Table 3: Resources
Reagent or resource Source Identifier Used for
Chemicals, solutions, peptides and recombinant proteins
Immersion Liquid Leica Microsystems
Cat11513859 CLSM
ProLongTM Diamond Antifade
Mountant with DAPI Invitrogen Cat#P36971 IF
T3* Sigma Aldrich Cat#T6397-250MG Cell culture
Albumin from Bovine Serum Sigma Aldrich Cat#A6003-25G IF, WB Bovine Serum Albumin (low
endotoxin)
Sigma Aldrich Cat#A8806-5G Lipolysis assay
Collagenase I Serva Cat#DS17461.02 Isolation of cells
Cortisol Sigma Aldrich Cat#C-106 Cell culture
Dexamethasone Sigma Aldrich Cat#D4902 Cell culture
DMEM/HAM’S F-12 Capricorn S. Cat#DMEM-12-HXA Cell culture
EBM-2* PromoCell Cat#C-22211 Cell culture
EGM-2 growth factors* PromoCell Cat#C-39311 Cell culture
MSC-FBS Gibco Cat#12662-029 Cell culture
Gentamycin Sulfate Lonza Cat#17-5182 Isolation of cells
Insulin Solution Human Sigma Aldrich Cat#I9278-5ML Cell culture
IBMX* Sigma Aldrich Cat#I5879-16 Cell culture
PBS* Capricorn S. Cat#PBS-1A Cell culture
dPBS* Sigma Aldrich Cat#D8662-500ML Cell culture
rh EGF Immunotools Cat#11343406 Cell culture
rhFGF-b/FGF-2 Immunotools Cat#11343623 Cell culture
Rosiglitazone Cayman Cat#122323-73-4 Adipogenesis
Transferrin human Sigma Aldrich Cat#T8158-1G Cell culture
0.05 % Trypsin in EDTA (1X) Gibco Cat#25300-062 Trypsinisation
hrVEGF-C (CHO cell derived) RD systems Cat#9199-VC Cell culture hrpro-dVEGF-C (S2 cell derived) Jeltsch lab. Kindly provided Cell culture hrpro-mVEGF-C (S2 cell derived) Jeltsch lab Kindly provided Cell culture
Tween 20 (0.05 %) Prepared aliquot (50 %) IF
TritonTM X-100 Sigma Aldrich Cat#T8787-50ML IF
16 % Formaldehyde solution Thermo F. S. Cat#28908 IF
L-Glutamine Solution 200mM Capricorn S. Cat#GLN-B Cell culture Penicillin/Streptomycin Solution
(100X) (Pen/Strep) Capricorn S. Cat#PS-B Cell culture
Biotin Sigma Aldrich Cat#B4639-1G Cell culture
D-Panthothenic Acid Sigma Aldrich Cat#P5155-100G Cell culture
dbcAMP* Sigma Aldrich Cat#D0627-100MG Lipolysis assay
Bradford Reagent Sigma Aldrich Cat#B6916-500ML Protein Assay
WST-1 Reagent Abcam Cat#ab155902 Cell culture
Commercial Assays
RNeasy Mini Kit (250) Qiagen Cat#74106 RNA isolation
mirVanaTM ParisTM Invitrogen Cat#AM1556 miRNA isolation
High Capacity cDNA Reverse
Transciption Kit Applied
Biosystems Cat#4368814 RT-qPCR
Cells-To-CTTM Kit Invitrogen Cat#A35374 RT-qPCR
miRCURY LNATM RT Kit Qiagen Cat#339340 RT-qPCR
miRCURY LNA miRNA PCR Assays
Qiagen Cat#YP00204243 Cat#YP00204499 Cat#YP00204679 Cat#YP00205892 Cat#YP0020431Ycat#YP00205911
Cat#P00204260
RT-qPCR
Randox GLY Randox Cat#GY105 Glycerol assay
Randox NEFA Randox Cat#FA115 NEFA assay
ELISA (Duosets) RD Systems Cytokines
Dnase I Amplification Grade Invitrogen Cat#18068-015 RT reaction TaqManTM Fast Advanced Master
Mix
Applied Biosystems
Cat#4444557 qPCR analysis
74
PowerUp Syber green Master Mix Applied
Biosystems Cat#A25742 qPCR analysis
TaqMan Pre Amp Master Mix Applied Biosystems
