Expression Of SDF-1 Research Articles

Biocompatibility and Angiogenic Effect of Chitosan/Graphene Oxide Hydrogel Scaffolds on EPCs.

Angiogenesis in the field of tissue engineering has attracted significant attention. Graphene oxide has become a promising nanomaterial in tissue engineering for its unique biochemical properties. Therefore, herein, a series of chitosan (CS)/graphene oxide (GO) hydrogel scaffolds were synthesized by crosslinking CS and GO at different concentrations (0.1, 0.5, and 1.0 wt.%) using genipin. Compared with the CS hydrogel scaffolds, the CS/GO hydrogel scaffolds have a better network structure and mechanical strength. Then, we used endothelial progenitor cells (EPCs) extracted from human umbilical cord blood and cocultured these EPCs with the as-prepared scaffolds. The scaffolds with 0.1 and 0.5 wt.%GO showed no considerable cytotoxicity, could promote the proliferation of EPCs and tube formation, and upregulated the expressions of CD34, VEGF, MMP9, and SDF-1 in EPCs compared to the case of the scaffold with 1.0 wt.%GO. This study shows that the addition of graphene oxide improves the structure of chitosan hydrogel and enhances the proliferation activity and angiogenic capacity of EPCs.

Platelet-derived biomaterials-mediated improvement of bone injury through migratory ability of embryonic fibroblasts: in vitro and in vivo evidence.

Bony injuries lead to compromised skeletal functional ability which further increase in aging population due to decreased bone mineral density. Therefore, we aimed to investigate the therapeutic potential of platelet-derived biomaterials (PDB) against bone injury. Specifically, we assessed the impact of PDB on osteo-inductive characteristics and migration of mouse embryonic fibroblasts (MEFs). Osteogenic lineage, matrix mineralization and cell migration were determined by gene markers (RUNX2, OPN and OCN), alizarin Red S staining, and migration markers (FAK, pFAK and Src) and EMT markers, respectively. The therapeutic impact of TGF-β1, a key component of PDB, was confirmed by employing inhibitor of TGF-β receptor I (Ti). Molecular imaging-based in vivo cellular migration in mice was determined by establishing bone injury at right femurs. Results showed that PDB markedly increased expression of osteogenic markers, matrix mineralization, migration and EMT markers, revealing higher osteogenic and migratory potential of PDB-treated MEFs. In vivo cell migration was manifested by expression of migratory factors, SDF-1 and CXCR4. Compared to control, PDB-treated mice exhibited higher bone density and volume. Ti treatment inhibited both migration and osteogenic potential of MEFs, affirming impact of TGF-β1. Collectively, our study clearly indicated PDB-rescued bone injury through enhancing migratory potential of MEFs and osteogenesis.

Measurement of 45 cytokine, chemokine and growth factors in established cell culture supernatants and autologous serum from advanced melanoma patients.

Melanoma is one of the most aggressive forms of human cancer and its incidence has significantly increased worldwide over the last decades. This neoplasia has been characterized by the release of a wide variety of soluble factors, which could stimulate tumor cell proliferation and survival in an autocrine and paracrine manner. Consequently, we sought to evaluate the pattern of soluble factors produced by pre-metastatic and metastatic melanoma established cultures, and to determine whether these factors can be detected in the autologous serum of malignant melanoma patients. Our results showed that both melanoma cultures had a common profile of 27 soluble factors mainly characterized by the high expression of VEGF-A, IL-6, MCP-1, IL-8, and SDF-1. In addition, when we compared supernatants, we observed significant differences in VEGF-A, BDNF, FGF-2, and NGF-β concentrations. As we found in melanoma cultures, serum samples also had their specific production pattern composed by 21 soluble factors. Surprisingly, PDGF-BB and EGF were only found in serum, whereas IL-2, IL-4, IL-8, IL31, FGF2, and GRO-α were only expressed in the supernatant. Significant differences in PDGF-BB, MIP-1β, HGF, PIGF-1, BDNF, EGF, Eotaxin, and IP-10 were also found after comparing autologous serum with healthy controls. According to this, no correlation was found between culture supernatants and autologous serum samples, which suggests that some factors may act locally, and others systemically. Nonetheless, after validation of our results in an independent cohort of patients, we concluded that PDGF-BB, VEGF-A, and IP-10 serum levels could be used to monitor different melanoma stages.

SDF Factor-1α Promotes the Migration, Proliferation, and Osteogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells Through the Wnt/β-Catenin Pathway.

