Abstract
The placenta has emerged as an attractive source of mesenchymal stem cells (MSCs) because of the absence of ethical issues, non-invasive access, and abundant yield. However, inflammatory cell invasion into grafts negatively impacts the survival and efficacy of transplanted cells. Previous studies have shown that synthetic C16 peptide can competitively block the transmigration of leukocytes into the central nerve system, while angiopoietin-1 (Ang-1) can inhibit inflammation-induced blood vessel leakage and inflammatory cell infiltration in rats with experimental allergic encephalomyelitis (EAE). In this study, we investigated the effects of intravenous administration of C16 and Ang-1 on the efficacy of placenta-derived MSC (PMSC) transplantation in a rat model of EAE. We found that, compared with PMSCs alone, treatment with PMSCs along with intravenously administered C16 and Ang-1 was more effective at ameliorating demyelination/neuronal loss and neurological dysfunction, reducing inflammatory cell infiltration, perivascular edema, and reactive astrogliosis (pā<ā0.05). Mechanistic studies revealed that intravenous C16 and Ang-1 increased PMSC engraftment in the central nervous system and promoted expression of the neurotropic proteins brain-derived neurotrophic factor, growth-associated protein 43, and p75 neurotrophin receptor as well as the neuronal-glial lineage markers neurofilament protein 200 and myelin basic protein in the engrafted PMSCs.
Similar content being viewed by others
Introduction
The autoimmune disease multiple sclerosis (MS) affects the central nervous system (CNS) and has great socio-economic impact in developed countries1. In MS, the immune system attacks the protective sheath (myelin) of nerve fibers, eventually leading to permanent nerve damage and neurological disability2. Mesenchymal stem cells (MSCs) have demonstrated immunoregulatory and neuroprotective functions in animal models of MS, and thus, are considered a new potential therapeutic modality for this disease3,4,5. Apart from their high proliferation and differentiation potential, embryonic MSCs (EMSCs) have been shown to exhibit superior immunoregulatory properties, and therefore, outperform bone marrow MSCs in the treatment of experimental allergic encephalomyelitis (EAE), a common model of MS6,7,8. However, the application of EMSCs is limited by ethical concerns. In recent years, placenta-derived MSCs (PMSCs) have emerged as an attractive alternate source of MSCs for their lack of ethical issues, non-invasive access, and abundant yield9. In a recent study, transplanted PMSCs were shown to reduce disease severity and improve survival in a mouse EAE model, presumably through the release of the anti-inflammatory protein tumor necrosis factor alpha (TNF-Ī±)-stimulated gene/protein 6 (TSG-6)10.
Increased bloodābrain barrier (BBB) permeability and infiltration of inflammatory cells into the CNS lead to demyelination and neuronal dysfunction in EAE11. In MSC treatment of EAE, inflammatory factors such as nuclear factor kappa-light chain-enhancer of activated B cells (NF-ĪŗB), tumor necrosis factor alpha (TNF-Ī±), and cyclooxygenase 2 (COX-2) in the inflamed CNS microenvironment can negatively impact the survival of grafted cells12. Thus, blocking inflammatory cell infiltration should protect not only the neurons within the CNS of transplant recipients but also the transplanted MSCs themselves. Angiopoietin-1 (Ang-1), an endothelial growth factor, is well documented to promote and maintain vascular maturation, homeostasis, and integrity13. It has been shown to inhibit inflammation-induced blood vessel leakage and inflammatory cell infiltration in a rat model of EAE14. C16 is a synthetic peptide that selectively binds the Ī±vĪ²3 and Ī±vĪ²1 integrins expressed on endothelial cells, and this binding has been shown to inhibit inflammatory cell transmigration by blocking leukocyteāendothelial interaction15. Furthermore, C16 and Ang-1 have been reported to work synergistically to mitigate vascular leakage and inflammation and protect against demyelination and axonal loss in rats with EAE14.
In the present study, we examined the effects of intravenous C16 and Ang-1 on the efficacy of PMSC transplantation for treating EAE in a rat model. The neurological functions, CNS infiltration of inflammatory cells, perivascular edema, white matter demyelination, axonal loss, neuronal apoptosis, and reactive astrogliosis were evaluated. The homing of transplanted PMSCs to the CNS as well as the expression of the neurotrophic proteins brain-derived neurotrophic factor (BDNF), growth-associated protein 43 (GAP-43), p75 neurotrophin receptor (p75NTR) and the neuronal-glial lineage markers neurofilament protein 200 (NF-200) and myelin basic protein (MBP) in the engrafted PMSCs were examined.
Results
Intravenous C16 and Ang-1 enhanced the efficacy of PMSC therapy for preventing neurological dysfunctions in rats with EAE
Neurological dysfunctions in rats with EAE started 1 week post immunization (pi) (clinical scoresā>ā2) and quickly progressed to the peak level (clinical scores ~3.7) by 2 weeks pi (Fig.Ā 1A). After that, the rats underwent spontaneous recovery and the clinical scores returned to 2 by 8 weeks pi (Fig.Ā 1A). Rats treated with PMSCs only also exhibited symptoms as early as 1 week pi, whereas those treated with PMSCs plus intravenous C16 and Ang-1 did not show neurological dysfunction until 2 weeks pi (Fig.Ā 1A). The symptoms peaked at 3 weeks pi in both the PMSCs and Pā+āCā+āA groups, which was 1 week later than in the vehicle-treated group (Fig.Ā 1A). The clinical scores of rats in the PMSCs group were significantly lower than those in the vehicle-treated group from 2 to 8 weeks pi, and the scores were lower still in the Pā+āCā+āA group from 1 to 3 weeks pi (the onset stage) and from 6 to 8 weeks pi (the recovery stage; Fig.Ā 1A, Pā<ā0.05).
