Preconditioning with Substance P Restores Therapeutic Efficacy of Aged ADSC by Elevating TNFR2 and Paracrine Potential.

Aging leads to a decline in stem cell activity by reducing the repopulation rate and paracrine potential, ultimately diminishing efficacy in vivo. TNF-α can exert inflammatory and cell death actions via Erk by binding to TNFR-1, and survival and tissue repair actions via Akt by binding to TNFR-2. Aged cells are reported to have insufficient expression of TNFR-2, indicating that aged adipose-derived stem cells (ADSCs-E) lack the ability for cell survival and immune control compared to young ADSCs (ADSCs-Y). This study aims to assess the preconditioning effect of SP on the response of ADSCs-E to inflammation. ADSCs-E were treated with SP and then exposed to a high dose of TNF-α for 24 h. Consequently, ADSC-E exhibited weaker viability and lower TNFR2 levels compared to ADSC-Y. In response to TNF-α, the difference in TNFR2 expression became more pronounced in ADSC-E and ADSC-Y. Moreover, ADSC-E showed a severe deficiency in proliferation and paracrine activity. However, preconditioning with SP significantly enhanced the viability of ADSCs-E and also restored TNFR2 expression and paracrine potential, similar to ADSC-Y under inflammatory conditions. Our findings support the idea that preconditioning with SP has the potential to restore the cellular function of senescent stem cells before transplantation.

Substance P-Mediated Vascular Protection Ameliorates Bone Loss.

Estrogen deficiency causes bone loss via diverse pathological cellular events. The involvement of the vasculature in bone formation has been widely studied, and type H vasculature has been found to be closely related to bone healing. Ovariectomy- (OVX-) induced estrogen deficiency reduces type H vessel density and promotes reduction of bone density. Analysis of early events after OVX showed that estrogen deficiency preferentially induces oxidative stress, which might provoke endothelial dysfunction and reduce angiogenic factors systemically and locally. The instability of the vascular potential is expected to promote bone loss under estrogen deficiency. Substance P (SP) is an endogenous neuropeptide that controls inflammation and prevents cell death under pathological conditions. SP can elevate nitric oxide production in endothelial cells and inhibit endothelial dysfunction. This study is aimed at investigating the preventive effects of systemically injected SP on OVX-induced vascular loss and osteoporosis onset. SP was systemically administered to OVX rats twice a week for 4 weeks, immediately after OVX induction. OVX conditions could decrease antioxidant enzyme activity, type H vessels, and angiogenic growth factors in the bone marrow, followed by inflammation and bone loss. However, pretreatment with SP could block type H vessel loss, accompanied by the enrichment of nitric oxide and sustained angiogenic factors. SP-mediated early vascular protection inhibits bone density reduction. Altogether, this study suggests that early administration of SP can block osteoporosis development by modulating oxidative stress and protecting the bone vasculature and angiogenic paracrine potential at the initial stage of estrogen deficiency.

Substance-P Inhibits Cardiac Microvascular Endothelial Dysfunction Caused by High Glucose-Induced Oxidative Stress.

Diabetes is characterized by high glucose (HG) levels in the blood circulation, leading to exposure of the vascular endothelium to HG conditions. Hyperglycemia causes oxidative stress via excessive reactive oxygen species (ROS) production in the endothelium, which leads to cellular dysfunction and the development of diabetic vascular diseases. Substance-P (SP) is an endogenous peptide involved in cell proliferation and migration by activating survival-related signaling pathways. In this study, we evaluated the role of SP in cardiac microvascular endothelial cells (CMECs) in HG-induced oxidative stress. CMECs were treated with diverse concentrations of glucose, and then the optimal dose was determined. Treatment of CMECs with HG reduced their viability and induced excessive ROS secretion, inactivation of PI3/Akt signaling, and loss of vasculature-forming ability in vitro. Notably, HG treatment altered the cytokine profile of CMECs. However, SP treatment inhibited the HG-mediated aggravation of CMECs by restoring viability, free radical balance, and paracrine potential. SP-treated CMECs retained the capacity to form compact and long stretching-tube structures. Collectively, our data provide evidence that SP treatment can block endothelial dysfunction in hyperglycemia and suggest the possibility of using SP for treating diabetic complications as an antioxidant.

