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Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. In this study, topically applied substance P (SP) significantly alleviated AD-like clinical symptoms in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced dermatitis in NC/Nga mice. This effect was nullified by pretreatment of the neurokinin-1 receptor (NK-1R) antagonist CP99994. SP treatment significantly reduced the infiltration of mast cells and CD3-positive T cells as well as inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and thymic stromal lymphopoietin (TSLP), in AD-like skin lesions and decreased the levels of IgE and thymus and activation-regulated chemokine in serum. This SP-induced alleviation of allergic inflammatory responses was also confirmed as reduced activation in the axillary lymph nodes (aLN) and spleen, suggesting the systemic effect of SP on immune responses in TNCB-induced NC/Nga mice. Furthermore, SP-mediated TSLP reduction was confirmed in human keratinocyte culture under pro-inflammatory TNF-α stimulation. Taken together, these results suggest that topically administered SP may have potential as a medication for atopic dermatitis.

Macrophage polarization plays an important role in tissue damage and repair. In this study, we show that Substance-P (SP) can directly induce M2 polarization of inflammatory macrophages. SP induced the differentiation of GM-CSF-differentiated pro-inflammatory macrophages into alternatively activated phagocytic M2 like macrophages (M2SP) through direct activation of the PI3K/Akt/mTOR/S6kinase pathway and induction of Arginase-1, CD163, and CD206, all of which were nullified by pretreatment with the neurokinin-1 receptor (NK-1R) antagonist RP67580 and specific signaling pathway inhibitors. M2SP were distinct from IL-4/IL-13-induced M2a and IL-10-induced M2c subtypes; they did not show STAT activation and exhibited high phagocytic and endothelial adhesive activity. Furthermore, SP had a dominant effect on M2 polarization over Interferon gamma (IFNγ), a potent M1-skewing cytokine, and effectively induced the M2 phenotype in monocytes and the human THP-1 cell line. Finally, adoptively transferred M2SP migrated to a spinal cord injury (SCI) lesion site and improved functional recovery. Collectively, our findings show that SP, a neuropeptide, plays a role as a novel cytokine by inducing tissue-repairing M2SP macrophages and thus may be developed for pharmacological intervention in diseases involving chronic inflammation and acute injury.

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture.

Dextran sulfate sodium (DSS) has been used to destroy intestinal epithelium structure and impair barrier function to mimic clinic intestine damages, which causes the intestinal inflammation and furthermore, tissue destruction. In this study, we have attempted to damage intestinal epithelium by treating DSS to Caco-2, human intestinal epithelial cell line, and elucidated the recovery effect of SP in vitro. DSS altered cellular morphology with the loss of TJ and reduced cell viability. Notably, DSS arrested cell cycle to decrease proliferating cell pool. However, SP treatment could preserve cellular morphology and TJ expression level similar to the normal even under DSS-induced toxicity. SP was also able to enhance cell survival, accompanied by the accelerated cell proliferation. Collectively, our study revealed SP can block the intestine damage due to toxicity of DSS. Our results provide the possibility of SP as a potential therapeutic for intestinal damage.

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.

Substance-P (SP) can function of mobilizing mesenchymal stem cells (MSCs) from the bone marrow to the circulation and exert anti-inflammatory effects by increasing anti-inflammatory M2-type macrophage and regulatory T cells in the circulation. The preclinical efficacy of SP was demonstrated in a variety of conditions including ischemia damages, but its safety was not assessed. In this study, we assessed the genotoxicity of SP by using in vitro bacterial reverse mutation assay, chromosomal aberration assay, and in vivo micronucleus test. The maximum test dose of SP was 250 μg/mL in a cell-based assay and 16.6 mg/kg for an in vivo test. Ames test revealed SP did not increase in the number of revertant colonies. An in vitro chromosomal aberration assay with Chinese hamster lung cells showed that SP was not clastogenic even at the maximum dose applied. In vivo micronucleus test also demonstrated that SP did not induce micronuclei formation in the bone marrow cells of male ICR mice. Taken together, our results suggest that SP is not mutagenic to bacterial cells. Moreover, SP does not cause chromosomal damage in mammalian cells either in vitro or in vivo.

