TBX20 Regulates Angiogenesis Through the Prokineticin 2-Prokineticin Receptor 1 Pathway

Abstract

Background: Angiogenesis is integral for embryogenesis, and targeting angiogenesis improves the outcome of many pathological conditions in patients. TBX20 is a crucial transcription factor for embryonic development, and its deficiency is associated with congenital heart disease. However, the role of TBX20 in angiogenesis has not been described.

Methods: Loss- and gain-of-function approaches were used to explore the role of TBX20 in angiogenesis both in vitro and in vivo. Angiogenesis gene array was used to identify key downstream targets of TBX20.

Results: Unbiased gene array survey showed that TBX20 knockdown profoundly reduced angiogenesis-associated PROK2 (prokineticin 2) gene expression. Indeed, loss of TBX20 hindered endothelial cell migration and in vitro angiogenesis. In a murine angiogenesis model using subcutaneously implanted Matrigel plugs, we observed that TBX20 deficiency markedly reduced PROK2 expression and restricted intraplug angiogenesis. Furthermore, recombinant PROK2 administration enhanced angiogenesis and blood flow recovery in murine hind-limb ischemia. In zebrafish, transient knockdown of tbx20 by morpholino antisense oligos or genetic disruption of tbx20 by CRISPR/Cas9 impaired angiogenesis. Furthermore, loss of prok2 or its cognate receptor prokr1a also limited angiogenesis. In contrast, overexpression of prok2 or prokr1a rescued the impaired angiogenesis in tbx20-deficient animals.

Conclusions: Our study identifies TBX20 as a novel transcription factor regulating angiogenesis through the PROK2-PROKR1 (prokineticin receptor 1) pathway in both development and disease and reveals a novel mode of angiogenic regulation whereby the TBX20-PROK2-PROKR1 signaling cascade may act as a "biological capacitor" to relay and sustain the proangiogenic effect of vascular endothelial growth factor. This pathway may be a therapeutic target in the treatment of diseases with dysregulated angiogenesis.

Keywords: G-protein-coupled; neovascularization, physiologic; peripheral arterial disease; receptors.

Figure 1. Tbx20 is essential for developmental angiogenesis but not vasculogenesis

a & b, Bright-field images showing the gross morphology of 30 hpf (a, upper panel) or 3 dpf (b, upper panel) Tg(fli1:EGFP)^y1 zebrafish embryos injected with CT-MO, tbx20-MO, or the combination of tbx20-MO and tbx20 mRNA. The boxed regions (red) indicate confocal imaging locations. Morphology of ISVs (boxed region) in 30 hpf (a, lower panel) or SIVs (boxed region) in 3 dpf (b, lower panel) embryos. c, Quantification of the ISV length in a (left) or SIV length in b (right). d. Diagram showing the position and DNA sequence (underline) of the CRISRP/Cas9 target site of zebrafish tbx20 gene locus. PAM sequence (CGG) is shown in red. e. Sanger sequencing results revealed a 4-bp genomic DNA fragment deletion from the target site in tbx20 mutants. f. An alignment of the protein sequences encoded by WT Tbx20 (446 AA) and Tbx20 mutant (193 AA). A 4-bp deletion caused frameshift of open reading frame leading to a premature stop codon (marked as *). g. Representative alkaline phosphatase staining of SIVs in WT animals or tbx20 mutants at 3 dpf. h. Quantification of WT animals or tbx20 mutants showing normal or inhibited SIV sprouting in g. Scale bar: a, 100 μm; b & g, 60 μm. All data are presented as mean ± S.E.M; numbers of animals analyzed are indicated in the bars of c & h. N.S., not significant. **p≤ 0.01, ****p≤ 0.0001, compared with CT-MO group.

