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Microglia are resident immune cells of the central nervous system such as brain-specific macrophages and also known to regulate the innate immune functions of astrocytes through secretory molecules. This conversation plays an important role in brain functions and homeostasis as well as in neuropathologic disease. In this study, we aimed to elucidate whether astrocytes and microglia can cross-talk to induce microglial polarization and proliferation, which can be further regulated under the brain stroke-mimic microenvironment. Microglia in mixed glial culture increased their survival and proliferation and altered to the M2 microglia, whose role was provided by CD11b-GFAP+ astrocytes by showing approximately tenfold increase in microglia cell proliferation after the astrocyte reconstitution. Furthermore, GM-CSF stimulated microglial proliferation approximately tenfold and induced to CCR7+ M1 microglia, whose phenotype could be suppressed by anti-inflammatory cytokines such as IL-4, IL-10, and Substance-P. Also, astrocyte in the microglia co-culture revealed A2 phenotype, which could be activated to A1 astrocyte by TNFα and IFNγ under the stroke-mimic condition. Altogether, astrocyte in the mixed glial culture stimulated the microglia proliferation and M2 polarization possibly through its acquisition of A2 phenotype, both of which could be converted to M1 microglia and A1 astrocytes under the inflammatory stroke-mimic environment. This study demonstrated that microglia and astrocyte can be polarized to M2 microglia and A2 astrocytes respectively through the cross-talk in vitro and provided a system to explore how microglia and astrocyte may behave in the inflammatory disease milieu after in vivo transplantation.

Recently, the stem cell-derived secretome, which is the set of proteins expressed by stem cells and secreted into the extracellular space, has been demonstrated as a critical contributor for tissue repair. In this study, we have produced two sets of high concentration secretomes from adipose-derived mesenchymal stem cells (ADSCs) that contain bovine serum or free of exogenous molecules. Through proteomic analysis, we elucidated that proteins related to extracellular matrix organization and growth factor-related proteins are highly secreted by ADSCs. Additionally, the application of ADSC secretome to full skin defect showed accelerated wound closure, enhanced angiogenic response, and complete regeneration of epithelial gaps. Furthermore, the ADSC secretome was capable of reducing scar formation. Finally, we show high-dose injection of ADSC secretome via intraperitoneal or transdermal delivery demonstrated no detectable pathological conditions in various tissues/organs, which supports the notion that ADSC secretome can be safely utilized for tissue repair and regeneration.

Near the bone remodeling compartments (BRC), extracellular calcium concentration (Ca2+o) is locally elevated and bone marrow stromal cells (BMSCs) close to the BRC can be exposed to high calcium concentration. The calcium-sensing receptor (CaSR) is known to play a key role in maintaining extracellular calcium homeostasis by sensing fluctuations in the levels of extracellular calcium (Ca2+o). When human BMSCs (hBMSCs) were exposed to various calcium concentrations (1.8, 3, 5, 10, 30 mM), moderate-high extracellular calcium concentrations (3&ndash, 5 mM) stimulated proliferation, while a high calcium concentration (30 mM) inhibited the proliferation. Exposure to various calcium concentrations did not induce significant differences in the apoptotic cell fraction. Evaluation of multi-lineage differentiation potential showed no significant difference among various calcium concentration groups, except for the high calcium concentration (30 mM) treated group, which resulted in increased calcification after in vitro osteogenic differentiation. Treatment of NPS2143, a CaSR inhibitor, abolished the stimulatory effect on hBMSCs proliferation and migration indicating that CaSR is involved. These results suggest that the calcium concentration gradient near the BRC may play an important role in bone remodeling by acting as an osteoblast&ndash, osteoclast coupling mechanism through CaSR.

Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-&beta, ), and stromal cell-derived factor-1 (SDF-1). SP, TGF-&beta, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-&beta, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-&beta, while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-&beta, TGF-&beta, pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-&beta, noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-&beta, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.

Endogenous bone marrow-derived mesenchymal stem cells (BM-MSCs) are mobilized into peripheral blood and injured tissues by various growth factors and cytokines that are expressed in the injured tissues, such as substance P (SP), stromal cell derived factor-1 (SDF-1), and transforming growth factor-beta (TGF-β). Extracellular bioactive lipid metabolites such as ceramide-1-phosphate and sphingosine-1-phosphate also modulate BM-MSC migration as SP, SDF-1, and TGF-β. However, the roles of intrinsic lipid kinases of BM-MSCs in the stem cell migration are unclear. Here, we demonstrated that ceramide kinase mediates the chemotactic migration of BM-MSCs in response to SP, SDF-1, or TGF-β. Furthermore, a specific inhibitor of ceramide kinase inhibited TGF-β-induced migration of BM-MSCs and N-cadherin that is necessary for BM-MSCs migration in response to TGF-β. Therefore, these results suggest that the intracellular ceramide kinase is required for the BM-MSCs migration and the roles of the intrinsic ceramide kinase in the migration are associated with N-cadherin regulation.

Aortic injuries, including aortic aneurysms and dissections, are fatal vascular diseases with distinct histopathological features in the aortic tissue such as inflammation-induced endothelial dysfunction, infiltration of immune cells, and breakdown of the extracellular matrix. Few treatments are available for treating aortic aneurysms and dissections; thus, basic and clinical studies worldwide have been attempted to inhibit disease progression. Substance P (SP) exerts anti-inflammatory effects and promotes restoration of the damaged endothelium, leading to vasculature protection and facilitation of tissue repair. This study was conducted to explore the protective effects of systemically injected SP on thoracic aortic injury (TAI). A TAI animal model was induced by orally administering β-aminopropionitrile to rats for 6 weeks. β-aminopropionitrile blocked crosslinking ECM in aorta to cause structural alteration with inflammation within 1 week and then, induced aortic dissection within 4 weeks of initiating treatment, leading to mortality within 6 weeks. Treatment of TAI rats with SP-induced anti-inflammatory responses systemically and locally, possibly by enriching anti-inflammatory M2 monocytes in the spleen and peripheral blood at early phase of aortic injury due to β-aminopropionitrile. SP-induced immune suppression finally prevented the development of aortic dissection by limiting inflammation-mediated aortic destruction. Taken together, these results suggest that SP treatment can block aortic injury by controlling the immune-cell profile and suppressing proinflammatory responses during the initial stage of vascular disease progression.

