Macrophages Aggravate Hypoxia-Induced Cardiac Microvascular Endothelial Cell Injury via Peroxynitrite: Protection by Tongxinluo

Figures & data

Table 1. Primers used for real time PCR detection.

	Primers	Annealing temperature	Product size
HIF-1α:Sense	5' CCTGCTTGGTGCTGATTTGTG 3'	55 °C	265 bp
Anti-sense	5' CTGTACTGTCCTGTGGTGACTTGTC 3'
HIF-2α:Sense	5' GAAATGGAATCCTGCTCACAAAATC 3'	60 °C	287 bp
Anti-sense	5' TGAAAGACCATCCGAGTCACATAGC 3'
VEGF:Sense	5' TAGACACACCCACCCACATACATAC 3'	55 °C	173 bp
Anti-sense	5' CCCAACTCAAGTCCACAGCAG 3'
iNOS:Sense	5' CCAGCCTCAAGTCTTATTTCCTC 3'	55 °C	177 bp
Anti-sense	5' GCAAGTTCCATCTTTCACCCAC 3'
eNOS:Sense	5' ACGAGACGCTGGTGCTGGTGGTAAC 3'	62 °C	274 bp
Anti-sense	5' GAGCCGAGCCCGAACACACAGAAC 3'
ECE-1: Sense	5' GACCTGGTGGACTCGCTCTC 3'	60 °C	296 bp
Anti-sense	5' TAGCTGAAGAAGTCATGGCAGG 3'
COX-2:Sense	5' CTTTGCCCAGCACTTCACGCATCAG 3'	65 °C	269 bp
Anti-sense	5' CCTGCCCCACAGCAAACCGTAGATG 3'
PGIS:Sense	5' GAGACGGAGTTTCACGCTTATTGC 3'	60 °C	228 bp
Anti-sense	5' AGGCAAATCACGAGGTCAGGAG 3'
β-actin:Sense	5' CTCCATCCTGGCCTCGCTGT 3'	60 °C	268 bp
Anti-sense	5' GCTGTCACCTTCACCGTTCC 3'
Mouse iNOS:
Sense	5' AGTAGCCTAGTCAACTGCAAGAG 3'	60 °C	267 bp
Anti-sense	5' TCCAGGGATTCTGGAACATTCTG 3'
Mouse β-actin:
Sense	5' TGGTAGGTATGGGTCAGAAGGA 3'	60 °C	229 bp
Anti-sense	5' GTCATCTTCTCCCGGTTAGCTT 3'

Figure 1. Macrophage co-culture aggravated gene expressions in hypoxia-stimulated HCMECs. The gene mRNAs were measured by real-time RT-PCR. (A) The changes of HIF-1α, HIF-2α and VEGF in control (Con), hypoxia (Hx), hypoxia plus macrophage co-culture (Hx + Mϕ) and macrophage co-culture (Mϕ) groups. (B) The changes of eNOS, iNOS and ECE-1 in Con, Hx, Hx + Mϕ and Mϕ groups. (C) The changes of COX-2 and PGIS in Con, Hx, Hx + Mϕ and Mϕ groups. **p < 0.01 or *p < 0.05, versus Con group. ##p < 0.01 or #p < 0.05, versus Hx group. (D) The time-dependent expression of iNOS in hypoxia-activated macrophages. **p < 0.01 or *p < 0.05, versus 0 h group. The data were repeated three times and represented as mean ± SD.

Figure 2. Gene expression of HIF-1α, HIF-2α and VEGF in treated HCMECs. **p < 0.01, versus Con group; ##p < 0.01 or #p < 0.05, versus Hx + Mϕ group; ††p < 0.01, versus Hx + Mϕ+ FeTPPS group. The data were repeated three times and represented as mean ± SD. Con, control; Hx + Mϕ, hypoxia plus macrophage co-culture; LT, low dose TXL; MT, middle dose TXL; HT, high dose TXL.

Figure 3. Gene expression of eNOS, iNOS and ECE-1 in treated HCMECs. **p < 0.01, versus Con group; ##p < 0.01 or #p < 0.05, versus Hx + Mϕ group; ††p < 0.01, versus Hx + Mϕ + FeTPPS group. The data were repeated three times and represented as mean ± SD. Con, control; Hx + Mϕ, hypoxia plus macrophage co-culture; LT, low dose TXL; MT, middle dose TXL; HT, high dose TXL.

Figure 4. Gene expression of COX-2 and PGIS in treated HCMECs. **p < 0.01, versus Con group; ##p < 0.01 or #p < 0.05, versus Hx + Mϕ group; ††p < 0.01, versus Hx + Mϕ + FeTPPS group. The data were repeated three times and represented as mean ± SD. Con, control; Hx + Mϕ, hypoxia plus macrophage co-culture; LT, low dose TXL; MT, middle dose TXL; HT, high dose TXL.

Figure 5. Protein levels of the remarkably altered genes in treated HCMECs. (A) Protein levels were detected by western blotting. (B) The changes of HIF-2α and VEGF in control (Con), hypoxia plus macrophage co-culture (Hx + Mϕ), SIN-1, Hx + Mϕ + FeTPPS and Hx + Mϕ + MT groups. (C) The changes of iNOS and ECE-1 in Con, Hx + Mϕ, SIN-1, Hx + Mϕ + FeTPPS and Hx + Mϕ + MT groups. (D) The changes of COX-2 and PGIS in Con, Hx + Mϕ, SIN-1, Hx + Mϕ + FeTPPS and Hx + Mϕ + MT groups. **p < 0.01 or *p < 0.05, versus Con group; ##p < 0.01 or #p < 0.05, versus Hx + Mϕ group. The data were repeated three times and represented as mean ± SD. MT, middle dose TXL.

Figure 6. TXL inhibited nitrotyrosine formation to attenuate HCMEC injury (400×). The nitrotyrosine content was evaluated by immunofluorescence. The upper ones were HCMEC morphological alterations and the lower showed nitrotyrosine fluorescence. Con, control; Hx + Mϕ, hypoxia plus macrophage co-culture; MT, middle dose TXL.