Lactate stimulates vasculogenic stem cells via the thioredoxin system and engages an autocrine activation loop involving hypoxia-inducible factor 1

Abstract

The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34(+) SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34(+) cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34(+) SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45(+) leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors.

FIG. 1.

Counts of CD34⁺ cells in Matrigel plugs. Plugs were digested with dispase, stained with fluorochrome-tagged antibodies, and suspended in PBS. The total number of cells in each sample was calculated based on the volume of the cell suspension and the rate at which fluid was taken up in the flow cytometer. *, significantly different from cell number in unsupplemented Matrigel harvested at 18 h (day 1); a, significantly different from cell numbers in unsupplemented Matrigel samples harvested at 5 and 10 days. Cell counts in Matrigel-lactide at 5 versus 10 days postimplantation were also significantly different. Numbers are means ± SE (n = 5) for all groups.

FIG. 2.

SPC surface marker expression and vascular channels identified by dextran and Nile red beads in Matrigel harvested 18 h postimplantation. Note that each row shows images from different samples. The bottom row shows images of Matrigel harvested at 18 h and incubated ex vivo for 7 days.

FIG. 3.

The relationship between Nile red bead fluorescence in Matrigel plugs and fluorescence from CD34⁺ cells is shown. Matrigel was stained with FITC-conjugated anti-CD34 antibody, and fluorescence in green and red channels was quantified as outlined in Materials and Methods. The six data points closest to the origin are for unsupplemented Matrigel, whereas the six in the upper right corner are for Matrigel with lactide. Values are relative fluorescence units.

FIG. 4.

SPC flow cytometry analysis protocol. Leukocytes in Matrigel with or without lactide were evaluated based on forward and side laser light scattering, and the circled populations were selected for further analysis. SPCs were enumerated based on the surface expression of CD34 and DNA staining using DRAQ5. CD34⁺ DRAQ5⁺ cells were then evaluated for the expression of CD31, Sca-1, CXCR4, CD133, and VEGFR2.

FIG. 5.

DCF fluorescence in CD34⁺ cell-lined channels within Matrigel. The top two rows of images show DCF fluorescence, as well as colocalization with the CD34⁺ cells in a Matrigel-lactide sample (M + L) with and without the topical addition of 24 mM KCl to cause cell depolarization. The bar graphs at the bottom show the ratios of DCF fluorescence (without versus with KCl) from different Matrigel samples (n = 3 different mice for each calculation). Where indicated, Matrigel samples were supplemented with siRNA specific to LDH (+siRNA-LDH). The fluorescence in the standard-preparation (standard prep.) Matrigel-lactide samples and Matrigel samples (M) was significantly greater than that in the siRNA samples.

FIG. 6.

Intracellular HIF-1 and β-actin in permeabilized CD34⁺ cells assessed by flow cytometry. (A) Shown are representative dot plots for cells stained for β-actin. Among samples in each group (blood, Matrigel with and without lactide, and bone marrow), there were no significant differences in the magnitude of β-actin detected, thus demonstrating comparable degrees of permeabilization in all samples. (B) Cells were probed for HIF-1, and histograms were generated. Representative examples are shown. Curves labeled a are for cells from normal wild-type mice, curves labeled b are for CD34⁺ cells from mice with conditionally HIF-1 null myeloid cells, and curves labeled c are for wild-type cells stained with nonspecific immunoglobulin G conjugated to APC.

FIG. 7.

Protein expression patterns in cells found in Matrigel. Cell lysates were subjected to Western blotting as outlined in Materials and Methods. All values were normalized to the protein concentration found in cells isolated from unsupplemented Matrigel. The actual values for each sample can be found in Table 5. Agents were added to Matrigel or Matrigel-lactide as indicated on the abscissas. Wild-type mice were used in all cases, except where the bars are indicated to represent results for mice with conditionally HIF-1 null myeloid cells. Agents included in the Matrigel samples were nonsilencing control siRNA; siRNA directed against LDH, TrxR, or HIF-1; apocynin; DTE; NAC; U0126; anti-VEGF antibody; or anti-SDF antibody. The contents of proteins (Trx1, TrxR, HIF-1, VEGF, and SDF) in cells were expressed as the ratios of the band densities on Western blots to the β-actin band densities on the same blots. In all cases, except those involving Matrigel containing nonsilencing control siRNA, values for cells isolated from Matrigel with inhibitors were lower than the values for cells isolated from Matrigel without inhibitors. In all cases, the protein contents of cells isolated from Matrigel-lactide were significantly greater than those of cells isolated from Matrigel that did not contain lactide. Phosphorylated enzymes were evaluated as ratios compared to the total amount of the enzymes in cells. The fractions in cells from Matrigel-lactide were significantly greater than those in cells from unsupplemented Matrigel. The fractions of phosphorylated enzymes in cells from samples with inhibitors were significantly reduced compared to those in the control cells, except for the fractions in cells containing nonsilencing control siRNA and the phosphorylated p38 content in cells from samples containing U0126. Numbers in parentheses are the numbers of samples in the indicated groups. pERK, pJNK, and pP38, phosphorylated ERK, JNK, and p38.

FIG. 8.

Representative Western blot demonstrating the presence of HIF-1, TrxR, and Trx1, as well as β-actin, in cell lysates from Matrigel implanted into wild-type (WT) mice, Matrigel containing either nonsilencing control siRNA or siRNA specific to HIF-1 implanted into wild-type mice, and Matrigel implanted into mice with conditionally HIF-1 null myeloid cells. M, unsupplemented Matrigel samples; M+L, Matrigel-lactide samples.

FIG. 9.

Representative Western blot demonstrating the presence of VEGF and SDF-1, as well as β-actin, in cell lysates from Matrigel implanted into wild-type (WT) mice, Matrigel containing either nonsilencing control siRNA or siRNA specific to HIF-1 implanted into wild-type mice, and Matrigel implanted into mice with conditionally HIF-1 null myeloid cells. M, unsupplemented Matrigel samples; M+L, Matrigel-lactide samples.

FIG. 10.

Diagram showing the hypothesized sequence of effects triggered by lactate that stimulate SPC recruitment to Matrigel based on experimental findings. Items in parentheses are inhibitors used in this investigation to assess the roles of various agents in the pathway. Trx-S₂, oxidized thioredoxin; Trx-SH₂, reduced thioredoxin; Ig's, immunoglobulin antibodies to VEGF or SDF-1.