Cat#4391128 qPCR analysis Software and databases
CellProfiler 52 Software Image analysis
BioRender Online Software https://biorender.com/ Image analysis
ImageJ-win32 Software Image analysis
LAS AF Lite_ Software Image analysis
GraphPad Prism 9 Software Results analysis
Endnote X9 3.3 Software Text processing
7500 Software v. 2.0.5 Software qPCR analysis
Gene Globe Online software https://geneglobe.qiagen.com/us/ miRNA analysis
GenEx Software miRNA analysis
miRDB Online database http://mirdb.org/ miRNA analysis
miRBase Online database https://mirbase.org/ miRNA analysis Single Cell Portal Online database https://singlecell.broadinstitute.org/ mRNA analysis
*EBM-2 (Endothelial Cell Basal Medium 2); *EGM-2 growth factors (SupplementPack Endothelial cell GM2); *dbcAMP (N6, 2-0-Dibutyryladenosine 3', 5'- cyclic monophosphate sodium salt); *T3 (3,3,5-Triiodo-L-thyronine sodium salt 3,3,5- Triiodo-L-thyronine sodium salt); DPBS* (Dulbecco's Phospahte Buffered Saline); PBS* (Dulbecco's PBS 1X). These abbreviations are not included in the list of abbreviations.
Media/Buffer
Basal medium
DMEM/HAM'S F-12 medium Biotine (33µM),
D-Panthotenate (17µM), Pen/Strep (100U/ml) Diffrentiation medium Basal medium
insulin (66nM) dexamethasone (1µM) T3 (1nM)
Transferrin human (0.1 ug/ml) IBMX (250 µM)
rosiglitazone (1µM) cortisol (0.1 µM) PM4 medium Basal Medium b-FGF (25 ug/ml) EGF (100 ug/ml) 2.5% MSC-qualified FBS Insulin (130 nM) EGM-2 medium EBM-2 medium
2 % fetal bovine serum-angio type and
EGM-2-MV SingleQuots R (R3-IGF, VEGF-A, FGF, EGF, ascorbic acid (AA) and hydrocortisone) ELB buffer
0,1 mM EDTA 0,15 M HN4Cl 5,6 mM K2HPO4
75 Primers related to miRNA analysis
miRCURY miRNA Focus panel PCR Panel plasma https://dataanalysis2.qiagen.com/miRCury/analysisjob/50446
Table 4: Primers related to AT and in vitro experiments
Gene symbol Assay ID Gene name in vitro AT
ADA Hs01110945_m1 adenosine deaminase ●
ADAMTS2 Hs01029111_m1 ADAM Metallopeptidase With Thrombospondin Type 1 Motif 2
●
ADAMTS3 Hs01046996_m1 ADAM Metallopeptidase With Thrombospondin Type 1 Motif 3
●
ADAMTS9 Hs00172025_m1 ADAM Metallopeptidase With Thrombospondin Type 1 Motif 9
●
ADIPOQ Hs00605917_m1 Adiponectin ● ●
ADRB2 Hs00240532_s1 Adrenergic, beta-2-, receptor, surface ●
AGO1 Hs01084661_m1 Argonaute RISC Component 1 ●
ANGPTL4 Hs01101127_m1 Angiopoietin-like 4 ● ●
APLN Hs00175572_m1 Apelin
BAMBI Hs03044164_m1 BMP and activin membrane bound inhibitor ● ● CCBE1 Hs00827104_m1 Collagen and Calcium Binding EGF Domains 1 ●
CCL5=RANTE S
Hs00174575_m1 chemokine (C-C motif) ligand 5 ●
CD14 Hs00169122_g1 CD14 molecule ●
CD36 Hs00354519_m1 CD36 molecule ● ●
CD3e Hs01062241_m1 CD3e molecule, epsilon ●
CD4 Hs01058407_m1 CD4 molecule ●
CD8a Hs00233520_m1 CD8a molecule ●
CHREBP Hs00975714_m1 MLX interacting protein-like ● ●
CPEB3 Hs00208640_m1 Cytoplasmic Polyadenylation Element Binding Protein 3
●
CPT1A Hs00912671_m1 Carnitine Palmitoyltransferase 1A ●