Changes in cortical somatosensory evoked potential (CSEP) and cortical motor evoked potential (CMEP) have been commonly used to assess the level of neural damage in MS patients16,17,18,19,20. Compared with normal rats, rats in which EAE was induced displayed prolonged latency to waveform initiation and lower peak amplitudes for both CSEP and CMEP at 3 and 8 weeks pi (TableĀ 1, Fig. S1), indicating a slower speed of conduction and loss of functioning nerve fibers, respectively, in these rats. These EAE-associated electrophysiological disturbances were significantly attenuated by treatment with PMSCs and PMSCs plus intravenous C16 and Ang-1 (TableĀ 1, Fig. S1; Pā<ā0.05).
Intravenous C16 and Ang-1 enhanced the efficacy of PMSC therapy for inhibiting inflammatory cell infiltration in rats with EAE
Immunostaining of tissues collected 3 weeks pi showed diffuse CNS infiltration of CD68+ cells around blood vessels, throughout brain tissue and spinal cord parenchyma, and below the meninges (Fig.Ā 1C,HāI,L,O). The perivascular and parenchymal infiltrates at 3 and 8 weeks pi were significantly reduced by PMSC transplantation (Fig.Ā 1D,E,J,M,P) and further decreased by treatment with PMSCs plus intravenous C16 and Ang-1 (Fig.Ā 1F,K,N,Q). These results were confirmed by inflammatory scores at 3 and 8 weeks pi (Fig.Ā 1V,W; Pā<ā0.05). CD68 was not detected in the engrafted PMSCs; however, significant CD68-positive cell infiltrates were detected in the areas surrounding the grafts and in the central region of the graft cell mass. Cell invasion into the grafts was less severe in the Pā+āCā+āA group than in the PMSCs group (Fig.Ā 1RāT).
Intravenous C16 and Ang-1 enhanced the anti-inflammatory and anti-astrogliosis effects of PMSCs in rats with EAE
At 3 and 8 weeks pi, expression of the pro-inflammatory factors NF-ĪŗB and COX-2 in the brain cortex and spinal cords was analyzed by RT-PCR (Fig. SP2), western blotting (Fig.Ā 2AāF) and immunostaining (Figs S4, S5). In addition, serum TNF-Ī±, IL-17, IFN-Ī³, and TGF-Ī² levels were measured by ELISA (Fig.Ā 3AāD). The levels of NF-ĪŗB, COX-2, TNF-Ī±, IL-17, and IFN-Ī³ were significantly higher in rats with EAE than in normal rats (FigsĀ 2, 3, S2ā5). The increases in these pro-inflammatory factors in rats with EAE were significantly reduced by PMSC therapy (Pā<ā0.05) and further decreased by treatment with PMSCs plus intravenous C16 and Ang-1 (Pā<ā0.05) expect for IFN-Ī³ (FigsĀ 2, 3, S2ā5). In contrast, the serum TGF-Ī² level was slightly lower in rats with EAE than in normal rats at 3 and 8 weeks pi (Fig.Ā 3D, Pā<ā0.05). PMSC transplantation restored the TGF-Ī² levels in rats with EAE, and treatment with PMSCs plus intravenous C16 and Ang-1 further increased the TGF-Ī² levels (Fig.Ā 3D, Pā<ā0.05).
Astrocyte proliferation in response to neuronal injury in EAE (reactive astrogliosis) drives CNS inflammation and can lead to the formation of glial scars at the lesion site, which in turn inhibits axonal regeneration21. In this study, we assessed the expression and distribution of the specific astrocyte marker GFAP in the brain cortex and spinal cord by immunofluorescence staining. The results revealed astrocyte proliferation from 3 weeks pi (Fig.Ā 4B,F), with noticeable glial scar formation at 8 weeks pi in rats with EAE (Fig.Ā 4I,L) but not in normal rats (Fig.Ā 4A,E). Astrocyte proliferation was significantly inhibited by treatment with PMSCs alone or in combination with intravenous C16 and Ang-1 at both 3 (Fig.Ā 4C,D,G,H) and 8 weeks (Fig.Ā 4J,K,M,N) PI. Moreover, Pā+āCā+āA rats exhibited significantly reduced astrocyte proliferation compared with PMSCs rats at 8 weeks pi (Fig.Ā 4S,T; Pā<ā0.05). These results were confirmed by western blot (Fig.Ā 4GāI) and RT-PCR (Fig. SP2) analysis of GFAP expression in the brain cortex.
GFAP was not detected in the engrafted PMSCs; however, astrocyte proliferation was observed in areas circumjacent to the grafts, which, in turn, obstructed the transmigration of PMSCs (Fig.Ā 4OāR). Consequently, the Pā+āCā+āA group exhibited more efficient PMSC homing to the CNS compared with the PMSCs group (Fig.Ā 4OāR).
