Introduction
Materials and methods
Patients and controls
Detection of circulating endothelial cells and progenitors by flow-cytometry
Isolation of CECs and EPCs from peripheral blood
RNA extraction
Gene array analysis
Real time RT-PCR
Statistical analysis
Results
CECs and EPCs in patients with SSc
SSc patients (50) | Healthy controls (50) | P value | |
---|---|---|---|
CECs/mmc | 689 ± 464 | 22 ± 17 | < 0,0001 |
EPCs/mms | 146 ± 92 | 1 ± 1 | < 0,0001 |
Skin ulcers - SSc patients (33) | Skin ulcers + SSc patients (17) | P value | |
---|---|---|---|
CECs/mmc | 600 ± 401 | 968 ± 553 | 0.05 |
EPCs/mms | 142 ± 93 | 158 ± 92 | 0.597 |
CECs/mmc | EPCs/mmc | |
---|---|---|
Data before iloprost infusion | 661 ± 404 | 152 ± 93 |
Data 72 h after one day iloprost therapy | 745 ± 453 | 186 ± 104¶ |
Data 72 h after five days iloprost therapy | 775 ± 382 | 206 ± 139¥ |
Gene array analysis of endothelial cells
Probe set ID | Gene Title | Gene symbol | F.C. SSc ulcers/healthy | F.C. SSc ulcers post-treatment/SSc ulcers pre-treatment | Representative Public ID |
---|---|---|---|---|---|
Positive regulation of anti-apoptosis | |||||
210621_s_at | RAS p21 protein activator (GTPase activating protein) 1 | RASA1 | 8.72 down | 4.28 up | M23612 |
214917_at | protein kinase, AMP-activated, alpha 1 catalytic subunit | PRKAA1 | 6.49 down | 6.60 up | AK024252 |
201849_at | BCL2/adenovirus E1B 19 kDa interacting protein 3 | BNIP3 | 69.04 down | 11.78 up | NM_004052 |
Response to stress
| |||||
200985_s_at | CD59 molecule, complement regulatory protein | CD59 | 18.76 down | 2.72 up | NM_000611 |
202906_s_at | nibrin | NBN | 12.38 down | 4.28 up | AF049895 |
206040_s_at | mitogen-activated protein kinase 11 | MAPK11 | 17.27 up | 10.62 down | NM_002751 |
209305_s_at | growth arrest and DNA-damage-inducible. beta | GADD45B | 13.58 down | 8.27 down | AF078077 |
210512_s_at | vascular endothelial growth factor | VEGF | 36.08 down | 5.58 up | AF022375 |
213756_s_at | heat shock transcription factor 1 | HSF1 | 8.12 up | 3.47 down | AI393937 |
217684_at | thymidylate synthetase | TYMS | 4.21 down | 3.30 up | BG281679 |
220038_at | serum/glucocorticoid regulated kinase family. member 3 | SGK3 | 8.24 down | 9.60 down | NM_013257 |
Response to wounding and wound healing
| |||||
209277_at | Tissue factor pathway inhibitor 2 | TFPI2 | 9.85 down | 2.62 up | AL574096 |
203294_s_at | lectin, mannose-binding, 1 | LMAN1 | 11.53 up | 9.84 down | U09716 |
205767_at | epiregulin | EREG | 5.13 down | 3.61 down | NM_001432 |
209101_at | connective tissue growth factor | CTGF | 595.44 down | 14.43 up | M92934 |
Wnt receptor activity
| |||||
203987_at | frizzled homolog 6 | FZD6 | 39.71 down | 2.18 up | NM_003506 |
Receptor complex
| |||||
201474_s_at | integrin. alpha 3 | ITGA3 | 5.21 down | 2.36 up | NM_002204 |
204625_s_at | integrin. beta 3 | ITGB3 | 2.17 up | 2.47 down | BF115658 |
206009_at | integrin. alpha 9 | ITGA9 | 3.18 down | 3.58 down | NM_002207 |