DGAT2 Hs01045913_m1 Diacylglycerol O-acyltransferase 2 ● ●
ELOVL6 Hs00225412_m1 ELOVL fatty acid elongase 6 ●
FABP4 Hs01086177_m1 Fatty acid binding protein 4 ●
FASN Hs01005622_m1 Fatty acid synthase ● ●
FN1 Hs00365052_m1 Fibronectin 1 ●
GDF15 Hs00171132_m1 Growth differentiation factor 15 ● ●
GLUT4 (SLC2A4)
Hs00168966_m1 Glucose transporter 4 ●
GOT2 Hs00905827_g1 Glutamic-oxaloacetic transaminase 2 ●
HAND2 Hs00232769_m1 heart and neural crest derivatives expressed 2 ● HIF1A Hs00153153_m1 Hypoxia inducible factor 1, alpha subunit ●
76
HSL (LIPE) Hs00943404_m1 Lipase, hormone-sensitive ● ●
IL1b Hs01555410_m1 Interleukin 1, beta ●
IL2RA (CD25) Hs00907779_m1 Interleukin 2 receptor, alpha ●
IL6 Hs00985639_m1 Interleukin 6 (interferon, beta 2)
IL8 Hs00174103_m1 Interleukin 8 ●
INHBA Hs01081598_m1 Inhibin beta A subunit ● ●
IRF1 Hs00971960_m1 Interferon regulatory factor 1 ● ●
IRF5 Hs00158114_m1 interferon regulatory factor 5 ●
ITGAX Hs00174217_m1 integrin, alpha X (complement component 3 receptor 4 subunit) (CD11C, SLEB6)
●
KLF4 Hs00358836_m1 Kruppel-like factor 4 ● ●
KLF6 Hs00810569_m1 Kruppel-like factor 6 ●
KLF9 Hs00230918_m1 Kruppel-like factor 9 ●
LEP Hs00174877_m1 Leptin ● ●
LUM Hs00929860_m1 Lumican ● ●
MCP1 Hs00234140_m1 Chemokine (C-C motif) ligand 2 ●
MRC1=CD206 Hs00267207_m1 Mannose receptore C type 1 ●
NDN Hs00267349_s1 Necdin, MAGE family member ● ●
PDPN Hs00366766_m1 Podoplanin ● ●
PECAM 1 Hs01065282_m1 Platelet/endothelial cell adhesion molecule 1 ● ●
PLIN Hs00160173_m1 Perilipin ● ●
PLIN2 Hs00605340_m1 Perilipin 2 ●
PLIN3 Hs00998416_m1 Perilipin 3 ●
PNPLA2 (ATGL)
Hs00982040_g1 Patatin-like phospholipase domain containing 2 ● ●
PPARG Hs01115513_m1 Peroxisome proliferator-activated receptor gamma ● ●
PROX1 Hs00896294_m1 prospero homeobox 1 ● ●
RPS13 Hs01011487_g1 Ribosomal protein S13 ● ●
RUNX2 Hs00231692_m1 Runt-related transcription factor 2 ● ●
SCD Hs01682761_m1 Stearoyl-CoA desaturase ● ●
SREBF1 Hs01088691_m1 Sterol regulatory element binding transcription factor 1
●
TBP Hs00427620_m1 TATA box binding protein ● ●
TGFb1 Hs00998133_m1 Transforming growth factor, beta 1 ● ●
TIMP1 Hs00171558_m1 Tissue inhibitor of metalloproteinase 1 ●
TIMP2 Hs00234278_m1 TIMP metallopeptidase inhibitor 2 ●
TLR4 Hs01060206_m1 Toll-like receptor 4 ●
TNC Hs01115665_m1 Tenascin C ● ●
VEGFA Hs00900055_m1 Vascular endothelial growth factor A ● ●
VEGFC Hs01099203_m1 Vascular endothelial growth factor C ● ●
VEGFD Hs01128657_m1 Vascular endothelial growth factor D ● ●
VEGFR2 Hs00911700_m1 kinase insert domain receptor ●
77
VEGFR3 Hs00176607_m1 fms-related tyrosine kinase 3 ●
WISP2 Hs00180242_m1 WNT1 inducible signaling pathway protein 2 ● ●
ZHX1 Hs00232545_m1 Zinc Fingers And Homeoboxes 1 ●
ZFP423/ZNF423 Hs00323880_m1 Zinc finger protein 423 ● ●
*Some of the abbreviations are not mentioned in the list of abbreviations because they were named only in the results section.