Given recent progress in regenerative medicine, we need a means to expand chondrocytes in quantity without losing their regenerative capability. Although many reports have shown that growth factor supplementation can have beneficial effects, the use of growth factor–supplemented basal media has widespread effect on the characteristics of chondrocytes. Chondrocytes were in vitro cultured in the 2 most widely used chondrocyte growth media, conventional chondrocyte culture medium and mesenchymal stem cell (MSC) culture medium, both with and without fibroblast growth factor-2 (FGF2) supplementation. Their expansion rates, expressions of extracellular matrix–related factors, senescence, and differentiation potentials were examined in vitro and in vivo. Our results revealed that chondrocytes quickly dedifferentiated during expansion in all tested media, as assessed by the loss of type II collagen expression. The 2 basal media (chondrocyte culture medium vs. MSC culture medium) were associated with distinct differences in cell senescence. Consistent with the literature, FGF2 was associated with accelerated dedifferentiation during expansion culture and superior redifferentiation upon induction. However, chondrocytes expanded in FGF2-containing conventional chondrocyte culture medium showed MSC-like features, as indicated by their ability to direct ectopic bone formation and cartilage formation. In contrast, chondrocytes cultured in FGF2-supplemented MSC culture medium showed potent chondrogenesis and almost no bone formation. The present findings show that the chosen basal medium can exert profound effects on the characteristics and activity of in vitro–expanded chondrocytes and indicate that right growth factor/medium combination can help chondrocytes retain a high-level chondrogenic potential without undergoing hypertrophic transition.

Microglia are resident immune cells of the central nervous system that act as brain-specific macrophages and are also known to regulate the innate immune functions of astrocytes through secretory molecules. This communication plays an important role in brain functions and homeostasis as well as in neuropathologic disease. In this study, we aimed to elucidate whether astrocytes and microglia could crosstalk to induce microglial polarization and proliferation, which can be further regulated under a microenvironment mimicking that of brain stroke. Microglia in a mixed glial culture showed increased survival and proliferation and were altered to M2 microglia, CD11b−GFAP+ astrocytes resulted in an approximately tenfold increase in microglial cell proliferation after the reconstitution of astrocytes. Furthermore, GM-CSF stimulated microglial proliferation approximately tenfold and induced them to become CCR7+ M1 microglia, which have a phenotype that could be suppressed by anti-inflammatory cytokines such as IL-4, IL-10, and substance P. In addition, the astrocytes in the microglial co-culture showed an A2 phenotype, they could be activated to A1 astrocytes by TNF-α and IFN-γ under the stroke-mimicking condition. Altogether, astrocytes in the mixed glial culture stimulated the proliferation of the microglia and M2 polarization, possibly through the acquisition of the A2 phenotype, both could be converted to M1 microglia and A1 astrocytes under the inflammatory stroke-mimicking environment. This study demonstrated that microglia and astrocytes could be polarized to M2 microglia and A2 astrocytes, respectively, through crosstalk in vitro and provides a system with which to explore how microglia and astrocytes may behave in the inflammatory disease milieu after in vivo transplantation.

Intravenous administration of mesenchymal stem cells (MSCs) has served as a clinical intervention for inflammatory diseases. Once entered to blood circulation, MSCs are exposed to a harsh environment which sharply decreases cell viability due to the fact that injected cells, being susceptible to shear stress, are subjected to the high velocities of the bloodstream and lack of proper mechanical support that keeping them in an attachment-deprived state. Here, we coated the nanofilm onto viable MSCs by depositing poly-l-lysine and hyaluronic acid molecules along with arginine-glycine-aspartic acid (RGD peptide) as building blocks to protect cells from shear stress and stabilize them in a single cell, suspension state. In this article, we found that nanofilm-coated cells showed significantly increased cell survival in vitro and in vivo, which was also supported by the activation of survival-related protein, Akt. The coated nanofilm did not interfere with the stemness of MSCs which was determined based on the ...

This study aimed to investigate the analgesic effect of substance P (SP) in an animal model of neuropathic pain. An experimental model of neuropathic pain, the chronic constriction injury (CCI) model, was established using ICR mice. An intravenous (i.v.) injection of SP (1 nmole/kg) was administered to the mice to examine the analgesic effects of systemic SP on neuropathic pain. Behavioral testing and immunostaining was performed following treatment of the CCI model with SP. SP attenuated mechanical allodynia in a time-dependent manner, beginning at 1 h following administration, peaking at 1 day post-injection, and decaying by 3 days post-injection. The second injection of SP also increased the threshold of mechanical allodynia, with the effects peaking on day 1 and decaying by day 3. A reduction in phospho-ERK and glial fibrillary acidic protein (GFAP) accompanied the attenuation of mechanical allodynia. We have shown for the first time that i.v. administration of substance P attenuated mechanical allodynia in the maintenance phase of neuropathic pain using von Frey's test, and simultaneously reduced levels of phospho-ERK and GFAP, which are representative biochemical markers of neuropathic pain. Importantly, glial cells in the dorsal horn of the spinal cord (L4- L5) of SP-treated CCI mice, expressed the anti-inflammatory cytokine, IL-10, which was not seen in vehicle saline-treated mice. Thus, i.v. administration of substance P may be beneficial for improving the treatment of patients with neuropathic pain, since it decreases the activity of nociceptive factors and increases the expression of anti-nociceptive factors.

Diabetic ulceration is one of the most debilitating complications of diabetes and is the main cause of amputation. The diabetic environment is characterized by prolonged inflammation and abnormal angiogenesis, leading to delayed wound healing. Thus, regulation of inflammation and neovascularization is considered a desirable target for diabetes. The critical purpose of this study was to determine whether systemically administered Substance P (SP) could promote wound healing in diabetic environments via suppression of inflammation, induction of angiogenesis, and mobilization of stem cells. The effect of SP was assessed by analyzing epidermal and dermal recovery, vessel formation, cytokine secretion profile, and the stem cell pool in the circulation and bone marrow. Compared with the vehicle-treated group, the SP-treated group exhibited more rapid wound coverage, reduced infiltration of leukocytes, suppression of injury-mediated enlargement of the spleen and mesenteric lymph nodes, reduced tumor necrosis factor-alpha levels, increased interleukin-10, elevated pool of M2 monocytes and vascular endothelial growth factor levels in the blood. Moreover, the stem cell pool in the bone marrow, which is very low in diabetes, was markedly restored by SP to normal levels, which could provide a favorable environment to facilitate wound healing in diabetes. This result demonstrates, for the first time, a possible application of SP for the treatment of diabetic complications, including diabetic ulcers.

Aims Arterial insufficiency ulcers are frequent complications of peripheral artery disease and infection or long-term neglect of the ulcer can eventually lead to amputation of the affected body part. An ischemic environment, caused by interrupted blood flow, affects the supply of nutrients and elongates the inflammation period, inducing tissue degeneration. Thus, the modulation of neovascularization and inflammation could be an ideal therapeutic strategy for ischemic wound healing. This study aimed to elucidate whether systemically administered substance P (SP) could promote ischemic wound repair in mice by restoring blood perfusion and suppressing inflammation. Main methods The effects of SP were assessed by analyzing wound size, blood flow, epidermal and dermal layer regeneration, vessel formation, and the inflammatory cytokine profiles in a hind-limb ischemia non-clinical mouse model. Key findings SP-treated mice exhibited dramatically rapid wound healing and restoration of blood flow within the ischemic zone, compared with saline-treated mice. Notably, SP-treated mice showed enhanced pericyte-covered vasculature compared to saline-treated mice. Moreover, anti-inflammatory effects were detected in mice in the SP-treated group, including suppression of inflammation-mediated spleen enlargement, reduction of tumor necrosis factor-alpha, and promotion of circulatory interleukin-10 levels. Significance These results suggest that SP could be a possible therapeutic candidate for patients with peripheral artery disease, including those with ischemic ulcers.

