- Email: [email protected]
Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome
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DIABET-842; No. of Pages 4 Diabetes & Metabolism xxx (2017) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Short Report
Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome I. Jialal a,b,*, B. Adams-Huet c, A. Major d, S. Devaraj d a
Department of Physiology and Metabolism, California North-state University, College of Medicine, 9700, West Taron Drive, Elk Grove, CA 95757, United States b Veterans Affairs Medical Center, Mather, CA, United States c Division of Biostatistics, Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX, United States d Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 August 2016 Received in revised form 7 December 2016 Accepted 15 December 2016 Available online xxx
Aims. – Metabolic syndrome (MetS) is globally a common disorder that predisposes to both diabetes and cardiovascular disease (CVD). There is a paucity of data on fibrosis and angiogenesis in adipose tissue (AT) in patients with nascent MetS uncomplicated by diabetes or CVD. Hence, we assayed various indices of fibrosis and angiogenesis in subcutaneous AT (SAT). Methods. – In both patients with MetS and matched controls, we determined fibrosis and the densities of CD31, VEGF and Angiopoietin (Angio) 2 and 1 by immunohistochemistry in gluteal SAT. Results. – The fibrosis score was significantly increased in SAT of Met S. Also, both CD31 and VEGF densities were significantly increased. Surprisingly, Angio-2 was not increased and the ratio of Angio2:1 was decreased. Both indices of fibrosis and angiogenesis correlated with biomediators of inflammation. Conclusions. – In conclusion, we report increased fibrosis and paradoxical increased angiogenesis in gluteal SAT and speculate that the increased angiogenesis is a protective mechanism in mitigating further adipose tissue dysregulation in this depot. Published by Elsevier Masson SAS.
Keywords: Adipose tissue Angiogenesis Fibrosis Inflammation Metabolic syndrome
Introduction
Research design and methods
Metabolic syndrome (MetS), which affects 35% of US adults, is globally a very common disorder and confers an increased risk for diabetes and cardiovascular disease [1]. There is sparse data on adipose tissue (AT) dysfunction in nascent MetS without the confounding of diabetes or cardiovascular disease. Previously, we have reported a dysregulation of adipokines and increased macrophage density in AT including crown–like structures [2,3]. Given the scanty data on adipose tissue fibrosis and angiogenesis in MetS and the conflicting data especially with respect to adipose tissue angiogenesis in obesity [4–8], we examined indices of both fibrosis and angiogenesis in subcutaneous AT (SAT) from patients with MetS to gain further insights.
Patients and methods
* Corresponding author. Department of Physiology and Metabolism, California North-state University, College of Medicine, 9700, West Taron Drive, Elk Grove, CA, 95757, United States. E-mail address: [email protected] (I. Jialal).
Subjects (aged 27–69 years) with MetS (n = 20) and healthy controls (n = 15) who had available adipose tissue and biochemical sample data were recruited from Sacramento County using procedures as described previously [2,3,9,10]. MetS was defined using the criteria of the NCEP ATP III [1]. Briefly, nascent MetS is defined as patients with MetS without diabetes (fasting plasma glucose < 125 mg/dL and HbA1C < 6.5% (< 47.5 mmol/mol) or clinical CVD (coronary artery disease, peripheral arterial disease, cerebrovascular disease) as reported previously [2,3,9]. Control subjects had to have 2 features of MetS and not be on blood pressure medications. Other exclusion criteria for control subjects were fasting plasma glucose (> 100 mg/dL) and triglycerides (TGs) (> 200 mg/dL). None of the subjects in both groups were on statins, angiotensin receptor blockers, fibrates, aspirin or PPAR (peroxisome proliferator activated receptor) gamma agonists or high dose antioxidant supplements. All subjects had a C-reactive protein (hsCRP) < 10 mg/L and normal white cell and had no acute or
http://dx.doi.org/10.1016/j.diabet.2016.12.004 1262-3636/Published by Elsevier Masson SAS.