In this study, we developed the disc-type bio-cartilage reconstruction strategies for transplantable hyaline cartilage for reconstructive surgery using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes. We compared chondrogenesis efficiency between different chondrogenic-induction methods such as micromass culture, pellet culture, and 3D-cell sheet culture. Among them, the 3D-cell sheet culture resulted in the best chondrogenesis with the disc-type bio-cartilage (>12 mm diameter in size) in vitro, but sometimes spontaneous curling and contraction of 3D-cell sheet culture resulted in the formation of bead-type cartilage, which was prevented by type I collagen coating or by culturing on amniotic membrane. Previously, it was reported that tissue-engineered cartilage reconstructed in vitro does not maintain its cartilage phenotype after transplantation but tends to transform to other tissue type such as bone or connective tissue. However, the disc-type bio-cartilage of 3D-cell sheet culture maintained its hyaline cartilage phenotype even after exposure to the osteogenic-induction condition in vitro for 3 weeks or after the transplantation for 4 weeks in mouse subcutaneous. Collectively, the disc-type bio-cartilage with 12 mm diameter can be reproducibly reconstructed by the 3D-cell sheet culture, whose hyaline cartilage phenotype and shape can be maintained under the osteogenic-induction condition as well as after the transplantation. This disc-type bio-cartilage can be proposed for the application to reconstructive surgery and repair of disc-type cartilage such as mandibular cartilage and digits.

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.

Since clinical application of MSCs requires long-term ex vivo culture inducing senescence in MSCs and reducing the therapeutic activity of transplanted MSCs, numerous efforts have been attempted to sustain the active state of MSCs. Substance P (SP) is a neuropeptide that functions to activate the cellular physiological responses of MSCs, including proliferation, migration, and secretion of specific cytokines. In this study, we explored the potential of SP to restore the weakened immune modulating activity of MSCs resulting from long-term culture by measuring T cell activity and interleukin-2 (IL-2) secretion of CD4(+) Jurkat leukemic T cells and primary CD4(+) T cells. As the number of cell passages increased, the immunosuppressive function of MSCs based on T cell activity decreased. This weakened activity of MSCs could be restored by SP treatment and nullified by co-treatment of an NK1 receptor blocker. Higher levels of transforming growth factor beta 1 (TGF-β1) secretion were noted in the medium of SP-treated late passage MSC cultures, but IL-10 levels did not change. SP-treated MSC-conditioned medium decreased T cell activity and IL-2/Interferon gamma (IFN-g) secretion in T cells even in the activation by lipopolysaccharide (LPS) or CD3/CD28 antibodies, both of which were successfully blocked by inhibiting the TGF beta signaling pathway. This stimulatory effect of SP on late passage MSCs was also confirmed in direct cell-cell contact co-culture of MSCs and CD4(+) Jurkat T cells. Collectively, our study suggests that SP pretreatment to MSCs may recover the immunosuppressive function of late passage MSCs by potentiating their ability to secrete TGF-β1, which can enhance the therapeutic activity of ex vivo expanded MSCs in long-term culture.