211772_x_at | cholinergic receptor. nicotinic. alpha 3 | CHRNA3 | 2.54 up | 4.46 down | BC006114 |
204773_at | interleukin 11 receptor. alpha | IL11RA | 15.25 down | 2.90 down | NM_004512 |
membrane
| |||||
202637_s_at | intercellular adhesion molecule 1 (CD54) | ICAM1 | 28.90 down | 6.61 up | AI608725 |
203699_s_at | deiodinase, iodothyronine, type II | DIO2 | 7.75 up | 10.10 down | U53506 |
203988_s_at | fucosyltransferase 8 (alpha (1,6) fucosyltransferase) | FUT8 | 17.44 down | 17.71 up | NM_004480 |
204273_at | endothelin receptor type B | EDNRB | 10.11 up | 2.75 down | NM_000115 |
205421_at | solute carrier family 22, member 3 | SLC22A3 | 6.50 up | 6.21 down | NM_021977 |
213856_at | CD47 molecule | CD47 | 12.43 down | 10.98 up | BG230614 |
Chemotaxis
| |||||
205242_at | chemokine (C-X-C motif) ligand 13 | CXCL13 | 3.03 down | 7.56 up | NM_006419 |
209687_at | chemokine (C-X-C motif) ligand 12 | CXCL12 | 24.98 down | 2.54 up | U19495 |
210845_s_at | plasminogen activator, urokinase receptor | PLAUR | 12.79 down | 2.05 up | U08839 |
207850_at | chemokine (C-X-C motif) ligand 3 | CXCL3 | 24.57 down | 3.15 down | NM_002090 |
210163_at | chemokine (C-X-C motif) ligand 11 | CXCL11 | 34.76 down | 5.60 up | AF030514 |
215723_s_at | phospholipase D1, phosphatidylcholine-specific | PLD1 | 12.37 down | 3.08 up | AJ276230 |
219825_at | cytochrome P450, family 26, subfamily B, polypeptide 1 | CYP26B1 | 17.74 down | 15.25 up | NM_019885 |
DNA-dependent DNA replication
| |||||
205085_at | origin recognition complex, subunit 1-like | ORC1L | 10.43 down | 7.25 up | NM_004153 |
208070_s_at | REV3-like, catalytic subunit of DNA polymerase zeta | REV3L | 38,90 down | 2.80 down | NM_002912 |
208808_s_at | high-mobility group box 2 | HMGB2 | 5.53 down | 2.41 up | BC000903 |
209084_s_at | RAB28, member RAS oncogene family | RAB28 | 23.27 down | 2.09 up | BE504689 |
210892_s_at | general transcription factor II, i | GTF2I | 3.72 down | 3.64 up | BC004472 |
DNA-dependent DNA replication
| |||||
205085_at | carbonyl reductase 1 | CBR1 | 2.39 up | 2.34 down | BC002511 |
G0 to G1 transition
| |||||
205655_at | Mdm4, p53 binding protein | MDM4 | 3.27 down | 6.07 up | NM_002393 |
platelet-derived growth factor beta-receptor activity
| |||||
205226_at | platelet-derived growth factor receptor-like | PDGFRL | 5.33 up | 9.50 down | NM_006207 |
actin cytoskeleton organization and biogenesis
| |||||
209209_s_at | pleckstrin homology domain containing, family C, member1 | PLEKHC1 | 51.02 down | 13.59 up | AW469573 |
216621_at | Rho-associated, coiled-coil containing protein kinase 1 | ROCK1 | 8.58 down | 8.52 up | AL050032 |
220997_s_at | diaphanous homolog 3 (Drosophila) | DIAPH3 | 3.72 down | 10.53 up | NM_030932 |
208614_s_at | filamin B, beta (actin binding protein 278) | FLNB | 59.49 down | 2.91 down | M62994 |
214925_s_at | spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) | SPTAN1 | 13.23 down | 2.62 down | AK026484 |