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
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2 Table 1 Salient baseline characteristics. Variable
Controls (n = 15)
MetS (n = 20)
P-value Controls vs. MetS
Sex, F/M (n) Age (yrs) Waist (cm) BMI (kg/m2) BP-systolic (mmHg) BP-diastolic (mmHg) Glucose (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) hsCRP (mg/L) HOMA-IR FFA Plasma IL-1b (pg/mL) Plasma IL-6 (pg/mL) Plasma IL-8 (pg/mL) Plasma chemerin (ng/mL) pp38/p38 MAPK sTNF-R1 (pg/mL) Adipose tissue Angiopoietins density (rau/hpf) Angio 1
14/1 47 14 94 14 31.3 4.7 124 12 78 7 87 8 186 30 83 (60, 97) 51 13 118 24 1.8 (0.4, 4.0) 1.1 (1.07, 2.8) 0.44 0.19 482 (256, 844) 874 (456, 1425) 625 (546, 746) 253 49 0.07 0.03 856 177
15/5 53 10 112 16 36.1 6.7 126 13 80 10 102 12 188 29 123 (96, 160) 42 12 121 21 4.0 (1.8, 6.0) 3.3 (2.4, 5.1) 0.79 0.12 875 (797, 1187) 1871 (1693, 2018) 1142 (814, 1887) 360 60 0.23 0.12 1227 219
0.21 0.12 0.002 0.02 0.56 0.56 0.0002 0.81 0.001 0.04 0.62 0.02 0.0003 0.0002 0.01 0.0002 0.02 0.0003 0.0002 0.0005
1.4 (0.9, 1.8)
3.1 (1.7, 5.1)
1.1 (0.9, 1.6)
1.1 (0.8, 1.2)
0.0002 0.0006* 0.66 0.78*
Angio 2
Results are presented as mean standard deviation or median (25th percentile, 75th percentile). Continuous variable comparisons between groups are made with two-sample ttests; transformations were applied for skewed distributions. rau: relative arbitrary units; hpf: high power field. * Adjusted for age and waist circumference.
chronic inflammatory diseases or recent infection. All other exclusion criteria including smoking, albuminuria > 30 mg/G creatinine, clinical cardiovascular diseases are detailed in previous publications [2,9]. Informed consent was obtained from participants in the study, which was approved by the institutional review board at the University of California Davis. After a 10-hour fast, blood was obtained for basic chemistries, hsCRP, and homeostasis model assessment (HOMA) calculation as described previously [2,9]. Plasma cytokines were assayed by multiplex ELISA as described previously [2,9]. Phospho-P38MAPKinase activity was assayed by a validated bioplex multiplex phospho-protein detection system [10]. Subcutaneous adipose tissue (SAT) biopsy was performed on all subjects as described previously [2], fixed in 10% buffered formalin after cleaning and processed for immunohistochemistry as described previously [11]. Briefly, sections were deparaffinized and boiled for 10 minutes in 10 mM sodium citrate buffer (pH 6.0), incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies or an appropriate isotype negative control. Sections were exposed to H2O2 for 5 min and then incubated with biotinylated anti-rat IgG (BD Biosciences Pharmingen, San Diego, CA) or anti-rabbit IgG (BD Biosciences Pharmingen, San Diego, CA) or anti-goat IgG respectively (Vector Laboratories Inc., Burlingame, CA). A Vector stain ABC kit (Vector Laboratories Inc., Burlingame, CA) was applied to the tissue followed by DAB solution (DAKO). The slides were counterstained with haematoxylin. Sirius red staining was performed for fibrosis. Quantification of immunostaining was assessed for all the images using ImageJ and expressed as relative absorbance units. Also, we have previously reported on adipokines and other inflammatory biomediators in our patients with Met S [2,3,9]. Statistical analysis Results are expressed as mean and standard deviation (SD) or as median and interquartile range. Log or square root transformations
