PKM2-Driven Lactate Overproduction Triggers Endothelial-To-Mesenchymal Transition in Ischemic Flap via Mediating TWIST1 Lactylation

Abstract

The accumulation of lactate is a rising risk factor for patients after flap transplantation. Endothelial-to-mesenchymal transition (EndoMT) plays a critical role in skin fibrosis. Nevertheless, whether lactate overproduction directly contributes to flap necrosis and its mechanism remain unknown. The current study reveals that skin flap mice exhibit enhanced PKM2 and fibrotic response. Endothelial-specific deletion of PKM2 attenuates flap necrosis and ameliorates flap fibrosis in mice. Administration of lactate or overexpressing PKM2 promotes dysfunction of endothelial cells and stimulates mesenchymal-like phenotype following hypoxia. Mechanistically, glycolytic-lactate induces a correlation between Twist1 and p300/CBP, leading to lactylation of Twist1 lysine 150 (K150la). The increase in K150la promotes Twist1 phosphorylation and nuclear translocation and further regulates the transcription of TGFB1, hence inducing fibrosis phenotype. Genetically deletion of endothelial-specific PKM2 in mice diminishes lactate accumulation and Twist1 lactylation, then attenuates EndoMT-associated fibrosis following flap ischemia. The serum lactate levels of flap transplantation patients are elevated and exhibit predictive value for prognosis. This findings suggested a novel role of PKM2-derived lactate in mediating Twist1 lactylation and exacerbates flap fibrosis and ischemia. Inhibition of glycolytic-lactate and Twist1 lactylation reduces flap necrosis and fibrotic response might become a potential therapeutic strategy for flap ischemia.

Keywords: Pyruvate kinase M2; endothelial‐to‐mesenchymal transition; lactate; lactylation; random‐pattern skin flap.

Figure 1

Enhanced expression of PKM2 is associated with lactate production and endothelial‐to‐mesenchymal transition in ischemic flaps. A) Metabolic pathway analysis exhibiting the top 20 pathways participated in differences between the skin from sham and skin flap mice. B) Heatmap showing differentially expressed genes involved in pyruvate metabolism in the skin from sham and skin flap mice. C) PKM1, PKM2 and HIF‐1α protein levels in the skin from sham and skin flap mice on postoperative day 7. D) Detection of dimeric and tetrameric PKM2 protein levels in the skin from sham and skin flap mice on postoperative day 7. E) Detection of p‐PKM2 (pY105) expression in the skin from sham and skin flap mice on postoperative day 7. F) Relative protein expression of PKM1, PKM2, HIF‐1α and p‐PKM2 in the skin from sham and skin flap mice on postoperative day 7. G) Immunofluorescence co‐staining of HIF‐1α and PKM2 of the dermal layer in the skin from sham and skin flap mice on postoperative day 7. Scale bar: 10 µm. H,I) Comparison of fluorescence intensity of HIF‐1α and PKM2 in the above two groups (n = 5). J) Schematic diagram of primary metabolic pathway for glucose flux through anaerobic glycolysis. K,L) Measurement of serum and skin lactate levels from sham and skin flap mice on postoperative day 7. (n = 5). M) Representative photomicrographs of H&E staining and Masson staining in the skin from sham and skin flap mice on postoperative day 7. Scale bar: 50 µm. N,O) Quantification of fibrotic area and number of vessels in the above two groups. (n = 5). P) Protein levels of endothelial marker CD31, VE‐cadherin, mesenchymal marker α‐SMA, Collagen1a1 and FSP1 in the skin from sham and skin flap mice on postoperative day 7. Q) Immunofluorescence staining of mesenchymal marker FSP1 in the skin from sham and skin flap mice on postoperative day 7. Scale bar: 10 µm. R) Comparison of fluorescence intensity of FSP1 in the above two groups (n = 5). Accurate P‐values are listed in the figures. Data is presented as mean±S.D. (F, H, I, K, L, N and R), unpaired two‐tailed t test.

