Int J Stem Cells.  2019 Mar;12(1):63-72. 10.15283/ijsc18097.

Perivascular Cells and NADPH Oxidase Inhibition Partially Restore Hyperglycemia-Induced Alterations in Hematopoietic Stem Cell and Myeloid-Derived Suppressor Cell Populations in the Bone Marrow

• Kim JY
• Lee JY
• Ha KS
• Han ET
• Park WS
• Min CK
• Hong SH

• KMID: 2447225
• DOI: http://doi.org/10.15283/ijsc18097

Abstract

BACKGROUND AND OBJECTIVES
Patients suffer from long-term diabetes can result in severe complications in multiple organs through induction of vascular dysfunctions. However, the effects of chronic hyperglycemic conditions on hematopoiesis and the microenvironment in the bone marrow (BM) are not yet well understood.
METHODS
BM cells were harvested from femurs of mice and analyzed using flow cytometry. Human PVCs were cultured in serum-free α-MEM. After 24hrs, PVC-CM was collected and filtered through a 0.22 μm filter.
RESULTS
In this study, we showed that hyperglycemia alters hematopoietic composition in the BM, which can partially be restored via paracrine mechanisms, including perivascular cells (PVCs) and NADPH oxidase (NOX) inhibition in mice with streptozotocin-induced diabetes. Prolonged hyperglycemic conditions resulted in an increase in the frequency and number of long-term hematopoietic stem cells as well as the number of total BM cells. The altered hematopoiesis in the BM was partially recovered by treatment with PVC-derived conditioned medium (CM). Long-term diabetes also increased the number of myeloid-derived suppressor cells in the BM, which was partially restored by the administration of PVC-CM and diphenyleneiodonium (DPI), a NOX inhibitor. We further showed the downregulation of ERK and p38 phosphorylation in BM cells of diabetic mice treated with PVC-CM and DPI. This may be associated with dysfunction of hematopoietic cells and promotion of subsequent diabetic complications.
CONCLUSIONS
Our data suggested that alterations in BM hematopoietic composition due to prolonged hyperglycemic conditions might be restored by improvement of the hematopoietic microenvironment and modulation of NOX activity.

Keyword

Hyperglycemia; Hematopoiesis; Perivascular niche; NOX; MDSCs

MeSH Terms

Animals
Bone Marrow*
Culture Media, Conditioned
Diabetes Complications
Down-Regulation
Femur
Flow Cytometry
Hematopoiesis
Hematopoietic Stem Cells*
Humans
Hyperglycemia
Mice
NADP*
NADPH Oxidase*
Phosphorylation
Culture Media, Conditioned
NADP
NADPH Oxidase

Figure

• 

  Fig. 1 Changes in body-weight and blood glucose levels in STZ-treated diabetic mice and control. (A) Protocols for the induction of hyperglycemia using STZ. (B) Blood glucose and (C) body weights measurements of STZ-treated mice for 100 days. WT (n=19), STZ-treated mouse (n=26). (D) BM total cell counts per femurs of STZ-treated and control mice. WT (n=5), STZ-treated mice (n=8).

• 

  Fig. 2 Characterization of the HSCs in the BM of STZ-treated diabetic mice and control. (A) Frequency of LT-HSC and ST-HSC in different phases of STZ-treated diabetic and control mice. WT (n=20), STZ-treated mouse (n=28). Data are mean±SD., *p<0.05. (B) The cell counts of LT- and ST-HSC of BM cells in different phases of STZ-treated diabetic and control mice. (C) CFU colony numbers of BM cells in different phases of STZ-treated diabetic and control mice. (D) Number of CFU-E,-G,-M per 6000 cells of BM cells in different phases of STZ-treated diabetic and control mice. (E) Distribution of CFU-E,-G,-M as a percentage of BM cells in different phases of STZ-treated diabetic and control mice. WT (n=6), STZ-treated mice (n=6).

• 

  Fig. 3 Effect of PVC-CM in the hematopoietic stem cell of STZ-treated diabetic mice. (A) Protocols for the PVC-CM treatment after induction of hyperglycemia using STZ. At 6weeks after PVC-CM treatment, (B) Blood glucose and body weights measurements of STZ-treated, STZ+ PVC-CM and STZ+DPI mice. WT (n=9), STZ-treated mice (n=9), STZ+PVC-CM (n=7), STZ+DPI (n=8). (C) BM total cell counts per femurs of STZ-treated, STZ+PVC-CM, STZ+DPI and control mice. (D) Frequency of LT-HSC and ST-HSC in each BM cells. (E) CFU colony numbers of BM cells in each mouse. (F) Number of CFU-E,-G,-M per 6000 cells of BM cells in each mouse. WT (n=16), STZ-treated mice (n=20), STZ+PVC-CM (n=17), STZ+DPI (n=17).

• 

  Fig. 4 PVC-CM treatment influence activated and differentiated mouse MDSCs in BM of STZ-treated diabetic mice. (A) Representative FACS plot of G-MDSCs and M-MDSCs in total BM. (B) Frequency of G-MDSC and M-MDSC in each BM cells. (C) M-MDSC/G-MDSC ratio in total BM cells of each mouse. (D) MDSC counts in total BM cells of each mouse. Data represent mean±SD, *p<0.05, **p<0.001. (E) Expression levels of ARG1 and iNOS in total BM cells. WT (n=5), STZ-treated mice (n=7), STZ+PVC-CM (n=5), STZ+DPI (n=6). Data are mean±SD., *p<0.05. (F) Western blot analysis for MAPK in total BM cells.

Reference

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