Bone marrow mesenchymal stem cells (BMSCs) are thought to have great potential in the treatment of many diseases and may serve as a cell source for tissue engineering. These cells may be regulated by stromal cell-derived factor-1α (SDF-1α), which has been shown to promote the migration, proliferation, and osteogenic differentiation of BMSCs in inflammation-associated diseases. However, the specific mechanism underlying this process remains unclear. We herein transduced lentivirus carrying SDF-1α, empty vector, or siRNA-SDF-1α into mouse BMSCs and then performed transwell, CCK-8, cell cycle, alkaline phosphatase activity, and Alizarin Red staining experiments on the three groups of samples. Overexpression of SDF-1α promoted the migration, proliferation, and osteogenic differentiation of BMSCs, and SDF-1α upregulated the expression of Wnt pathway-related factors and downstream target genes as determined by western blot, real-time polymerase chain reaction, and immunofluorescence. The effect of low SDF-1α expression on BMSCs was significantly weakened. In addition, we transduced lentivirus carrying siRNA-Wnt3a into BMSCs and treated them with SDF-1 drugs. After inhibiting the Wnt pathway, SDF-1 significantly weakened the migration, proliferation, and osteogenic differentiation of BMSCs. From this, we concluded that high SDF-1 expression can promote the migration, proliferation, and osteogenic differentiation of BMSCs, at least in part by activating the Wnt pathway.

Assessing the effect and related mechanism of naringenin on the proliferation, osteogenic differentiation and endothelial differentiation of human periodontal ligament stem cells

Naringenin (NAR) is a natural flavonoid which exerts extensive biological activity, including anti-oxidation, anti-inflammation, anti-cancer, immune regulation and so on. However, the effect and mechanism of NAR in the alveolar bone regeneration are still unclear, which limits its clinical use. Hence, we investigated the effects of NAR in the proliferation, osteogenic and endothelial differentiation of human periodontal ligament stem cells (hPDLSCs) and explore the possible mechanism. The results showed that the proper concentrations (100 nM–10 μM) of NAR can promote the proliferation rate, osteogenic and endothelial differentiation of hPDLSCs. And the 1 μM NAR had the best proliferation promoting effect, while the 10 μM NAR had the best ability of promoting osteogenic and endothelial differentiation. NAR also promoted the mRNA expression of SDF-1 in a concentration dependent manner in PDLSCs. After adding the selective CXCR4 antagonist AMD3100, the osteogenic effect of NAR on PDLSCs is slightly enhanced, while the endothelial differentiation effect of NAR on hPDLSCs is attenuated. In summary, these results indicated that NAR promoted the proliferation of hPDLSCs, and promoted endothelial differentiation of hPDLSCs via SDF-1 to activate SDF-1/CXCR4 signaling pathway. However, the mechanism of which SDF-1 related signaling pathway is activated by NAR to enhance the osteogenic differentiation of hPDLSCs still needs to be investigated.

Abstract 14306: Mural Cell SDF1 Signaling is Associated With the Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pericytes and pulmonary artery smooth muscle cells (PASMCs) are NG2+ mural cells that provide structural support to pulmonary vessels. In pulmonary arterial hypertension (PAH), both mural cell types contribute to PA muscularization, but whether distinct mechanisms that are responsible for their behavior is unclear. Methods: RNA-Seq was used to compare the gene profile of pericytes and PASMCs isolated from PAH and healthy lungs. NG2-CreER mice were used to generate tamoxifen-inducible NG2-selective reporter mice (NG2tdT fl/+ ) for cell lineage identification and for NG2-selective SDF1 knockout (SDF1 NG2-KO ). Results: Hierarchical clustering of RNA-seq data demonstrated that the genetic profile of PAH pericytes and PASMCs is significantly similar. Cellular lineage staining studies on NG2tdT mice in chronic hypoxia showed that tdT+ cells accumulate in muscularized arteries. RNAseq analysis performed on these hypoxic tdT+ cells demonstrated significant upregulation of SDF1, a chemokine involved in chemotaxis and angiogenesis. Compared to controls, SDF1 NG2-KO mice in chronic hypoxia had reduced RVSP, fulton index, less muscularization and lower abundance of NG2+ cells around microvessels (Fig. 1). In addition, SDF1 stimulation in human healthy pericytes induced greater contractility, impaired their capacity to establish endothelial-pericyte tube formation and increased expression of SMC markers SMA, Calponin and SM22. In contrast, SDF1 knockdown reduced PAH pericyte proliferation, contractility and improved endothelial cell tube formation on matrigel. Conclusions: SDF1 is upregulated in NG2+ mural cells. High expression of SDF1 could drive pericyte detachment and gain an SMC-like phenotype that contributes to abnormal muscularization and vascular obstruction. Targeting SDF1 could help prevent and reverse muscularization in PAH.

SDF1α/CXCR4 axis may be associated with the malignant progression of gastric cancer in the hypoxic tumor microenvironment.

