HUCBCs increase angiopoietin 1 and induce neurorestorative effects after stroke in T1DM rats

Abstract

Background and purpose: We investigated the neurorestorative effects and underlying mechanisms of stroke treatment with human umbilical cord blood cells (HUCBCs) in Type one diabetes mellitus (T1DM) rats.

Methods: Type one diabetes mellitus rats were subjected to middle cerebral artery occlusion (MCAo) and 24 h later were treated with: (1) phosphate-buffered-saline; (2) HUCBCs. Brain endothelial cells (MBECs) were cultured and capillary tube formation was measured.

Results: Human umbilical cord blood cells treatment significantly improved functional outcome and promoted white matter (WM) remodeling, as identified by Bielschowsky silver, Luxol fast blue and SMI-31 expression, increased oligodendrocyte progenitor cell and oligodendrocyte density after stroke in T1DM rats. HUCBC also promoted vascular remodeling, evident from enhanced vascular and arterial density and increased artery diameter, and decreased blood-brain barrier leakage. HUCBC treatment also increased Angiopoietin-1 and decreased receptor for advanced glycation end-products (RAGE) expression compared to T1DM-MCAo control. In vitro analysis of MBECs demonstrated that Ang1 inversely regulated RAGE expression. HUCBC and Ang1 significantly increased capillary tube formation and decreased inflammatory factor expression, while anti-Ang1 attenuated HUCBC-induced tube formation and antiinflammatory effects.

Conclusion: Human umbilical cord blood cells is an effective neurorestorative therapy in T1DM-MCAo rats and the enhanced vascular and WM remodeling and associated functional recovery after stroke may be attributed to increasing Angiopoietin-1 and decreasing RAGE.

Keywords: HUCBC; Neurorestorative therapy; Stroke; T1DM; Vascular remodeling; White matter remodeling.

Figure 1

Human umbilical cord blood cells (HUCBC) treatment (5 × 10⁶ cells) 24‐h poststroke in Type 1 DM (T1DM)‐MCAo rats, significantly improved functional outcome. (A) Foot fault test (B) modified neurological severity score (mNSS) test evaluated before MCAo, 1 day after MCAo before treatment, and on days 7, 14 after MCAo.

Figure 2

Human umbilical cord blood cells (HUCBC) treatment (5 × 10⁶ cells) 24‐h poststroke in T1DM‐MCAo rats, significantly increased white matter remodeling, axon density and oligodendrocytes number in the ischemic brain 14 days later. (A) Immunostaining with Luxol fast blue (B) Bielschowsky silver (C) SMI‐31 (D) Immunostaining with NG2, an oligodendrocyte progenitor cell marker (E) CNPase, an oligodendrocytes marker and quantification data in the IBZ (F) shows the immunostaining measurement area in the ischemic border zone (IBZ). Scale bars in A–E = 0.1 mm.

Figure 3

Human umbilical cord blood cells (HUCBC) treatment (5 × 10⁶ cells), 24‐h poststroke in Type 1 DM (T1DM)‐MCAo rats promoted vascular remodeling and vascular integrate in the ischemic brain 14 days later. (A) FITC‐Albumin staining (B) vWF staining (C) α‐SMA staining and quantification data for vascular density, artery density and artery wall thickness in the ischemic brain. Scale bars in A–C = 0.1 mm.

Figure 4

Human umbilical cord blood cells (HUCBC) treatment (5 × 10⁶ cells), 24‐h poststroke in T1DM‐MCAo rats, significantly increased Ang‐1 and decreased receptor for advanced glycation end‐products (RAGE) expression in the ischemic brain 14 days later. (A) Ang‐1 immunostaining and quantification data in the ischemic border zone (IBZ) (B) RAGE immunostaining and quantification data in the IBZ (C) HUCBC treatment in T1DM rats increases Ang1 but decreases RAGE expression in the ischemic brain after stroke as shown by Western Blot assay (D) HUCBC treatment in T1DM rats increases Ang1 but decreases RAGE gene expression in the ischemic brain after stroke as shown by real time PCR assay. Scale bars in (A–B) = 0.1 mm.

Figure 5

Ang‐1 treatment significantly decreases receptor for advanced glycation end‐products (RAGE) expression in cultured brain endothelial cells. (A) RAGE Western blot assay (B) Western blot quantitative data.

Figure 6

Human umbilical cord blood cells (HUCBC) and Ang1 increases capillary tube formation and decreases inflammatory factor expression, inhibition of Ang1 attenuates HUCBC‐induced tube formation and antiinflammatory effects. (A) Brain endothelial cell (BECs) capillary tube formation assay with the treatment of control, HUCBC, Ang1 and anti‐Ang‐1 antibody (B) BECs capillary tube formation quantitative data (C) Real time PCR for receptor for advanced glycation end‐products (RAGE) (D) Real time PCR for TLR2 measurement.