Biological aging (BA) is a tool for comprehensive assessment of individual health status. A rat model was developed for measuring BA by intravenously administering adipose-derived stem cells (ADSCs) into rats and evaluating several biochemical parameters. In addition, the effect of basic fibroblast growth factor (bFGF) on the differentiation potential of ADSCs was analyzed. A total of 12 male Sprague Dawley rats were divided into autologous ADSC administration (n=6) and saline administration (n=6) groups. The ADSC administration group was further divided into the bFGF supplemented (n=3) and bFGF non-supplemented (n=3) groups. Biochemical parameters and antioxidant potential were evaluated prior to fat harvest and ADSC administration, as well as 1, 3, and 5 weeks following ADSC administration. ADSC administration regulated inflammation, renal and hepatic functions, and levels of antioxidant enzymes. The cell doubling time of the bFGF-supplemented group was shorter (P=0.0001) than that of the bFGF non-supplemented group. Renal and hepatic functions were maintained with bFGF supplementation, which possibly enhanced the effect of ADSCs. The rat model developed in the present study may promote better understanding of BA in the context of bFGF-supplemented ADSC administration.

Background Substance P (SP) may attenuate ischemia-reperfusion injury by reducing inflammation. We assessed cardioprotective effect of SP in a porcine model of acute myocardial infarction (AMI). Methods AMI was induced by occlusion of the left anterior descending artery on 28 swine, randomized to SP 5 nmol/kg (group 1, n = 14) and normal saline (group 2, n = 14) given intravenously 5 min before reperfusion. Blood samples were collected at baseline, 3 days and 4 weeks. Echocardiography and myocardial perfusion single photon emission computed tomography (SPECT) were performed at 1 week and 4 weeks. Histomorphometric infarct size assessment was done at 4 weeks. Results Left ventricular (LV) ejection fraction (EF) (LVEF) after AMI induction was higher in group 1 than group 2 (37.9 ± 4.6% vs. 29.4 ± 3.2%, p = 0.001) but not different at 4 weeks. No significant difference was observed in perfusion defect extent and total perfusion defect on SPECT at 1 week and 4 weeks. Pathologic infarct size (% LV) was significantly smaller in group 1 than group 2 (2.4 ± 2.3% vs. 5.7 ± 2.5%, p = 0.020). The ratio of neutrophil to lymphocyte on day 3 and serum creatinine concentration at 4 weeks after AMI were lower in group 1. Conclusions In a porcine model of AMI, SP improved LVEF early post-MI and reduced infarct size. SP may be beneficial in reducing inflammation and ischemia-reperfusion injury after AMI.

Preservation of pancreatic β-cells is required for the development of therapies for type 1 and type 2 diabetes (T1D and T2D, respectively). Our previous study demonstrated that substance P (SP) preserves β-cell populations in mice with streptozotocin-induced T1D. Here, we demonstrated that chronic systemic treatment with SP restored the mass of β-cells both in nonobese diabetic (NOD) mice with T1D or db/db mice with T2D. SP delayed the onset of T1D in NOD mice via immune modulation. SP inhibited immune infiltration into islets and the salivary glands of NOD mice. In db/db mice, SP treatment rescued glucose intolerance. Moreover, SP inhibited apoptosis, as well as the activation of pancreatic stellate cells in pancreatic islets of db/db mice. SP downregulated the number of α-smooth muscle actin (α-SMA) expressing cells in db/db pancreatic islets. Cleaved-caspase-3 expression was reduced in islets of SP-treated db/db mice compared to that in the control. Therefore, these results suggested that SP may preserve pancreatic β-cells through immune modulation and protection from the stimulated activation of pancreatic stellate cells and apoptosis in T1D and T2D, respectively.

Toxicity study evaluating the effects of intravenous injection of SP with single- and repeated-dose in rats was performed. Single 100, 50, and 10 mg/kg doses of SP were administered and animals were monitored for 14 days. Although 10 mg/kg dose did not affect behavior and survival, 100 and 50 mg/kg doses elicited mortality, suggesting the minimum lethal dose (MLD) of SP to be between 10 and 50 mg/kg. To evaluate the toxicity of repeated dosing, rats were injected twice first two days per week with 30, 10, and 3 mg/kg of SP over 2 weeks and evaluated for survival, behavior, hematological factors, and organ weight. All rats administered with 10 and 3 mg/kg SP survived with minor changes in behavior and hematological indices. Thus, certain poison dose was estimated to be approximately 3 mg/kg, indicating that application of SP below 3mg/kg may not be associated with toxicity.

Retinal degeneration is caused by neovascularization and persistent inflammation in the retinal pigment epithelium (RPE) and choroid, and causes serious eye disease including age-related macular degeneration (AMD). Thus, inhibiting inflammation and neovascularization may be a primary approach to protect the retina from degeneration. The purpose of this study was to determine whether substance P (SP), which can suppress inflammation and mobilize stem cells, can protect the RPE from degeneration. The effect of SP was evaluated by analyzing systemic inflammation, cell survival, and neovascularization within the argon laser-injured retina of mice. At 1 week postinjury, the SP-treated group had lower tumor necrosis factor-alpha and higher interleukin-10 serum concentrations, and a more intact retinal structure compared to the vehicle-treated group. In mice administered SP repeatedly for 4 weeks, the retinal structure appeared normal and showed sparse neovascularization, whereas the vehicle-treated group showed severe retinal destruction and dense neovascularization. Moreover, the efficacy of SP was identical to that of mesenchymal stem cells that were transplanted into the vitreous after retinal injury. This study highlights the potential for the endogenous neuropeptide SP as a treatment for retinal damage to prevent conditions such as AMD.

Chemical agents that can potentially cause skin irritation are typically tested in animal models or in vitro assays of cell viability or cytokine expression. However, these methods do not always provide translatable results and are not sufficiently sensitive for subtoxicity detection. Here, we introduce the mechanical properties of keratinocytes as novel endpoints for the safety assessment of chemical agents at the subtoxicity level. Human primary keratinocytes were treated with various concentrations of sodium lauryl sulfate (SLS) and their biological properties (proliferation, membrane integrity, inflammatory response, and morphology) were observed. Their biomechanical and geometrical parameters (stiffness and surface roughness) were also investigated by atomic force microscopy. Keratinocyte morphophysiological changes and inflammatory responses were significant at ≥25 μM SLS. The keratinocytes became less stiff due to changes in the distribution of F-actin filaments and α-tubulin; these changes were significant even at lower doses of SLS (≤10 μM). The morphophysiological changes of keratinocytes were clearly seen at a relatively high dose of SLS, while the mechanical properties of keratinocytes responded linearly to SLS at lower doses. Therefore, changes in mechanical properties can be used as new endpoints for in vitro toxicity testing with keratinocytes.