Substance P ameliorates TNF-α-mediated impairment of human aortic vascular cells in vitro.

Vascular diseases are caused by endothelial dysfunction due to inflammation. On endothelial injury, the expression of extracellular matrix (ECM) is enhanced and nitric oxide (NO) bioavailability becomes deficient. This condition affects endothelial metabolism and leads to vascular destruction. The aim of this investigation was to determine whether substance P (SP) is able to protect the endothelium against inflammatory stress. To this end, aortic endothelial cells were pre-treated with SP, followed by tumour necrosis factor α (TNF-α), and cellular responses were evaluated using a combination of cell biology and quantification assays, as well as western blot analyses. Our results show that TNF-α enhanced ECM expression and reduced NO production within 4 hours, promoting immune cell adhesion to the endothelium and monocyte chemoattractant protein-1 (MCP-1) secretion from aortic smooth muscle cells. However, SP treatment ameliorated TNF-α-induced endothelial impairment by maintaining low ECM levels. Our data suggest that this protective effect is mediated by Akt activation and NO-enriched conditions. The inhibition of aortic endothelial cell injury by SP also reduced MCP-1 production in aortic smooth muscle cells. Together, our data indicate that SP can protect aortic endothelial and smooth muscle cells from inflammatory injury, which suggests that SP may prevent cardiovascular disease.

Substance P blocks ß-aminopropionitrile-induced aortic injury through modulation of M2 monocyte-skewed monocytopoiesis.

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 ß-aminopropionitrile to rats for 6 weeks. ß-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 ß-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.

Substance P blocks ovariectomy-induced bone loss by modulating inflammation and potentiating stem cell function.

Osteoporosis is an age-related disease caused by imbalanced bone remodeling resulting from excessive bone resorption. Osteoporosis is tightly linked with induction of chronic inflammation, which activates osteoclasts and impairs osteoprogenitor in bone marrow. T helper 17 (Th17) cells have been recently recognized as one of major inducers in the pathophysiology of bone loss by secreting IL-17. Thus, modulation of Th17 development is anticipated to affect the progression of osteoporosis. Substance P (SP) is reported to provide anti-inflammatory effects by controlling immune cell profile and also, promote restoration of damaged stem cell niches-the bone marrow-by repopulating BMSCs or potentiating its paracrine ability. This study aimed to explore the therapeutic effects of systemically injected SP on ovariectomy (OVX)-induced osteoporosis. Resultantly, SP injection obviously blocked OVX-induced impairment of bone microarchitecture and reduction of the mineral density. In osteoporotic condition, SP could ameliorate chronic inflammation by promoting Treg cell polarization and inhibiting the development of osteoclastogenic Th17 cells. Moreover, SP could rejuvenate stem cell and enable stem cells to repopulate and differentiate into osteoblast. Collectively, our study strongly suggests that SP treatment can block osteoporosis and furthermore, SP treatment provides therapeutic effect on chronic disease with inflammation and stem cell dysfunction.

Substance-P Restores Cellular Activity of ADSC Impaired by Oxidative Stress.