Stromal cell-derived factor 1α (SDF1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) have been reported to form an important chemokine signaling pathway. Our previous study reported that SDF1α from tumor stromal cells may stimulate the proliferation of gastric cancer (GC) cells through the CXCR4 axis in a hypoxic microenvironment. However, a limited number of studies have addressed the clinicopathological significance of the expression of SDF1α and CXCR4 in GC, particularly at hypoxic regions. Immunohistochemistry was used to investigate the expression levels of SDF1α, CXCR4 and the hypoxic marker carbonic anhydrase 9 (CA9) in 185 patients with stage II and III GC. The results demonstrated that CA9 was expressed on cancer and stromal cells in hypoxic lesions, CXCR4 was mainly expressed in cancer cells, and SDFα was mainly expressed in stromal cells. CXCR4 expression in cancer cells and SDFα expression in stromal cells were associated with the hypoxic regions with CA9 expression. The CA9 and CXCR4 expression in the cancer cells, and the SDF1α expression in the stromal cells (CA9/CXCR4/SDF1α) was significantly associated with macroscopic type 4 tumor (P=0.012) and the pattern of tumor infiltration into the surrounding tissue (P<0.001). The prognosis of the all CA9/CXCR4/SDF1α-positive patients was significantly poorer compared with that of patients with CA9-, CXCR4- or SDF1α-negative GC at Stage III (P=0.041). These results indicated that hypoxia may upregulate SDFα production in stromal cells and CXCR4 expression in cancer cells. The SDF1α/CXCR4 axis may serve an important role in the progression of GC.

Loss of smooth muscle SDF-1/CXCL12 leads to cardiac hypertrophy and aortic valve stenosis

Abstract Background Stromal cell-derived factor-1 (SDF-1 or CXCL12) and its receptors CXCR4/CXCR7 have prominent role in cardiovascular development and myocardial repair following ischemic injury. Nevertheless, detailed mechanisms of the cell specific role of SDF-1 are poorly understood. Since SDF-1-EGFP lineage tracking revealed high expression of SDF-1 in smooth muscle cells, we aimed to investigate the cell specific role by generating a smooth muscle cell specific SDF-1 (SM-SDF-1−/−) knockout mouse model. Methods SDF-1 expression was analyzed utilizing SDF-1-EGFP reporter mice. Conditional SM-SDF-1 KO mice were generated using Tagln-Cre; SDF-1fl/fl mice. Hearts were analysed with histology and high-resolution episcopic microscopy. Cardiac function was assessed utilizing echocardiography. RNAseq, qRT-PCR, flow cytometry and western blotting were performed. Cardiac fibrosis, apoptotic index, cell proliferation, aortic valve calcification were analyzed. SM-SDF-1−/− mice were treated with the CXCR7 agonist TC14012 (10mg/kg/I.P). Results SDF-1-EGFP lineage tracking and immunofluorescence revealed high expression of SDF-1 particularly in smooth muscle cells and less frequently in perivascular and endothelial cells. Conditional SM-SDF-1−/− mice showed a high pre- and perinatal mortality (50%). Immunohistochemistry of SM-SDF-1−/− mice revealed severe cardiac hypertrophy, associated with increased cardiac fibrosis, apoptotic cell death, thinned and dilated arteries and significantly decreased M2 like CD11b+/CD206+ cells. Echocardiography confirmed concentric hypertrophy, with decreased stroke volume. As a possible reason for cardiac hypertrophy, SDF-1 mutants exhibited aortic stenosis due to aortic valve thickening associated with downregulation of the SDF-1 co-receptor CXCR7. We further noticed increased plasma levels of SDF-1 in aortic stenosis patients suggesting a cardioprotective role. Transcriptome analyses from KO hearts showed an abnormal extracellular matrix (ECM) remodelling with a specific upregulation of the important valve related proteoglycans Versican, Glycan. Western blot analysis revealed activation of AKT and ERK, whereas CXCR7 expression was significantly downregulated in KO mice. To rescue the phenotype we treated KO mice with the CXCR7 agonist (TC14012) which partially attenuated aortic valve remodelling through activation of the ERK signalling pathway. Conclusion Our data suggest that SDF-1 is critically involved in maintaining the homeostasis of the aortic valve by regulating CXCR7 signalling. Pharmacological activation of CXCR7 might be a promising therapeutic target to limit the progression of aortic valve stenosis. Ghadge_SM-SDF-1−/− Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund, Austrian research promotion agency

Rescue of cardiac function in obese type-2 diabetic mice by transfer of a human longevity gene

