FGF2/HGF priming facilitates adipose-derived stem cell-mediated bone formation in osteoporotic defects.

Aims: The activity of adipose-derived stem cells (ADSCs) is susceptible to the physiological conditions of the donor. Therefore, employing ADSCs from donors of advanced age or with diseases for cell therapy necessitates a strategy to enhance therapeutic efficacy before transplantation. This study aims to investigate the impact of supplementing Fibroblast Growth Factor 2 (FGF2) and Hepatocyte Growth Factor (HGF) on ADSC-mediated osteogenesis under osteoporotic conditions and to explore the underlying mechanisms of action. Main methods: Adipose-derived stem cells (ADSCs) obtained from ovariectomized (OVX) rats were cultured ex vivo. These cells were cultured in an osteogenic medium supplemented with FGF2 and HGF and subsequently autologously transplanted into osteoporotic femur defects using Hydroxyapatite-Tricalcium Phosphate. The assessment of bone formation was conducted four weeks post-transplantation. Key findings: Osteoporosis detrimentally affects the viability and osteogenic differentiation potential of ADSCs, often accompanied by a deficiency in FGF2 and HGF signaling. However, priming with FGF2 and HGF facilitated the formation of immature osteoblasts from OVX ADSCs in vitro, promoting the expression of osteoblastogenic proteins, including Runx-2, osterix, and ALP, during the early phase of osteogenesis. Furthermore, FGF2/HGF priming augmented the levels of VEGF and SDF-1α in the microenvironment of OVX ADSCs under osteogenic induction. Importantly, transplantation of OVX ADSCs primed with FGF2/HGF for 6 days significantly enhanced bone formation compared to non-primed cells. The success of bone regeneration was confirmed by the expression of type-1 collagen and osteocalcin in the bone tissue of the deficient area. Significance: Our findings corroborate that priming with FGF2/HGF can improve the differentiation potential of ADSCs. This could be applied in autologous stem cell therapy for skeletal disease in the geriatric population.

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.

Priming with a Combination of FGF2 and HGF Restores the Impaired Osteogenic Differentiation of Adipose-Derived Stem Cells.

Classical aging-associated diseases include osteoporosis, diabetes, hypertension, and arthritis. Osteoporosis causes the bone to become brittle, increasing fracture risk. Among the various treatments for fractures, stem cell transplantation is currently in the spotlight. Poor paracrine/differentiation capacity, owing to donor age or clinical history, limits efficacy. Lower levels of fibroblast growth factor 2 (FGF2) and hepatocyte growth factor (HGF) are involved in cell repopulation, angiogenesis, and bone formation in the elderly ADSCs (ADSC-E) than in the young ADSCs (ADSC-Y). Here, we study the effect of FGF2/HGF priming on the osteogenic potential of ADSC-E, determined by calcium deposition in vitro and ectopic bone formation in vivo. Age-induced FGF2/HGF deficiency was confirmed in ADSCs, and their supplementation enhanced the osteogenic differentiation ability of ADSC-E. Priming with FGF2/HGF caused an early shift of expression of osteogenic markers, including Runt-related transcription factor 2 (Runx-2), osterix, and alkaline phosphatase (ALP) during osteogenic differentiation. FGF2/HGF priming also created an environment favorable to osteogenesis by facilitating the secretion of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF). Bone tissue of ADSC-E origin was observed in mice transplanted with FGF/HGF-primed ADSC-E. Collectively, FGF2/HGF priming could enhance the bone-forming capacity in ADSC-E. Therefore, growth factor-mediated cellular priming can enhance ADSC differentiation in bone diseases and thus contributes to the increased efficacy in vivo.

Substance P Hinders Bile Acid-Induced Hepatocellular Injury by Modulating Oxidative Stress and Inflammation.

