears to be largely mediated via an effect on vascular smooth muscle cell function, whereas the role of TGF- in the later feature of diabetic glomerular disease is likely via affecting cell hypertrophy and mesangial matrix accumulation.
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8 X9 ?1 t& ?4 H- _- bThe cellular mechanisms mediating impaired vasoconstrictor-induced [Ca 2  ] c transients in vascular smooth muscle cells exposed to the diabetic milieu and/or TGF- are unclear. Surface ANG II-binding sites are reduced in vascular smooth muscle cells in early diabetes ( 68 ) and TGF- has been described to downregulate AT 1 R in proximal tubular cells under high-glucose conditions ( 47 ). Our results in aortic tissue of diabetic rats demonstrate an increase in AT 1 R mRNA levels, suggesting that impaired ANG II binding is not responsible for the impaired ANG II effect. However, we do find that anti-TGF- treatment further increases AT 1 R, suggesting that TGF- may play a role to decrease ANG II receptor binding. In addition to altered plasma membrane binding of vasoconstrictors, intracellular defects are also likely to play a role. TGF- downregulates IP 3 RI in mesangial cells and impairs IP 3 sensitivity in permeabilized cells ( 55, 56 ). Furthermore, there is a decrease of IP 3 RI in vascular arteriolar structures in the diabetic rat kidney ( 57 ). In the present study, we find that diabetic aortic tissue has reduced IP 3 RI and the reduction is under the control of TGF-. In addition, alterations in calcium influx ( 7 ), protein kinase C ( 41, 66, 68 ), aldose-reductase ( 14, 49 ), reactive oxygen species ( 44 ), and arachidonic acid metabolites ( 20, 31, 33, 36, 40, 43 ) have all been implicated in mediating glucose toxicity in a variety of cell types including endothelial and vascular smooth muscle cells. The relative roles of each of these pathways and possible cross talk between pathways will need to be elucidated." O. _  t8 {  I  {& s" p& i. M

1 L( d+ L& o3 p* `* WOur studies demonstrate that vascular smooth muscle cell dysfunction is an important feature of early diabetic vascular dysfunction. Numerous studies have found that endothelial dysfunction with impaired nitric oxide (NO) release is a characteristic finding in chronic diabetes ( 12, 29, 48 ). Although defective endothelial derived NO release would contribute to vasoconstriction during chronic diabetes, it is unlikely to explain the vasodilation noted in diabetic microvessels ( 7, 20 ) during the early stages. It is possible that TGF- may also play a role in endothelial dysfunction by inhibiting calcium mobilization in endothelial cells and thus impairing endothelial NO synthase activity. Studies to evaluate this possibility would provide further insight into the role of TGF- in vascular dysfunction of chronic diabetes.
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$ V4 n, X* r* D8 u7 JInterestingly, TGF- 2 was the isoform that we identified to be upregulated in aortic tissue, whereas TGF- 1 was increased in glomerular tissue from diabetic rats. In addition, TGF- 2 had a more potent effect to impair ANG II-induced calcium mobilization compared with TGF- 1 in the cell culture studies. The lack of increase of the TGF- 1 isoform with high glucose in aortic vascular smooth muscle cells was also reported previously ( 71 ) and contrasts with published results showing stimulation of TGF- 1 by high glucose in glomerular mesangial cells ( 23, 70 ). The signaling pathways underlying high-glucose-induced stimulation of TGF- 2 by high glucose are likely to be different than for TGF- 1 as the two isoforms are stimulated by different conditions and their respective promoters share little homology ( 52, 53 ).. I: q8 b" N3 e4 s0 |% F8 w! H( ~
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In conclusion, our studies implicate TGF- as a key mediator of diabetes-induced alteration of calcium transients in vascular smooth muscle cells. In addition to the well-described role of TGF- in stimulating matrix accumulation during diabetic kidney disease ( 54, 74 ), TGF- may also mediate diabetic complications via its effects on vascular smooth muscle cell function.$ W. X4 @4 a% A0 J

2 m/ k5 [: c& ], UDISCLOSURES
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# ]: n5 t9 m) m4 S+ s; UThis work was supported by grants from the Juvenile Diabetes Research Foundation, American Diabetes Association, and National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-53867) to K. Sharma.
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ACKNOWLEDGMENTS5 A2 x; K+ i9 T, I# V. m1 y

8 _; Z# }, [; M$ k* oWe appreciate the advice of Dr. M. Steffes of the University of Minnesota and Dr. P. McCue of Thomas Jefferson University for the image analysis of glomerular compartments. We also thank S. R. Dunn for assistance in manuscript preparation.
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7 q8 p8 S* d* ~7 t  _Rebolledo A, Ayala-Paredes F, Milesi V, Grassi A, and Rinaldi G. Short-term streptozotocin-induced diabetes induces blood-pressure decrease associated with reduced aortic ( 45 ) Ca 2  uptake and selective depression of the sustained noradrenergic contraction. Diabetes Metab 27: 40-48, 2001.
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( M6 h' v* b0 X9 ~, NSharma K, McGowan TA, Wang L, Madesh M, Kaspar V, Szalai G, Thomas AP, and Hajnoczky G. Inhibition of type I and III IP 3 Rs by TGF- is associated with impaired calcium release in mesangial cells. Am J Physiol Renal Physiol 278: F1022-F1029, 2000.* x( ]3 a* z/ H) [* `

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造血干细胞

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发表于 2015-6-25 17:18 |显示全部帖子
爷爷都是从孙子走过来的。  

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干细胞之家微信公众号
不错,看看。  

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勤奋真能造就财富吗?  

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观看中  

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发表于 2015-8-8 18:28 |显示全部帖子
青春就像卫生纸。看着挺多的,用着用着就不够了。  

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原来是这样  

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哈哈,看的人少,回一下  

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我在顶贴~!~  
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