PATIENTS AND METHODS Subjects Twenty-nine morbidly obese subjects involved in a gastric surgery program were enrolled in the study between October 25 and March 21. Bariatric surgery was performed in patients meeting the criteria recommended by consensus conferences, as previously described (1,2), i. e. BMI 4 or 35 kg/m 2 and at least two significant comorbidities. Diabetic subjects were excluded, because diabetes itself alters angiogenesis. Finally, as it is a contraindication to bariatric surgery, no patient had evidence of acute or chronic inflammatory disease, infectious disease or cancer. Twenty-two subjects underwent gastric by-pass, 5 adjustable gastric banding, and 2 gastric banding withdrawal. Informed consent was required and obtained before inclusion, and the local ethics committee approved the protocol. Clinical and fasting biological data were recorded less than 3 months before surgery as previously described (3,4). Criteria for the metabolic syndrome were those of the NCEP-ATP III (5). Weight was prospectively measured 12, 6 months and 24 h before surgery, and then 6 and 12 months after in the 22 subjects who underwent gastric bypass. Adipose tissues samples Paired subcutaneous WAT from abdominal wall, at the same site in all the patients, and from omental WAT were collected during the surgical procedure and were immediately transported at 2 C to the laboratory in Dulbecco s modified Eagle s medium (DMEM)/F12 (Invitrogen, Cergy pontoise, France) containing 2% BSA (Sigma, Saint-Quentin Fallavier, France). Aliquots of the samples were fixed in 4% paraformaldehyde and then processed for standard paraffin embedding. The other part of each WAT biopsy was frozen in liquid nitrogen and stored at -8 C. Histochemistry Sections of 7 µm were stained as described below and observed under a Leica DM4 microscope. Digital images were captured by a Leica DFC42 camera. Fat-cell sizes were measured on sections stained with.2% toluidine blue. Immunohistochemical detection of vessels and inflammatory cells was performed by immunohistochemistry using the avidin-biotin peroxidase method. The vascular density was measured on slides that were stained with anti von Willebrand antibody (1:6; Dako, Trappes, France), after antigen unmasking by trypsin,25%. The inflammatory infiltrate was assessed using an anti-cd45 antibody (1:5; Dako), after antigen unmasking by microwave washing in citrate buffer. Adipocyte area and vessel density were determined on at least three sections at X5 magnification, and CD45+ cells were counted on at least five sections at X1 magnification, using computer-assisted morphometric analysis with them IVision program.
Real-time quantitative RT-PCR Angiogenic factor mrnas were quantified by real-time quantitative RT-PCR. Total RNA from the 58 frozen samples was extracted with TriZol (2.5 ml/5 mg tissue) and purified using the RNeasy Lipid Tissue kit (Qiagen, Valencia, CA) according to the manufacturer s instructions. The purified RNA was then used to synthesize cdna (Quantitect Rev. Qiagen kit). Expression levels of vascular endothelial growth factor VEGF-A, VEGF-C, VEGF-R1, VEGF-R2, Placental growth factor (PlGF) and TSP1 were assessed with icycler iq TM Real time PCR detection system (Biorad, U.S.) using SybR Green (Bio-Rad). The PCR were performed in triplicate. The 2(- Ct) method was used to quantify the mrna level relative to -actin, which was determined to be a stable housekeeping gene in this model. Primers were designed using Primer Blast NCBI and are presented in supplemental table 1. Statistical analysis Data are expressed as mean ± SD for continuous data and number and percentages for categorical data. They were analyzed with the Sigmastat program. The Wilcoxon signed rank test was used to compare numerical data of paired and. Correlations were examined with the non parametric Spearman rank order test. Results were considered statistically significant at a P value <.5. References (1) Ledoux S, Msika S, Moussa F, Larger E, Boudou P, Salomon L, Roy C, Clerici C 26 Comparison of nutritional consequences of conventional therapy of obesity, adjustable gastric banding, and gastric bypass. Obes Surg 16:141-149 (2) Ledoux S, Coupaye M, Essig M, Msika S, Roy C, Queguiner I, Clerici C, Larger E 21 Traditional anthropometric parameters still predict metabolic disorders in women with severe obesity. Obesity (Silver Spring) 18:126-132 (3) Ledoux S, Queguiner I, Msika S, Calderari S, Rufat P, Gasc JM, Corvol P, Larger E 28 Angiogenesis associated with visceral and subcutaneous adipose tissue in severe human obesity. Diabetes 57:3247-3257 (4) Ledoux S, Msika S, Moussa F, Larger E, Boudou P, Salomon L, Roy C, Clerici C 26 Comparison of nutritional consequences of conventional therapy of obesity, adjustable gastric banding, and gastric bypass. Obes Surg 16:141-149 (5) Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM 24 Prevalence of overweight and obesity among US children, adolescents, and adults, 1999-22. Jama 291:2847-285
Supplemental figure 1. Adipocyte size, vascular density, inflammatory infiltrate and expression of angiogenesis related genes in and. (A) Mean adipocyte area in (open bar) and (dark bar). (B) Representative photography of (left) and (right) samples stained with toluidin blue for the measurement of adipocyte area. (C) Capillary vessel density measured after staining with anti von Willebrand antibody on sections of (open bar) and (dark bar). (D) Capillary vessel density, normalized per adipocyte number, expressed as number of vessels per 1 adipocytes in (open bar) and (black bar). (E) Density of inflammatory cells stained with anti-cd45 antibody in (open bar) and (dark bar). (F) Number of inflammatory cells per 1 adipocytes in (open bar) and (black bar). (G) Relative mrna expression of selected angiogenic factors and VEGF receptors in SAT and VAT. Dark bar indicate higher expression in, and open bar higher expression in. * p <.5, ** p <.1, *** p <.1, Wilcoxon signed rank test.