Table 5: Antibodies related to FACS analysis
Name of antibody Clone Conjugates Supplier
CD45 2D PerCP BD Biosciences
CD206 clone 19.2 BV 421 BD Biosciences
CD206 clone 19.2 APC BD Biosciences
CD16 clone 3G8 APC-Cy7 BD Biosciences
CD4 RPAT4 FITC BD Biosciences
CD3 clone UCHT1 PE BD Biosciences
CD8 RPAT8 Alexa 647 BD Biosciences
CD45Ro clone UCHL1 APC H7 BD Biosciences
CD45Ra clone HI100 BV510 BD Biosciences
CD11c clone B-ly6 BV510 BD Biosciences
CD127 clone hIL-7R-M21 PE BD Biosciences
CD25 clone M-A251 PE-Cy7 BD Biosciences
CD196 clone 11A9 APC BD Biosciences
CD183 clone 1C6/CXCR3 BV421 BD Biosciences
CD194 clone 1G1 BV510 BD Biosciences
CD163 clone GHI/61 PE Exbio
CD11c clone Bu15 Pacific Blue Exbio
CCR2 clone REA264 Pe-Vio770 Miltenyi Biotec
Table 6: Antibodies related to IF analysis
Name of antibody Identifier Supplier
Polyclonal goat anti-hVE-cadherin (1:20) Cat#AF938-SP Biotechne Goat polyclonal anti-hPROX(1:10) Cat#AF2727-SP Biotechne Donkey anti-goat (488) (1:200) Cat#A11055 Invitrogen
HCS Lipidtox Red (1:200) Cat#H34476 Invitrogen
78
6 RESULTS
Aim 1: To compare the impact of varying degrees of lymphatic drainage on circulating lipolytic products and lipolytic activity of femoral AT in obese women.
To test the hypothesis that the variations in lymphatic drainage could be one of the factors affecting metabolism, and particularly lipolysis, in GAT in women, we compared the lipolytic activity of femoral AT and markers of lipolysis in plasma and ISF in two groups of premenopausal women that differed in the efficiency of lymphatic drainage of the lower body.
Subjects characteristics
Women from both groups were metabolically healthy and the groups were not different in respect of indices of carbohydrate and lipid metabolism. Relative fat mass in total body and lower limbs was similar in both groups (Table 7). Volume of acquired blister ISF per subject was similar in both groups (1306 ± 135.1 µl in NLD vs.1580 ± 109.4 µl in WLD). Leptin and adiponectin concentration in blister ISF and plasma did not differ between groups as shown in Table 7.