Intravenous C16 and Ang-1 enhanced the efficacy of PMSC therapy for preventing demyelination, vascular leakage, and neuronal loss in rats with EAE
EAE is characterized by demyelination, perivascular edema, and neuronal cell death in the CNS. In this study, demyelination was assessed by western blotting and immunofluorescence staining for MBP, a specific marker of myelination. In addition, morphological changes in the myelin sheath, neurons, and blood vessels in the CNS were examined by TEM. Western blotting revealed significantly decreased MBP expression in the brain cortex of rats with EAE at 3 and 8 weeks pi (Fig.Ā 5AāC, Pā<ā0.05). Immunofluorescence staining for MBP showed myelin disruption and demyelination in the brain cortex and spinal cords of rats with EAE (Fig.Ā 6B,E,I,L). EAE-associated MBP loss and demyelination were ameliorated by treatment with PMSCs, either alone or in combination with intravenous C16 and Ang-1 (Fig.Ā 6AāU, IāIII; Pā<ā0.05), with the Pā+āCā+āA group showing more effective prevention of MBP loss and demyelination than PMSC treatment alone (Fig.Ā 6I-III, Pā<ā0.05). Engrafted PMSCs (green) were detected in the subarachnoid space, with infiltration into the parenchyma (Fig.Ā 6VāY). MBP, a marker of the oligodendrocyte lineage, was detected in the engrafted PMSCs in both the PMSCs and Pā+āCā+āA groups, with relatively higher levels detected in the Pā+āCā+āA group (Fig.Ā 6VāY, I; Pā<ā0.05). LFB staining for myelin revealed loss of myelin where inflammatory cell infiltration was observed in the brain cortex and spinal cords of rats with EAE, and these EAE-associated changes were reversed by treatment with PMSCs, alone or in combination with intravenous C16 and Ang-1 (Fig. S6).
TEM images of the brain cortex and spinal cord of normal rats showed neurons with an intact myelin sheath as well as well-defined nuclei (Fig.Ā 7A,B). However, neurons in rats with EAE began to display noticeable myelin sheath splitting as well as vacuolar changes by 3 weeks pi (Fig.Ā 7C). At 8 weeks pi, myelin lamellae disintegration had worsened, with partial or complete loss of the nerve fibers (Fig.Ā 7L; arrow points to loss of nerve fiber). Neuronal apoptosis (Fig.Ā 7F) and inflammatory cell infiltration into the parenchyma (Fig.Ā 7G; arrow indicates an extravasated inflammatory cell in edema tissue) were detected at 3 weeks pi. Some neurons displayed morphological signs of necrosis, including the presence of large vacuoles, degenerated organelles, ruptured cytoplasmic membranes, and oncolytic chromatin (Fig.Ā 7N; arrow indicates oncolytic chromatin). Perivascular edema was observed at 3 weeks pi and worsened over time (Fig.Ā 7D,E,M; arrows in E and M indicate perivascular edema). The reduced extracellular space surrounding the vessels at 3 and 8 weeks pi in PMSCs-treated rats, especially those co-treated with C16 and Ang-1, indicated alleviation of perivascular edema (Fig.Ā 7Q,T,U,V; Pā<ā0.05). Moreover, treatment with PMSCs, either alone or in combination with C16 and Ang-1, effectively prevented myelin sheath splitting (Fig.Ā 7H,J) and neuronal apoptosis at 3 weeks pi (Fig.Ā 7I,K). At 8 weeks pi, rats in the PMSCs and Pā+āCā+āA groups displayed only mild demyelination along with formation of new myelin sheaths around intact axons (Fig.Ā 7OāT; arrow in O indicates newly formed myelin sheath). The morphological changes in subcellular organelles and nuclei were also largely reversed, especially in the Pā+āCā+āA group (Fig.Ā 7PāT).
Intravenous C16 and Ang-1 enhanced the efficacy of PMSCs for preventing axonal loss in rats with EAE
Changes in the morphology of axons were examined by immunofluorescence staining for NF-200. Axonal degeneration was assessed by Bielschowskyās silver staining as well as western blot analysis of NF-200 expression. Immunofluorescence staining for NF-200 and Bielschowskyās silver staining revealed reduced axonal density as well as degeneration of the remaining axons in rats with EAE at 3 weeks pi, which worsened over time (Fig.Ā 8B,E,I,L). Treatment with PMSCs, alone (Fig.Ā 8C,F,J,M; Fig. S7C,G,J,M) or in combination with intravenous C16 and Ang-1 (Fig.Ā 8D,G,K,N; Fig. S7D,H,K,N), restored axonal density and morphology, with the Pā+āCā+āA treatment showing better efficacy than PMSC therapy alone (Fig. S7O,P; Pā<ā0.05). Moreover, western blot and RT-PCR analysis revealed diminished NF-200 levels in the brain cortex of rats with EAE, and treatment with PMSCs, alone or in combination with intravenous C16 and Ang-1, ameliorated NF-200 loss, with the Pā+āCā+āA treatment showing a relatively more prominent effect at later stage of clinic process (Fig.Ā 5DāF, Fig. SP3C,D: Pā<ā0.05). NF-200 was detected in the engrafted PMSCs in both PMSCs and Pā+āCā+āA groups (Fig.Ā 8SāV), with the Pā+āCā+āA group showing a higher percentage of NF-200-positive cells (Fig.Ā 8W, Pā<ā0.05).