were applied to variables with skewed distributions prior to parametric analyses. Comparisons between the control and MetS groups were made with two-sample t-tests and analysis of covariance to control for age and waist circumference (WC). Significance was defined as a P value < 0.05. The association of these indices with number of features of MetS and P for trend was derived using Jonckheere– Terpstra test. Combining the control and metabolic syndrome groups, Spearman rank correlation coefficients were computed to assess the association between adipose tissue CD31, VEGF, fibrosis and metabolic and other variables. Data were analyzed using SAS version 9.4 (SAS Institute, Cary, NC). Results As shown in Table 1, the cardio-metabolic features were significantly abnormal in patients with MetS as in our previous reports [2,9]. In addition, hsCRP, HOMA-IR, phospho-p38MAPkinase, and IL1, IL6, IL-8 chemerin and sTNFR-1 levels were significantly increased. There was increase in the fibrosis score in SAT of patients with MetS compared to controls, even following adjustment for waist circumference (WC) as shown by the Sirius red staining (Fig. 1). With respect to angiogenesis, there was increased staining for both CD31 and VEGF in SAT following adjustment for WC. Whilst there was a significant increase in staining for Angiopoietin-1, there was no difference in Angiopoietin-2 staining resulting in a significant decrease in Angio2/Angio1 ratio in MetS. Representative staining is shown in Supplementary data, Fig. S1; see supplementary material associated with this article online. There were significant increases in CD31, VEGF and Fibrosis Score in SAT with increasing features of MetS (P < 0.001). Also, there was a significant increase in Angio-1 (P = 0.03) and a decrease in Angio-2/Angio-1 ratio with increasing features of MetS (P = 0.04). Relevant correlations were undertaken with the above markers with a P value < 0.01 defined as significant. CD31 significantly
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
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10 p < 0.0001
p < 0.0001
VEGF (rau/hpf)
CD31 (rau/hpf)
60 50 40 30 20 10 0
6 4 2
MetSyn
Control
25
MetSyn
3.0 p < 0.0001
Angio-2/Angio-1
Fibrosis score (rau/hpf)
8
0
Control
20
3
15 10 5 0
p = 0.002
2.5 2.0 1.5 1.0 0.5 0.0
Control
MetSyn
Control
MetSyn
Fig. 1. Fibrosis Score, CD31, VEGF and Angio2/1 ratio in SAT from MetS and Controls. P values adjusted for adiposity are shown.
correlated with FFA (r = 0.66, P = 0.001), sTNFR1 (r = 0.70, P = 0.0007), chemerin (r = 0.56, P = 0.008), IL-1 (r = 0.54, P = 0.004), IL-8 (r = 0.58, P = 0.001), pp38MAPK (r = 0.68, P = 0.0003), Fibrosis Score (r = 0.63, P < 0.0001) and VEGF (r = 0.57, P = 0.0007). VEGF significantly correlated with HOMA-IR (r = 0.54, P = 0.005), FFA (r = 0.64, P = 0.007), sTNFR1 (r = 0.65, P = 0.0002), chemerin (r = 0.60, P = 0.006), IL-1 (r = 0.61, P = 0.0001), Fibrosis Score (r = 0.51, P = 0.002) and Leptin (r = 0.46, P = 0.008). Fibrosis Score significantly correlated also with sTNFR1 (r = 0.70, P = 0.0006), sTNFR2 (0.78, P < 0001) IL-1b (r = 0.55, P = 0.004), IL-8 (r = 0.66, P = 0.0002), phospho-p38MAPKinase (r = 0.55, P = 0.007).