Oxidative stress induces cellular damage, which accelerates aging and promotes the development of serious illnesses. Adipose-derived stem cells (ADSCs) are novel cellular therapeutic tools and have been applied for tissue regeneration. However, ADSCs from aged and diseased individuals may be affected in vivo by the accumulation of free radicals, which can impair their therapeutic efficacy. Substance-P (SP) is a neuropeptide that is known to rescue stem cells from senescence and inflammatory attack, and this study explored the restorative effect of SP on ADSCs under oxidative stress. ADSCs were transiently exposed to H2O2, and then treated with SP. H2O2 treatment decreased ADSC cell viability, proliferation, and cytokine production and this activity was not recovered even after the removal of H2O2. However, the addition of SP increased cell viability and restored paracrine potential, leading to the accelerated repopulation of ADSCs injured by H2O2. Furthermore, SP was capable of activating Akt/GSK-3ß signaling, which was found to be downregulated following H2O2 treatment. This might contribute to the restorative effect of SP on injured ADSCs. Collectively, SP can protect ADSCs from oxidant-induced cell damage, possibly by activating Akt/GSK-3ß signaling in ADSCs. This study supports the possibility that SP can recover cell activity from oxidative stress-induced dysfunction.

Osteoporotic Conditions Influence the Activity of Adipose-Derived Stem Cells.

BACKGROUND: Estrogen deficiency decreases bone density and increases the risk of osteoporosis and fracture, thereby necessitating reconstruction of bone regeneration. As bone marrow mesenchymal stem cell (BMSCs) lose viability and differentiation potential under osteoporotic conditions, it is impossible to use autologous BMSCs for osteoporosis treatment. As an alternative, adipose-derived stem cells (ADSCs) may serve as the source of therapeutic cells. METHOD: We evaluated the effects of osteoporosis on the functional characteristics of ADSCs. Osteoporosis was induced in ovariectomy (OVX) rat model, and the ADSCs from Sham and OVX groups were cultured and analyzed comparatively. RESULTS: As a result, the viability was higher for the ADSCs from Sham group than those from OVX group. The analysis of the paracrine potential of ADSCs revealed the elevated levels of inflammatory and cellular senescence factors in the ADSCs from OVX group. The ADSCs from OVX group had much higher differentiation potential into adipocytes than those from the Sham group. Osteoporotic environment had no effect on the osteogenic potential of ADSCs. CONCLUSION: Osteoporosis may reduce the activity and influence immune response of ADSCs by modulating paracrine action and adipogenic potential. These characteristics of ADSCs should be given consideration for therapeutic purpose.

Substance P accelerates wound repair by promoting neovascularization and preventing inflammation in an ischemia mouse model.

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.

Substance P Promotes Liver Sinusoidal Endothelium-Mediated Hepatic Regeneration by NO/HGF Regulation.

Liver sinusoidal endothelial cells (LSECs) are highly specialized and involved in hepatic regeneration by interacting with hepatic stellate cells (HSCs) and hepatocytes in a paracrine manner. However, hepatic injury can impair cellular activity and lead to endothelial dysfunction, eventually inducing the development of critical hepatic disease, including cirrhosis. Because LSECs exert their effects through paracrine factors, maintenance of paracrine potentials and survival activity in LSECs under injury stress is a critical strategy for inhibiting disease progression. This study explored the effect of Substance-P (SP) on cell viability, proliferation, and nitric oxide (NO)/hepatocyte growth factor (HGF) production in LSECs. Under noninjured conditions, SP treatment enhanced cell viability, cell proliferation, and HGF/NO secretion in LSECs. In the presence of tumor necrosis factor (TNF)-α-induced inflammatory stress, SP blocked TNF-α-induced endothelial dysfunction, accompanied by elevated cell viability and NO/HGF secretion. Interestingly, SP-primed LSEC-conditioned medium accelerated hepatocyte repopulation without causing morphological alterations. The primitive effect of SP was reversed by endothelial nitric oxide synthase inhibitor or HGF/c-MET inhibitor, indicating the importance of the NO/HGF combination in hepatic regeneration by SP. Taken together, these results suggest SP can protect LSECs from inflammatory stress by NO/HGF, which contributes to hepatocyte repopulation.

Substance P ameliorates tumor necrosis factor-alpha-induced endothelial cell dysfunction by regulating eNOS expression in vitro.

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.

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro.

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.