215602_at | FYVE, RhoGEF and PH domain containing 2 | FGD2 | 12.94 up | 2.60 down | AK024456 |
Innate immune response
| |||||
204924_at | toll-like receptor 2 | TLR2 | 5.94 down | 3.16 up | NM_003264 |
206271_at | toll-like receptor 3 | TLR3 | 55.81 down | 6.59 down | NM_003265 |
206206_at | CD180 molecule | CD180 | 23.53 down | 10.73 up | NM_005582 |
210166_at | toll-like receptor 5 | TLR5 | 27.49 down | 2.03 up | AF051151 |
215388_s_at | complement factor H | CFH | 561.66 down | 2.36 down | X56210 |
206157_at | pentraxin-related gene, rapidly induced by IL-1 beta | PTX3 | 5.17 down | 12.45 up | NM_002852 |
206693_at | interleukin 7 | IL7 | 3.06 down | 11.97 up | NM_000880 |
206727_at | complement component 9 | C9 | 6.43 up | 9.24 down | K02766 |
Probe Set ID | Gene Title | Gene symbol | FC SSc/healthy | FC SSc post-treatment/SSc pre-treatment | Representative Public ID |
---|---|---|---|---|---|
Positive regulation of anti-apoptosis | |||||
201849_at | BCL2/adenovirus E1B 19 kDa interacting protein 3 |
BNIP3
| 19.65 down | 5.93 up | NM_004052 |
210621_s_at | RAS p21 protein activator (GTPase activating protein) 1 |
RASA1
| 5.35 down | 2.02 up | M23612 |
214917_at | protein kinase, AMP-activated, alpha 1 catalytic subunit |
PRKAA1
| 2.88 down | 3.50 up | AK024252 |
Response to stress
| |||||
202906_s_at | nibrin |
NBN
| 3.83 down | 4.33 up | AF049895 |
206040_s_at | mitogen-activated protein kinase 11 |
MAPK11
| 3.53 up | 2.15 up | NM_002751 |
209305_s_at | growth arrest and DNA-damage-inducible, beta |
GADD45B
| 11.23 down | 4.25 up | AF078077 |
210512_s_at | vascular endothelial growth factor |
VEGF
| 7.03 down | 2.38 up | AF022375 |
217684_at | thymidylate synthetase |
TYMS
| 2.76 down | 5.84 up | BG281679 |
Response to wounding and wound healing
| |||||
209101_at | connective tissue growth factor |
CTGF
| 1912.1 down | 11.18 up | M92934 |
209277_at | Tissue factor pathway inhibitor 2 |
TFPI2
| 3.36 down | 8.90 down | AL574096 |
Wnt receptor activity
| |||||
203987_at | frizzled homolog 6 |
FZD6
| 19.85 down | 3.25 up | NM_003506 |
Receptor complex
| |||||
201474_s_at | integrin, alpha 3 |
ITGA3
| 3.37 down | 2.04 up | NM_002204 |
206009_at | integrin, alpha 9 |
ITGA9
| 2.05 down | 2.02 up | NM_002207 |
211772_x_at | cholinergic receptor, nicotinic, alpha 3 |
CHRNA3
| 2.10 up | 12.73 down | BC006114 |
204773_at | interleukin 11 receptor, alpha |
IL11RA
| 10.76 down | 6.66 up | NM_004512 |
Membrane
| |||||
202638_s_at | intercellular adhesion molecule 1 (CD54) |
ICAM1
| 21.89 down | 2.00 up | NM_000201 |
204273_at | endothelin receptor type B |
EDNRB
| 12.18 up | 9.07 up | NM_000115 |
205421_at | solute carrier family 22, member 3 |
SLC22A3
| 14.37 up | 8.31 down | NM_021977 |
213857_s_at | CD47 molecule |
CD47
| 6.17 down | 3.51 up | BG230614 |
Chemotaxis
| |||||
207850_at | chemokine (C-X-C motif) ligand 3 |
CXCL3
| 35.50 down | 2.32 up | NM_002090 |
