Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors

Table 1 Primer sequences used for RT-PCR.

Genes	Primer (upstream)	Primer (downstream)	Length (bp) of PCR product	Annealing(temperature)
CYR61	5’-GAATGGAGCCTCGCATCCGATA-3’	5’-GTGGCTGCATTAGTGTCCATCC-3’	703	60
CD31	5’-ATGTGGCTTGGAGTCCTGCTGA-3’	5’-GTGTAGTTGCCACTGTGCTCCA-3’	878	60
RANTES	5’-GGATGGAGAGTCCTTGAACCTG-3’	5’-TTGAGACGGAGTCTCGCTCTGT-3’	441	60
PRSS3	5’-AGCAGCTCACTGCTACAAGACC-3’	5’-AGACCTTGGTGTAGACTCCAGG-3’	518	60
PTGS2	5’-CGGTGAAACTCTGGCTAGACAG-3’	5’-TTGGCTTCCAGTAGGCAGGAGA-3’	941	60
EBI3	5’-CCTTCATTGCCACGTACAGGCT-3’	5’-AGTGACTGGGATTACAGGTGCG-3’	845	60
ANXA1	5’-GTGAAGTCATCCAAAGGTGGTCC-3’	5’-CCTTATGGCGAGTTCCAACACC-3’	811	60
EGR1	5’-AGTGCCATCCAACGACAGCAGT-3’	5’-AGGACTTGGCTCTGAGAACCTC-3’	1 183	60
EDN1	5’-GCTGTTTGTGGCTTGCCAAGGA-3’	5’-GACCTTCGTCAGAAACTCCACC-3’	956	60
ADAM15	5’-TTCCAGCACCTGCTAAACCGCA-3’	5’-CAACTGAGCAGAGACAGGCTGT-3’	708	60
Rac1	5’-TGGTGGGAGACGGAGCTGTA-3’	5’-GGATCGCTTCGTCAAACACTG-3’	791	60
TIMP-1	5’-CACCAGAGAACCCACCATG-3’	5’-GCAGGCTTCAGCTTCCACTC-3’	669	57
TIMP-2	5’-TTTGCAATGCAGATGTAGTG-3’	5’-TCGAGAAACTCCTGCTTGG-3’	534	56
GAPDH	5’-GCCAAAAGGGTCATCATCTC-3’	5’-GTAGAGGCAGGGATGATGTTC-3’	286	60

Table 2 Patterns of gene expression observed during capillary morphogenesis in 3-D MC-based angiogenesis system.

Patterns	Gene symbol
1	CDCA8, IRTA2, FLJ23548, ZNF83, ZNF131, ARPC4, COL13A1, LRRFIP1, FLJ90806, FACL4, AZ2, TNFRSF6B, GBP3, FLJ11029, MGC39350, FMO3, POLA2, PLEC1, DHX29, SLC20A1, FLJ21908, WSX1, CIRBP, CDC20, NRCAM, PRC1, LOC85028, TUBGCP5, C1orf33, CDC2, ADAM15
2	LOC51659, CCL5, GLRA3, CCNF, HELLS, WBSCR20A, C14orf80, EBI3, UBD, MCM4, SQLE, CDH2, TAGLN, HYAL3, FLJ22215, CDCA5, S100A3, NAV1, NFKB2, COL22A1, FRA, TP53I5, AURKB, URP2, TRAF1, ARHGDIA, STARD10, C22orf18, CX3CL1, CDT1, PRSS3
3	CAMK2G, PSMD5, MRPL35, RGS4, PXK, UBXD2, CGI-116, NET1, FLJ30707, CPD, DKFZp434L142, TRIP12, MGC11324, MAN2A1, CCL21, PRCP, CDA08, TPK1, SAS10, PTGDS, TRA1, ZNF317, ANGPT2, ADAMTS1, GPR116, GEMIN6, LEREPO4, RBMS1, ANGPT2, CARD10
4	OSR1, SKIL, SPATA1, RASA1, NFATC3, HDAC9, NYD-SP20, MGC8685, DPT, THAP6, BGN, COL4A3, SDCCAG33, KIAA1387, UBE2D3, CHD3, TNRC9, GATM, KERA, KCNF1, LOC90378, AEBP1, MFGE8, WISP3, CDC2L5, FBLN1, KIAA0014, GJA4, CPLX2, KIAA1365
5	FOS, FOSB, DUSP2, EGR4, ATF3, EGR3, EGR1, EGR2, NR4A1, NR4A2, CEBPA, PTGS2, MYLK2, SLC30A3, ZFP36, PTBP2, MYNN, JUNB, CDKN1C, ID2, GADD45B, TM4SF12, GLRA3, KLF4, SOCS5, SKIL, NR4A2, ID2, SUI1, FLJ13615, CYR61, EDN1
6	BHLHB2, KLF4, LOC92906, FXR1, DUSP6, NRP1, MIR, C6orf166, DUSP1, XLKD1, MAF, C11orf15, LRRC1, JUN, DLG1, FLJ90798, SPRY2, NPEPPS, PARVA, MGC9850, DKFZP564B1162, SEPP1, RGS2, HSA9761, ALDH1A1, DKFZP434F0318, LZK1, PDK4, dJ55C23.6, HES1, ANXA1, CD31

