Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

Endothelial progenitor cells (EPCs) are involved in vascular repair and modulate properties of smooth muscle cells (SMCs) relevant for their contribution to neointima formation following injury. Considering the relevant role of the CXCL12–CXCR4 axis in vascular homeostasis and the potential of EPCs and SMCs to release CXCL12 and express CXCR4, we analyzed the engagement of the CXCL12–CXCR4 axis in various modes of EPC–SMC interaction relevant for injury- and lipid-induced atherosclerosis. We now demonstrate that the expression and release of CXCL12 is synergistically increased in a CXCR4-dependent mechanism following EPC–SMC interaction during co-cultivation or in response to recombinant CXCL12, thus establishing an amplifying feedback loop Additionally, mechanical injury of SMCs induces increased release of CXCL12, resulting in enhanced CXCR4-dependent recruitment of EPCs to SMCs. The CXCL12–CXCR4 axis is crucially engaged in the EPC-triggered augmentation of SMC migration and the attenuation of SMC apoptosis but not in the EPC-mediated increase in SMC proliferation. Compared to EPCs alone, the alliance of EPC–SMC is superior in promoting the CXCR4-dependent proliferation and migration of endothelial cells. When direct cell–cell contact is established, EPCs protect the contractile phenotype of SMCs via CXCL12–CXCR4 and reverse cholesterol-induced transdifferentiation toward a synthetic, macrophage-like phenotype. In conclusion we show that the interaction of EPCs and SMCs unleashes a CXCL12–CXCR4-based autoregulatory feedback loop promoting regenerative processes and mediating SMC phenotype control to potentially guard vascular homeostasis.

CXCL12/CXCR4 Mediates Orthodontic Root Resorption via Regulating the M1/M2 Ratio

Mechanical force–induced external root resorption is a major clinical side effect of orthodontic treatment. Recent work has revealed that M1 macrophages play a vital role in promoting orthodontic root resorption (ORR), but the mechanism of how mechanical force stimulation increases the M1/M2 macrophage ratio in periodontal tissue is poorly understood. In the current study, we showed that C-X-C motif chemokine 12 (CXCL12)+ periodontal ligament cells (PDLCs) and C-X-C chemokine receptor type 4 (CXCR4)+ monocytes in the periodontal ligament (PDL) were significantly increased after force application with ongoing root resorption, and these effects were partially rescued after force removal in mice. The expression of CXCL12 in PDLCs was increased by force stimulation in a time- and intensity-dependent manner in vitro. Blockage of the CXCL12/CXCR4 axis using CXCR4 antagonist AMD3100 was sufficient to alleviate ORR and reverse the force-enhanced M1/M2 macrophage ratio. Further mechanism exploration showed that Ly6Chi inflammatory monocytes homed in a CXCL12/CXCR4 axis-dependent manner. The number and proportion of CD11b+ Ly6Chi inflammatory monocytes in cervical lymph nodes were significantly increased by force loading, accompanied by decreased CD11b+ Ly6Chi monocytes in the blood. These changes were blunted by intraperitoneal injection of AMD3100. In addition, blockage of the CXCL12/CXCR4 axis effectively reversed M2 suppression and promoted M1 polarization. Collectively, results indicate that force-induced CXCL12/CXCR4 axis mediates ORR by increasing the M1/M2 ratio in periodontal tissues through attracting Ly6Chi inflammatory monocytes and modulating macrophage polarization. The results also imply that AMD3100 is potentially inhibitory to root resorption.

CXCL12/CXCR4 Axis Drives Mitochondrial Trafficking in Tumor Myeloma Microenvironment

Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) also transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. As metabolic rewiring is involved in the regulation of MSC phenotype, we first analyzed metabolic profile of healthy control (HC-) and MM-MSCs. NAD +/NADH ratio was decreased in MM-MSCs (n=8) as compared with HC-MSCs (n=4, p<0.05), meanwhile ATP/ADP ratio was not significantly different between the two groups. This led us to analyze whether MM-MSCs were much prone in transferring mitochondria than HC-MSCs. We first labeled HC- and MM-MSCs with Mitotracker Red CMXRos before co-culture with MM cells. After 24h of coculture, we quantified mitochondria transfer by flow cytometry. The obtained values were significantly higher in MM cells co-cultured with MM-MSCs (n=10) as compared to PCs co-cultured with HC-MSCs (n=5, p<0.01). In the cell-to-cell contact the gap junction-forming protein CX43 has been found critical for mitochondria uptake in lung and brain injury and it also can regulate CXCL12 secretion by MSCs. We found that MM-MSCs showed a significantly up-regulated CXCL12 expression as compared to HC-MSCs (p<0.001). Therefore, we co-cultured HS-5 cells with myeloma cell lines and observed that significantly increased CXCL12-CX43 colocalization in healthy MSCs. To evaluate the selective PC-induced activation of CXCL12 expression via CX43 in MSCs, we co-cultured HS-5 cells with MM cell lines and exposed cocultures to ioxynil octanoate (IO), a selective inhibitor of CX43-based gap junctions. We found that the up-regulation of CXCL12 induced by MM cells was reverted by exposition to the CX43 inhibitor, thereby indicating that CX43 activated by PCs regulates CXCL12 production in MSCs. Given that CX43 is involved in mitochondria trafficking, we subsequently cocultured MM cells with HS-5 in presence or not of IO. Our data showed that mitochondrial transfer was abolished by CX43 inhibitor. Given that MM PCs induced increased CX43 and CXCL12 colocalization in HS-5 cells, we supposed that CXCL12/CXCR4 signaling could regulate mitochondria trafficking throughout this axis. For this reason, we analyzed the kinetic of mitochondria uptake of several HMCLs and related their CXCR4 expression with the percentage of transferred mitochondria. Our data demonstrated that HMCLs with higher expression of CXCR4 had also higher percentage of transferred mitochondria both in time lapse and flow cytometry. The correlation between CXCR4 expression and the percentage of mitochondria uptake in HMCLs was also confirmed in primary myeloma PCs. Furthermore, plerixafor, a selective inhibitor of CXCR4, significantly reduced mitochondrial transfer from MSCs to myeloma PCs further establishing mechanistically that CXCR4/CXCL12 is directly involved in mitochondrial trafficking. Next, we investigated whether combination of plerixafor with bortezomib or carfilzomib interferes with mitochondrial transfer from MSCs to PCs. Interestingly, we found that the proteasome inhibitors promoted mitochondrial transfer while their combination with plerixafor inhibited mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. In conclusion, we have shown that MM-MSCs are relatively low dependent on mitochondria metabolism and are inclined to transfer mitochondria to MM tumor cells. Furthermore, tumor PCs increase the expression of CX43 in MSCs leading to an increased levels of CXCL12 and stimulation of its corresponding receptor expressed on MM cells. The resulting CX43/CXCL12/CXCR4 interplay enhances mitochondrial trafficking from MSCs to myeloma PCs and can protect cancer cells against anti-myeloma agents. Understanding pro-tumorigenic phenotype of MSCs and mechanisms of adhesion and heterocellular communication favoring their interaction with cancer PCs, will allow to manipulate critical pathways, including CXCL12/CXCR4 axis, thus improving disease outcome. Disclosures Di Raimondo: Pfizer: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Jazz Pharmaceutical: Honoraria; Janssen Pharmaceuticals: Honoraria; Amgen: Honoraria.

Mesenchymal Stem Cells With Cancer-Associated Fibroblast-Like Phenotype Stimulate SDF-1/CXCR4 Axis to Enhance the Growth and Invasion of B-Cell Acute Lymphoblastic Leukemia Cells Through Cell-to-Cell Communication

Background: Bone marrow mesenchymal stem cells (BM-MSCs) are the stromal cells in the leukemia microenvironment, and can obtain cancer-associated fibroblast (CAF)-like phenotype under certain conditions to further promote leukemia progression. However, the mechanism of MSCs with CAF-like phenotype interacting with leukemia cells in B-cell acute lymphoblastic leukemia (B-ALL) and promoting the progression of B-ALL remains unclear.Methods: Mesenchymal stem cells with CAF-like phenotype were obtained by treating MSCs with recombinant human transforming growth factor-β (rhTGF-β), hereafter referred to as TGF-β conditioned MSCs. In vivo mouse model experiments, in vitro transwell chamber experiments, three-dimensional (3D) cell culture models, lentiviral transfection and other experimental methods were used to investigate the possible mechanism of the interaction between TGF-β conditioned MSCs and leukemia cells in promoting the growth, migration and invasion of B-ALL cells.Results: Compared with untreated MSCs, TGF-β conditioned MSCs significantly promoted the growth and proliferation of leukemia cells in mice, and increased the expression of CXCR4 in tumor tissues. In vitro cell experiments, TGF-β conditioned MSCs obviously promoted the migration and invasion of Nalm-6/RS4;11 cells, which were effectively blocked by the CXCR4 inhibitor AMD3100, thereby inhibiting the secretion of MMP-9 in TGF-β conditioned MSCs and inhibiting the activation of the PI3K/AKT signaling pathway in leukemia cells. Further, findings were made that the interaction between TGF-β conditioned MSCs and leukemia cells were mediated by the interaction between the integrin receptor α5β1 on the surface of leukemia cells and the increased expression of fibronectin on TGF-β conditioned MSCs. AMD3100 could weaken such effect by reducing the expression of integrin α5β1 on leukemia cells. Further regulation of integrin β1 could effectively interfere with the interaction between TGF-β conditioned MSCs and leukemia cells.Conclusion: Mesenchymal stem cells with CAF-like phenotype could be a key factor in promoting the growth and invasion of B-ALL cells, and the SDF-1/CXCR4 axis might be a significant factor in mediating the communication of MSCs with CAF-like phenotype and leukemia cells. To prevent the progression of B-ALL cells, blocking the SDF-1/CXCR4 axis with AMD3100 or targeting integrin β1 might be a potential therapeutic strategy.