Here we compared MSCs isolated from bone marrow (BMSC) and peripheral blood at 2 days after intravenous substance P (SP) injection (MSCSP) to define specific roles of circulating MSCs in tissue repair process. The mass analysis of changed genes over and under log2 fold showed 97% of genes were identical in these two groups. We found MSCSP increased the molecular signatures related to homing and engraftment (tetraspanin 8, tetraspanin 12), extracellular matrix (ECM) (collagen type IV α1, nidogen 2, laminin-α5, basement membrane components; collagen type XII α1, collagen XV α1, ECM components linking basement membrane to stroma), transmigration (ICAM-1, P-selectin, endothelin), motility (Rho GTPase activating protein 8, myosin heavy chain 10 and 11, troponin C type 1 and 2), transdifferentiation potential (keratin 19, CMTM 8), immunity (IL-1α, IL-33, IL-1R type 1), and angiogenesis (endothelin1, VEGF-C, IL-33, laminin-α5) according to functional classification of the 3 % of changed genes. Functional differences were also shown in MSCSP in regulation of viability of immune cells, Jurkat T cells and U937 monocytes. In addition, a-smooth muscle actin (α-SMA) significantly increased in MSCSP compared with BMSC, demonstrating committed differentiation to myofibroblast participated in tissue repair. However, intrinsic differentiation potential to mesenchymal tissues, adipogenesis and osteogenesis, was not altered in MSCSP. From the molecular and cellular phenotyping of MSCSP, circulating MSCSP seems to be a distinct population mobilized from heterogenous BMSC with well adapted for the participation in tissue repair process by enhancing homing, engraftment, migration, and angiogenesis with multipotent differentiation potential to either myofibroblasts or epithelial-like cells.

In almost all human tissues and organs, adult stem cells or tissue stem cells are present in a unique location, the so-called stem cell niche or its equivalent, continuously replenishing functional differentiated cells. Those endogenous stem cells can be expanded for cell therapeutics using ex vivo cell culture or recalled for tissue repair in situ through cell trafficking and homing. In the aging process, inefficiency in the endogenous stem cell–mediated healing mechanism can emerge from a variety of impairments that accumulate in the processes of stem cell self-renewal, function, differentiation capacity, and trafficking through cell autonomous intrinsic pathways (such as epigenetic alterations) or systemic extrinsic pathways. This review examines the homeostasis of endogenous stem cells, particularly bone marrow stem cells, and their dysregulation in disease and aging and discusses possible intervention strategies. Several systemic pro-aging and rejuvenating factors, recognized in heterochronic parabiosis or premature aging progeroid animal models, are reviewed as possible anti-aging pharmaceutical targets from the perspective of a healthy environment for endogenous stem cells. A variety of epigenetic modifications and chromosome architectures are reviewed as an intrinsic cellular pathway for aging and senescence. A gradual increase in inflammatory burden during aging is also reviewed. Finally, the tissue repair and anti-aging effects of Substance-P, a peptide stimulating stem cell trafficking from the bone marrow and modifying the inflammatory response, are discussed as a future anti-aging target.

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.

Mesenchymal stem cells (MSCs), which are multipotent and have self-renewal ability, support the regeneration of damaged normal tissue. A number of external stimuli promote migration of MSCs into peripheral blood and support their participation in wound healing. In an attempt to harness the potential beneficial effects of such external stimuli, we exposed human MSCs (hMSCs) to one such stimulus—low-dose ionizing radiation (LDIR)—and examined their biological properties. To this end, we evaluated differences in proliferation, cell cycle, DNA damage, expression of surface markers (CD29, CD34, CD90, and CD105), and differentiation potential of hMSCs before and after irradiation with γ-rays generated using a 137CS irradiator. At doses less than 50 mGy, LDIR had no significant effect on the viability or apoptosis of hMSCs. Interestingly, 10 mGy of LDIR increased hMSC viability by 8% (p

Purpose. The fate and function of the induced Schwann cells (iSCs) like cells from adipose tissue have not been critically evaluated in vivo after transplantation. The objective of this study is to compare the fate of iSCs with naïve SCs (nSCs) after transplantation into the lesion sites of sciatic nerve, respectively. Methods. Adipose-derived stem cells from eGFP-expressing transgenic rat’s subcutaneous fat were induced to iSCs in vitro. iSCs were injected to the sciatic nerve lesion area after crush injury and the cells fate was comparatively analyzed with that of nSCs from the same rat. Results. At 12 weeks after transplantation, nSCs were detected only in the restricted area of cell transplantation site but iSCs were widely distributed all over the sciatic nerve. Based on double fluorescence observations, both iSCs and naïve ones were colocalized with P0-expressing myelin sheath, outbound by laminin-expressing basal membrane, and terminated at contactin-associated protein-expressing doublets. However, some of iSCs were also differentiated to the fibrocyte/fibroblast-like cells. In the histological analysis of repaired sciatic nerves, axon density was higher in iSC-received group than in the nSCs group and normal sciatic nerve. Conclusion. iSCs induced from subcutaneous fat tissues have higher engraftment and migration capacity than nSCs.