Abstract Background Healthy longevity is the result of the interaction between favourable environment and unique genetic makeup. We showed that horizontal transfer of a longevity-associated gene variant (LAV-BPIFB4) improves endothelial function and accelerates the recovery from ischemia. Purpose To determine if the benefit of LAV-BPIFB4 gene therapy can be extended to diabetic cardiomyopathy. Methods and results We confirmed that human diabetic patients with heart failure (n=13) show a decreased cardiac expression of BPIFB4 compared with healthy subjects (n=10). Obese db/db mice received a systemic injection of adeno-associated viral vector (AAV9)-LAV-BPIFB4, AAV9-wild type (WT)-BPIFB4 (both 100 μL at 1×1012 GC/mL) or vehicle before the onset of cardiomyopathy, and were euthanised four weeks later for histological, metabolic and transcriptional analyses. Echocardiographic evaluation (n=8/group), performed at baseline and after gene therapy, showed that LAV-BPIFB4 treatment, despite not resolving hyperglycaemia, improved left ventricular function compared with the other groups. Histological analyses of the hearts (n=5 to 10/group) revealed that LAV-BPIFB4 reduced myocardial fibrosis and increased angiogenesis compared with vehicle and WT-hearts; moreover, LAV increased the expression of the alpha-isoform of the cardiac myosin heavy chain, which is associated with a superior cardiomyocyte contractility. Interestingly, LAV-BPIFB4 treatment induced an increase in cardiac SDF1 expression compared with WT and vehicle, despite the mechanism linking the two events is still unknown. The oral administration of the CXCR4 antagonist AMD-070, given at 2 mg/kg/day for four weeks, abolished several of the beneficial effects exerted by the LAV-BPIFB4 therapy in the obese diabetic mice, as assessed by echocardiography and histology (n=7/group). At the molecular level, next-generation RNA sequencing (n=3 to 4 /group) showed 8 genes were differentially expressed by LAV-BPIFB4-hearts compared with vehicle-hearts. These genes are associated with mitochondrial and metabolic functions. Among them, changes in the UCP3, HMGCS2, CS, ATPB and TOMM20 expression were also validated at the protein level by western blotting. Lipidomics using ultrahigh-performance liquid chromatography-mass spectrometry (n=6 or 7/group) showed 63 metabolites differentially expressed by LAV-BPIFB4- compared with vehicle-hearts, with only 3 (two cardiolipins and one glycerophospholipid) returning close to the non-diabetic phenotype following LAV-BPIFB4 treatment. Conclusions This study newly shows the possibility of transferring the benefit of salutary polymorphic gene variants to protect the cardiovascular system from metabolic pressure. Rather than combating pathogenic mechanisms, the strategy activates alternative pathways overriding disease risk factors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): British Heart Foundation project grant “Longevity-associated BPIFB4 gene therapy for treatment of ischemic disease”

CREB activation affects mesenchymal stem cell migration and differentiation in periodontal tissues due to orthodontic force.

During the orthodontic tooth movement, cells in periodontal ligament could differentiate into osteoblasts to synthesize alveolar bone as well as affect the proliferation, migration and differentiation of mesenchymal stem cells, which also contribute to bone remodeling. However, the mechanism is still largely elusive. Here, we evaluated the expression of CREB at the tension site of mouse periodontal ligament under orthodontic mechanical strain and in the cyclic tension strain treated human periodontal ligament cells. Then, through gain and loss of function analysis, we revealed that CREB in PDLCs promotes SDF-1 and FGF2 secretion, which enhance the migration and osteoblastic differentiation of BMSCs. We further discovered that CREB transcriptionally activates FGF2 and SDF-1 expressions by binding to the promoter regions.In conclusion, this study confirms that CREB is an upregulated gene in periodontal ligament under orthodontic tension strain stimulation and plays an important role in regulating BMSCs' physiological activity in orthodontic tension strain-induced bone formation.

Short-wave enhances mesenchymal stem cell recruitment in fracture healing by increasing HIF-1 in callus

BackgroundAs a type of high-frequency electrotherapy, a short-wave can promote the fracture healing process; yet, its underlying therapeutic mechanisms remain unclear.PurposeTo observe the effect of Short-Wave therapy on mesenchymal stem cell (MSC) homing and relative mechanisms associated with fracture healing.Materials and methodsFor in vivo study, the effect of Short-Wave therapy to fracture healing was examined in a stabilized femur fracture model of 40 SD rats. Radiography was used to analyze the morphology and microarchitecture of the callus. Additionally, fluorescence assays were used to analyze the GFP-labeled MSC homing after treatment in 20 nude mice with a femoral fracture. For in vitro study, osteoblast from newborn rats simulated fracture site was first irradiated by the Short-Wave; siRNA targeting HIF-1 was used to investigate the role of HIF-1. Osteoblast culture medium was then collected as chemotaxis content of MSC, and the migration of MSC from rats was evaluated using wound healing assay and trans-well chamber test. The expression of HIF-1 and its related factors were quantified by q RT-PCR, ELISA, and Western blot.ResultsOur in vivo experiment indicated that Short-Wave therapy could promote MSC migration, increase local and serum HIF-1 and SDF-1 levels, induce changes in callus formation, and improve callus microarchitecture and mechanical properties, thus speeding up the healing process of the fracture site. Moreover, the in vitro results further indicated that Short-Wave therapy upregulated HIF-1 and SDF-1 expression in osteoblast and its cultured medium, as well as the expression of CXCR-4, β-catenin, F-actin, and phosphorylation levels of FAK in MSC. On the other hand, the inhibition of HIF-1α was significantly restrained by the inhibition of HIF-1α in osteoblast, and it partially inhibited the migration of MSC.ConclusionsThese results suggested that Short-Wave therapy could increase HIF-1 in callus, which is one of the crucial mechanisms of chemotaxis MSC homing in fracture healing.

