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. 2014 Jul 14;20(10):935–944. doi: 10.1111/cns.12307

HUCBCs Increase Angiopoietin 1 and Induce Neurorestorative Effects after Stroke in T1DM Rats

Tao Yan 1,2, Poornima Venkat 1,3, Xinchun Ye 1,4, Michael Chopp 1,3, Alex Zacharek 1, Ruizhuo Ning 1, Yisheng Cui 1, Cynthia Roberts 1, Nicole Kuzmin‐Nichols 5, Cyndy Davis Sanberg 5, Jieli Chen 1,2,✉
PMCID: PMC4180763  NIHMSID: NIHMS622528  PMID: 25042092

Summary

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

Introduction

Diabetic Mellitus (DM) is a chronic health concern that elevates the risk of ischemic stroke 1. DM patients rapidly develop vascular disorders and suffer significantly worse outcomes poststroke with poor long term recovery hindered by recurrent strokes 2, 3. Our earlier studies with bone‐marrow‐stromal cells indicated that treatment initiated at 24‐h poststroke improved functional recovery in non‐DM rats, but not in Type 1 DM (T1DM) rats 4. This indicates that effective therapy for stroke in non‐DM patients may not necessarily transfer to DM stroke patients. Therefore, investigating and developing new therapeutic approaches specifically targeting the diabetic stroke population is of prime research interest.

Human umbilical cord blood cells (HUCBCs) are an effective treatment for hematological disorders, and researchers are investigating HUCBCs as a treatment for nonhematological disorders like diabetes 5, 6, 7. HUCBCs have the potential to reverse impending cell death by inhibiting the spread of apoptosis, regulating inflammatory and immune responses, and improving behavioral recovery in non‐DM MCAo rats 8, 9. Apart from cell replacement, HUCBCs produce neurotrophic and antiinflammatory factors to promote functional recovery poststroke 10. However, the neurorestorative effect of HUCBCs in stroke with DM has not been investigated.

Diabetic stroke animals exhibited significantly suppression of Ang‐1 expression and elevation of inflammatory factors such as receptor for advanced glycation end‐products (RAGE) in the ischemic brain correlates with functional deficit after stroke 11. Ang‐1 is a protein with potent roles in vascular remodeling, protection and angiogenesis 12. Ang‐1 is a primary physiological ligand for TIE2 and plays a vital role in the migration, adhesion and survival of endothelial cells and in vessel maturation 13. Ang1 regulates in the organization and maturation of new blood vessels. It also boasts the capability to suppress vascular inflammation, leakage and endothelial death 12. Ang1 plays an important role in promoting vascular maturation and stabilization as well as increasing angiogenesis after stroke 14. Decreased Ang1 is related with increased blood brain‐barrier (BBB) leakage and brain hemorrhagic transformation after stroke in DM mice 15. Ang1 also inhibits pro‐inflammatory mediators (Tumor necrosis factor alpha, IL‐6 and IL‐8) 16 that exacerbate vascular and white matter (WM) damage after stroke in diabetic populations 4, 17.

Receptor for advanced glycation end‐products is a primary receptor for high‐mobility group protein B1 (HMGB1), and can initiate and sustain a proinflammatory phenotype upon activation by HMGB1 18. RAGE has been implicated in the pathogenesis of diabetic complications, inflammatory disorders and neurodegenerative diseases 19, 20. In our previous studies, we have found that T1DM‐stroke rats exhibit significantly increased HMGB1 and RAGE expression in the ischemic brain 21. The RAGE expression is closely related with inflammation in the ischemic brain and correlated with neurological deficit after stroke in DM animals 21.

In this study, we investigated the therapeutic effect of HUCBC treatment in T1DM stroke rats and the underlying mechanisms of HUCBC treatment induced neurorestorative effects in T1DM rats after stroke. Ang1 and inflammatory factor RAGE expression will be measured.