Figure 2

PKM2 regulates lactate production and EndoMT in endothelial cells following hypoxia. HUVECs were transfected with PKM2 siRNA after hypoxic or normoxic challenge. A) Knockdown efficiencies of PKM2 in HUVECs using western blotting. B) Western blotting detection of endothelial marker CD31 and mesenchymal marker α‐SMA under normoxia or hypoxia. C) PKM2 knockdown following hypoxia decreased glucose uptake in HUVECs (n = 3). D) PKM2 knockdown following hypoxia down‐regulated lactate production in HUVECs (n = 3). E–G) Glycolysis Stress, Glycolytic Rate and Mito Stress assays of HUVECs cultured and transfected as in panel. H) H&E staining, Masson staining and Immunofluorescence co‐staining of PKM2 and mesenchymal marker FSP1 from the above groups on postoperative day 7. I,J) Quantification of fibrotic area and number of vessels in the above four groups (n = 5). Scale bar: upper panel: 50 µm, middle panel: 50 µm, lower panel: 10 µm. K,L) Comparison of fluorescence intensity of FSP1 and PKM2 in the above four groups (n = 5). Accurate P‐values are listed in the figures. Data is presented as mean±S.D. C–G), Two‐way ANOVA; I–L), unpaired two‐tailed t test.

Figure 3

Supplemental lactate accelerates EndoMT progression in endothelial cells following hypoxia. HUVECs were cultured with 10 mM lactate after exposed to normoxia or hypoxia. A) Representative images of morphology of HUVECs. Scale bar: 50 µm, 25 µm. B) Immunofluorescence co‐staining of endothelial marker VE‐cadherin and mesenchymal marker FSP1 (n = 3). Scale bar: 25 µm. C) Western blotting measurement of mesenchymal marker α‐SMA and endothelial marker VE‐cadherin in endothelial cells. D) qRT‐PCR detection of mesenchymal marker α‐SMA and endothelial marker VE‐cadherin in endothelial cells. (n = 3). E) Angiogenesis of endothelial cells was determined by tube formation assay (n = 3). Scale bar: 200 µm. F) Migration of endothelial cells were detected using transwell assay (n = 3). Scale bar: 200 µm. G) Endothelial cell contractility was determined by collagen gel contraction assay (n = 3). Accurate P‐values are listed in the figures. Data is presented as mean±S.D. B and D–G), Two‐way ANOVA.

Figure 4

Lactate induces TGF‐β/Smad2 pathway following flap ischemia/hypoxia. A–D) HUVECs were stimulated with 10 mM lactate following exposed to normoxia or hypoxia. qRT‐PCR was employed to detect the mRNA levels of TGFB1, SMAD2 and SMAD3 (n = 3). Western blotting was conducted to measure the expression of TGF‐β, phospho(p)–SMAD2, phospho‐SMAD3. E–G) qRT‐PCR was employed to measure the mRNA expression of TGFB1, SMAD2 and SMAD3 in skin from sham and skin flap mice on postoperative day 7 (n = 5). Western blotting was employed to detect the protein levels of phospho‐SMAD2 and TGF‐β in the above groups (n = 5). H) Immunofluorescence co‐staining of PKM2 and TGF‐β in the above groups (n = 5). Scale bar, 10 µm. I) Immunofluorescence co‐staining of PKM2 and phospho‐SMAD2 in the above groups (n = 5). Scale bar, 10 µm. Accurate P‐values are listed in the figures. Data is presented as mean±S.D. (A‐C), Two‐way ANOVA; (E and G‐I), unpaired two‐tailed t test.

Figure 5

Lactate accelerates Twist1 nuclear translocation and lactylation after hypoxia. HUVECs were cultured with 10 mM lactate after exposed to normoxia or hypoxia. A,B) Western blotting and immunofluorescent staining were employed to detect the expression levels of cytoplasmic Twist1 and nuclear Twist1 in endothelial cells. Scale bar, 20 µm. C,D) Western blotting and immunofluorescent staining were employed to detect the expression levels of cytoplasmic Twist1 and nuclear Twist1 in skin from sham and skin flap mice on postoperative day 7 (n = 5). Scale bar, 10 µm. E) Western blotting was conducted to assess the expression of phospho(p)‐Twist1 (pS68) and Twist1. F) Lactylation and acetylation of Twist1, as well as the interaction between Twist1 and p300/CBP were detected using Immunoprecipitation (IP). G–K) MCT inhibitor CHC was utilized to inhibit intracellular lactate of endothelial cells before lactate treatment. Lactylation and acetylation of Twist1, and the interaction between Twist1 and p300/CBP were examined by immunoprecipitation. Accurate P‐values are listed in the figures. Data is presented as mean±S.D. A,B), unpaired two‐tailed t test; C,D), Two‐way ANOVA.