Single-cell RNA-seq analysis reveals aberrant CSF1 expression in disease-causing synovial fibroblasts of pigmented villonodular synovitis

Objectives: Although the role of the CSF1/CSF1R axis in pigmented villous synovitis (PVNS) has been confirmed, the cells that express CSF1 and CSF1R and the underlying mechanism remain unclear. Single-cell RNA sequencing (scRNA-seq) of PVNS obtained through biopsies depicted the cellular diversity of PVNS, revealed specific CSF1/CSF1R-expressing cells and further identified novel gene expression that is associated with the development of PVNS. Methods: scRNA-seq was performed on tissues obtained from the 6 biopsies of 3 patients with PVNS. Flow cytometry, immunofluorescence and western blot validated the transcriptional results, while co-culture systems revealed the cross talk between fibroblasts and macrophages. Results: 8 subsets of fibroblasts and 5 subsets of macrophages were identified from the synovium of patients with PVNS and were found to be related to distinct signaling pathways. The cellular components of localized and diffuse PVNS are overall similar. Moreover, the synovium and nodule of PVNS share similar composition. The specific cells expressing CSF1/CSF1R were also identified. Other than that, unique CXCL12+CSF1+ fibroblasts were revealed to attract macrophages as disease-causing synovial fibroblasts, leading to the formation of masses in PVNS. Conclusions: PVNS consists of macrophages, fibroblasts, T cells, endothelial cells and mast cells. Among them, the CSF1-expressing fibroblasts appeared to be tumor-like cells that attract macrophages, subsequently forming tumor-like mass in PVNS. This paves the path for novel treatments of PVNS by targeting CXCL12+CSF1+ fibroblasts and the CXCL12-CXCR4 axis.

Important Role of SDF-1/CXCR4 Axis in the Homing of Systemically Transplanted Human Amnion-Derived Mesenchymal Stem Cells (Had-Mscs) to Ovaries in Rats with Chemotherapy-Induced Premature Ovarian Insufficiency (POI)

Background: Chemotherapy can induce premature ovarian insufficiency (POI). POI causes multiple sequelae and is currently incurable. Our previous studies have demonstrated that systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) can home to chemotherapy-induced POI ovaries, and thus reduce ovarian injury and improve ovarian function in rats with POI. However, the cellular mechanisms that direct the migration and homing of hAD-MSCs to chemotherapy-induced POI ovaries are barely understood. This study was to investigate the role of SDF-1/CXCR4 axis in the migration and homing of systemically transplanted hAD-MSCs to chemotherapy-induced POI ovaries and its relevant downstream signaling pathways.Methods: CXCR4 expression in hAD-MSCs was tested by western blot and immunofluorescence assay. hAD-MSC migration was tested by transwell migration assay. SDF-1 level in rats was detected by ELISA. 72 of female SD rats were randomly divided into the control, POI, hAD-MSCs and hAD-MSCs+AMD3100 groups. POI rat models were established by intraperitoneal injection of cyclophosphamide. For the inhibitor treatment, hAD-MSCs were pretreated with AMD3100 before transplantation. hAD-MSCs labeled with PKH26 were injected into the tail vein of POI rats at 24 h after chemotherapy. After hAD-MSC transplantation, the homing of hAD-MSCs in ovaries, and ovarian function and pathological changes were examined. To further investigate molecular mechanisms, PI3K/Akt and ERK1/2 signaling pathways were detected. Results: hAD-MSCs expressed CXCR4. SDF-1 induced hAD-MSC migration in vitro. SDF-1 levels in ovaries and serum significantly increased in POI rats induced by chemotherapy, and POI ovaries attracted the homing of hAD-MSCs expressing CXCR4. The block of SDF-1/CXCR4 axis with AMD3100 can significantly reduce the number of hAD-MSCs homing to the POI ovaries, and further reduce their efficacy in POI treatment. The binding of SDF-1 to CXCR4 activated PI3K/Akt signaling pathway, and LY294002 significantly inhibited hAD-MSC migration induced by SDF-1 in vitro. Moreover, inhibition of PI3K/Akt signaling pathway significantly reduced the number of systemically transplanted hAD-MSCs homing to chemotherapy-induced POI ovaries in rats.Conclusions: SDF-1/CXCR4 axis partially mediates the migration and homing of systemically transplanted hAD-MSCs to chemotherapy-induced POI ovaries in rats, and PI3K/Akt signaling pathway might be involved in the migration and homing of hAD-MSCs mediated by SDF-1/CXCR4 axis.