Materials and Methods

All experiments were conducted in accordance with the standards and procedures of the American Council on Animal Care and Institutional Animal Care and Use Committee of Henry Ford Health System.

Diabetes Induction

A single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg; Sigma Chemical Co., St. Louis, MO, USA) was given to adult male Wistar rats (225–250 g; Charles River, Wilmington, MA, USA) for diabetes induction. Two weeks later, the fasting blood glucose level was tested using a glucose analyzer (Accu‐Chek Compact System; Roche Diagnostics, Indianapolis, IN, USA) and animals with fasting blood glucose >300 mg/dL underwent 2 h transient MCAo 21.

MCAo Model and Experiment Groups

Transient (2 h) MCAo was performed on anesthetized T1DM rats via intraluminal vascular occlusion as previously described 22, 23. Rats were then randomly assigned to different groups and treated with: (1) PBS (Phosphate‐Buffered‐Saline) as vehicle control (n = 9); (2) HUCBCs (5 × 106, n = 6) (Saneron CCEL Therapeutics) via tail‐vein injection starting at 24 h post‐MCAo. Sham surgery was performed in the same way as the MCAo model without inserting the nylon suture. Sham controls were also treated with either PBS (n = 4) or HUCBCs (5 × 106, n = 4) at 24 h postsham surgery via tail‐vein injection. Rats were sacrificed at 14 days after MCAo for immunostaining quantification analysis.

Neurological Functional Tests

An investigator was blinded to the experimental groups to perform a battery of functional tests including Foot fault, and a modified neurological severity score (mNSS) evaluation before MCAo, at 1 day after MCAo before treatment, and at 7, 14 days after MCAo 22, 24. mNSS is a composite of motor, sensory, balance and reflex tests. Neurological function was assessed and each animal received a score between 0 and 18 (normal score 0; maximal deficit score 18). The absence of a tested reflex or abnormal response received one point. Hence, higher the score, greater is the impairment of normal function. Rats with score <6 or over 13 at 24 h after MCAo (prior to treatment) were not included.

In the foot fault test 25, animals were placed on an elevated grid floor (45 cm × 30 cm), 2.5 cm higher than a solid base floor, with 2.5 cm × 2.5 cm diameter grid spacings. While moving on the wire frame using their paws, an inaccurate forelimb placement leads to a fall/slip through one of the grid openings and is recorded as a foot fault. A total of 100 forelimb movements were counted, and the total number of foot faults for the left forelimb was recorded. The percentage of foot faults of the left paw of total steps was calculated.

Histological and Immunohistochemical Assessment

Transcardial perfusion with saline was used to fix brains, followed by perfusion and immersion in 4% paraformaldehyde. The brains were then embedded in paraffin and a standard block was obtained from the center of the lesion (bregma −1 mm ~ +1 mm). A series of 6‐μm‐thick sections was cut from the block. Hematoxylin and eosin (H&E) stained seven coronal sections of tissue were used for lesion volume calculation and presented as a percentage of lesion compared with the contralateral hemisphere.

Brain coronal tissue sections were prepared and antibody against α‐smooth muscle actin (α‐SMA, mouse monoclonal IgG, 1:800; Dako, Carpenteria, CA, USA); Von Willebrand Factor (vWF, 1:400; Dako); NG2 (oligodendrocyte progenitor cell [OPC] marker, 1:100; Chemicon, CA, USA); 2′,3′‐Cyclic‐nucleotide 3′‐phosphodiesterase (CNPase, an oligodendrocyte marker, 1:200; Millipore, Billerica, MA, USA), SMI‐31 (Neurofilaments, phosphorylated monoclonal antibody, 1:1000, Covance, CA), RAGE (1:400; Dako), Angiopoietin‐1 (Ang1; 1:2000; Abcam, Cambridge, MA, USA) were employed. Bielschowsky‐silver (BS) immunostaining was used to demonstrate axons; luxol fast blue (LFB) staining was used to demonstrate myelin and antibody against albumin (albumin‐FITC, polyclonal, 1:500; Abcam) and was used to demonstrate BBB leakage. Control experiments consisted of staining brain coronal tissue sections as outlined above, but nonimmune serum was substituted for the primary antibody.