Intravenous C16 and Ang-1 upregulated neurotropic protein expression and enhanced the efficacy of PMSC therapy for preventing neuronal apoptosis in rats with EAE
BDNF supports neuronal differentiation, growth, and survival22,23, and GAP-43 is a crucial component of the axon and presynaptic terminal24,25. p75NTR is implicated in the regulation of both synaptic transmission and axonal elongation26. To further elucidate the molecular mechanisms underlying the neuroprotective effects of PMSCs, we examined the expression of BDNF, p75NTR, and GAP-43 as well as expression of the apoptosis marker caspase-3 by western blotting, RT-PCR and immunofluorescence staining. The results revealed increased GAP-43 and p75NTR but decreased BDNF levels in the CNS of rats with EAE (Fig.Ā 5GāO; Fig. S3EāJ, Fig. S8ā10). Treatment with PMSCs, alone or in combination with intravenous C16 and Ang-1, significantly upregulated GAP-43, p75NTR and BDNF expression, with relatively more prominent effects detected in the Pā+āCā+āA group (FigsĀ 5GāO, S3EāJ; S8ā10; Pā<ā0.05). Western blotting revealed drastically increased caspase-3 levels in the brain cortex of rats with EAE (Fig.Ā 5PāR), and immunofluorescence staining revealed elevated caspase-3 expression in the multipolar motor neurons of the spinal cord anterior horn as well as the pyramid-shaped motor neurons of the precentral gyrus (Fig. S11). Treatment with PMSCs, either alone or in combination with intravenous C16 and Ang-1, inhibited caspase-3 upregulation in rats with EAE, with the Pā+āCā+āA treatment showing a more potent effect (Fig.Ā 5PāR, S11; Pā<ā0.05). Nissl staining showed visible neuronal loss in the CNS of rats with EAE that progressed over time (Fig.Ā 9AāN). In the PMSCs and Pā+āCā+āA groups, the numbers of surviving neurons in the brain cortex and spinal cord were increased (Fig.Ā 9AāN), with the Pā+āCā+āA treatment showing a relatively more potent neuronal cell-preservation effect (Fig.Ā 9O,P; Pā<ā0.05).
Intravenous C16 and Ang-1 upregulated the expression of the neuronalāglial lineage markers in engrafted PMSCs
We detected expression of the neuronal-glial lineage markers NF-200 and MBP (FigsĀ 6VāY and 8OāR) but not the astrocyte marker GFAP (Fig.Ā 4OāR) in PMSCs homing to the CNS of rats with EAE. Intravenous C16 and Ang-1 increased the expression of NF-200 and MBP in these cells (FigsĀ 6I and 8S), suggesting that the combinatorial treatment can promote differentiation of engrafted PMSCs along neuronalāglial lineages. We next examined the expression of the neurotropic proteins GAP-43, p75NTR, and BDNF; the proinflammatory factors NF-ĪŗB and COX-2; and the apoptosis marker caspase-3 in engrafted PMSCs by immunofluorescence staining. NF-ĪŗB and COX-2 were not detected (Fig.Ā 10A,C,E,G), but caspase-3 was detected at low levels at 8 weeks pi (Fig.Ā 10I,K). GAP-43, p75NTR, and BDNF were all detected in the engrafted PMSCs (Fig.Ā 10M,O,Q,S,U,W). Intravenous C16 and Ang-1 upregulated GAP-43, p75NTR, and BDNF (Fig.Ā 10IIāIV, Pā<ā0.05) and downregulated caspase-3 (Fig.Ā 10I, Pā<ā0.05) in the engrafted cells.
Discussion
In a recent side-by-side comparison of PMSC versus EMSC therapy for EAE, PMSCs demonstrated similar efficacy to EMSCs at inhibiting inflammatory cell infiltration, demyelination, axonal loss, and neurological dysfunction12. Moreover, both PMSCs and EMSCs demonstrated the ability to migrate into the inflamed CNS tissues as well as the potential to differentiate along the neuralāglial lineage12. These findings supported PMSCs as an ideal cell-based therapy for MS. However, significant inflammatory cell infiltration and inflammation of the CNS remained after PMSC transplantation, which limited the therapeutic potential of this treatment modality12.
C16 and Ang-1 have been reported to block inflammatory cell migration by promoting vascular integrity and interfering with leukocyteāendothelial interactions, respectively13,14,15. In addition, because these two molecules act through different mechanisms, they have been shown to work synergistically to mitigate inflammatory responses in EAE14. In the present study, intravenous administration of C16 and Ang-1 significantly increased the efficacy of PMSC therapy for reducing CNS inflammation, neuronal injury, and neurological dysfunction in a rat model of EAE.
Similar to our previous findings12, transplantation of PMSCs downregulated the proinflammatory molecules TNF-Ī±, IFN-Ī³, IL-17, NF-ĪŗB, and COX-2 and upregulated the anti-inflammatory cytokine TGF-Ī². The perivascular/parenchymal infiltration of CD68+ microglia/macrophages was also attenuated by treatment with PMSCs. These anti-inflammatory effects of PMSCs were enhanced by intravenous administration of C16 and Ang-1.
Our mechanistic studies suggested that C16 and Ang-1 enhanced the efficacy of PMSC therapy by promoting homing of these cells to the inflamed tissues in the CNS as well as enhancing their differentiation/regenerative capacity. Reactive astrogliosis in rats with EAE, which obstructed PMSC transmigration, was attenuated by combinatorial treatment with C16 and Ang-1, leading to more effective PMSC engraftment in the CNS. Transplanted stem cells can produce neurotrophins to stimulate neuronal cell growth in animals with EAE. They can also differentiate along the neuronalāglial lineage to replace damaged neurons27,28,29,30. C16 and Ang-1 boosted the ability of the engrafted PMSCs to differentiate down the neuronalāglial lineage as assessed by the expression of the neuronalāglial markers NF-200 and MBP in these cells. Moreover, C16 and Ang-1 upregulated BDNF (a key player in neuron survival and differentiation22,23), GAP-43 (a driving factor of axonal sprouting and restoration24,25), and p75NTR (a regulator of neuron proliferation and maturation31,32) and downregulated the apoptosis marker caspase-3 in the engrafted PMSCs. Invasion of inflammatory cells into MSC grafts is considered to negatively impact the survival and function of the grafted cells12. In this study, we detected infiltration of CD68+ microglia/macrophages into the PMSC grafts in the CNS, mainly localized in the central part of the cellular mass as described in our previous reports12. Combinatorial treatment with the C16 and Ang-1 markedly reduced inflammatory cell infiltration into the grafts, which, we believe, contributed to the improved retention, differentiation, and neuroprotective function of the transplanted PMSCs.