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.

Tissue repair post injury is a multi-step wound healing process and consists of inflammatory, proliferative, and remodeling phases, which are coordinated by interplays between many resident and infiltrating cells as well as growth factors and extracellular matrix proteins. Macrophages are major cell populations regulating inflammation by initiation, propagation, and resolution. Activated macrophages that are differentially polarized, either classically or alternatively, are also called pro-inflammatory M1 and anti-inflammatory M2, respectively. These macrophages participate in the whole process of tissue repair. The contribution of multi-potent mesenchymal stem cells (MSCs) in tissue repair, either endogenous or therapeutically injected, has been addressed in a variety of degenerative and inflammatory disease models. Initially, the role of MSCs in the tissue repair process was restricted to the replacement of dysfunctional cells by either mesenchymal differentiation or transdifferentiation into other lineages. However, growing evidence has clearly defined MSCs as beneficial cells in tissue repair via immunomodulatory functions. In this review, we intend to summarize recent important observations in two essential cell components, MSCs and macrophages, in the context of tissue repair. Individual roles of MSCs and macrophages as well as the crosstalk between these two types of cells are discussed. The roles of both MSC-educated macrophages and macrophage-associated MSCs during tissue repair are also discussed.

We previously reported that wound fluid generated from injured cornea at 5 day after injury had ability to induce mesenchymal to epithelial transition (MET) of circulating mesenchymal stem cells (MSCs). In the present study, we further analyzed wound fluid by cytokine array. Comparing with normal cornea, increased expression of chemokines CXCL2, CXCL3, CXCL5, CCL2, anti-inflammatory cytokine IL-10, growth factors VEGF and β-NGF, protease inhibitor TIMP-1 were observed in the wound fluid of injured cornea. Interestingly, the inductions of inflammatory cytokines, IL-1α, IL-1β, IL-4, IL-6, and TNFα were not detected. It suggests wound fluid at 5 day after injury consisted of beneficial mediators for wound healing. In particular, chemokine CXCL5 increased over 6 fold. We further analyzed the roles of CXCL5 using MSCs, isolated from bone marrow and human umbilical vein endothelial cells (HUVECs). Administration of CXCL5 significantly enhanced motility of MSCs as well as showed chemotactic effects on MSCs. Moreover, CXCL5 increased the endothelial proliferation and vascular density and spouting determined by vessel formation assay. Taken together, wound fluids at day 5 after injury are the pool of beneficial mediators for wound healing process.

Mesenchymal stem cells (MSCs) have been used to treat patients with incurable diseases. However, the low yield of MSCs even after long periods of ex vivo culture remains a serious problem. Several attempts were made to increase cell expansion rate of MSCs in cultures. In this study, we examined the stimulatory effect of monocytes on the proliferation and colony forming efficiency of MSCs by co-culturing CD29+/CD45-MSCs with CD45+/ CD11b+ monocytes. In presence of CD45+/CD11b+ monocytes, CD29+/CD45-MSCs proliferated rapidly to form large and compact colonies within 10 days. In the absence of CD45+/CD11b+ monocytes, CD29+/CD45-MSC colony formation was much delayed, indicating that the presence of CD45+/CD11b+ monocytes is required for CD29+/ CD45-MSC proliferation and stemness in vitro. Similar growth-stimulating effect on MSC culture was also found when conditioned medium from CD45+/CD11b+ monocyte cultures was supplied instead of CD45+/CD11b+ monocytes. These results suggest that secretory molecules of CD45+/CD11b+ monocytes may mainly mediate the enhanced growth of CD29+/CD45-MSCs. Collectively, this study reveals that co-culture of CD29+/CD45-MSCs with CD45+/CD11b+ monocytes stimulate MSC growth via paracrine factors, which could improve colony forming capacity of MSC. Furthermore, conditioned medium of CD45+/CD11b+ monocytes may also be developed for cell culture supplement for MSC cell therapeutics.