Quantification Analysis

An investigator blind to the experimental groups performed all immunostaining quantification analysis. Five slides from each brain, with each slide containing eight fields from striatum of the ischemic border zone (IBZ) were digitized under a 20× objective (Olympus BX40; Olympus America, Center Valley, PA, USA) using a 3‐CCD color video camera (Sony DXC‐970MD; Tokyo, Japan) interfaced with an MCID image analysis system (Imaging Research, St. Catharines, ON, Canada). For BS and LFB measurements, positive areas of immunoreactive cells were measured in the WM bundles of the stratum in the IBZ. For other immunostaining (NG2, CNPase, SMI‐31, Ang1, RAGE and albumin), positive areas of immunoreactive cells were measured in the IBZ (Figure 2F).

Figure 2.

Figure 2

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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.

Vascular Density Measurement

To measure the vascular density in the IBZ, eight fields of view of vWF immunostaining from the IBZ were digitized using a 20× objective via the MCID software 26.

αSMA‐Positive Coated Arterial Diameter and Wall Thickness

The α‐SMA stained vessels were analyzed with regard to small and large vessels (≥10 μm diameter). The arterial density in the IBZ was measured 27. In addition, the 10 largest arterial wall thicknesses and internal arterial diameters were measured.

Western Blot Assay

From an additional set of rats sacrificed at 14 days after MCAo (n = 4/group), ischemic brain tissues were extracted from the IBZ 28. Protein was isolated from samples using Trizol (Invitrogen, Carlsbad, CA, USA) following standard protocol. Protein concentration was measured using the BCA (Thermo Scientific, Waltham, MA, USA) kit. Forty micrograms of protein/lane in a 10% SDS PAGE precast gel (Invitrogen). Gel was transferred to a nitrocellulose membrane (Bio Rad, Hercules, CA, USA) by running the transfer at 400 mA for 2 h. Nitrocellulose membrane was blocked in 2% I‐Block (Applied Biosystems, Foster City, CA, USA) in 1× TBS‐T for 1 h, and then either anti‐β‐actin (1:10,000; Abcam), anti‐Ang1 (1:1000; Abcam). RAGE (1:500; R&D Systems, Minneapolis, MN, USA) primary antibodies were added in 2% I‐Block in TBS‐T, and incubated on a shaker overnight at 4°C. Following morning, the membranes were washed three times for 5 min with 1× TBS‐T. Secondary antibodies (anti‐mouse, anti‐rabbit, or anti‐rat, Jackson ImmunoResearch, West Grove, PA, USA) were added at 1:1000 dilution in 2% I‐Block in 1× TBS‐T on a room temperature shaker for 1 h. After the incubation, the membranes were washed three times for 5 min with 1× TBS‐T. After the final wash, Luminol Reagent (Santa Cruz, Dallas, TX, USA) was added and allowed to react with the membranes for 2 min. The membranes were then developed using a FluorChem E Imager system (ProteinSimple, Santa Clara, CA, USA) exposing them for 1–30 min, depending on the intensity of the band.

Real Time PCR Assay

Total RNA was isolated with TRIzol (Invitrogen), following standard protocol. Two micrograms of total RNA was used to make cDNA using the M‐MLV (Invitrogen) standard protocol. Of this cDNA, 2 μL was used to run a quantitative PCR using the SYBR Green real time PCR method. Quantitative PCR was performed on a ViiA 7 PCR instrument (Applied Biosystems) using three‐stage program parameters provided by the manufacturer, as follows; 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 seconds at 95°C and 1 min at 60°C. Each sample was tested in triplicate, and analysis of relative gene expression data using the 2−∆∆CT method.