79
Subjects with normal
lymph drainage (n=12) Subjects with worsened lymph drainage (n=16)
Age (year) 32.4±6.5 39.0±7.7*
Weight (kg) 75.8±7.6 75.0±13.6
Fat mass (%) bioimpedance 30.4±5.9 29.4±6.4
Fat mass (kg) bioimpedance 23.3± 6.4 22.5± 8.2
Fat mass (%) calculated 33.9±5.3 34.0± 5.8
Fat mass (kg) calculated 25.9±6.4 26.1±9.5
ECW/TBW % 42.6±1.4 43.5±1.7
Hip circumference (cm) 97.3±6.0 96.1±10.4
Waist to Hip ratio 0.78±0.02 0.81±0.04*
Thigh circumference (cm) 56.4±5.1 54.5±5.4
Volume of lower limb (l) 13.1±1.3 12.8±2.2
Fat mass in lower limbs % 35.8±3.4 36.0±4.8
BMI (kg/m2) 27.4±3.8 26.9±3.6
Cholesterol (mmol/l) 4.12±0.75 4.49±0.68
HDL cholesterol (mmol/l) 1.61±0.28 1.76±0.37
LDL cholesterol (mmol/l) 2.29±0.50 2.51±0.58
Triglycerides (mmol/l) 0.85±.31 0.67± 0.19
Fasting glucose (mmol/l) 5.21±0.40 5.11±0.24
Fasting insulin (mU/l) 8.20±3.33 6.17±2.75
HOMA-IR 1.93±0.87 1.42±0.66
Leptin (ng/ml) in plasma 21.29±9.41 17.77±9.43
Leptin (ng/ml) in blister ISF 8.55±4.55 8.78±5.09 Adiponectin (µg/ml) in plasma 7.07±2.39 6.14±3.23 Adiponectin (µg/ml) in blister 1.69±0.69 1.56±0.80
Table 7: Anthropometric and biochemical characteristics of the subjects. ECW/TBW-ratio of extracellular and total body water. Dara are shown as mean ± SD (standard deviation). * p <0.05.
80 Lipolytic markers in basal and dynamic conditions
Compared with NLD group, plasma levels of glycerol were lower in the WLD group, the same trend (p = 0.07) was seen for FFA (Fig. 16A). Similarly, in vivo lipolysis index was lower in women with WLD (Fig. 16B). Concentration of glycerol and FFA in ISF/MD were not different between the groups. The ratio of glycerol and FFA levels in plasma vs. blister ISF were significantly lower in the WLD group. (Fig. 16B). The ratio of FFA in plasma vs.
blister ISF correlated with a total protein concentration in blister ISF (Fig. 16C). In response to adrenaline stimulation, concentrations of glycerol in MD increased within 30min and remained elevated until the end of the experiment in both groups. Glycerol levels in 75–90 min of the experiment were significantly higher in NLD compared with the WLD group (Fig. 16D).
0 200 400 600 800
M FFA
plasma blister NLD n=12 WLD n=16 0.07
MD
A
0 20 40 60 80 100
M glycerol
plasma blister
**
NLD n=12 WLD n=16
MD
0 1 2 3 4 5
0 1 2 3 4 5
ratio plasma/ISF in vivo lipolysis index
NLD n=12 WLD n=16 0.08
FFA glycerol
*
**
B
10 20 30 40
-2 0 2 4 6 8
total protein ISF (mg/ml)
FFA plasma/ISF ratio
R=-0.5929 p=0.0009
C
time (minutes)
M glycerol
0 15 30 45 60 75 90 105 120 0
50 100 150 200
NLD n=9 WLD n=14 adrenalin
** * D
Figure 16: Lipolytic markers in two groups of women differing in the efficiency of lymphatic drainage. A. Concentrations of FFA and glycerol in plasma, blister ISF and MD. FFA in MD could not be assessed (unpaired t test). B. Ratio between plasma and ISF levels calculated for FFA and glycerol. Right Y axis represents calculated “in vivo lipolysis index” (Mann–Whitney test). C.
Correlation between total protein content in blister ISF and ratio FFA plasma/ISF (Spearman). D.
Time-course of glycerol levels in microdialysate of femoral AT (two-way ANOVA, with Sidak posthoc tests). Data are means ± SE, *p < 0.05, **p < 0.01.
81
Aim 2: To examine and compare adipogenic, lipogenic, lipolytic, immune fibrotic and angiogenic parameters of healthy and lymphedema associated SAT in women
In order to comprehensively evaluate AT alterations induced by massive damage to LS, we analysed several parameters of LAT reflecting adipocyte qualities - adipocyte size, adipogenic, lipogenic, and lipolytic signature. Moreover, immune, fibrotic, (lymph)angiogenic paramters of LAT were assesed and compared it to properties of AT from healthy women and/or AT from healthy limb of LYM women.