A B Mean adipocyte area (µm 2 ) C 4 3 2 1 Vessel density (n/1 4 µm 2 ) 5 4 3 2 1 * * * D 1 5 Vessels (n/1 adipocytes)15 E CD45+ cells (n/1 5 µm 2 ) 5 4 3 2 1 ** F CD45+ cells (n/1 adipocyte) 15 1 5 ** G VAT>SAT mrna expression (fold difference) SAT>VAT 3 *** 3 2 1 VEGFR1 VEGFR2 VEGFA PLGF -1 1-2 2-3 3 ** * * TSP1 * *** VEGFC
Supplemental table 1. Primers for RT- quantitative PCR Gene Primer Tm hbéta-actine Fw hbéta-actine RV hvegf-r1 Fw hvegf-r1 RV hvegf-r2 Fw hvegf-r2 RV hvegf-a Fw hvegf-a RV hvegf-c Fw hvegf-c RV hplgf Fw hplgf RV 5'- CCA ACC GCG AGA AGA TGA-3' 5'-CCA GAG GCG TAC AGG GAT AG-3' 5'-TCA ATG TTT CCC TGC AA-3' 5'-GGA GGT ATG GTG CTT CCT GA-3' 5'-CAC CAC TCA AAC GCT GAC ATG TA-3' 5'-GCT CGT TGG CGC ACT CTT-3' 5'-TGC CGC CAC ACC ATC AC-3' 5'-GTC TCG CCC TCC GGA CCC AA-3' 5'-TTC ATT CCA TTA TTA GAC GTT CCC T- 3' 5'-GAT TAT TCC ACA TGT AAT TGG TGG G- 3' 5'-CCT ACG TGG AGC TGA CGT TCT-3' 5'-TCC TTT CCG GCT TCA TCT TCT-3' htsp1 Fw 5 - CAC CGG CTC ACA GCC CTT CG -3 htsp1 RV 5 - GCA GGG GTT ACG GGG CTT GC - 3
Supplemental table 2. Clinical and biological parameters n 29 Age (years) 41 ± 11 M/F (n) 3/26 Current smokers (n(%)) 1 (34%) Weight (kg) 117 ± 17 BMI (kg/m²) 45 ± 6 Waist circumference (cm) 121 ± 14 Hip circumference (cm) 13 ± 15 Waist to hip ratio.9 ±.1 Glucose homeostasis Glucose (mmol/l) 5.39 ±.41 Insulin (µu/ml) 16.8 ± 8. HOMA index 4.1 ± 2.3 Lipid homeostasis Cholesterol (mmol/l) 5.29 ±.83 HDL cholesterol (mmol/l) 1.19 ±.29 Triglycerides (mmol/l) 1.6 ±.95 Metabolic syndrome criteria 3(n(%)) 14 (45) Blood pressure 13/85 mmhg 17 (58) Triglycerides 1.7 mmol/l 8 (28) HDL < 1.3 mmol/l (M). 129 mmol/l (F) 19 (66) Glucose 6 mmol/l 6 (21) Liver function tests Aspartate aminotransferase (IU/l) 2.38 ± 9.34 Alanine aminotransferase (IU/l) 34.21 ± 26.39 -glutamyl transferase (IU/l) 58.1 ± 67.7 Inflammatory markers High sensitivity CRP (mg/dl) 9.97 ± 6.59 Fibrinogen (g/l) 3.93 ±.75 Obstructive sleep apnea syndrome (n(%)) 25 (86) Apnea-hypopnea index (n/h) 33 ± 26 HOMA: homeostasis minimal assessment. HDL: high density lipoprotein The metabolic syndrome components are those defined by the ATP-NCP III. Apnea-hypopnea index >1 indicates obstructive sleep apnea syndrome.
Supplemental table 3. Correlations of vascular density with clinical and biological parameters Vascular density(n/1 4 µm²) N = 29 Spearman Spearman p r r p Age.16.5 -.4.82 Weight.4.3.43.2 BMI.41.2.29.12 Waist circumference.29.13.47.1 Fasting blood glucose.33.7.28.12 Fasting serum insulin -.7.72.1.99 HOMA index -.1.94.8.65 Triglycerides -.15.41 -.1.57 HDL-cholesterol.3.85 -.19.32 ASAT -.15.42 -.4.3 ALAT -.2.9 -.27.14 -GT -.13.46 -.31.1 CRP.14.45.5.78 Fibrinogen.15.41.1.92 CRP: C-reactive protein, HDL: high density lipoprotein, ASAT: Aspartate aminotransferase, ALAT: Alanine aminotransferase, γgt: γglutamyl transferase. HOMA: homeostasis minimal assessment..
Supplemental table 4. Correlations of excess of weight loss (EWL) in % at 6 and 12 months after bypass surgery with parameters of adipose tissue: adipocyte area, vessel density, CD45+cell density and VEGF-A expression. The table shows the Spearman correlation coefficients of significant correlations. Correlations that were found significant (p<.5) in multivariate analysis taking into account the same parameters adjusted for BMI, age and gender are marked with an * Adipocyte area Vessel density CD45+ density VEGF-A EWL 6 months -,51* - - - - - -,54 EWL 1 year -,53* - -.38 -,66 -,58*,47,7*