The injection of antigens into the anterior chamber of the eye induces a systemic suppression of cell-mediated and humoral immune responses to the antigen. This so called āanterior chamber associated-immune deviation (ACAID)ā is largely mediated by TGF-Ī²2 in the aqueous humor acting on ocular antigen-presenting cells (APCs), eventually leading to activation of antigen-specific T regulatory cells (Tregs)33. Previous studies have shown that intravenous injection of in vitro-generated APCs specific to the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG) and/or MBP induces antigen-specific tolerance33,34,35. These encephalitogenic antigen-specific APCs might also augment the efficacy of PMSC and Pā+āCā+āA therapy for EAE, especially in a MOG/MBP-induced chronic EAE model with pathological features typical of chronic, progressive MS36.
Granulocyte macrophage colony-stimulating factor (GM-CSF) has emerged as a putative therapeutic target in MS37. Macrophage infiltration is considered a major contributing factor to demyelination in both clinical MS and animal models of EAE. GM-CSF stimulates proliferation and activation of macrophages, monocytes, neutrophils, eosinophils, dendritic cells, and microglia with subsequent induction of pro-inflammatory mediators, and evidence suggests that this cytokine may be involved in the inflammatory processes related to MS38,39,40. Indeed, GM-CSFā/ā mice are resistant to EAE and immune cell infiltration in the CNS37. However, GM-CSF has also been known to suppress autoimmune diseases such as Crohnās disease, type-1 diabetes, Myasthenia gravis, and experimental autoimmune thyroiditis by promoting Treg expansion and/or modulating phenotype-specific differentiation of precursor immune cells41,42. Thus, GM-CSF could function as a double-edged sword in EAE development. The effects of GM-CSF on PMSC and Pā+āCā+āA therapy for EAE warrant further investigation.
Conclusion
Intravenous C16 and Ang-1 increased the efficacy of PMSC therapy for preventing demyelination/neuronal loss and ameliorating neurological dysfunction in a rat model of EAE by inhibiting inflammatory cell infiltration and enhancing PMSC engraftment, survival, differentiation, and neurotrophin production. Further pharmacokinetic and pharmacodynamic studies, preferably in primates, are needed to evaluate the therapeutic potential of this treatment regimen for MS.
Methods
Isolation and culture of GFP-expressing rat PMSCs
Green fluorescent protein (GFP)-expressing PMSCs were isolated from rats as previously described12. The cells were maintained at 37āĀ°C in a humidified incubator with 5% CO2 for 4ā5 weeks prior to experimental studies. The medium was exchanged every 3ā4 days.
Animal model of EAE
A total of 77 adult male Lewis rats (9ā10 weeks of age, 200ā250āg) were obtained from the Center of Laboratory Animal Services at Zhejiang University. The rats were randomly assigned to four groups: normal control group (normal, nā=ā11), vehicle-treated EAE group (vehicle, nā=ā22), PMSC-treated EAE group (PMSCs, nā=ā22), and EAE group treated with PMSCs plus intravenous C16 and Ang-1 (Pā+āCā+āA, nā=ā22). EAE was induced as previously described12 by subcutaneous injection of guinea pig spinal cord homogenate (GPSCH) emulsified at a 1:1 ratio with complete Freund adjuvant (CFA) containing heat killed Mycobacterium tuberculosis. Rats in the normal group received an injection of CFA emulsified at a 1:1 ratio in 0.9% saline. Each rat received an intraperitoneal injection of 300 ng Pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) in 0.1āml distilled water immediately after the subcutaneous injection and again 48āh later. Rats in the Pā+āCā+āA group also received once daily intravenous administration of C16 (2āmg) and Ang-1 (400āĀµg) until the time of sacrifice, starting immediately after EAE induction. C16 and Ang-1 were purchased from Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China).
The clinical manifestations of EAE were assessed daily until the time of sacrifice. Disease severity was scored on a 5-point scale: 0ā=āno signs, 1ā=āpartial loss of tail tonicity, 2ā=āloss of tail tonicity, 3ā=āunsteady gait and mild paralysis, 4ā=āhind limb paralysis and incontinence, and 5ā=āmoribund or death15. Disease scoring was performed by pathologists blinded to treatment conditions.
All animal studies were approved by the Animal Ethics Committee of Zhejiang University and carried out in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.
PMSC transplantation
One week after EAE induction, rats in the PMSCs and Pā+āCā+āA groups received intrathecal infusion of 1āĆā106 PMSCs into the subarachnoid space as previously described12. Rats in the vehicle group received injections of phosphate-buffered saline (PBS) instead.