Brain Endothelial Cell Culture

To test the relationship of Ang1 with RAGE, brain endothelial cell (BEC) (cell line from ATCC) culture was performed in vitro. BECs were subjected to 2 h oxygen‐glucose deprivation (OGD). Culture media was removed and replaced with serum and glucose free media. Cells were placed in a hypoxia chamber (Forma Anaerobic System; Thermo Scientific) with 37°C incubator for 2 h. After 2 h, the cells were removed and then cultured in high glucose (HG, 37.5 mM) DMEM media with 10% FBS (Life Technologies, Grand Island, NY, USA) and treated with: (1) nontreatment control; (2) 200 ng Ang1 (Millipore) for 24 h. RAGE expression was measured by Western blot.

Brain Endothelial Cell Capillary Tube Formation Assay

Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) was diluted to 75% with SF‐DMEM and 100 μL was added per well in a 96‐well plate before being incubated at 37°C for 30 min. Meanwhile, BEC were harvested and counted to give 22,500 cells/well and were suspended in SF‐DMEM and cultured in high glucose (HG, 37.5 mM) condition with: (1) no‐treatment control; (2) +HUCBC (22,500 cells/well); (3) +Ang1 (200 ng/mL, Millipore); and (4) +HUCBC+Ang1 inhibitor antibody (0.625 μg/mL, Millipore). A total of 100 μL of cell suspension with treatment was added to each well (n = 4) and allowed to incubate for 3 h. After 3 h, the Matrigel wells were digitized under a 10× objective (Olympus BX40) for measurement of total tube length of capillary tube formation. Tracks of endothelial cells organized into networks of cellular cords (tubes) were counted and averaged in four randomly selected microscopic fields.

Inflammatory Factor Gene Expression Assay

To test whether HUCBC and Ang1 regulates inflammatory factor expression, BECs were cultured in the HG (37.5 mM) condition and treated with: (1) no‐treatment control; (2) +HUCBC; (3) +Ang1 (200 ng/mL); and (4) +HUCBC+Ang1 inhibitor antibody (0.625 μg/mL, Millipore) for 24 h. RAGE and TLR2 gene expression was measured by real time PCR.

Statistical Analysis

One‐way Analysis of Variance (ANOVA) was used for the evaluation of functional outcome and histology, respectively. “Contract/estimate” statement was used to test the group difference. If an overall treatment group effect was detected at P < 0.05, pair‐wise comparisons were made. All data are presented as mean ± standard error (SE).

Results

HUCBC Treatment Significantly Improved Functional Outcome After Stroke Without Reduction of Lesion Volume or Blood Glucose Level

To assess the effect of HUCBC treatment on long term functional outcome after stroke in T1DM rats, a set of behavioral tests was performed. Figure 1 presents foot fault (Figure 1A) and mNSS evaluation (Figure 1B) data for 14 days after stroke that indicate significantly improved functional outcome in T1DM MCAo rats treated with HUCBCs compared to control (P < 0.05). However, HUCBC treatment did not decrease lesion volume (T1DM MCAo control: 28.9 ± 3.4%, T1DM MCAo with HUCBC treatment: 31.2 ± 2.8%, sham surgery control: 0%) and blood glucose (T1DM MCAo control: d1: 440.7 ± 50.3 mg/dL, d14: 500.1 ± 40.7 mg/dL; T1DM MCAo with HUCBC treatment: d1: 540.2 ± 50.6 mg/dL, d14: 440.8 ± 60.1 mg/dL). No significant change of blood glucose level was observed in sham control rats (PBS treated group: 518.7 ± 49.3 mg/dL day 1 to 579.5 ± 21.2 mg/dL on day 14; HUCBC treated group: 587.2 ± 13.9 mg/dL day 1 to 585.1 ± 15.7 mg/dL on day 14). It can be inferred that HUCBC treatment induces neurorestorative effects which are not related to blood glucose level.

Figure 1.

Figure 1

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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.