Preservation of the pancreatic β-cell population is required for the development of therapies for diabetes, which is caused by a decrease in β-cells. Here, we demonstrate the antidiabetic effects of substance P (SP) in type 1 diabetes (T1D) mice induced with streptozotocin. SP enhanced the compensatory proliferation of β-cells in order to restore β-cells in response to acute injury induced by a single high-dose of streptozotocin. However, SP affected neither the basal proliferation of β-cells nor their apoptosis. In vitro studies by using the INS-1 pancreatic β-cell line showed that SP mediated the increase in the proliferation of β-cells via the activation of Akt. Chronic systemic treatment with SP restored the mass of β-cells and inhibited insulitis in T1D mice induced with multiple low-doses of streptozotocin. Therefore, systemic treatment with SP may be a promising therapeutic strategy for treating diabetes in patients with loss of functional β-cells.

Lysyl oxidase (LOX) is a cell-secreted amine oxidase that crosslinks collagen and elastin in extracellular microenvironment. LOX-traceable nanoparticles (LOXab-NPs) consisting of LOX antibodies (LOXab) and paclitaxel, can accumulate at high concentrations at radiation-treated target sites, as a tumor-targeting drug carrier for chemotherapy. Tumor-targeting and anticancer effects of PLGA based LOXab-NPs in vitro and in vivo were evaluated at radiation-targeted site. In the in vivo A549 lung carcinoma xenograft model, we showed highly specific tumor targeting (above 7.0 times higher) of LOXab-NPs on irradiated tumors. Notably, systemically administered NPs delayed tumor growth, reducing tumor volumes by more than 2 times compared with non-irradiated groups (222% vs. >500%) over 2 weeks. Radiotropic LOXab-NPs can serve as chemotherapeutic vehicles for combined targeted chemo-radiotherapy in clinical oncology.

The retinal pigment epithelium (RPE) is a monolayer to function as a compact barrier between photoreceptor and choroid and also a regulator of the overlying photoreceptor layer by providing nutrients. If RPE is damaged, choroidal neovascularization is occurred, accompanied by inflammation, which leads to degeneration of whole retina layer and furthermore, loss of vision. Thus, the preservation of RPE layer is very critical treatment to block the progression and occurrence of diverse ocular disease, inducing age-related macular degeneration (AMD). In this study, we explored the new role of SP in RPE regeneration. After confirming the expression of NK-1R on ARPE-19 cells, we treated SP to ARPE-19 cells in vitro to evaluate the effect of SP on proliferation rate and migratory capacity. SP could promote proliferation and migration of ARPE-19 in dose-dependent manner in vitro. Based on the in vitro data, 5nmole/kg of SP was intravitreally treated to laser-induced RPE damaged mice in vivo, showing that PBS-injected mice had severed disturbed retina layer, whereas SP-injected mice maintained retina layer more intact without detachment of RPE. In addition to histological comparison, the efficacy of SP was assessed according to the score that indicate the severity of retina degeneration. This confirmed that SP can exert the beneficial effect on laser-induced RPE damage. Collectively, our findings suggest that SP can stimulate the regeneration of RPE layer and thus, it may be possible that SP is used as the new therapeutics for RPE-damaged disease including AMD.

Current rheumatoid arthritis (RA) therapies such as biologics inhibiting pathogenic cytokines substantially delay RA progression. However, patient responses to these agents are not always complete and long lasting. This study explored whether substance P (SP), an 11 amino acids long endogenous neuropeptide with the novel ability to mobilize mesenchymal stem cells (MSC) and modulate injury-mediated inflammation, can inhibit RA progression. SP efficacy was evaluated by paw swelling, clinical arthritis scoring, radiological analysis, histological analysis of cartilage destruction, and blood levels of tumor necrosis factor-alpha (TNF-α) interleukin (IL)-10, and IL-17 in vivo. SP treatment significantly reduced local inflammatory signs, mean arthritis scores, degradation of joint cartilage, and invasion of inflammatory cells into the synovial tissues. Moreover, the SP treatment markedly reduced the size of spleens enlarged by excessive inflammation in CIA, increased IL-10 levels, and decreased TNF-α and IL-17 levels. Mobilization of stem cells and induction of T(reg) and M2 type macrophages in the circulation were also increased by the SP treatment. These effect of SP might be associated with the suppression of inflammatory responses in RA and, furthermore, blockade of RA progression. Our results propose SP as a potential therapeutic for autoimmune-related inflammatory diseases.

Reconstruction of elastic cartilage requires a source of chondrocytes that display a reliable differentiation tendency. Predetermined tissue progenitor cells are ideal candidates for meeting this need; however, it is difficult to obtain donor elastic cartilage tissue because most elastic cartilage serves important functions or forms external structures, making these tissues indispensable. We found vestigial cartilage tissue in xiphoid processes and characterized it as hyaline cartilage in the proximal region and elastic cartilage in the distal region. Xiphoid process-derived chondrocytes (XCs) showed superb in vitro expansion ability based on colony-forming unit fibroblast assays, cell yield, and cumulative cell growth. On induction of differentiation into mesenchymal lineages, XCs showed a strong tendency toward chondrogenic differentiation. An examination of the tissue-specific regeneration capacity of XCs in a subcutaneous-transplantation model and autologous chondrocyte implantation model confirmed reliable regeneration of elastic cartilage regardless of the implantation environment. On the basis of these observations, we conclude that xiphoid process cartilage, the only elastic cartilage tissue source that can be obtained without destroying external shape or function, is a source of elastic chondrocytes that show superb in vitro expansion and reliable differentiation capacity. These findings indicate that XCs could be a valuable cell source for reconstruction of elastic cartilage.

Bone marrow derived mesenchymal stem cells (MSCs) have self-renewal characteristics and are able to differentiate into various cell types. These cells can mobilize to peripheral tissues in order to participate in important biological processes such as wound healing. They are also able to regulate hematopoietic stem cells (HSCs) trafficking between the bone marrow and the periphery through the regulation of intercellular interactions and the expression of cytokines, such as SDF-1. Previous studies demonstrated that substance P (SP) induces the proliferation of MSCs in vitro and mediates the migration of MSCs to injury sites to promote healing. We found that SP increases CFU-F of the bone marrow total cells and up-regulates the osteogenic differentiation potential of MSCs without affecting their adipogenic differentiation potential. We also found that SP increases the mRNA levels of SDF-1 and N-cadherin in total bone marrow. Therefore, it is very likely that SP modulates the properties of MSCs linked to their therapeutic potential and their role in trafficking of stem cells.