Role of the SDF-1/CXCR4 signaling pathway in cartilage and subchondral bone in temporomandibular joint osteoarthritis induced by overloaded functional orthopedics in rats

ObjectivesTo (i) use a mandibular advancement appliance in rats to investigate the role of the stromal cell-derived factor/CXC receptor 4 (SDF-1/CXCR4) signaling pathway in temporomandibular joint osteoarthritis (TMJ OA) induced by overloaded functional orthopedics (OFO) and (ii) provide a cellular and molecular basis for efficacious treatment of skeletal class-II malocclusion and avoidance of TMJ OA.MethodMale Sprague-Dawley rats (6 weeks) were divided randomly into control + normal saline (NS), EXP + ADM3100 (SDF-1 antagonist), EXP + NS, and control + ADM3100 groups. Changes in articular cartilage and subchondral bone after TMJ OA in these four groups were observed by hematoxylin and eosin (H&E), immunofluorescence double staining (IDS), Safranin-O staining, immunohistochemical (IHC) staining, real-time polymerase chain reaction, and micro-computed tomography at 2, 4, and 8 weeks.ResultsOFO led to increased expression of SDF-1, CXCR4, and matrix metalloproteinase (MMP) 13 and decreased expression of collagen II. The thickness of the hypertrophic cartilage layer was reduced at 4 weeks in the EXP + NS group, and damage to subchondral bone was observed at 2 weeks. Using ADM3100 to inhibit SDF-1 signaling could attenuate expression of MMP13, cartilage damage, and osteoblast differentiation. IDS showed that the areas of expression of SDF-1 and OSX in subchondral bone overlapped.ConclusionsOverloaded functional orthopedics (OFO) induced TMJ OA. The destruction of subchondral bone in TMJ OA caused by OFO occurred before damage to cartilage. SDF-1/CXCR4 may induce the osteogenic differentiation and cause cartilage degradation in TMJ OA caused by OFO.

Abstract 1106: Cisplatin-induced activation of SDF-1/CXCR4 axis sustains lung cancer metastasis by promoting co-recruitment of metastasis initiating cells and inflammatory monocytes

Abstract Background: Platinum-based treatment of locally advanced non-small cell lung cancer (NSCLC) provides some clinical benefit in controlling local disease but is ineffective on metastatic dissemination. Rising evidence suggests that chemotherapy can promote an inflammatory reaction supportive of metastasis growth.We previously demonstrated that cisplatin treatment of mouse xenografts enriches for the chemoresistant fraction of CD133+CXCR4+ lung cancer metastasis initiating cells (MICs), increasing distant metastasis development. We hypothesize here that the SDF-1/CXCR4 axis, implied in MICs migration and in stromal cells trafficking, could play a critical role in cisplatin-induced pro-metastatic effects. Results: To study the effects of cisplatin in promoting a pre-metastatic niche, tumor-free naïve SCID mice were treated with cisplatin plus/minus peptide R, a novel inhibitor of CXCR4. Cisplatin resulted in a rapid expansion of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IM) in the bone marrow, concomitantly with their recruitment to murine lungs guided by enhanced endothelial release of SFD-1. Tail-vein injection of H460 human lung cancer cells 72h after cisplatin administration resulted in augmented number of lung metastases enriched in CD133+CXCR4+ MICs. We proposed that the abundance of CXCR4+IM together with increased endothelial permeability caused by cisplatin favors tumor cells extravasations and the expansion of MICs through SDF-1/CXCR4 axis activation. Combination treatment with a CXCR4 inhibitor prevented IM recruitment, MICs expansion and consequently abolished metastasis overgrowth induced by cisplatin. Combination treatment of H460 subcutaneous xenografts in a regimen mimicking clinical setting revealed that cisplatin also caused tumor release of SDF-1 able to trigger local expansion of MICs subset and to recruit CXCR4+tumor associated macrophages involved in tumor cells intravasation. Stromal SDF-1 induced by cisplatin at distant site also co-recruited MICs and CCR2+CXCR4+IM, promoting spontaneous metastasis formation that can be counteracted by CXCR4 blockade. We confirmed in clinical setting that platinum-based neoadjuvant treatment of NSCLC patients significantly increases tumor SDF-1 expression. Moreover, an higher expression of tumor SDF-1 after cisplatin neo-adjuvant treatment was associated with patients' shorter DFS (p=0,0056; Hazards Ratio= 3,1) and poor OS (p=0,029; Hazards Ratio= 3,46). Conclusions: Our data reveal a paradoxical pro-metastatic effect of cisplatin that fosters MIC-IM recruitment and cross-talk via SDF-1/CXCR4 axis activation. A new combination strategy based on CXCR4 inhibition may disrupt these interactions, providing more effective results for lung cancer treatment Citation Format: Giulia Bertolini, Valeria Cancila, Orazio Fortunato, Giuseppe Lo Russo, Monica Tortoreto, Giovanni Centonze, Massimo Milione, Claudio Tripodo, Stefania Scala, Gabriella Sozzi, Luca Roz. Cisplatin-induced activation of SDF-1/CXCR4 axis sustains lung cancer metastasis by promoting co-recruitment of metastasis initiating cells and inflammatory monocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1106.