Stewartia koreana (S. koreana) has been used in the treatment of inflammatory diseases, such as acute gastroenteritis and aches, in Korean folk medicine and has been reported to have a number of biological activities, such as anti-inflammatory activity and the promotion of angiogenesis. In this study, we aimed to determine the effects of S. koreana extract (SKE) and its components on dermal fibroblast growth and migration, and to investigate the wound healing activity of the extract in mice. In vitro experiments revealed that the numbers of SKE-treated cells increased by approximately 2.5 - and 3.7-fold with 50 and 100 µg/ml of SKE, respectively. 5-bromo-2'-deoxy-uridine (BrdU) incorporation was also increased in the SKE-treated cells by 2.3-fold. SKE promoted the migration of human skin fibroblasts and, among the isolated compounds, hyperin increased the proliferation and migration of the fibroblasts to almost the same degree as SKE. Western blot analysis demon- strated that SKE stimulated the MEK/ERK1/2 and PI3K/Akt signaling pathways. In in vivo experiments, the SKE-treated wound lesions of mice decreased by approximately 7% in diameter after 2 days of treatment with SKE compared with the wound lesions on the 1st day of the experiment. On the 9th day of treatment, the diameter of the lesions was further reduced by approximately 83% in the SKE-treated wound areas compared with the wound areas on the 1st day of treatment. Our results demonstrate that methanol extracts of S. koreana leaves promote the proliferation and migration of skin fibroblasts and possess effective wound healing activity through the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways. Hyperin was identified as an active compound responsible for the stimulation of fibroblast growth and migration.

We developed in vitro test model for assessing skin inflammation. This model consisted with co-culture of a stratified 3D skin and RAW264.7 cells in transwell. The defined expression of Keratin 5 and 10 was observed in the basal layer and suprabasal layer of the epidermis in the 3D skin, respectively. Skin barrier, determined by immunoreactivity of involucrin and filaggrin was well developed in the 3D skin. Importantly, elevated IL-1α which plays an essential role in the maintenance of skin barrier function and TNF-α in the dermal layer was detected, suggesting immunologically active reconstitution of the skin. RAW264.7 cells were morphologically transformed in response to lipopolysaccharides (LPS) either vacuolated or spindle shaped, which was accompanied with the induced expression of inducible nitric oxide synthase (iNOS), cycloxygenase-2 (COX-2), and cytokines. However, topical administration of LPS to the co-culture system reduced LPS-responding cells, which accompanied with COX-2 reduction. In addition, impaired production of TNF-α and reduction of IL-1α were observed in co-culture system. It represents stratified 3D skin actively played as a skin barrier and provided innate immunity in response to LPS. Eventually, the immune modulating activity of 3D skin alleviated LPS-induced immune responses of RAW264.7 cells alone. We propose that this co-culture system for assessing skin inflammation in vitro and this model could serve as an alternative to animal experiments for predicting skin sensitization potential.

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.

We previously reported that substance-P (SP) mobilized mesenchymal stem cells (MSCSP) from bone marrow and recruited them to the injured site, which then participated in wound healing process by incorporation into the corneal epithelium in a rabbit alkali burn model. In the present study, we investigated if the injured microenvironment could stimulate the recruited MSCSP to lose their mesenchymal phenotype and then acquire epithelial phenotype or not. Especially, we focused on the effects of secreted soluble tissue factors of wound microenvironment in the rat cornea wound model. Priming the circulating MSCSP with conditioned medium of the injured tissue at day 5 elicited morphological and molecular changes similar to those noted during mesenchymal to epithelial transition (MET), which was accompanied by the reduction of alpha-smooth muscle actin (α-SMA), a typical mesenchymal marker, and induction of E-cadherin, a typical epithelial marker. Furthermore, these primed MSCSP rearranged their actin cytoskeletal system from distinct actin stress fiber projection to the cell-to-cell-contact-enriched actin microfilament. The primed cells also had increased survival in keratinocyte growth medium (KGM). In conclusion, MSCSP could be differentiated in vitro to the epithelial cells under the injured microenvironment, which may also occur in vivo during tissue repair.