HUCBC Treatment Significantly Increased WM Remodeling

To test whether HUCBC treatment regulates WM remodeling, BS (Axon marker), LFB (myelin marker) and SMI‐31 (pan‐axonal neurofilament marker) staining were performed. Figure 2 shows that in T1DM‐MCAo rats, compared to controls, HUCBC treatment significantly increased myelin (Figure 2A), and axonal (Figure 2B,C) density in the IBZ (P < 0.05). To test whether stroke treatment with HUCBC can regulate oligodendrocyte density in the ischemic brain, immunostaining with NG2 (OPC marker) and CNPase (OL marker) were performed. Treatment with HUCBCs significantly increased OPC (NG2, Figure 2D) and OL (CNPase, Figure 2E) in the IBZ of striatum compared to control T1DM‐MCAo rats.

HUCBC Treatment Promoted Vascular Remodeling in the Ischemic Brain

To evaluate the effects of HUCBC treatment on vascular remodeling, FITC‐albumin levels and vascular and arterial density were measured in the IBZ. In T1DM‐MCAo rats, treatment with HUCBCs significantly decreased BBB leakage demonstrated by FITC‐albumin staining in comparison with control rats (Figure 3A). HUCBC treatment also increased vascular density (vWF, Figure 3B) and the density and diameter of arteries (a‐SMA, Figure 3C) in the IBZ compared to T1DM‐MCAo controls (P < 0.05). Hence, HUCBC treatment not only promotes WM remodeling, but also increases vascular remodeling and decreases BBB leakage in the ischemic brain.

Figure 3.

Figure 3

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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.

HUCBC Treatment Significantly Increased Ang‐1 and Decreased RAGE Expression in the IBZ in T1DM Rats

To understand the underlying mechanisms of HUCBC‐induced functional improvement in T1DM‐MCAo rats, the angiogenic factor Ang‐1 and inflammatory factor RAGE expressions were quantified in the IBZ. Figure 4 shows that treatment with HUCBCs significantly increased Ang‐1 (Figure 4A) and decreased RAGE (Figure 4B, P < 0.05) expression compared to T1DM‐MCAo control. Consistent with the immunostaining results, the Western blot data and real time PCR (Figure 4C,D) also suggest increased Ang1 and decreased RAGE expression in the IBZ upon HUCBC treatment compared to control T1DM‐MCAo rats.

Figure 4.

Figure 4

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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.

Ang1 Treatment Decreased RAGE Expression in Cultured BECs

Earlier studies have reported that high glucose significantly decreased Ang1 and increased RAGE expression 15, 21. Presently, we have found that HUCBC treatment increased Ang1, but significantly decreased RAGE expression in the ischemic brain. To test the relationship of Ang‐1 and RAGE, BECs were treated with or without Ang1 under OGD and high glucose conditions. Figure 5 shows that Ang1 treatment significantly decreased RAGE expression in cultured BECs. These data indicate that increasing Ang1 induced by HUCBC treatment down‐regulates RAGE expression.

Figure 5.

Figure 5

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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.

HUCBCs Increase Angiopoietin 1 and Induce Neurorestorative Effects After Stroke in T1DM Rats

To test whether increased Ang1 expression mediates HUCBC‐induced vascular remodeling and antiinflammatory effects, an in vitro brain endothelial cell capillary tube formation and real time PCR for inflammatory factor expression were performed. Figure 6 shows that HUCBC and Ang1 treatment significantly increases capillary tube formation (6A,B) and decreases inflammatory factor RAGE and TLR2 expression (6C,D, P < 0.05), while inhibition of Ang1 with the anti‐Ang1 antibody partially attenuates HUCBC‐induced capillary tube formation and antiinflammatory effects (vs. HUCBC treatment, P < 0.05). Therefore, increasing Ang1 may contribute to HUCBC treatment induced vascular remodeling and antiinflammatory effects.

Figure 6.

Figure 6

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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.