Subjects characteristics
3 weight-matched groups of women were recruited: 11 women with unilateral lymphedema in the upper extremity (non-pitting breast cancer-related lymphedema)-LYM group; 11 healthy women without lymphedema undergoing elective liposuction-Healthy group; 11 women- breast cancer survivors without lymphedema-NOLYM group. Women from LYM group developed lymphedema 0–13 years after primary anti-tumour treatment (median 2.5 years) and lymphedema lasted for 2–9 years (median 4.5 years) until they were indicated for liposuction. None of the women had active cancer. The amount of fat removed by liposuction was 869-223 ml (median 1181 ml) and it represented a 26-103 % reduction of diseased limb volume compared to the volume of the healthy limb (median 68 %). Despite all efforts to achieve initial aim of matching, healthy women undergoing elective liposuction were younger and less obese than cancer survivors (both the LYM and NOLYM groups) (Tab. 8).
Healthy and LYM groups were matched according to relative fat mass measured by bioimpedance, but discordant in calculated relative fat mass (Tab. 8). This discrepancy apparently reflects a higher volume of extracellular water in patients with lymphedema, which has a direct impact on bioimpedance measurements. Healthy women had similar profile of blood lipids as LYM and NOLYM group but better indices of glucose metabolism (fasting glucose, insulin levels, HOMA-IR, Tab. 8), which could be related to the age and adiposity.
82 Table 8: Anthropometric and biochemical characteristics of the individual group. *Healthy group (n = 11), * Non-LYM group (n=11), *LYM group (n=11). Data are shown as mean ± SD. * p
<0.05, ** p <0.01, *** p<0.001 (difference related to healthy subjects).
Adipocyte-related characteristics of SAT from arm depot
Previous studies evaluating histological sections of LAT showed hypertrophy of lymphedema adipocytes in both upper and lower extremities [185, 258]. Nevertheless, our analysis of isolated adipocytes did not show any significant difference in the mean diameter of adipocytes in between healthy and LYM groups (88.8 ± 5.6 lymphedema, vs. 97.4 ± 3.3 um healthy. Even more, comparison of frequency distribution of adipocytes revealed higher proportion of larger adipocytes (100–120 µm) in healthy AT compared to LAT (Fig. 17).
Thus, results of size assessment of isolated adipocytes did not confirm hypertrophic pattern of adipocytes of lymphedema-AT
Healthy group*
NONLYM group*
LYM group*
Age (year) 45.0±8.1 60.4±10.3*** 66.1±5.9 ***
Weight (kg) 69.2±8.3 78.8±13.1 78.3±7.6
BMI (kg/m2) 25.2±3.7 29.4±4.0* 29.4±3.5*
Fat mass (%) 32.5±7.8 36.9±5.3 41.1±3.3**
Fat mass (%) calculated 32.1±6.1 40.4±5.4** 41.2±5.4**
Fat mass (kg) 22.6±7.1 28.7± 6.2 32.4±5.9**
Fat mass (kg) calculated 22.5±6.2 32.4±9.2* 32.4±6.6*
Waist circumference (cm) 83.6±9.8 100.2±10.4** 94.0±11.0 Hip circumference (cm) 104.1±6.8 112.1±8.9 106.6±7.6 Waist to Hip ratio 0.80±0.07 0.89±0.05* 0.88±0.06*
NEFA (mmol/l) 0.81±0.29 0.80±0.31 0.87±0.29
Glycerol (µmol/l) 55.0±31.9 78.8±42.9 81.0±43.2 Cholesterol (mmol/l) 4.90±0.73 4.95±0.72 5.27±0.74 HDL cholesterol (mmol/l) 1.84±0.41 1.50±0.39 1.45±0.27*
LDL cholesterol (mmol/l) 2.66±0.78 2.81±0.172 3.33±0.65 Triglycerides (mmol/l) 0.88±0.38 1.60±0.56 1.79±1.03*
Fasting glucose (mmol/l) 4.8±0.4 5.9±1.0** 5.9±0.7**
Fasting insulin (mU/l) 4.3±3.2 12.8±6.2** 13.0±5.3**
HOMA-IR 0.85±0.79 3.53±2.40** 3.52±1.72***
Uric acid μmol/l 252.6±59.9 339.1±39.2* 295.7±103.9
83
30 50 70 90 110 130 150 170 190 0
10 20 30
bin centers (m)
% of adipocytes LAT (n=9)healthy AT (n=11)
*
Figure 17: Adipocyte size assessment. Comparison of frequency distribution of adipocytes revealed higher proportion of larger adipocytes (100 µm – 120 µm) in healthy AT compared to LAT. Dara are shown as mean ± SD. * p <0.05.