Discussion Due to the paucity of data, we report on indices of both fibrosis and angiogenesis in subcutaneous AT (SAT) from patients with nascent MetS and make the novel observation that there is increased fibrosis and paradoxical increased angiogenesis in MetS even following adjustment for adiposity arguing that they are also features of MetS, per se. Whilst both CD31 and VEGF density were significant increased, we failed to see an increase in Angio-2 and the Angio-2/Angio1 ratio. We base our conclusion of increased angiogenesis on the better studied biomediators, VEGF and CD31 [4–7]. The only other published study on adipose tissue biology in an equivalent patient sample is the report by Walton et al. [8] who compare insulin sensitive (IS) (n = 12) and insulin resistant (IR) (n = 15) patients. Interestingly, they showed a decrease in VEGF
mRNA but no changes in vessels per adipocyte, Tie1, CD31 and the Angio-2 or the ratio to Angio-1. Thus, whilst both groups show similar data with respect to Angio1 and 2, we, unlike Walton et al., show increased angiogenesis based on increased CD31 and VEGF densities. Previously, we have reported increased levels of circulating VEGF in MetS and suggest that AT is a contributor [12]. Also, other biomediators that support increased angiogenesis in these patients include significantly increased levels IL-8 and sTNFR-1, [2,9]. The only major difference between the 2 studies is the site of biopsy, we studied gluteal SAT from nascent MetS with the cardio-metabolic cluster, and they reported on abdominal SAT from insulin resistant and sensitive individuals. Thus, our thesis of increased angiogenesis in SAT is supported by several lines of evidence but further work by other groups is needed to establish increased angiogenesis in SAT and visceral fat depot in MetS. We speculate that the paradoxical increased angiogenesis is a function of the adipose tissue depot i.e. gluteal fat also termed ‘‘innocent fat’’ and potentially provides a protective mechanism against further dysregulation of gluteal SAT. It would be most interesting to determine if this protective mechanism is lost in visceral fat depots in MetS, which is considered the major contributor in AT dysregulation with concomitant increased insulin resistance and increased inflammation [2]. Lemoine et al. [13] suggested that angiogenesis may influence white adipose tissue in severely obese subjects. In their study, expression of the receptor for VEGF2 correlated with adipocyte area in both subcutaneous and omental fat. In human adipose tissue, Pasarica et al. [14] reported a positive link between insulin sensitivity (suppression of lipolysis) with VEGF mRNA and vessel density. Notably, their study was on patients with obesity and type
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
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4
2 diabetic subjects and in abdominal fat, while ours was in adipose tissue from gluteal region and in nascent MetS without diabetes. Whilst we undertook multiple correlations, the reasonable simple deduction is that both fibrosis and angiogenesis correlate with biomarkers of inflammation in human MetS also [4,7]. The correlations between fibrosis and both CD31 and VEGF are novel and suggest that they might both derive from a proximal signal i.e., inflammation. The other correlation worthy of comment is that between FFA and both VEGF and CD31 suggesting increased vascularity promoting lipolysis. The correlation of CD31 with VEGF suggests cross talk between AT and endothelial cells and this autocrine-paracrine loop may result in increased angiogenesis in AT and insulin resistance also in MetS. In conclusion, we make the novel observation of increased fibrosis and paradoxical increased angiogenesis in gluteal fat from MetS, the latter finding we speculate affords a protection of gluteal fat from further dysregulation. Studies confirming or refuting this paradox in abdominal fat and visceral fat depots are urgently required. Author Contributions I.J. and S.D. participated in the study design, wrote the manuscript and read and approved the final manuscript, B.AH. performed all the statistical analyses and read and approved the final manuscript. I.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure of interest The authors declare that they have no competing interest. Acknowledgments This study was supported by an ADA grant (IJ).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.diabet.2016.12. 004. References [1] Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, et al. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society and International Association for the Study of Obesity. Circulation 2009;120:1640–5. [2] Bremer A, Deveraj S, Afify A, Jialal I. Adipose tissue dysregulation in patients with metabolic syndrome. J Clin Endocrinol Metab 2011;96:E1782–8. [3] Jialal I, Devaraj S, Kaur H, Adams-Huet B, Bremer A. Increased chemerin and decreased omentin-1 in both adipose tissue and plasma in nascent metabolic syndrome. J Clin Endocrinol Metab 2013;98:E514–7. [4] Cao Y. Angiogenesis and vascular functions in modulation of obesity, adipose metabolism, and insulin sensitivity. Cell Metab 2013;18:478–89. [5] Sun K, Tordjman J, Cle´ment K, Scherer PE. Fibrosis and adipose tissue dysfunction. Cell Metab 2013;18:470–7. [6] Villaret A, Galitzky J, Decaunes P, Este`ve D, Marques MA, Sengene`s C, et al. Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic and inflammatory gene expression and cellular senescence. Diabetes 2010;59:2755–63. [7] Ledoux S, Queguiner I, Msika S, Calderari S, Rufat P, Gasc JM, et al. Angiogenesis associated with visceral and subcutaneous adipose tissue in severe human obesity. Diabetes 2008;57:3247–57. [8] Walton RG, Finlin BS, Mula J, Long DE, Zhu B, Fry CS, et al. Insulin-resistant subjects have normal angiogenic response to aerobic exercise training in skeletal muscle, but not in adipose tissue. Physiol Rep 2015;3 [pii: e12415]. [9] Jialal I, Huet BA, Kaur H, Chien A, Devaraj S. Increased toll-like receptor activity in patients with metabolic syndrome. Diabetes Care 2012;35:900–4. [10] Jialal I, Adams-Huet B, Pahwa R. Selective increase in monocyte p38MAPkinase activity in metabolic syndrome. Diab Vasc Dis Res 2016;13:93–6. [11] Jialal I, Major AM, Devaraj S. Global Toll-like receptor 4 knockout results in decreased renal inflammation, fibrosis and podocytopathy. J Diabetes Complications 2014;28:755–61. [12] Jialal I, Fadini GP, Pollock K, Devara JS. Circulating levels of endothelial progenitor cell mobilizing factors in metabolic syndrome. Am J Cardiol 2010;106:1606–8. [13] Lemoine AY, Ledoux S, Que´guiner I, Calde´rari S, Mechler C, Msika S, et al. Link between adipose tissue angiogenesis and fat accumulation in severely obese subjects. J Clin Endocrinol Metab 2012;97:E775–80. [14] Pasarica M, Rood J, Ravussin E, Schwarz JM, Smith SR, Redman LM. Reduced oxygenation in human obese adipose tissue is associated with impaired insulin suppression of lipolysis. J Clin Endocrinol Metab 2010;95:4052–5.
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
DIABET-842; No. of Pages 4 Diabetes & Metabolism xxx (2017) xxx–xxx
Available online at
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Short Report
Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome I. Jialal a,b,*, B. Adams-Huet c, A. Major d, S. Devaraj d a
Department of Physiology and Metabolism, California North-state University, College of Medicine, 9700, West Taron Drive, Elk Grove, CA 95757, United States b Veterans Affairs Medical Center, Mather, CA, United States c Division of Biostatistics, Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX, United States d Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 August 2016 Received in revised form 7 December 2016 Accepted 15 December 2016 Available online xxx
Aims. – Metabolic syndrome (MetS) is globally a common disorder that predisposes to both diabetes and cardiovascular disease (CVD). There is a paucity of data on fibrosis and angiogenesis in adipose tissue (AT) in patients with nascent MetS uncomplicated by diabetes or CVD. Hence, we assayed various indices of fibrosis and angiogenesis in subcutaneous AT (SAT). Methods. – In both patients with MetS and matched controls, we determined fibrosis and the densities of CD31, VEGF and Angiopoietin (Angio) 2 and 1 by immunohistochemistry in gluteal SAT. Results. – The fibrosis score was significantly increased in SAT of Met S. Also, both CD31 and VEGF densities were significantly increased. Surprisingly, Angio-2 was not increased and the ratio of Angio2:1 was decreased. Both indices of fibrosis and angiogenesis correlated with biomediators of inflammation. Conclusions. – In conclusion, we report increased fibrosis and paradoxical increased angiogenesis in gluteal SAT and speculate that the increased angiogenesis is a protective mechanism in mitigating further adipose tissue dysregulation in this depot. Published by Elsevier Masson SAS.
Keywords: Adipose tissue Angiogenesis Fibrosis Inflammation Metabolic syndrome
Introduction
Research design and methods
Metabolic syndrome (MetS), which affects 35% of US adults, is globally a very common disorder and confers an increased risk for diabetes and cardiovascular disease [1]. There is sparse data on adipose tissue (AT) dysfunction in nascent MetS without the confounding of diabetes or cardiovascular disease. Previously, we have reported a dysregulation of adipokines and increased macrophage density in AT including crown–like structures [2,3]. Given the scanty data on adipose tissue fibrosis and angiogenesis in MetS and the conflicting data especially with respect to adipose tissue angiogenesis in obesity [4–8], we examined indices of both fibrosis and angiogenesis in subcutaneous AT (SAT) from patients with MetS to gain further insights.
Patients and methods
* Corresponding author. Department of Physiology and Metabolism, California North-state University, College of Medicine, 9700, West Taron Drive, Elk Grove, CA, 95757, United States. E-mail address: [email protected] (I. Jialal).
