Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

Abstract

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student's T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

Keywords: Angiogenic genes; HIF-1a; Pedicle flap; mRNA transfection.

Figure 1

Schematic representation of HIF-1-mediated gene regulation under hypoxia. HIF-1 is a heterodimeric transcription factor composed of an O₂-regulated HIF-1α subunit and a constitutively expressed HIF-1β subunit. Under hypoxic conditions, HIF-1α is stabilized and binds to hypoxia response elements (HREs) in the promoter regions of target genes, leading to their upregulation. HIF-1 mediates the expression of hundreds of genes, including those involved in angiogenesis.

Figure 2

Isolation, Cloning, and Production In Vitro of Four HIF-1α mRNA Transcript Isoforms. (A) lanes show the isolation of a full-length “predominant’ form as well as three transcript variants. Sanger DNA sequencing covered 100% of each HIF-1α isoform. The full-length predominant form contained an open reading frame of 826 amino acids, while variants 1, 2, and 3 contained 424, 516, and 785 amino acids respectively. The HiScribe T7 ARCA mRNA kit (NEB, Ipswich, MA) yielded transcripts with a 5’-methyl-cap and a 3’ poly-A tail. Each mRNA reaction was purified using RNA Cleanup (NEB, Ipswich, MA), quantitated, and visualized on an agarose gel. (B) lanes on the left represent the samples before purification; lanes on the right represent the samples after purification.

Figure 3

Structural motifs and domains of HIF-1α protein and six isoforms and mutants. HIF-1α mRNAs that encode the full-length protein (P1, P2, P3) and the three shortened splice variants (V1, V2, V3) are shown. P402A and P564A represent Prolines involved in HIF-1α degradation. bHLH is basic helix-loop-helix, PASa (Per-Arnt-Sim) and PASb are molecular sensors, ODD is oxygen-dependent degradation, ID is an inhibitory domain, N-TAD, and C-TAD are amino- and carboxy-transactivation domains.

Figure 4

HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for HIF-1α expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

Figure 5

HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for VEGF, PGF, or ANGPT1, expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.

Figure 6

HIF-1α mRNAs (P1, P2, P3. V1, V2, V3) were transfected into primary dermal fibroblasts. At 3, 6, or 10 h, total RNA was extracted and assayed for FLT-1, or EDN1 expression levels by qRT-PCR. Values were normalized to beta-actin levels. “Lipo.” is lipofectamine alone control. The Student’s T-test was used to determine significance and the mean and standard deviation of independent triplicate wells is shown. *p = 0.05 or lower, **p = 0.01 or lower, and ***p = 0.001 or lower.