Figure 6

Lactate‐mediated Twist1 nuclear translocation in turn activates TGFB1 expression. HUVECs were cultured with 10 mM lactate after exposed to normoxia or hypoxia. A) ChIP experiment was conducted with anti‐Twist1 antibody and subjected to qRT‐PCR utilizing primers specific for the promoter of TGF‐β in endothelial cells (n = 3). B) Transcription factor binding sites of Twist1 using JASPAR database. C,D) The direct binding of Twist1 to the TGFB1 promoter was identified using ChIP assays in endothelial cells, including nonspecific control (ChIP NC) (a sequence located 3000 bp upstream from the transcription start site), TGFB1‐ChIP1 and TGFB1‐ChIP2. E) The direct binding capability of Twist1 to the TGFB1 promoter was confirmed using qRT‐PCR in endothelial cells (n = 3). F) Dual‐luciferase reporter gene assay was employed to assess the regulation of Twist1 to TGFB1 transcription in a ChIP 1 and ChIP 2 element‐dependent manner (n = 3). Accurate P‐values are listed in the figures. Data is presented as mean±S.D. A), Two‐way ANOVA; E,F), unpaired two‐tailed t test;.

Figure 7

Twist1 K150 is the key site by which lactate drives Twist1 lactylation and exacerbates EndoMT. HUVECs were cultured with 10 mM lactate after exposed to hypoxia. A,B) Mass spectrometry spectrum of the lactylated Twist1 K73, K76 and K150 sites. C) Flowchart of in vitro experiments to validate lactate‐mediated lactylation of Twist1. D) Western blotting analysis of Twist1 knockout in endothelial cells. E) Western blotting analysis of Flag‐tag protein and Pan‐Kla expression levels after transfection of Flag‐Twist1, Flag‐Twist1 (K73A), Flag‐Twist1 (K75A) and Flag‐Twist1 (K150A) respectively. F) Schematic Representations of Twist1 protein domain and protein multiple sequence alignment analysis of Twist1 K150 sites. G) Western blotting analysis of TGF‐β, p‐SMAD2, SMAD2, p‐Twist1 (pSer68) and Flag‐tag. Twist1 KO endothelial cells was transfected with Flag‐Twist1 or Flag‐Twist1 (K150A) respectively. H) Under the same conditions as G), immunofluorescence staining of Flag‐tag (n = 3). Scale bar: 25 µm. I) Under the same conditions as G) Endothelial cell contractility was determined by collagen gel contraction assay (n = 3). Accurate P‐values are listed in the figures. Data is presented as mean±S.D. H,I), unpaired two‐tailed t test.

Figure 8

Overexpression of Twist1 accelerated EndoMT and necrosis in ischemic flap. PKM2^fl/fl , and PKM2^fl/fl , Tie2‐Cre mice were subjected to skin flap surgery and were sacrificed on postoperative day 7. A) Representative images of random‐pattern skin flaps on mice accompanied with Twist1 AAV or vector injection at different periods (1,4 and 7 days after operation). B) Analysis of the survival area of flaps on postoperative day 7 (n = 5). C,D) LDBF Photographing of skin flaps on postoperative day 7 (n = 5). E) Comparison of fibrotic area and number of vessels in the skin from skin flap mice accompanied with Twist1 AAV or vector injection on postoperative day 7 by Masson staining and H&E staining (n = 5). Scale bar: 500 µm, 100 µm. F) Analysis of PECAM1‐ACTA2‐positive blood vessels by immunofluorescence co‐staining of ACTA2 and PECAM1 in skin from the above groups (n = 5). Scale bar: 100 µm. Accurate P‐values are listed in the figures. Data is presented as mean±S.D. B,C and E,F), unpaired two‐tailed t test.

Figure 9

Serum lactate in patients is predictive for flap ischemia diagnosis. A) Schematic diagram indicating retrospective analysis for lactate in serum of patients with flap ischemia. B) Clinical analysis of patients including body weight, height, BMI, sex and age. C) Representative photographing of ischemic flaps with different degree. D) Representative photographing of ischemic flaps at different periods after flap transplantation (1, 4, 7 days). E) Serum lactate levels of patients with mild, middle and severe flap ischemia on postoperative day 7. F–H) Serum lactate levels were detected at 1, 3, 5, 7 days after flap transplantation. I) Comparison of fibrotic area and number of blood vessels in the skin from normal skin or severe ischemic skin flap utilizing H&E and Masson staining (n = 3). Scale bar: 500 µm, 200 µm. J) Immunohistochemistry of PKM2 and Twist1 in the skin from normal skin or severe ischemic skin flap (n = 3). Scale bar: 200 µm, 50 µm. Accurate P‐values are listed in the figures. Data is presented as mean±S.D. E and I,J), unpaired two‐tailed t test.