Previously, we have reported that substance P (SP) enhanced functional recovery from spinal cord injury (SCI) possibly by the anti-inflammatory modulation associated with the induction of M2-type macrophages at the injured lesion. In this study, we explored the cytokine expression profiles and apoptotic cell death in the lesion site of the SCI after an immediate intravenous injection of SP. SP injection increased the levels of interleukin-4 (IL-4), IL-6, and IL-10 at day 1 after the SCI approximately by 2-, 9-, and 10-folds when compared with the control SCI, respectively. On the basis of double immunofluorescence staining with IL-10 and CD11b, activated macrophages or microglia expressing IL-10 appeared in the margin of the lesion site at day 1 only after the SP injection. This SP-mediated alteration in the lesion microenvironment was shown to be associated with the lower cell death of neuronal cells at day 1 and oligodendrocytes at day 5 by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which was also accompanied by a decrease in caspase-3 activation. These findings suggest that SP may reduce the inflammation-induced secondary cell death, possibly through immune modulation at an early stage after the SCI.

Human dermal fibroblasts were inoculated into chitosan sponge scaffolds coated with type I collagen and it might be developed as a dermal substitute and/or dressing material. The application of 14% uniaxial cyclic strain to the cellular scaffolds affected the characteristics of the seeded human dermal fibroblasts. Cyclic strain enhanced cellular proliferation, the activity of metalloproteinase-2, and the expression of extracellular matrix proteins such as fibronectin. Moreover, cyclic strain increased the expression of vascular endothelial growth factor (VEGF) and interleukin (IL)-6, which are critical to wound healing. Even under static culture (strain, 0%) following 14% cyclic strain, the expression of VEGF and IL-6, which had increased under 14% strain, was amplified or maintained for at least 3 days. Uniaxial cyclic strain may enhance the wound-healing potential of human dermal fibroblasts seeded on chitosan scaffolds through the changes in the cellular characteristics of the fibroblasts when the cellular scaffold is transplanted into skin wounds, especially chronic wounds such as diabetic wounds.

Background Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. Substance P (SP) is an 11-amino-acid endogenous neuropeptide that belongs to the tachykinin family and several reports recently have supported the anti-inflammatory and tissue repairing roles of SP. Objective In this study, we investigated whether SP can improve AD symptoms, especially the impaired skin barrier function, in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced chronic dermatitis of NC/Nga mice or not. Method AD-like dermatitis was induced in NC/Nga mice by repeated sensitization with TNCB for 5 weeks. The experimental group designations and topical treatments were as follows: vehicle group (AD-VE); SP group (AD-SP); and SP with NK1R antagonist CP99994 (AD-SP-A) group. Histological analysis was performed to evaluate epidermal differentiation, dermal integrity, and epidermal nerve innervation in AD-like lesions. The skin barrier functions and pruritus of NC/Nga mice were evaluated by measuring transepidermal water loss (TEWL) and scratching behavior, respectively. Result Topical SP treatment resulted in significant down-regulation of Ki67 and the abnormal-type keratins (K) K6, K16, and K17, restoration of filaggrin and claudin-1, marked reduction of TEWL, and restoration of basement membrane and dermal collagen deposition, even under continuous sensitization of low dose TNCB. In addition, SP significantly reduced innervation of itch-evoking nerve fibers, gelatinase activity and nerve growth factor (NGF) expression in the epidermis but upregulated semaphorin-3A (Sema3A) expression in the epidermis, along with reduced scratching behavior in TNCB-treated NC/Nga mice. All of these effects were completely reversed by co-treatment with the NK1R antagonist CP99994. In cultured human keratinocytes, SP treatment reduced expression of TGF-α, but upregulated TGF-β and Sema3A. Conclusion Topically administered SP can restore normal skin barrier function, reduce epidermal infiltration of itch-evoking nerve fibers in the AD-like skin lesions, and alleviate scratching behavior. Thus, SP may be proposed as a potential medication for chronic dermatitis and AD.

Methanol extract of Koreana stewartia leaves (SKE) stimulated collagen production in ultraviolet-B (UVB)-irradiated human fibroblast cells. An active compound was isolated from SKE by successive partitioning and chromatography, and the chemical structure was determined to be 3-O-β-D-glucopyranosylspinasterol (spinasterol-Glc) by spectroscopic characterization. Spinasterol-Glc increased collagen production in the supernatant of UVB-irradiated dermal fibroblast cell cultures in a dose-dependent manner. The effects of spinasteol-Glc on expression of procollagen and matrix metalloproteinase-1 (MMP-1) were further evaluated. We found that the compound stimulated collagen production in UVB-treated fibroblasts than in vehicle-treated control cells by about 3-fold. In addition, we also demonstrate that the compound increased the mRNA and protein levels of procollagen in UVB-treated fibroblast cells, while it inhibited expression of MMP-1. These results indicate that spinasterol-Glc protects fibroblast cells from the adverse effects of UV radiation via stimulation of procollagen synthesis as well as inhibition of MMP-1 expression. Spinasterol-Glc may be useful in the future development of therapeutic and cosmetic applications.

OBJECTIVE The aim of this study was to explore the beneficial effects of SP on NO production and inflammation-induced vascular endothelium cell death. METHODS To mimic the inflammatory environment, TNF-α was treated with HUVECs, and SP was added prior to TNF-α to determine its protective effect. WST-1 assay was performed to detect cell viability. NO level in conditioned medium was measured by Griess Reagent System. The protein level of cleaved caspase-3, eNOS, and phosphorylated Akt was detected by Western blot analysis. RESULTS TNF-α declined endothelial cell viability by downregulating Akt and NO production. TNF-α-induced cell death was reliably restored by NO, confirming the requirement of NO for cell survival. By contrast, pretreatment of SP attenuated TNF-α-induced cellular apoptosis, accompanied by an increase in the phosphorylation of Akt, eNOS expression, and NO production. Blockage of NK-1R, phosphorylated Akt or eNOS by CP-96345, A6730, or L-NAME entirely eliminated the effect of SP. CONCLUSIONS SP can protect the vascular endothelium against inflammation-induced damage through modulation of the Akt/eNOS/NO signaling pathway.

TGFbeta is known to transactivate EGFR. However, the signaling component involved in this crosstalk has yet to be revealed. Here, we found that TGFbeta(1) phosphorylated EGFR in a dose-dependent manner in SCC13 and A431 cells, and it was not blocked by EGF-neutralizing antibody. H(2)O(2) was increased by TGFbeta(1) treatment in the same time-kinetics as EGFR activation. Pretreatment of N-acetyl cysteine abolished TGFbeta(1)-induced H(2)O(2) induction and EGFR activation. Direct treatment of H(2)O(2) phosphorylated EGFR and catalase inhibitor prolonged TGFbeta(1)-induced EGFR activation. These results show that TGFbeta(1) activates EGFR via an H(2)O(2)-dependent mechanism, which subsequently leads to the activation of Erk(1/2).