Discussion

In this study, we have demonstrated for the first time, to our knowledge, that HUCBC treatment of T1DM stroke improves functional outcome without reducing blood glucose or lesion volume with treatment initiated at 24 h after MCAo. HUCBC treatment also increases Ang1, decreases RAGE as well as promotes WM and vascular remodeling in the ischemic brain.

Human umbilical cord blood cells are an abundant source of immature progenitor cells with the potential to differentiate into cells of multiple cell lineages 29. Our earlier study has demonstrated that HUCBCs administered poststroke in non‐DM‐MCAo rats have the potential to migrate into the ischemic brain and promote neurological functional recovery 24. However, only a few HUCBCs can migrate into the ischemic brain and differentiate into neural cells 9, 24. Therefore, it is unlikely that the HUCBC‐induced neurorestorative effects are due to their differentiation into neural cells and replacement of the damaged brain tissue. In this study, HUCBC treatment enhanced poststroke functional outcome in diabetic animals without altering the lesion volume by enhancing vascular and WM remodeling.

Vascular remodeling via angiogenesis in the IBZ can induce neurorestorative effects poststroke 30 by improving blood circulation, and oxygen and nutrition supply to ischemic tissue 31. Diabetic MCAo animals are more prone to vascular injury, arteriosclerosis and have decreased tight junction protein expression, increased BBB leakage and poor functional outcomes compared to wild‐type MCAo animals 4, 15, 17. Our data shows that HUCBC treatment significantly increased vascular and arterial density with larger diameters as well as promoted vascular integrity and decreased BBB leakage in T1DM‐MCAo rats. Therefore, increasing vascular remodeling may contribute to HUCBC‐induced neurorestorative effects in T1DM rats.

Under stroke conditions, oligovascular coupling is interrupted, and this contributes to WM damage 32. In comparison to gray matter, WM with its limited blood flow is more susceptible to stroke. DM stroke mice exhibit significantly increased WM damage and have worse functional outcome compared to the non‐DM stroke mice 17. Oligodendrocytes and OPCs play critical roles in WM homeostasis. In the central nervous system, WM is primarily constituted by axonal bundles and their myelin sheath. OPC's differentiate into oligodendrocytes and form this myelin sheaths 32. Oligodendrocytes also are highly susceptible to focal cerebral ischemia yet, poststroke increase in oligodendrocyte numbers can occur through axon sprouting or regeneration in the peri‐infarct areas of ischemic brain and stimulate brain repair 33. Oligodendrocytes promote WM and facilitate neurological functional recovery 34. We found that HUCBC treatment of stroke in T1DM rats significantly increased WM remodeling identified by increased oligodendrocyte and OPC numbers as well as upregulation of axon and myelin density in the WM of the ischemic brain. HUCBC treatment significantly increased WM remodeling which may play an important role in mediating neurorestorative effects after stroke.

The current data indicated that HUCBC treatment of stroke in T1DM rats significantly promotes vascular and WM remodeling. The underlying mechanisms of HUCBC‐induced neurorestorative effects are not fully understood. Angiopoietins stimulate new blood vessel formation from preexisting vessels primarily in the IBZ, playing an important role in angiogenesis and vascular development in the injured areas 35. Transgenic over‐expression of Ang1 reduces BBB leakage and increases vascular stabilization in the ischemic brain 36, 37, 38. The induction of Ang‐1 in the IBZ increases the supply of oxygen and nutrition to damaged tissue and also enhances neurogenesis and synaptogenesis 39. T1DM and T2DM stroke animals exhibit significantly decreased brain Ang‐1 expression and have increased vascular damaged in the ischemic brain 15. HUCBC treatment significantly increased Ang1 gene and protein expression in the ischemic brain which may be associated enhanced vascular remodeling and vascular integrity and stabilization in T1DM‐MCAo rats. In addition to regulating vascular remodeling, Ang1 up‐regulation also appears to play an important role in WM remodeling, and decreases overall inflammation 30, 40. Ang‐1 treatment increases neurite outgrowth in cultured primary cortical neuron 30, 40. In this study, we found that HUCBC treatment in T1DM rats significantly increases Ang1 gene and protein level in the ischemic brain. HUCBC and Ang1 treatment both significantly increase capillary tube formation, while inhibition of Ang1 attenuates HUCBC‐induced capillary tube formation and antiinflammatory factor expression. These data indicate that increasing Ang1 expression may contribute to HUCBC‐induced neurorestorative effects in T1DM.