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.

Bone marrow is a reservoir for hematopoietic stem cells, endothelial precursor cells, and bone marrow stromal cells (also generally called mesenchymal stem cells), whose positive role in tissue repair is highly anticipated. In this report, we introduce a novel function of substance-P (SP), an 11-amino-acid peptide, as an injury-inducible messenger to mobilize bone marrow stem cells to the blood and finally to engage in tissue repair. This new drug may substitute for ex vivo cell culture of therapeutic cells by stimulating cell proliferation in the bone marrow in vivo and mobilizing those therapeutic cells to the patient's own blood stream. Again, the additional role of SP in mitigating inflammation-mediated tissue damage can further rationalize the clinical development of SP-peptide as a stem cell stimulant.

The promoter of pre-B cell stage-specific Crlz1 gene, whose protein translocates the cytoplasmic core binding factor β (CBFβ) into the nucleus and thereby allows its heterodimerization with Runx, has a very strong activity, which is about 25% of cytomegalovirus (CMV) promoter activity and comparable to the EF-1α promoter activity. Its transcription start site was mapped at 155 nt upstream of translation initiation codon. 5'-truncation analysis of charged amino acids rich leucine zipper 1 (Crlz1) promoter revealed that one distal region from -612 to -536 and one proximal region from -198 to -100 as numbered from the transcription start site were critical for the promoter activity. The 3'-truncation analysis of the promoter revealed that the basal promoter sequence around the transcription start site, which should be necessary for the assembly of transcription initiation complex and the start of RNA polymerase II, was also essential, although not sufficient by itself. When transcription factor binding sites within those two critical regions were searched by in vivo footprinting, one distal LEF-1 and multiple proximal Ets consensus-like sites were found to be footprinted. Indeed, the protein causing a footprint over the distal region was found to be LEF-1, and the ones causing three footprints over the proximal region were found to be such Ets family members as Fli-1 and GABP, as verified by EMSA and ChIP analyses. Furthermore, those LEF-1 and Ets sites were shown to drive additively a strong transcription of Crlz1 gene.

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.

In the present study, we found that nestin-expressing spheroid cells derived from multipotent adipose stem cells of subcutaneous fat tissue could efficiently differentiate into Schwann cells (SCs) in vitro based on expression of SC markers such as A2B5, GFAP, O4, p75, S100, Sox10, Krox-20, and L1. The induced SC is engrafted to spinal cord injury lesions and formed a peripheral nervous system (PNS)-type myelin sheath on central nervous system (CNS) axons. PNS-type myelin sheath formation in repaired tissue was confirmed by transplantation of both induced PKH26-labeled SC and induced EGFP-expressing SC generated from EGFP transgenic rats. In addition to direct participation as myelin sheath-forming SC in repaired tissue, the induced SC also expressed several neurotrophic factors, as did native SC, which may suggest an additional role for induced SC in stimulation of endogenous healing responses. Thus, spheroid-forming cells from subcutaneous fat tissue demonstrated rapid and efficient induction into SC, and such cells show therapeutic promise for repair of damage to the CNS and PNS.

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.

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.

Endogenous adult or tissue stem cells reserved in the tissue and organs of the body may be utilized for tissue regeneration in situ, without elaborate ex vivo cell culture if small molecules or growth factors regulating stem cell self-renewal and trafficking are identified. Those candidate molecules may be more efficiently and immediately treated to the patients with emergency care, such as strokes and acute myocardial infarction, by utilizing patients' own stem cells through a mechanism of stem cell mobilization and homing. Several growth factors and peptides, such as stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), and Substance-P, known to mobilize progenitor cells like hematopoietic stem cells (HSCs), endothelial precursor cells (EPCs), and bone marrow stromal cells (BMSCs) from the bone marrow and facilitate tissue repair in well-defined animal models, will be reviewed in the context of stem cell trafficking and tissue repair, and a couple of experimental strategies to monitor kinetics of stem cell mobilization and homing using animal injury models will be introduced.