Hyperbaric oxygen potentiates diabetic wound healing by promoting fibroblast cell proliferation and endothelial cell angiogenesis

BackgroundDiabetic foot ulcer (DFU), one of the diabetic complications, brings high burden to diabetic patients. Hyperbaric oxygen therapy (HBOT) has been proven to be an effective clinical method for the treatment of DFU. However, the mechanisms still to be elucidated. MethodsDiabetic foot mice model was established, and treated with hyperbaric oxygen. Haematoxylin & eosin (H&E) staining and Masson's trichrome staining were used for the analysis of wound healing. Human skin fibroblast (HSF) and human umbilical vein endothelial cell (HUVECS) were exposed to high glucose and hyperbaric oxygen for studying the mechanism of hyperbaric oxygen promoted wound healing in vitro. Wound healing assay, reactive oxygen species (ROS) assay, cell proliferation assay and tube formation assay were used for the analysis of wound healing. Quantitative-polymerase chain reaction (Q-PCR), Western blotting and enzyme-linked immunosorbent assay (ELISA) were used for the analysis of gene expression. ResultsHBOT facilitated wound healing in DFU mice model, and promoted the expression of HIF-1α, NF-κB, VEGFA, SDF-1, VEGFR2 and CXCR4. Hyperbaric oxygen promoted the proliferation, migration and ROS production, as well as the expression of SDF-1 and VEGFA in HSF. HBOT stimulated the proliferation, migration and tube formation, as well as the expression of CXCR4 and VEGFR2 in HUVECS. ConclusionHyperbaric oxygen potentiates angiogenesis and diabetic wound healing by activating HIF-1α signaling, so as to promote the expression of VEGF/SDF-1 in HSF and the expression of VEGFR/CXCR4 in HUVECS, ultimately to promote the proliferation of HSF and the angiogenesis of HUVECS.

Etoposide-mediated interleukin-8 secretion from bone marrow stromal cells induces hematopoietic stem cell mobilization

BackgroundWe assessed the mechanism of hematopoietic stem cell (HSC) mobilization using etoposide with granulocyte-colony stimulating factor (G-CSF), and determined how this mechanism differs from that induced by cyclophosphamide with G-CSF or G-CSF alone.MethodsWe compared the clinical features of 173 non-Hodgkin’s lymphoma patients who underwent autologous peripheral blood stem cell transplantation (auto-PBSCT). Additionally, we performed in vitro experiments to assess the changes in human bone marrow stromal cells (hBMSCs), which support the HSCs in the bone marrow (BM) niche, following cyclophosphamide or etoposide exposure. We also performed animal studies under standardized conditions to ensure the following: exclude confounding factors, mimic the conditions in clinical practice, and identify the changes in the BM niche caused by etoposide-induced chemo-mobilization or other mobilization methods.ResultsRetrospective analysis of the clinical data revealed that the etoposide with G-CSF mobilization group showed the highest yield of CD34+ cells and the lowest change in white blood cell counts during mobilization. In in vitro experiments, etoposide triggered interleukin (IL)-8 secretion from the BMSCs and caused long-term BMSC toxicity. To investigate the manner in which the hBMSC-released IL-8 affects hHSCs in the BM niche, we cultured hHSCs with or without IL-8, and found that the number of total, CD34+, and CD34+/CD45- cells in IL-8-treated cells was significantly higher than the respective number in hHSCs cultured without IL-8 (p = 0.014, 0.020, and 0.039, respectively). Additionally, the relative expression of CXCR2 (an IL-8 receptor), and mTOR and c-MYC (components of IL-8-related signaling pathways) increased 1 h after IL-8 treatment. In animal studies, the etoposide with G-CSF mobilization group presented higher IL-8-related cytokine and MMP9 expression and lower SDF-1 expression in the BM, compared to the groups not treated with etoposide.ConclusionCollectively, the unique mechanism of etoposide with G-CSF-induced mobilization is associated with IL-8 secretion from the BMSCs, which is responsible for the enhanced proliferation and mobilization of HSCs in the bone marrow; this was not observed with mobilization using cyclophosphamide with G-CSF or G-CSF alone. However, the long-term toxicity of etoposide toward BMSCs emphasizes the need for the development of more efficient and safe chemo-mobilization strategies.