Figure 2. TBX20 modulates adult angiogenesis and endothelial migration

a. Bright field image of VEGF-induced angiogenesis in matrigel plugs that were premixed with CT or Tbx20 siRNA (Tbx20 KD). b. Representative images of HE staining of matrigel plugs with the indicated treatment. c. FACS analysis of CD144⁺ cells in CD11b⁻ population in digested matrigel plugs. d. Quantification of c. e. Quantitative RT-PCR analysis of Tbx20 gene expression in matrigel plugs retrieved from mice. Quantitative RT-PCR examination of TBX20 mRNA (f) and western blot analysis of TBX20 protein (g) in CT or TBX20 KD HUVECs. h. Representative images of in vitro tube formation of CT or TBX20 KD HUVECs. i. Representative images of cell migration after scratch. j. Quantification of cell migration in i. Scale bar: 100 μm. All data are expressed as mean ± S.E.M; n=3. *p≤ 0.05, **p≤ 0.01, ****p≤ 0.0001 vs CT.

Figure 3. PROK2 mediates the effect of TBX20 on angiogenesis and cell migration

a. Lists of angiogenesis-related genes that were downregulated over two-fold in TBX20 KD HUVECs compared with CT KD cells. b. Representative tube formation images of TBX20 or CT KD HUVECs with or without recombinant PROK2 supplement. Quantitative RT-PCR analysis of TBX20 mRNA (c) and western blot assessment of TBX20 protein (d) in TBX20 OE HUVECs. e. Quantification of cell migration after scratch assay of CT and TBX20 OE HUVECs. f. PROK2 gene expression in TBX20 OE or CT HUVECs. g. Prok2 gene expression of the matrigel plugs implanted in mice. h. Trunk regions of zebrafish injected with CT or tbx20 MO were dissected to analyze prok2 gene expression. i & j. TBX20 and PROK2 gene expression in HUVECs stimulated with or without VEGFA165 50 ng/ml for 4 hr after scratch assay. k & l. PROK2 andTBX20 gene expression in stable TBX20 KD or CT HUVEC treated as in i. Scale bar: 100 μm. All data are expressed as mean ± S.E.M; n=3. *p≤ 0.05, **p≤ 0.01, ***p≤ 0.0001 vs CT.

Figure 4. Effect of Prok2 deficiency on zebrafish developmental angiogenesis

a & b, Tg(fli1:EGFP)^y1 zebrafish embryos were injected with CT-MO, prok2-MO, or the combination of prok2-MO and prok2 mRNA, and ISV and SIV was imaged at 30 hpf (a) and at 3 dpf (b), respectively. c, Quantification of the ISV length in 30 hpf (left) or SIV length in 3 dpf animals (right) with the indicated treatment. d. Diagram showing the position and DNA sequence (underline) of the CRISRP/Cas9 target site of zebrafish prok2 gene locus. PAM sequence (CGG) is shown in red. e. Sanger sequencing revealed a 5-bp genomic DNA fragment insertion from the target site in prok2 mutants. f. An alignment of the protein sequences of WT Prok2 (107 AA) and Prok2 mutant protein (64 AA). A 5-bp insertion caused frameshift leading to a premature stop codon (marked as *). g. Alkaline phosphatase staining of SIVs in WT animals or prok2 mutants at 3 dpf. h. Quantification of WT animals or prok2 mutants showing normal or inhibited SIV sprouting in g. Scale bars, a, 100 μm; b & g, 60 μm. All data are expressed as mean ± S.E.M; numbers of animals analyzed are shown in the bars of c & h. N.S., not significant. **p≤ 0.01, ****p≤ 0.0001, compared with CT group.