Subjects (aged 27–69 years) with MetS (n = 20) and healthy controls (n = 15) who had available adipose tissue and biochemical sample data were recruited from Sacramento County using procedures as described previously [2,3,9,10]. MetS was defined using the criteria of the NCEP ATP III [1]. Briefly, nascent MetS is defined as patients with MetS without diabetes (fasting plasma glucose < 125 mg/dL and HbA1C < 6.5% (< 47.5 mmol/mol) or clinical CVD (coronary artery disease, peripheral arterial disease, cerebrovascular disease) as reported previously [2,3,9]. Control subjects had to have 2 features of MetS and not be on blood pressure medications. Other exclusion criteria for control subjects were fasting plasma glucose (> 100 mg/dL) and triglycerides (TGs) (> 200 mg/dL). None of the subjects in both groups were on statins, angiotensin receptor blockers, fibrates, aspirin or PPAR (peroxisome proliferator activated receptor) gamma agonists or high dose antioxidant supplements. All subjects had a C-reactive protein (hsCRP) < 10 mg/L and normal white cell and had no acute or
http://dx.doi.org/10.1016/j.diabet.2016.12.004 1262-3636/Published by Elsevier Masson SAS.
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
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DIABET-842; No. of Pages 4 I. Jialal et al. / Diabetes & Metabolism xxx (2017) xxx–xxx
2 Table 1 Salient baseline characteristics. Variable
Controls (n = 15)
MetS (n = 20)
P-value Controls vs. MetS
Sex, F/M (n) Age (yrs) Waist (cm) BMI (kg/m2) BP-systolic (mmHg) BP-diastolic (mmHg) Glucose (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) hsCRP (mg/L) HOMA-IR FFA Plasma IL-1b (pg/mL) Plasma IL-6 (pg/mL) Plasma IL-8 (pg/mL) Plasma chemerin (ng/mL) pp38/p38 MAPK sTNF-R1 (pg/mL) Adipose tissue Angiopoietins density (rau/hpf) Angio 1
14/1 47 14 94 14 31.3 4.7 124 12 78 7 87 8 186 30 83 (60, 97) 51 13 118 24 1.8 (0.4, 4.0) 1.1 (1.07, 2.8) 0.44 0.19 482 (256, 844) 874 (456, 1425) 625 (546, 746) 253 49 0.07 0.03 856 177
15/5 53 10 112 16 36.1 6.7 126 13 80 10 102 12 188 29 123 (96, 160) 42 12 121 21 4.0 (1.8, 6.0) 3.3 (2.4, 5.1) 0.79 0.12 875 (797, 1187) 1871 (1693, 2018) 1142 (814, 1887) 360 60 0.23 0.12 1227 219
0.21 0.12 0.002 0.02 0.56 0.56 0.0002 0.81 0.001 0.04 0.62 0.02 0.0003 0.0002 0.01 0.0002 0.02 0.0003 0.0002 0.0005
1.4 (0.9, 1.8)
3.1 (1.7, 5.1)
1.1 (0.9, 1.6)
1.1 (0.8, 1.2)
0.0002 0.0006* 0.66 0.78*
Angio 2
Results are presented as mean standard deviation or median (25th percentile, 75th percentile). Continuous variable comparisons between groups are made with two-sample ttests; transformations were applied for skewed distributions. rau: relative arbitrary units; hpf: high power field. * Adjusted for age and waist circumference.