Background Atopic dermatitis (AD) is a Th2-type disease. Keratinocytes, a major type in the skin, produce Th2 chemokines such as thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22, which play pivotal roles in the development of Th2-dominant inflammatory skin diseases. Recently, it was reported that 5,6-dihydroergosterol-glucoside (DHE-Glc) was synthesized and exhibited strong anti-inflammatory activity. Objective We aimed to investigate the effects of DHE-Glc, a synthetic molecule derived from ergosterol, on AD-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) in mice and to elucidate the effects of DHE-Glc on TNF-α/IFN-γ-induced production of CCL17 and CCL22 in human keratinocytes (HaCaTs) and DNCB induced skin inflammation mice model. Method Mice were sensitized and challenged on the skin of their backs with DNCB. At 30–60 days after sensitization, mice were treated with cutaneous administration of DHE-Glc by skin smear. HaCaT cells were used to evaluate the effects of DHE-Glc on production of CCL17 and CCL22 and investigate mechanisms of action by RT-PCR, ELISA, Western blot, and reporter assays. Result Topical administration of DHE-Glc attenuated AD-like skin inflammatory symptoms. DHE-Glc decreased infiltration of epidermal eosinophils and mast cells, and reduced levels of IgE, histamine, and mRNA expression and protein levels of CCL17/CCL22 in the plasma of DNCB-treated animals. In addition, DHE-Glc suppressed TNF-α/IFN-γ-induced expression of the Th2 chemokines CCL17 and CCL22 by inhibiting NF-κB and STAT activation in TNF-α/IFN-γ-induced HaCaT cells. Conclusion DHE-Glc improved AD-like skin inflammatory symptoms on the backs of DNCB-induced mice, partly by suppressing production of Th2 chemokines, CCL17 and CCL22 in inflamed skin. Therefore, DHE-Glc is a potential therapeutic agent for skin inflammatory diseases such as AD.

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.

Six6, a sine oculis homeobox protein, plays a crucial and conserved role in the development of the forebrain and eye. To understand how the expression of Six6 is regulated during embryogenesis, we screened ∼250 kb of genomic DNA encompassing the Six6 locus for cis-regulatory elements capable of directing reporter gene expression to sites of Six6 transcription in transgenic mouse embryos. Here, we describe two novel enhancer elements, that are highly conserved in vertebrate species and whose activities recapitulate Six6 expression in the ventral forebrain and eye, respectively. Cross-species comparisons of the Six6 forebrain enhancer sequences revealed highly conserved binding sites matching the consensus for homeodomain and SoxB1 transcription factors. Deletion of either of the binding sites resulted in loss of the forebrain enhancer activity in the ventral forebrain. Moreover, our studies show that members of the SoxB1 family, including Sox2 and Sox3, are expressed in the overlapping region of the ventral forebrain with Six6 and can bind to the Six6 forebrain enhancer. Loss of function of SoxB1 genes in vivo further emphasizes their role in regulating Six6 forebrain enhancer activity. Thus, our data strongly suggest that SoxB1 transcription factors are direct activators of Six6 expression in the ventral forebrain.

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.

Adipose stem cells (ASCs) are one of heterogeneous mixed populations in the stromal vascular fraction of adipose tissue. Recently, ASCs become attractive alternative for autologous neural cells due to its abundance, easy accessibility, and several successful inductions to neural and glial cells. In this review, we summarized recent progresses in identification of primary cell types in adipose tissue and their developmental origin, a variety of different induction protocols, characterization of induced neural cells and glial cells, and the fate and outcomes of transplanted induced cells in the injured tissue of the peripheral nerve system and central nerve system. Although the differentiation of ASCs to neuron did not achieve remarkable success, Schwann cell differentiation of ASCs in vitro and in vivo and beneficial roles of ASCs in reduction of inflammation and apoptosis, enhanced angiogenesis, and nerve regeneration promise future alternatives to naive neural and glial cells for neural disease or disorder treatments.