In addition to Ang‐1, our results here indicate that T1DM‐MCAo rats treated with HUCBCs have significantly decreased RAGE expression in the ischemic brain. RAGE is a multiligand receptor of the immunoglobulin superfamily that functions as a cell surface binding site for Aβ and cytokine‐like mediators exerting proinflammatory activity. Enhanced expression of RAGE is critical for neurodegenerative pathology and immune and inflammatory responses 41. The RAGE signaling pathway has been implicated in promoting inflammation, neuronal death, vascular injury and brain damage following ischemia 42, 43. RAGE expression is significantly increased in diabetic stroke animals 21, and is dramatically increased at sites of WM damage in regions of demyelination 44. We found that HUCBC treatment significantly decreased RAGE expression in the IBZ of T1DM‐MCAo rats. Ang‐1 treatment is significantly correlated with decreased RAGE expression in cultured brain endothelial cells. HUCBC treatment induces antiinflammatory effects, such as, decreasing RAGE and TLR2 expression in cultured BECs, while inhibition of Ang1 attenuates HUCBC‐induced decreased RAGE expression. Therefore, decreased RAGE expression in the ischemic brain by HUCBC treatment may be regulated by increasing Ang1 level in the ischemic brain.

The interaction between these various factors; BBB leakage, inflammation, WM and vascular remodeling are likely important in stroke pathogenesis and stroke recovery. BBB integrity is central to the maintenance of brain homeostasis and its disruption is among the initial steps that precede many neurological disorders 45. A permeable BBB allows the infiltration of inflammatory factors from the circulation into the brain and exacerbates brain damage. In addition, an increase in proinflammatory factors can promote BBB disruption and even lead to hemorrhagic transformations in diabetic stroke animals 4, 46, 47. Systemic inflammation has been shown to worsen BBB disruption and exacerbate functional deficits after stroke in mice 48, 49. Activation of immune system has been implicated in neurovascular dysfunction which manifests itself in the form of BBB disruption 50. Progressive axonal and myelin damage are associated with a significant increase in neuroinflammation characterized by elevated expression levels of activated microglia and macrophages 51, 52. Hence, there are links among the inflammatory factors, BBB leakage and vascular and WM changes 53, 54. Thus, it may be important for stroke therapies to collectively target all aspects of neurorestoration. As a caveat to this study, long‐term survival experiments, beyond the present 14‐day survival, are warranted.

Conclusions

Human umbilical cord blood cells treatment increases Ang‐1 and concomitantly decreases inflammatory factor RAGE expression in the ischemic brain in T1DM rats, which may in concert at least partially contribute toward improving functional recovery by promoting WM and vascular remodeling in the ischemic brain.

Conflict of Interest

JC is a consultant to Saneron CCEL Therapeutics, Inc. In addition, CDS & NKN are inventor on cord blood patents/applications. CDS is Sr. VP of R&D, and NKN is President & COO at Saneron CCEL Therapeutics, Inc.

Acknowledgments

This work was supported by National Institute on Aging RO1 AG031811 (JC), National institute of neurological disorder and stroke (NINDS) R01NS083078‐01A1 (JC), National Institute on Aging RO1 AG 037506 (MC) and R41NS080329‐01A1. The authors wish to thank Qinge Lu, Sutapa Santra, Yihan Hong, Xinchu Tian and David Wu for the technical assistance.

The first two authors contributed equally to this work.

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