chronic inflammatory diseases or recent infection. All other exclusion criteria including smoking, albuminuria > 30 mg/G creatinine, clinical cardiovascular diseases are detailed in previous publications [2,9]. Informed consent was obtained from participants in the study, which was approved by the institutional review board at the University of California Davis. After a 10-hour fast, blood was obtained for basic chemistries, hsCRP, and homeostasis model assessment (HOMA) calculation as described previously [2,9]. Plasma cytokines were assayed by multiplex ELISA as described previously [2,9]. Phospho-P38MAPKinase activity was assayed by a validated bioplex multiplex phospho-protein detection system [10]. Subcutaneous adipose tissue (SAT) biopsy was performed on all subjects as described previously [2], fixed in 10% buffered formalin after cleaning and processed for immunohistochemistry as described previously [11]. Briefly, sections were deparaffinized and boiled for 10 minutes in 10 mM sodium citrate buffer (pH 6.0), incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies or an appropriate isotype negative control. Sections were exposed to H2O2 for 5 min and then incubated with biotinylated anti-rat IgG (BD Biosciences Pharmingen, San Diego, CA) or anti-rabbit IgG (BD Biosciences Pharmingen, San Diego, CA) or anti-goat IgG respectively (Vector Laboratories Inc., Burlingame, CA). A Vector stain ABC kit (Vector Laboratories Inc., Burlingame, CA) was applied to the tissue followed by DAB solution (DAKO). The slides were counterstained with haematoxylin. Sirius red staining was performed for fibrosis. Quantification of immunostaining was assessed for all the images using ImageJ and expressed as relative absorbance units. Also, we have previously reported on adipokines and other inflammatory biomediators in our patients with Met S [2,3,9]. Statistical analysis Results are expressed as mean and standard deviation (SD) or as median and interquartile range. Log or square root transformations
were applied to variables with skewed distributions prior to parametric analyses. Comparisons between the control and MetS groups were made with two-sample t-tests and analysis of covariance to control for age and waist circumference (WC). Significance was defined as a P value < 0.05. The association of these indices with number of features of MetS and P for trend was derived using Jonckheere– Terpstra test. Combining the control and metabolic syndrome groups, Spearman rank correlation coefficients were computed to assess the association between adipose tissue CD31, VEGF, fibrosis and metabolic and other variables. Data were analyzed using SAS version 9.4 (SAS Institute, Cary, NC). Results As shown in Table 1, the cardio-metabolic features were significantly abnormal in patients with MetS as in our previous reports [2,9]. In addition, hsCRP, HOMA-IR, phospho-p38MAPkinase, and IL1, IL6, IL-8 chemerin and sTNFR-1 levels were significantly increased. There was increase in the fibrosis score in SAT of patients with MetS compared to controls, even following adjustment for waist circumference (WC) as shown by the Sirius red staining (Fig. 1). With respect to angiogenesis, there was increased staining for both CD31 and VEGF in SAT following adjustment for WC. Whilst there was a significant increase in staining for Angiopoietin-1, there was no difference in Angiopoietin-2 staining resulting in a significant decrease in Angio2/Angio1 ratio in MetS. Representative staining is shown in Supplementary data, Fig. S1; see supplementary material associated with this article online. There were significant increases in CD31, VEGF and Fibrosis Score in SAT with increasing features of MetS (P < 0.001). Also, there was a significant increase in Angio-1 (P = 0.03) and a decrease in Angio-2/Angio-1 ratio with increasing features of MetS (P = 0.04). Relevant correlations were undertaken with the above markers with a P value < 0.01 defined as significant. CD31 significantly
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
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10 p < 0.0001
p < 0.0001
VEGF (rau/hpf)
CD31 (rau/hpf)
60 50 40 30 20 10 0
6 4 2
MetSyn
Control
25
MetSyn
3.0 p < 0.0001
Angio-2/Angio-1
Fibrosis score (rau/hpf)
8
0
Control
20
3
15 10 5 0
p = 0.002
2.5 2.0 1.5 1.0 0.5 0.0
Control
MetSyn
Control
MetSyn
Fig. 1. Fibrosis Score, CD31, VEGF and Angio2/1 ratio in SAT from MetS and Controls. P values adjusted for adiposity are shown.
correlated with FFA (r = 0.66, P = 0.001), sTNFR1 (r = 0.70, P = 0.0007), chemerin (r = 0.56, P = 0.008), IL-1 (r = 0.54, P = 0.004), IL-8 (r = 0.58, P = 0.001), pp38MAPK (r = 0.68, P = 0.0003), Fibrosis Score (r = 0.63, P < 0.0001) and VEGF (r = 0.57, P = 0.0007). VEGF significantly correlated with HOMA-IR (r = 0.54, P = 0.005), FFA (r = 0.64, P = 0.007), sTNFR1 (r = 0.65, P = 0.0002), chemerin (r = 0.60, P = 0.006), IL-1 (r = 0.61, P = 0.0001), Fibrosis Score (r = 0.51, P = 0.002) and Leptin (r = 0.46, P = 0.008). Fibrosis Score significantly correlated also with sTNFR1 (r = 0.70, P = 0.0006), sTNFR2 (0.78, P < 0001) IL-1b (r = 0.55, P = 0.004), IL-8 (r = 0.66, P = 0.0002), phospho-p38MAPKinase (r = 0.55, P = 0.007).