Table 3 Chemokines/chemokine receptors detected during capillary morphogenesis.

Sequence derived from	Gene symbol	24 h/0.5 h log ratio	24 h/0.5 h change	24 h/0.5 h change P	72 h/24 h log ratio	72 h/24 h change	72 h/24 h change P	72 h/0.5 h log ratio	72 h/0.5 h change	72 h/0.5 h change P
Pattern 1
NM_001511.1	CXCL1	1.1	I	0.00004	-0.1	NC	0.5	0.8	I	0.00002
AK026546.1	CXCL5	1.2	I	0.009292	-0.4	NC	0.506476	0.6	I	0.00249
Pattern 2
U84487	CX3CL1	1.5	I	0.000389	-1.9	D	0.999562	-0.8	NC	0.911062
NM_002985.1	CCL5	3.4	I	0.00002	-3.9	D	0.99998	-0.4	D	0.998799
S69738.1	CCL2	2	I	0.00002	-1.3	D	0.99998	0.5	I	0.00002
Pattern 3
NM_002989.1	CCL21	-0.1	NC	0.596188	2.8	I	0.00002	2.1	I	0.000023
U58913.1	CCL23	-0.3	NC	0.5	1.4	I	0.000692	0.9	I	0.000147
Pattern 4
NM_000609.1	CXCL12	0.4	NC	0.118009	-0.8	D	0.999727	-0.4	NC	0.777251
NM_002993.1	CXCL6	0.3	NC	0.088938	-1	D	0.999226	-0.6	NC	0.930187
NM_013246.1	CLC	-0.1	NC	0.5	-0.1	D	0.996959	-1.2	NC	0.98579
Pattern 5
NM_006273.2	CCL7	-0.6	NC	0.5	-0.8	NC	0.977068	-0.7	D	0.999759
M57731.1	CXCL2	-1	D	0.99288	-0.5	NC	0.900148	-1.5	D	0.995927
Pattern 6
AF348491.1	CXCR4	-1.7	D	0.99998	1.8	I	0.00002	0.1	NC	0.094279
NM_000584.1	IL8	-0.9	D	0.999965	1.1	I	0.001486	-0.2	NC	0.5
NM_004166.1	CCL15	-1.4	D	0.99998	0.4	I	0.000552	-0.9	D	0.99998
Pattern 0
NM_016602.1	CCR10	1.1	NC	0.028766	-0.4	NC	0.62112	0.4	NC	0.019624
NM_002416.1	CXCL9	0.1	NC	0.5	-0.4	NC	0.814019	-0.2	NC	0.692594
AF275260.1	CXCL16	0.5	NC	0.5	-0.4	NC	0.5	0.1	NC	0.013078
AF030514.1	CXCL11	-1	NC	0.692594	0.9	NC	0.018128	0.2	NC	0.078937
AF145439.1	CCR9	0.3	NC	0.5	-0.7	NC	0.973302	-0.9	NC	0.938478
NM_001295.1	CCR1	0.4	NC	0.5	-0.8	NC	0.822588	-0.3	NC	0.881991
NM_002983.1	CCL3	0.3	NC	0.5	-0.5	NC	0.506476	-0.5	NC	0.60871
AF266504.1	CCL28	0.3	NC	0.177412	-0.9	NC	0.987976	-0.6	NC	0.854318

D: decrease; I: increase; NC: no change.