Figure 5. Effect of Prokr1a deficiency on zebrafish angiogenesis

a & b. Tg(fli1:EGFP)^y1 zebrafish embryos were injected with CT-MO, prokr1a-MO, or the combination of prokr1a-MO and prokr1a mRNA, and ISV and SIV was imaged at 30 hpf (a) and at 3 dpf (b), respectively. C. Quantification of the ISV length in 30 hpf (left) or SIV length in 3 dpf embryos (right). d. Diagram showing the position and DNA sequence (underline) of the CRISRP/Cas9 target site of zebrafish prokr1a gene locus. PAM sequence (TGG) is shown in red. e. Sanger sequencing results revealed a 4-bp genomic DNA fragment deletion from the target site in prokr1a mutants. f. An alignment of the protein sequences of WT Prokr1a (385 AA) and Prokr1a mutant protein (130 AA). A 4-bp deletion caused frameshift leading to a premature stop codon (marked as *). g. Alkaline phosphatase staining of SIVs in WT animals or prokr1a mutants at 3 dpf. h. Quantification of WT animals or prokr1a mutants showing normal or inhibited SIV sprouting in g. Scale bar: a, 100 μm; b & g, 60 μm. All data are presented as mean ± S.E.M; numbers of animals analyzed are indicated in the bars of c & h. N.S., not significant. **p≤ 0.01, ****p≤ 0.0001, compared with CT-MO group.

Figure 6. PROK2 controls angiogenesis and cell migration through PROKR1

a. FACS analysis of cell surface expression of PROKR1 and PROKR2 in HUVECs. b. PROKR1 and PROKR2 mRNA fold change of HUVECs treated with CT siRNA, PROKR1 siRNA, or PROKR2 siRNA. c. Representative image of tube formation of HUVECs transfected with CT siRNA, PROKR1 siRNA, or PROKR2 siRNA and stimulated with 60 ng/ml recombinant PROK2. d. Migration distance of HUVECs treated as in c and subjected to scratch assay. e & f. Western blot analysis of p-ERK1/2 and total ERK1/2 (e) or p-Akt and total Akt (f) in HUVECs transfected with CT siRNA or PROKR1 siRNA and stimulated with recombinant PROK2. g. Quantitative data of e & f. The intensity of pERK1/2, total ERK1/2, pAkt, and total Akt immunoblotting signals was measured and normalized with baseline level at 0 min. Scale bar: 100 μm. All data are presented as mean ± S.E.M; n=3. *p≤ 0.05, **p≤ 0.01.

Figure 7. Prok2-Prokr1a is downstream of Tbx20 in controlling angiogenesis

a. Morphology of ISVs in 30 hpf (upper panel) or SIVs in 3 dpf (lower pannel) animals with the indicated treatment. b. Quantification of ISV length in 30 hpf (left) or SIV length in 3 dpf animals (right) from a. prokr1a gene expression in CT or prok2 morphants (c) and in CT or tbx20 morphants (d). e. prokr1b gene expression in CT, Tbx20, or Prok2 knockdown animals, 50 embryos were pooled in each group. Scale bar: a (upper),100 μm; a (lower), 60 μm. All data are presented as mean ± S.E.M; numbers of animals analyzed are indicated in the bars of b. N.S., not significant. *p≤ 0.05, **p≤ 0.01.

Figure 8. PROK2 enhances recovery from hindlimb ischemia

Ten months old mice were subjected to hindlimb ischemia, and recombinant PROK2 (0.1 μg/g body weight) or PBS was injected i.m. right after the surgery and at day 7 after surgery. Blood perfusion was monitored before, immediately after, and 4, 7, 11 and 14 days after the surgery. Limb muscles were dissected at day 14 for histological analysis. a–c. Gene expression of Tbx20 (a), Prok2 (b) and Prokr1 (c) of limb muscles dissected at 0, 4, 14, and 21 days after surgery in CT group. d. Mean perfusion ratio of ischemic to un-operated limb at different time points after injury. e. Hindlimb ischemia score at day 14. f. Ultrasound imaging of blood perfusion in CT or PROK2-treated mice 14 days after ischemia. g. CD31 immunohistochemistry analysis of microvessels in the hindlimb muscle of CT or PROK2-treated animals. All data are presented as mean ± S.E.M; n=10 per group. *p≤ 0.05 compared to CT group.