Discussion Due to the paucity of data, we report on indices of both fibrosis and angiogenesis in subcutaneous AT (SAT) from patients with nascent MetS and make the novel observation that there is increased fibrosis and paradoxical increased angiogenesis in MetS even following adjustment for adiposity arguing that they are also features of MetS, per se. Whilst both CD31 and VEGF density were significant increased, we failed to see an increase in Angio-2 and the Angio-2/Angio1 ratio. We base our conclusion of increased angiogenesis on the better studied biomediators, VEGF and CD31 [4–7]. The only other published study on adipose tissue biology in an equivalent patient sample is the report by Walton et al. [8] who compare insulin sensitive (IS) (n = 12) and insulin resistant (IR) (n = 15) patients. Interestingly, they showed a decrease in VEGF
mRNA but no changes in vessels per adipocyte, Tie1, CD31 and the Angio-2 or the ratio to Angio-1. Thus, whilst both groups show similar data with respect to Angio1 and 2, we, unlike Walton et al., show increased angiogenesis based on increased CD31 and VEGF densities. Previously, we have reported increased levels of circulating VEGF in MetS and suggest that AT is a contributor [12]. Also, other biomediators that support increased angiogenesis in these patients include significantly increased levels IL-8 and sTNFR-1, [2,9]. The only major difference between the 2 studies is the site of biopsy, we studied gluteal SAT from nascent MetS with the cardio-metabolic cluster, and they reported on abdominal SAT from insulin resistant and sensitive individuals. Thus, our thesis of increased angiogenesis in SAT is supported by several lines of evidence but further work by other groups is needed to establish increased angiogenesis in SAT and visceral fat depot in MetS. We speculate that the paradoxical increased angiogenesis is a function of the adipose tissue depot i.e. gluteal fat also termed ‘‘innocent fat’’ and potentially provides a protective mechanism against further dysregulation of gluteal SAT. It would be most interesting to determine if this protective mechanism is lost in visceral fat depots in MetS, which is considered the major contributor in AT dysregulation with concomitant increased insulin resistance and increased inflammation [2]. Lemoine et al. [13] suggested that angiogenesis may influence white adipose tissue in severely obese subjects. In their study, expression of the receptor for VEGF2 correlated with adipocyte area in both subcutaneous and omental fat. In human adipose tissue, Pasarica et al. [14] reported a positive link between insulin sensitivity (suppression of lipolysis) with VEGF mRNA and vessel density. Notably, their study was on patients with obesity and type
Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004
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2 diabetic subjects and in abdominal fat, while ours was in adipose tissue from gluteal region and in nascent MetS without diabetes. Whilst we undertook multiple correlations, the reasonable simple deduction is that both fibrosis and angiogenesis correlate with biomarkers of inflammation in human MetS also [4,7]. The correlations between fibrosis and both CD31 and VEGF are novel and suggest that they might both derive from a proximal signal i.e., inflammation. The other correlation worthy of comment is that between FFA and both VEGF and CD31 suggesting increased vascularity promoting lipolysis. The correlation of CD31 with VEGF suggests cross talk between AT and endothelial cells and this autocrine-paracrine loop may result in increased angiogenesis in AT and insulin resistance also in MetS. In conclusion, we make the novel observation of increased fibrosis and paradoxical increased angiogenesis in gluteal fat from MetS, the latter finding we speculate affords a protection of gluteal fat from further dysregulation. Studies confirming or refuting this paradox in abdominal fat and visceral fat depots are urgently required. Author Contributions I.J. and S.D. participated in the study design, wrote the manuscript and read and approved the final manuscript, B.AH. performed all the statistical analyses and read and approved the final manuscript. I.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure of interest The authors declare that they have no competing interest. Acknowledgments This study was supported by an ADA grant (IJ).
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Please cite this article in press as: Jialal I, et al. Increased fibrosis and angiogenesis in subcutaneous gluteal adipose tissue in nascent metabolic syndrome. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2016.12.004