211122_s_at | chemokine (C-X-C motif) ligand 11 |
CXCL11
| 2.98 down | 2.32 down | AF002985 |
215723_s_at | phospholipase D1, phosphatidylcholine-specific |
PLD1
| 7.72 down | 2.07 up | AJ276230 |
219825_at | cytochrome P450, family 26, subfamily B, polypeptide 1 |
CYP26B1
| 25.09 down | 3.98 down | NM_019885 |
203218_at | mitogen-activated protein kinase 9 | MAPK9 | 9.53 down | 7.02 up | W37431 |
DNA-dependent DNA replication
| |||||
208070_s_at | REV3-like, catalytic subunit of DNA polymerase zeta |
REV3L
| 21.36 down | 8.90 up | NM_002912 |
208808_s_at | high-mobility group box 2 |
HMGB2
| 2.78 down | 3.18 up | BC000903 |
209084_s_at | RAB28, member RAS oncogene family |
RAB28
| 7.25 down | 3.79 up | BE504689 |
213336_at | General transcription factor II, i |
GTF2I
| 4.51 down | 4.52 up | AI826454 |
Prostaglandin-E2 9-reductase activity
| |||||
50221_at | transcription factor EB | TFEB | 6.09 down | 11.09 up | AI524138 |
G0 to G1 transition
| |||||
210386_s_at | metaxin 1 | MTX1 | 6.35 down | 4.85 up | BC001906 |
platelet-derived growth factor beta-receptor activity
| |||||
205226_at | platelet-derived growth factor receptor-like |
PDGFRL
| 5.04 up | 2.72 up | NM_006207 |
actin cytoskeleton organization and biogenesis
| |||||
208614_s_at | filamin B, beta (actin binding protein 278) |
FLNB
| 92.27 down | 2.75 up | M62994 |
214925_s_at | spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) |
SPTAN1
| 17.80 down | 8.95 up | AK026484 |
215602_at | FYVE, RhoGEF and PH domain containing 2 |
FGD2
| 15.22 down | 5.67 up | AK024456 |
Innate immune response
| |||||
206271_at | toll-like receptor 3 |
TLR3
| 8.66 down | 10.24 down | NM_003265 |
210166_at | toll-like receptor 5 |
TLR5
| 7.83 down | 2.95 up | AF051151 |
215388_s_at | complement factor H |
CFH
| 295.64 down | 3.15 down | X56210 |
206693_at | interleukin 7 |
IL7
| 10.94 down | 2.96 up | NM_000880 |
203854_at | complement factor I | CFI | 578.23 down | 16.50 up | NM_000204 |
206727_at | complement component 9 |
C9
| 11.81 down | 10.01 up | K02766 |
Biological process | Number of probe sets | P-value |
---|---|---|
Immune response | 210 | 0.0000000009 |
Defense response | 221 | 0.0000000145 |
Response to wounding | 109 | 0.0000012730 |
Signal transducer activity | 472 | 0.0000206037 |
Response to stress | 236 | 0.0000215677 |
Regulation of cell proliferation | 89 | 0.0000403309 |
Positive regulation of nitric oxide biosynthesis | 8 | 0.0001150065 |
Positive regulation of biosynthesis | 16 | 0.0001813324 |
Receptor activity | 290 | 0.0003570920 |
Cell activation | 36 | 0.0005153290 |
Cell proliferation | 137 | 0.001020579 |
Positive regulation of innate immune response | 3 | 0.001302911 |
Regulation of immune response | 27 | 0.001521656 |
Inflammatory response | 47 | 0.002011958 |
Positive regulation of immune response | 20 | 0.002900865 |
Cell adhesion | 136 | 0.006908177 |
Cytokine activity | 47 | 0.017808983 |