BMP-2 Variants in Breast Epithelial to Mesenchymal Transition and Microcalcifications Origin.

This study aims to investigate the possible different roles of the BMP-2 variants, cytoplasmic and nuclear variant, in both epithelial to mesenchymal transition and in microcalcifications origin in human breast cancers. To this end, the in situ expression of cytoplasmic and nuclear BMP-2 was associated with the expression of the main epithelial to mesenchymal transition biomarkers (e-cadherin and vimentin) and molecules involved in bone metabolisms (RUNX2, RANKL, SDF-1) by immunohistochemistry. In addition, the expression of cytoplasmic and nuclear BMP-2 was associated with the presence of microcalcifications. Our data showed a significant association among the number of cytoplasmic BMP-2-positive cells and the number of both vimentin (positive association) and e-cadherin (negative association) positive breast cells. Conversely, no associations were found concerning the nuclear BMP-2-positive breast cells. Surprisingly, the opposite result was obtained by analyzing the variants of BMP-2 and both the expression of RANKL and SDF-1 and the presence of microcalcifications. Specifically, the presence of microcalcifications was related to the expression of nuclear BMP-2 variant rather than the cytoplasmic one, as well as a strong association between the number of nuclear BMP-2 and the expression of the main breast osteoblast-like cells (BOLCs) biomarkers. To further corroborate these data, an in vitro experiment for demonstrating the co-expression of nBMP-2 and RANKL or vimentin or SDF-1 in breast cancer cells that acquire the capability to produce microcalcifications was developed. These investigations confirmed the association between the nBMP-2 expression and both RANKL and SDF-1. The data supports the idea that whilst cytoplasmic BMP-2 can be involved in epithelial to mesenchymal transition phenomenon, the nuclear variant is related to the essential mechanisms for the formation of breast microcalcifications. In conclusion, from these experimental and translational perspectives, the complexity of BMP-2 signaling will require a detailed understanding of the involvement of specific BMP-2 variants in breast cancers.

589-P: Impaired Endothelial Nrf2 Expression Reduces Wound Neovascularization in Diabetic Mice

The global cost of diabetes in the U.S. is 1.3 trillion dollars, with chronic diabetic foot ulcers accounting for up to 13 billion dollars annually. An underlying factor that contributes to the chronicity of these wounds is poor vascular flow, which impedes physiologic wound healing. Here, using an in vivo murine model of wound healing, we demonstrate that Nrf2 expression in endothelial cells is necessary for physiologic neovascularization. Full-thickness 10 mm stented excisional wounds were created on the dorsa of double transgenic mouse with inducible conditional deletion of Nrf2 in cadherin 5 expressing endothelial cells (KO), wildtype (WT), and heterozygous mice. Time to wound closure was delayed in the KO compared to WT mice (KO 33.0±1.73 days vs. WT 14.0±0 days) and Nrf2 KO healed similarly to diabetic mice (KO 33.0±1.73 days vs. Diabetic 31.0±1.41 days). Endothelial cells isolated from the wounds of KO mice via flow cytometry demonstrated significantly decreased angiogenic functionality when compared to heterozygous controls. KO endothelial cells formed significantly fewer branches (KO 64.75±14.81 branches/cm2 vs. control 282.8±46.7 branches/cm2, p=0.014) and fewer branching points (KO 39.0±5.18 branching points/cm2 vs. Control 150.8±15.34 branching points/cm2, p=0.0031), indicating impaired angiogenic activity. Immunofluorescent staining of endothelial cells within the wound beds of KO also demonstrate decreased vascularity compared to WT controls. We looked at regulation of the CXCR4/SDF-1 axis, key to angiogenesis, using C166 mouse endothelial cells. Nrf2 knockdown cells in hypoxia, results in reduced Nrf2 levels by 76.5% (p=0.001) and that of downstream marker NQO1 by 66.4% (p=0.02). Expression of CXCR4 and SDF-1 decreased 61.7% (p=0.001) and 47.6% (p=0.02) respectively. These findings suggest that the Nrf2 dysregulation associated with diabetes may contribute to the impaired angiogenesis and decreased revascularization present in chronic diabetic wounds. Disclosure J. Lee: None. A.P. Villarreal Ponce: None. J.A. David: None. J. Kuhn: None. J. Kwong: None. D. Ceradini: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-ACE-08 to D.C.)