Neurophysiological testing
Cortical somatosensory evoked potentials (CSEPs) were recorded as previously described16,17,18 at 3 weeks (peak stage of clinical manifestations) and 8 weeks (recovery stage) pi (nā=ā5 per group). For measurement of CSEPs, rats were fixed to a stereotaxic frame. A constant current stimulator (Digitimer, Welwyn Garden City, UK) was used to deliver positive current pulses (15āV, 40āms duration) to produce a maximum SEP (averaged over 30 stimuli). The SEPs from three series of stimulations were amplified, filtered, digitally converted, and stored for post-hoc analysis. Peak positive and negative values were recorded. The results are presented as the meanāĀ±āstandard deviation (SD) of voltage amplitude (ĀµV) and latency (ms).
Cortical motor evoked potentials (CMEPs) were recorded following previously reported procedures19,20 at the same time points (nāt 5 per group). Following anesthesia, a midline incision was made on the scalp. The tissues underneath were cleaned, and the cranium exposed. Screw electrodes were implanted to a depth of 0.75āmm over the primary somatomotor cortical areas, gently touching the dura mater. A needle electrode was inserted into the muscle of the hindlimb, and an inactive reference electrode was inserted under the skin, 2āmm from the screw electrode. The somatomotor cortex was stimulated with a train of 10ā25 pulses at 10āHz that evoked visible contralateral hindlimb movement. The signals were recorded and CEMPs were calculated over three independent experiments.
Tissue collection and processing
Rats in each group were sacrificed at 3 and 8 weeks after immunization (nā=ā5 per group at each time point). Rats were anesthetized with an intraperitoneal injection of 1% Nembutal (40āmg/kg) and perfused intracardially with cold saline followed by 4% paraformaldehyde in 0.1āM phosphate buffer (PBS, pH 7.4). The spinal cord and brain tissues were carefully dissected. One centimeter of the lumbar spinal cord and half of the brain of each animal were fixed in the same fixative for 4āh and then transferred to 30% sucrose in PBS until the tissue sunk to the bottom of the container. Twenty-micrometer-thick sections were cut on a freezing microtome through the coronal plane of the brain and transverse plane of the spinal cord using a Leica cryostat and then mounted onto 0.02% poly-L-lysine-coated slides. All sections were collected for histological assessment and immunohistological and immunofluorescent staining. The remains of the CNS tissue were fixed in 2.5% glutaraldehyde solution and examined by transmission electron microscopy (TEM).
Histological assessment
Extravasated macrophages were detected by immunofluorescence staining for CD68, a specific macrophage marker. In digital photomicrographs taken at 200x magnification in three fields per tissue section, the severity of inflammatory cell infiltration was scored from 0ā443: 0ā=āno infiltration, 1ā=āinfiltration only around blood vessels and meninges, 2ā=ālight infiltration in the parenchyma (1ā10 cells per section), 3ā=āmoderate infiltration in the parenchyma (11ā100 cells per section), and 4ā=āsevere infiltration in the parenchyma (100ā+ācells per section). Neuronal loss was assessed by Cresyl Violet staining (Nissl staining). Neuronal counts were restricted to cells that displayed a well-defined nucleolus as well as adequate amounts of endoplasmic reticulum.
The severity of axon demyelination was assessed by Luxol fast blue (LFB) staining and immunofluorescence staining for MBP. Demyelination was scored from 0ā543: 0ā=āno demyelination, 1ā=ārare foci of demyelination, 2ā=ālight demyelination, 3ā=āconfluent perivascular or subpial demyelination, 4ā=āsubstantial perivascular and subpial demyelination in at least one half of the spinal cord together with inflammatory cell infiltration in the CNS parenchyma, and 5ā=āmassive perivascular and subpial demyelination across the entire spinal cord along with inflammatory cell infiltration in the CNS parenchyma.
Axonal loss was assessed by Bielschowsky silver staining and scored from 0ā344: 0ā=āno loss, 1ā=āsuperficial loss in less than 25% of tissue, 2ā=ādeep loss in over 25% of tissue, and 3ā=ādeep loss encompassing the entire tissue.
Immunofluorescence staining
Five sections of the brain cortex and anterior horns of the spinal cord from each rat were randomly selected and subjected to immunofluorescence staining. The tissue sections were incubated overnight at 4āĀ°C with anti-NF-200 (1:500; Abcam, Cambridge, MA, USA), anti-GAP-43 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-BDNF (1:500; Abcam), anti-NF-ĪŗB p65 (1:500; Abcam), Anti-p75NTR (1:500; Abcam), anti-MBP (1:500; Abcam), anti-glial fibrillary acidic protein (GFAP, 1:200, Thermo Fisher Scientific, Waltham, MA, USA), anti-COX-2 (1:1000; BioVision, Milpitas, CA, USA), and anti-CD68 (1:100; Santa Cruz Biotechnology) antibodies, individually. After washing in PBS, the sections were incubated with TRITC/FITC-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1āh at 37āĀ°C. The sections were subsequently mounted on glass slides and coverslipped with Antifade Gel Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL, USA). Stained sections were viewed under a microscope at 200x magnification, and images were taken of three fields per tissue section. Areas stained positively for GFAP, MBP, and CD68 were analyzed using NIH Image software. The numbers of cells stained positively for GAP43, caspase-3, NF-200, BDNF, NF-kB p65, COX-2, Caspase-3, and p75NTR were counted.
TEM analysis
Sections of the brain cortex and lumbar spinal cord were examined by TEM as described previously12,13,14,15. Both low and high magnification images were recorded. The extracellular space surrounding the vessels were calculated with NIH Image software.
