CXC motif chemokine receptor type 4 (CXCR4) is associated with normal and abnormal development, including oncogenesis. The ligand of CXCR4 is stromal cell-derived factor (SDF), also known as CXC motif ligand (CXCL) 12. Through the SDF-1/CXCR4 axis, both homing and migration of hematopoietic (stem) cells are regulated through niches in the bone marrow. Outside of the bone marrow, however, SDF-1 can recruit CXCR4-positive cells from the bone marrow. SDF/CXCR4 has been implicated in the maintenance and/or differentiation of stemness, and tissue-derived stem cells can be associated with SDF-1 and CXCR4 activity. CXCR4 plays a role in multiple pathways involved in carcinogenesis and other pathologies. Here, we summarize reports detailing the functions of CXCR4. We address the molecular signature of CXCR4 and how this molecule and cells expressing it are involved in either normal (maintaining stemness or inducing differentiation) or abnormal (developing cancer and other pathologies) events. As a constituent of stem cells, the SDF-1/CXCR4 axis influences downstream signal transduction and the cell microenvironment.

Id is one of the helix-loop-helix family of proteins that regulate differentiation in several types of cells, including myoblasts. We found that Id mRNA was constitutively expressed in ROS17/2.8 rat osteoblastic osteosarcoma cells and that the level of Id message in these cells was suppressed by 1,25-dihydroxyvitamin D3 (vitamin D), a calcitropic hormone known to enhance expression of differentiation-related phenotypes in these cells. The vitamin D suppression was dose-dependent, starting at 10(-11) M and saturating at 10(-8) M. This vitamin D effect was seen within 6 hr after the initiation of the treatment and lasted at least 48 hr. Cycloheximide did not block the vitamin D suppression of Id message level. Vitamin D reduced the rate of Id gene transcription by approximately 80% as estimated by nuclear run-on assay. Electrophoretic mobility-shift assay indicated the specific binding of nuclear factors from ROS17/2.8 cells to an E-box DNA sequence, and the binding signal was enhanced in nuclear extracts of the cells treated with vitamin D. The suppressive effect was specific to vitamin D, since another calcitropic hormone, the synthetic compound dexamethasone, did not suppress Id message expression. These observations indicate the presence of helix-loop-helix proteins in osteoblastic cells and indicate that vitamin D, a potent regulator of differentiation in several types of cells, controls the expression of the helix-loop-helix molecules.

3D cultures have gained attention in the field of regenerative medicine for their usefulness asin vitromodel of solid tissues. Bottom-up technology to generate artificial tissues or organs is prospective and an attractive approach that will expand as the field of regenerative medicine becomes more translational. We have characterized c-kit positive cardiac stem cells after long-term cultures and established a 3D-nanoculture system using collagen scaffolds. By combining informatics-based studies, including proteomic analyses and microarrays, we sought to generate methods that modeled cardiac regeneration which can ultimately be used to build artificial hearts. Here, we describe the use of biodegradable beads or 3D cultures to study cardiac regeneration. We summarize recent work that demonstrates that, by using a combination of molecular analyses with 3D cultures, it is possible to evaluate concise mechanisms of solid tissue stem cell biology.

C-X-C chemokine receptor type 4 (CXCR4) may be involved in the development of pulmonary arterial hypertension (PAH). CXCR4 inhibitor AMD3100 was described to have a positive effect on the prevention of pulmonary arterial muscularization in PAH models. Silibinin is a traditional medicine that has an antagonistic effect on CXCR4. We investigated the effect of silibinin using rat models of PAH. PAH was induced by a single subcutaneous injection of monocrotaline. The rats were maintained in a chronic hypoxic condition (10% O2) with or without silibinin. To evaluate the efficacy of silibinin on PAH, right ventricular systolic pressure (RVSP), Fulton index (weight ratio of right ventricle to the left ventricle and septum), percent medial wall thickness (% MT), and vascular occlusion score (VOS) were measured and calculated. Immunohistochemical analysis was performed targeting CXCR4 and c-Kit. Reverse transcription-quantitative polymerase chain reaction was performed for the stem cell markers CXCR4, stromal cell derived factor-1 (SDF-1), c-Kit, and stem cell factor (SCF), and the inflammatory markers monocyte chemoattractant protein 1 (MCP1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα). Statistical analyses were performed using t-test and one-way analysis of variance with Bonferroni’s post hoc test. Silibinin treatment for 1 week reduced RVSP and Fulton index. Treatment for 2 weeks reduced RVSP, Fulton index, % MT, and VOS, as well as downregulating the expression of CXCR4, SDF-1, and TNFα in pulmonary arteries. In contrast, treatment for 3 weeks failed to ameliorate PAH. The time-course study demonstrated that RVSP, Fulton index, % MT, and VOS gradually increased over time, with a decrease in the expression of CXCR4 and TNFα occurring after 2 weeks of PAH development. After 3 weeks, SDF-1, c-Kit, and SCF began to decrease and, after 5 weeks, MCP1 and IL-6 gradually accumulated. The CXCR4 inhibitor silibinin can ameliorate PAH, possibly through the suppression of the CXCR4/SDF-1 axis, until the point where PAH becomes a severe and irreversible condition. Silibinin results in reduced pulmonary arterial pressure and delays pulmonary arteriolar occlusion and pulmonary vascular remodeling.

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.