In the present study, we investigated the ex vivo expansion of human adipose tissue-derived mesenchymal stromal cells (ATSCs) to identify factors that promoted efficient expansion while preserving stem cell potential. We examined several growth factors and steroids, and found that the combination of a low concentration of fibroblast growth factor-2 (FGF-2) (1 ng/mL) and dexamethasone (DEX) or betamethasone (BET) enhanced the proliferation of ATSCs by approximately 30-60% as compared to control. Enhanced proliferation under these conditions was confirmed using ATSCs isolated from three independent donors. ATSCs that were expanded in the presence of FGF-2 and DEX for 5 days were capable of differentiating into either osteoblastic or adipogenic cells, and the cells were positive for the mesenchymal stem cell markers such as CD29, CD44, CD90, CD105, and CD146, suggesting that the stem cell potential of the ATSCs was preserved. Analysis of signaling pathway revealed that tyrosine phosphorylation of Src kinase was dramatically increased in response to FGF-2 and DEX, suggesting the involvement of Src-dependent pathways in the stimulatory mechanism of proliferation of ATSCs by FGF-2 and DEX. Moreover, Src family kinase inhibitors (SU6656 and Src kinase inhibitor I) substantially reduced the FGF-2 and DEX-induced proliferation of ATSCs. SU6656 also inhibited the osteogenic and adipogenic differentiation of ATSCs. The results of the current study demonstrate that FGF-2 in combination with DEX stimulates the proliferation and osteoblastic and adipogenic differentiation of ATSCs through a Src-dependent mechanism, and that FGF-2 and DEX promote the efficient ex vivo expansion of ATSCs.

Tissue injury may create a specific microenvironment for inducing the systemic participation of stromal-like cells in the repair process. Here we show that substance P is an injury-inducible factor that acts early in the wound healing process to induce CD29(+) stromal-like cell mobilization. Likewise, mobilization of such cells also occurs in uninjured mice, rats and rabbits if substance P is intravenously injected. Upon further characterization these substance P-mobilized CD29(+) cells were found to be similar to stromal cells from a number of connective tissues, including bone marrow (that is, bone marrow stromal cells, or BMSCs). Both substance P injection and transfusion of autologously derived substance P-mobilized CD29(+) cells from uninjured rabbits accelerated wound healing in an alkali burn model. Also, epithelial engraftment of the transfused cells into the injured tissue occurred during the wound healing. Finally, using human BMSCs as a test population, we show that substance P stimulates transmigration, cell proliferation, activation of the extracellular signal-related kinases (Erk) 1 and 2 and nuclear translocation of beta-catenin in vitro. This finding highlights a previously undescribed function of substance P as a systemically acting messenger of injury and a mobilizer of CD29(+) stromal-like cells to participate in wound healing.

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation-induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP-induced cellular signaling in radiation-induced damage of the intestine. Exposure of Caco-2 cells to SP increases cell proliferation and Erk phosphorylation in a time- and dose-dependent manner. The proliferation of cells exposed to gamma-irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase-3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation-damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay reveals fewer cells stained in SP-treated mice compared with untreated controls. These findings show the potential for SP-induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase-3 cleavage.

Although adipose-derived stem cells (ADSCs) have many advantageous traits compared with other postnatal stem cells, the consensus is that their differentiation potential must be improved to allow their practical utilization. During the in vitro expansion of human ADSCs (hADSCs), pre-treatment of fibroblast growth factor 2 (FGF2) not only induced an increase of approximately 44-fold in cell number at passage 7 but also augmented the differentiation potential of hADSCs. The effect of FGF2-induced cell preconditioning was evaluated by in vitro and in vivo osteogenesis after pre-treatment with various concentrations of FGF2 (0, 5, 25 ng/ml). FGF2-pre-treated hADSCs showed enhanced in vitro osteogenesis. An evaluation of in vivo osteogenic potential with an ectopic bone model showed that FGF2-preconditioned hADSCs produced an abundant osteoid/bone matrix and the effect was dependent on the concentration of FGF2 pre-treatment; bone matrix formation by control hADSCs was virtually non-existent. FGF2-pre-treated hADSCs also showed enhanced in vitro chondrogenesis, whereas no significant difference was observed in adipogenic potential. Pre-treatment of hADSCs with FGF2 induced an increase in the expression of osteogenic markers such as Cbfa1/Runx2 and alkaline phosphatase and in the expression of β-catenin. These results suggest that FGF2 plays a highly beneficial role in the preconditioning of ADSCs for musculoskeletal tissue engineering.

The effects of heat treatment of chitosan film on the proliferation of vascular cells were examined in vitro. Chitosan film was heat treated at 110 °C in vacua for 1∼3 days. Heat treatment slightly improved the hydrophilicity of chitosan, although the cross-linking of chitosan took place after heat treatment. Chitosan films were cytocompatible toward smooth muscle cells (SMCs) and human dermal microvascular endothelial cells (HDMECs) before and after heat treatment of chitosan. In particular, the heat treated chitosan supported the proliferation of both vascular cell types for one day. The growth of HDMECs was greater than that of SMCs on heat treated chitosan film for one day. Heat treated chitosan has intrinsic biological activity which could contribute to a retardation of SMC growth relative to HDMECs.

Objective The aim of this study was to evaluate the angiogenicity of a combination of BM‐EPCs and BM‐MSCs in vitro in the presence of SP and its working mechanism. Methods BM‐MSCs and BM‐EPCs were cocultured with or without SP. ELISA and RT‐PCR were performed to detect angiogenic factors such as VEGF and PDGF‐BB. N‐cadherin was detected by Western blot analysis. The tubular network‐forming ability was evaluated by a Matrigel tube‐forming assay. Results BM‐EPCs coculture with BM‐MSCs strongly stimulated the recruitment of BM‐MSCs onto the BM‐EPC‐generated endothelial tubular network. Upon SP treatment, endothelial branching point, tubule length, and tubular recruitment of BM‐MSCs were further increased and stabilized. The coculture of BM‐EPCs and BM‐MSCs synergistically stimulated expression of VEGF, VEGF receptor, N‐cadherin, and PDGF‐BB, all of which were further enhanced by SP treatment. Blockade of PDGF‐BB by its functional blocking antibodies markedly reduced the BM‐MSC incorporation into the endothelial tubules. SP‐pretreated BM‐MSCs were preferentially incorporated into the preformed BM‐EPC tubular network. Conclusions BM‐EPCs along with SP promote the pericyte‐like coverage of BM‐MSCs on endothelial tubules possibly through the induction of PDGF‐BB.