Enzyme-linked immunosorbent assay (ELISA)
Peripheral blood samples were collected at 3 and 8 weeks pi (nā=ā5 per group at each time point). Concentrations of TNF-Ī±, IL-17, and TGF-Ī² were determined using ELISA kits from Abcam. Concentrations of IFN-Ī³ were determined using an ELISA kit from BioLegend Inc. (San Diego, CA, USA). Optical density at 450 nm was recorded, and the data were tabulated using GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA, USA).
RT-PCR
Rats were sacrificed by decapitation at 3 and 8 weeks pi (nā=ā5 per group at each time point). Total RNA was extracted from brain tissues using TRIzol reagents (Invitrogen, CA, USA) according to the manufacturerās instructions. cDNA was synthesized from 2āĪ¼l RNA using a cDNA Reverse Transcription Kit (Thermo Fisher Scientific, CA). The expression of NF-200, GAP-43, caspase-3, NF-ĪŗB p65, p75NTR, MBP, GFAP, COX-2 and BDNF was determined by PCR amplification followed by agarose gel electrophoresis. The results were normalized to those for GAPDH. All experiments were performed in triplicate.
Western blotting
Rats were sacrificed by decapitation at 3 and 8 weeks pi (nā=ā5 per group at each time point). Brain cortex tissues and 10-mm lumbar spinal cord segments were homogenized and analyzed by western blotting as previously described12,13,14,15.
Statistical analysis
Data were analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Differences between groups were identified by two-way analysis of variance (ANOVA) followed by post-hoc Tukey t-tests. A P value of <0.05 was considered statistically significant. All statistical graphs were created using GraphPad Prism Version 4.0.
Change history
12 May 2020
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
References
Conlon, P., Oksenberg, J. R., Zhang, J. & Steinman, L. The immunobiology of multiple sclerosis: an autoimmune disease of the central nervous system. Neurobiol Dis. 6, 149ā166 (1999).
Compston, A. & Coles, A. Multiple sclerosis. Lancet. 359, 1221ā1231 (2002).
Payne, N. L. et al. Distinct immunomodulatory and migratory mechanisms underpin the therapeutic potential of human mesenchymal stem cells in autoimmune demyelination. Cell Transplant. 22, 1409ā1425 (2013).
Cobo, M. et al. Mesenchymal stem cells expressing vasoactive intestinal peptide ameliorate symptoms in a model of chronic multiple sclerosis. Cell Transplant. 22, 839ā854 (2013).
Payne, N. L. et al. Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination. Cell Adhesion & Migration. 6, 179ā189 (2012).
Giuliani, M. et al. Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation. PLoS One. 6, e19988 (2011).
Wang, X. et al. Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis. Stem Cell Reports. 3, 115ā130 (2014).
Drukker, M. et al. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells. 24, 221ā229 (2006).
Diaz-Prado, S. et al. Isolation and characterization of mesenchymal stem cells from human amniotic membrane. Tissue Eng Part C Methods. 17, 49ā59 (2011).
Fisher-Shoval, Y. et al. Transplantation of placenta-derived mesenchymal stem cells in the EAE mouse model of MS. J Mol Neurosci. 48, 176ā184 (2012).
Frohman, E. M., Racke, M. K. & Raine, C. S. Multiple sclerosisāthe plaque and its pathogenesis. N Engl J Med. 354, 942ā955 (2006).
Jiang, H., Zhang, Y., Tian, K., Wang, B. & Han, S. Amelioration of experimental autoimmune encephalomyelitis through transplantation of placental derived mesenchymal stem cells. Sci Rep. 7, 41837 (2017).
Jiang, H., Zhang, F., Yang, J. & Han, S. Angiopoietin-1 ameliorates inflammation-induced vascular leakage and improves functional impairment in a rat model of acute experimental autoimmune encephalomyelitis. Exp Neurol. 261, 245ā257 (2014).
Wang, B., Tian, K. W., Zhang, F., Jiang, H. & Han, S. Angiopoietin-1 and C16 Peptide Attenuate Vascular and Inflammatory Responses in Experimental Allergic Encephalomyelitis. CNS Neurol Disord Drug Targets. 15, 496ā513 (2016).
Fang, M. et al. C16 peptide shown to prevent leukocyte infiltration and alleviate detrimental inflammation in acute allergic encephalomyelitis model. Neuropharmacology. 70, 83ā99 (2013).
All, A. H. et al. Effect of MOG sensitization on somatosensory evoked potential in Lewis rats. J Neurol Sci. 284, 81ā89 (2009).
Troncoso, E., Muller, D., Czellar, S. & Zoltan Kiss, J. Epicranial sensory evoked potential recordings for repeated assessment of cortical functions in mice. J Neurosci Methods. 97, 51ā58 (2000).
Troncoso, E. et al. Recovery of evoked potentials, metabolic activity and behavior in a mouse model of somatosensory cortex lesion: role of the neural cell adhesion molecule (NCAM). Cereb Cortex. 14, 332ā341 (2004).
Bolay, H., Gursoy-Ozdemir, Y., Unal, I. & Dalkara, T. Altered mechanisms of motor-evoked potential generation after transient focal cerebral ischemia in the rat: implications for transcranial magnetic stimulation. Brain Res. 873, 26ā33 (2000).
Amadio, S. et al. Motor evoked potentials in a mouse model of chronic multiple sclerosis. Muscle Nerve. 33, 265ā273 (2006).
Brambilla, R. et al. Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination. Glia. 62, 452ā467 (2014).
Makar, T. K. et al. Brain derived neurotrophic factor treatment reduces inflammation and apoptosis in experimental allergic encephalomyelitis. J Neurol Sci. 270, 70ā76 (2008).