Liver failure is an outcome of chronic liver disease caused by steatohepatitis and cholestatic injury. This study examined substance P (SP) effect on liver injury due to cholestatic stress caused by excessive bile acid (BA) accumulation. Chenodeoxycholic acid (CDCA) was added to HepG2 cells to induce hepatic injury, and cellular alterations were observed within 8 h. After confirming BA-mediated cellular injury, SP was added, and its restorative effect was evaluated through cell viability, reactive oxygen species (ROS)/inflammatory cytokines/endothelial cell media expression, and adjacent liver sinusoidal endothelial cell (LSEC) function. CDCA treatment provoked ROS production, followed by IL-8 and ICAM-1 expression in hepatocytes within 8 h, which accelerated 24 h post-treatment. Caspase-3 signaling was activated, reducing cell viability and promoting alanine aminotransferase release. Interestingly, hepatocyte alteration by CDCA stress could affect LSEC activity by decreasing cell viability and disturbing tube-forming ability. In contrast, SP treatment reduced ROS production and blocked IL-8/ICAM-1 in CDCA-injured hepatocytes. SP treatment ameliorated the effect of CDCA on LSECs, preserving cell viability and function. Collectively, SP could protect hepatocytes and LSECs from BA-induced cellular stress, possibly by modulating oxidative stress and inflammation. These results suggest that SP can be used to treat BA-induced liver injury.

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.

Age affects the paracrine activity and differentiation potential of human adipose­derived stem cells.

Stem cell therapy is considered a novel treatment modality for critical diseases. Adipose tissue is a rich and easily accessible source of stem cells. Adipose­derived stem cells (ADSCs) can be expanded ex vivo and possess characteristics similar to those derived from the bone marrow. However, the quality of ADSCs can be affected by age, underlying disease or the lifestyle of individuals. The aim of the present study was to explore the association between age and ADSC activity, including paracrine and differentiation potential. Adipose tissues from young (age <30 years) and elderly (age >70 years) groups were obtained, and ADSCs from each group were cultured in vitro. The effect of age on ADSC activity was investigated in vitro by evaluating the proliferation rate, adipo/osteogenic differentiation potential and cytokine profile using ELISA. The results revealed that increased age reduced cell activity and increased the doubling time of ADSCs, without causing profound morphological changes. The paracrine action of ADSCs was markedly altered by increased age, as demonstrated by reduced expression levels of vascular endothelial growth factor, stromal cell­derived factor­1α and hepatocyte growth factor. Differentiation of ADSCs into osteoblasts or adipocytes rarely occurred in the elderly group compared with the young group. Overall, these results indicate that age may affect the cellular function of ADSCs and should be considered prior to ADSC transplantation.

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 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.

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.

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.

Impact of fish consumption by subjects with prediabetes on the metabolic risk factors: using data in the 2015 (6th) Korea National Health and Nutrition Examination Surveys.

BACKGROUND/OBJECTIVES: The effects of fish consumption by subjects with prediabetes on the metabolic risk factors were examined based on the data from the 6th Korea National Health and Nutrition Examination Surveys in 2015. SUBJECTS/METHODS: A total of 1,520 subjects who agreed to participate in a blood test and dietary intake survey were divided into a prediabetes group and normal blood glucose group, and the level of the subjects' fish consumption was divided into ≤ 17.0 g/day, 18.0-93.0 g/day, and ≥ 94 g/day. The correlation between the level of fish intake and the metabolic risk factors was evaluated by multinomial logistic regression analysis. RESULTS: A significant difference in the gender distribution was observed in the prediabetes group, which is a group with a high risk of non-communicable diseases, according to the fish intake, and there were significant differences in the total energy intake, protein intake, n-3 fatty acids intake, and the intakes of sodium and micro-nutrients according to the intake group (P < 0.05). In addition, the blood total cholesterol (TC) decreased 0.422 fold in model 1 (unadjusted) [95% confidence interval (CI): 0.211-0.845] and 0.422 fold in model 2 (adjusted for sex) (95% CI: 0.210-0.846) in those with a fish intake of 18.0-93.0 g/day (P < 0.05) compared to those with a fish intake of ≤ 17.0 g/day. The blood TC decreased 0.555 fold (95% CI: 0.311-0.989) in model 1 and 0.549 fold (95% CI: 0.302-0.997) in model 2 in those with a fish intake of ≥ 94 g/day compared to those with a fish intake of ≤ 17.0 g/day (P < 0.05). CONCLUSIONS: Subjects with prediabetes or the metabolic risk factors can maintain their blood low density lipoprotein cholesterol (LDL-C) and blood TC concentrations at the optimal level by consuming fish (18.0-93.0 g/day).