Bone marrow stem cells, especially, endothelial precursor cells (EPC), mesenchymal stem cells (MSC) or hematopoietic stem cell (HSC) are expected as reparative cells for the repair of a variety of tissue damages such as stroke and myocardial infarction, even though their role in the repair is not demonstrated. This report was investigated to find a role of Substance-p (SP) as a reparative agent in the tissue repair requiring EPC and MSC. In order to examine EPC (EPC SP ) and MSC (MSC SP ) mobilized by SP, we injected SP intravenously for consecutive 2 days and saline was injected as a vehicle. At 3 post injection, peripheral blood (PB) was collected.To get mesenchymal stem cells or endothelial progenitor cells, MNCs were incubated in MSCGM or EGM-2 respectively for 10 days. Functional characteristics of the EPC SP were proven by the capacity to form endothelial tubule network in the matrigel in vitro and in the matrigel plug assay in vivo. In contrast, MSC SP did not form a tube-like structure but formed a pellet-structure on matrigel. However, when both cells were premixed before the matrigel assay, much longer and branched tubular network was formed, in which a-SMA expressing MSC SP were decorating outside of the endothelial tube, especially enriched at the bifurcating point. MSC SP may contribute and reinforce elaborate vascular network formation in vivo by working as pericyte-like cells. Thus, the EPC SP and MSC SP were labeled with PKH green and PKH red respectively and their tubular network was examined. Well organized tubular network was formed, which was covered by PKH green labeled cells and was decorated in a punctate pattern by PKH red labeled cells. In order to investigate the role of EPC SP and MSC SP specifically in vivo, rabbit EPC SP and MSC SP were transplanted to full thickness skin wound. The vessel of EPC SP -transplanted groups was UEA-lectin+, which was not covered with a-SMA+ pericytes but EPC SP + MSC SP -transplanted groups showed, in part, a-SMA+ pericyte-encircled UEA-lectin+ vessels. This proved the specific role of MSC SP as pericytes. From these data, we have postulated that the collaboration of MSC and EPC is essential for normal vessel structure and furthermore, accelerated wound healing as ischemia diseases, which can be stimulated through by SP injection.

Mechanical stimulation is a known modulator of survival and proliferation for many cells, including endothelial cells, smooth muscle cells, and bone marrow-derived mesenchymal stem cells. In this study, we found that mechanical strain prevents apoptosis and increases the adhesive ability of dermal fibroblasts in vitro and thus confers the survival advantage in vivo after transplantation of fibroblasts into the full-thickness wound of diabetic mice. Cyclic stretch at a frequency of 0.5 Hz and maximum elongation of 20% stimulates cellular survival mediated by the activation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the serine/threonine kinase Akt (AKT). Stretching of the fibroblasts increases the synthesis of extracellular matrix proteins and the formation of denser focal adhesion structures, both of which are required for fibroblast adhesion. The stretched fibroblasts also upregulate the expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), which enhanced wound healing in vivo. Indeed, preconditioning with mechanical stretch allows better survival of the transplanted fibroblasts, when compared to unstretched control cells, in the wound environment of mice with streptozotocin-induced diabetes and thus accelerates the wound-healing process in these mice.

This study was conducted to investigate the protective role of vitamin E against the formations of lipid peroxides in plasma and tissues and of 8-hydroxydeoxyguanosine (8-OHdG) in livers of rats fed fish oil. Six-week-old male Sprague-Dawley rats were divided into four groups and fed experimental diets for 8 weeks. Three fish oil (F) groups were fed a menhaden fish oil and soybean oil (9:1) mixture as 10% (wt/wt) of their diet. These three groups (F0, FI, and FII) were provided with < or = 3, 45, and 209 IU of vitamin E/kg diet, respectively. The SI group was fed soybean oil with < or = 45 IU of vitamin E/kg diet. The F0 group had the highest levels of thiobarbituric acid-reactive substances (TBARS) in plasma (per milligram lipid), and liver, lung, heart, and kidney. The FI group had higher levels of TBARS than the SI group in plasma and tissues except the lung. In liver, the TBARS levels of the FII group were also higher than those of SI group, but in other tissues, similar levels were observed in the FII and SI groups. Plasma level of vitamin E was lowest in F0 group and vitamin E levels were generally lower in F groups than in SI group. These levels were expressed as vitamin E per milliliter of plasma. However, plasma E levels were similar when expressed per milligram plasma lipid. The liver 8-OHdG concentration tended to decrease as dietary vitamin E increased in the F groups, but there was no difference in the level of 8-OHdG between the FI and SI groups. These results suggest that vitamin E should be supplemented in fish oil feeding to prevent the enhanced lipid peroxidation and the formation of 8-OHdG in the body.

A phytosterol derivative, 3-O-β-D-glucopyanosylspinasterol (spinasterol-Glc) isolated from leaves of Stewartia koreana was reported to inhibit LPS-induced cytokine production in macrophage cells. Thymus and activation regulated chemokine (TARC/CCL17) is produced in response to pro-inflammatory cytokines in keratinocytes, which is implicated in the development of inflammatory skin diseases. In present study, we investigated the effect of spinasterol-Glc on production of TARC/CCL17 induced by TNF-α and IFN-γ in human HaCaT keratinocytes. Spinasterol-Glc inhibited the mRNA and protein expression of TARC/CCL17 induced by TNF-α/IFN-γ in a dose-dependent manner. Inhibitors of c-Raf-1, p38 MAPK, and JAK2, suppressed the TNF-α/IFN-γ-induced production of TARC/CCL17, and phosphorylation of these signaling molecules were attenuated by spinasterol-Glc. The compound also inhibited phosphorylation of IKKα/β and IκB-α, and reduced translocation of NF-κB to the nucleus. We demonstrated that spinasterol-Glc suppressed the NF-κB-driven and the GAS-driven expression of luciferase reporter gene induced by TNF-α and IFN-γ. In addition, spinasterol-Glc inhibited the DNA binding of NF-κB and STAT1 to its cognate binding site. These results suggest that spinasterol-Glc has effective inhibitory effects on production of TARC/CCL17 in keratinocytes via inhibition of NF-κB as well as STAT activation, and could be utilized for development of a potential therapeutic agent against skin inflammatory diseases.

The potential benefits or the tissue-damaging effects of inflammatory response after central nervous system injuries have long been disputed. Recent studies have noted that substance P (SP), a neuropeptide, plays an important role in the wound-healing process by recruiting bone marrow stem cells to the injured tissue. In this study, we examined whether SP can enhance recovery from spinal cord injury (SCI) in Sprague-Dawley rats through its known function of stem cell mobilization and/or through the modulation of inflammation. We examined proinflammatory and anti-inflammatory cytokines and markers for macrophage subtypes. SP treatment modulated the SCI microenvironment toward a more anti-inflammatory and reparative one by inducing interleukin-10 and M2 macrophages and suppressing inducible nitric oxide synthase and tumor necrosis factor-α. This modulation was achieved at 1 day much earlier than SP-stimulated bone marrow stem cells' mobilization. Early intervention of the devastating inflammatory response by SP treatment caused the lesion cavity to become filled with robust axonal outgrowth that overlaid the M2 macrophages at 2 weeks--all of which culminated in tissue sparing and improvement in functional recovery from the SCI. SP is therefore a potential anti-inflammatory modulator for the treatment of injury-induced inflammatory central nervous system disorders.