Mariga, A., Mitre, M. & Chao, M. V. Consequences of brain-derived neurotrophic factor withdrawal in CNS neurons and implications in disease. Neurobiol Dis. 97, 73ā79 (2017).
Jacobson, R. D., Virag, I. & Skene, J. H. A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS. J Neurosci. 6, 1843ā1855 (1986).
Benowitz, L. I. & Routtenberg, A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 20, 84ā91 (1997).
Dechant, G. & Barde, Y. A. The neurotrophin receptor p75 (NTR): novel functions and implications for diseases of the nervous system. Nat Neurosci. 5, 1131ā1136 (2002).
Covacu, R. & Brundin, L. Endogenous spinal cord stem cells in multiple sclerosis and its animal model. J Neuroimmunol (2016).
Ghasemi, N. et al. Transplantation of human adipose-derived stem cells enhances remyelination in lysolecithin-induced focal demyelination of rat spinal cord. Mol Biotechnol. 56, 470ā478 (2014).
Yu, J. W. et al. Synergistic and Superimposed Effect of Bone Marrow-Derived Mesenchymal Stem Cells Combined with Fasudil in Experimental Autoimmune Encephalomyelitis. J Mol Neurosci. 60, 486ā497 (2016).
Leite, C. et al. Differentiation of human umbilical cord matrix mesenchymal stem cells into neural-like progenitor cells and maturation into an oligodendroglial-like lineage. PLoS One. 9, e111059 (2014).
Li, H. Y. & Zhou, X. F. Potential conversion of adult clavicle-derived chondrocytes into neural lineage cells in vitro. J Cell Physiol. 214, 630ā644 (2008).
Wang, W. X. et al. Nerve growth factor induces cord formation of mesenchymal stem cell by promoting proliferation and activating the PI3K/Akt signaling pathway. Acta Pharmacol Sin. 32, 1483ā1490 (2011).
Farooq, S. M. & Ashour, H. M. In vitro-induced cell-mediated immune deviation to encephalitogenic antigens. Brain Behav Immun. 35, 64ā69 (2014).
Farooq, S. M., Elkhatib, W. F. & Ashour, H. M. The in vivo and in vitro induction of anterior chamber associated immune deviation to myelin antigens in C57BL/6 mice. Brain Behav Immun. 42, 118ā122 (2014).
Farooq, S. M. & Ashour, H. M. Eye-mediated induction of specific immune tolerance to encephalitogenic antigens. CNS Neurosci Ther. 19, 503ā510 (2013).
Berard, J. L., Wolak, K., Fournier, S. & David, S. Characterization of relapsing-remitting and chronic forms of experimental autoimmune encephalomyelitis in C57BL/6 mice. Glia. 58, 434ā445 (2010).
McQualter, J. L. et al. Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. J Exp Med. 194, 873ā882 (2001).
Croxford, A. L., Spath, S. & Becher, B. GM-CSF in Neuroinflammation: Licensing Myeloid Cells for Tissue Damage. Trends Immunol. 36, 651ā662 (2015).
Reddy, P. H. et al. Granulocyte-macrophage colony-stimulating factor antibody suppresses microglial activity: implications for anti-inflammatory effects in Alzheimerās disease and multiple sclerosis. J Neurochem. 111, 1514ā1528 (2009).
Schottelius, A. The role of GM-CSF in multiple sclerosis. Drug Res (Stuttg). 63(Suppl 1), S8 (2013).
Bhattacharya, P. et al. Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy. J Interferon Cytokine Res. 35, 585ā599 (2015).
Bhattacharya, P. et al. GM-CSF: An immune modulatory cytokine that can suppress autoimmunity. Cytokine. 75, 261ā271 (2015).
Ma, X. et al. Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice. PLoS One. 5, e13489 (2010).
Yin, J. X. et al. Centrally administered pertussis toxin inhibits microglia migration to the spinal cord and prevents dissemination of disease in an EAE mouse model. PLoS One. 5, e12400 (2010).
Acknowledgements
This work was funded by the Zhejiang Provincial Natural Science Foundation of China (Project No. LY16H090002), Foundation of Zhejiang Provincial Education Department (Y201431129) and by the National Natural Science Foundation of China (Project No. 81271333). It was partially supported by the āDouble First-rateā project initiative.
Author information
Authors and Affiliations
Contributions
Conception and design: Shu Han, Financial support: Shu Han, Administrative support: Shu Han, Provision of study material or patients: Ke-wei Tian, Collection and/or assembly of data: Ke-wei Tian and Yuan-yuan Zhang, Data analysis and interpretation: Ke-wei Tian and Yuan-yuan Zhang, Manuscript writing: Shu Han and Hong Jiang, Final approval of manuscript: Shu Han and Hong Jiang.
Corresponding author
Ethics declarations
Competing Interests
The authors declare no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the articleās Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the articleās Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Tian, Kw., Zhang, Yy., Jiang, H. et al. Intravenous C16 and angiopoietin-1 improve the efficacy of placenta-derived mesenchymal stem cell therapy for EAE. Sci Rep 8, 4649 (2018). https://doi.org/10.1038/s41598-018-22867-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-018-22867-9
This article is cited by
-
The therapeutic applications of mesenchymal stromal cells from human perinatal tissues in autoimmune diseases
Stem Cell Research & Therapy (2021)
-
Adjusting vascular permeability, leukocyte infiltration, and microglial cell activation to rescue dopaminergic neurons in rodent models of Parkinsonās disease
npj Parkinson's Disease (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.