As previously documented, experimentally induced lymphedema in mice was connected with increased protein levels of proadipogenic transcription factors PPARɤ and C/EBPα [259]. Therefore we have evaluated mRNA expression of 9 factors (GLUT4, FASN, SCD, ELOVL6, DGAT2, ADIPOQ, LEP, CHREBP, SREBF1) implicated in human AT mass regulation and 7 genes involved in AT lipogenesis (Fig. 18). This analysis revealed 5 genes that were altered in LAT compared to AT from both healthy subjects and healthy limb of LYM subject. Expression of adipogenic regulators revealed mixed adipogenic response to lymphedema, as mRNA expression of both proadipogenic (ZNF423) and anti-adipogenic (WISP2, GOT2) factors was higher in AT from diseased limb of LYM women when compared to AT from paired healthy limb and also to AT from healthy women. Moreover, expression of INHBA and KLF9 was enhanced while KLF4 and RUNX2 was decreased in paired comparison between healthy and diseased limb of LYM patients. Nevertheless, expression of adiposity marker (leptin) was increased in lymphedema limb compared to both healthy subjects and healthy limb of LYM subject. Similarly, this mixed response was observed also in the set of lipogenic markers. The expression of two analysed lipogenic markers (ChREBP, ELOVL6) was significantly lower in AT from diseased limb of LYM women when compared to AT from paired healthy limb and to AT from healthy women, while expression of FASN was higher in paired comparison between healthy and diseased limb of LYM patients (Fig. 18).
84
0.0 0.5 1.0 1.5 2.0
Adipogenesis and lipogenesis
mRNA, fold change
ADIPOQ DGAT2 ELOVL6 FASN
GLUT4 CHREBP
b
LEP SCD
a *a
SREBF1
*
*
*
a
***
healthy AT
LYM - healthy limb AT LYM - lymphedema limb AT
0.0 0.5 1.0 1.5 2.0 2.5
Regulators of adipogenesis
mRNA, fold change
BAMBI GOT2 INHBA KLF4 KLF9 PPAR
b
**
RUNX2 WISP2 ZNF423
a a
b
** *
**
a
a b
***
* healthy AT
LYM -healthy limb AT LYM - lymphedema limb AT
Figure 18: LAT qualities related to adipocytes I. mRNA levels of markers of adipogenesis, lipogenesis and regulators of adipogenesis in whole AT expressed as fold change over the mean expression of healthy group (Kruskal–Wallis test of 2ΔCt values, Dunn’s correction, comparison of 3 groups, *p < 0.05, **p < 0.01, ***p < 0.001; Wilcoxon test of paired healthy and diseased limb of LYM subjects, a- p < 0.05, b- p < 0.01, c- p < 0.001).
Since altered lipolysis could contribute to AT expansion in lymphedema, we analysed basal and isoproterenol induced lipolysis rate ex vivo in AT explants and isolated adipocytes.
Explants from lymphedema AT released more glycerol and FFA in both basal and isoproterenol stimulated conditions (Fig. 19 A). The same trend for higher basal and isoproterenol stimulated glycerol levels in LYM compared to healthy was observed in isolated adipocytes from a subset of subjects (n = 6 healthy, n = 7 LYM) (not shown).
Although isoproterenol induced lipolysis expressed as a fold change of basal lipolysis did not differ between the groups for glycerol, fold change of FFA was lower in LYM group compared to healthy. Moreover, baseline FFA:glycerol ratio in explants (but not isolated adipocytes) from LAT was much higher compared to healthy AT, suggestive of lower re- esterification or utilization of FFA by LAT (adipocytes or SVF cells) within relatively intact AT structure. We also analysed whether higher basal lipolysis in LAT is based on the alteration of expression of genes involved in lipolysis and lipid handling. Nevertheless, contrary to the expectations stemming from ex vivo results, ATGL mRNA expression was
85 lower while expression of lipid droplet coating PLIN1 was higher in diseased limb compared to healthy and healthy limb of LYM (Fig. 19 B).