CCL23 is a CC chemokine and exerts its biological activities on endothelial cells as well as on immune cells through CCR1. We investigated the potential effect of CCL23 on expression of KDR/Flk-1 receptor in endothelial cells. PCR, confocal microscope and Western blot analysis revealed that CCL23 up-regulated KDR/Flk-1 mRNA and protein levels in endothelial cells. A reporter assay indicated that CCL23-induced KDR/Flk-1 expression primarily occurred at the transcriptional level. In addition, CCL23 stimulated phosphorylation of SAPK/JNK, and an inhibitor of SAPK/JNK blocks the CCL23-induced KDR/Flk-1 expression. Furthermore, VEGF-induced ERK phosphorylation was stimulated by CCL23. Finally, CCL23 promoted VEGF-induced endothelial proliferation and migration, which were correlated with the maximal stimulation of KDR/Flk-1 expression by CCL23. Taken together, these findings suggest that CCL23 results in up-regulation of KDR/flk-1 receptor gene transcription and protein expression and that KDR/Flk-1 up-regulation induced by CCL23 may contribute to potentiation of VEGF action in angiogenesis.

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.

PURPOSE Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding. METHODS To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semi-dry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding. RESULTS The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% +/- 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornea-like structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells. CONCLUSIONS The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.

Corrigendum Corrigendum to ‘‘CCL23 up-regulates expression of KDR/Flk-1 and potentiates VEGF-induced proliferation and migration of human endothelial cells” [Biochem. Biophys. Res. Commun. 382 (2009) 124–128] Kyu Yeon Han, Chan Woo Kim, Tae Hoon Lee, Youngsook Son, Jiyoung Kim * Graduate School of Biotechnology, College of Life Science, Kyung Hee University, 1 Seocheon-Ri Giheung-Eup, Yongin 446-701, Republic of Korea

The mammalian thalamus is an essential diencephalic derivative that plays unique roles in processing and relaying sensory and motor information to and from the cerebral cortex. The profile of transcription factors and lineage tracing experiments revealed a spatiotemporal relationship between diencephalic progenitor domains and discrete differentiated neurons contributing to thalamic nuclei. However, the precise molecular mechanisms by which heterogeneous thalamic neurons become specified and assemble into distinct thalamic nuclei are still poorly understood. Here, we show that a combinatorial interaction between the bHLH transcription factors Ascl1 and Helt is required for acquiring thalamic progenitor identity. Surprisingly, in the combined absence of Ascl1 and Helt, rostral thalamic progenitors (TH-R) adopt a molecular profile of a more rostral diencephalic derivative, the prethalamus. Furthermore, we show that the prethalamic factors Dlxs upregulated by Ascl1/Helt deficiency play unique roles in regulating thalamic progenitor specification, and that derepression of Dlx2 and Dlx5 suppress generation of TH-R neurons. Taken together, our results suggest a model whereby the combined activity of two distinct bHLH factors plays a key role in the development of discrete classes of thalamic interneurons.

The SRY-related HMG box transcription factor Sox2 plays critical roles throughout embryogenesis. Haploinsufficiency for SOX2 results in human developmental defects including anophthalmia, microphthalmia and septo-optic dysplasia, a congenital forebrain defect. To understand how Sox2 plays a role in neurogenesis, we combined genomic and in vivo transgenic approaches to characterize genomic regions occupied by Sox2 in the developing forebrain. Six3, a homeobox gene associated with holoprosencephaly, a forebrain midline defect, was identified as a Sox2 transcriptional target. This study shows that Sox2 directly regulates a previously unidentified long-range forebrain enhancer to activate Six3 expression in the rostral diencephalon. Further biochemical and genetic evidences indicated a direct regulatory link between Sox2 and Six3 during forebrain development, providing a better understanding of a common molecular mechanism underlying these forebrain defects.