We induced human melanocyte dedifferentiation to Schwann cell-like cells in vitro by a combination of forskolin, neuregulin-β1, neurotrophin-3, platelet-derived growth factor-aa, basic fibroblast growth factor, laminin, and heparin. Cultured human melanocytes constitutively expressed neural cell and melanocyte markers but melanocyte-specific marker, including microphthalmia-associated transcription factor and tyrosinase, expression was selectively lost after induction. In the sciatic nerve injury site, the induced cells were engrafted and closely aligned to axons and P0-expressing myelin sheaths, whereas uninduced cells were not colocalized with axons and myelin sheaths and reexpressed melanocyte-specific tyrosinase activity in vivo. Human melanocytes lose their melanocyte phenotype and transdifferentiate into Schwann cells under specific induction conditions and display their Schwann cell phenotype after transplantation to injured sciatic nerve tissue.

Tissue injury may bring up the systemic participation of bone marrow stem cells in the repair. In this study, we report that substance-p is a systemically acting messenger to mobilize mesenchymal stem cell (MSC) probably from the bone marrow and be engaged in the wound healing process. This was supported by the injury-inducible substance-p detection in the tissue and the peripheral blood and subsequent MSC mobilization in the alkali-burn rabbit eye model, whose kinetics were dependent on the wound size. Furthermore, i.v. injection of exogenous substance-p stimulated MSC mobilization in the uninjured rabbits and those mobilized cells showed multipotent differentiation capacity. We demonstrated that earlier substance-p elevation in the circulation by substance-p injection or transfusion of autologous PKH-labeled MSC mobilized by substance-p strongly accelerated the wound healing of the alkali-burned rabbit eye and the transfused MSCs were engrafted to the epithelial and stromal layer of the injured tissue, suggesting their epithelial transdifferentiation in the regenerating tissue. Finally, we showed that substance-p stimulated transmigration of human MSC with concomitant induction of MMPs and inhibition of their inhibitors, cell proliferation, ERK1/2 activation, and nuclear translocation of [beta]-catenin in vitro. In conclusion, substance-p plays a role as a systemic wound-messenger to promote the MSC mobilization from the marrow and their participation in the wound healing possibly through its stimulant effect on MSC repopulation as well.

Costal cartilage has been proposed as an alternative donor of chondrocytes for articular-cartilage repair. In the present study we compared the initial cell yield of chondrocytes from rabbit costal cartilage and their cell expansion rates in monolayer culture with those of articular cartilage. Costal cartilage gave an approx. 2.6-fold higher cell yield than did articular cartilage. During in vitro culture, CCs (costal chondrocytes) grew faster and displayed approx. 3-fold more cell expansion up to P4 (passage 4) than did ACs (articular chondrocytes). In order to match the degree of dedifferentiation during serial cultivation with the cells' expansion rate, type II collagen expression and the emergence of fibroblastic morphology were monitored at each cell passage. Both ACs and CCs gradually lost their chondrocytic phenotype, changed to fibroblast-like cells and displayed a reduced expression of type II collagen. We then also evaluated the redifferentiation capacity of the expanded ACs and CCs by culturing them at high density in collagen gel. Almost fully dedifferentiated CCs at P4 were successfully redifferentiated into hyaline cartilage, which showed the expression of glycosaminoglycan and type II collagen as well as the formation of lacunae and a territorial matrix. In conclusion, costal cartilage may have advantages over articular cartilage as an alternative donor tissue for autologous chondrocytes on the basis of its higher cell yield, higher cell expansion and successful reversion into hyaline cartilage without ossification in vitro. However, although this experiment with a rabbit model gave a better insight into the problem than other experiments have done, it does not answer definitively the question as to which cells are most appropriate for articular cartilage repair in humans.

We recently showed that intravenous treatment of Substance P (SP) after spinal cord injury increased the number of alternatively activated M2 macrophages at injured lesion of spinal cord and eventually promoted tissue repair. However, it was unclear enhanced M2 polarization of differentiated macrophages is resulted from SP-mediated signaling pathway or accompanied by immune modulatory actions of SP. In the present study, we aim to elucidate the direct effect of SP in M2 polarization in tissue macrophages. Rat macrophages (MO), differentiated with GM-CSF treatment for 5 days on bone marrow-derived monocytes (MO) were prepared. Immunofluorescence staining with specific marker for monocytes and macrophages, MO was characterized as ED-1 (CD68, a marker for macrophages) and CCR7-positive M1-like precursor differentiated from CD14 and CD11b-positive bone marrow monocytes. Administration of SP in MO enhanced the expression of M2 marker, CD206 and CD163 concurrently with the reduction of M1 marker, CCR7 and CD68. M2 polarization of MO in response to SP was completely blocked by NK1-R antagonist, RP67580, and PI3K inhibitor, LY294002. Furthermore, SP also showed transforming ability of M1-polarized MO induced by IFN toward M2 type with induction of CD163 expression. In summary, the results of the present study demonstrate that SP directly polarizes M2 macrophages of MO and IFN-primed M1 type and acts as a Th2 cytokine.

8-Hydroxydeoxyguanosine (8-OHdG), an oxygen radical induced modification of purine residue in DNA, was measured in the liver, pancreas, and kidney of streptozotocin-induced diabetic rats (STZR) exhibiting microalbuminuria. At 4 weeks after the injection of streptozotocin (50 mg/kg, i.v.), the rate of urinary albumin excretion was 0.5 +/- 0.1 and 2.0 +/- 0.2 mg/24 h in age-matched control rats (CR) and STZR, respectively. Compared to CR, STZR also showed a significantly increased level of 8-OHdG in the kidney but not the liver and pancreas. Amounts of 8-OHdG/10(5) dG for CR and STZR were 3.4 +/- 0.3 and 5.1 +/- 0.2 for renal cortices, and 4.1 +/- 0.2 and 20.0 +/- 3.7 for renal papillae. Daily injection of insulin (2 U, SC) starting on the third day after streptozotocin treatment significantly reduced both urinary albumin excretion and papillary 8-OHdG formation, which suggests that these are associated with the diabetic state induced by streptozotocin rather than a direct nephrotoxic effect of the drug. This study suggests that formation of 8-OHdG and, therefore, oxidative damage are closely related in the process of diabetic nephropathy.