basal ISO 0
4 8 12 16
glycerol (M)/mg lipids
***
*
healthy AT LAT
A
basal ISO 0
2 4 6 8
glycerol fold change
basal ISO 0
10 20 30
FFA mM/mg lipids
***
*
basal ISO 0
20 40 60
FFA fold change
***
basal ISO 0
1 2 3
FFA:GLY ratio ***
0.0 0.5 1.0 1.5 2.0 2.5
Lipolysis and lipid handling
mRNA, fold change
ADA ADRB2 ATGL CD36 HSL PLIN PLIN2 PLIN3 b
*
***
****
* ***
****
healthy AT
LYM -healthy limb AT LYM - lymphedema limb AT
****
b
b
c
B
Figure 19: LAT qualities related to adipocytes II. A. Ex vivo lipolysis in AT explants. Cells were exposed to basal conditions or 1 µM isoproterenol (ISO) for 4 h. Concentrations of glycerol and FFA normalized to mg of lipids and fold change over the basal conditions are shown (Two-way ANOVA of LN transformed data, Sidak post-hoc analysis, *p < 0.05, ***p < 0.001). B. mRNA levels of markers of lipolysis and lipid handling in whole AT expressed as fold change over the mean expression of healthy group (Kruskal–Wallis test of 2ΔCt values, Dunn’s correction, comparison of 3 groups, *p < 0.05, **p < 0.01, ***p < 0.001; Wilcoxon test of paired healthy and diseased limb of LYM subjects, a- p < 0.05, b- p < 0.01, c- p < 0.001).
86 Adipose progenitors-related characteristics of SAT from arm depot
Since reprogramming of adipose precursors by lymph stasis towards higher adipogenic potential could partially explain observed LAT expansion, we analysed numbers and behaviour of adipose precursors from both healthy and lymphedema AT. First, we compared relative numbers of mesenchymal stem cells in AT from healthy subjects and lymphedema patients by flow cytometry (method described in Annexe II). Within CD45 negative population, relative content of MSC defined as CD34+ or CD31− was found to be lower in LAT. However, more detailed analysis of MSC utilizing combination of other MSC markers (CD90, CD73, CD271, CD29 and CD105) did not confirm these differences (Fig. 20A).
Thus, abnormal AT accumulation in lymphedema cannot be explained by higher numbers of MSC in lymphedema patients. Next, we analysed in vitro proliferation of MSC/preadipocytes isolated from AT of healthy and LYM subjects. Preadipocytes from LAT exhibited similar proliferation rate as cells from healthy subjects (Fig. 20B), also population doubling time at passage 3 and passage 10 was not different between the groups (passage 3, healthy AT- 40.6 ± 2.0 h, LAT 44.8 ± 3.7 h; passage 10, healthy AT 106.7 ± 19.0 h, LAT 131.2 ± 22.8 h). Thus, abnormal accumulation of AT in lymphedema is probably not caused by faster proliferation of adipose precursors that have comparable proliferative capacity to control cells at least when kept under standard in vitro conditions.
Therefore, we compared the adipogenic capacity of these precursors in response to four different media imitating optimal and suboptimal adipogenic conditions-standard serum free differentiation medium, serum free medium with 10 times lower concentration of components of hormonal mix (rosiglitazone, T3, dexamethasone, IBMX) and these two media supplemented with 2.5% human serum (Fig. 20D). Neutral lipid accumulation measured by Oil Red O assay was not different between the cells from lymphedema patients and healthy controls in any of the tested media (Fig. 20C). We also analysed the degree of differentiation at the mRNA expression levels of typical adipogenic markers (PPARγ, perilipin, FAS, DGAT2, ATGL)-but we could not find major differences between cells derived from lymphedema patients and control subjects (not shown). Nevertheless, preadipocytes derived from lymphedema subjects exhibited differential expression of KLF4, KLF6, INHBA and ZNF423, i.e. genes implicated in adipogenesis, when compared with cells from healthy women (Fig. 20E). Futhermore, expression levels of KLF4 and KLF6 were correlated (not shown).