Silibinin Upregulates CXCR4 Expression in Cultured Bone Marrow Cells (BMCs) Especially in Pulmonary Arterial Hypertension Rat Model.

Previously we reported that silibinin ameliorated pulmonary arterial hypertension (PAH) in rat PAH models, possibly through the suppression of the CXCR4/SDF-1, until the point where PAH became a severe and irreversible condition. To further investigate how silibinin ameliorates PAH, we first attempted to clarify its effect on bone marrow cells (BMCs), since the CXCR4/SDF-1 axis is known to regulate stem cell migration and attachment in BM niches. Rat PAH models were established through a combination of a single subcutaneous injection of monocrotaline (MCT) and chronic hypoxic conditions (10% O2). BMCs were harvested and cultured, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry (FCM) were performed to investigate whether silibinin affected CXCR4 expression. Silibinin upregulated the gene expression of stem cell related markers CXCR4, SDF-1, SCF, and c-Kit, inflammatory markers IL-6 and TNFα, mesenchymal stem cell (MSC)-related markers CD44 and CD29, and the granulocyte/monocyte-macrophage marker CD14 in cultured BM in PAH rats, but not in normal rats, except CXCR4. FCM showed that silibinin increased the CXCR4-positive cell population in a granulocyte fraction of cultured BMCs. However, immunohistochemical (IHC) staining showed no significant change in CXCR4 expression in the BM of the tibias. These results suggest that silibinin increases the expression of CXCR4 in BM, and the increased CXCR4-positive cells could be granulocytes/monocyte-macrophages.

Effect of isofebrifugine on the proliferation and invasion of human gastric cancer cells via MMP

Gastric cancer (GC) is one of the most common fatal cancers among gastrointestinal malignancies. At present, the treatment of gastric cancer involves a combination of surgery and chemotherapy. Isofebrifugine (IFE) is an alkaloid with many biological properties. In this study, results from MTT, scratch and invasion assays showed that IFE significantly inhibited the proliferation, migration and invasion of SGC7901 gastric cancer cells. Through RT-PCR and Western blot experiments, it was revealed that IFE significantly inhibited the mRNA and protein expressions of MMP-2, MMP-9 and SDF-1 which are closely related to cancer invasion and metastasis. Thus, IFE possesses anti-gastric cancer properties.

Inhibition of CXCR4 and CXCR7 Is Protective in Acute Peritoneal Inflammation.

Our previous studies revealed a pivotal role of the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 on migratory behavior of polymorphonuclear granulocytes (PMNs) in pulmonary inflammation. Thereby, the SDF-1-CXCR4/CXCR7-axis was linked with adenosine signaling. However, the role of the SDF-1 receptors CXCR4 and CXCR7 in acute inflammatory peritonitis and peritonitis-related sepsis still remained unknown. The presented study provides new insight on the mechanism of a selective inhibition of CXCR4 (AMD3100) and CXCR7 (CCX771) in two models of peritonitis and peritonitis-related sepsis by injection of zymosan and fecal solution. We observed an increased expression of SDF-1, CXCR4, and CXCR7 in peritoneal tissue and various organs during acute inflammatory peritonitis. Selective inhibition of CXCR4 and CXCR7 reduced PMN accumulation in the peritoneal fluid and infiltration of neutrophils in lung and liver tissue in both models. Both inhibitors had no anti-inflammatory effects in A2B knockout animals (A2B–/–). AMD3100 and CCX771 treatment reduced capillary leakage and increased formation of tight junctions as a marker for microvascular permeability in wild type animals. In contrast, both inhibitors failed to improve capillary leakage in A2B–/– animals, highlighting the impact of the A2B-receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A2B. The CXCR4- and CXCR7-inhibitor reduced the release of cytokines in wild type animals via decreased intracellular phosphorylation of ERK and NFκB p65. In vitro, CXCR4 and CXCR7 antagonism diminished the chemokine release of human cells and increased cellular integrity by enhancing the expression of tight junctions. These protective effects were linked with functional A2B-receptor signaling, confirming our in vivo data. In conclusion, our study revealed new protective aspects of the pharmacological modulation of the SDF-1-CXCR4/CXCR7-axis during acute peritoneal inflammation in terms of the two hallmarks PMN migration and barrier integrity. Both anti-inflammatory effects were linked with functional adenosine A2B-receptor signaling.